@phdthesis{Halboth2018,
  author    = {Halboth, Florian},
  title     = {Building behavior and nest climate control in leaf-cutting ants: How environmental cues affect the building responses of workers of \(Atta\) \(vollenweideri\)},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-161701},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {The present work investigates the influence of environmental stimuli on the building behavior of workers of the leaf-cutting ant Atta vollenweideri. It focuses on cues related to the airflow-driven ventilation of their giant underground nests, i.e., air movements and their direction, carbon dioxide concentrations and humidity levels of the nest air. First, it is shown that workers are able to use airflow and its direction as learned orientation cue by performing learning experiments with individual foragers using a classical conditioning paradigm. This ability is expected to allow workers to also navigate inside the nest tunnels using the prevailing airflow directions for orientation, for example during tasks related to nest construction and climate control. Furthermore, the influence of carbon dioxide on the digging behavior of workers is investigated. While elevated CO2 levels hardly affect the digging rate of the ants, workers prefer to excavate at locations with lower concentrations and avoid higher CO2 levels when given a choice. Under natural conditions, shifting their digging activity to soil layers containing lower carbon dioxide levels might help colonies to excavate new or to broaden existing nest openings, if the CO2 concentration in the underground rises. It is also shown that workers preferably transport excavated soil along tunnels containing high CO2 concentrations, when carbon dioxide levels in the underground are elevated as well. In addition, workers prefer to carry soil pellets along outflow tunnels instead of inflow tunnels, at least for high humidity levels of the air. The material transported along tunnels providing outflow of CO2-rich air might be used by workers for the construction of ventilation turrets on top of the nest mound, which is expected to promote the wind-induced ventilation and the removal of carbon dioxide from the underground. The climatic conditions inside the nest tunnels also influence the structural features of the turrets constructed by workers on top the nest. While airflow and humidity have no effect on turret structure, outflow of CO2-rich air from the nest causes workers to construct turrets with additional openings and increased aperture, potentially enhancing the airflow-driven gas exchanges within the nest. Finally, the effect of airflow and ventilation turrets on the gas exchanges in Atta vollenweideri nests is tested experimentally on a physical model of a small nest consisting of a single chamber and two nest tunnels. The carbon dioxide clearance rate from the underground was measured depending on both the presence of airflow in the nest and the structural features of the built turrets. Carbon dioxide is removed faster from the physical nest model when air moves through the nest, confirming the contribution of wind-induced flow inside the nest tunnels to the ventilation of Atta vollenweideri nests. In addition, turrets placed on top of one of the tunnel openings of the nest further enhance the CO2 clearance rate and the effect is positively correlated with turret aperture. Taken together, climatic variables like airflow, carbon dioxide and humidity levels strongly affect the building responses of Atta vollenweideri leaf-cutting ants. Workers use these environmental stimuli as orientation cue in the nest during tasks related to excavation, soil transport and turret construction. Although the effects of these building responses on the microclimatic conditions inside the nest remain elusive so far, the described behaviors are expected to allow ant colonies to restore and maintain a proper nest climate in the underground.},
  subject      = {Verhalten},
  language  = {en}
}
@phdthesis{Koenig2018,
  author    = {K{\"o}nig, Julia Maria},
  title     = {Fungal grass endophytes and their dependence on land-use intensity},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-163890},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Plant-associated fungi can affect the plants' interaction with herbivores and other microorganisms. For example, many common forage grasses are infected with Epichlo{\"e} endophytes. The endophytes systemically colonize the aerial parts of the plants. They produce bioprotective alkaloids that can negatively affect insects and livestock feeding on the grasses, and interact with other fungal species which living from the plants' nutrients. Environmental conditions strongly influence Epichlo{\"e} endophytes. Endophyte-mediated effects on herbivores are more pronounced under increased temperatures and the endophytes may benefit from land use in managed grasslands. Under the framework of the large-scale German project "Biodiversity Exploratories", I investigated whether infection rates and alkaloid concentrations of Epichlo{\"e} festucae var. lolii in Lolium perenne (Chapter I) and Epichlo{\"e} endophytes (E. uncinata, E. siegelii) in Festuca pratensis (Chapter II) depend on land use and season. Further I analysed, whether foliar fungal assemblages of L. perenne are affected by the presence of Epichlo{\"e} endophytes (Chapter IV).},
  subject      = {Endophytische Pilze},
  language  = {en}
}
@phdthesis{Boeck2018,
  author    = {B{\"o}ck, Julia},
  title     = {Differenzielle Methylierungsanalysen mittels verschiedener Next-Generation Sequencing-basierter Techniken: Die Bedeutung von differenziell methylierten Regionen in der menschlichen Hirnevolution und bei der Krebsentstehung},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-164220},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Die Evolution der Primaten zeigt eine Verbindung zwischen der zunehmenden Komplexit{\"a}t des sozialen Verhaltens und der Vergr{\"o}ßerung des humanen Gehirns, insbesondere des pr{\"a}frontalen Cortex. Deshalb stellt der pr{\"a}frontale Cortex bez{\"u}glich der Evolution des Menschen eine der interessantesten Strukturen im humanen Gehirn dar. Es wird angenommen, dass nicht allein die Gr{\"o}ße, sondern auch die Funktion, vor allem das Zusammenspiel von Neuronen und nicht-neuronalen Zellen, wie z.B. Gliazellen, zur Differenzierung des menschlichen Gehirns von dem rezenter Primaten gef{\"u}hrt hat. Daraus l{\"a}sst sich schließen, dass die Gehirnfunktionen {\"u}ber eine ausgeglichene und gut aufeinander abgestimmte transkriptionelle Landschaft kontrolliert werden, die durch ein zugrundeliegendes genetisches und epigentisches R{\"u}ckgrat organisiert ist. In dieser Studie wurden das Methylierungsprofil neuronaler und nicht-neuronaler Zellen des pr{\"a}frontalen Cortex (Brodmann-Areal 10) von drei Menschen und drei Schimpansen miteinander verglichen. Die intra- und interspezifischen differenziell methylierten Regionen (DMRs) waren in bestimmten genomischen Regionen angereichert. Intraspezifische Methylierungsunterschiede zwischen neuronalen und nicht-neuronalen Zellen konnten dreimal h{\"a}ufiger beobachtet werden als interspezifische Unterschiede in den einzelnen Zelltypen. Rund 90\% der humanen intraspezifischen DMRs wiesen eine Hypomethylierung in den neuronalen Zellen im Vergleich zu den nicht-neuronalen Zellen auf. In den intraspezifischen DMRs (Mensch und Schimpanse) waren Gene angereichert, die mit verschiedenen neuropsychiatrischen Erkrankungen assoziiert sind. Der Vergleich zwischen Menschen und Schimpanse in den neuronalen und nicht-neuronalen Zelltypen zeigte eine Anreicherung von Genen mit human-spezifischer Histonsignatur. In den nicht-neuronalen Zellen konnten mehr interspezifische DMRs (n=666) detektiert werden als in den neuronalen Zellen (n=96). Ungef{\"a}hr 95\% der nicht-neuronalen interspezifischen DMRs waren im Menschen, im Vergleich zum Schimpansen, hypermethyliert. Daraus ergibt sich der Eindruck, dass mehrere hundert der nicht-neuronalen Gene w{\"a}hrend der humanen Gehirnevolution einer Methylierungswelle unterlagen. Dies f{\"u}hrt zu der Annahme, dass der Einfluss dieser Ver{\"a}nderungen in den nicht-neuronalen Zellen auf die Verg{\"o}ßerung des menschlichen Gehirns bisher stark untersch{\"a}tzt wurde. Die bekannteste genetische Ursache f{\"u}r erblichen Brust- und Eierstockkrebs sind Mutationen in den Tumorsuppressorgenen (TSG) BRCA1 und BRCA2. Dennoch k{\"o}nnen nur rund 20-25\% der famili{\"a}ren Brustkrebserkrankungen {\"u}ber Keimbahnmutationen in BRCA1/BRCA2 erkl{\"a}rt werden, besonders bei Frauen, deren Erkrankung vor dem vierzigsten Lebensjahr auftritt. Epigenetische Ver{\"a}nderungen, die zu einer aberranten Genexpression f{\"u}hren, spielen ebenfalls eine wichtige Rolle bei der Karzinogenese und der Entwicklung einer Brustkrebserkrankung. Es ist bekannt, dass TSG nicht nur durch den Verlust der Heterozygotie (engl. loss of heterozygosity, LOH) oder homozygote Deletionen, sondern auch durch transkriptionelle Stilllegung via DNA-Methylierung inaktiviert werden k{\"o}nnen. Im Rahmen dieser Arbeit wurde {\"u}berpr{\"u}ft, welchen Einfluss aberrante Methylierungsmuster im Promotorbereich von TSG auf die Brustkrebskarzinogenese und die Expression der Gene haben. F{\"u}r die Quantifizierung der Epimutationen wurden die Promotorbereiche von acht TSG (BRCA1, BRCA2, RAD51C, ATM, PTEN, TP53, MLH1, RB1) und des estrogene receptor (ESR1) Gens, welches eine Rolle in der Tumorprogression spielt, mittels Deep Bisulfite Amplicon Sequencing (DBAS) analysiert. Es wurden Blutproben von zwei unabh{\"a}ngigen BRCA1/BRCA2-mutationsnegativen Brustkrebs (BC)-Patientenkohorten, sowie von zwei unabh{\"a}ngigen alters-gematchten, gesunden Kontrollkohorten untersucht. BC-Kohorte 1 beinhaltet early-onset (EO) BC-Patientinnen. Kohorte 2 enth{\"a}lt BC-Patientinnen mit einem Risiko von >95\% eine heterozygote Mutation in BRCA1/BRCA2 (high-risk, HR) zu tragen. Allele mit >50\% methylierten CpGs werden als funktionell relevante Epimutationen erachtet, da bekannt ist, dass TSG {\"u}ber eine Methylierung im Promotorbereich transkriptionell stillgelegt werden. Im Vergleich zu ESR1 ({\O} Methylierung, 3\%), welches die Methylierungslevel eines durchschnittlichen Promotors wiederspiegelt, zeigten die TSG sehr geringe durchschnittliche Methylierungswerte von weniger als 1\%. Zudem waren die durchschnittlichen Epimutationsraten (EMR; <0,0001-0,1\%) der TSG sehr gering. Mit der Ausnahme von BRCA1, welches eine erh{\"o}hte EMR in der BC-Kohorte verglichen zu den Kontrollen (0,31\% gegen 0,06\%) zeigte, gab es keine signifikanten Gruppenunterschiede zwischen BC-Patientinnen und Kontrollen. Eine von 36 HR BC-Patientinnen zeigte im Vergleich zu den restlichen Proben eine stark erh{\"o}hte EMR von 14,7\% in BRCA1. Rund ein Drittel (15/44) der EO BC-Patientinnen wiesen eine erh{\"o}hte Rate an Einzel-CpG Fehlern in mehreren TSG auf. Die nachfolgenden Expressionsanalysen ergaben eine erniedrigte Expression vieler TSG je analysierter Patientin. Diese Ergebnisse f{\"u}hren zu der Annahme, dass epigenetische Ver{\"a}nderungen in normalen K{\"o}rperzellen als ein m{\"o}glicher Indikator f{\"u}r einen gest{\"o}rten Mechanismus, der f{\"u}r die Aufrechterhaltung des unmethylierten Status und der daraus resultierenden normalen Genexpression zust{\"a}ndig ist, angesehen werden k{\"o}nnen. Dies kann mit einem erh{\"o}hten BC-Risiko assoziiert werden.},
  subject      = {Epigenetik},
  language  = {de}
}
@phdthesis{Kropf2018,
  author    = {Kropf, Jan},
  title     = {The Dual Olfactory Pathway in the Honeybee Brain: Sensory Supply and Electrophysiological Properties},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-108369},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {The olfactory sense is of utmost importance for honeybees, Apis mellifera. Honeybees use olfaction for communication within the hive, for the identification of nest mates and non-nest mates, the localization of food sources, and in case of drones (males), for the detection of the queen and mating. Honeybees, therefore, can serve as excellent model systems for an integrative analysis of an elaborated olfactory system. To efficiently filter odorants out of the air with their antennae, honeybees possess a multitude of sensilla that contain the olfactory sensory neurons (OSN). Three types of olfactory sensilla are known from honeybee worker antennae: Sensilla trichoidea, Sensilla basiconica and Sensilla placodea. In the sensilla, odorant receptors that are located in the dendritic arborizations of the OSNs transduce the odorant information into electrical information. Approximately 60.000 OSN axons project in two parallel bundles along the antenna into the brain. Before they enter the primary olfactory brain center, the antennal lobe (AL), they diverge into four distinct tracts (T1-T4). OSNs relay onto ~3.000-4.000 local interneurons (LN) and ~900 projection neurons (PN), the output neurons of the AL. The axons of the OSNs together with neurites from LNs and PNs form spheroidal neuropil units, the so-called glomeruli. OSN axons from the four AL input tracts (T1-T4) project into four glomerular clusters. LNs interconnect the AL glomeruli, whereas PNs relay the information to the next brain centers, the mushroom body (MB) - associated with sensory integration, learning and memory - and the lateral horn (LH). In honeybees, PNs project to the MBs and the LH via two separate tracts, the medial and the lateral antennal-lobe tract (m/lALT) which run in parallel in opposing directions. The mALT runs first to the MB and then to the LH, the lALT runs first to the LH and then to the MB. This dual olfactory pathway represents a feature unique to Hymenoptera. Interestingly, both tracts were shown to process information about similar sets of odorants by extracting different features. Individual mALT PNs are more odor specific than lALT PNs. On the other hand, lALT PNs have higher spontaneous and higher odor response action potential (AP) frequencies than mALT PNs. In the MBs, PNs form synapses with ~184.000 Kenyon cells (KC), which are the MB intrinsic neurons. KCs, in contrast to PNs, show almost no spontaneous activity and employ a spatially and temporally sparse code for odor coding. In manuscript I of my thesis, I investigated whether the differences in specificity of odor responses between m- and lALT are due to differences in the synaptic input. Therefore, I investigated the axonal projection patterns of OSNs housed in S. basiconica in honeybee workers and compared them with S. trichoidea and S. placodea using selective anterograde labeling with fluorescent tracers and confocal- microscopy analyses of axonal projections in AL glomeruli. Axons of S. basiconica-associated OSNs preferentially projected into the T3 input-tract cluster in the AL, whereas the two other types of sensilla did not show a preference for a specific glomerular cluster. T3- associated glomeruli had previously been shown to be innervated by mALT PNs. Interestingly, S. basiconica as well as a number of T3 glomeruli lack in drones. Therefore I set out to determine whether this was associated with the reduction of glomeruli innervated by mALT PNs. Retrograde tracing of mALT PNs in drones and counting of innervated glomeruli showed that the number of mALT-associated glomeruli was strongly reduced in drones compared to workers. The preferential projections of S. basiconica-associated OSNs into T3 glomeruli in female workers together with the reduction of mALT-associated glomeruli in drones support the presence of a female-specific olfactory subsystem that is partly innervated by OSNs from S. basiconica and is associated with mALT projection neurons. As mALT PNs were shown to be more odor specific, I suppose that already the OSNs in this subsystem are more odor specific than lALT associated OSNs. I conclude that this female-specific subsystem allows the worker honeybees to respond adequately to the enormous variety of odorants they experience during their lifetime. In manuscript II, I investigated the ion channel composition of mALT and lALT PNs and KCs in situ. This approach represents the first study dealing with the honeybee PN and KC ion channel composition under standard conditions in an intact brain preparation. With these recordings I set out to investigate the potential impact of intrinsic neuronal properties on the differences between m- and lALT PNs and on the sparse odor coding properties of KCs. In PNs, I identified a set of Na+ currents and diverse K+ currents depending on voltage and Na+ or Ca2+ that support relatively high spontaneous and odor response AP frequencies. This set of currents did not significantly differ between mALT and lALT PNs, but targets for potential modulation of currents leading to differences in AP frequencies were found between both types of PNs. In contrast to PNs, KCs have very prominent K+ currents, which are likely to contribute to the sparse response fashion observed in KCs. Furthermore, Ca2+ dependent K+ currents were found, which may be of importance for coincidence detection, learning and memory formation. Finally, I conclude that the differences in odor specificity between m- and lALT PNs are due to their synaptic input from different sets of OSNs and potential processing by LNs. The differences in spontaneous activity between the two tracts may be caused by different neuronal modulation or, in addition, also by interaction with LNs. The temporally sparse representation of odors in KCs is very likely based on the intrinsic KC properties, whereas general excitability and spatial sparseness are likely to be regulated through GABAergic feedback neurons.},
  subject      = {Voltage-Clamp-Methode},
  language  = {en}
}
@phdthesis{Jung2018,
  author    = {Jung, Jamin},
  title     = {Precise timing of the trypanosome cell division cycle},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-114932},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {African trypanosomes are the causative agents of fatal diseases in humans and livestock. Trypanosomes show a complex lifecycle and shuttle between the transmitting vector, the tsetse (Glossina spec.), and the mammalian host. As a result of this the parasite undergoes tremendous changes in morphology and metabolism to adapt to the different living environments. The two best-studied lifecycle stages are the procyclic forms (PCF) that live in the tsetse fly and the proliferative bloodstream form (BSF) that resides in the mammalian blood. The most conspicuous weapon that trypanosomes use to evade the host immune attack is a dense layer of a single protein type, the variant surface glycoprotein (VSG), which shields the entire cell surface. Immune evasion required high rates of surface membrane turnover and surface coat recycling. Trypanosomes show highly polarised cell architecture with all major eukaryotic organelles (endoplasmic reticulum, Golgi apparatus, endosomal apparatus, lysosome, mitochondrion and peroxisome-like glycosomes) generally present in single copy. Furthermore, trypanosomes possess a single flagellum, which is important not only for cellular motility but also for cell division. How the duplication of all these cellular components is coordinated in order to progresss through the cell division cycle is poorly understood. We used trypanosomes as a model organism due to the relative simplicity and the polarised nature of their cell architecture and determined the duplication of all their compartments. This was only possible due to a new synchronisation approach developed during this project. In the first part of the thesis a precise temporal map of the cell division cycle of the BSF T. brucei cell division cycle was generated. By the use of well-described morphological markers (K/N status, new flagellum outgrowth and DNA synthesis) the position of individual cells was determined with high temporal resolution; this allowed us for the first time to synchronise a cell population in silico without affecting the naturally asynchronous growth. In the second part of the thesis we used this tool to follow duplication events of the Major organelles during progression through the cell division cycle. We precisely determined the time points of organelle duplication and found that it is ordered in trypanosomes. Furthermore we found that BSF T. brucei cells do not grow continuously, cell size start to increase rapidly, during a short period of time, late in the cell division cycle. We speculate that the initiation of cell volume increase is temporally separated from the formation of all secretory organelles in order to ensure maintenance of the protective coat, which must remain intact at all times in order for BSF trypanosomes to be able to evade the host immune response.},
  subject      = {Zellteilung},
  language  = {en}
}
@phdthesis{Bargul2018,
  author    = {Bargul, Joel Ltilitan},
  title     = {Characterization of motility and erythrocyte adherence as virulence factors in African trypanosomes},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-115053},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Pathogens causing African animal trypanosomiasis (AAT), the major livestock disease in sub-Saharan Africa, belong to the salivarian group of the African trypanosomes, which are transmitted by the bite of the tsetse fly (Glossina spec.). T. vivax, T. congolense and T. brucei brucei are major pathogens of cattle in particular, causing nagana, with dramatic socio-economic consequences for the affected regions. The parasites additionally have a huge reservoir of other livestock and wild animal hosts. T. brucei, the species which also includes the subspecies pathogenic to humans causing sleeping sickness, has been extensively studied as the cultivatable model trypanosome. But less is known about the other salivarian species, which are not routinely held in culture, if at all possible. A hallmark of trypanosomal lifestyle is the protozoan flagellates incessant motility, which enables them to populate an enormous range of habitats in very diverse hosts. We were now able to characterize, for the first time with high spatiotemporal resolution microscopy, the swimming behaviour and mechanism of the most relevant salivarian species isolated directly from blood. We show the influence of viscosity on the motility of bloodstream form (BSF) cells and simulate their movement between erythrocytes, giving a clear picture of how all analyzed species move under varying environmental conditions. We show that although the basic mechanism of flagellar motility applies to all analyzed species, there are clear morphological differences that produce different reactions to the physical environment. We could define specific conditions for highly increased swimming persistence and speed for compared to the behaviour in standard culture. These results have important implications for the parasites survival strategies in the host, e.g. regarding the capacity for antibody clearance. Although we show all species to effectively remove antibodies from the cell surface, T. congolense differed markedly in its motility behaviour, which gives rise to interesting questions about this species behaviour in the bloodstream. Most of the T. congolense parasites (and to a lesser extent T. vivax) adhere to sheep erythrocytes. Further in vitro studies showed that T. congolense and T. vivax adhered to rabbit, goat, pig and cattle erythrocytes- but binding behaviour was absent in murine blood. Notably, both T. brucei and T. evansi lacked adherence to all studied host erythrocytes. Generally, attachment to blood cells caused reduction of swimming velocities. Judging from its cell architecture, as well as the motility studies in higher media viscosity and in micropillar arrays, T. congolense is not adapted to swim at high speeds in the mammalian bloodstream. Low swimming speeds could allow these purely intravascular parasites to remain bound to the host erythrocytes.},
  subject      = {Motili{\"a}t},
  language  = {en}
}
@phdthesis{Herweg2018,
  author    = {Herweg, Jo-Ana},
  title     = {Die Simkania-Vakuole: Die Rolle von ER, retro-/anterograden Protein- und Lipidtransport},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-136844},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Simkania negevensis (Sn) is a Chlamydia-like obligate intracellular bacterium which replicates within a membrane bound vacuole, termed SCV (Simkania-containing vacuole). The SCV is a unique compartment closely associated with ER-membranes, consequently ER-stress is blocked by the bacteria. SCV morphology is similar among epithelial cells (HeLa229, A549, HEp-2) and macrophages (THP1). The SCV represents the first intracellular interface between the host and pathogen which serves as a replication niche. Identifying human and bacterial factors associated with ER-SCV-membranes should contribute towards the understanding of SCV composition and formation as well as interactions with ER or transports. Comparative studies of the SCV should indicate similarities to the chlamydial inclusion since some host cell factors are already known for Chlamydia. In this thesis, a purification protocol has been established that is applicable to HeLa229 and THP1 ER-SCV-membranes and has been further utilized for proteome and lipidome analyses. 302 bacterial and 1178 human proteins composing ER-SCV-membranes and 885 bacterial proteins composing purified Sn have been identified by using label-free mass spectrometry measurements. Among the human factors of non or Sn infected ER-(SCV-) membranes we found 51 enriched or depleted proteins in addition to 57 transport associated ones that indicated infection induced differences among intracellular protein transport. Contrary regulation of retrograde and anterograde transported proteins could be confirmed by using RNA interference and inhibitor tests, whereby Clathrin-associated and COPI vesicles seem to play a central role. Application of Retro-inhibitors, which interfered with retrograde transport processes between endosome to Golgi or early to late endosomes, as well as Bafilomycin A1 (retrograde, late endosomes and lysosomes) and Brefeldin A (anterograde, ER and Golgi) exerted a strong influence on SCV formation, morphology and intracellular lipid transport. By using label-free mass spectrometry measurements and thin layer chromatography we could determine differences in lipid levels within Sn infected cells, ER-SCV-membranes and purified Sn in comparison to uninfected cells. In addition to lipid enrichment or depletion in whole-cell extracts and ER-SCV-membranes, we identified two infection-specific lipids, cholesterol-ß-Dglucoside and PE 30:0. Further, high-throughput RNA interference tests indicated a dependence of Sn infections on endosome to Golgi and Clathrin-associated vesicle transports. Taken together, we were able to identify initial potential SCV-associated proteins and lipids that were connected to bacterial infection. Furthermore, SCV formation and Sn infectiousness depends on retrograde transport processes and therefore also on acquisition of nutrients, such as lipids.},
  subject      = {Simkania},
  language  = {de}
}
@phdthesis{Schuecker2018,
  author    = {Sch{\"u}cker, Katharina},
  title     = {The molecular architecture of the meiotic chromosome axis as revealed by super-resolution microscopy},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-144199},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {During meiosis proteins of the chromosome axis are important for monitoring chromatin structure and condensation, for pairing and segregation of chromosomes, as well as for accurate recombination. They include HORMA-domain proteins, proteins of the DNA repair system, synaptonemal complex (SC) proteins, condensins and cohesins. To understand more about their function in shaping the meiotic chromosome it is crucial to establish a defined model of their molecular architecture. Up to now their molecular organization was analysed using conventional methods, like confocal scanning microscopy (CLSM) and transmission electron microscopy (TEM). Unfortunately, these techniques are limited either by their resolution power or their localization accuracy. In conclusion, a lot of data on the molecular organization of chromosome axis proteins stays elusive. For this thesis the molecular structure of the murine synaptonemal complex (SC) and the localization of its proteins as well as of three cohesins was analysed with isotropic resolution, providing new insights into their architecture and topography on a nanoscale level. This was done using immunofluorescence labelling in combination with super-resolution microscopy, line profiles and average position determination. The results show that the murine SC has a width of 221.6 nm ± 6.1 nm including a central region (CR) of 148.2 nm ± 2.6 nm. In the CR a multi-layered organization of the central element (CE) proteins was verified by measuring their strand diameters and strand distances and additionally by imaging potential anchoring sites of SYCP1 (synaptonemal complex protein 1) to the lateral elements (LEs). We were able to show that the two LEs proteins SYCP2 and SYCP3 do co-localize alongside their axis and that there is no significant preferential localization towards the inner LE axis of SYCP2. The presented results also predict an orderly organization of murine cohesin complexes (CCs) alongside the chromosome axis in germ cells and support the hypothesis that cohesins in the CR of the SC function independent of CCs. In the end new information on the molecular organization of two main components of the murine chromosome axis were retrieved with nanometer precision and previously unknown details of their molecular architecture and topography were unravelled.},
  subject      = {Meiose},
  language  = {en}
}
@phdthesis{SchenkneeWolf2018,
  author    = {Schenk [n{\´e}e Wolf], Mariela},
  title     = {Timing of wild bee emergence: mechanisms and fitness consequences},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-161565},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Solitary bees in seasonal environments have to align their life-cycles with favorable environmental conditions and resources. Therefore, a proper timing of their seasonal activity is highly fitness relevant. Most species in temperate environments use temperature as a trigger for the timing of their seasonal activity. Hence, global warming can disrupt mutualistic interactions between solitary bees and plants if increasing temperatures differently change the timing of interaction partners. The objective of this dissertation was to investigate the mechanisms of timing in spring-emerging solitary bees as well as the resulting fitness consequences if temporal mismatches with their host plants should occur. In my experiments, I focused on spring-emerging solitary bees of the genus Osmia and thereby mainly on O. cornuta and O. bicornis (in one study which is presented in Chapter IV, I additionally investigated a third species: O. brevicornis). Chapter II presents a study in which I investigated different triggers solitary bees are using to time their emergence in spring. In a climate chamber experiment I investigated the relationship between overwintering temperature, body size, body weight and emergence date. In addition, I developed a simple mechanistic model that allowed me to unite my different observations in a consistent framework. In combination with the empirical data, the model strongly suggests that solitary bees follow a strategic approach and emerge at a date that is most profitable for their individual fitness expectations. I have shown that this date is on the one hand temperature dependent as warmer overwintering temperatures increase the weight loss of bees during hibernation, which then advances their optimal emergence date to an earlier time point (due to an earlier benefit from the emergence event). On the other hand I have also shown that the optimal emergence date depends on the individual body size (or body weight) as bees adjust their emergence date accordingly. My data show that it is not enough to solely investigate temperature effects on the timing of bee emergence, but that we should also consider individual body conditions of solitary bees to understand the timing of bee emergence. In Chapter III, I present a study in which I investigated how exactly temperature determines the emergence date of solitary bees. Therefore, I tested several variants degree-day models to relate temperature time series to emergence data. The basic functioning of such degree-day models is that bees are said to finally emerge when a critical amount of degree-days is accumulated. I showed that bees accumulate degree-days only above a critical temperature value (~4°C in O. cornuta and ~7°C in O. bicornis) and only after the exceedance of a critical calendar date (~10th of March in O. cornuta and ~28th of March in O. bicornis). Such a critical calendar date, before which degree-days are not accumulated irrespective of the actual temperature, is in general less commonly used and, so far, it has only been included twice in a phenology model predicting bee emergence. Furthermore, I used this model to retrospectively predict the emergence dates of bees by applying the model to long-term temperature data which have been recorded by the regional climate station in W{\"u}rzburg. By doing so, the model estimated that over the last 63 years, bees emerged approximately 4 days earlier. In Chapter IV, I present a study in which I investigated how temporal mismatches in bee-plant interactions affect the fitness of solitary bees. Therefore, I performed an experiment with large flight cages serving as mesocosms. Inside these mesocosms, I manipulated the supply of blossoms to synchronize or desynchronize bee-plant interactions. In sum, I showed that even short temporal mismatches of three and six days in bee-plant interactions (with solitary bee emergence before flower occurrence) can cause severe fitness losses in solitary bees. Nonetheless, I detected different strategies by solitary bees to counteract impacts on their fitness after temporal mismatches. However, since these strategies may result in secondary fitness costs by a changed sex ratio or increased parasitism, I concluded that compensation strategies do not fully mitigate fitness losses of bees after short temporal mismatches with their food plants. In the event of further climate warming, fitness losses after temporal mismatches may not only exacerbate bee declines but may also reduce pollination services for later-flowering species and affect populations of animal-pollinated plants. In conclusion, I showed that spring-emerging solitary bees are susceptible to climate change as in response to warmer temperatures bees advance their phenology and show a decreased fitness state. As spring-emerging solitary bees not only consider overwintering temperature but also their individual body condition for adjusting emergence dates, this may explain differing responses to climate warming within and among bee populations which may also have consequences for bee-plant interactions and the persistence of bee populations under further climate warming. If in response to climate warming plants do not shift their phenologies according to the bees, bees may experience temporal mismatches with their host plants. As bees failed to show a single compensation strategy that was entirely successful in mitigating fitness consequences after temporal mismatches with their food plants, the resulting fitness consequences for spring-emerging solitary bees would be severe. Furthermore, I showed that spring-emerging solitary bees use a critical calendar date before which they generally do not commence the summation of degree-days irrespective of the actual temperature. I therefore suggest that further studies should also include the parameter of a critical calendar date into degree-day model predictions to increase the accuracy of model predictions for emergence dates in solitary bees. Although our retrospective prediction about the advance in bee emergence corresponds to the results of several studies on phenological trends of different plant species, we suggest that more research has to be done to assess the impacts of climate warming on the synchronization in bee-plant interactions more accurately.},
  subject      = {wild bees},
  language  = {en}
}
@phdthesis{Mildner2018,
  author    = {Mildner, Stephanie},
  title     = {Temporal organization in \(Camponotus\) \(ants\): endogenous clocks and zeitgebers responsible for synchronization of task-related circadian rhythms in foragers and nurses},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-149382},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {The rotation of the earth around its axis causes recurring and predictable changes in the environment. To anticipate those changes and adapt their physiology and behavior accordingly, most organisms possess an endogenous clock. The presence of such a clock has been demonstrated for several ant species including Camponotus ants, but its involvement in the scheduling of daily activities within and outside the ant nest is fairly unknown. Timing of individual behaviors and synchronization among individuals is needed to generate a coordinated collective response and to maintain colony function. The aim of this thesis was to investigate the presence of a circadian clock in different worker castes, and to determine the daily timing of their behavioral tasks within the colonies of two nectar-collecting Camponotus species. In chapter I, I describe the general temporal organization of work throughout the worker life in the species Camponotus rufipes. Continuous tracking of behavioral activity of individually- marked workers for up to 11 weeks in subcolonies revealed an age-dependent division of labor between interior and exterior workers. After eclosion, the fairly immobile young ants were frequently nurtured by older nurses, yet they started nursing the brood themselves within the first 48 hours of their life. Only 60\% of workers switched to foraging at an age range of one to two weeks, likely because of the reduced needs within the small scale of the subcolonies. Not only the transition rates varied between subcolonies, but also the time courses of the task sequences between workers did, emphasizing the timed allocation of workers to different tasks in response to colony needs. Most of the observed foragers were present outside the nest only during the night, indicating a distinct timing of this behavioral activity on a daily level as well. As food availability, humidity and temperature levels were kept constant throughout the day, the preference for nocturnal activity seems to be endogenous and characteristic for C. rufipes. The subsequent monitoring of locomotor activity of workers taken from the subcolonies revealed the presence of a functional endogenous clock already in one-day old ants. As some nurses displayed activity rhythms in phase with the foraging rhythm, a synchronization of these in-nest workers by social interactions with exterior workers can be hypothesized. Do both castes use their endogenous clock to schedule their daily activities within the colony? In chapter II, I analyzed behavioral activity of C. rufipes foragers and nurses within the social context continuously for 24 hours. As time-restricted access to food sources may be one factor affecting daily activities of ants under natural conditions, I confronted subcolonies with either daily pulses of food availability or ad libitum feeding. Under nighttime and ad libitum feeding, behavioral activity of foragers outside the nest was predominantly nocturnal, confirming the results from the simple counting of exterior workers done in chapter I. Foragers switched to diurnality during daytime feeding, demonstrating the flexible and adaptive timing of a daily behavior. Because they synchronized their activity with the short times of food availability, these workers showed high levels of inactivity. Nurses, in contrast, were active all around the clock independent of the feeding regime, spending their active time largely with feeding and licking the brood. After the feeding pulses, however, a short bout of activity was observed in nurses. During this time period, both castes increasingly interacted via trophallaxis within the nest. With this form of social zeitgeber, exterior workers were able to entrain in-nest workers, a phenomenon observed already in chapter I. Under the subsequent monitoring of locomotor activity under LD conditions the rhythmic workers of both castes were uniformly nocturnal independent of the feeding regime. This endogenous activity pattern displayed by both worker castes in isolation was modified in the social context in adaption to task demands. Chapter III focuses on the potential factors causing the observed plasticity of daily rhythms in the social context in the ant C. rufipes. As presence of brood and conspecifics are likely indicators of the social context, I tested the effect of these factors on the endogenous rhythms of otherwise isolated individuals. Even in foragers, the contact to brood triggered an arrhythmic activity pattern resembling the arrhythmic behavioral activity pattern seen in nurses within the social context. As indicated in chapter I and II, social interaction could be one crucial factor for the synchronization of in nest activities. When separate groups were entrained to phase-shifted light-dark-cycles and monitored afterwards under constant conditions in pairwise contact through a mesh partitioning, both individuals shifted parts of their activity towards the activity period of the conspecific. Both social cues modulated the endogenous rhythms of workers and contribute to the context dependent plasticity in ant colonies. Although most nursing activities are executed arrhythmically throughout the day (chapter II), previous studies reported rhythmic translocation events of the brood in Camponotus nurses. As this behavior favors brood development, the timing of the translocations within the dark nest seems to be crucial. In chapter IV, I tracked translocation activity of all nurses within subcolonies of C. mus. Under the confirmed synchronized conditions of a LD-cycle, the daily pattern of brood relocation was based on the rhythmic, alternating activity of subpopulations with preferred translocation direction either to the warm or to the cold part of the temperature gradient at certain times of the day. Although the social interaction after pulse feeding had noticeable effects on the in-nest activity in C. rufipes (chapter I and II), it was not sufficient to synchronize the brood translocation rhythm of C. mus under constant darkness (e.g. when other zeitgebers were absent). The free-running translocation activity in some nurses demonstrated nevertheless the involvement of an endogenous clock in this behavior, which could be entrained under natural conditions by other potential non-photic zeitgebers like temperature and humidity cycles. Daily cycling of temperature and humidity could not only be relevant for in-nest activities, but also for the foraging activity outside the nest. Chapter V focuses on the monitoring of field foraging rhythms in the sympatric species C. mus and C. rufipes in relation to abiotic factors. Although both species had comparable critical thermal limits in the laboratory, foragers in C. mus were strictly diurnal and therefore foraged under higher temperatures than the predominant nocturnal foragers in C. rufipes. Marking experiments in C. rufipes colonies with higher levels of diurnal activity revealed the presence of temporally specialized forager subpopulations. These results suggest the presence of temporal niches not only between the two Camponotus species, but as well between workers within colonies of the same species. In conclusion, the temporal organization in colonies of Camponotus ants involves not only the scheduling of tasks performed throughout the worker life, but also the precise timing of daily activities. The necessary endogenous clock is already functioning in all workers after eclosion. Whereas the light-dark cycle and food availability seem to be the prominent zeitgebers for foragers, nurses may rely more on non-photic zeitgeber like social interaction, temperature and humidity cycles.},
  subject      = {circadian clocks},
  language  = {en}
}
@phdthesis{Bartossek2018,
  author    = {Bartossek, Thomas},
  title     = {Structural and functional analysis of the trypanosomal variant surface glycoprotein using x-ray scattering techniques and fluorescence microscopy},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-144775},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Trypanosoma brucei is an obligate parasite and causative agent of severe diseases affecting humans and livestock. The protist lives extracellularly in the bloodstream of the mammalian host, where it is prone to attacks by the host immune system. As a sophisticated means of defence against the immune response, the parasite's surface is coated in a dense layer of the variant surface glycoprotein (VSG), that reduces identification of invariant epitopes on the cell surface by the immune system to levels that prevent host immunity. The VSG has to form a coat that is both dense and mobile, to shield invariant surface proteins from detection and to allow quick recycling of the protective coat during immune evasion. This coat effectively protects the parasite from the harsh environment that is the mammalian bloodstream and leads to a persistent parasitemia if the infection remains untreated. The available treatment against African Trypanosomiasis involves the use of drugs that are themselves severely toxic and that can lead to the death of the patient. Most of the drugs used as treatment were developed in the early-to-mid 20th century, and while developments continue, they still represent the best medical means to fight the parasite. The discovery of a fluorescent VSG gave rise to speculations about a potential interaction between the VSG coat and components of the surrounding medium, that could also lead to a new approach in the treatment of African Trypanosomiasis that involves the VSG coat. The initially observed fluorescence signal was specific for a combination of a VSG called VSG'Y' and the triphenylmethane (TPM) dye phenol red. Exchanging this TPM to a bromo-derivative led to the observation of another fluorescence effect termed trypanicidal effect which killed the parasite independent of the expressed VSG and suggests a structurally conserved feature between VSGs that could function as a specific drug target against T. b. brucei. The work of this thesis aims to identify the mechanisms that govern the unique VSG'Y' fluorescence and the trypanocidal effect. Fluorescence experiments and protein mutagenesis of VSG'Y' as well as crystallographic trials with a range of different VSGs were utilized in the endeavour to identify the binding mechanisms between TPM compounds and VSGs, to find potentially conserved structural features between VSGs and to identify the working mechanisms of VSG fluorescence and the trypanocidal effect. These trials have the potential to lead to the formulation of highly specific drugs that target the parasites VSG coat. During the crystallographic trials of this thesis, the complete structure of a VSG was solved experimentally for the first time. This complete structure is a key component in furthering the understanding of the mechanisms governing VSG coat formation. X-ray scattering techniques, involving x-ray crystallography and small angle x-ray scattering were applied to elucidate the first complete VSG structures, which reveal high flexibility of the protein and supplies insight into the importance of this flexibility in the formation of a densely packed but highly mobile surface coat.},
  subject      = {Trypanosoma brucei brucei},
  language  = {en}
}
@phdthesis{Joschinski2018,
  author    = {Joschinski, Jens},
  title     = {Is the phenology of pea aphids (\(Acyrthosiphon\) \(pisum\)) constrained by diurnal rhythms?},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-148099},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {The rotation of the earth leads to a cyclic change of night and day. Numerous strategies evolved to cope with diurnal change, as it is generally advantageous to be synchronous to the cyclic change in abiotic conditions. Diurnal rhythms are regulated by the circadian clock, a molecular feedback loop of RNA and protein levels with a period of circa 24 hours. Despite its importance for individuals as well as for species interactions, our knowledge of circadian clocks is mostly confined to few model organisms. While the structuring of activity is generally adaptive, a rigid temporal organization also has its drawbacks. For example, the specialization to a diurnal pattern limits the breadth of the temporal niche. Organisms that are adapted to a diurnal life style are often poor predators or foragers during night time, constraining the time budget to only diurnal parts of the day/night cycle. Climate change causes shifts in phenology (seasonal timing) and northward range expansions, and changes in season or in latitude are associated with novel day length - temperature correlations. Thus, seasonal organisms will have some life history stages exposed to novel day lengths, and I hypothesized that the diurnal niche determines whether the day length changes are beneficial or harmful for the organism. I thus studied the effects of day length on life-history traits in a multi-trophic system consisting of the pea aphid Acyrthosiphon pisum and predatory larvae of Chrysoperla carnea (common green lacewing) and Episyrphus balteatus (marmalade hoverfly). In order to identify the mechanisms for phenological constraints I then focused on diurnal rhythms and the circadian clock of the pea aphid. Aphids reacted to shorter days with a reduced fecundity and shorter reproductive period. Short days did however not impact population growth, because the fitness constraints only became apparent late in the individual's life. In contrast, E. balteatus grew 13\% faster in the shorter day treatment and preyed on significantly more aphids, whereas C. carnea grew 13\% faster under longer days and the elevation of predation rates was marginally significant. These results show that day length affects vital life-history traits, but that the direction and effect size depends on species. I hypothesized that the constraints or fitness benefits are caused by a constricted or expanded time budget, and hence depend on the temporal niche. E. balteatus is indeed night-active and C. carnea appears to be crepuscular, but very little data exists for A. pisum. Hence, I reared the pea aphid on an artificial diet and recorded survival, moulting and honeydew excretion. The activity patterns were clearly rhythmic and molting and honeydew excretion were elevated during day-time. Thus, the diurnal niche could explain the observed, but weak, day length constraints of aphids. The diurnal niche of some organisms is remarkably flexible, and a flexible diurnal niche may explain why the day length constrains were relatively low in A. pisum. I thus studied its circadian clock, the mechanism that regulates diurnal rhythms. First, I improved an artificial diet for A. pisum, and added the food colorant Brilliant Blue FCF. This food colorant stained gut and honeydew in low concentration without causing mortalities, and thus made honeydew excretion visible under dim red light. I then used the blue diet to raise individual aphids in 16:08 LD and constant darkness (DD), and recorded honeydew excretion and molting under red light every three hours. In addition, we used a novel monitoring setup to track locomotor activity continuously in LD and DD. Both the locomotor rhythm and honeydew excretion of A. pisum appeared to be bimodal, peaking in early morning and in the afternoon in LD. Both metabolic and locomotor rhythm persisted also for some time under constant darkness, indicating that the rhythms are driven by a functional circadian clock. However, the metabolic rhythm damped within three to four days, whereas locomotor rhythmicity persisted with a complex distribution of several free-running periods. These results fit to a damped circadian clock that is driven by multiple oscillator populations, a model that has been proposed to link circadian clocks and photoperiodism, but never empirically tested. Overall, my studies integrate constraints in phenological adaptation with a mechanistic explanation. I showed that a shorter day length can constrain some species of a trophic network while being beneficial for others, and linked the differences to the diurnal niche of the species. I further demonstrated that a flexible circadian clock may alleviate the constraints, potentially by increasing the plasticity of the diurnal niche.},
  subject      = {Tagesrhythmus},
  language  = {en}
}
@phdthesis{Dejure2018,
  author    = {Dejure, Francesca Romana},
  title     = {Investigation of the role of MYC as a stress responsive protein},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-158587},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {The transcription factor MYC is deregulated in over 70\% of all human tumors and, in its oncogenic form, plays a major role in the cancer metabolic reprogramming, promoting the uptake of nutrients in order to sustain the biosynthetic needs of cancer cells. The research presented in this work aimed to understand if MYC itself is regulated by nutrient availability, focusing on the two major fuels of cancer cells: glucose and glutamine. Initial observations showed that endogenous MYC protein levels strongly depend on the availability of glutamine, but not of glucose. Subsequent analysis highlighted that the mechanism which accounts for the glutamine-mediated regulation of MYC is dependent on the 3´-untranslated region (3´-UTR) of MYC. Enhanced glutamine utilization by tumors has been shown to be directly linked to MYC oncogenic activity and MYC-dependent apoptosis has been observed under glutamine starvation. Such effect has been described in experimental systems which are mainly based on the use of MYC transgenes that do not contain the 3´-UTR. It was observed in the present study that cells are able to survive under glutamine starvation, which leads to cell cycle arrest and not apoptosis, as previously reported. However, enforced expression of a MYC transgene, which lacks the 3´-UTR, strongly increases the percentage of apoptotic cells upon starvation. Evaluation of glutamine-derived metabolites allowed to identify adenosine nucleotides as the specific stimulus responsible for the glutamine-mediated regulation of MYC, in a 3´-UTR-dependent way. Finally, glutamine-dependent MYC-mediated effects on RNA Polymerase II (RNAPII) function were evaluated, since MYC is involved in different steps of global transcriptional regulation. A global loss of RNAPII recruitment at the transcriptional start site results upon glutamine withdrawal. Such effect is overcome by enforced MYC expression under the same condition. This study shows that the 3´UTR of MYC acts as metabolic sensor and that MYC globally regulates the RNAPII function according to the availability of glutamine. The observations presented in this work underline the importance of considering stress-induced mechanisms impinging on the 3´UTR of MYC.},
  subject      = {Myc},
  language  = {en}
}
@phdthesis{Burgert2018,
  author    = {Burgert, Anne},
  title     = {Untersuchung von Sphingolipiden und anderen Membrankonjugaten mittels hochaufl{\"o}sender Fluoreszenzmikroskopie},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-145725},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Methoden der Fluoreszenz-Lokalisationsmikroskopie (engl. single-molecule localization microscopy, SMLM) erm{\"o}glichen es Molek{\"u}le zu quantifizieren und deren Verteilung zu analysieren. Im Rahmen dieser Arbeit wurden verschiedene Membranmolek{\"u}le auf unterschiedlichen eukaryotischen Zellen, aber auch auf Prokaryoten mit dSTORM (engl. direct stochastic optical reconstruction microscopy) oder PALM (engl.: photoactivated localization microscopy) aufgenommen und quantifiziert. Bevor jedoch diese hochaufl{\"o}sende fluoreszenzbasierte Technik f{\"u}r biologische Fragestellungen angewendet werden konnten, mussten zun{\"a}chst potentielle Artefakt-ausl{\"o}sende Quellen identifiziert und Strategien gefunden werden, um diese zu eliminieren. Eine m{\"o}gliche Artefakt-Quelle ist eine zu niedrige Photonenzahl, die von Fluorophoren emittiert wird. Werden zu wenige Photonen detektiert, kann die Lokalisation eines Fluorophors weniger pr{\"a}zise bestimmt werden. Dies kann zu einer falschen Abbildung von Strukturen f{\"u}hren oder zu falschen R{\"u}ckschl{\"u}ssen {\"u}ber die Verteilung von Molek{\"u}len. Eine M{\"o}glichkeit die Anzahl der emittierten Photonen zu erh{\"o}hen, ist chemische Additive als Triplettl{\"o}scher einzusetzen. Sie bewirken, dass die Fluorophore wieder in den Grundzustand relaxieren und somit wieder angeregt werden k{\"o}nnen. Es wurden verschiedene Additive, die in der Literatur als Triplettl{\"o}scher beschrieben sind, getestet. Dazu wurden zun{\"a}chst ihre Auswirkungen auf den Triplettzustand verschiedener Fluorophore (Alexa Fluor (Al) 488, 532 und 647 und Atto655) mit Hilfe von Fluoreszenzkorrelationsspektroskopie (FCS) untersucht. Cyclooctatetraen (COT) bewirkte dabei eine Abnahme der Triplettausbeute von Al488, Al532 und Al647 um ~ 40-60\%, bei Atto655 ver{\"a}nderte sie sich nicht. Obwohl die Ergebnisse der FCS-Messungen darauf hindeuten, dass COT in einer erh{\"o}hten Anzahl an emittierten Photonen resultiert, konnte dies bei dSTORM-Messungen nicht best{\"a}tigt werden. Hier hatte COT nur einen gr{\"o}ßeren positiven Effekt auf das Fluorophor Al647 (Zunahme um ~ 60\%). Eine Erkl{\"a}rung f{\"u}r diese Widerspr{\"u}chlichkeit zu den Ergebnissen aus den FCS-Messungen, k{\"o}nnte das Vorhandensein des Schaltpuffers bei dSTORM-Messungen sein. Dieser bewirkt den {\"U}bergang der Fluorophore in den Aus-Zustand bzw. entzieht dem Puffer Sauerstoff. Bei der Zugabe von 5 mM Kaliumiodid (KI) nahm die Triplettamplitude bei FCS-Messungen nur bei Al488 ab (um ~ 80\%). Eine geringe Steigerung (um ~ 10\%) der Intensit{\"a}t von Al488 mit KI konnte bei dSTORM-Messungen mit niedrigen Konzentrationen (~ 0,5 mM) erzielt werden. Bei einer Konzentration von 5 mM sank die Intensit{\"a}t jedoch wieder um 40\%. Deuteriumoxid (D2O) soll, anders als die Triplettl{\"o}scher, eine Verbesserung der Photonenausbeute dadurch bewirken, dass strahlungslose Relaxationsprozesse minimiert werden. Mit dSTORM-Messungen konnte gezeigt werden, dass Atto655 und Al647 in D2O zwar pro An-Zustand mehr Photonen emittieren als in Schaltpuffer ohne D2O, da die Fluorophore hier jedoch schneller bleichen, letztendlich die gleiche Anzahl an Photonen detektiert werden. Um die Anzahl an emittierten Photonen zu erh{\"o}hen, eignet sich also nur COT bei dSTORM-Messungen mit AL647 und KI in sehr geringen Konzentrationen bei Al488. D2O kann eingesetzt werden, wenn eine Probe schnell vermessen werden muss, wie zum Beispiel bei Lebendzellmessungen. Nicht nur eine zu niedrige Photonenzahl, auch eine zu geringe Photoschaltrate kann Artefakte bei dSTORM-Messungen erzeugen. Dies wurde anhand von verschiedenen biologischen Strukturen, die mit unterschiedlichen Anregungsintensit{\"a}ten aufgenommen wurden, deutlich gemacht. Besonders die Aufnahmen von Plasmamembranen sind anf{\"a}llig f{\"u}r die Generierung von Artefakten. Sie weisen viele inhomogene und lokal dichte Regionen auf. Wenn nun mehr als ein Emitter pro µm² gleichzeitig an ist, erzeugt das Auswertungsprogramm große artifizielle Cluster. Die hier durchgef{\"u}hrten Messungen machen deutlich, wie wichtig es ist, dSTORM-Bilder immer auf m{\"o}gliche Artefakte hin zu untersuchen, besonders wenn Molek{\"u}le quantifiziert werden sollen. Daf{\"u}r m{\"u}ssen die unbearbeiteten Rohdaten sorgf{\"a}ltig gesichtet werden und notfalls die Messungen mit einer h{\"o}heren Laserleistung wiederholt werden. Da dSTORM mittlerweile immer mehr zur Quantifizierung eingesetzt wird und Clusteranalysen durchgef{\"u}hrt werden, w{\"a}re es sinnvoll bei Ver{\"o}ffentlichungen die Rohdaten von entscheidenden Aufnahmen der {\"O}ffentlichkeit zur Verf{\"u}gung zu stellen. Die F{\"a}rbemethode ist ein weiterer Punkt, durch den Artefakte bei der Abbildung von Molek{\"u}len mittels SMLM entstehen k{\"o}nnen. H{\"a}ufig werden Antik{\"o}rper zum Markieren verwendet. Dabei sollte darauf geachtet werden, dass m{\"o}glichst kleine Antik{\"o}rper oder Antik{\"o}rperfragmente verwendet werden, besonders wenn Clusteranalysen durchgef{\"u}hrt werden sollen. Anderenfalls leidet die Aufl{\"o}sung darunter, bzw. erh{\"o}ht sich die Gefahr der Kreuzvernetzung von Molek{\"u}len. Im zweiten Teil der vorliegenden Arbeit, wurden Plasmamembran-Ceramide untersucht. Ceramide geh{\"o}ren zu den Sphingolipiden und regulieren diverse zellul{\"a}re Prozesse. Verschiedene Stimuli bewirken eine Aktivierung von Sphingomyelinasen (SMasen), die Ceramide in der Plasmamembran synthetisieren. Steigt die Konzentration von Ceramiden in der Plasmamembran an, kondensieren diese zu Ceramid-reichen Plattformen (CRPs). Bisher ist noch wenig {\"u}ber die Verteilung der Ceramide und die Gr{\"o}ße der CRPs bekannt. Sie wurden hier {\"u}ber IgG-Antik{\"o}rper in der Plasmamembran von Jurkat-, U2OS-, HBME- und prim{\"a}ren T-Zellen angef{\"a}rbt und erstmals mit dSTORM hochaufgel{\"o}st, um sie dann zu quantifizieren. Unabh{\"a}ngig von der Zelllinie befanden sich 50\% aller Ceramidmolek{\"u}le in ~ 75 nm großen CRPs. Im Mittel bestanden die CRPs aus ~ 20 Ceramiden. Mit Hilfe einer Titrationsreihe konnte ausgeschlossen werden, dass diese Cluster nur durch die Antik{\"o}rper-F{\"a}rbung artifiziell erzeugt wurden. Bei Inkubation der Zellen mit Bacillus cereus Sphingomyelinase (bSMase) stieg die Gesamtkonzentration der Ceramide in der Plasmamembran an, ebenso wie die Ceramidanzahl innerhalb der CRPs, außerdem die Anzahl und Gr{\"o}ße der CRPs. Dies k{\"o}nnte zu einer Ver{\"a}nderung der L{\"o}slichkeit von Membrankomponenten f{\"u}hren, was wiederum eine Akkumulation bestimmter Rezeptoren oder eine Kompartimentierung bestimmter Proteine erleichtern k{\"o}nnte. Die Anh{\"a}ufung der Ceramide in den CRPs k{\"o}nnte ebenfalls die lokale Interaktion mit anderen Membranmolek{\"u}len erleichtern und dadurch m{\"o}glicherweise die Reaktivit{\"a}t von Rezeptoren ver{\"a}ndern. Mittels Azid-modifizierten Ceramidanaloga und kupferfreier Click-Chemie wurden Plasmamembran-Ceramide auch in lebenden Jurkat-Zellen mit Hilfe konfokaler Laser-Raster-Mikroskopie (CLSM, engl. confocal laser scanning microscopy) und Strukturierter Beleuchtungsmikroskopie (SIM, engl. structured illumination microscopy) untersucht. Dabei konnte gezeigt werden, dass die Fetts{\"a}ure-Kettenl{\"a}nge und die Position des Azids bei den Ceramidanaloga eine entscheidende Rolle spielt, wie hoch das detektierte Signal in der Plasmamembran letztendlich ist. Die Versuche machen auch deutlich, dass die klickbaren Ceramidanaloga lebendzellkompatibel sind, sodass sie eine hervorragende M{\"o}glichkeit darstellen, zellul{\"a}re Reaktionen zu verfolgen. Es wurden hier nicht nur Ceramide in eukaryotischen Zellen analysiert, sondern auch in Bakterien. Neisseria meningitidis (N. meningitidis) sind gramnegative Bakterien, die im Menschen eine Sepsis oder eine Meningitis ausl{\"o}sen k{\"o}nnen. Es wurde mittels immunhistochemischen F{\"a}rbungen mit dem anti-Ceramid IgG-Antik{\"o}rper, aber auch mit den klickbaren Ceramidanaloga, ein Signal in der Membran erhalten, was mit dSTORM hochaufgel{\"o}st wurde. In anderen Bakterien wurden ebenfalls schon Sphingolipide nachgewiesen. Studien zu Ceramiden in N. meningitidis wurden bisher jedoch noch nicht ver{\"o}ffentlicht. Im Rahmen dieser Arbeit konnten erstmals Ergebnisse erhalten werden, die darauf hinweisen, dass N. meningitidis ebenfalls Ceramide besitzen k{\"o}nnten. In einem dritten Projekt wurde die Interaktion zwischen NK-Zellen und Aspergillus fumigatus untersucht. Der Schimmelpilz kann eine Invasive Aspergillose in immunsupprimierten Menschen ausl{\"o}sen, was zum Tod f{\"u}hren kann. Verschiedene Studien konnten schon zeigen, dass NK-Zellen eine wichtige Rolle bei der Bek{\"a}mpfung des Pilzes spielen. Der genaue Mechanismus ist jedoch noch unbekannt. Im Rahmen dieser Arbeit konnte nachgewiesen werden, dass der NK-Zell-Marker CD56 entscheidend f{\"u}r die Pilzerkennung ist. Mit immunhistochemischen F{\"a}rbungen und LSM-, aber auch dSTORM-Messungen, konnte gezeigt werden, dass die normalerweise homogen verteilten CD56-Rezeptoren auf der Plasmamembran von NK-Zellen aktiv an die Interaktionsstelle zu A. fumigatus transportiert werden. Mit der Zeit akkumulieren hier immer mehr CD56-Proteine, w{\"a}hrend das Signal in der restlichen Membran immer weiter abnimmt. Es konnte erstmals CD56 als wichtiger Erkennungsrezeptor f{\"u}r A. fumigatus identifiziert werden. In dem letzten bearbeiteten Projekt, wurde die Bindung von Anti-N-Methyl-D-Aspartat (NMDA)-Rezeptor Enzephalitis Autoantik{\"o}rper an Neuronen untersucht. Bei einer Anti-NMDA-Rezeptor Enzephalitis bilden die Patienten Autoantik{\"o}rper gegen die NR1-Untereinheit ihrer eigenen postsynaptischen NMDA-Rezeptoren. Da die Krankheit oft sehr sp{\"a}t erkannt wird und die Behandlungsm{\"o}glichkeiten noch sehr eingeschr{\"a}nkt sind, f{\"u}hrt sie noch oft zum Tod. Sie wurde erst vor wenigen Jahren beschrieben, sodass der genaue Mechanismus noch unbekannt ist. Im Rahmen dieser Arbeit, konnten erste F{\"a}rbungen mit aufgereinigten Antik{\"o}rper aus Anti-NMDA-Rezeptor Enzephalitis Patienten an NMDA-Rezeptor-transfizierte HEK-Zellen und hippocampalen Maus-Neuronen durchgef{\"u}hrt und mit dSTORM hochaufgel{\"o}st werden. Mit den Messungen der HEK-Zellen konnte best{\"a}tigt werden, dass die Autoantik{\"o}rper an die NR1-Untereinheit der Rezeptoren binden. Es konnten erstmals auch die Bindung der Antik{\"o}rper an Neuronen hochaufgel{\"o}st werden. Dabei wurde sichtbar, dass die Antik{\"o}rper zum einen dicht gepackt in den Synapsen vorliegen, aber auch d{\"u}nner verteilt in den extrasynaptischen Regionen. Basierend auf der Ripley's H-Funktion konnten in den Synapsen große Cluster von ~ 90 nm Durchmesser und im Mittel ~ 500 Lokalisationen und extrasynaptisch kleinere Cluster mit einem durchschnittlichen Durchmesser von ~ 70 nm und ~ 100 Lokalisationen ausgemacht werden. Diese ersten Ergebnisse legen den Grundstein f{\"u}r weitere Messungen, mit denen der Mechanismus der Krankheit untersucht werden kann.},
  subject      = {Ceramide},
  language  = {de}
}
@phdthesis{Kay2018,
  author    = {Kay, Janina},
  title     = {The circadian clock of the carpenter ant \(Camponotus\) \(floridanus\)},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-158061},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Due to the earth´s rotation around itself and the sun, rhythmic daily and seasonal changes in illumination, temperature and many other environmental factors occur. Adaptation to these environmental rhythms presents a considerable advantage to survival. Thus, almost all living beings have developed a mechanism to time their behavior in accordance. This mechanism is the endogenous clock. If it fulfills the criteria of (1) entraining to zeitgebers (2) free-running behavior with a period of ~ 24 hours (3) temperature compensation, it is also referred to as "circadian clock". Well-timed behavior is crucial for eusocial insects, which divide their tasks among different behavioral castes and need to respond to changes in the environment quickly and in an orchestrated fashion. Circadian rhythms have thus been studied and observed in many eusocial species, from ants to bees. The underlying mechanism of this clock is a molecular feedback loop that generates rhythmic changes in gene expression and protein levels with a phase length of approximately 24 hours. The properties of this feedback loop are well characterized in many insects, from the fruit fly Drosophila melanogaster, to the honeybee Apis mellifera. Though the basic principles and components of this loop are seem similar at first glance, there are important differences between the Drosophila feedback loop and that of hymenopteran insects, whose loop resembles the mammalian clock loop. The protein PERIOD (PER) is thought to be a part of the negative limb of the hymenopteran clock, partnering with CRYPTOCHROME (CRY). The anatomical location of the clock-related neurons and the PDF-network (a putative in- and output mediator of the clock) is also well characterized in Drosophila, the eusocial honeybee as well as the nocturnal cockroach Leucophea maderae. The circadian behavior, anatomy of the clock and its molecular underpinnings were studied in the carpenter ant Camponotus floridanus, a eusocial insect Locomotor activity recordings in social isolation proved that the majority of ants could entrain to different LD cycles, free-ran in constant darkness and had a temperature-compensated clock with a period slightly shorter than 24 hours. Most individuals proved to be nocturnal, but different types of activity like diurnality, crepuscularity, rhythmic activity during both phases of the LD, or arrhythmicity were also observed. The LD cycle had a slight influence on the distribution of these activities among individuals, with more diurnal ants at shorter light phases. The PDF-network of C. floridanus was revealed with the anti-PDH antibody, and partly resembled that of other eusocial or nocturnal insects. A comparison of minor and major worker brains, only revealed slight differences in the number of somata and fibers crossing the posterior midline. All in all, most PDF-structures that are conserved in other insects where found, with numerous fibers in the optic lobes, a putative accessory medulla, somata located near the proximal medulla and many fibers in the protocerebrum. A putative connection between the mushroom bodies, the optic lobes and the antennal lobes was found, indicating an influence of the clock on olfactory learning. Lastly, the location and intensity of PER-positive cell bodies at different times of a 24 hour day was established with an antibody raised against Apis mellifera PER. Four distinct clusters, which resemble those found in A. mellifera, were detected. The clusters could be grouped in dorsal and lateral neurons, and the PER-levels cycled in all examined clusters with peaks around lights on and lowest levels after lights off. In summary, first data on circadian behavior and the anatomy and workings of the clock of C. floridanus was obtained. Firstly, it´s behavior fulfills all criteria for the presence of a circadian clock. Secondly, the PDF-network is very similar to those of other insects. Lastly, the location of the PER cell bodies seems conserved among hymenoptera. Cycling of PER levels within 24 hours confirms the suspicion of its role in the circadian feedback loop.},
  subject      = {Chronobiologie},
  language  = {en}
}
@phdthesis{Aufmkolk2018,
  author    = {Aufmkolk, Sarah},
  title     = {Super-Resolution Microscopy of Synaptic Proteins},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-151976},
  school      = {Universit{\"a}t W{\"u}rzburg},
  pages     = {X, 97},
  year      = {2018},
  abstract  = {The interaction of synaptic proteins orchestrate the function of one of the most complex organs, the brain. The multitude of molecular elements influencing neurological correlations makes imaging processes complicated since conventional fluorescence microscopy methods are unable to resolve structures beyond the diffraction-limit. The implementation of super-resolution fluorescence microscopy into the field of neuroscience allows the visualisation of the fine details of neural connectivity. The key element of my thesis is the super-resolution technique dSTORM (direct Stochastic Optical Reconstruction Microscopy) and its optimisation as a multi-colour approach. Capturing more than one target, I aim to unravel the distribution of synaptic proteins with nanometer precision and set them into a structural and quantitative context with one another. Therefore dSTORM specific protocols are optimized to serve the peculiarities of particular neural samples. In one project the brain derived neurotrophic factor (BDNF) is investigated in primary, hippocampal neurons. With a precision beyond 15 nm, preand post-synaptic sites can be identified by staining the active zone proteins bassoon and homer. As a result, hallmarks of mature synapses can be exhibited. The single molecule sensitivity of dSTORM enables the measurement of endogenous BDNF and locates BDNF granules aligned with glutamatergic pre-synapses. This data proofs that hippocampal neurons are capable of enriching BDNF within the mature glutamatergic pre-synapse, possibly influencing synaptic plasticity. The distribution of the metabotropic glutamate receptor mGlu4 is investigated in physiological brain slices enabling the analysis of the receptor in its natural environment. With dual-colour dSTORM, the spatial arrangement of the mGlu4 receptor in the pre-synaptic sites of parallel fibres in the molecular layer of the mouse cerebellum is visualized, as well as a four to six-fold increase in the density of the receptor in the active zone compared to the nearby environment. Prior functional measurements show that metabotropic glutamate receptors influence voltage-gated calcium channels and proteins that are involved in synaptic vesicle priming. Corresponding dSTORM data indeed suggests that a subset of the mGlu4 receptor is correlated with the voltage-gated calcium channel Cav2.1 on distances around 60 nm. These results are based on the improvement of the direct analysis of localisation data. Tools like coordinated based correlation analysis and nearest neighbour analysis of clusters centroids are used complementary to map protein connections of the synapse. Limits and possible improvements of these tools are discussed to foster the quantitative analysis of single molecule localisation microscopy data. Performing super-resolution microscopy on complex samples like brain slices benefits from a maximised field of view in combination with the visualisation of more than two targets to set the protein of interest in a cellular context. This challenge served as a motivation to establish a workflow for correlated structured illumination microscopy (SIM) and dSTORM. The development of the visualisation software coSIdSTORM promotes the combination of these powerful super-resolution techniques even on separated setups. As an example, synapses in the cerebellum that are affiliated to the parallel fibres and the dendrites of the Purkinje cells are identified by SIM and the protein bassoon of those pre-synapses is visualised threedimensionally with nanoscopic precision by dSTORM. In this work I placed emphasis on the improvement of multi-colour super-resolution imaging and its analysing tools to enable the investigation of synaptic proteins. The unravelling of the structural arrangement of investigated proteins supports the building of a synapse model and therefore helps to understand the relation between structure and function in neural transmission processes.},
  subject      = {Hochaufl{\"o}sende Mikroskopie},
  language  = {en}
}
@phdthesis{Bucher2018,
  author    = {Bucher, Hannes},
  title     = {Pre-clinical modeling of viral- and bacterial-induced exacerbations of chronic obstructive pulmonary disease},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-144368},
  school      = {Universit{\"a}t W{\"u}rzburg},
  pages     = {XIII, 105},
  year      = {2018},
  abstract  = {Chronic Obstructive Pulmonary Disease (COPD) exacerbations are a considerable reason for increased morbidity and mortality in patients. Infections with influenza virus (H1N1), respiratory syncytial virus (RSV) or nontypeable Haemophilus influenzae (NTHi) are important triggers of exacerbations. To date, no treatments are available which can stop the progression of COPD. Novel approaches are urgently needed. Pre-clinical models of the disease are crucial for the development of novel therapeutic options. In order to establish pre-clinical models which mimic aspects of human COPD exacerbations, mice were exposed to cigarette smoke (CS) and additionally infected with H1N1, RSV and/or NTHi. Clinically relevant treatments such as the corticosteroids Fluticasone propionate and Dexamethasone, the phosphodiesterase-4 (PDE-4) inhibitor Roflumilast and the long-acting muscarinic receptor antagonist Tiotropium were tested in the established models. Furthermore, a novel treatment approach using antibodies (Abs) directed against IL-1α, IL-1β or IL-1R1 was examined in the established CS/H1N1 model. Levels of IFN-γ, IL-1β, IL-2, IL-6, KC, TNF-α, RANTES, IL-17, MCP-1, MIP 1α and MIP-1β were measured in lung homogenate. Numbers of total cells, neutrophils and macrophages were assessed in bronchoalveolar lavage (BAL) fluid. Hematoxylin- and eosin- (H\&E-) stained lung slices were analyzed to detect pathological changes. Quantitative polymerase-chain-reaction (qPCR) was used to investigate gene expression of ICAM-1 and MUC5 A/C. The viral/bacterial load was investigated in lung homogenate or BAL fluid. In addition to the in vivo studies, the effects of the above mentioned treatments were investigated in vitro in H1N1, RSV or NTHi-infected (primary) human bronchial epithelial cells using submerged or air-liquid-interface (ALI) cell culture systems. Four pre-clinical models (CS/H1N1, CS/RSV, CS/NTHi, CS/H1N1/NTHi) were established depicting clinically relevant aspects of COPD exacerbations such as increased inflammatory cells and cytokines in the airways and impaired lung function. In the CS/H1N1 model, Tiotropium improved lung function and was superior in reducing inflammation in comparison to Fluticasone or Roflumilast. Moreover, Fluticasone increased the loss of body-weight, levels of IL-6, KC and TNF-α and worsened lung function. In CS/RSV-exposed mice Tiotropium but not Fluticasone or Roflumilast treatment reduced neutrophil numbers and IL-6 and TNF α levels in the lung. The viral load of H1N1 and RSV was significantly elevated in CS/virus-exposed mice and NCI-H292 cells after Fluticasone and Dexamethasone treatment. The results from these studies demonstrate that Tiotropium has anti-inflammatory effects on CS/virus-induced inflammation and might help to explain the observed reduction of exacerbation rates in Tiotropium-treated COPD patients. Furthermore, the findings from this work indicate that treatment with Fluticasone or Dexamethasone might not be beneficial to reduce inflammation in the airways of COPD patients and supports clinical studies that link treatment with corticosteroids to an increased risk for pneumonia. Testing of anti-IL-1α, anti-IL-1β or anti-IL-1R1 Abs in the CS/H1N1 model suggests that, in line with clinical data, antagonization of IL-1β is not sufficient to reduce pulmonary inflammation and indicates a predominant role of IL-1α in CS/virus-induced airway inflammation. In line with the in vivo findings, anti-IL-1α but not anti-IL-1β Abs reduced levels of TNF-α and IL-6 in H1N1-infected primary human bronchial epithelial ALI cell culture. Blocking the IL-1R1 provided significant inhibitory effects on inflammatory cells in vivo but was inferior compared to inhibiting both its soluble ligands IL-1α and IL-1β. Concomitant usage of Abs against IL-1α/IL-1β revealed strong effects and reduced total cells, neutrophils and macrophages. Additionally, levels of KC, IL-6, TNF-α, MCP-1, MIP-1α and MIP-1β were significantly reduced and ICAM-1 mRNA expression was attenuated. These results suggest that combined inhibition of IL-1α/IL-1β might be beneficial to reduce inflammation and exacerbations in COPD patients. Moreover, combined targeting of both IL-1α/IL-1β might be more efficient compared to inhibition of the IL-1R1. As in the CS/virus models, corticosteroid treatment failed to reduce inflammatory cells in the CS/NTHi and CS/H1N1/NTHi models, increased the loss of body-weight and the bacterial load. Furthermore, Roflumilast administration had no significant effects on cell counts or cytokines. However, it improved compliance in the CS/NTHi model. Treatment with Azithromycin reduced the bacterial load in the CS/NTHi model and reduced numbers of total cells, neutrophils, macrophages and levels of KC and TNF-α in the CS/H1N1/NTHi model. In conclusion, the established CS/H1N1, CS/RSV, CS/NTHi, CS/H1N1/NTHi models depict clinically relevant aspects of human COPD exacerbations in mice and provide the opportunity to investigate underlying disease mechanisms and to test novel therapies.},
  subject      = {Obstruktive Ventilationsst{\"o}rung},
  language  = {en}
}
@phdthesis{Bemm2018,
  author    = {Bemm, Felix Mathias},
  title     = {Genetic foundation of unrivaled survival strategies - Of water bears and carnivorous plants -},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-157109},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {All living organisms leverage mechanisms and response systems to optimize reproduction, defense, survival, and competitiveness within their natural habitat. Evolutionary theories such as the universal adaptive strategy theory (UAST) developed by John Philip Grime (1979) attempt to describe how these systems are limited by the trade-off between growth, maintenance and regeneration; known as the universal three-way trade-off. Grime introduced three adaptive strategies that enable organisms to coop with either high or low intensities of stress (e.g., nutrient deficiency) and environmental disturbance (e.g., seasons). The competitor is able to outcompete other organisms by efficiently tapping available resources in environments of low intensity stress and disturbance (e.g., rapid growers). A ruderal specism is able to rapidly complete the life cycle especially during high intensity disturbance and low intensity stress (e.g., annual colonizers). The stress tolerator is able to respond to high intensity stress with physiological variability but is limited to low intensity disturbance environments. Carnivorous plants like D. muscipula and tardigrades like M. tardigradum are two extreme examples for such stress tolerators. D. muscipula traps insects in its native habitat (green swamps in North and South Carolina) with specialized leaves and thereby is able to tolerate nutrient deficient soils. M. tardigradum on the other side, is able to escape desiccation of its terrestrial habitat like mosses and lichens which are usually covered by a water film but regularly fall completely dry. The stress tolerance of the two species is the central study object of this thesis. In both cases, high througput sequencing data and methods were used to test for transcriptomic (D. muscipula) or genomic adaptations (M. tardigradum) which underly the stress tolerance. A new hardware resource including computing cluster and high availability storage system was implemented in the first months of the thesis work to effectively analyze the vast amounts of data generated for both projects. Side-by-side, the data management resource TBro [14] was established together with students to intuitively approach complex biological questions and enhance collaboration between researchers of several different disciplines. Thereafter, the unique trapping abilities of D. muscipula were studied using a whole transcriptome approach. Prey-dependent changes of the transcriptional landscape as well as individual tissue-specific aspects of the whole plant were studied. The analysis revealed that non-stimulated traps of D. muscipula exhibit the expected hallmarks of any typical leaf but operates evolutionary conserved stress-related pathways including defense-associated responses when digesting prey. An integrative approach, combining proteome and transcriptome data further enabled the detailed description of the digestive cocktail and the potential nutrient uptake machinery of the plant. The published work [25] as well as a accompanying video material (https://www.eurekalert.org/pub_releases/ 2016-05/cshl-fgr042816.php; Video credit: S{\"o}nke Scherzer) gained global press coverage and successfully underlined the advantages of D. muscipula as experimental system to understand the carnivorous syndrome. The analysis of the peculiar stress tolerance of M. tardigradum during cryptobiosis was carried out using a genomic approach. First, the genome size of M. tardigradum was estimated, the genome sequenced, assembled and annotated. The first draft of M. tardigradum and the workflow used to established its genome draft helped scrutinizing the first ever released tardigrade genome (Hypsibius dujardini) and demonstrated how (bacterial) contamination can influence whole genome analysis efforts [27]. Finally, the M. tardigradum genome was compared to two other tardigrades and all species present in the current release of the Ensembl Metazoa database. The analysis revealed that tardigrade genomes are not that different from those of other Ecdysozoa. The availability of the three genomes allowed the delineation of their phylogenetic position within the Ecdysozoa and placed them as sister taxa to the nematodes. Thereby, the comparative analysis helped to identify evolutionary trends within this metazoan lineage. Surprisingly, the analysis did not reveal general mechanisms (shared by all available tardigrade genomes) behind the arguably most peculiar feature of tardigrades; their enormous stress tolerance. The lack of molecular evidence for individual tardigrade species (e.g., gene expression data for M. tardigradum) and the non-existence of a universal experimental framework which enables hypothesis testing withing the whole phylum Tardigrada, made it nearly impossible to link footprints of genomic adaptations to the unusual physiological capabilities. Nevertheless, the (comparative) genomic framework established during this project will help to understand how evolution tinkered, rewired and modified existing molecular systems to shape the remarkable phenotypic features of tardigrades.},
  subject      = {B{\"a}rtierchen},
  language  = {en}
}
@phdthesis{Das2018,
  author    = {Das, Sudip},
  title     = {Genome-wide identification of virulence-associated genes in Staphylococcus aureus using Transposon insertion-site deep sequencing},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-143362},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Staphylococcus aureus asymptomatically colonises one third of the healthy human population, finding its niche in the nose and on skin. Apart from being a commensal, it is also an important opportunistic human pathogen capable of destructing tissue, invading host cells and killing them from within. This eventually contributes to severe hospital- and community-acquired infections. Methicillin-resistant Staphylococcus aureus (MRSA), resistant to commonly used antibiotics are protected when residing within the host cell. This doctoral thesis is focused on the investigation of staphylococcal factors governing intracellular virulence and subsequent host cell death. To initiate an unbiased approach to conduct this study, complex S. aureus mutant pools were generated using transposon insertional mutagenesis. Genome-wide infection screens were performed using these S. aureus transposon mutant pools in vitro and in vivo, followed by analysis using Transposon insertion site deep sequencing (Tn-seq) technology. Amongst several other factors, this study identified a novel regulatory system in S. aureus that controls pathogen-induced host cytotoxicity and intra-host survival. The primary components of this system are an AraC-family transcription regulator called Repressor of surface proteins (Rsp) and a virulence associated non-coding RNA, SSR42. Mutants within rsp exhibit enhanced intra-host survival in human epithelial cells and delayed host cytotoxicity. Global gene-expression profiling by RNA-seq demonstrated that Rsp controls the expression of SSR42, several cytotoxins and other bacterial factors directed against the host immune system. Rsp enhances S. aureus toxin response when triggered by hydrogen peroxide, an antimicrobial substance employed by neutrophils to destroy pathogens. Absence of rsp reduces S. aureus-induced neutrophil damage and early lethality during mouse pneumonia, but still permits blood stream infection. Intriguingly, S. aureus lacking rsp exhibited enhanced survival in human macrophages, which hints towards a Trojan horse-like phenomenon and could facilitate dissemination within the host. Hence, Rsp emerged as a global regulator of bacterial virulence, which has an impact on disease progression with prolonged intra-cellular survival, delayed-lethality but allows disseminated manifestation of disease. Moreover, this study exemplifies the use of genome-wide approaches as useful resources for identifying bacterial factors and deduction of its pathogenesis.},
  subject      = {Staphylococcus aureus},
  language  = {en}
}
@phdthesis{Chen2018,
  author    = {Chen, Jiangtian},
  title     = {Functions of allatostatin A (AstA) and myoinhibitory peptides (MIPs) in the regulation of food intake and sleep in Drosophila},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-156838},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Neuropeptides and peptide hormones carrying neural or physiological information are intercellular signalling substances. They control most if not all biological processes in vertebrates and invertebrates by acting on specific receptors on the target cell. In mammals, many different neuropeptides and peptide hormones are involved in the regulation of feeding and sleep. In \textit{Drosophila}, allatostatin A (AstA) and myoinhibitory peptides (MIPs) are brain-gut peptides. The AstA receptors are homologues of the mammalian galanin receptors and the amino acid sequences of MIPs are similar to a part of galanin, which has an orexigenic effect and is implicated in the control of sleep behaviour in mammals. I am interested in dissecting pleiotropic functions of AstA and MIPs in the regulation of food intake and sleep in \textit{Drosophila}. \par In the first part of the dissertation the roles of brain-gut peptide allatostatin A are analysed. Due to the genetic and molecular tools available, the fruit fly \textit{Drosophila melanogaster} is chosen to investigate functions of AstA. The aims in this part are to identify pleiotropic functions of AstA and assign specific effects to the activity of certain subsets of AstA expressing cells in \textit{Drosophila} adults. A new and restricted \textit{AstA\textsuperscript{34}-Gal4} line was generated. The confocal imaging result showed that AstA neurons are located in the posterior lateral protocerebrum (PLP), the gnathal ganglia (GNG), the medullae, and thoracic-abdominal ganglion (TAG). AstA producing DLAa neurons in the TAG innervate hindgut and the poterior part of midgut. In addition, AstA are detected in the enteroendocrine cells (EECs).\par Thermogenetic activation and neurogenetic silencing tools with the aid of the \textit{UAS/Gal4} system were employed to manipulate the activity of all or individual subsets of AstA cells and investigate the effects on food intake, locomotor activity and sleep. Our experimental results showed that thermogenetic activation of two pairs of PLP neurons and/or AstA expressing EECs reduced food intake, which can be traced to AstA signalling by using \textit{AstA} mutants. In the locomotor activity, thermogenetic activation of two pairs of PLP neurons and/or AstA expressing EECs resulted in strongly inhibited locomotor activity and promoted sleep without sexual difference, which was most apparent during the morning and evening activity peaks. The experimental and control flies were not impaired in climbing ability. In contrast, conditional silencing of the PLP neurons and/or AstA expressing EECs reduced sleep specifically in the siesta. The arousal experiment was employed to test for the sleep intensity. Thermogenetically activated flies walked significantly slower and a shorter distance than controls for all arousal stimulus intensities. Furthermore, PDF receptor was detected in the PLP neurons and the PLP neurons reacted with an intracellular increase of cAMP upon PDF, only when PDF receptor was present. Constitutive activation of AstA cells by tethered PDF increased sleep and thermogenetic activation of the PDF producing sLNvs promoted sleep specifically in the morning and evening. \par The study shows that the PLP neurons and/or EECs vis AstA signalling subserve an anorexigenic and sleep-regulating function in \textit{Drosophila}. The PLP neurons arborise in the posterior superior protocerebrum, where the sleep relevant dopaminergic neurons are located, and EECs extend themselves to reach the gut lumen. Thus, the PLP neurons are well positioned to regulate sleep and EECs potentially modulate feeding and possibly locomotor activity and sleep during sending the nutritional information from the gut to the brain. The results of imaging, activation of the PDF signalling pathway by tethered PDF and thermoactivation of PDF expressing sLNvs suggest that the PLP neurons are modulated by PDF from sLNv clock neurons and AstA in PLP neurons is the downstream target of the central clock to modulate locomotor activity and sleep. AstA receptors are homologues of galanin receptors and both of them are involved in the regulation of feeding and sleep, which appears to be conserved in evolutionary aspect.\par In the second part of the dissertation, I analysed the role of myoinhibitory peptides. MIPs are brain-gut peptides in insects and polychaeta. Also in \textit{Drosophila}, MIPs are expressed in the CNS and EECs in the gut. Previous studies have demonstrated the functions of MIPs in the regulation of food intake, gut motility and ecdysis in moths and crickets. Yet, the functions of MIPs in the fruit fly are little known. To dissect effects of MIPs regarding feeding, locomotor activity and sleep in \textit{Drosophila melanogater}, I manipulated the activity of MIP\textsuperscript{W{\"U}} cells by using newly generated \textit{Mip\textsuperscript{W{\"U}}-Gal4} lines. Thermogenetical activation or genetical silencing of MIP\textsuperscript{W{\"U}} celles did not affect feeding behaviour and resulted in changes in the sleep status. \par My results are in contradiction to a recent research of Min Soohong and colleagues who demonstrated a role of MIPs in the regulation of food intake and body weight in \textit{Drosophila}. They showed that constitutive silencing of MIP\textsuperscript{KR} cells increased food intake and body weight, whereas thermogenetic activation of MIP\textsuperscript{KR} cells decreased food intake and body weight by using \textit{Mip\textsuperscript{KR}-Gal4} driver. Then I repeated the experiments with the \textit{Mip\textsuperscript{KR}-Gal4} driver, but could not reproduce the results. Interestingly, I just observed the opposite phenotype. When MIP\textsuperscript{KR} cells were silenced by expressing UAS-tetanus toxin (\textit{UAS-TNT}), the \textit{Mip\textsuperscript{KR}\$>\$TNT} flies showed reduced food intake. The thermogenetic activation of MIP\textsuperscript{KR} cells did not affect food intake. Furthermore, I observed that the thermogenetic activation of MIP\textsuperscript{KR} cells strongly reduced the sleep duration.\par In the third part of the dissertation, I adapted and improved a method for metabolic labelling for \textit{Drosophila} peptides to quantify the relative amount of peptides and the released peptides by mass spectrometry under different physiological and behavioural conditions. qRT-PCR is a practical technique to measure the transcription and the corresponding mRNA level of a given peptide. However, this is not the only way to measure the translation and production of peptides. Although the amount of peptides can be quantified by mass spectrometry, it is not possible to distinguish between peptides stored in vesicles and released peptides in CNS extracts. I construct an approach to assess the released peptides, which can be calculated by comparing the relative amount of peptides between two timepoints in combination with the mRNA levels which can be used as semiquantitative proxy reflecting the production of peptides during this period. \par After optimizing the protocol for metabolic labelling, I carried out a quantitative analysis of peptides before and after eclosion as a test. I was able to show that the EH- and SIFa-related peptides were strongly reduced after eclosion. This is in line with the known function and release of EH during eclosion. Since this test was positive, I next used the metabolic labelling in \textit{Drosophila} adult, which were either fed \textit{ad libitum} or starved for 24 hrs, and analysed the effects on the amount of AstA and MIPs. In the mRNA level, my results showed that in the brain \textit{AstA} mRNA level in the 24 hrs starved flies was increased compared to in the \textit{ad libitum} fed flies, whereas in the gut the \textit{AstA} mRNA level was decreased. Starvation induced the reduction of \textit{Mip} mRNA level in the brain and gut. Unfortunately, due to technical problems I was unable to analyse the metabolic labelled peptides during the course of this thesis.\par},
  subject      = {AstA},
  language  = {en}
}
@phdthesis{KissnergebStenger2018,
  author    = {Kißner [geb. Stenger], Stefanie Martina},
  title     = {Morphologische Untersuchungen an Myoblasten von Patienten, die an facioscapulohumeraler Muskeldystrophie (FSHD) leiden},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-156676},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Die autosomal-dominant vererbte facioscapulohumerale Muskeldystrophie (FSHD) ist mit einer Pr{\"a}valenz von etwa 1:20.000 die dritth{\"a}ufigste Form der heredit{\"a}ren Myopathien. Erste Beschwerden werden meist in der zweiten Lebensdekade beobachtet. Betroffen sind vor allem die Muskulatur von Gesicht, Schultern, Oberarmen, die Fußhebermuskulatur und die Muskeln des H{\"u}ftg{\"u}rtels. FSHD wird durch einen Gendefekt ausgel{\"o}st, der den langen Arm des Chromosoms vier (4q35) betrifft, wobei es zur teilweisen Deletion des polymorphen Abschnitts D4Z4, der f{\"u}r das Protein DUX4 codiert, kommt. Dabei treten unter anderem St{\"o}rungen in der DUX4-Expression, Ver{\"a}nderungen der myogenen Genexpression, eine Unterdr{\"u}ckung der Muskelzelldifferenzierung und eine Inhibition der Muskelbildung auf. FSHD und eine andere Form der Muskeldystrophie, die Emery-Dreifuss-Muskeldystrophie (EDMD), zeigen trotz unterschiedlicher genetischer Ursachen ph{\"a}notypisch {\"A}hnlichkeiten in der Auspr{\"a}gung der Erkrankungen. In fr{\"u}heren Studien zeigte die Kernh{\"u}lle von EDMD-Myoblasten morphologische Auff{\"a}lligkeiten. In anderen Untersuchungen waren morphologische Ver{\"a}nderungen der Mitochondrien von FSHD-Patienten festzustellen. Daher wurden elektronenmikroskopische Untersuchungen der Kernh{\"u}lle und der Mitochondrien von FSHD-Myoblasten durchgef{\"u}hrt und mit der entsprechenden Kontrolle verglichen. Hierf{\"u}r wurden drei verschiedene Zelllinien-Paare in unterschiedlichen Passagen, das heißt unterschiedlicher Anzahl an Subkultivierungen, eingesetzt, wobei in den h{\"o}heren Passagen vermehrt morphologische Atypien beobachtet werden konnten. Die eingesetzten Zelllinien differenzieren sich durch verschiedene Parameter wie beispielsweise Alter und Geschlecht der Patienten. Dabei zeigten sich sowohl zwischen den Kontrollzellen als auch zwischen den FSHD-Myoblasten Unterschiede. Im Rahmen der Probenvorbereitung f{\"u}r die Elektronenmikroskopie kamen zwei verschiedene Fixierungsmethoden zum Einsatz: die konventionelle chemische Fixierung, Entw{\"a}sserung und Flacheinbettung von Kulturzellen und die Hochdruckgefrierung mit anschließender Gefriersubstitution. In Bezug auf die Qualit{\"a}t des Strukturerhalts, die beim Hochdruckgefrieren erreicht wird, wird dieser Art der Fixierung eine {\"U}berlegenheit gegen{\"u}ber allen anderen Verfahren zugeschrieben. Diese allgemeine Aussage kann nicht vollst{\"a}ndig auf die Untersuchungen an den Myoblasten {\"u}bertragen werden. F{\"u}r die Untersuchung der Kernmembranen sind beide Methoden geeignet, wobei der Abstand zwischen innerer und {\"a}ußerer Kernmembran nach der HPF-Fixierung sch{\"a}rfer abgebildet wurde. Bei der Darstellung der Mitochondrien zeigten die elektronenmikroskopischen Aufnahmen nach dem Hochdruckgefrieren bessere und sch{\"a}rfere Ergebnisse. Die Kernporen waren bei beiden Fixierungsmethoden gut erkennbar. Beim Vergleich der gesunden und erkrankten Myoblasten wiesen die Kontrollzellen deutlich weniger Auff{\"a}lligkeiten auf als die Myoblasten von FSHD-Patienten. Innere und {\"a}ußere Kernmembran verliefen bei den Kontrollzellen meist parallel und die Mitochondrien zeigten in den meisten F{\"a}llen eine typische wurmartige, l{\"a}ngliche Form mit Cristae. Dies traf sowohl f{\"u}r die konventionelle Fixierung als auch f{\"u}r das Hochdruckgefrieren zu. Die erkrankten Myoblasten wiesen im Vergleich zur Kontrolle bei beiden Fixierungsmethoden deutliche Auff{\"a}lligkeiten in der Mitochondrien-Morphologie auf. Neben einer oft großen Variationsbreite hinsichtlich Form und L{\"a}nge war auch das teilweise Fehlen der Cristae festzustellen. Bei Betrachtung der Kernh{\"u}lle fielen jedoch deutliche Unterschiede zwischen konventioneller und HPF-Fixierung auf. Die {\"a}ußere Kernmembran der konventionell fixierten FSHD-Myoblasten verlief unregelm{\"a}ßig und gewellt. Im Gegensatz dazu wies die Kernh{\"u}lle der HPF-fixierten erkrankten Myoblasten einen erstaunlich parallelen Verlauf auf. Da bei EDMD in vorangegangenen Untersuchungen auch fluoreszenzmikroskopisch Ver{\"a}nderungen der erkrankten Zellen auff{\"a}llig waren, wurde neben den Methoden der Elektronenmikroskopie das Vorliegen und die Verteilung verschiedener Proteine in FSHD-Myoblasten mittels indirekter Immunfluoreszenz untersucht und mit den Kontrollzellen verglichen. Zur Beurteilung der Kernh{\"u}lle wurden Antik{\"o}rper gegen Lamin A/C und Nukleoporine eingesetzt. Die Mitochondrien wurden mithilfe des Antik{\"o}rpers ANT1/2, der an den Adenin-Nukleotid-Translokator der inneren Mitochondrienmembran bindet, untersucht. Im Gegensatz zu den Untersuchungen an EDMD-Myoblasten waren die Lamine A und C sowie die Kernporen sowohl bei den Myoblasten der FSHD-Patienten als auch bei den Kontrollzellen nachweisbar und gleichm{\"a}ßig verteilt. Bei der indirekten Immunfluoreszenz mit ANT1/2 zeigten sich Unterschiede zwischen den untersuchten Myoblasten-Paaren. Durch die vorliegenden Ergebnisse ist darauf zu schließen, dass die Myoblasten von FSHD-Patienten Ver{\"a}nderungen Mitochondrien aufweisen. Die Untersuchungen der Kernh{\"u}lle liefern abh{\"a}ngig von der Fixierungsmethode unterschiedliche Ergebnisse.},
  subject      = {Landouzy-D{\´e}jerine-Atrophie},
  language  = {de}
}
@phdthesis{Carstensen2018,
  author    = {Carstensen, Anne Carola},
  title     = {Identification of novel N-MYC interacting proteins reveals N-MYC interaction with TFIIIC},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-143658},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {N-MYC is a member of the human MYC proto-oncogene family, which comprises three transcription factors (C-, N- and L-MYC) that function in multiple biological processes. Deregulated expression of MYC proteins is linked to tumour initiation, maintenance and progression. For example, a large fraction of neuroblastoma displays high N-MYC levels due to an amplification of the N-MYC encoding gene. MYCN-amplified neuroblastoma depend on high N-MYC protein levels, which are maintained by Aurora-A kinase. Aurora-A interaction with N-MYC interferes with degradation of N-MYC via the E3 ubiquitin ligase SCFFBXW7. However, the underlying mechanism of Aurora-A-mediated stabilisation of N-MYC remains to be elucidated. To identify novel N-MYC interacting proteins, which could be involved in N-MYC stabilisation by Aurora-A, a proteomic analysis of purified N-MYC protein complexes was conducted. Since two alanine mutations in MBI of N-MYC, T58A and S62A (N-MYC mut), disable Aurora-A-mediated stabilisation of N-MYC, N-MYC protein complexes from cells expressing either N-MYC wt or mut were analysed. Proteomic analysis revealed that N-MYC interacts with two deubiquitinating enzymes, USP7 and USP11, which catalyse the removal of ubiquitin chains from target proteins, preventing recognition by the proteasome and subsequent degradation. Although N-MYC interaction with USP7 and USP11 was confirmed in subsequent immunoprecipitation experiments, neither USP7, nor USP11 was shown to be involved in the regulation of N-MYC stability. Besides USP7/11, proteomic analyses identified numerous additional N-MYC interacting proteins that were not described to interact with MYC transcription factors previously. Interestingly, many of the identified N-MYC interaction partners displayed a preference for the interaction with N-MYC wt, suggesting a MBI-dependent interaction. Among these were several proteins, which are involved in three-dimensional organisation of chromatin domains and transcriptional elongation by POL II. Not only the interaction of N-MYC with proteins functioning in elongation, such as the DSIF component SPT5 and the PAF1C components CDC73 and CTR9, was validated in immunoprecipitation experiments, but also with the POL III transcription factor TFIIIC and topoisomerases TOP2A/B. ChIP-sequencing analysis of N-MYC and TFIIIC subunit 5 (TFIIIC5) revealed a large number of joint binding sites in POL II promoters and intergenic regions, which are characterised by the presence of a specific motif that is highly similar to the CTCF motif. Additionally, N-MYC was shown to interact with the ring-shaped cohesin complex that is known to bind to CTCF motifs and to assist the insulator protein CTCF. Importantly, individual ChIP experiments demonstrated that N-MYC, TFIIIC5 and cohesin subunit RAD21 occupy joint binding sites comprising a CTCF motif. Collectively, the results indicate that N-MYC functions in two biological processes that have not been linked to MYC biology previously. Furthermore, the identification of joint binding sites of N-MYC, TFIIIC and cohesin and the confirmation of their interaction with each other suggests a novel function of MYC transcription factors in three-dimensional organisation of chromatin.},
  subject      = {Biologie},
  language  = {en}
}
@phdthesis{Ankenbrand2018,
  author    = {Ankenbrand, Markus Johannes},
  title     = {Squeezing more information out of biological data - development and application of bioinformatic tools for ecology, evolution and genomics},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-156344},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {New experimental methods have drastically accelerated the pace and quantity at which biological data is generated. High-throughput DNA sequencing is one of the pivotal new technologies. It offers a number of novel applications in various fields of biology, including ecology, evolution, and genomics. However, together with those opportunities many new challenges arise. Specialized algorithms and software are required to cope with the amount of data, often requiring substantial training in bioinformatic methods. Another way to make those data accessible to non-bioinformaticians is the development of programs with intuitive user interfaces. In my thesis I developed analyses and programs to tackle current problems with high-throughput data in biology. In the field of ecology this covers the establishment of the bioinformatic workflow for pollen DNA meta-barcoding. Furthermore, I developed an application that facilitates the analysis of ecological communities in the context of their traits. Information from multiple public databases have been aggregated and can now be mapped automatically to existing community tables for interactive inspection. In evolution the new data are used to reconstruct phylogenetic trees from multiple genes. I developed the tool bcgTree to automate this process for bacteria. Many plant genomes have been sequenced in current years. Sequencing reads of those projects also contain data from the chloroplasts. The tool chloroExtractor supports the targeted extraction and analysis of the chloroplast genome. To compare the structure of multiple genomes specialized software is required for calculation and visualization of the relationships. I developed AliTV to address this. In contrast to existing programs for this task it allows interactive adjustments of produced graphics. Thus, facilitating the discovery of biologically relevant information. Another application I developed helps to analyze transcriptomes even if no reference genome is present. This is achieved by aggregating the different pieces of information, like functional annotation and expression level, for each transcript in a web platform. Scientists can then search, filter, subset, and visualize the transcriptome. Together the methods and tools expedite insights into biological systems that were not possible before.},
  language  = {en}
}
@phdthesis{Danner2017,
  author    = {Danner, Nadja},
  title     = {Honey bee foraging in agricultural landscapes},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-139322},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {1. Today honey bee colonies face a wide range of challenges in modern agricultural landscapes which entails the need for a comprehensive investigation of honey bees in a landscape context and the assessment of environmental risks. Within this dissertation the pollen foraging of honey bee colonies is studied in different agricultural landscapes to gain insight into the use of pollen resources and the influence of landscape structure across the season. General suggestions for landscape management to support honey bees and other pollinators are derived. 2. Decoding of waggle dances and a subsequent spatial foraging analysis are used as methods in Chapters 4 and 5 to study honey bee colonies in agricultural landscapes. The recently developed metabarcoding of mixed pollen samples was applied for the first time in honey bee foraging ecology and allowed for a detailed analysis of pollen, that was trapped from honey bees in front hive entrances (Chapter 6). 3. Pollen identification through molecular sequencing and DNA barcoding has been proposed as an alternative approach to light microscopy, which still is a tedious and error-prone task. In this study we assessed mixed pollen probes through next-generation sequencing and developed a bioinformatic workflow to analyse these high-throughput data with a newly created reference database. To evaluate the feasibility, we compared results from classical identification based on light microscopy from the same samples with our sequencing results. Abundance estimations from sequencing data were significantly correlated with counted abundances through light microscopy. Next-generation sequencing thus presents a useful and efficient workflow to identify pollen at the genus and species level without requiring specialized palynological expert knowledge. 4. During maize flowering, four observation hives were placed in and rotated between 11 landscapes covering a gradient in maize acreage. A higher foraging frequency on maize fields compared to other landuse types showed that maize is an intensively used pollen resource for honey bee colonies. Mean foraging distances were significantly shorter for maize pollen than for other pollen origins, indicating that effort is put into collecting a diverse pollen diet. The percentage of maize pollen foragers did not increase with maize acreage in the landscape and was not reduced by grassland area as an alternative pollen resource. Our findings allow estimating the distance-related exposure risk of honey bee colonies to pollen from surrounding maize fields treated with systemic insecticides. 5. It is unknown how an increasing area of mass-flowering crops like oilseed rape (OSR) or a decrease of semi-natural habitats (SNH) change the temporal and spatial availability of pollen resources for honey bee colonies, and thus foraging distances and frequency in different habitat types. Sixteen observation hives were placed in and rotated between 16 agricultural landscapes with independent gradients of OSR and SNH area within 2 km to analyze foraging distances and frequencies. SNH and OSR reduced foraging distance at different spatial scales and depending on season, with possible benefits for the performance of honey bee colonies. Frequency of pollen foragers per habitat type was equally high for SNH, grassland and OSR fields, but lower for other crops and forest. In landscapes with a small proportion of SNH a significantly higher density of pollen foragers on SNH was observed, indicating the limitation of pollen resources in simple agricultural landscapes and the importance of SNH. 6. Quantity and diversity of collected pollen can influence the growth and health of honey bee colonies, but little is known about the influence of landscape structure on pollen diet. In a field experiment we rotated 16 honey bee colonies across 16 agricultural landscapes (see also Chapter 5), used traps to get samples of collected pollen and observed the intra-colonial dance communication to gain information about foraging distances. Neither the amount of collected pollen nor pollen diversity were related to landscape diversity. The revealed increase of foraging distances with decreasing landscape diversity suggests that honey bees compensate for a lower landscape diversity by increasing their pollen foraging range in order to maintain pollen amount and diversity. 7. Our results show the importance of diverse pollen resources for honey bee colonies in agricultural landscapes. Beside the risk of exposure to pesticides honey bees face the risk of nutritional deficiency with implications for their health. By modifying landscape composition and therefore availability of resources we are able to contribute to the wellbeing of honey bees. Agri-environmental schemes aiming to support pollinators should focus on possible spatial and temporal gaps in pollen availability and diversity in agricultural landscapes.},
  subject      = {Apis mellifera},
  language  = {en}
}
@phdthesis{Kuen2017,
  author    = {Kuen, Janina},
  title     = {Influence of 3D tumor cell/fibroblast co-culture on monocyte differentiation and tumor progression in pancreatic cancer},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-156226},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {Pancreatic cancer (PC) remains one of the most challenging solid tumors to treat with a high unmet medical need as patients poorly respond to standard-of-care-therapies. Prominent desmoplastic reaction involving cancer-associated fibroblasts (CAFs) and the immune cells in the tumor microenvironment (TME) and their cross-talk play a significant role in tumor immune escape and progression. To identify the key cellular mechanisms induce an immunosuppressive tumor microenvironment, we established 3D co-culture model with pancreatic cancer cells, CAFs, monocyte as well as T cells. Using this model, we analysed the influence of tumor cells and fibroblasts on monocytes and their immune suppressive phenotype. Phenotypic characterization of the monocytes after 3D co-culture with tumor/fibroblast spheroids was performed by analysing the expression of defined cell surface markers and soluble factors. Functionality of these monocytes and their ability to influence T cell phenotype and proliferation was investigated. 3D co-culture of monocytes with pancreatic cancer cells and fibroblasts induced the production of immunosuppressive cytokines which are known to promote polarization of M2 like macrophages and myeloid derived suppressive cells (MDSCs). These co-culture spheroid polarized monocyte derived macrophages (MDMs) were poorly differentiated and had an M2 phenotype. The immunosuppressive function of these co-culture spheroids polarized MDMs was demonstrated by their ability to inhibit autologous CD4+ and CD8+ T cell activation and proliferation in vitro, which we could partially reverse by 3D co-culture spheroid treatment with therapeutic molecules that are able to re-activate spheroid polarized MDMs or block immune suppressive factors such as Arginase-I. In conclusion, we generated a physiologically relevant 3D co-culture model, which can be used as a promising tool to study complex cell-cell interactions between different cell types within the tumor microenvironment and to support drug screening and development. In future, research focused on better understanding of resistance mechanisms to existing cancer immunotherapies will help to develop new therapeutic strategies in order to combat cancer.},
  subject      = {monocyte},
  language  = {en}
}
@phdthesis{Ritter2017,
  author    = {Ritter, Cathrin},
  title     = {Scientific basics for new immunotherapeutic approaches towards Merkel cell carcinoma},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-124162},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {Merkel cell carcinoma (MCC) is an aggressive neuroendocrine skin cancer that has been associated with the Merkel cell polyomavirus (MCPyV). Indeed, MCC is one of the cancers with the best-established viral carcinogenesis. Despite persistence of the virus in MCC cells and the subsequent expression of viral antigens, the majority of MCC tumors are able to escape the surveillance of the immune system. Therefore the aim of the here presented thesis was to scrutinize immune escape mechanisms operative in MCC. A better understanding of their underlying molecular processes should allow to improve immunotherapeutic treatment strategies for MCC patients. The manuscripts included in this thesis characterize three novel immune evasion strategies of MCC. I) the epigenetic silencing of the NKG2D ligands MICA and MICB via histone H3 hypoacetylation II) reduced HLA class I surface expression via epigenetic silencing of the antigen processing machinery (APM) III) the activation of the PI3K-AKT pathway in a mutation independent manner as potential immune escape strategy MCC tumors and MCC cell lines were analyzed for their expression of MICA/B, HLA and components of the antigen processing machinery as well as for the activation of the PI3K-AKT pathway in situ and in vitro. These analysis reviled MICA and MICB, as well as HLA class I were not expressed or at least markedly reduced in ~80\% of MCCs in situ. The PI3K-AKT pathway, that had only recently been demonstrated to play a significant role in tumor immune escape, was activated in almost 90\% of MCCs in situ. To determine the underlying molecular mechanisms of these aberrations well characterized MCC cell lines were further analyzed in vitro. The fact that the PI3K-AKT pathway activation was due to oncogenic mutations in the PIK3CA or AKT1 gene in only 10\% of MCCs, suggested an epigenetic regulation of this pathway in MCC. In line with this MICA/B as well as components of the APM were indeed silenced epigenetically via histone hypoacetylation in their respective promoter region. Notably MICA/B and HLA class I expression on the cell surface of MCC cells could be restored after treatment with HDAC inhibitors in combination with the Sp1 inhibitor Mithramycin A in all analyzed MCC cell lines in vitro and in a xenotransplantation mouse model in vivo. Moreover inhibition of HDACs increased immune recognition of MCC cell lines in a MICA/B and HLA class I dependent manner. Several studies have accumulated evidence that immunotherapy is a promising treatment option for MCC patients due to the exquisite immunogenicity of this malignancy. However, current immunotherapeutic interventions towards solid tumors like MCC have to account for the plentitude of tumor immune escape strategies, in order to increase response rates. The immune escape mechanisms of MCC described in this thesis can be reverted by HDAC inhibition, thus providing the rationale to combine 'epigenetic priming' with currently tested immunotherapeutic regimens.},
  subject      = {Merkel-Zellkarzinom},
  language  = {en}
}
@phdthesis{Borst2017,
  author    = {Borst, Andreas},
  title     = {Apoptosis \& senescence: cell fate determination in inhibitor-treated melanoma cells},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-155085},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {Neoplasms of the skin represent the most frequent tumors worldwide; fortunately, most of them are benign or semi-malignant and well treatable. However, the two most aggressive and deadly forms of malignant skin-neoplasms are melanoma and Merkel cell carcinoma (MCC), being responsible for more than 90\% of skin-cancer related deaths. The last decade has yielded enormous progress in melanoma therapy with the advent of targeted therapies, like BRAF or MEK inhibitors, and immune-stimulating therapies, using checkpoint antibodies targeting CTLA- 4, PD-1 or PD-L1. Very recent studies suggest that also MCC patients benefit from a treatment with checkpoint antibodies. Nevertheless, in an advanced metastatic stage, a cure for both of these aggressive malignancies is still hard to achieve: while only a subset of patients experience durable benefit from the immune-based therapies, the widely applicable targeted therapies struggle with development of resistances that inevitably occur in most patients, and finally lead to their death. The four articles included in this thesis addressed current questions concerning therapy and carcinogenesis of melanoma and MCC. Moreover, they are discussed in the light of the up-to-date research regarding targeted and immune-based therapies. In article I we demonstrated that besides apoptosis, MAPK pathway inhibition in BRAF-mutated melanoma cells also induces senescence, a permanent cell cycle arrest. These cells may provide a source for relapse, as even permanently arrested cancer cells can contribute to a pro-tumorigenic milieu. To identify molecular factors determining the differential response, we established M14 melanoma cell line derived single cell clones that either undergo cell death or arrest when treated with BRAF/MEK inhibitors. Using these single cell clones, we demonstrated in article IV that downregulation of the pro-apoptotic BH3-only protein BIK via epigenetic silencing is involved in apoptosis deficiency, which can be overcome by HDAC inhibitors. These observations provide a possible explanation for the lack of a complete and durable response to MAPK inhibitor treatment in melanoma patients, and suggest the application of HDAC inhibitors as a complimentary therapy to MAPK pathway inhibition. Concerning MCC, we scrutinized the interactions between the Merkel cell polyomavirus' (MCV) T antigens (TA) and the tumor suppressors p53 and Rb in article II and III, respectively. In article III, we demonstrated that the cell cycle master regulator Rb is the crucial target of MCV large T (LT), while it - in contrast to other polyomavirus LTs - exhibits much lower affinity to the related proteins p107 and p130. Knockdown of MCV LT led to proliferation arrest in MCC cells, which can be rescued by knockdown of Rb, but not by knockdown of p107 and p130. Contrary to Rb, restriction of p53 in MCC seems to be independent of the MCV TAs, as we demonstrated in article II. In conclusion, the presented thesis has revealed new molecular details, regarding the response of melanoma cells towards an important treatment modality and the mechanisms of viral carcinogenesis in MCC.},
  subject      = {Melanom},
  language  = {en}
}
@phdthesis{Scholz2017,
  author    = {Scholz, Nicole},
  title     = {Genetic analyses of sensory and motoneuron physiology in Drosophila melanogaster},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-123249},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {During my PhD I studied two principal biological aspects employing Drosophila melanogaster. Therefore, this study is divided into Part I and II. Part I: Bruchpilot and Complexin interact to regulate synaptic vesicle tethering to the active zone cytomatrix At the presynaptic active zone (AZ) synaptic vesicles (SVs) are often physically linked to an electron-dense cytomatrix - a process referred to as "SV tethering". This process serves to concentrate SVs in close proximity to their release sites before contacting the SNARE complex for subsequent fusion (Hallermann and Silver, 2013). In Drosophila, the AZ protein Bruchpilot (BRP) is part of the proteinous cytomatrix at which SVs accumulate (Kittel et al., 2006b; Wagh et al., 2006; Fouquet et al., 2009). Intriguingly, truncation of only 1\% of the C-terminal region of BRP results in a severe defect in SV tethering to this AZ scaffold (hence named brpnude; Hallermann et al., 2010b). Consistent with these findings, cell-specific overexpression of a C-terminal BRP fragment, named mBRPC-tip (corresponds to 1\% absent in brpnude; m = mobile) phenocopied the brpnude mutant in behavioral and functional experiments. These data indicate that mBRPC-tip suffices to saturate putative SV binding sites, which induced a functional tethering deficit at motoneuronal AZs. However, the molecular identity of the BRP complement to tether SVs to the presynaptic AZ scaffold remains unknown. Moreover, within larval motoneurons membrane-attached C-terminal portions of BRP were sufficient to tether SVs to sites outside of the AZ. Based on this finding a genetic screen was designed to identify BRP interactors in vivo. This screen identified Complexin (CPX), which is known to inhibit spontaneous SV fusion and to enhance stimulus evoked SV release (Huntwork and Littleton, 2007; Cho et al., 2010; Martin et al., 2011). However, so far CPX has not been associated with a function upstream of priming/docking and release of SVs. This work provides morphological and functional evidence, which suggests that CPX promotes recruitment of SVs to the AZ and thereby curtails synaptic short-term depression. Together, the presented findings indicate a functional interaction between BRP and CPX at Drosophila AZs. Part II: The Adhesion-GPCR Latrophilin/CIRL shapes mechanosensation The calcium independent receptor of α-latrotoxin (CIRL), also named Latrophilin, represents a prototypic Adhesion class G-protein coupled-receptor (aGPCR). Initially, Latrophilin was identified based on its capacity to bind the α-component of latrotoxin (α-LTX; Davletov et al., 1996; Krasnoperov et al., 1996), which triggers massive exocytotic activity from neurons of the peripheral nervous system (Scheer et al., 1984; Umbach et al., 1998; Orlova et al., 2000). As a result Latrophilin is considered to play a role in synaptic transmission. Later on, Latrophilins have been associated with other biological processes including tissue polarity (Langenhan et al., 2009), fertility (Pr{\"o}mel et al., 2012) and synaptogenesis (Silva et al., 2011). However, thus far its subcellular localization and the identity of endogenous ligands, two aspects crucial for the comprehension of Latrophilin's in vivo function, remain enigmatic. Drosophila contains only one latrophilin homolog, named dCirl, whose function has not been investigated thus far. This study demonstrates abundant dCirl expression throughout the nervous system of Drosophila larvae. dCirlKO animals are viable and display no defects in development and neuronal differentiation. However, dCirl appears to influence the dimension of the postsynaptic sub-synaptic reticulum (SSR), which was accompanied by an increase in the postsynaptic Discs-large abundance (DLG). In contrast, morphological and functional properties of presynaptic motoneurons were not compromised by the removal of dCirl. Instead, dCirl is required for the perception of mechanical challenges (acoustic-, tactile- and proprioceptive stimuli) through specialized mechanosensory devices, chordotonal organs (Eberl, 1999). The data indicate that dCirl modulates the sensitivity of chordotonal neurons towards mechanical stimulation and thereby adjusts their input-output relation. Genetic interaction analyses suggest that adaption of the molecular mechanotransduction machinery by dCirl may underlie this process. Together, these results uncover an unexpected function of Latrophilin/dCIRL in mechanosensation and imply general modulatory roles of aGPCR in mechanoception.},
  subject      = {Drosophila},
  language  = {en}
}
@phdthesis{Fuchs2017,
  author    = {Fuchs, Benjamin Felix},
  title     = {Effects of timing and herbivory on a grass-endophyte association and its trophic interactions},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-141465},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {I.) Plant associated microorganisms can affect the plant`s interaction with herbivores and higher trophic levels. For instance, endophytic fungi infecting aerial plant parts of grass species produce bioactive alkaloids that can negatively affect species from higher trophic levels, indicating a defensive mutualism between the grass and the endophyte. However, beneficial insects can also be negatively affected by the endophyte, which might question the mutualistic effect of endophytic fungi. On the other hand, grass-endophytes are affected by environmental conditions and species interactions. Grazing can increase endophyte frequencies in natural habitats. Furthermore, endophyte mediated effects on herbivores are most pronounced during warm summers following rainy springs. In this study, we investigated whether endophyte derived alkaloids cascade up a food chain (chapter II) and whether their concentrations depend on plant age and season (chapter III). Further we analysed, whether altered herbivore phenology affects the endophytic fungus (chapter IV) and whether endophyte derived alkaloid production is induced by different herbivore species (chapter V). II.) In our first experimental study we analysed whether grass-endophyte derived alkaloids decreased the performance of two ladybird species feeding on aphids exclusively reared on endophyte infected grass (6 weeks young grass). Further, we screened species from three trophic levels (grass, herbivores and aphid predators) for their alkaloid content using two year old infected grass as diet for herbivores. We established an UPLC-MS method to detect and quantify the amount of the endophyte derived alkaloids peramine and lolitrem B extracted from the organic plant and insect material. Performance parameters of ladybirds revealed little differences between ladybirds fed on aphids reared on endophyte infected and non-infected grass, which probably resulted from low alkaloid concentrations in the young (6-weeks old) endophyte infected grass used in this part of the study. Alkaloid quantification of the two year old endophyte infected grass, herbivores and aphid predators revealed similar concentrations between grass and aphids, while aphid predators contained approximately half of that amount which still exceeded the bioactive threshold. We conclude that alkaloids produced by grass-endophytes cascade up the food chain and are responsible for fitness disadvantages of higher trophic levels. III.) In the second study we investigated the impact of plant age and seasonal timing on grass-endophyte growth and alkaloid production. Plants were sown in April of 2013 and sampled monthly over 30 consecutive months. Endophyte growth was quantified with real-time PCR (qPCR) and alkaloid concentrations with UPLC-MS. We showed that alkaloid concentrations and fungal growth followed a seasonal rhythmicity and that alkaloid concentrations increased with plant age. Alkaloid concentrations peak during summer, when also herbivore abundances are high. Consequently, we conclude that plant age and season contribute to the toxicity of endophytes on grass herbivores IV.) In the third study we simulated earlier spring arrival of aphids by enhancing aphid abundance on endophyte infected and endophyte-free grass in spring and analysed responses across three trophic levels. Enhanced aphid abundance in spring caused higher aphid abundances during the study period. Predators stayed unaffected by increased herbivore abundances; however they did level aphid numbers within two weeks after arrival on the plants, independent of aphid abundance. Grass-endophyte showed a time delayed growth, two weeks after aphid abundance peak and after predators already controlled aphid infestations on the plants. We conclude that phenology shifts of herbivorous insects can affect multi-trophic interactions leading to desynchronizations between phenologies of interacting species and mismatches in food-webs. V.) In the fourth study we analysed whether herbivores induce endophyte growth and alkaloid production and whether different types of herbivores induce specific alkaloid production. We applied three different herbivore treatments on endophyte infected grass over 18 weeks. Locust herbivory increased the insect deterring alkaloid peramine and clipping of plants (simulation of grazing livestock) increased the vertebrate toxic alkaloid lolitrem B. Aphid herbivory did not affect endophyte derived alkaloid concentrations. Endophyte responses to herbivory were species specific which indicates a primarily plant protecting role of alkaloid synthesis in endophyte infected plants and a close chemical crosstalk between interacting species. VI.) In summary, we showed that endophyte derived alkaloids affect higher trophic levels and that alkaloid concentrations in the plant depend on prevalent herbivore species, plant age and seasonal timing. Our results indicate a close chemical crosstalk between the host plant and the endophytic fungus which is susceptible to environmental changes altering the endophyte`s alkaloid production in plants. We gained insights into the grass-endophyte symbiosis in ecological contexts and conclude that several factors determine the herbivore toxic potential of endophytic fungi and thereby their plant mutualistic or parasitic character. Future studies should investigate the mechanisms behind the herbivore induced alkaloid concentration increase, shown in this thesis, especially whether plant signals mediate the endophyte response. Furthermore it would be interesting to study the induction of indirect endophyte mediated defence and how it affects multi-trophic level interactions.},
  language  = {en}
}
@phdthesis{Gupta2018,
  author    = {Gupta, Shishir Kumar},
  title     = {Re-annotation of Camponotus floridanus Genome and Characterization of Innate Immunity Transcriptome Responses to Bacterial Infections},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-140168},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {The sequencing of several ant genomes within the last six years open new research avenues for understanding not only the genetic basis of social species but also the complex systems such as immune responses in general. Similar to other social insects, ants live in cooperative colonies, often in high densities and with genetically identical or closely related individuals. The contact behaviours and crowd living conditions allow the disease to spread rapidly through colonies. Nevertheless, ants can efficiently combat infections by using diverse and effective immune mechanisms. However, the components of the immune system of carpenter ant Camponotus floridanus and also the factors in bacteria that facilitate infection are not well understood. To form a better view of the immune repository and study the C. floridanus immune responses against the bacteria, experimental data from Illumina sequencing and mass-spectrometry (MS) data of haemolymph in normal and infectious conditions were analysed and integrated with the several bioinformatics approaches. Briefly, the tasks were accomplished in three levels. First, the C. floridanus genome was re-annotated for the improvement of the existing annotation using the computational methods and transcriptomics data. Using the homology based methods, the extensive survey of literature, and mRNA expression profiles, the immune repository of C. floridanus were established. Second, large-scale protein-protein interactions (PPIs) and signalling network of C. floridanus were reconstructed and analysed and further the infection induced functional modules in the networks were detected by mapping of the expression data over the networks. In addition, the interactions of the immune components with the bacteria were identified by reconstructing inter-species PPIs networks and the interactions were validated by literature. Third, the stage-specific MS data of larvae and worker ants were analysed and the differences in the immune response were reported. Concisely, all the three omics levels resulted to multiple findings, for instance, re-annotation and transcriptome profiling resulted in the overall improvement of structural and functional annotation and detection of alternative splicing events, network analysis revealed the differentially expressed topologically important proteins and the active functional modules, MS data analysis revealed the stage specific differences in C. floridanus immune responses against bacterial pathogens. Taken together, starting from re-annotation of C. floridanus genome, this thesis provides a transcriptome and proteome level characterization of ant C. floridanus, particularly focusing on the immune system responses to pathogenic bacteria from a biological and a bioinformatics point of view. This work can serve as a model for the integration of omics data focusing on the immuno-transcriptome of insects.},
  subject      = {Camponotus floridanus},
  language  = {en}
}
@phdthesis{Iltzsche2017,
  author    = {Iltzsche, Fabian},
  title     = {The Role of DREAM/MMB-mediated mitotic gene expression downstream of mutated K-Ras in lung cancer},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-154108},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {The evolutionary conserved Myb-MuvB (MMB) multiprotein complex has an essential role in transcriptional activation of mitotic genes. MMB target genes as well as the MMB associated transcription factor B-Myb and FoxM1 are highly expressed in a range of different cancer types. The elevated expression of these genes correlates with an advanced tumor state and a poor prognosis. This suggests that MMB could contribute to tumorigenesis by mediating overexpression of mitotic genes. Although MMB has been extensively characterized biochemically, the requirement for MMB to tumorigenesis in vivo remains largely unknown and has not been tested directly so far. In this study, conditional knockout of the MMB core member Lin9 inhibits tumor formation in vivo in a mouse model of lung cancer driven by oncogenic K-Ras and loss of p53. The incomplete recombination observed within tumors points towards an enormous selection pressure against the complete loss of Lin9. RNA interference (RNAi)-mediated depletion of Lin9 or the MMB associated subunit B-Myb provides evidence that MMB is required for the expression of mitotic genes in lung cancer cells. Moreover, it was demonstrated that proliferation of lung cancer cells strongly depends on MMB. Furthermore, in this study, the relationship of MMB to the p53 tumor suppressor was investigated in a primary lung cancer cell line with restorable p53 function. Expression analysis revealed that mitotic genes are downregulated after p53 re-expression. Moreover, activation of p53 induces formation of the repressive DREAM complex and results in enrichment of DREAM at mitotic gene promoters. Conversely, MMB is displaced at these promoters. Based on these findings the following model is proposed: In p53-negative cells, mitogenic stimuli foster the switch from DREAM to MMB. Thus, mitotic genes are overexpressed and may promote chromosomal instability and tumorigenesis. This study provides evidence that MMB contributes to the upregulation of G2/M phase-specific genes in p53-negative cells and suggests that inhibition of MMB (or its target genes) might be a strategy for treatment of lung cancer.},
  subject      = {Nicht-kleinzelliges Bronchialkarzinom (NSCLC)},
  language  = {en}
}
@phdthesis{Kunz2017,
  author    = {Kunz, Meik},
  title     = {Systembiologische Analysen von Interaktionen: Zytokinine (Pflanzenpathogene), 3D-Zellkulturen (Krebstherapie) und Drugtargets},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-134911},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {Der Einsatz von computergest{\"u}tzten Analysen hat sich zu einem festen Bestandteil der biowissenschaftlichen Forschung etabliert. Im Rahmen dieser vorliegenden Arbeit wurden systembiologische Untersuchungen auf verschiedene biologische Themengebiete und Organismen angewendet. In diesem Zusammenhang liefert die Arbeit einen innovativen und interdisziplin{\"a}ren methodischen Ansatz. Die grundlegende Frage lautet: Wie verstehe und beschreibe ich Signalwege und wie kann ich sie beeinflussen? Der Ansatz verkn{\"u}pft verschiedene biologische Datens{\"a}tze und Datenebenen miteinander, beginnend vom Genom und Interaktionskontext {\"u}ber semiquantitative Simulationen hin zu neuen Interventionen und Experimenten, welche therapeutisch und biotechnologisch genutzt werden k{\"o}nnen. Die Analysen k{\"o}nnen auf diese Weise - zu einem besseren Verst{\"a}ndnis experimenteller Daten und biologischer Fragestellungen beitragen und erm{\"o}glichen ein systematisches Verst{\"a}ndnis der zugrunde liegenden Signalwege und Netzwerkeffekte (z.B. in Pflanzen). - Dar{\"u}ber hinaus erm{\"o}glichen sie die Identifizierung wichtiger funktioneller Hubproteine und die Entwicklung neuer therapeutischer Strategien f{\"u}r weitere experimentelle Testungen (z.B. Tumormodelle), - stellen zudem einen hilfreichen Schritt auf dem Weg zur personalisierten Medizin (z.B. lncRNAs und Tumormodelle) und Medikamentenentwicklung (z.B. Datenbank DrumPID) dar. (i) Als Grundlage wurde hierzu eine integrierte systembiologische Methode entwickelt, welche experimentelle Daten (z.B. Transkriptomdaten) hinsichtlich ihrer biologischen Funktionen untersucht und die Identifizierung relevanter funktioneller Cluster und Hubproteine erm{\"o}glicht. In einem ersten Teil wurden Analysen zum pflanzlichen Immunsystem durchgef{\"u}hrt. Mithilfe der entwickelten Methode wurden Genexpressionsdatens{\"a}tze von A. thaliana, die mit dem Pathogen Pst DC3000 infiziert wurden, untersucht, um den Einfluss verschiedener Virulenzfaktoren auf das Interaktom der Wirtspflanze zu untersuchen und neue Modulatoren einer CK-vermittelten Immunabwehr zu finden. In diesem Zusammenhang konnte gezeigt werden, dass die von Pst DC3000 sekretierten Abwehrstoffe wichtige pflanzliche Hormonsignalwege f{\"u}r die Immunabwehr in A. thaliana beeinflussen. Die Ergebnisse zeigen zudem, dass sich der Einfluss auf das Netzwerkverhalten der Effektorproteine und COR-Phytotoxine von dem der PAMPs unterscheidet, sich jedoch auch eine Regulierung gemeinsamer Signalwege und eine {\"U}berlappung der beiden Phasen der Immunantwort (PTI und ETI) in A. thaliana finden lassen. Die komplexe Immunantwort auf eine Infektion spiegelt sich zudem in einer h{\"o}heren Anzahl an funktionellen Clustern und Hubproteinen in Pst DC3000 gegen{\"u}ber den beiden untersuchten Mutanten wider, wobei sich f{\"u}r Pst DC3000 insbesondere ein stark vernetztes immunrelevantes Cluster um den JA-Signalweg zeigt. Weiterhin wurden anhand der entwickelten Methode wichtige Hubproteine f{\"u}r die Immunabwehr identifiziert. Als bedeutende Vertreter sind AHK2 und AAR14 zu nennen, welche Teil des Zweikomponentensystems der Signal{\"u}bertragung von CK sind und hierbei wichtige Modulatoren f{\"u}r eine CK-vermittelte Immunabwehr darstellen. (ii) Im zweiten Teil der Arbeit schließen sich Untersuchungen an einem in vitro-Experiment einer 2D- und 3D-Zellkultur einer HSP90-Behandlung in einem Lungentumormodell an. In diesem Zusammenhang wurden mithilfe der entwickelten Methode Unterschiede zwischen den beiden Zellkultursystemen gefunden, die das unterschiedliche Behandlungsansprechen erkl{\"a}ren, und f{\"u}r die beiden KRAS-mutierten Zelllinien A549 und H441 des 3D-Testsystems neue prognostische und therapeutische Kandidaten identifiziert. Hierbei haben die durchgef{\"u}hrten Analysen zwei funktionelle Cluster von Protein-Interaktionen um p53 und die STAT-Familie gefunden, welche eine Verbindung zu HSP90 haben und die entsprechenden Behandlungsunterschiede nach einer HSP90-Inhibierung zwischen den beiden Zellkultursystemen erkl{\"a}ren k{\"o}nnen. Unter Ber{\"u}cksichtigung des zelllinien-spezifischen Mutationshintergrunds wurde eine prognostische Markersignatur und daraus abgeleitet HIF1A f{\"u}r die H441-Zelllinie und AMPK f{\"u}r die A549-Zelllinie als neue therapeutische Targets gefunden, wobei die anschließend durchgef{\"u}hrten in silico-Simulationen einen potentiellen therapeutischen Effekt aufzeigen konnten. Weiterhin wurden wichtige experimentelle Readout-Parameter in ein in silico-Lungentumormodell integriert, wobei unter Einbeziehung des Mutationshintergrunds f{\"u}r die verwendeten Zelllinien die HSP90-Behandlung des 3D-Testsystems computergest{\"u}tzt abgebildet werden konnte. Im weiteren Verlauf wurden im in silico-Lungentumormodell Resistenzmechanismen nach einer Gefitinib-Behandlung mit bekanntem Mutationsstatus f{\"u}r die Zelllinien HCC827 und A549 untersucht und daraus folgend neue Therapieans{\"a}tze abgeleitet, die von potentieller klinischer Bedeutung sein k{\"o}nnen. Die durchgef{\"u}hrten in silico-Simulationen f{\"u}r HCC827 konnten hierbei zeigen, dass eine EGFR- und c-MET-Koaktivierung zu einer Gefitinib-Resistenz f{\"u}hren kann, wohingegen bei den A549 eine Komutation von KRAS und IGF-1R zu einem geringen Behandlungsansprechen beitr{\"a}gt. Die Simulationen lassen zudem erkennen, dass eine direkte Inhibierung der an der Resistenzentwicklung beteiligten Rezeptoren c-MET und IGF-1R in beiden F{\"a}llen nicht die bestm{\"o}gliche Therapiestrategie darstellt. In beiden Zelllinien konnte gezeigt werden, dass eine kombinierte Inhibierung von PI3K und MEK den bestm{\"o}glichen therapeutischen Effekt liefert, was demnach einen vielversprechenden Therapieansatz bei Gefitinib-resistenten Lungentumorpatienten darstellt. In einem weiteren Schritt wurde das therapeutische Potential der miRNA-21 im in silico-Modell f{\"u}r die HCC827-Zelllinie untersucht. Die durchgef{\"u}hrten Simulationen zeigen, dass eine miRNA-21-{\"U}berexpression zu einer Resistenzentwickung nach Gefitinib-Behandlung beitragen kann, wobei eine Inhibierung der miRNA-21 diesen Effekt umkehren kann. Die Ergebnisse lassen zudem erkennen, dass eine PTEN-Aktivierung als potentieller Marker einer erfolgreichen therapeutischen Inhibierung der miRNA-21 fungieren kann, wohingegen eine reduzierte miRNA-21-Expression als m{\"o}glicher Marker f{\"u}r eine erfolgreiche Gefitinib-Behandlung dienen kann. (iii) Im dritten Teil der Arbeit wurden systematisch RNA- und Protein-Interaktionen untersucht. Hierzu wurden integrierte systembiologische Analysen an neu identifizierten und funktionell bislang unbekannten lncRNAs durchgef{\"u}hrt. Die Analysen f{\"u}r die infolge einer Herzhypertrophie hochregulierte lncRNA Chast haben umfassend gezeigt, dass diese Proteine und Transkriptionsfaktoren regulieren und binden kann, welche die Signal{\"u}bertragung und Genexpression regulieren, aber auch eine Verbindung zum kardiovaskul{\"a}ren System und stressinduzierter Herzhypertrophie besitzt. Anhand der Ergebnisse l{\"a}sst sich schlussfolgern, dass Chast direkt und indirekt (a) Proteine binden und die Translation beeinflussen kann, zudem eine Chromatin-modifizierende Funktion besitzt und so die Transkription, z.B. f{\"u}r herz- und stress-assoziierte Gene, reguliert, und/oder (b) in einem negativen Feedbackloop seine eigene Transkription reguliert. Obwohl lncRNAs meist eine geringe Konservierung aufweisen, konnten die durchgef{\"u}hrten Analysen f{\"u}r Chast eine Sequenz-Struktur-Konservierung in S{\"a}ugetieren aufzeigen. Weiterhin haben die Untersuchungen an zwei hypoxie-induzierten lncRNAs in Endothelzellen gezeigt, dass die lncRNA MIR503HG eine hohe Sequenz-Struktur-Konservierung in S{\"a}ugetieren besitzt, wohingegen die LINC00323-003 eine geringe Konservierung aufzeigt. Dies untermauert die Tatsache, dass lncRNAs h{\"a}ufig eine geringe Konservierung aufweisen, was Untersuchungen in Modellorganismen hinsichtlich einer therapeutischen Nutzung schwierig machen. Da sich zahlreiche Untersuchungen auf Interaktionen und Signalwege konzentriert haben, wurde abschließend eine Datenbank entwickelt, welche Analysen von Protein-Interaktionen und Signalwegen nachhaltig voranbringt. Die entwickelte DrumPID-Datenbank stellt insbesondere die Interaktion zwischen einem Medikament und seinem Target in den Fokus und erm{\"o}glicht Analysen einzelner Interaktionen und beteiligter Signalwege, bietet zus{\"a}tzlich aber auch verschiedene Links zu anderen Datenbanken f{\"u}r individuelle weiterf{\"u}hrende Analysen. DrumPID erm{\"o}glicht ein geeignetes Medikament u. a. f{\"u}r ein vorgegebenes Zielprotein zu finden und dessen Wirkmechanismus und Interaktionskontext zu untersuchen, was zu einem besseren experimentellen Verst{\"a}ndnis beitragen kann. Zudem erlaubt DrumPID eine potentielle chemische Leitstruktur f{\"u}r ein Zielprotein zu entwickeln, was z.B. spezifisch ein parasitisches Protein inhibiert, ohne dabei einen toxischen Effekt im Menschen zu haben. Zahlreiche weitere Pharmakabeispiele belegen, dass DrumPID f{\"u}r den t{\"a}glichen wissenschaftlichen Gebrauch auf dem Gebiet der Analyse von Protein-Pharmaka-Interaktionen und der Medikamentenentwicklung geeignet ist. Die beschriebenen Ergebnisse der Promotionsarbeit wurden in f{\"u}nf Originalarbeiten, zwei {\"U}bersichtsartikeln und einem Buchteil, u. a. in Science Translational Medicine, ver{\"o}ffentlicht, sechs dieser Publikationen erfolgten im Rahmen von Erstautorschaften.},
  subject      = {Systembiologie},
  language  = {de}
}
@phdthesis{Faist2017,
  author    = {Faist, Hanna},
  title     = {Bedeutung und Charakterisierung der bakteriellen Flora in Vitis vinifera mit und ohne Wurzelhalsgallen},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-154359},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {Am Rebstock werden in der Natur von Agrobacterium vitis, dem Ausl{\"o}ser Wurzelhalsgallenerkrankung, charakteristische Wurzelhalsgallentumore induziert. Virulente Vertreter der Gattung der Agrobacteria schleusen bakterielle DNA in das pflanzliche Genom ein, wodurch die Pflanze Tumore produziert. Die Wurzelhalsgallenerkrankung wird seit einem Jahrhundert als ein Beispiel der Pflanzen-Pathogen-Interaktion untersucht. Die Rolle der bakteriellen Flora im Zusammenhang mit der Wurzelhalsgallenerkrankung beim Rebstock wurde bisher kaum betrachtet. Um dieser Frage nachzugehen, habe ich die endophytische mikrobielle Zusammensetzung von Rebst{\"o}cken mit und ohne Wurzelhalsgalle analysiert. Es werden Proben von drei Zeitpunkten einer Wachstumsperiode (Fr{\"u}hling, Sommer und Herbst) und von den Organen der Rebst{\"o}cke (Wurzeln, Pfropfstelle und einj{\"a}hrige Triebe) sowie dem Boden in einer Weinanlage bei Himmelstadt in Unterfranken genommen. Die Bakterienflora dieser Umweltproben wird mit kultivierungsabh{\"a}ngigen (Isolierung von Bakterien) und kultivierungsunabh{\"a}ngigen (Hochdurchsatzsequenzierungen) Methoden untersucht. Zudem werden i) die Virulenz der verschiedenen Agrobacterium-Isolate in Tumorassays bestimmt, ii) synthetische Bakteriengemeinschaften von in vitro kultivierten Weinpfl{\"a}nzchen mit Wurzelhalsgallen analysiert, iii) die Genome von einem virulenten und einem nicht-virulenten Agrobacteria-Isolat aus der Wurzelhalsgalle verglichen, iv) erste Interaktionsstudien auf festen N{\"a}hrmedien durchgef{\"u}hrt und v) virulente Agrobacteria mittels bildgebender Fluoreszenz-Lebenszeit-Mikroskopie (FLIM) in Wurzelhalsgallen lokalisiert. Die Rebst{\"o}cke dieser Studie haben eine organspezifische Bakterienflora, die innerhalb einer Wachstumsperiode variiert. Nur die Bakterienflora der Pfropfstelle (mit oder ohne Wurzelhalsgalle) aber nicht die des Bodens, der Wurzeln, und der einj{\"a}hrigen Triebe unterscheidet sich strukturell zwischen gesunden und erkrankten Rebst{\"o}cken. Mikroskopisch konnten virulente Agrobacteria punktuell in Interzellularen, sklerenchymatischen Geweben und assoziiert mit Leitgef{\"a}ßen nachgewiesen werden. Dadurch ist ausreichend Lebensraum vorhanden, der zus{\"a}tzlich von tumorspezifischen Bakterien besiedelt werden kann. Im Gegensatz zur gesunden Pfropfstelle ist in der Wurzelhalsgalle eine saisonal stabile Kernmikroflora, bestehend aus Vertreter von A. vitis, Pseudomonas, Enterobacteriaceae, Agrobacterium tumefaciens, Gammaproteobacteria und Burkholderiales, vorhanden. Diese Bakterien werden {\"u}berwiegend aus dem Boden rekrutiert und profitieren von der N{\"a}hrstoffsituation in der Wurzelhalsgalle. Wurzelhalsgallen enthalten Opine, die nur von der transformierten Pflanzenzelle produziert werden. Interessanterweise hat in dieser Arbeit ein Agrobacterium-Isolat Gene, die zum Opinkatabolismus beitragen und ein Pseudomonas-Isolat kann Opine als einzige Kohlenstoffquelle nutzen. Trotzdem sind beide Isolate weder virulent noch verdr{\"a}ngen sie die virulenten A. vitis, die ebenso Opine nutzen, aus der Wurzelhalsgalle. In synthetischen Bakteriengemeinschaften an in vitro kultivierten Weinpfl{\"a}nzchen konnte gezeigt werden, dass diese und weitere tumorspezifischen Bakterien, neben A. vitis, nicht essentiell zur Entstehung der Wurzelhalsgalle n{\"o}tig sind aber unterschiedliche Funktionen in der Wurzelhalsgalle {\"u}bernehmen. Ein Serratia-Isolat hemmt das Wachstum von A. vitis auf festen N{\"a}hrmedium, andere f{\"o}rdern oder hemmen das Wachstum der Wurzelhalsgalle. Nach Studien in der Literatur erh{\"o}hen weitere Bakterien die Resistenz des Rebstocks gegen{\"u}ber biotischem und abiotischem Stress. Zusammengefasst identifizierten und isolierte ich in dieser Studie unter 150 unterschiedlichen Bakterien in der Wurzelhalsgalle jene Bakterien, die neben A. vitis von der neuen {\"o}kologischen Nische profitieren und somit wahrscheinlich Opportunisten mit unterschiedlichen Funktionen sind. In Folge von multiplen Interaktionen in der Wurzelhalsgalle entsteht ein {\"o}kologisches Gleichgewicht zwischen den opportunistischen Bakterien, der Wurzelhalsgalle und dem Rebstock, das den Fortbestand des Rebstocks mit Wurzelhalsgalle erm{\"o}glicht.},
  subject      = {Wurzelhalsgalle},
  language  = {de}
}
@phdthesis{Hagen2017,
  author    = {Hagen, Franziska},
  title     = {Sphingolipids in gonococcal infection},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-153852},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {Neisseria gonorrhoeae, the causative agent of the sexually transmitted disease gonorrhea, has the potential to spread in the human host and cause a severe complication called disseminated gonococcal infection (DGI). The expression of the major outer membrane porin PorBIA is a characteristic of most gonococci associated with DGI. PorBIA binds to the scavenger receptor expressed on endothelial cells (SREC-I), which mediates the so-called low phosphate-dependent invasion (LPDI). This uptake mechanism enables N. gonorrhoeae to rapidly invade epithelial and endothelial cells in a phosphate-sensitive manner. We recently demonstrated that the neutral sphingomyelinase, which catalyses the hydrolysis of sphingomyelin to ceramide and phosphorylcholine, is required for the LPDI of gonococci in non-phagocytic cells. Neutral sphingomyelinase 2 (NSM2) plays a key role in the early PorBIA signaling by recruiting the PI3 kinase to caveolin. The following activation of the PI3 kinase-dependent downstream signaling leads to the engulfment of the bacteria. As a part of this work, I could confirm the involvement of the NSM2. The role of the enzyme was further elucidated by the generation of antibodies directed against NSM2 and the construction of an epithelium-based NSM2 knockout cell line using CRISPR/Cas9. The knockout of the NSM2 strongly inhibits the LPDI. The invasion could be, however, restored by the complementation of the knockout using an NSM2-GFP construct. However, the results could not be reproduced. In this work, I could show the involvement of further members of the sphingolipid pathway in the PorBIA-mediated invasion. Lipidome analysis revealed an increase of the bioactive molecules ceramide and sphingosine due to gonococcal infection. Both molecules do not only affect the host cell, but seem to influence the bacteria as well: while ceramide seems to be incorporated by the gonococci, sphingosine is toxic for the bacteria. Furthermore, the sphingosine kinase 2 (SPHK2) plays an important role in invasion, since the inhibition and knockdown of the enzyme revealed a negative effect on gonococcal invasion. To elucidate the role of the sphingosine kinases in invasion in more detail, an activity assay was established in this study. Additionally, the impact of the sphingosine-1-phosphate lyase (S1PL) on invasion was investigated. Inhibitor studies and infection experiments conducted with a CRISPR/Cas9 HeLa S1PL knockout cell line revealed a role of the enzyme not only in the PorBIA-mediated invasion, but also in the Opa50/HSPG-mediated gonococcal invasion. The signaling experiments allowed the categorization of the SPHK and S1PL activation in the context of infection. Like the NSM2, both enzymes play a role in the early PorBIA signaling events leading to the uptake of the bacteria. All those findings indicate an important role of sphingolipids in the invasion and survival of N. gonorrhoeae. In the last part of this work, the role of the NSM2 in the inhibition of apoptosis in neutrophils due to gonococcal infection was investigated. It could be demonstrated that the delayed onset of apoptosis is independent of neisserial porin and Opa proteins. Furthermore, the influence of neisserial peptidoglycan on PMN apoptosis was analysed using mutant strains, but no connection could be determined. Since the NSM2 is the most prominent sphingomyelinase in PMNs, fulfils manifold cell physiological functions and has already been connected to apoptosis, the impact of the enzyme on apoptosis inhibition due to gonococcal infection was investigated using inhibitors, with no positive results.},
  subject      = {gonococcal},
  language  = {en}
}
@phdthesis{Tsoneva2017,
  author    = {Tsoneva, Desislava},
  title     = {Humanized mouse model: a system to study the interactions of human immune system with vaccinia virus-infected human tumors in mice},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-118983},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {Ein vielversprechender neuer Ansatz zur Behandlung von Krebs beim Menschen ist die Verwendung von onkolytischen Viren, die einen Tumor-spezifischen Tropismus aufweisen. Einer der Top-Kandidaten in diesem Bereich ist das onkolytische Vaccinia Virus (VACV), das bereits vielversprechende Ergebnisse in Tierversuchen und in klinischen Studien gezeigt hat. Aber die von den in vivo in tierischen Modellen erhaltenen Resultate k{\"o}nnten ungenaue Informationen wegen der anatomischen und physiologischen Unterschiede zwischen den Spezies liefern. Andererseits sind Studien in Menschen aufgrund ethischer Erw{\"a}gungen und potenzieller Toxizit{\"a}t nur limitiert m{\"o}glich. Die zahlreichen Einschr{\"a}nkungen und Risiken, die mit den Humanstudien verbunden sind, k{\"o}nnten mit der Verwendung eines humanisierten Mausmodells vermieden werden. Die LIVP-1.1.1, GLV-2b372, GLV-1h68, GLV-1h375, GLV-1h376 and GLV-1h377 VACV St{\"a}mmen wurden von der Genelux Corporation zur Verf{\"u}gung gestellt. GLV-2b372 wurde durch Einf{\"u}gen der TurboFP635 Expressionskassette in den J2R Genlocus des parentalen LIVP-1.1.1-Stammes konstruiert. GLV-1h375, -1h376 and -1h377 kodiert das Gen f{\"u}r den menschlichen CTLA4-blockierenden Einzelketten-Antik{\"o}rper (CTLA4 scAb). Befunde aus Replikations- and Zytotoxizit{\"a}tsstudien zeigten, dass alle sechs Viren Tumorzellen infizieren, sich in ihnen replizieren und sie in Zellkultur schließlich ebenso dosis- und zeitabh{\"a}ngig effizient abt{\"o}ten konnten. CTLA4 scAb und β-Glucuronidase (GusA) Expression sowie Virus Titer in GLV-1h376-infizierten A549-Zellen wurde anhand von ELISA-, β-Glucuronidase- and Standard Plaque-Assays bestimmt. Hierbei zeigte sich eine ausgezeichnete Korrelation mit Korrelationskoeffizienten R2>0.9806. Der durch das GLV-1h376 kodierte CTLA4 scAb wurde erfolgreich aus {\"U}berst{\"a}nden von infizierten CV-1-Zellen gereinigt. CTLA4 scAb hat eine hohe in-vitro-Affinit{\"a}t zu seinem menschlichen CTLA4-Zielmolek{\"u}l sowie abwesende Kreuzreaktivit{\"a}t gegen{\"u}ber murine CTLA4 gezeigt. CTLA4 scAb Funktionalit{\"a}t wurde in Jurkat-Zellen best{\"a}tigt. LIVP-1.1.1, GLV-2b372, GLV-1h68 und GLV-1h376 wurden auch in nicht-tumor{\"o}sen und/oder tumortragenden humanisierten M{\"a}usen getestet. Zun{\"a}chst wurde gezeigt, dass die Injektion von menschlichen CD34+ Stammzellen in die Leber von vorkonditionierten neugeborenen NSG M{\"a}usen zu einer erfolgreichen systemische Rekonstitution mit menschlichen Immunzellen gef{\"u}hrt hat. CD19+-B-Zellen, CD4+- und CD8+-CD3+-T-Zellen, NKp46+CD56- und NKp46+CD56+-NK-Zellen sowie CD33+-myeloischen Zellen wurden detektiert. Die Mehrheit der nachgewisenen humanen h{\"a}matopoetischen Zellen im M{\"a}useblut in den ersten Wochen nach der Humanisierung waren CD19+-B-Zellen, und nur ein kleiner Teil waren CD3+-T-Zellen. Mit der Zeit wurde eine signifikante Ver{\"a}nderung in CD19+/CD3+-Verh{\"a}ltnis beobachtet, die parallel zur Abnahme der B-Zellen und einem Anstieg der T-Zellen kam. Die Implantation von A549-Zellen unter die Haut dieser M{\"a}use f{\"u}hrte zu einem progressiven Tumorwachstum. Bildgebende Verfahren zur Detektion von Virus-vermittelter TurboFP635- und GFP-Expression, Standard Plaque Assays sowie immunohistochemische Analysen best{\"a}tigten die erfolgreiche Invasion der Viren in die subkutanen Tumoren. Die humane CD45+-Zellpopulation in Tumoren wurde haupts{\"a}chlich durch NKp46+CD56bright-NK-Zellen und einen hohen Anteil von aktivierten CD4+- und zytotoxische CD8+-T-Zellen dargestellt. Es wurden jedoch keine signifikanten Unterschiede zwischen den Kontroll- und LIVP-1.1.1-infizierten Tumoren beobachtet, was darauf hindeutete, dass die Rekrutierung von NK- und aktivierten T-Zellen, mehr Tumorgewebe-spezifisch als Virus-abh{\"a}ngig waren. Die GLV-1h376-vermittelten CTLA4 scAb-Expression in den infizierten Tumoren war ebenfalls nicht in der Lage, die Aktivierung von Tumor-infiltrierenden T-Zellen im Vergleich zur Kontrolle und GLV-1h68-behandelten M{\"a}usen, signifikant zu erh{\"o}hen. ELISA-, β-Glucuronidase- and Standard Plaque-Assays zeigten eine eindeutige Korrelation mit den Korrelationskoeffizienten R2>0,9454 zwischen CTLA4 scAb- und GusA-Konzentrationen und Virus Titer in Tumorproben von GLV-1h376-behandelten M{\"a}usen. T-Zellen, die aus der Milz dieser Tumor-tragenden M{\"a}use isoliert wurden, waren funktionell und konnten erfolgreich mit Beads aktiviert werden. Mehr CD25+ und IFN-ɣ+ T-Zellen wurden in der GLV-1h376-Gruppe gefunden, wahrscheinlich aufgrund der CTLA4-Blockade durch die Virus-vermittelte CTLA4 scAb-Expression in den M{\"a}usen. Außerdem wurde eine h{\"o}here Konzentration von IL-2 in dem Kultur{\"u}berstand von diesen Splenozyten im Vergleich zu Kontrollproben nachgewiesen. Im Gegensatz zu der Aktivierung mit Beads konnten T-Zellen von allen drei Maus-Gruppen nicht durch A549 Tumorzellen ex vivo aktiviert werden. Unser Mausmodell hat den besonderen Vorteil, dass sich Tumoren unter der Haut der humanisierten M{\"a}use entwickeln, was eine genaue {\"U}berwachung des Tumorwachstums und Auswertung der onkolytischen Virotherapie erm{\"o}glicht.},
  subject      = {Vaccinia virus},
  language  = {en}
}
@phdthesis{Reis2017,
  author    = {Reis, Helena},
  title     = {Characterization of telomere protein complexes in Trypanosoma brucei},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-151323},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {African trypanosomiasis is a disease endemic to sub-Saharan Africa. It affects humans as well as wild and domestic animals. The human form of the disease is known as sleeping sickness and the animal form as nagana, which are usually fatal if left untreated. The cause of African trypanosomiasis is the unicellular parasite Trypanosoma brucei. During its life cycle, Trypanosoma brucei shuttles between a mammalian host and the tsetse fly vector. In the mammalian host the parasite multiplies as bloodstream form (BSF) extracellularly in the bloodstream or the lymphatic system. Survival of BSF parasites relies on immune evasion by antigenic variation of surface proteins because its extracellular lifestyle leads to direct exposure to immune responses. At any given time each BSF cell expresses a single type of variant surface glycoprotein (VSG) on its surface from a large repertoire. The active VSG is transcribed from one of 15 specialized subtelomeric domains, termed bloodstream expression sites (BESs). The remaining 14 BESs are silenced. This monoallelic expression and periodic switching of the expressed VSG enables to escape the immune response and to establish a persistent infection in the mammalian host. During developmental differentiation from BSF to the insect vector-resident procyclic form (PCF), the active BES is transcriptionally silenced to stop VSG transcription. Thus, all 15 BESs are inactive in the PCF cells as surface protein expression is developmentally regulated. Previous reports have shown that the telomere complex components TbTRF, TbRAP1 and TbTIF2 are involved in VSG transcriptional regulation. However, the precise nature of their contribution remains unclear. In addition, no information is available about the role of telomeres in the initiation and regulation of developmental BES silencing. To gain insights into the regulatory mechanisms of telomeres on VSG transcription and developmental repression it is therefore essential to identify the complete composition of the trypanosome telomere complex. To this end, we used two complementary biochemical approaches and quantitative label-free interactomics to determine the composition of telomere protein complexes in T. brucei. Firstly, using a telomeric pull-down assay we found 17 potential telomere-binding proteins including the known telomere-binding proteins TbTRF and TbTIF2. Secondly, by performing a co-immunoprecipitation experiment to elucidate TbTRF interactions we co-purified five proteins. All of these five proteins were also enriched with telomeric DNA in the pull-down assay. To validate these data, I characterized one of the proteins found in both experiments (TelBP1). In BSF cells, TelBP1 co-localizes with TbTRF and interacts with already described telomere-binding proteins such as TbTRF, TbTIF2 and TbRAP1 indicating that TelBP1 is a novel component of the telomere complex in trypanosomes. Interestingly, protein interaction studies in PCF cells suggested a different telomere complex composition compared to BSF cells. In contrast to known members of the telomere complex, TelBP1 is dispensable for cell viability indicating that its function might be uncoupled from the known telomere-binding proteins. Overexpression of TelBP1 had also no effect on cell viability, but led to the discovery of two additional shorter isoforms of TelBP1. However, their source and function remained elusive. Although TelBP1 is not essential for cell viability, western blot analysis revealed a 4-fold upregulation of TelBP1 in the BSF stage compared to the PCF stage supporting the concept of a dynamic telomere complex composition. We observed that TelBP1 influences the kinetics of transcriptional BES silencing during developmental transition from BSF to PCF. Deletion of TelBP1 caused faster BES silencing compared to wild-type parasites. Taken together, TelBP1 function illustrates that developmental BES silencing is a fine-tuned process, which involves stage-specific changes in telomere complex formation.},
  subject      = {Trypanosoma brucei},
  language  = {en}
}
@phdthesis{ContarAdolfi2017,
  author    = {Contar Adolfi, Mateus},
  title     = {Sex determination and meiosis in medaka: The role of retinoic acid},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-136335},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {Sex determination (SD) is a complex and diverse developmental process that leads to the decision whether the bipotential gonad anlage will become a testis or an ovary. This mechanism is regulated by gene cascades, networks and/or chromosomal systems, and can be influenced by fluctuations of extrinsic factors like temperature, exposure to hormones and pollution. Within vertebrates, the group of fish show the widest variety of sex determination mechanism. This whole diversity of processes and mechanisms converges to the formation of two different gametes, the eggs and the sperm, the first bigger and static, and the second smaller and motile. Meiosis is crucial for the formation of both types of gametes, and the timing of meiosis entry is one of the first recognizable differences between male and female in vertebrates. The germ cells go into meiosis first in female than in male, and in mammals, this event has been shown to be regulated by retinoic acid (RA). This small polar molecule induces in the germ cells the expression of the pre-meiotic marker Stra8 (stimulated by retinoic acid gene 8), which is necessary for meiosis initiation. Interestingly, genome analyzes have shown that the majority of fish (including medaka) lack the stra8 gene, adding a question mark to the role of RA in meiosis induction in this group. Since a role of RA in entry of meiosis and sexual development of fish is still far from being understood, I investigated in medaka (Oryzias latipes) a possible signaling function of RA during the SD period in embryos and in reproductively active gonads of adults. I generated a transgenic medaka line that reports responsiveness to RA in vivo. With this tool, I compared RA responsiveness with the expression of the main gene involved in the synthesis of RA. My results show that there is a de-correlation between the action of RA with its source. In adults, expression of the RA metabolizing enzymes show sexually dimorphic RA levels, with aldh1a2 levels being higher in testis, and cyp26a1 stronger in female gonad. In ovary, the responsiveness is restricted to the early meiotic oocytes. In testis, RA is acting directly in the pre-meiotic cells, but also in Sertoli and Leydig cells. Treatment experiments on testis organ culture showed that RA pathway activation leads to a decrease in meiosis markers expression levels. During the development, RA responsiveness in the germ cells was observed in both sexes much earlier than the first female meiosis entry. Treatments with RA-synthesis inhibitor show a decrease in meiosis markers expression levels only after the sex differentiation period in female. Expression analyzes of embryos treated with exogenous RA showed induction of dmrt1a at the gonad levels and an increase of amh levels. Both genes are not only involved in male formation, but also in the regulation of germ cell proliferation and differentiation. RA is important in meiosis induction and gametogenesis in adult medaka. However, there is no evidence for a similar role of RA in initiating the first meiosis in female germ cells at the SD stage. Moreover, contrary to common expectation, RA seems to induce sex related genes that are involved indirectly in meiosis inhibition. In this thesis, I showed for the first time that RA can be involved in both induction and inhibition of meiosis entry, depending on the sex and the developmental stage in a stra8-independent model organism.},
  subject      = {Japank{\"a}rpfling},
  language  = {en}
}
@phdthesis{Zimnol2017,
  author    = {Zimnol, Anna},
  title     = {Relevance of angiotensin II type 1a receptor and NADPH oxidase for the formation of angiotensin II-mediated DNA damage},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-137469},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {Das Renin-Angiotensin-Aldosteron-System (RAAS) reguliert den Blutdruck sowie den Elektrolyt- und Wasserhaushalt. Das aktive Peptid, Angiotensin II (AngII), f{\"u}hrt dabei zur Vasokonstriktion und in h{\"o}heren Konzentrationen zu Bluthochdruck. Hypertensive Patienten haben ein erh{\"o}htes Risiko an Krebs zu erkranken, vor allem an Nierenkrebs. Wir konnten bereits in vivo zeigen, dass AngII in der Lage ist, den Blutdruck zu steigern und dosisabh{\"a}ngig zu DNA-Sch{\"a}den {\"u}ber den Angiotensin II Typ 1-Rezeptor (AT1R) f{\"u}hrt. Ein stimuliertes RAAS kann ferner {\"u}ber die Aktivierung der NADPH-Oxidase, einer Hauptquelle der Generierung reaktiver Sauerstoffspezies (ROS) in der Zelle, zu oxidativem Stress f{\"u}hren. Zielsetzung dieser Arbeit war es zum einen, mit Hilfe von AT1a-Rezeptor-defizienten M{\"a}usen in vivo zu pr{\"u}fen, ob die Bildung von ROS, sowie die Bildung von DNA-Sch{\"a}den in der Niere und im Herzen unabh{\"a}ngig von einem erh{\"o}hten Blutdruck auftreten. Zum anderen sollte, ebenfalls in vivo, untersucht werden, ob eine oder beide von zwei untersuchten Isoformen der NADPH-Oxidase (Nox) f{\"u}r die Ausl{\"o}sung oxidativen Stresses in der Niere verantwortlich ist. Zun{\"a}chst wurden f{\"u}r den Versuch zur {\"U}berpr{\"u}fung der Abh{\"a}ngigkeit AngII-induzierter DNA-Sch{\"a}den vom Blutdruck m{\"a}nnliche C57BL/6-M{\"a}use und AT1a-Knockout (KO)-M{\"a}use mit osmotischen Minipumpen ausgestattet, die AngII in einer Konzentrationen von 600 ng/kg min {\"u}ber einen Zeitraum von 28 Tagen abgaben. Zus{\"a}tzlich wurde eine Gruppe von AngII-behandelten Wildtyp (WT)-M{\"a}usen mit dem AT1-Rezeptor-Blocker Candesartan (Cand) behandelt. W{\"a}hrend des Versuchszeitraumes fanden regelm{\"a}ßige, nicht-invasive Blutdruckmessungen an den wachen M{\"a}usen statt. In WT-M{\"a}usen induzierte AngII Bluthochdruck, verursachte erh{\"o}hte Albumin-Level im Urin und f{\"u}hrte zur Bildung von ROS in Niere und im Herzen. Außerdem traten in dieser Gruppe DNA-Sch{\"a}den in Form von Einzel- und Doppelstrangbr{\"u}chen auf. All diese Reaktionen auf AngII konnten jedoch durch gleichzeitige Behandlung mit Cand verhindert werden. AT1a-KO-M{\"a}use hatten, verglichen mit WT-Kontrollm{\"a}usen, einen signifikant niedrigeren Blutdruck und normale Albumin-Level im Urin. In AT1a-KO-M{\"a}usen, die mit AngII behandelt wurden, konnte kein Anstieg des systolischen Blutdrucks sowie kein Einfluss auf die Nierenfunktion gefunden werden. Jedoch f{\"u}hrte AngII in dieser Gruppe zu einer Steigerung von ROS in der Niere und im Herzen. Zus{\"a}tzlich wurden genomische Sch{\"a}den, vor allem in Form von Doppelstrangbr{\"u}chen signifikant in dieser Gruppe induziert. Auch wenn AT1a-KO-Tiere, unabh{\"a}ngig von einer AngII-Infusion, keine eingeschr{\"a}nkte Nierenfunktion zeigten, so wiesen sie erhebliche histopathologische Sch{\"a}den im Hinblick auf die Glomeruli und das Tubulussystem auf. Diese Art von Sch{\"a}den deuten auf eine besondere Bedeutung des AT1aR im Hinblick auf die embryonale Entwicklung der Niere hin. Zusammenfassend beweisen die Ergebnisse dieses Experiments eindeutig, dass eine AngII-induzierte ROS-Produktion und die Induktion von DNA-Sch{\"a}den unabh{\"a}ngig von einem erh{\"o}hten Blutdruck auftreten. Da in der AngII-behandelten AT1a-KO-Gruppe eine signifikant h{\"o}here Expression des AT1b-Rezeptors zu finden war und die Blockade von beiden Rezeptorsubtypen mit Cand zu einer Verhinderung der sch{\"a}dlichen Effekte durch AngII f{\"u}hrte, scheint der AT1bR im Falle einer AT1aR-Defizienz f{\"u}r die Entstehung der Sch{\"a}den zust{\"a}ndig zu sein. Ziel des zweiten Experimentes war es, den Beitrag der Nox2 und Nox4 zum oxidativen DNA-Schaden in vivo zu untersuchen. Hierf{\"u}r wurden m{\"a}nnliche C57BL/6-M{\"a}use und Nox2- oder Nox4-defiziente M{\"a}use mit osmotischen Minipumpen ausgestattet, die AngII in einer Konzentration von 600 ng/kg min {\"u}ber einen Zeitraum von 28 Tagen abgaben. Im WT-Stamm und in beiden Nox-defizienten St{\"a}mmen induzierte AngII Bluthochdruck, verursachte erh{\"o}hte Albumin-Level im Urin und f{\"u}hrte zur Bildung von ROS in der Niere. Außerdem waren in allen AngII-behandelten Gruppen genomische Sch{\"a}den, vor allem in Form von Doppelstrangbr{\"u}chen, erh{\"o}ht. Auch in Abwesenheit von AngII wiesen Nox2- und Nox4-defiziente M{\"a}use mehr Doppelstrangbr{\"u}che im Vergleich zu WT-Kontrollm{\"a}usen auf. Interessanterweise kompensieren allerdings weder Nox2 noch Nox4 das Fehlen der jeweils anderen Isoform auf RNA-Basis. Aufgrund dieser Ergebnisse schließen wir, dass bislang keine Isoform alleine f{\"u}r die Generierung von oxidativen DNA-Sch{\"a}den in der Niere verantwortlich gemacht werden kann und dass eine Beteiligung einer weiteren Nox-Isoform sehr wahrscheinlich ist. M{\"o}glicherweise k{\"o}nnten aber auch andere ROS-generierende Enzyme, wie Xanthinoxidase oder Stickoxidsynthase involviert sein. Da genomische Sch{\"a}den in Nieren von Nox2- und Nox4-defizienten M{\"a}usen in Abwesenheit von AngII gegen{\"u}ber den Sch{\"a}den in WT-Kontrollm{\"a}usen erh{\"o}ht waren, k{\"o}nnten die beiden Isoformen auch eine sch{\"u}tzende Funktion im Bereich von Nierenkrankheiten {\"u}bernehmen. Da dies aber bislang nur f{\"u}r Nox4 beschrieben ist, ist es wahrscheinlicher, dass das Fehlen von einer der beiden Isoformen eher einen Einfluss auf die Embryonalentwicklung hat. Um dies jedoch abschließend zu kl{\"a}ren w{\"a}re es sinnvoll mit induzierbaren Knockout-Modellen zu arbeiten, bei denen m{\"o}gliche entwicklungsbedingte Effekte minimiert werden k{\"o}nnen.},
  subject      = {Angiotensin II},
  language  = {de}
}
@phdthesis{Hofmann2017,
  author    = {Hofmann, Stefan},
  title     = {Hybridisierungsbasierte Multiplex-Detektion von microRNA mit CMOS-Technologie f{\"u}r die Anwendung in der Point-of-Care-Diagnostik},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-150949},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {MicroRNAs sind kurze, nicht-kodierende Ribonukleins{\"a}uren, die eine wichtige Rolle bei der Genregulation spielen. Sie sind an vielen physiologischen Prozessen beteiligt und werden als vielversprechende Kandidaten f{\"u}r eine neue Generation von Biomarkern gehandelt. Die Quantifizierung von miRNAs aus Blut oder anderen K{\"o}rperfl{\"u}ssigkeiten verspricht eine fr{\"u}he Diagnose verschiedener Krankheitsbilder. Dazu z{\"a}hlen neben zahlreichen Krebsformen unter anderem auch Autoimmun- oder Herz-Kreislauferkrankungen. Um diese Biomarker schnell, sensitiv und spezifisch detektieren zu k{\"o}nnen, werden geeignete Detektionssysteme ben{\"o}tigt. Dabei liegt ein besonderer Fokus auf der Entwicklung von Point-of-Care-Systemen, die eine automatisierte Durchf{\"u}hrung mit einfacher Handhabung verlangen. Mikrochips k{\"o}nnen als leistungsf{\"a}hige technische Hilfsmittel f{\"u}r eine robuste und miniaturisierte Signalerfassung an biochemischen Grenzfl{\"a}chen dienen. Auf der Grundlage eines CMOS-Chips mit einem Sensorarray aus interdigitalen Gold-Elektroden sollte in dieser Arbeit eine quantitative und multiplexf{\"a}hige miRNA-Detektionsmethode mit elektrochemischer Signaltransduktion entworfen und untersucht werden. Weitere wichtige Zielfaktoren waren eine einfache und schnelle Durchf{\"u}hrbarkeit, eine hohe Spezifit{\"a}t und eine gute Sensitivit{\"a}t bei gleichzeitigem Verzicht auf Amplifikation und Vormarkierung des Ausgangsmaterials. Es wurden verschiedene Methoden entworfen, {\"u}berpr{\"u}ft, untersucht, optimiert und weiterentwickelt. Das beste Ergebnis wurde letztlich mit einem als Sandwich-Ligations-Methode bezeichneten Verfahren erzielt. Dabei wird zun{\"a}chst ein aus zwei doppelstr{\"a}ngigen Assay-Komponenten und der Ziel-miRNA bestehender dreiteiliger Hybridisierungskomplex gebildet, der eine beidseitige spezifische Ligation der miRNA mit einem auf der Sensoroberfl{\"a}che immobilisierten F{\"a}ngerstrang und einem enzymmarkierten Reporterstrang vermittelt. Durch einen anschließenden Waschschritt werden alle {\"u}bersch{\"u}ssigen Markierungen vom Detektionsbereich entfernt, so dass bei der Detektion nur Reporterenzyme ausgelesen werden, die {\"u}ber die Ziel-miRNA kovalent mit dem immobilisierten Strang verbunden sind. Dieses Signal ist daher proportional zur Ausgangskonzentration der gesuchten miRNA. Die Methode wurde mit Hilfe von synthetischen miRNAs etabliert und optimiert. Sie erreichte eine analytische Sensitivit{\"a}t von unter 1 pM Ziel-Nukleins{\"a}ure bei einer Gesamt-Versuchsdauer von nur 30 Minuten. Konzentrationsreihen demonstrierten einen linearen dynamischen Messbereich zwischen 1 pM und 1 nM, der eine verl{\"a}ssliche Quantifizierung der detektierten miRNAs in diesem Bereich erm{\"o}glicht. Die sehr gute Spezfifit{\"a}t des Assays zeigte sich bei der Untersuchung des Einflusses verschiedener IsomiRs auf das Messergebnis sowie im Rahmen von Experimenten mit miRNAs der let-7-Familie. Dabei konnten Ziel-Nukleins{\"a}uren mit Einzelbasenunterschieden klar differenziert werden. Die Multiplexf{\"a}higkeit der vorgestellten Methode wurde durch die gleichzeitige Quantifizierung von bis zu acht miRNAs auf einem CMOS-Chip demonstriert, zuz{\"u}glich Kontrollen. Die Validierung der Detektionsmethode erfolgte mit Gesamt-RNA-Extrakten aus Vollblutproben. Dazu wurde ein kardiales Panel aus acht miRNAs, die auf Basis von Studien zu zirkulierenden miRNAs bei Herzerkrankungen ausgew{\"a}hlt wurden, festgelegt. Mit Hilfe der entsprechenden optimierten Detektionskomponenten wurden aus Spenderblut gewonnene endogene miRNAs analysiert. Dabei zeigte sich f{\"u}r f{\"u}nf der acht Kandidaten sowohl eine solide Korrelation zwischen eingesetzter Gesamt-RNA-Menge und Messsignal, als auch eine gute Reproduzierbarkeit der Ergebnisse. Die Konzentrationen der {\"u}brigen drei miRNAs lagen nah am unteren Detektionslimit und lieferten daher keine verl{\"a}sslichen Daten. Mit Hilfe sogenannter branched DNA zur Signalamplifikation k{\"o}nnte bei Bedarf die Sensitivit{\"a}t des Assays noch verbessert werden, was durch weitere Experimente dieser Arbeit demonstriert wurde. Ein Vergleichsexperiment zwischen der Sandwich-Ligations-Methode und qRT-PCR zeigte nur eine schwache Korrelation der Messergebnisse. Dies ist jedoch konsistent mit anderen Studien zur Vergleichbarkeit unterschiedlicher Detektionsmethoden. Abschließend wurden die miRNAs des kardialen Panels in Gesamt-RNA-Extrakten aus Vollblut von Herzinfarktpatienten und Kontrollen mit der entwickelten Detektionsmethode analysiert und die Ergebnisse verglichen. Dabei konnten Abweichungen in den Konzentrationen von miR-15a und miR-425 aufgedeckt werden. Eine entsprechende diagnostische Untersuchung mit der hier vorgelegten und validierten Detektionsmethode k{\"o}nnte eine Alternative oder Erg{\"a}nzung zu aktuell eingesetzten proteinbasierten Tests bieten.},
  subject      = {miRNS},
  language  = {de}
}
@phdthesis{Bertho2016,
  author    = {Bertho, Sylvain},
  title     = {Biochemical and molecular characterization of an original master sex determining gene in Salmonids},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-139130},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {Sexual development is a fundamental and versatile process that shapes animal morphology, physiology and behavior. The underlying developmental process is composed of the sex determination and the sex differentiation. Sex determination mechanisms are extremely labile among taxa. The initial triggers of the sex determination process are often genetics called sex determining genes. These genes are expressed in the bipotential gonad and tilt the balance to a developmental program allowing the differentiation of either a testis or an ovary. Fish represent a large and fascinating vertebrate group to study both sex determination and sex differentiation mechanisms. To date, among the known sex determining genes, three gene families namely sox, dmrt and TGF-β factors govern this developmental program. As exception to this rule, sdY "sexually dimorphic on the Y" does not belong to one of these families as it comes from the duplication / evolution of an ancestor gene related to immunity, i.e., the interferon related factor 9, irf9. sdY is the master sex determining gene in salmonids, a group of fishes that include species such as rainbow trout and Atlantic salmon. The present study was aimed to firstly characterize the features of SdY protein. Results indicate that SdY is predominantly localized in the cytoplasm tested in various fish and mammalian cell lines and confirmed by different methods. Predictive in silico analysis revealed that SdY is composed of a β-sandwich core surrounded by three α-helices as well specific characteristics conferring a putative protein-protein interaction site. Secondly, the study was aimed to understand how SdY could trigger testicular differentiation. SdY is a truncated divergent version of Irf9 that has a conserved protein-protein domain but lost the DNA interaction domain of its ancestor gene. It was then hypothesized that SdY could initiate testicular differentiation by protein-protein interactions. To evaluate this we first conducted a yeast-two-hybrid screen that revealed a high proportion of transcription factors including fox proteins. Using various biochemical and cellular methods we confirm an interaction between SdY and Foxl2, a major transcription factor involved in ovarian differentiation and identity maintenance. Interestingly, the interaction of SdY with Foxl2 leads to nuclear translocation of SdY from the cytoplasm. Furthermore, this SdY translocation mechanism was found to be specific to fish Foxl2 and to a lesser extend Foxl3 and not other Fox proteins or mammalian FoxL2. In addition, we found that this interaction allows the stabilization of SdY and prevents its degradation. Finally, to better decipher SdY action we used as a model a mutated version of SdY that was identified in XY females of Chinook salmon natural population. Results show that this mutation induces a local conformation defect obviously leading to a misfolded protein and a quick degradation. Moreover, the mutated version compromised the interaction with Foxl2 defining a minimal threshold to induce testicular differentiation. Altogether results from my thesis propose that SdY would trigger testicular differentiation in salmonids by preventing Foxl2 to promote ovarian differentiation. Further research should be now carried out on how this interaction of SdY and Foxl2 acts in-vivo.},
  subject      = {Lachsartige <Familie>},
  language  = {en}
}
@phdthesis{Winkler2015,
  author    = {Winkler, Ann-Cathrin Nicole},
  title     = {Identification of human host cell factors involved in \(Staphylococcus\) \(aureus\) 6850 infection},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-114300},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {Staphylococcus aureus is both a human commensal and a pathogen. 20\%-30\% of all individuals are permanently or occasionally carriers of S. aureus without any symptoms. In contrast to this, S. aureus can cause life-threatening diseases e.g. endocarditis, osteomyelitis or sepsis. Here, the increase in antibiotic resistances makes it more and more difficult to treat these infections and hence the number of fatalities rises constantly. Since the pharmaceutical industry has no fundamentally new antibiotics in their pipeline, it is essential to better understand the interplay between S. aureus and the human host cell in order to find new, innovative treatment options. In this study, a RNA interference based whole genome pool screen was performed to identify human proteins, which play a role during S. aureus infections. Since 1,600 invasion and 2,271 cell death linked factors were enriched at least 2 fold, the big challenge was to filter out the important ones. Here, a STRING pathway analysis proved to be the best option. Subsequently, the identified hits were validated with the help of inhibitors and a second, individualised small interfering RNA-based screen. In the course of this work two important steps were identified, that are critical for host cell death: the first is bacterial invasion, the second phagosomal escape. The second step is obligatory for intracellular bacterial replication and subsequent host cell death. Invasion in turn is determining for all following events. Accordingly, the effect of the identified factors towards these two crucial steps was determined. Under screening conditions, escape was indirectly measured via intracellular replication. Three inhibitors (JNKII, Methyl-beta-cyclodeytrin, 9-Phenantrol) could be identified for the invasion process. In addition, siRNAs targeted against 16 different genes (including CAPN2, CAPN4 and PIK3CG), could significantly reduce bacterial invasion. Seven siRNAs (FPR2, CAPN4, JUN, LYN, HRAS, AKT1, ITGAM) were able to inhibit intracellular replication significantly. Further studies showed that the IP3 receptor inhibitor 2-APB, the calpain inhibitor calpeptin and the proteasome inhibitor MG-132 are able to prevent phagosomal escape and as a consequence intracellular replication and host cell death. In this context the role of calpains, calcium, the proteasome and the mitochondrial membrane potential was further investigated in cell culture. Here, an antagonistic behaviour of calpain 1 and 2 during bacterial invasion was observed. Intracellular calcium signalling plays a major role, since its inhibition protects host cells from death. Beside this, the loss of mitochondrial membrane potential is characteristic for S. aureus infection but not responsible for host cell death. The reduction of membrane potential can be significantly diminished by the inhibition of the mitochondrial Na+/Ca2+ exchanger. All together, this work shows that human host cells massively contribute to different steps in S. aureus infection rather than being simply killed by bacterial pore-forming toxins. Various individual host cell factors were identified, which contribute either to invasion or to phagosomal escape and therefore to S. aureus induced cytotoxicity. Finally, several inhibitors of S. aureus infection were identified. One of them, 2-APB, was already tested in a sepsis mouse model and reduced bacterial load of kidneys. Thus, this study shows valuable evidence for novel treatment options against S. aureus infections, based on the manipulation of host cell signalling cascades.},
  subject      = {Staphylococcus aureus},
  language  = {en}
}
@phdthesis{Maurus2016,
  author    = {Maurus, Katja},
  title     = {Melanoma Maintenance by the AP1 Transcription Factor FOSL1},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-142995},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {Identifying novel driver genes in cancer remains a crucial step towards development of new therapeutic approaches and the basic understanding of the disease. This work describes the impact of the AP1 transcription activator component FOSL1 on melanoma maintenance. FOSL1 is strongly upregulated during the progression of melanoma and the protein abundance is highest in metastases. I found that the regulation of FOSL1 is strongly dependent on ERK1/2- and PI3K- signaling, two pathways frequently activated in melanoma. Moreover, the involvement of p53 in FOSL1 regulation in melanoma was investigated. Elevated levels of the tumor suppressor led to decreased FOSL1 protein levels in a miR34a/miR34c- dependent manner. The benefit of elevated FOSL1 amounts in human melanoma cell lines was analyzed by overexpression of FOSL1 in cell lines with low endogenous FOSL1 levels. Enhanced levels of FOSL1 had several pro-tumorigenic effects in human melanoma cell lines. Besides increased proliferation and migration rates, FOSL1 overexpression induced the colony forming ability of the cells. Additionally, FOSL1 was necessary for anchorage independent growth in 3D cell cultures. Microarray analyses revealed novel downstream effectors of FOSL1. On the one hand, FOSL1 was able to induce the transcription of different neuron-related genes, such as NEFL, NRP1 and TUBB3. On the other hand, FOSL1 influenced the transcription of DCT, a melanocyte specific gene, in dependence of the differentiation of the melanoma cell line, indicating dedifferentiation. Furthermore, FOSL1 induced the transcription of HMGA1, a chromatin remodeling protein with reprogramming ability, which is characteristic for stem cells. Consequently, the influence of HMGA1 on melanoma maintenance was investigated. In addition to decreased proliferation and reduced anoikis resistance, HMGA1 knockdown reduced melanoma cell survival. Interestingly, the FOSL1 induced pro-tumorigenic effects were demonstrated to be dependent on the HMGA1 level. HMGA1 manipulation reversed FOSL1 induced proliferation and colony forming ability, as well as the anchorage independent growth effect. In conclusion, I could show that additional FOSL1 confers a clear growth benefit to melanoma cells. This benefit is attributed to the induction of stem cell determinants, but can be blocked by the inhibition of the ERK1/2 or PI3K signaling pathways.},
  subject      = {Melanom},
  language  = {en}
}
@phdthesis{Beck2016,
  author    = {Beck, Katherina},
  title     = {Einfluss von RSK auf die Aktivit{\"a}t von ERK, den axonalen Transport und die synaptische Funktion in Motoneuronen von \(Drosophila\) \(melanogaster\)},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-130717},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {In dieser Arbeit sollte die Funktion von RSK in Motoneuronen von Drosophila untersucht werden. Mutationen im RSK2-Gen verursachen das Coffin-Lowry-Syndrom (CLS), das durch mentale Retardierung charakterisiert ist. RSK2 ist haupts{\"a}chlich in Regionen des Gehirns exprimiert, in denen Lernen und Ged{\"a}chtnisbildung stattfinden. In M{\"a}usen und Drosophila, die als Modellorganismen f{\"u}r CLS dienen, konnten auf makroskopischer Ebene keine Ver{\"a}nderungen in den Hirnstrukturen gefunden werden, dennoch wurden in verschiedenen Verhaltensstudien Defekte im Lernen und der Ged{\"a}chtnisbildung beobachtet. Die synaptische Plastizit{\"a}t und die einhergehenden Ver{\"a}nderungen in den Eigenschaften der Synapse sind fundamental f{\"u}r adaptives Verhalten. Zur Analyse der synaptischen Plastizit{\"a}t eignet sich das neuromuskul{\"a}re System von Drosophila als Modell wegen des stereotypen Innervierungsmusters und der Verwendung ionotroper Glutamatrezeptoren, deren Untereinheiten homolog sind zu den Untereinheiten der Glutamatrezeptoren des AMPA-Typs aus S{\"a}ugern, die wesentlich f{\"u}r die Bildung von LTP im Hippocampus sind. Zun{\"a}chst konnte gezeigt werden, dass RSK in den Motoneuronen von Drosophila an der pr{\"a}synaptischen Seite lokalisiert ist, wodurch RSK eine Synapsen-spezifische Funktion aus{\"u}ben k{\"o}nnte. Morphologische Untersuchungen der Struktur der neuromuskul{\"a}ren Synapsen konnten aufzeigen, dass durch den Verlust von RSK die Gr{\"o}ße der neuromuskul{\"a}ren Synapse, der Boutons sowie der Aktiven Zonen und Glutamatrezeptorfelder reduziert ist. Obwohl mehr Boutons gebildet werden, sind weniger Aktive Zonen und Glutamatrezeptorfelder in der neuromuskul{\"a}ren Synapse enthalten. RSK reguliert die synaptische Transmission, indem es die postsynaptische Sensitivit{\"a}t, nicht aber die Freisetzung der Neurotransmitter an der pr{\"a}synaptischen Seite beeinflusst, obwohl in immunhistochemischen Analysen eine postsynaptische Lokalisierung von RSK nicht nachgewiesen werden konnte. RSK ist demnach an der Regulation der synaptischen Plastizit{\"a}t glutamaterger Synapsen beteiligt. Durch immunhistochemische Untersuchungen konnte erstmals gezeigt werden, dass aktiviertes ERK an der pr{\"a}synaptischen Seite lokalisiert ist und diese synaptische Lokalisierung von RSK reguliert wird. Dar{\"u}ber hinaus konnte in dieser Arbeit nachgewiesen werden, dass durch den Verlust von RSK hyperaktiviertes ERK in den Zellk{\"o}rpern der Motoneurone vorliegt. RSK wird durch den ERK/MAPK-Signalweg aktiviert und {\"u}bernimmt eine Funktion sowohl als Effektorkinase als auch in der Negativregulation des Signalwegs. Demnach dient RSK in den Zellk{\"o}rpern der Motoneurone als Negativregulator des ERK/MAPK-Signalwegs. Dar{\"u}ber hinaus k{\"o}nnte RSK die Verteilung von aktivem ERK in den Subkompartimenten der Motoneurone regulieren. Da in vorangegangenen Studien gezeigt werden konnte, dass ERK an der Regulation der synaptischen Plastizit{\"a}t beteiligt ist, indem es die Insertion der AMPA-Rezeptoren zur Bildung der LTP reguliert, sollte in dieser Arbeit aufgekl{\"a}rt werden, ob der Einfluss von RSK auf die synaptische Plastizit{\"a}t durch seine Funktion als Negativregulator von ERK zustande kommt. Untersuchungen der genetischen Interaktion von rsk und rolled, dem Homolog von ERK in Drosophila, zeigten, dass die durch den Verlust von RSK beobachtete reduzierte Gesamtzahl der Aktiven Zonen und Glutamatrezeptorfelder der neuromuskul{\"a}ren Synapse auf die Funktion von RSK als Negativregulator von ERK zur{\"u}ckzuf{\"u}hren ist. Die Gr{\"o}ße der neuromuskul{\"a}ren Synapse sowie die Gr{\"o}ße der Aktiven Zonen und Glutamatrezeptorfelder beeinflusst RSK allerdings durch seine Funktion als Effektorkinase des ERK/MAPK-Signalwegs. Studien des axonalen Transports von Mitochondrien zeigten, dass dieser in vielen neuropathologischen Erkrankungen beeintr{\"a}chtigt ist. Die durchgef{\"u}hrten Untersuchungen des axonalen Transports in Motoneuronen konnten eine neue Funktion von RSK in der Regulation des axonalen Transports aufdecken. In den Axonen der Motoneurone von RSK-Nullmutanten wurden BRP- und CSP-Agglomerate nachgewiesen. RSK k{\"o}nnte an der Regulation des axonalen Transports von pr{\"a}synaptischem Material beteiligt sein. Durch den Verlust von RSK wurden weniger Mitochondrien in anterograder Richtung entlang dem Axon transportiert, daf{\"u}r verweilten mehr Mitochondrien in station{\"a}ren Phasen. Diese Ergebnisse zeigen, dass auch der anterograde Transport von Mitochondrien durch den Verlust von RSK beeintr{\"a}chtigt ist.},
  subject      = {Taufliege},
  language  = {de}
}
@phdthesis{Pischimarov2016,
  author    = {Pischimarov, Jordan Ivanov},
  title     = {Bioinformatische Methoden zur Identifizierung und Klassifizierung somatischer Mutationen in h{\"a}matologischen Erkrankungen},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-147773},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {Die Sequenzierungstechnologien entwickeln sich stetig weiter, dies erm{\"o}glicht eine zuvor nicht erreichte Ausbeute an experimentellen Daten und auch an Neuentwicklungen von zuvor nicht realisierbaren Experimenten. Zugleich werden spezifische Datenbanken, Algorithmen und Softwareprogramme entwickelt, um die neu entstandenen Daten zu analysieren. W{\"a}hrend der Untersuchung bioinformatischer Methoden f{\"u}r die Identifizierung und Klassifizierung somatischer Mutationen in h{\"a}matologischen Erkrankungen, zeigte sich eine hohe Vielfalt an alternativen Softwaretools die f{\"u}r die jeweiligen Analyseschritte genutzt werden k{\"o}nnen. Derzeit existiert noch kein Standard zur effizienten Analyse von Mutationen aus Next-Generation-Sequencing (NGS)-Daten. Die unterschiedlichen Methoden und Pipelines generieren Kandidaten, die zum gr{\"o}ßten Anteil in allen Ans{\"a}tzen identifiziert werden k{\"o}nnen, jedoch werden Software spezifische Kandidaten nicht einheitlich detektiert. Um eine einheitliche und effiziente Analyse von NGS-Daten durchzuf{\"u}hren war im Rahmen dieser Arbeit die Entwicklung einer benutzerfreundlichen und einheitlichen Pipeline vorgesehen. Hierf{\"u}r wurden zun{\"a}chst die essentiellen Analysen wie die Identifizierung der Basen, die Alignierung und die Identifizierung der Mutationen untersucht. Des Weiteren wurden unter Ber{\"u}cksichtigung von Effizienz und Performance diverse verf{\"u}gbare Softwaretools getestet, ausgewertet und sowohl m{\"o}gliche Verbesserungen als auch Erleichterungen der bisherigen Analysen vorgestellt und diskutiert. Durch Mitwirken in Konsortien wie der klinischen Forschergruppe 216 (KFO 216) und International Cancer Genome Consortium (ICGC) oder auch bei Haus-internen Projekten wurden Datens{\"a}tze zu den Entit{\"a}ten Multiples Myelom (MM), Burkitt Lymphom (BL) und Follikul{\"a}res Lymphom (FL) erstellt und analysiert. Die Selektion geeigneter Softwaretools und die Generierung der Pipeline basieren auf komparativen Analysen dieser Daten, sowie auf geteilte Ergebnisse und Erfahrungen in der Literatur und auch in Foren. Durch die gezielte Entwicklung von Skripten konnten biologische und klinische Fragestellungen bearbeitet werden. Hierzu z{\"a}hlten eine einheitliche Annotation der Gennamen, sowie die Erstellung von Genmutations-Heatmaps mit nicht Variant-Calling-File (VCF)-Syntax konformen Dateien. Des Weiteren konnten nicht abgedeckte Regionen des Genoms in den NGS-Daten identifiziert und analysiert werden. Neue Projekte zur detaillierten Untersuchung der Verteilung von wiederkehrender Mutationen und Funktionsassays zu einzelnen Mutationskandidaten konnten basierend auf den Ergebnissen initiiert werden. Durch eigens erstellte Python-Skripte konnte somit die Funktionalit{\"a}t der Pipeline erweitert werden und zu wichtigen Erkenntnissen bei der biologischen Interpretation der Sequenzierungsdaten f{\"u}hren, wie beispielsweise zu der Detektion von drei neuen molekularen Subgruppen im MM. Die Erweiterungen, der in dieser Arbeit entwickelten Pipeline verbesserte somit die Effizienz der Analyse und die Vergleichbarkeit unserer Daten. Des Weiteren konnte durch die Erstellung eines eigenen Skripts die Analyse von unbeachteten Regionen in den NGS-Daten erfolgen.},
  subject      = {Pipeline-Rechner},
  language  = {de}
}
@phdthesis{Bruehlmann2017,
  author    = {Br{\"u}hlmann, David},
  title     = {Tailoring Recombinant Protein Quality by Rational Media Design},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-147345},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {Nowadays, more than half of the biotherapeutics are produced in mammalian cell lines as a result of correct protein folding and assembly as well as their faculty to bring about a variety of post-translational modifications. The widespread progression of biosimilars has moved the focus in mammalian cell-culture process development. Thereby, the modulation of quality attributes of recombinant therapeutic proteins has increasingly gained importance from early process development stages. Protein quality directly shapes the clinical efficacy and safety in vivo, and therefore, the control of the complex post-translational modifications, such as glycosylation (e.g. high mannose, fucosylation, galactosylation and sialylation), charge variants, aggregates and low-molecular-weight species formation, is pivotal for efficient receptor binding and for triggering the desired immune responses in patients. In the frame of biosimilar development, product quality modulation methods using the potential of the host cell line are particularly sought after to match the quality profile of the targeted reference medicinal product (RMP) as closely as possible. The environment the cell is dwelling in directly influences its metabolism and the resulting quality profile of the expressed protein. Thereby the cell culture medium plays a central role in upstream manufacturing. In this work, concentration adjustment of selected media components and supplementation with a variety of compounds was performed to alter various metabolic pathways, enzyme activities and in some cases the gene expression levels of Chinese Hamster Ovary (CHO) cells in culture. The supplementation of cell culture medium with the trisaccharide raffinose in fed-batch cultures entailed an increase of the abundance of high mannose glycans in two different CHO cell lines. Raffinose especially favored mannose 5 glycans. At the same time, it impaired cell culture performance, induced changes on the intracellular nucleotide levels and even varied the expression levels of glycosylation-related genes. Supplementation with a number of galactosyltransferase inhibiting compounds, in particular fluorinated galactose analogs (alpha- and beta-2F-peracetyl-galactose), consistently decreased the production of galactosylated monoclonal antibodies (mAb). By means of targeted addition during the culture rather than at the beginning, the inhibition was further increased, while limiting detrimental effects on both growth and productivity. High-throughput screening in 96-deepwell plates showed that spermine and L-ornithine also reduced the level of galactosylation. On the other hand, exploratory screening of a variety of potentially disulfide-bridge-reducing agents highlighted that the inherent low-molecular-species level of the proprietary platform cell culture process was likely due to favored reduction. This hypothesis was reinforced by the observation that supplementation of cysteine and N-acetylcysteine promoted fragmentation. Additionally, fragmentation decreased with higher protein expression. At that point, aiming to improve the efficiency in process development, a rational experimental design method was developed to identify and to define the optimal concentration range of quality modulating compounds by calling on a combination of high throughput fed-batch testing and multivariate data analysis. Seventeen medium supplements were tested in five parallel 96-deepwell plate experiments. The selection process of promising modulators for the follow-up experiment in shake tubes consisted in a three-step procedure, including principal component analysis, quantitative evaluation of their performance with respect to the specifications for biosimilarity and selection following a hierarchical order of decisions using a decision tree. The method resulted in a substantial improvement of the targeted glycosylation profile in only two experimental rounds. Subsequent development stages, namely validation and transfer to industrial-scale facilities require tight control of product quality. Accordingly, further mechanistic understanding of the underlying processes was acquired by non-targeted metabolomic profiling of a CHO cell line expressing a mAb cultured in four distinct process formats. Univariate analysis of intra- and extracellular metabolite and temporal glycosylation profiles provided insights in various pathways. The numerous of parameters were the main driver to carry out principal component analysis, and then, using the methodology of partial-least-square (PLS) projection on latent structures, a multivariate model was built to correlate the extracellular data with the distinct glycosylation profiles. The PLS observation model proved to be reliable and showed its great benefit for glycan pattern control in routine manufacturing, especially at large scale. Rather than relying on post-production interpretation of glycosylation results, glycosylation can be predicted in real-time based on the extracellular metabolite levels in the bioreactor. Finally, for the bioactivity assessment of the glycan differences between the biosimilar and the reference medicinal product (RMP), the health agencies may ask for in the drug registration process, extended ranges of glycan variants need to be generated so that the in vitro assays pick up the changes. The developed glycosylation modulator library enabled the generation of extreme glycosylation variants, including high mannose, afucosylated, galactosylated as well as sialic acid species of both a mAb and an antibody fusion molecule with three N-glycosylation sites. Moreover, to create increased variety, enzymatic glycoengineering was explored for galactosylation and sialylation. The glyco variants induced significant responses in the respective in vitro biological activity assays. The data of this work highlight the immense potential of cell culture medium optimization to adjust product quality. Medium and feed supplementation of a variety of compounds resulted in reproducible and important changes of the product quality profile of both mAbs and a fusion antibody. In addition to the intermediate modulation ranges that largely met the requirements for new-biological-entity and biosimilar development, medium supplementation even enabled quick and straightforward generation of extreme glycan variants suitable for biological activity testing.},
  subject      = {Zellkultur},
  language  = {en}
}
@phdthesis{Ruf2016,
  author    = {Ruf, Franziska},
  title     = {The circadian regulation of eclosion in \(Drosophila\) \(melanogaster\)},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-146265},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {Eclosion is the emergence of an adult insect from the pupal case at the end of development. In the fruit fly Drosophila melanogaster, eclosion is a circadian clock-gated event and is regulated by various peptides. When studied on the population level, eclosion reveals a clear rhythmicity with a peak at the beginning of the light-phase that persists also under constant conditions. It is a long standing hypothesis that eclosion gating to the morning hours with more humid conditions is an adaption to reduce water loss and increase the survival. Eclosion behavior, including the motor pattern required for the fly to hatch out of the puparium, is orchestrated by a well-characterized cascade of peptides. The main components are ecdysis-triggering hormone (ETH), eclosion hormone (EH) and crustacean cardioactive peptide (CCAP). The molt is initiated by a peak level and pupal ecdysis by a subsequent decline of the ecdysteroid ecdysone. Ecdysteroids are produced by the prothoracic gland (PG), an endocrine tissue that contains a peripheral clock and degenerates shortly after eclosion. Production and release of ecdysteroids are regulated by the prothoracicotropic hormone (PTTH). Although many aspects of the circadian clock and the peptidergic control of the eclosion behavior are known, it still remains unclear how both systems are interconnected. The aim of this dissertation research was to dissect this connection and evaluate the importance of different Zeitgebers on eclosion rhythmicity under natural conditions. Potential interactions between the central clock and the peptides regulating ecdysis motor behavior were evaluated by analyzing the influence of CCAP on eclosion rhythmicity. Ablation and silencing of CCAP neurons, as well as CCAP null-mutation did not affect eclosion rhythmicity under either light or temperature entrainment nor under natural conditions. To dissect the connection between the central and the peripheral clock, PTTH neurons were ablated. Monitoring eclosion under light and temperature entrainment revealed that eclosion became arrhythmic under constant conditions. However, qPCR expression analysis revealed no evidence for cycling of Ptth mRNA in pharate flies. To test for a connection with pigment-dispersing factor (PDF)-expressing neurons, the PDF receptor (PDFR) and short neuropeptide F receptor (sNPFR) were knocked down in the PTTH neurons. Knockdown of sNPFR, but not PDFR, resulted in arrhythmic eclosion under constant darkness conditions. PCR analysis of the PTTH receptor, Torso, revealed its expression in the PG and the gonads, but not in the brain or eyes, of pharate flies. Knockdown of torso in the PG lead to arrhythmicity under constant conditions, which provides strong evidence for the specific effect of PTTH on the PG. These results suggest connections from the PDF positive lateral neurons to the PTTH neurons via sNPF signaling, and to the PG via PTTH and Torso. This interaction presumably couples the period of the peripheral clock in the PG to that of the central clock in the brain. To identify a starting signal for eclosion and possible further candidates in the regulation of eclosion behavior, chemically defined peptidergic and aminergic neurons were optogenetically activated in pharate pupae via ChR2-XXL. This screen approach revealed two candidates for the regulation of eclosion behavior: Dromyosuppressin (DMS) and myo-inhibitory peptides (MIP). However, ablation of DMS neurons did not affect eclosion rhythmicity or success and the exact function of MIP must be evaluated in future studies. To assess the importance of the clock and of possible Zeitgebers in nature, eclosion of the wildtype Canton S and the clock mutant per01 and the PDF signaling mutants pdf01 and han5304 was monitored under natural conditions. For this purpose, the W{\"u}rzburg eclosion monitor (WEclMon) was developed, which is a new open monitoring system that allows direct exposure of pupae to the environment. A general decline of rhythmicity under natural conditions compared to laboratory conditions was observed in all tested strains. While the wildtype and the pdf01 and han5304 mutants stayed weakly rhythmic, the per01 mutant flies eclosed mostly arrhythmic. PDF and its receptor (PDFR encoded by han) are required for the synchronization of the clock network and functional loss can obviously be compensated by a persisting synchronization to external Zeitgebers. The loss of the central clock protein PER, however, lead to a non-functional clock and revealed the absolute importance of the clock for eclosion rhythmicity. To quantitatively analyze the effect of the clock and abiotic factors on eclosion rhythmicity, a statistical model was developed in cooperation with Oliver Mitesser and Thomas Hovestadt. The modelling results confirmed the clock as the most important factor for eclosion rhythmicity. Moreover, temperature was found to have the strongest effect on the actual shape of the daily emergence pattern, while light has only minor effects. Relative humidity could be excluded as Zeitgeber for eclosion and therefore was not further analyzed. Taken together, the present dissertation identified the so far unknown connection between the central and peripheral clock regulating eclosion. Furthermore, a new method for the analysis of eclosion rhythms under natural conditions was established and the necessity of a functional clock for rhythmic eclosion even in the presence of multiple Zeitgebers was shown.},
  subject      = {Taufliege},
  language  = {en}
}
@phdthesis{Blaettner2016,
  author    = {Bl{\"a}ttner, Sebastian},
  title     = {The role of the non-ribosomal peptide synthetase AusAB and its product phevalin in intracellular virulence of Staphylococcus aureus},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-146662},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {Staphylococcus aureus is a prevalent commensal bacterium which represents one of the leading causes in health care-associated bacterial infections worldwide and can cause a variety of different diseases ranging from simple abscesses to severe and life threatening infections including pneumonia, osteomyelitis and sepsis. In recent times multi-resistant strains have emerged, causing severe problems in nosocomial as well as community-acquired (CA) infection settings, especially in the United States (USA). Therefore S. aureus has been termed as a superbug by the WHO, underlining the severe health risk originating from it. Today, infections in the USA are dominated by S. aureus genotypes which are classified as USA300 and USA400, respectively. Strains of genotype USA300 are responsible for about 70\% of the CA infections. The molecular mechanisms which render S. aureus such an effective pathogen are still not understood in its entirety. For decades S. aureus was thought to be a strictly extracellular pathogen relying on pore-forming toxins like α-hemolysin to damage human cells and tissue. Only recently it has been shown that S. aureus can enter non-professional phagocytes, using adhesins like the fibronectin-binding proteins which mediate an endocytotic uptake into the host cells. The bacteria are consequently localized to endosomes, where the degradation of enclosed bacterial cells through phagosome maturation would eventually occur. S. aureus can avoid degradation, and translocate to the cellular cytoplasm, where it can replicate. The ability to cause this so-called phagosomal escape has mainly been attributed to a family of amphiphilic peptides called phenol soluble modulins (PSMs), but as studies have shown, they are not sufficient. In this work I used a transposon mutant library in combination with automated fluorescence microscopy to screen for genes involved in the phagosomal escape process and intracellular survival of S. aureus. I thereby identified a number of genes, including a non-ribosomal peptide synthetase (NRPS). The NRPS, encoded by the genes ausA and ausB, produces two types of small peptides, phevalin and tyrvalin. Mutations in the ausAB genes lead to a drastic decrease in phagosomal escape rates in epithelial cells, which were readily restored by genetic complementation in trans as well as by supplementation of synthetic phevalin. In leukocytes, phevalin interferes with calcium fluxes and activation of neutrophils and promotes cytotoxicity of intracellular bacteria in both, macrophages and neutrophils. Further ausAB is involved in survival and virulence of the bacterium during mouse lung pneumoniae. The here presented data demonstrates the contribution of the bacterial cyclic dipeptide phevalin to S. aureus virulence and suggests, that phevalin directly acts on a host cell target to promote cytotoxicity of intracellular bacteria.},
  subject      = {Staphylococcus aureus},
  language  = {en}
}
@phdthesis{Jung2016,
  author    = {Jung, Lisa Anna},
  title     = {Targeting MYC Function as a Strategy for Tumor Therapy},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-146993},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {A large fraction of human tumors exhibits aberrant expression of the oncoprotein MYC. As a transcription factor regulating various cellular processes, MYC is also crucially involved in normal development. Direct targeting of MYC has been a major challenge for molecular cancer drug discovery. The proof of principle that its inhibition is nevertheless feasible came from in vivo studies using a dominant-negative allele of MYC termed OmoMYC. Systemic expression of OmoMYC triggered long-term tumor regression with mild and fully reversible side effects on normal tissues. In this study, OmoMYC's mode of action was investigated combining methods of structural biology and functional genomics to elucidate how it is able to preferentially affect oncogenic functions of MYC. The crystal structure of the OmoMYC homodimer, both in the free and the E-box-bound state, was determined, which revealed that OmoMYC forms a stable homodimer, and as such, recognizes DNA via the same base-specific DNA contacts as the MYC/MAX heterodimer. OmoMYC binds DNA with an equally high affinity as MYC/MAX complexes. RNA-sequencing showed that OmoMYC blunts both MYC-dependent transcriptional activation and repression. Genome-wide DNA-binding studies using chromatin immunoprecipitation followed by high-throughput sequencing revealed that OmoMYC competes with MYC/MAX complexes on chromatin, thereby reducing their occupancy at consensus DNA binding sites. The most prominent decrease in MYC binding was seen at low-affinity promoters, which were invaded by MYC at oncogenic levels. Strikingly, gene set enrichment analyses using OmoMYC-regulated genes enabled the identification of tumor subgroups with high MYC levels in multiple tumor entities. Together with a targeted shRNA screen, this identified novel targets for the eradication of MYC-driven tumors, such as ATAD3A, BOP1, and ADRM1. In summary, the findings suggest that OmoMYC specifically inhibits tumor cell growth by attenuating the expression of rate-limiting proteins in cellular processes that respond to elevated levels of MYC protein using a DNA-competitive mechanism. This opens up novel strategies to target oncogenic MYC functions for tumor therapy.},
  subject      = {Myc},
  language  = {en}
}
@phdthesis{Reimer2017,
  author    = {Reimer, Anastasija},
  title     = {Search for novel antimicrobials against \(Neisseria\) \(gonorrhoeae\) and \(Chlamydia\) \(trachomatis\)},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-143168},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {The obligate human pathogen Neisseria gonorrhoeae is responsible for the widespread sexually transmitted disease gonorrhoea, which in rare cases also leads to the development of disseminated gonococcal infection (DGI). DGI is mediated by PorBIA-expressing bacteria that invade host cells under low phosphate condition by interaction with the scavenger receptor-1 (SREC-I) expressed on the surface of endothelial cells. The interaction of PorBIA and SREC-I was analysed using different in vitro approaches, including surface plasmon resonance experiments that revealed a direct phosphate-independent high affinity interaction of SREC-I to PorBIA. However, the same binding affinity was also found for the other allele PorBIB, which indicates unspecific binding and suggests that the applied methods were unsuitable for this interaction analysis. Since N. gonorrhoeae was recently classified as a "super-bug" due to a rising number of antibiotic-resistant strains, this study aimed to discover inhibitors against the PorBIA-mediated invasion of N. gonorrhoeae. Additionally, inhibitors were searched against the human pathogen Chlamydia trachomatis, which causes sexually transmitted infections as well as infections of the upper inner eyelid. 68 compounds, including plant-derived small molecules, extracts or pure compounds of marine sponges or sponge-associated bacteria and pipecolic acid derivatives, were screened using an automated microscopy based approach. No active substances against N. gonorrhoeae could be identified, while seven highly antichlamydial compounds were detected. The pipecolic acid derivatives were synthesized as potential inhibitors of the virulence-associated "macrophage infectivity potentiator" (MIP), which exhibits a peptidyl prolyl cis-trans isomerase (PPIase) enzyme activity. This study investigated the role of C. trachomatis and N. gonorrhoeae MIP during infection. The two inhibitors PipN3 and PipN4 decreased the PPIase activity of recombinant chlamydial and neisserial MIP in a dose-dependent manner. Both compounds affected the chlamydial growth and development in epithelial cells. Furthermore, this work demonstrated the contribution of MIP to a prolonged survival of N. gonorrhoeae in the presence of neutrophils, which was significantly reduced in the presence of PipN3 and PipN4. SF2446A2 was one of the compounds that had a severe effect on the growth and development of C. trachomatis. The analysis of the mode of action of SF2446A2 revealed an inhibitory effect of the compound on the mitochondrial respiration and mitochondrial ATP production of the host cell. However, the chlamydial development was independent of proper functional mitochondria, which excluded the connection of the antichlamydial properties of SF2446A2 with its inhibition of the respiratory chain. Only the depletion of cellular ATP by blocking glycolysis and mitochondrial respiratory chain inhibited the chlamydial growth. A direct effect of SF2446A2 on C. trachomatis was assumed, since the growth of the bacteria N. gonorrhoeae and Staphylococcus aureus was also affected by the compound. In summary, this study identified the severe antichlamydial activity of plant-derived naphthoquinones and the compounds derived from marine sponges or sponge-associated bacteria SF2446A2, ageloline A and gelliusterol E. Furthermore, the work points out the importance of the MIP proteins during infection and presents pipecolic acid derivatives as novel antimicrobials against N. gonorrhoeae and C. trachomatis.},
  subject      = {Neisseria gonorrhoeae},
  language  = {en}
}
@phdthesis{Stangler2015,
  author    = {Stangler, Eva},
  title     = {Effects of habitat fragmentation on trap-nesting bees, wasps and their natural enemies in small secondary rainforest fragments in Costa Rica},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-108254},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {Summary (English) I. Human induced global change threatens biodiversity and trophic interactions. Fragmentation is considered as one of the major threats to biodiversity and can cause reduced species richness, population declines, loss of genetic diversity and disruption of trophic interactions such as predation and parasitism. However forest fragmentation effects can be eclectic due to species specific traits. Specialist species with narrower niches or at higher trophic levels may be in danger of extinction whereas generalist species with less specific habitat requirements may even profit from fragmentation. In the tropics, known as "the" terrestrial biodiversity hotspots, even biodiversity inventories are often lacking, especially in forest canopies. Ongoing deforestation and resulting fragmentation in tropical regions are expected to heavily affect ecosystem functions by changes in biodiversity, community compositions and disruption of trophic interactions. It is even less unknown in what extent different global change drivers for example climate change and fragmentation interact. It is unlikely that deforestation will end, so that small secondary forest fragments will be important habitat elements that must be investigated to optimize their potential contribution to biodiversity conservation. This dissertation aimed to disentangle the effects of forest fragmentation on trap-nesting bee and wasp communities in small secondary forest fragments addressing the following main questions: 1) Are there interactive effects between microclimate and fragmentation on the abundance of bees and wasps, their mortality - and parasitism rates (Chapter II)? 2) How does fragmentation affect bee biodiversity from canopy to the understory with considerations of single species patterns (Chapter III)? 3) How is fragmentation affecting diversity and community composition of different trophic levels between understory and canopy with emphasis on the host-antagonist relation? (Chapter IV). II. A variety of global change drivers affect biodiversity and trophic interactions. The combined effects of habitat fragmentation and climate change are poorly understood and with ongoing deforestation and agricultural intensification secondary rainforest fragments might contribute to biodiversity conservation and mitigation of climate warming. This chapter investigated the interactive effects of habitat fragmentation and microclimate on the abundance and biotic interactions of trap-nesting bees and wasps in secondary forest fragments in the Northeastern lowlands of Costa Rica. Habitat area did not affect hymenopteran abundance, parasitism and mortality rates, but tree location- from the forest border to the forest center- influenced all variables. Interactive effects were found such as in the higher mortality rates at interior locations in larger fragments. Mean temperature at edge and interior locations led to significant effects on all tested variables and interactive effects between temperature and tree locations were found. Abundances at interior locations were significantly higher with increasing temperatures. Mortality rates at interior location increased at lower mean temperatures, whereas higher temperatures at edges marginally increased mortality rates. Our results indicate, that edge effects, mediated by altered microclimatic conditions, significantly change biotic interactions of trap-nesting hymenopterans in small secondary fragments. III. This chapter focusses on the vertical distribution of bees, their parasitism and mortality rates as well as single species patterns in relation to fragment size and edge effects in secondary rainforest remnants. No size effects on bee abundance, bee diversity and on parasitism- and mortality rates were found. Bees were least abundant at the intermediate height and were most abundant in the understory; whereas the highest diversity was found in the canopy. Tree location had no effect on bee abundance, but on bee diversity since most species were found in the forest interior. The cuckoo bees Aglaomelissa duckei and Coelioxys sp. 1 only partly followed the patterns of their hosts, two Centris species. Edge effects greatly influenced the bee community, so that the amount of edge habitat in secondary forest fragments will influence the conservation value for bees. IV. In this section the effects of habitat fragmentation on biodiversity, on community structure of hosts and natural enemies as well as the relation of hosts and antagonists were investigated from the understory to the canopy. The results stress the importance to monitor biodiversity, community composition and trophic interactions from the understory to the canopy. The higher trophic level of the antagonists was found to be more sensitive to fragment size compared to their hosts. Again edge effects were found to be the dominant driver since both host and antagonist richness, as well as community compositions were strongly affected. Ongoing fragmentation and increased amount of edge habitat could favor few abundant disturbance-adapted species over the rare and more diverse forest-adapted species. A positive-density dependent parasitism rate was demonstrated, as well as an increase of the parasitism rate not only with antagonist abundance but also diversity. Small secondary forest fragments surely can contribute to the conservation of biodiversity and trophic interactions, but increase of edge habitat will have negative consequences on above-ground nesting Hymenoptera, so that important interactions such as pollination, predation and parasitism could be disrupted. Therefore small forest fragments could contribute to biodiversity conservation but will not be able to compensate for the loss of large areas of primary forests. V. This dissertation contributes to the understanding of habitat area - and edge effects as well as the interaction of those with microclimatic conditions in small secondary rainforest fragments. As study system trap nests inhabited by solitary above-ground nesting bees, wasps and their natural enemies were chosen because they allow to study trophic interactions along their whole vertical distribution from the understory to the canopy. The effect of fragment size was rather weak, however, larger sizes affected the diversity of natural enemies positively, proofing the hypothesis that higher trophic levels react more sensitive to habitat loss. Edge effects heavily affected the abundance, diversity and community composition of hosts and their natural enemies as well as parasitism and mortality rates. Increased edge conditions resulting from ongoing fragmentation and deforestation will therefore negatively affect bees, wasps and their trophic interactions with natural enemies. Those changes affect important processes such as pollination, predation and parasitism, which could result in changes of ecosystem functioning. This study showed the importance to include all strata in biodiversity monitoring since height did matter for the trap-nesting communities. Diversity was shown to be higher in the canopy and community composition did change significantly. To conclude we could show that secondary forest fragments can sustain a trap-nesting bee and wasp community, but the amount of interior habitat is highly important for the conservation of forest-adapted species. Probably the conservation of large primary forest in combination with a high habitat connectivity, for example with small secondary forest fragments, will help to sustain biodiversity and ecosystem functioning better than the mere presence of small forest fragments.},
  subject      = {Costa Rica},
  language  = {en}
}
@phdthesis{Mattern2016,
  author    = {Mattern, Felix},
  title     = {Alterungsbedingte Effekte auf DNA-Methylierungsprofile entwicklungsrelevanter Gene in Eizellen und Embryonen am Modellorganismus Bos taurus},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-144562},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {Die postovulatorische Alterung sowie die ovarielle Alterung konnten bei der Anwendung assistierter Reproduktionstechniken (ARTs) als entscheidende Faktoren identifiziert werden, die den Reproduktionserfolg nachhaltig beeintr{\"a}chtigen. Die postovulatorische Alterung tritt ein, sobald die reife Eizelle nicht mehr innerhalb ihres physiologischen Zeitfensters befruchtet wird. Die ovarielle Alterung beschreibt hingegen die Abnahme des Follikel-Vorrats mit zunehmendem Alter des weiblichen Individuums bzw. des Ovars. Sowohl die postovulatorische Alterung als auch die ovarielle Alterung f{\"u}hren u.a. zu einer reduzierten Oozytenqualit{\"a}t und einer geringeren Blastozystenrate. Die Zielsetzung dieser Arbeit bestand darin, den Einfluss der postovulatorischen Alterung und der ovariellen Alterung im Holstein-Rind (Bos taurus) auf die DNA-Methylierung entwicklungsrelevanter Gene in Eizellen und Embryonen zu untersuchen. Aus Schlachthof-Ovarien wurden Antralfollikeln unterschiedlicher Gr{\"o}ße (<2 mm, 3-5 mm und >6 mm) isoliert. Eizellen aus Follikeln der Gr{\"o}ße 3-5 mm wurden f{\"u}r 24h (physiologisch) und 48h (gealtert) in vitro gereift (IVM). Die gereiften Oozyten wurden anschließend in vitro fertilisiert und Embryonen im 4-6 Zellstadium generiert. Sowohl in den unreifen Eizellen aus Antralfollikeln unterschiedlicher Gr{\"o}ße als auch in den gereiften Oozyten und den Embryonen wurde die Promotormethylierung der Gene bH19, bSNRPN, bZAR1, bDNMT3A, bOCT4, bDNMT3Lo und bDNMT3Ls analysiert. Zur Untersuchung der ovariellen Alterung wurden mittelgroßen Antralfollikel aus Ovarien lebender Rinder (in vivo) unterschiedlichen Alters (9-12 Monate, 3-7 Jahre und 8-11 Jahre) gewonnen. In den daraus isolierten unreifen Eizellen wurde die DNA-Methylierung der Promotorregionen der Gene bTERF2, bREC8, bBCL-XL, bPISD, bBUB1, bDNMT3Lo, bH19 und bSNRPN bestimmt. Als Methode zur Analyse der Promotormethylierung wurde die Limiting Dilution Bisulfit-Sequenzierung angewendet. In unreifen Eizellen aus Antralfollikeln unterschiedlicher Gr{\"o}ße (<2 mm, 3-5 mm und >6 mm) konnte ein erh{\"o}htes Auftreten abnormal methylierter Allele in den gepr{\"a}gten Genen bH19 und bSNRPN von Eizellen kleiner Follikel (<2 mm) identifiziert werden. Dieses Ergebnis k{\"o}nnte eine m{\"o}gliche Ursache einer bereits bekannten und mehrfach beschriebenen geringeren Entwicklungskompetenz von Eizellen kleiner Follikel (<2 mm) auf epigenetischer Ebene darstellen. Die verl{\"a}ngerte Reifungsdauer der IVM-Eizellen hatte eine signifikante Hypermethylierung in der Promotorregion des Gens DNMT3Lo von 48h-gereiften Eizellen zur Folge. Beim {\"U}bergang von 48h-gereiften Eizellen zum Embryo konnte eine signifikante Hypomethylierung von CpG7 des stammzellspezifischen Transkripts DNMT3Ls beobachtet werden. Diese CpG-Stelle wies ebenfalls einen signifikanten Anstieg von CpGs mit nicht-eindeutigem Methylierungszustand in unreifen Eizellen mit steigender Follikelgr{\"o}ße auf. Da sich die CpG-Position innerhalb eines Sequenz-Motivs einer Bindungsstelle des Transkriptionsfaktors CREB befindet, k{\"o}nnten die Methylierungsdaten auf eine Interaktion zwischen dem Transkriptionsfaktor CREB und der DNA-Methylierung w{\"a}hrend der Entwicklung und Reifung der Eizelle sowie der Transition von der Eizelle zum Embryo hindeuten. Die DNA-Methylierungsprofile der untersuchten Gene in unreifen Eizellen aus K{\"u}hen unterschiedlichen Alters (9-12 Monate, 3-7 Jahre und 8-11 Jahre) wiesen keine signifikanten Unterschiede zwischen den Altersgruppen auf. Die ovarielle Alterung bei Rindern zwischen 9 Monaten und 11 Jahren zeigte damit keinen Effekt auf die DNA-Methylierung der untersuchten Promotorregionen der Gene bTERF2, bREC8, bBCL-XL, bPISD, bBUB1, bDNMT3Lo, bH19 und bSNRPN. Nach einer simulierten postovulatorischen Alterung durch eine in vitro Reifung f{\"u}r 48h konnte eine Ver{\"a}nderung der DNA-Methylierung der Oozyten-spezifischen (DNMT3Lo) und Stammzell-spezifischen (DNMT3Ls) Promotoren des katalytisch inaktiven Cofaktors von DNMT3A, DNMT3L, beobachtet werden. Die ver{\"a}nderte DNA-Methylierung von DNMT3Ls tritt dabei erst im fr{\"u}hen Embryo in Erscheinung und interagiert vermutlich mit dem Transkriptionsfaktor CREB. Die Ver{\"a}nderungen von DNMT3Lo in Eizellen und DNMT3Ls in den daraus generierten Embryonen l{\"a}sst vermuten, dass es sich hierbei um eine dynamische Anpassung des Embryos auf {\"a}ußere Umweltbedingungen der Eizelle {\"u}ber die Methylierung der DNA handelt.},
  subject      = {Oozyte},
  language  = {de}
}
@phdthesis{Sickel2016,
  author    = {Sickel, Wiebke},
  title     = {High-throughput biodiversity assessment - Powers and limitations of meta-barcoding},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-144573},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {Traditional species identification based on morphological characters is laborious and requires expert knowledge. It is further complicated in the case of species assemblages or degraded and processed material. DNA-barcoding, species identification based on genetic data, has become a suitable alternative, yet species assemblages are still difficult to study. In the past decade meta-barcoding has widely been adopted for the study of species communities, due to technological advances in modern sequencing platforms and because manual separation of individual specimen is not required. Here, meta-barcoding is put into context and applied to the study of bee-collected pollen as well as bacterial communities. These studies provide the basis for a critical evaluation of the powers and limitations of meta-barcoding. Advantages identified include species identification without the need for expert knowledge as well as the high throughput of samples and sequences. In microbiology, meta-barcoding can facilitate directed cultivation of taxa of interest identified with meta-barcoding data. Disadvantages include insufficient species resolution due to short read lengths and incomplete reference databases, as well as limitations in abundance estimation of taxa and functional profiling. Despite these, meta-barcoding is a powerful method for the analysis of species communities and holds high potential especially for automated biomonitoring.},
  subject      = {Biodiversit{\"a}t},
  language  = {en}
}
@phdthesis{Mueller2015,
  author    = {M{\"u}ller, Elisabeth},
  title     = {Pan-Raf-Inhibition als neue therapeutische Strategie im Multiplen Myelom},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-124666},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {Das Multiple Myelom (MM) ist eine durch monoklonale Vermehrung terminal differenzierter Antik{\"o}rper-produzierender B-Lymphozyten (Plasmazellen) im Knochenmark charakterisierte maligne Krankheit, die sich v.a. in osteolytischen Knochendestruktionen, h{\"a}matopoetischer und Niereninsuffizienz {\"a}ußert. Verbesserte Therapieans{\"a}tze wie die Hochdosis-Chemotherapie mit Melphalan und anschließender autologer Stammzelltransplantation sowie die Einf{\"u}hrung neuer pharmakologischer Substanzklassen (Proteasom-Inhibitoren, Cereblon-bindende Thalidomidderivate) f{\"u}hrten zu einer Verl{\"a}ngerung der durchschnittlichen {\"U}berlebenszeit, f{\"u}r die meisten der Patienten ist die Erkrankung jedoch derzeit unheilbar. Die Erforschung neuer potenzieller therapeutischer Angriffspunkte auf Grund pathobiologischer Erkenntnisse bleibt daher unabdingbar. Ein Ansatz zur Verbesserung des Verst{\"a}ndnisses der Pathogenese ist die funktionelle, molekulare und genetische Analyse des Signalnetzwerkes im MM. Im Zusammenhang mit diesem Konzept wurde entdeckt, dass wachstums-regulierende Signalwege in MM Zellen aktiviert oder dereguliert sind und zum {\"U}berleben und der Proliferation des Tumors beitragen. So konnte beispielsweise von unserer Arbeitsgruppe bereits gezeigt werden, dass onkogenes Ras essentiell zum {\"U}berleben der MM Zellen beitr{\"a}gt. Da Ras derzeit mangels spezifischer Inhibitoren pharmakologisch nicht angreifbar ist, stellen weitere funktionelle Bestandteile des Signalweges eine potenzielle therapeutische Zielstruktur dar. W{\"a}hrend die Blockade von MEK1/2 in MM Zellen keinen Einfluss auf das {\"U}berleben hatte, konnte durch die Blockade von Raf in ersten Tests unserer Arbeitsgruppe Apoptose hervorgerufen werden. Aus diesem Grund habe ich in der vorliegenden Arbeit zur Evaluation eines neuen Therapieansatzes die Rolle der Raf-abh{\"a}ngigen Signaltransduktion eingehend untersucht. Als Grundlage diente dabei die Hypothese, dass die Raf-Kinasen entscheidende Effektoren der durch onkogenes Ras vermittelten apoptotischen Effekte darstellen. In einem ersten Schritt konnte ich nachweisen, dass alle drei Raf-Isoformen (A-, B- und C-Raf) in humanen MM Zelllinien und in prim{\"a}ren MM Zellen aktiviert sind. Mittels shRNA-vermittelter, Isoform-spezifischer Raf-Knockdown-Experimente konnte ich zeigen, dass nur ein simultaner Knockdown aller Isoformen, d.h. ein Pan-Raf-Knockdown, zu einer De-Phosphorylierung von MEK1/2 und ERK1/2 f{\"u}hrte. Dieser Versuch ließ sich mittels pharmakologischer Raf-Inhibition, bei der ebenfalls nur eine Pan-Raf-Blockade zu einer Herunterregulation von MEK1/2 und ERK1/2 in MM Zellen f{\"u}hrte, best{\"a}tigen. Das MEK/ERK-Modul stellte somit einen hervorragenden Surrogat- und Biomarker f{\"u}r die Pan-Raf-Aktivit{\"a}t dar. Im Gegensatz zur Blockade des MEK/ERK-Moduls f{\"u}hrte eine Hemmung der Pan-Raf-Aktivit{\"a}t mittels shRNA oder pharmakologischer Inhibitoren in allen untersuchten Zelllinien und in der Mehrheit der prim{\"a}ren MM Zellen zu einer starken Induktion von Apoptose. Da das Ansprechen auf eine Pan-Raf-Blockade nicht mit dem Ras-Mutationsstatus korrelierte, k{\"o}nnten die Raf-Kinasen eine von onkogenem Ras unabh{\"a}ngie Qualit{\"a}t als therapeutische Zielstruktur aufweisen. Zur Untersuchung m{\"o}glicher MEK/ERK-unabh{\"a}ngiger Effektormechanismen der Pan-Raf-Inhibition habe ich die mRNA-basierten Genexpressionsprofile von INA-6 Zellen nach pharmakologischer Pan-Raf- oder MEK-Inhibition verglichen. Dabei f{\"u}hrte die Pan-Raf-Inhibition zu einer Regulation von wesentlich mehr Genen, wobei sich auch die Art der regulierten Gene unterschied, darunter Gene mit tumorrelevanten Funktionen wie Regulation von Proliferation, Zellzyklus und Apoptose. F{\"u}r eine dieser Gengruppen, die Gruppe der PI3K-abh{\"a}ngigen, mTOR-assoziierten Gene, konnte ich eine Regulation auch auf der Proteinebene nachweisen: die Phosphorylierungen von mTOR, p70S6K, Rb und AKT und die Expression von CyclinD1 und PDK1 waren nach Pan-Raf-Inhibition, nicht jedoch nach MEK-Blockade herunterreguliert. Dieses Ergebnis deutet auf eine Ko-Regulation der PI3K-abh{\"a}ngigen Signaltransduktion durch die Raf-kinasen hin. Mittels spezifischer PI3K-Inhibitoren ließ sich sowohl bei der Regulation der untersuchten Proteine als auch bei der Induktion von Apoptose eine deutliche Verst{\"a}rkung der Pan-Raf-Inhibition in HMZL und in prim{\"a}ren Zellen erzielen. Zusammengefasst zeigt diese Arbeit, dass die Pan-Raf-Blockade eine neue Therapiem{\"o}glichkeit darstellt, die durch Kombination mit einer PI3K/AKT-Inhibition noch verst{\"a}rkt werden kann.},
  subject      = {Plasmozytom},
  language  = {de}
}
@phdthesis{Cicova2016,
  author    = {Cicova, Zdenka},
  title     = {Characterization of a novel putative factor involved in host adaptation in Trypanosoma brucei},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-142462},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {Trypanosomes are masters of adaptation to different host environments during their complex life cycle. Large-scale proteomic approaches provide information on changes at the cellular level in a systematic way. However, a detailed work on single components is necessary to understand the adaptation mechanisms on a molecular level. Here we have performed a detailed characterization of a bloodstream form (BSF) stage-specific putative flagellar host adaptation factor (Tb927.11.2400) identified previously in a SILAC-based comparative proteome study. Tb927.11.2400 shares 38\% amino acid identity with TbFlabarin (Tb927.11.2410), a procyclic form (PCF) stage specific flagellar BAR domain protein. We named Tb927.11.2400 TbFlabarin like (TbFlabarinL) and demonstrate that it is a result of a gene duplication event, which occurred in African trypanosomes. TbFlabarinL is not essential for growth of the parasites under cell culture conditions and it is dispensable for developmental differentiation from BSF to the PCF in vitro. We generated a TbFlabarinL-specific antibody and showed that it localizes in the flagellum. The co-immunoprecipitation experiment together with a biochemical cell fractionation indicated a dual association of TbFlabarinL with the flagellar membrane and the components of the paraflagellar rod.},
  subject      = {Trypanosoma brucei},
  language  = {en}
}
@phdthesis{Schoenwaelder2016,
  author    = {Sch{\"o}nw{\"a}lder, Sina Maria Siglinde},
  title     = {Entwicklung und Charakterisierung von Gelatine-basierten Hydrogelen und PLGA-basierten Janus-Partikeln},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-142636},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {Zusammenfassung In der Regenerativen Medizin sind polymerbasierte Biomaterialien von großer Bedeutung f{\"u}r die Entwicklung und Anwendung verbesserter bzw. neuer Therapien. Die Erforschung der Oberfl{\"a}cheneigenschaften von Biomaterialien, welche als Implantate eingesetzt werden, ist eine grundlegende Voraussetzung f{\"u}r deren erfolgreichen Einsatz. Die Protein-Oberfl{\"a}chen- Interaktion geschieht initial, sobald ein Implantat mit K{\"o}rperfl{\"u}ssigkeiten oder mit Gewebe in Kontakt kommt, und tr{\"a}gt maßgeblich zur direkten Wechselwirkung von Implantat und umgebenden Zellen bei. Dieser Prozess wird in der vorliegenden Arbeit an Gelatine untersucht. Daher bestand ein Ziel darin, stabile, nanometerd{\"u}nne Gelatineoberfl{\"a}chen herzustellen und darauf die Adsorption von humanen Plasmaproteinen und bakteriellen Proteinen zu analysieren. Die Abscheidung der Gelatinefilme in variabler Schichtdicke auf zuvor mit PPX-Amin modifizierten Oberfl{\"a}chen wurde unter Verwendung eines Rotationsbeschichters durchgef{\"u}hrt. Um stabile Hydrogelfilme zu erhalten, wurden die Amingruppen der disaggregierten Gelatinefibrillen untereinander und mit denen der Amin-Modifizierung durch ein biokompatibles Diisocyanat quervernetzt. Dieser Prozess lieferte einen reproduzierbaren und chemisch stabilen Gelatinefilm, welcher durch die substratunabh{\"a}ngige Amin-Modifizierung kovalent auf unterschiedlichste Oberfl{\"a}chen aufgebracht werden konnte. Die durch den Herstellungsprozess pr{\"a}zise eingestellte Schichtdicke (Nano- bzw. Mikrometermaßstab) wurde mittels Ellipsometrie und Rasterkraftmikroskopie ermittelt. Die ebenso bestimmte Rauheit war unabh{\"a}ngig von der Schichtdicke sehr gering. Gelatinefilme, die auf funktionalisierte und strukturierte Proben aufgebracht wurden, konnten durch Elektronenmikroskopie dargestellt werden. Mit Hilfe der Infrarot-Reflexions-Absorptions-Spektroskopie wurden die Gelatinefilme im Hinblick auf ihre Stabilit{\"a}t chemisch charakterisiert. Zur Quantifizierung der Adsorption humaner Plasmaproteine (Einzelproteinl{\"o}sungen) und komplexer Proteingemische aus steril filtrierten Kultur{\"u}berst{\"a}nden des humanpathogenen Bakteriums Pseudomonas aeruginosa wurde die Quarzkristall-Mikrowaage mit Dissipations{\"u}berwachung eingesetzt. Hiermit konnte nicht nur die adsorbierte Menge an Proteinen auf dem Gelatinehydrogel bzw. Referenzoberfl{\"a}chen (Gold, PPX-Amin, Titan), sondern auch die viskoelastischen Eigenschaften des adsorbierten Proteinfilms bestimmt werden. Allgemein adsorbierte auf dem Gelatinehydrogel eine geringere Proteinmasse im Vergleich zu den Referenzoberfl{\"a}chen. Circa ein Viertel der adsorbierten Proteine migrierte in die Poren des gequollenen Gels und ver{\"a}nderte dessen viskoelastische Eigenschaften. Durch anschließende MALDI-ToF/MS- und MS/MS-Analyse konnten die bakteriellen Proteine auf den untersuchten Oberfl{\"a}chen identifiziert und untereinander verglichen werden. Hierbei zeigten sich nur geringf{\"u}gige Unterschiede in der Proteinzusammensetzung. Zudem wurde eine Sekund{\"a}rionenmassenspektrometrie mit Flugzeitanalyse an reinen Gelatinefilmen und an mit humanen Plasmaproteinen beladenen Gelatinefilmen durchgef{\"u}hrt. Durch eine anschließende multivariante Datenanalyse konnte zwischen den untersuchten Proben eindeutig differenziert werden. Dieser Ansatz erm{\"o}glicht es, die Adsorption von unterschiedlichen Proteinen auf proteinbasierten Oberfl{\"a}chen markierungsfrei zu untersuchen und kann zur Aufkl{\"a}rung der in vivo-Situation beitragen. Dar{\"u}ber hinaus bietet dieser Untersuchungsansatz neue Perspektiven f{\"u}r die Gestaltung und das schnelle und effiziente Screening von unterschiedlichen Proteinzusammensetzungen. Biomaterialien k{\"o}nnen jedoch nicht nur als Implantate oder Implantatbeschichtungen eingesetzt werden. Im Bereich des drug delivery und der Depotarzneimittel sind biologisch abbaubare Polymere, aufgrund ihrer variablen Eigenschaften, von großem Interesse. Die Behandlung von bakteriellen und fungalen Pneumonien stellt insbesondere bei Menschen mit Vorerkrankungen wie Cystische Fibrose oder prim{\"a}re Ziliendyskinesie eine große Herausforderung dar. Oral oder intraven{\"o}s applizierte Wirkstoffe erreichen die Erreger aufgrund der erh{\"o}hten Z{\"a}higkeit des Bronchialsekretes oft nicht in ausreichender Konzentration. Daher besteht ein weiteres Ziel der vorliegenden Arbeit darin, mittels electrohydrodynamic cojetting mikrometergroße, inhalierbare, wirkstoffbeladene Partikel mit zwei Kompartimenten (Janus-Partikel) herzustellen und deren Eignung f{\"u}r die therapeutische Anwendung bei Lungeninfektionen zu untersuchen. Durch das in dieser Arbeit entwickelte L{\"o}sungsmittelsystem k{\"o}nnen Janus-Partikel aus biologisch abbaubaren Co-Polymeren der Polymilchs{\"a}ure (Poly(lactid-co-glycolid), PLGA) hergestellt und mit verschiedenen Wirkstoffen beladen werden. Darunter befinden sich ein Antibiotikum (Aztreonam, AZT), ein Antimykotikum (Itraconazol, ICZ), ein Mukolytikum (Acetylcystein, ACC) und ein Antiphlogistikum (Ibuprofen, IBU). Die Freisetzung der eingelagerten Wirkstoffe, mit Ausnahme von ICZ, konnte unter physiologischen Bedingungen mittels Dialyse und anschließender Hochleistungsfl{\"u}ssigkeitschromatographie gemessen werden. Die Freisetzungsrate wird von der Kettenl{\"a}nge des Polymers beeinflusst, wobei eine k{\"u}rzere Kettenl{\"a}nge zu einer schnelleren Freisetzung f{\"u}hrt. Das in die Partikel eingelagerte Antimykotikum zeigte in vitro eine gute Wirksamkeit gegen Aspergillus nidulans. Durch das Einlagern von ICZ in die Partikel ist es m{\"o}glich diesen schlecht wasserl{\"o}slichen Wirkstoff in eine f{\"u}r Patienten zug{\"a}ngliche und wirksame Applikationsform zu bringen. In Interaktion mit P. aeruginosa erzielten die mit Antibiotikum beladenen Partikel in vitro bessere Ergebnisse als der Wirkstoff in L{\"o}sung, was sich in einem in vivo-Infektionsmodell mit der Wachsmotte Galleria mellonella best{\"a}tigte. AZT-beladene Partikel hatten gegen{\"u}ber einer identischen Wirkstoffmenge in L{\"o}sung eine 27,5\% bessere {\"U}berlebensrate der Wachsmotten zur Folge. Des Weiteren hatten die Partikel keinen messbaren negativen Einfluss auf die Wachsmotten. Dreidimensionale Atemwegsschleimhautmodelle, hergestellt mit Methoden des Tissue Engineerings, bildeten die Basis f{\"u}r Untersuchungen der Partikel in Interaktion mit humanen Atemwegszellen. Die Untersuchung von Apoptose- und Entz{\"u}ndungsmarkern im {\"U}berstand der 3D-Modelle zeigte diesbez{\"u}glich keinen negativen Einfluss der Partikel auf die humanen Zellen. Diese gut charakterisierten und standardisierten in vitro-Testsysteme machen es m{\"o}glich, Medikamentenuntersuchungen an menschlichen Zellen durchzuf{\"u}hren. Hinsichtlich der histologischen Architektur und funktionellen Eigenschaften der 3D-Modelle konnte eine hohe in vitro-/in vivo-Korrelation zu menschlichem Gewebe festgestellt werden. Humane Mucine auf den 3D-Modellen dienten zur Untersuchung der schleiml{\"o}senden Wirkung von ACC-beladenen Partikeln. Standen diese in r{\"a}umlichem Kontakt zu den Mucinen, wurde deren Z{\"a}higkeit durch das freigesetzte ACC herabgesetzt, was qualitativ mittels histologischen Methoden best{\"a}tigt werden konnte. Die in dieser Arbeit entwickelten Herstellungsprotokolle dienen als Grundlage und k{\"o}nnen f{\"u}r die Synthese {\"a}hnlicher Systeme, basierend auf anderen Polymeren und Wirkstoffen, modifiziert werden. Gelatine und PLGA erwiesen sich als vielseitig einsetzbare Werkstoffe und bieten eine breite Anwendungsvielfalt in der Regenerativen Medizin, was die erzielten Resultate bekr{\"a}ftigen.},
  subject      = {Gelatine},
  language  = {de}
}
@phdthesis{Kupper2016,
  author    = {Kupper, Maria},
  title     = {The immune transcriptome and proteome of the ant Camponotus floridanus and vertical transmission of its bacterial endosymbiont Blochmannia floridanus},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-142534},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {The evolutionary success of insects is believed to be at least partially facilitated by symbioses between insects and prokaryotes. Bacterial endosymbionts confer various fitness advantages to their hosts, for example by providing nutrients lacking from the insects' diet thereby enabling the inhabitation of new ecological niches. The Florida carpenter ant Camponotus floridanus harbours endosymbiotic bacteria of the genus Blochmannia. These primary endosymbionts mainly reside in the cytoplasm of bacteriocytes, specialised cells interspersed into the midgut tissue, but they were also found in oocytes which allows their vertical transmission. The social lifestyle of C. floridanus may facilitate the rapid spread of infections amongst genetically closely related animals living in huge colonies. Therefore, the ants require an immune system to efficiently combat infections while maintaining a "chronic" infection with their endosymbionts. In order to investigate the immune repertoire of the ants, the Illumina sequencing method was used. The previously published genome sequence of C. floridanus was functionally re-annotated and 0.53\% of C. floridanus proteins were assigned to the gene ontology (GO) term subcategory "immune system process". Based on homology analyses, genes encoding 510 proteins with possible immune function were identified. These genes are involved in microbial recognition and immune signalling pathways but also in cellular defence mechanisms, such as phagocytosis and melanisation. The components of the major signalling pathways appear to be highly conserved and the analysis revealed an overall broad immune repertoire of the ants though the number of identified genes encoding pattern recognition receptors (PRRs) and antimicrobial peptides (AMPs) is comparatively low. Besides three genes coding for homologs of thioester-containing proteins (TEPs), which have been shown to act as opsonins promoting phagocytosis in other insects, six genes encoding the AMPs defesin-1 and defensin-2, hymenoptaecin, two tachystatin-like peptides and one crustin-like peptide are present in the ant genome. Although the low number of known AMPs in comparison to 13 AMPs in the honey bee Apis mellifera and 46 AMPs in the wasp Nasonia vitripennis may indicate a less potent immune system, measures summarised as external or social immunity may enhance the immune repertoire of C. floridanus, as it was discussed for other social insects. Also, the hymenoptaecin multipeptide precursor protein may be processed to yield seven possibly bioactive peptides. In this work, two hymenoptaecin derived peptides were heterologously expressed and purified. The preliminary antimicrobial activity assays indicate varying bacteriostatic effects of different hymenoptaecin derived peptides against Escherichia coli D31 and Staphylococcus aureus which suggests a functional amplification of the immune response further increasing the antimicrobial potency of the ants. Furthermore, 257 genes were differentially expressed upon immune challenge of C. floridanus and most of the immune genes showing differential expression are involved in recognition of microbes or encode immune effectors rather than signalling components. Additionally, genes coding for proteins involved in storage and metabolism were downregulated upon immune challenge suggesting a trade-off between two energy-intensive processes in order to enhance effectiveness of the immune response. The analysis of gene expression via qRT-PCR was used for validation of the transcriptome data and revealed stage-specific immune gene regulation. Though the same tendencies of regulation were observed in larvae and adults, expression of several immune-related genes was generally more strongly induced in larvae. Immune gene expression levels depending on the developmental stage of C. floridanus are in agreement with observations in other insects and might suggest that animals from different stages revert to individual combinations of external and internal immunity upon infection. The haemolymph proteome of immune-challenged ants further established the immune-relevance of several proteins involved in classical immune signalling pathways, e.g. PRRs, extracellularly active proteases of the Toll signalling pathway and effector molecules such as AMPs, lysozymes and TEPs. Additionally, non-canonical proteins with putative immune function were enriched in immune-challenged haemolymph, e.g. Vitellogenins, NPC2-like proteins and Hemocytin. As known from previous studies, septic wounding also leads to the upregulation of genes involved in stress responses. In the haemolymph, proteins implicated in protein stabilisation and in the protection against oxidative stress and insecticides were enriched upon immune challenge. In order to identify additional putative immune effectors, haemolymph peptide samples from immune-challenged larvae and adults were analysed. The analysis in this work focussed on the identification of putative peptides produced via the secretory pathway as previously described for neuropeptides of C. floridanus. 567 regulated peptides derived from 39 proteins were identified in the larval haemolymph, whereas 342 regulated peptides derived from 13 proteins were found in the adult haemolymph. Most of the peptides are derived from hymenoptaecin or from putative uncharacterised proteins. One haemolymph peptide of immune-challenged larvae comprises the complete amino acid sequence of a predicted peptide derived from a Vitellogenin. Though the identified peptide lacks similarities to any known immune-related peptide, it is a suitable candidate for further functional analysis. To establish a stable infection with the endosymbionts, the bacteria have to be transmitted to the next generation of the ants. The vertical transmission of B. floridanus is guaranteed by bacterial infestation of oocytes. This work presents the first comprehensive and detailed description of the localisation of the bacterial endosymbionts in C. floridanus ovaries during oogenesis. Whereas the most apical part of the germarium, which contains the germ-line stem cells, is not infected by the bacteria, small somatic cells in the outer layers of each ovariole were found to be infected in the lower germarium. Only with the beginning of cystocyte differentiation, endosymbionts are exclusively transported from follicle cells into the growing oocytes, while nurse cells were never infected with B. floridanus. This infestation of the oocytes by bacteria very likely involves exocytosis-endocytosis processes between follicle cells and the oocytes. A previous study suggested a down-modulation of the immune response in the midgut tissue which may promote endosymbiont tolerance. Therefore, the expression of several potentially relevant immune genes was analysed in the ovarial tissue by qRT-PCR. The relatively low expression of genes involved in Toll and IMD signalling, and the high expression of genes encoding negative immune regulators, such as PGRP-LB, PGRP-SC2, and tollip, strongly suggest that a down-modulation of the immune response may also facilitate endosymbiont tolerance in the ovaries and thereby contribute to their vertical transmission. Overall, the present thesis improves the knowledge about the immune repertoire of C. floridanus and provides new candidates for further functional analyses. Moreover, the involvement of the host immune system in maintaining a "chronic" infection with symbiotic bacteria was confirmed and extended to the ovaries.},
  subject      = {Camponotus floridanus},
  language  = {en}
}
@phdthesis{Schwarze2014,
  author    = {Schwarze, Simone},
  title     = {Untersuchung von Faltungs- und Funktionsdynamik isolierter Proteindom{\"a}nen mittels Fluoreszenzl{\"o}schung},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-107080},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {Proteine bestehen aus einer spezifischen Sequenz verschiedener Aminos{\"a}uren, die ihre charakteristische Funktion bestimmt. Die große Variabilit{\"a}t an Aminos{\"a}uresequenzen erm{\"o}glichte die Evolution einer nahezu unbegrenzten Anzahl an Proteinen. Meistens nehmen diese Schl{\"u}sselpositionen ein, von robusten Baustoffen bis hin zu molekularen Maschinen. Daher kann eine Fehlfunktion gravierende Auswirkungen auf das Leben haben, z.B. Krankheiten wie Alzheimer oder Epilepsi. Um die Funktionen und Fehlfunktionen zu verstehen, ist eine umfassende Kenntnis der Proteinfaltung, der Protein-Protein Assoziation, sowie den Dynamiken innerhalb von Proteinen erforderlich. Diese Vorg{\"a}nge wurden in dieser Arbeit an drei isolierten Proteindom{\"a}nen durch die Anwendung der Fluoreszenzl{\"o}schmechanismen der H-Dimerbildung und des photoinduzierten Elektronentransfers untersucht. Der entfaltete Zustand der Bindungsdom{\"a}ne BBL, das Teil des 2-oxo-acid Dehydrogenasekomplexes ist, wurde unter physiologischen Bedingungen mit Zirkulardichroismus (CD) und einer Kombination aus photoinduziertem Elektronentransfer und Fluoreszenzkorrelationsspektroskopie analysiert. Beide Methoden zeigten {\"u}bereinstimmend anhand von 20 in BBL einzeln eingef{\"u}gten konservativen Punktmutationen, dass Seitenketteninteraktionen keine Auswirkungen auf die Sekund{\"a}rstruktur des denaturierten Zustandes, den Ausgangspunkt der Faltung, haben. Mit Hilfe der Dekonvolation der CD-Spektren wurde zudem gezeigt, dass die Reststruktur im denaturierten Zustand der helikalen Proteindom{\"a}ne von β-Str{\"a}ngen und β-Kehren dominiert wird, die eine entscheidende Funktion bei der Faltung in den nativen Zustand haben k{\"o}nnten. Die N-terminale Dom{\"a}ne (NTD), der f{\"u}r die Materialforschung hochinteressanten Spinnen-seidenfaser, ist f{\"u}r die Polymerisation des Spinnenseidenfadens auf den pH-Wechsel von pH 7 auf pH 6 hin verantwortlich. Dieser f{\"u}r die Proteinfunktion wichtige Prozess wurde durch die Einbringung eines extrinsischen Fluoreszenzschalters, basierend auf der H-Dimerbildung, mit der Stopped-Flow-Technik untersucht. Es wurde gezeigt, dass die NTDs 104 mit einer Rate von 3 x 10^8 M-1 s-1 assoziieren und somit nahezu das Geschwindigkeitslimit der Protein-Protein Assoziation erreicht wird. Zwei geladenen Seitenketten, der D39 und D40, kommt eine entscheidende Funktion in dem Prozess zu, da eine Mutation dieser die Assoziation verhindert. Des Weiteren wurde gezeigt, dass sich die NTD auf eine Erh{\"o}hung der Ionenst{\"a}rke entgegengesetzt zu anderen Proteinen verh{\"a}lt: die Dissoziation wird beschleunigt, die Assoziation nicht beeinflusst. Gleiches Verhalten wurde auf den einzelnen Austausch der {\"u}brigen protonierbaren Aminos{\"a}ureseitenketten hin beobachtet, ausgenommen die Mutation der E119, welche die Dissoziation verlangsamt. Daher scheint der makromolekulare Dipol, der auf Grund der Ladungsverteilung in der NTD entsteht, die Assoziation maßgeblich zu beeinflussen. Glutamatrezeptoren sind an der schnellen synaptischen Signalweiterleitung im Nervensys-tem von Vertebraten beteiligt. Die Konformationen der Ligandenbindungsdom{\"a}ne (LBD) haben dabei entscheidende Auswirkungen auf die Funktion des Gesamtrezeptors. Diese wurden mit einer Kombination aus photoinduziertem Elektronentransfer und Fluoreszenzkorrelationsspektroskopie untersucht. Mit dieser Methode wurde ein dynamisches Bild der gebundenen sowie ungebundenen Form der AMPA-spezifischen Glutamatrezeptor 2-LBD gezeigt. Es wurde zudem gezeigt, dass sich die Dynamiken in Abh{\"a}ngigkeit der Bindung von den Agonisten Glutamat und AMPA, dem partiellen Agonisten Kainate oder Cyclothiazid (CTZ), welches eine Dimerisierung der LBDs bewirkt, unterschiedlich ver{\"a}ndern. Dies k{\"o}nnte eine Auswirkung auf die Funktion der Rezeptoren haben. Die Anwendung der Fluoreszenzl{\"o}schmechanismen der H-Dimerbildung und des photoinduzierten Elektronentransfers in dieser Arbeit hat gezeigt, dass diese die M{\"o}glichkeit bieten, unterschiedlichste Fragestellungen zu beantworten und so Einblicke in dynamische Funktionsweisen von Proteinen er{\"o}ffnen. Kombiniert mit etablierten Fluoreszenzmethoden ist es so m{\"o}glich quantitativ Kinetiken auf unterschiedlichen Zeitskalen zu untersuchen.},
  subject      = {Protein-Protein-Wechselwirkung},
  language  = {de}
}
@phdthesis{Grosz2015,
  author    = {Grosz, Magdalena Urszula},
  title     = {Identification of phagosomal escape relevant factors in Staphylococcus aureus infection},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-121981},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {Staphylococcus aureus is a facultative Gram-positive human pathogen which can cause different severe infections. Staphylococci are phagocytosed by professional and non-professional phagocytes; they are strongly cytotoxic against eukaryotic cells and have been proposed to play a role in immune evasion by spreading within migrating phagocytes. This study investigated the post invasive events upon S. aureus infection. Strains which are able to escape the phagosome were identified and the responsible toxins were determined. Thereby innovative insights into host pathogen interaction were obtained. A novel class of small amphipathic peptides with strong surfactant-like properties, the phenol soluble modulins, particularly PSMα as well as the leukocidin LukAB, are involved in phagosomal escape of the clinical S. aureus strains LAC, MW2 and 6850 in non-professional and professional phagocytes. Whereas, PSMβ, δ-toxin, α-toxin, β-toxin or phosphatidyl inositol-dependent phospholipase C did not affect phagosomal escape. By blocking the bacterial DNA-dependent RNA polymerase with rifampicin phagosomal escape is determined to start approximately 2.5 hours post infection. Phagosomal escape further was required for intracellular replication of S. aureus. Strains which are not able to escape cannot replicate in the acidic vacuole, whereas, the host cytoplasm offers a rich milieu for bacterial replication. Additionally, phagosomal escape, with intracellular bacterial replication induces the subsequent host cell death. This could be confirmed by an infection assay including S. aureus knockout mutants in psmα or lukAB which were significantly less cytotoxic, compared with those infected with escape-positive wild type strains. Further, this study showed that phagosomal escape is not only mediated by bacterial toxins. Since, the phagocyte-specific cognate receptors for both escape relevant toxins, FPR2 (PSMα receptor) and CD11b (LukAB receptor) are produced in epithelial and endothelial cells only after infection with S. aureus in a calcium dependent fashion. The knockdown of both receptors using siRNA prevents S. aureus to escape the phagosome. Furthermore, blocking intracellular calcium release with the inositol trisphosphate receptor (IP3R) inhibitor 2-APB prohibits upregulation of fpr2 and cd11b and subsequently phagosomal escape of S. aureus. To conclude, the current study clarifies that phagosomal escape and host cell death are interplay of both, bacterial toxins and host cell factors. Staphylococcus aureus ist ein fakultativ Gram-positives Humanpathogen, dass verschiedene schwerwiegende Infektionen verursachen kann. Staphylokokken werden von professionellen und nicht-professionellen Phagozyten (Fresszellen) zu gleich aufgenommen. Desweitern sind sie stark zytotoxisch f{\"u}r eukaryotische Zellen. Außerdem wird vermutet, dass sie sich mittels migrierender Phagozyten dem angeborenen Immunsystem entziehen k{\"o}nnen. In dieser Studie werden die post-invasiven Ereignisse w{\"a}hrend einer Staphylokokken Infektion untersucht. Im Detail wurden St{\"a}mme identifiziert die aus den Phagosomen entkommen k{\"o}nnen und die daf{\"u}r verantwortlichen Toxine. Im Zuge dessen wurden neue Erkenntnisse der Interaktion zwischen Bakterien und Wirtszellen gewonnen. Eine neue Klasse von kleinen amphiphatischen Peptiden mit starken grenzfl{\"a}chenaktiven Eigenschaften (Surfactant), die sogenannten Phenol soluble modulins (PSMs) im Besonderen PSMα sowie das Leukozidin LukAB, sind am phagosomalen Ausbruch der klinisch relevanten S. aureus St{\"a}mmen LAC, MW2 und 6850 in nicht professionellen und professionellen Phagozyten involviert. Hingegen, sind PSMβ, δ-toxin, α-toxin, β-toxin oder Phosphatidylinositol abh{\"a}ngige Phospholipase C nicht am phagosomalen Ausbruch beteiligt. Durch die Hemmung der bakteriellen DNA-abh{\"a}ngigen RNA Polymerase mit Rifampicin wurde der Zeitpunkt f{\"u}r den Ausbruch auf etwa 2,5 Stunden nach der Infektion eingegrenzt. Der phagosomale Ausbruch ist weiterhin f{\"u}r die intrazellul{\"a}re Replikation von S. aureus notwendig. W{\"a}hrend St{\"a}mme, die nicht ausbrechen k{\"o}nnen in der anges{\"a}uerten Vakuole nicht replizieren k{\"o}nnen, bietet das Zytoplasma ein reichhaltiges Milieu f{\"u}r die Vermehrung. Zudem wird der Pathogen induzierte Zelltod erst nach dem phagosomalen Ausbruch und mit anschließender Vermehrung erm{\"o}glicht. Nachgewiesen wurde dies mittels psmα und lukAB defizienten Mutanten welche signifikant weniger zytotoxisch waren als der Wildtyp Stamm. Diese Studie zeigt dar{\"u}ber hinaus, dass der phagosomale Ausbruch nicht nur durch bakterielle Toxine vermittelt wird. Sondern, dass die Phagozyten-spezifischen Rezeptoren f{\"u}r beide relevanten Toxine, FPR2 (PSMα Rezeptor) und CD11b (LukAB Rezeptor), in Epithel- und Endothelzellen nach Infektion mit S. aureus calciumabh{\"a}ngig produziert werden und f{\"u}r den Ausbruch notwendig sind. Der knockdown beider Rezeptoren mittels siRNA verhindert den Ausbruch. Wird der intrazellul{\"a}re Calciumstrom mittels des Inositoltrisphosphat Rezeptor (IP3R) Inhibitor 2-APB blockiert k{\"o}nnen die Gene fpr2 und cd11b nicht hochreguliert werden und der Ausbruch wird ebenfalls verhindert. Folglich zeigt diese Studie, dass der phagosomale Ausbruch und Pathogen induzierte Zelltod sowohl durch bakterielle Toxine als auch Wirtsfaktoren vermittelt wird.},
  subject      = {Phagosom},
  language  = {en}
}
@phdthesis{Costea2016,
  author    = {Costea, Paul Igor},
  title     = {Stratification and variation of the human gut microbiota},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-139649},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {The microbial communities that live inside the human gastrointestinal tract -the human gut microbiome- are important for host health and wellbeing. Characterizing this new "organ", made up of as many cells as the human body itself, has recently become possible through technological advances. Metagenomics, the high-throughput sequencing of DNA directly from microbial communities, enables us to take genomic snapshots of thousands of microbes living together in this complex ecosystem, without the need for isolating and growing them. Quantifying the composition of the human gut microbiome allows us to investigate its properties and connect it to host physiology and disease. The wealth of such connections was unexpected and is probably still underestimated. Due to the fact that most of our dietary as well as medicinal intake affects the microbiome and that the microbiome itself interacts with our immune system through a multitude of pathways, many mechanisms have been proposed to explain the observed correlations, though most have yet to be understood in depth. An obvious prerequisite to characterizing the microbiome and its interactions with the host is the accurate quantification of its composition, i.e. determining which microbes are present and in what numbers they occur. Historically, standard practices have existed for sample handling, DNA extraction and data analysis for many years. However, these were generally developed for single microbe cultures and it is not always feasible to implement them in large scale metagenomic studies. Partly because of this and partly because of the excitement that new technology brings about, the first metagenomic studies each took the liberty to define their own approach and protocols. From early meta-analysis of these studies it became clear that the differences in sample handling, as well as differences in computational approaches, made comparisons across studies very difficult. This restricts our ability to cross-validate findings of individual studies and to pool samples from larger cohorts. To address the pressing need for standardization, we undertook an extensive comparison of 21 different DNA extraction methods as well as a series of other sample manipulations that affect quantification. We developed a number of criteria for determining the measurement quality in the absence of a mock community and used these to propose best practices for sampling, DNA extraction and library preparation. If these were to be accepted as standards in the field, it would greatly improve comparability across studies, which would dramatically increase the power of our inferences and our ability to draw general conclusions about the microbiome. Most metagenomics studies involve comparisons between microbial communities, for example between fecal samples from cases and controls. A multitude of approaches have been proposed to calculate community dissimilarities (beta diversity) and they are often combined with various preprocessing techniques. Direct metagenomics quantification usually counts sequencing reads mapped to specific taxonomic units, which can be species, genera, etc. Due to technology-inherent differences in sampling depth, normalizing counts is necessary, for instance by dividing each count by the sum of all counts in a sample (i.e. total sum scaling), or by subsampling. To derive a single value for community (dis-)similarity, multiple distance measures have been proposed. Although it is theoretically difficult to benchmark these approaches, we developed a biologically motivated framework in which distance measures can be evaluated. This highlights the importance of data transformations and their impact on the measured distances. Building on our experience with accurate abundance estimation and data preprocessing techniques, we can now try and understand some of the basic properties of microbial communities. In 2011, it was proposed that the space of genus level variation of the human gut microbial community is structured into three basic types, termed enterotypes. These were described in a multi-country cohort, so as to be independent of geography, age and other host properties. Operationally defined through a clustering approach, they are "densely populated areas in a multidimensional space of community composition"(source) and were proposed as a general stratifier for the human population. Later studies that applied this concept to other datasets raised concerns about the optimum number of clusters and robustness of the clustering approach. This heralded a long standing debate about the existence of structure and the best ways to determine and capture it. Here, we reconsider the concept of enterotypes, in the context of the vastly increased amounts of available data. We propose a refined framework in which the different types should be thought of as weak attractors in compositional space and we try to implement an approach to determining which attractor a sample is closest to. To this end, we train a classifier on a reference dataset to assign membership to new samples. This way, enterotypes assignment is no longer dataset dependent and effects due to biased sampling are minimized. Using a model in which we assume the existence of three enterotypes characterized by the same driver genera, as originally postulated, we show the relevance of this stratification and propose it to be used in a clinical setting as a potential marker for disease development. Moreover, we believe that these attractors underline different rules of community assembly and we recommend they be accounted for when analyzing gut microbiome samples. While enterotypes describe structure in the community at genus level, metagenomic sequencing can in principle achieve single-nucleotide resolution, allowing us to identify single nucleotide polymorphisms (SNPs) and other genomic variants in the gut microbiome. Analysis methodology for this level of resolution has only recently been developed and little exploration has been done to date. Assessing SNPs in a large, multinational cohort, we discovered that the landscape of genomic variation seems highly structured even beyond species resolution, indicating that clearly distinguishable subspecies are prevalent among gut microbes. In several cases, these subspecies exhibit geo-stratification, with some subspecies only found in the Chinese population. Generally however, they present only minor dispersion limitations and are seen across most of our study populations. Within one individual, one subspecies is commonly found to dominate and only rarely are several subspecies observed to co-occur in the same ecosystem. Analysis of longitudinal data indicates that the dominant subspecies remains stable over periods of more than three years. When interrogating their functional properties we find many differences, with specific ones appearing relevant to the host. For example, we identify a subspecies of E. rectale that is lacking the flagellum operon and find its presence to be significantly associated with lower body mass index and lower insulin resistance of their hosts; it also correlates with higher microbial community diversity. These associations could not be seen at the species level (where multiple subspecies are convoluted), which illustrates the importance of this increased resolution for a more comprehensive understanding of microbial interactions within the microbiome and with the host. Taken together, our results provide a rigorous basis for performing comparative metagenomics of the human gut, encompassing recommendations for both experimental sample processing and computational analysis. We furthermore refine the concept of community stratification into enterotypes, develop a reference-based approach for enterotype assignment and provide compelling evidence for their relevance. Lastly, by harnessing the full resolution of metagenomics, we discover a highly structured genomic variation landscape below the microbial species level and identify common subspecies of the human gut microbiome. By developing these high-precision metagenomics analysis tools, we thus hope to contribute to a greatly improved understanding of the properties and dynamics of the human gut microbiome.},
  subject      = {Mensch},
  language  = {en}
}
@phdthesis{Classen2014,
  author    = {Claßen, Alice},
  title     = {Diversity, traits and ecosystem services of pollinators along climate and land use gradients on Mount Kilimanjaro},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-101292},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {Since more than two centuries naturalists are fascinated by the profound changes in biodiversity observed along climatic gradients. Although the theories explaining changes in the diversity and the shape of organisms along climatic gradients belong to the foundations of modern ecology, our picture on the spatial patterns and drivers of biodiversity is far from being complete. Ambiguities in theory and data are common and past work has been strongly concentrated on plants and vertebrates. In the last two decades, interest in the fundamental processes structuring diversity along climatic gradients gained new impetus as they are expected to improve our understanding about how ecosystems will respond to global environmental changes. Global temperatures are rising faster than ever before; natural habitats are transformed into agricultural land and existing land use systems get more and more intensified to meet the demands of growing human populations. The fundamental shifts in the abiotic and biotic environment are proclaimed to affect ecosystems all over the world; however, precise predictions about how ecosystems respond to global changes are still lacking. We investigated diversity, traits and ecosystem services of wild bees along climate and land use gradients on Mount Kilimanjaro (Tanzania, East Africa). Wild bees play a major role in ecosystems, as they contribute to the reproduction and performance of wild and crop plants. Their responsiveness to environmental changes is therefore of high ecological and economic importance. Temperature and energy resources have often been suggested to be the main determinants of global and local species richness, but the mechanisms behind remain poorly understood. In the study described in chapter II we analyzed species richness patterns of wild bees along climate and land use gradients on Mount Kilimanjaro and disentangled the factors explaining most of the changes in bee richness. We found that floral resources had a weak but significant effect on pollinator abundance, which in turn was positively related to species richness. However, temperature was the strongest predictor of species richness, affecting species richness both directly and indirectly by positively influencing bee abundances. We observed higher levels of bee-flower-interactions at higher temperatures, independently of flower and bee abundances. This suggests that temperature restricts species richness by constraining the exploitation of resources by ectotherms. Current land use did not negatively affect species richness. We conclude that the richness of bees is explained by both temperature and resource availability, whereas temperature plays the dominant role as it limits the access of ectotherms to floral resources and may accelerate ecological and evolutionary processes that drive the maintenance and origination of diversity. Not only species numbers, but also morphological traits like body size are expected to be shaped by both physiological and energetic constraints along elevational gradients. Paradoxically, Bergmann´s rule predicts increases of body sizes in cooler climates resulting from physiological constraints, while species-energy theory suggests declines in the mean body size of species caused by increased extinction probabilities for large-bodied species in low-energy habitats. In chapter III we confronted this ambiguity with field data by studying community-wide body size variation of wild bees on Mt. Kilimanjaro. We found that along a 3680 m elevational gradient bee individuals became on average larger within species, while large species were increasingly absent from high-elevational communities. This demonstrates, on the one hand, how well-established, but apparently contrasting ecological theories can be merged through the parallel consideration of different levels of biological organization. On the other hand it signals that the extinction risk in the course of environmental change is not equally distributed among species within a community. Land use intensification is known to threaten biodiversity, but the consequences for ecosystem services are still a matter of debate. In chapter IV, we experimentally tested the single and combined contributions of pest predators and pollinators to coffee production along a land use intensification gradient on Mount Kilimanjaro. We found that pest predation increased fruit set by on average 9\%, while pollination increased fruit weight of coffee by on average 7.4\%. Land use had no significant effect on both ecosystem services. However, we found that in coffee plantations with most intensified land use, pollination services were virtually exclusively provided by the honey bee (Apis mellifera). The reliance on a single pollinator species is risky, as possible declines of that species may directly lower pollination services, resulting in yield losses. In contrast, pollination services in structurally complex homegardens were found to be provided by a diverse pollinator community, increasing the stability of pollination services in a long term. We showed that on Mount Kilimanjaro pollinator communities changed along elevational gradients in terms of species richness (chapter II) and trait composition (chapter III). Temperature and the temperature-mediated accessibility of resources were identified as important predictors of these patterns, which contributes to our fundamental understanding about the factors that shape ectothermic insect communities along climatic gradients. The strong temperature-dependence of pollinators suggests that temperature shifts in the course of global change are likely to affect pollinator communities. Pollinators might either profit from rising temperatures, or shift to higher elevations, which could result in related biotic attrition in the lowland with consequences for the provision of ecosystem services in cropping systems. Up to now, land use intensification had no significant impact on the diversity of pollinator communities and their ecosystem services. Pollinators might profit from the strong landscape heterogeneity in the region and from the amount of flower resources in the understory of cropping systems. However,progressing homogenization of the landscape and the pronounced application of pesticides could result in reduced diversity and dominance of single species, as we already found in sun coffee plantations. Such shifts in community compositions could threaten the stability of ecosystem services within cropping and natural systems in a long term.},
  subject      = {Kilimandscharo},
  language  = {en}
}
@phdthesis{Kindeketa2014,
  author    = {Kindeketa, William Joseph},
  title     = {Pollination in wild plant communities along altitudinal and land use gradients Mount Kilimanjaro, Tanzania},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-100136},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {1. Pollination of sexually reproducing plants requires pollen transfer agents, which can be biotic, abiotic or a combination of biotic and abiotic agents. The dominance of one of pollination system in wild plant communities depends on climatic factors and/or degrees of anthropogenic influences, which have effects on pollinator diversity and pollination function. Anthropogenic activities and climate change are also considered as main causes of ongoing invasion of invasive species into wild and managed habitats which can bring up competition for pollinators with possible negative consequences for the reproduction of co-occurring native plant species. 2. The study aimed to determine pollination systems and pollination limitation of invasive and native plant communities in natural savannah between 870 - 1130 m and semi-natural (managed) grassland between 1300 - 1750 m above sea level; effects of flower density and pollinator abundance on seed production of cross-pollinated and self-pollinated plants; and relationships of bee abundance and the proportion of cross- pollinated plants at the southern slope of Mount Kilimanjaro, Tanzania. 3. Pollinator-exclusion, open pollination and supplemental hand-pollination treatments were applied to 27 plant species in savannah and grassland habitats. Flowers were counted in each clusters based upon their species. Pollinators were sampled by using pan traps. Information-theory-based multi-model averaging and generalized linear mixed effects models were used to identify and analyze the effects of flower density, pollinator abundance, pollination treatments and habitat types on seed production. Regression models were used to determine relationships of altitude with bee abundance, and with proportion of cross-pollinated plants. 4. My results show that mean seed numbers of native plants were significantly lower in pollinator-exclusion treatments than in open-pollination treatments, indicating their reliance on pollinators for reproductive success. In contrast, seed numbers of invasive plants were similar in pollinator-exclusion and open-pollination treatments, demonstrating an ability of reproduction without pollinators. Despite of higher levels of self-pollination in invasive plants, supplemental hand-pollination treatments revealed pollen limitation in grassland and marginally in savannah habitats. There were no significant difference in seed numbers between supplemental hand pollination and open pollination treatments of native plant communities in savannah and grassland, which indicates no pollination limitation in the studied ecological system for native communities. Besides, grassland plants produced comparatively more seeds than savannah plants, however seeds in grasslands were lighter than those of the savannah which may be due to nutrient limitation in grassland. 5. I found 12 cross-pollinated and 15 self-pollinated plants along altitudinal gradient after comparing seeds from pollinator-excluded and open-pollinated experiments. I also found that proportions of cross-pollinated plants and bee abundance simultaneously decreased with increasing altitude. All cross-pollinated plants were native and grew in savannah habitats, with an exception of one species. 6. Neither effects of focal flower density nor a significant interaction between focal flower densities and bee abundance for self-pollinated plants were observed. However, there were effects of focal flower densities and interactions of flower density with bee abundance for cross-pollinated plants. Non-focal flower density has no significant effects on seed production of cross-pollinated and self-pollinated plants. 7. The results show that native plants depend more on cross-pollination than invasive plants, despite of most native plants in managed habitat (grassland) rely on self-pollination for reproduction. The tendency of having more cross-pollinated plants in natural savannah which are in low altitude coincides with other finding that the cross-pollinated plants and bee abundance simultaneously decrease with increasing altitude. Therefore, our findings support the hypotheses that self-fertilization of flowering plants increases with increasing altitude, and pollinator limitation is most pronounced in managed or disturbed habitats. Despite of reduction of pollinators in grassland, only invasive plants experience pollen limitation, which may be due to poor integration with available pollinator networks. 8. I also found bee abundance and flower density are not the main pollination factors required by self-pollinated plants during reproduction. However, focal flower density, which influences pollinator diversity, is more applicable to cross-pollinated plants. Climate change and anthropogenic activities in natural habitats are factors that influence pollinator abundance and functioning, which lead to a shift of mating systems in plant communities so as to assure their reproduction.},
  subject      = {Best{\"a}ubungs{\"o}kologie},
  language  = {en}
}
@phdthesis{Pasch2016,
  author    = {Pasch, Elisabeth},
  title     = {The role of SUN4 and related proteins in sperm head formation and fertility},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-139092},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {Spermiogenesis describes the differentiation of haploid germ cells into motile, fertilization-competent spermatozoa. During this fundamental transition the species-specific sperm head is formed, which necessitates profound nuclear restructuring coincident with the assembly of sperm-specific structures and chromatin compaction. In the case of the mouse, it is characterized by reshaping of the early round spermatid nucleus into an elongated sickle-shaped sperm head. This tremendous shape change requires the transduction of cytoskeletal forces onto the nuclear envelope (NE) or even further into the nuclear interior. LINC (linkers of nucleoskeleton and cytoskeleton) complexes might be involved in this process, due to their general function in bridging the NE and thereby physically connecting the nucleus to the peripheral cytoskeleton. LINC complexes consist of inner nuclear membrane integral SUN-domain proteins and outer nuclear membrane KASH-domain counterparts. SUN- and KASH-domain proteins are directly connected to each other within the perinuclear space, and are thus capable of transferring forces across the NE. To date, these protein complexes are known for their essential functions in nuclear migration, anchoring and positioning of the nucleus, and even for chromosome movements and the maintenance of cell polarity and nuclear shape. In this study LINC complexes were investigated with regard to their potential role in sperm head formation, in order to gain further insight into the processes occurring during spermiogenesis. To this end, the behavior and function of the testis-specific SUN4 protein was studied. The SUN-domain protein SUN4, which had received limited characterization prior to this work, was found to be exclusively expressed in haploid stages during germ cell development. In these cell stages, it specifically localized to the posterior NE at regions decorated by the manchette, a spermatid-specific structure which was previously shown to be involved in nuclear shaping. Mice deficient for SUN4 exhibited severely disorganized manchette residues and gravely misshapen sperm heads. These defects resulted in a globozoospermia-like phenotype and male mice infertility. Therefore, SUN4 was not only found to be mandatory for the correct assembly and anchorage of the manchette, but also for the correct localization of SUN3 and Nesprin1, as well as of other NE components. Interaction studies revealed that SUN4 had the potential to interact with SUN3, Nesprin1, and itself, and as such is likely to build functional LINC complexes that anchor the manchette and transfer cytoskeletal forces onto the nucleus. Taken together, the severe impact of SUN4 deficiency on the nucleocytoplasmic junction during sperm development provided direct evidence for a crucial role of SUN4 and other LINC complex components in mammalian sperm head formation and fertility.},
  subject      = {Maus},
  language  = {en}
}
@phdthesis{Hafen2015,
  author    = {Hafen, Bettina},
  title     = {Physical contact between mesenchymal stem cells and endothelial precursors induces distinct signatures with relevance to tissue regeneration and engineering},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-119417},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {The goal of the project VascuBone is to develop a tool box for bone regeneration, which on one hand fulfills basic requirements (e.g. biocompatibility, properties of the surface, strength of the biomaterials) and on the other hand is freely combinable with what is needed in the respective patient's situation. The tool box will include a variation of biocompatible biomaterials and cell types, FDA-approved growth factors, material modification technologies, simulation and analytical tools like molecular imaging-based in vivo diagnostics, which can be combined for the specific medical need. This tool box will be used to develop translational approaches for regenerative therapies of different types of bone defects. This project receives funding from the European Union's Seventh Framework Program (VascuBone 2010). The present study is embedded into this EU project. The intention of this study is to assess the changes of the global gene expression patterns of endothelial progenitor cells (EPCs) and mesenchymal stem cells (MSCs) after direct cell-cell contact as well as the influence of conditioned medium gained from MSCs on EPCs and vice versa. EPCs play an important role in postnatal vasculogenesis. An intact blood vessel system is crucial for all tissues, including bone. Latest findings in the field of bone fracture healing and repair by the use of tissue engineering constructs seeded with MSCs raised the idea of combining MSCs and EPCs to enhance vascularization and therefore support survival of the newly built bone tissue. RNA samples from both experimental set ups were hybridized on Affymetrix GeneChips® HG-U133 Plus 2.0 and analyzed by microarray technology. Bioinformatic analysis was applied to the microarray data and verified by RT-PCR. This study gives detailed information on how EPCs and MSCs communicate with each other and therefore gives insights into the signaling pathways of the musculoskeletal system. These insights will be the base for further functional studies on protein level for the purpose of tissue regeneration. A better understanding of the cell communication of MSCs and EPCs and subsequently the targeting of relevant factors opens a variety of new opportunities, especially in the field of tissue engineering. The second part of the present work was to develop an ELISA (enzyme-linked immunosorbent assay) for a target protein from the lists of differentially expressed genes revealed by the microarray analysis. This project was in cooperation with Immundiagnostik AG, Bensheim, Germany. The development of the ELISA aimed to have an in vitro diagnostic tool to monitor e.g. the quality of cell seeded tissue engineering constructs. The target protein chosen from the lists was klotho. Klotho seemed to be a very promising candidate since it is described in the literature as anti-aging protein. Furthermore, studies with klotho knock-out mice showed that these animals suffered from several age-related diseases e.g. osteoporosis and atherosclerosis. As a co-receptor for FGF23, klotho plays an important role in bone metabolism. The present study will be the first one to show that klotho is up-regulated in EPCs after direct cell-cell contact with MSCs. The development of an assay with a high sensitivity on one hand and the capacity to differentiate between secreted and shedded klotho on the other hand will allow further functional studies of this protein and offers a new opportunity in medical diagnostics especially in the field of metabolic bone disease.},
  subject      = {Vorl{\"a}uferzelle},
  language  = {en}
}
@phdthesis{Fackler2014,
  author    = {Fackler, Marc},
  title     = {Biochemical characterization of GAS2L3, a target gene of the DREAM complex},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-103394},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {GAS2L3 was identified recently as a target gene of the DREAM complex (Reichert et al., 2010; Wolter et al., 2012). It was shown that GAS2L3 is expressed in a cell cycle specific manner and that depletion of the protein leads to defects in cytokinesis and genomic instability (Wolter et al., 2012). Major aim of this thesis was, to further characterize the biochemical properties and physiological function of GAS2L3. By in vitro co-sedimentation and bundling assays, GAS2L3 was identified as a cytoskeleton associated protein which bundles, binds and crosslinks F-actin and MTs. GST pulldown assays and co-immunoprecipitation experiments revealed that GAS2L3 interacts in vitro and in vivo with the chromosomal passenger complex (CPC), a very important regulator of mitosis and cytokinesis, and that the interaction is mediated by the GAR domain of GAS2L3 and the C-terminal part of Borealin and the N-terminal part of Survivin. Kinase assays showed that GAS2L3 is not a substrate of the CPC but is strongly phosphorylated by CDK1 in vitro. Depletion of GAS2L3 by shRNA influenced protein stability and activity of the CPC. However pharmacological studies showed that the decreased CPC activity is not responsible for the observed cytokinesis defects upon GAS2L3 depletion. Immunofluorescence experiments revealed that GAS2L3 is localized to the constriction zone by the CPC in a GAR dependent manner and that the GAR domain is important for proper protein function. New interacting proteins of GAS2L3 were identified by stable isotope labelling by amino acids in cell culture (SILAC) in combination with tandem affinity purification and subsequent mass spectrometrical analysis. Co-immunoprecipitation experiments further confirmed the obtained mass spectrometrical data. To address the physiological function of GAS2L3 in vivo, a conditional and a non-conditional knockout mouse strain was established. The non-conditional mouse strain showed a highly increased mortality rate before weaning age probably due to heart failure. The physiological function of GAS2L3 in vivo as well as the exact reason for the observed heart phenotype is not known at the moment.},
  subject      = {Zellzyklus},
  language  = {en}
}
@phdthesis{Kober2015,
  author    = {Kober, Christina},
  title     = {Characterization of Murine GL261 Glioma Models for Oncolytic Vaccinia Virus Therapy},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-118556},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {Glioblastoma multiforme (GBM) is one of the most frequent and malignant forms of brain cancer in adults. The prognosis is poor with a median survival time of 12-15 months. There is a broad range of alternative treatment options studied in preclinical and clinical trials for GBM. One alternative treatment option is oncolytic virotherapy, defined as the use of replication-competent viruses that selectively infect and destroy cancer cells while leaving, non-transformed cells unharmed. Vaccinia virus (VACV) is one favorable candidate. Although oncolytic viruses can kill tumor cells grown in vitro with high efficiency, they often exhibit reduced replication capacity in vivo suggesting that physiological aspects of the tumor microenvironment decrease the virus' therapeutic potential. The percentage and composition of immune cells varies between cancer types and patients and is investigated as a biomarker in several studies. Making oncolytic virotherapy successful for GBM, it is necessary to understand the individual tumor biology, the interaction with the microenvironment and immune system. It was demonstrated that the attenuated VACV wild-type (wt) isolate LIVP 1.1.1 replicate and lyse the murine GL261 glioma cell line in vitro. In the following, the replication efficacy was characterized in a comparative approach in vivo. Immunocompetent C57BL/6 (wt) mice and immunodeficient mouse strains of different genetic background C57BL/6 athymic and Balb/c athymic mice were used. In addition, subcutaneous and intracranial locations were compared. The results revealed viral replication exclusively in Balb/c athymic mice with subcutaneous tumors but in none of the other models. In the following, the tumor microenvironment of the subcutaneous tumor models at the time of infection was performed. The study showed that implantation of the same tumor cells in different mouse strains resulted in a different tumor microenvironment with a distinct composition of immune cells. Highest differences were detected between immunodeficient and immunocompetent mice. The study showed major differences in the expression of MHCII with strongest expression in C57BL/6 wt and weakest in Balb/c athymic tumors. In the following, the influence of the phenotypic change associated with the upregulation of MHCII on GL261 tumor cells on viral replication was analyzed. Comparison of C57BL/6 wt and C57BL/6 IFN-γ knockout mice revealed endogenous IFN-γ levels to upregulate MHCII on GL261 tumor cells and to reduce viral replication in C57BL/6 wt mice. Analysis of single cell suspensions of tumor homogenates of C57BL/6 and Balb/c athymic mice showed that the IFN-γ-mediated anti-tumor effect was a reversible effect. Furthermore, reasons for inhibition of virus replication in orthotopic glioma models were elucidated. By immunohistochemical analysis it was shown that intratumoral amounts of Iba1+ microglia and GFAP+ astrocytes in Gl261 gliomas was independent from intratumoral VACV injection. Based on these findings virus infection in glioma, microglia and astrocytes was compared and analyzed in cell culture. In contrast to the GL261 glioma cells, replication was barely detectable in BV-2 microglia and IMA2.1 astrocytic cells. Co-culture experiments revealed that microglia compete for virus uptake in cell culture. It was further shown that BV-2 cells showed apoptotic characteristics after VACV infection while GL261 cells showed signs of necrotic cell death. Additionally, in BV-2 cells with M1-phenotype a further reduction of viral replication and inhibition of cell lysis was detected. Infection of IMA 2.1 cells was independent of the M1/M2-phenotype. Application of BV-2 microglia with M1-phenotype onto organotypic slice cultures with implanted GL261 tumors resulted in reduced infection of BV-2 cells with LIVP 1.1.1, whereas GL261 cells were significantly infected. Taken together, the analyzed GL261 tumors were imprinted by the immunologic and genetic background in which they grow. The experimental approach applied in this thesis can be used as suitable model which reflects the principles of personalized medicine In an additional project, based on gene expression data and bioinformatic analyses, the biological role and function of the anti-apoptotic factor AVEN was analyzed with regard to oncolytic VACV therapy. Besides a comparison of the replication efficacy of GLV-1h68 and VACV-mediated cell killing of four human tumor cell lines, it was shown that AVEN was expressed in all analyzed cells. Further, shown for HT-29 and 1936-MEL, the knockdown of AVEN by siRNA in cell culture resulted in an increase of apoptotic characteristics and a decrease of VACV infection. These findings provide essential insights for future virus development.},
  subject      = {Krebs <Medizin>},
  language  = {en}
}
@phdthesis{MeirgebRother2015,
  author    = {Meir [geb. Rother], Juliane},
  title     = {Influence of oncolytic vaccinia viruses on metastases of human and murine tumors},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-118530},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {Cancer is one of the leading causes of death. 90\% of all deaths are caused by the effects of metastases. It is of major importance to successfully treat the primary tumor and metastases. Tumors and metastases often differ in their properties and therefore, treatment is not always successful. In contrast, those therapeutic agents can even promote formation and growth of metastases. Hence, it is indispensable to find treatment options for metastatic disease. One promising candidate represents the oncolytic virus therapy with vaccinia viruses. The aim of this work was to analyze two cell lines regarding their metastatic abilities and to investigate whether oncolytic vaccinia viruses are useful therapy options. The cell lines used were the human cervical cancer cell line C33A implanted into immune-compromised mice and the murine melanoma cell line B16F10, implanted into immune-competent mice. The initial point of the investigations was the observation of enlarged lumbar und renal lymph nodes in C33A tumor-bearing mice 35 days post implantation of C33A cells subcutaneously into immune-compromised nude mice. Subsequently, the presence of human cells in enlarged lymph nodes was demonstrated by RT-PCR. To facilitate the monitoring of cancer cell spreading, the gene encoding for RFP was inserted into the genome of C33A cells. In cell culture experiments, it was possible to demonstrate that this insertion did not negatively affect the susceptibility of the cells to virus infection, replication and virus-mediated cell lysis. The analysis of the metastatic process in a xenografted mouse model revealed the continuous progression of lumbar (LN) and renal (RN) lymph node metastasis after C33A-RFP tumor cell implantation. The lymph node volume and the amount of RFP-positive LNs and RNs was increasing from week to week in accordance with the gain of the primary tumor volume. Moreover, the metastatic spread of cancer cells in lymph vessels between lumbar and renal lymph nodes was visualized. Additionally, the haematogenous way of cancer cell migration was demonstrated by RFP positive cancer cells in blood vessels. The haematogenous route of spreading was confirmed by detecting micrometastases in lungs of tumor bearing mice. The next step was to investigate whether the recombinant oncolytic vaccinia virus GLV-1h68 is a suitable candidate to cure the primary tumor and metastases. Therefore, GLV-1h68 was systemically injected into C33A-RFP tumor bearing mice 21 days after tumor cell implantation. It was demonstrated that the volume of the primary tumor was drastically reduced, and the volume and the amount of RFP positive lumbar and renal lymph nodes were significantly decreasing compared to the untreated control group. Subsequently, this process was analyzed further by investigating the colonization pattern in the C33A-RFP model. It was shown that first the primary tumor was colonized with highest detectable virus levels, followed by LN and RN lymph nodes. Histological analyses revealed the proliferative status of tumor cells in the tumor and lymph nodes, the amount of different immune cell populations and the vascular permeability in primary tumors and lymph nodes having an influence on the colonization pattern of the virus. Whereby, the vascular permeability seems to have a crucial impact on the preferential colonization of tumors compared to lymph node metastases in this tumor model. C33A turned out to be a useful model to study the formation and therapy of metastases. However, a metastatic model in which the influence of the immune system on tumors and especially on tumor therapy can be analyzed would be preferable. Therefore, the aim of the second part was to establish a syngeneic metastatic mouse model. Accordingly, the murine melanoma cell line B16F10 was analyzed in immunocompetent mice. First, the highly attenuated GLV 1h68 virus was compared to its parental strain LIVP 1.1.1 concerning infection, replication and cell lysis efficacy in cell culture. LIVP 1.1.1 was more efficient than GLV-1h68 and was subsequently used for following mouse studies. Comparative studies were performed, comparing two different implantation sites of the tumor cells, subcutaneously and footpad, and two different mouse strains, FoxN1 nude and C57BL/6 mice. Implantation into the footpad led to a higher metastatic burden in lymph nodes compared to the subcutaneous implantation site. Finally, the model of choice was the implantation of B16F10 into the footpad of immune-competent C57BL/6 mice. Furthermore, it was inevitable to deliver the virus as efficient as possible to the tumor and metastases. Comparison of two different injection routes, intravenously and intratumorally, revealed, that the optimal injection route was intratumorally. In summary, the murine B16F10 model is a promising model to study the effects of the immune system on vaccinia virus mediated therapy of primary tumors and metastases.},
  subject      = {Krebs <Medizin>},
  language  = {en}
}
@phdthesis{Andronic2014,
  author    = {Andronic, Joseph},
  title     = {Volumenregulatorische Transportwege von anorganischen und organischen Osmolyten in S{\"a}ugetierzellen},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-103255},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {Die Aufrechterhaltung des Zellvolumens unter variablen osmotischen Bedingungen stellt f{\"u}r nahezu alle tierischen Zellen eine essenzielle Aufgabe dar. Um regulatorische Volumenanpassungen vorzunehmen besitzen sie daher effektive Mechanismen, mit deren Hilfe der zellul{\"a}re Gehalt an organischen und anorganischen Osmolyten erh{\"o}ht (= regulatorische Volumenzunahme; RVI) oder gesenkt (= regulatorische Volumenabnahme; RVD) werden kann. Trotz langj{\"a}hriger Forschung auf diesem Gebiet konnten die hieran beteiligten Transportwege f{\"u}r Osmolyte bisher nur unvollst{\"a}ndig aufgekl{\"a}rt werden. Insbesondere bei T-Lymphozyten sind wichtige Zellfunktionen wie die Proliferation, Migration und die T-Zell-Aktivierung eng mit volumenregulatorischen Mechanismen verbunden. Bei all diesen Prozessen sind u. a. unterschiedliche Kaliumkan{\"a}le beteiligt, die insbesondere f{\"u}r die pharmakologische Manipulation von Immunsystemprozessen von wissenschaftlichem Interesse sind. Bisherige Modelle der hypotonen Volumenregulation von T-Lymphozyten ber{\"u}cksichtigen lediglich den spannungsabh{\"a}ngigen KV1.3 sowie den Ca2+-aktivierten IKCa1-Kanal, die zur Klasse der 6TM/P-K+-Kan{\"a}le geh{\"o}ren. Im ersten Teil der vorliegenden Arbeit wurde eine potentielle Rolle von k{\"u}rzlich entdeckten Zwei-Poren Dom{\"a}nen Kaliumkan{\"a}len (K2P) am RVD von murinen und humanen prim{\"a}ren CD4+-T-Lymphozyten untersucht. In einem kombinierten genetischen und pharmakologischen Ansatz mittels knockout-Tiermodellen und dem Einsatz kanalspezifischer Inhibitoren konnte mithilfe zellvolumetrischer Analysen gezeigt werden, dass die K2P-Vertreter TASK1, TASK2, TASK3 und TRESK maßgeblich am schwellungsaktivierten Efflux von K+ beteiligt sind. Beurteilt an den Ergebnissen dieser Untersuchung sind der spannungsabh{\"a}ngige TASK2- und der Ca2+-aktivierte TRESK-Kanal f{\"u}r die hypotone Volumenregulation in T-Zellen deutlich bedeutender als TASK1 und TASK3. Der Beitrag der Kan{\"a}le TASK2 und TRESK am RVD-Prozess war {\"u}ber dies vergleichbar mit dessen des bisher bekannten KV1.3-Kanals. In dieser Arbeit wurde damit erstmals eine Beteiligung der K2P-Kan{\"a}le am RVD muriner und humaner CD4+-Lymphozyten identifiziert. Aufgrund der engen Verbindung zwischen T-Zell-Funktion und der Volumenregulation k{\"o}nnen Zwei-Poren Dom{\"a}nen K+-Kan{\"a}le damit in den engeren Kreis potentieller immunmodulierende Angriffspunkte aufgefasst werden. Im zweiten und umfangreicheren Teil dieser Arbeit wurden dar{\"u}ber hinaus die schwellungsaktivierten Transportwege f{\"u}r organische Osmolyte (small organic osmolytes; SOOs) untersucht. SOOs stellen chemisch inerte Verbindungen dar, zu denen vor allem Polyole (Sorbitol, myo-Inositol), Methylamine (Betain, α-Glycerophosphocholin) sowie Aminos{\"a}uren (α- bzw. β-Alanin und Prolin) und deren Derivate (Taurin) z{\"a}hlen. Da SOOs weder die zellul{\"a}re Struktur noch die Funktion von Makromolek{\"u}len beeintr{\"a}chtigen, sind sie wichtige Instrumente der Volumenregulation, die sich in hohen Konzentrationen im Zytosol nahezu aller Zellen wiederfinden. Werden tierische Zellen mit hypotonen Bedingungen konfrontiert, dann ist bei nahezu allen Zellen die Freisetzung organischer Osmolyte zu beobachten, wodurch die zellul{\"a}re Osmolarit{\"a}t unabh{\"a}ngig von Elektrolyten angepasst werden kann. Trotz der wichtigen Funktion der SOOs in der Osmoregulation tierischer Zellen konnte die molekulare Identit{\"a}t beteiligter Effluxwege (Kan{\"a}le bzw. Transporter) bisher nicht aufgekl{\"a}rt werden. Ungeachtet der molekularen Identit{\"a}t der SOO-Effluxwege war es aus zahlreichen biotechnologischen Anwendungen zu Beginn dieser Arbeit bekannt, dass die schwellungsaktivierten Transportwege f{\"u}r organische Osmolyte eine gr{\"o}ßenselektive Permeabilit{\"a}t f{\"u}r eine Reihe monomerer Zucker und verwandter Verbindungen aufweisen. Um diese Gr{\"o}ßenselektivit{\"a}t n{\"a}her zu charakterisieren, wurde im ersten Schritt die schwellungsaktivierte Membranpermeabilit{\"a}t f{\"u}r eine Reihe strukturell homogener Polyethylenglykole unterschiedlicher Polymerl{\"a}nge (PEG200-1500; hydrodynamische Radien zwischen ~0,5-1,5 nm) unter iso- und hypotonen Bedingungen in Jurkat-Lymphozyten untersucht. Unter milden hypotonen Bedingungen (200 mOsm) war die Plasmamembran der untersuchten Lymphozyten f{\"u}r PEG300-1500 undurchl{\"a}ssig, was aus der F{\"a}higkeit der Zellen zur hypotonen Volumenregulation geschlossen werden konnte. Dar{\"u}ber hinaus wurde RVD in stark hypotonen L{\"o}sungen (100 mOsm) mit PEG600-1500 beobachtet, w{\"a}hrend PEG300-400 unter vergleichbaren osmotischen Bedingungen die Volumenregulation der Zellen inhibierten. Dieses Ergebnis deutet darauf hin, dass starkes hypotones Zellschwellen der Lymphozyten zur Permeabilisierung der Plasmamembran f{\"u}r PEG300-400, nicht jedoch f{\"u}r PEG600-1500, f{\"u}hrt. Anhand der hydrodynamischen Radien Rh der verwendeten PEGs konnte ein cutoff-Radius von ~0,74 nm f{\"u}r schwellungsaktivierte Transportwege organischer Osmolyte bestimmt werden. Da diese schwellungsaktivierten Transportwege vielf{\"a}ltig f{\"u}r Zellbeladungstechniken verwendet werden, k{\"o}nnte dieses Ergebnis f{\"u}r zahlreiche biotechnologische und biomedizinische Anwendungen von Interesse sein. Im zweiten Schritt wurde der Versuch unternommen, potentielle Transportwege f{\"u}r organische Osmolyte im RVD-Prozess molekular zu identifizieren. Da es grundlegend ungekl{\"a}rt war, wie viele unterschiedliche Transporter bzw. Kan{\"a}le am Efflux der zahlreichen organischen Osmolyte beteiligt sind, erfolgte zun{\"a}chst die vergleichende Analyse des schwellungsaktivierten Membrantransports strukturell verschiedener SOOs einschließlich der Aminosulfons{\"a}ure Taurin und des Polyols myo-Inositol. Hierbei wurde erstmals gezeigt, dass die schwellungsaktivierten Transportwege f{\"u}r Taurin und myo-Inositol deutlich unterschiedliche Aktivit{\"a}tsprofile aufweisen. W{\"a}hrend der Taurintransport bereits unter milden hypotonen Bedingungen, d.h. nach einer geringen Absenkung der Osmolalit{\"a}t von 300 auf ~230 mOsm, aktiviert wurde, erfolgte die Aktivierung der Membranpermeabilit{\"a}t f{\"u}r myo-Inositol bei einer viel niedrigeren Osmolalit{\"a}t von ~150 mOsm. Dar{\"u}ber hinaus wiesen die beiden Transportwege unter vergleichbarem hypotonen Stress von 100 mOsm deutlich unterschiedliche Aktivit{\"a}tsdauern auf (Transport von Taurin ~95 min und myo-Inositol ~40 min). Somit deuteten diese Ergebnisse erstmals auf substrat-spezifische Transportwege f{\"u}r SOOs hin, die voneinander stark abweichende osmotische Aktivierungsprofile besitzen. Als aussichtsreiche Kandidaten f{\"u}r diese Transportwege wurden zwei Mitglieder der Gruppe der Solute Carrier (SLC) untersucht, die klare {\"U}bereinstimmungen mit den gesuchten Transportern f{\"u}r SOOs aufweisen. Daher wurde im Weiteren eine RVD-Beteiligung dieser Transportergruppe mit einer Kombination aus molekularbiologischer und konventioneller bzw. hochaufgel{\"o}ster mikroskopischen Techniken {\"u}berpr{\"u}ft. Die semiqantitativen RT-PCR-Ergebnisse dieser Arbeit zeigen dabei, dass die Gentranskription der potentiellen SOO-Transporter SLC5A3 und SLC6A6 in den untersuchten Zelllinien Jurkat, HEK wie auch HepG2-Zellen durch hypotone Bedingungen deutlich verst{\"a}rkt wird. Hierbei nimmt der zellul{\"a}re mRNA-Gehalt der Gene SLC5A3 zwischen 20-60\% und SLC6A6 um 30-100\% innerhalb von 10-20 min zu, was auf eine potentielle RVD-Beteiligung von SLC-Transportern hindeutet. Ausgehend von diesem Ergebnis wurde daraufhin die zellul{\"a}re Lokalisation des SLC5A3-Transporters unter isotonen und hypotonen Bedingungen mikroskopisch untersucht. Wie anhand der konfokalen lasermikroskopischen Untersuchung zu erkennen ist, findet unter hypotoner Stimulation eine zellul{\"a}re Umverteilung des mit EGFP fluoreszenzmarkierten Proteins SLC5A3 statt. Innerhalb von 10 min wird der Transporter dabei von intrazellul{\"a}ren Regionen in Richtung Plasmamembran verlagert. Dar{\"u}ber hinaus konnte mit Hilfe der hochaufl{\"o}senden Mikroskopie-Technik dSTORM gezeigt werden, dass der Transporter SLC5A3 unter hypotoner Stimulation verst{\"a}rkt mit der Plasmamembran assoziiert vorliegt. Diese verst{\"a}rkte Membranassoziation des SLC5A3-Proteins deutet damit auf einen schwellungsinduzierten exozytotischen Einbau des Transporters hin. Die Ergebnisse dieser Arbeit zeigen damit erstmals, dass SLC-Transporter wie SLC5A3, SLC6A6 und vermutlich andere Vertreter der SLC-Superfamilie potentiell am Mechanismus der hypotonen Volumenregulation beteiligt sind. Da SLC-Transporter als wichtige Transportsysteme f{\"u}r Therapeutika angesehen werden und die Mechanismen der Volumenregulation bereits in zahlreichen biotechnologischen Anwendungen implementiert sind, k{\"o}nnte der hier aufgedeckte Zusammenhang einen Erkenntnisgewinn f{\"u}r zahlreiche biomedizinische Forschungsgebiete darstellen.},
  subject      = {S{\"a}ugetiere},
  language  = {de}
}
@phdthesis{Simann2015,
  author    = {Simann, Meike},
  title     = {Aufkl{\"a}rung der Effekte von Fibroblasten-Wachstumsfaktor 1 und 2 auf die Adipogenese und Osteogenese von prim{\"a}ren humanen Knochenmark-Stroma-Zellen},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-119322},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {Regulating and reverting the adipo-osteogenic lineage decision of trabecular human bone marrow stromal cells (hBMSCs) represents a promising approach for osteoporosis therapy and prevention. Fibroblast growth factor 1 (FGF1) and its subfamily member FGF2 were scored as lead candidates to exercise control over lineage switching processes (conversion) in favor of osteogenesis previously. However, their impact on differentiation events is controversially discussed in literature. Hence, the present study aimed to investigate the effects of these FGFs on the adipogenic and osteogenic differentiation and conversion of primary hBMSCs. Moreover, involved downstream signaling mechanisms should be elucidated and, finally, the results should be evaluated with regard to the possible therapeutic approach. This study clearly revealed that culture in the presence of FGF1 strongly prevented the adipogenic differentiation of hBMSCs as well as the adipogenic conversion of pre-differentiated osteoblastic cells. Lipid droplet formation was completely inhibited by a concentration of 25 ng/µL. Meanwhile, the expression of genetic markers for adipogenic initiation, peroxisome proliferator-activated receptor gamma 2 (PPARg2) and CCAAT/enhancer binding protein alpha (C/EBPa), as well as subsequent adipocyte maturation, fatty acid binding protein 4 (FABP4) and lipoprotein lipase (LPL), were significantly downregulated. Yet, the genetic markers of osteogenic commitment and differentiation were not upregulated during adipogenic differentiation and conversion under FGF supplementation, not supporting an event of osteogenic lineage switching. Moreover, when examining the effects on the osteogenic differentiation of hBMSCs and the osteogenic conversion of pre-differentiated adipocytic cells, culture in the presence of FGF1 markedly decreased extracellular matrix (ECM) mineralization. Additionally, the gene expression of the osteogenic marker alkaline phosphatase (ALP) was significantly reduced and ALP enzyme activity was decreased. Furthermore, genetic markers of osteogenic commitment, like the master regulator runt-related transcription factor 2 (RUNX2) and bone morphogenetic protein 4 (BMP4), as well as markers of osteogenic differentiation and ECM formation, like collagen 1 A1 (COL1A1) and integrin-binding sialoprotein (IBSP), were downregulated. In contrast, genes known to inhibit ECM mineralization, like ANKH inorganic pyrophosphate transport regulator (ANKH) and osteopontin (OPN), were upregulated. ANKH inhibition revealed that its transcriptional elevation was not crucial for the reduced matrix mineralization, perhaps due to decreased expression of ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) that likely annulled ANKH upregulation. Like FGF1, also the culture in the presence of FGF2 displayed a marked anti-adipogenic and anti-osteogenic effect. The FGF receptor 1 (FGFR1) was found to be crucial for mediating the described FGF effects in adipogenic and osteogenic differentiation and conversion. Yet, adipogenic conversion displayed a lower involvement of the FGFR1. For adipogenic differentiation and osteogenic differentiation/conversion, downstream signal transduction involved the extracellular signal-regulated kinases 1 and 2 (ERK1/2) and the mitogen-activated protein kinase (MAPK)/ERK kinases 1 and 2 (MEK1/2), probably via the phosphorylation of FGFR docking protein FGFR substrate 2a (FRS2a) and its effector Ras/MAPK. The c-Jun N-terminal kinase (JNK), p38-MAPK, and protein kinase C (PKC) were not crucial for the signal transduction, yet were in part responsible for the rate of adipogenic and/or osteogenic differentiation itself, in line with current literature. Taken together, to the best of our knowledge, our study was the first to describe the strong impact of FGF1 and FGF2 on both the adipogenic and osteogenic differentiation and conversion processes of primary hBMSCs in parallel. It clearly revealed that although both FGFs were not able to promote the differentiation and lineage switching towards the osteogenic fate, they strongly prevented adipogenic differentiation and lineage switching, which seem to be elevated during osteoporosis. Our findings indicate that FGF1 and FGF2 entrapped hBMSCs in a pre-committed state. In conclusion, these agents could be applied to potently prevent unwanted adipogenesis in vitro. Moreover, our results might aid in unraveling a pharmacological control point to eliminate the increased adipogenic differentiation and conversion as potential cause of adipose tissue accumulation and decreased osteoblastogenesis in bone marrow during aging and especially in osteoporosis.},
  subject      = {Mesenchymzelle},
  language  = {en}
}
@phdthesis{Imes2016,
  author    = {Imes, Dennis},
  title     = {Aufkl{\"a}rung der molekularen Struktur und Funktion des R-Typ Anionenkanals QUAC1 in Schließzellen},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-136860},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {Zum Gasaustausch mit Ihrer Umgebung besitzen h{\"o}here Pflanzen stomat{\"a}re Komplexe. Die Turgor-getrieben Atmungs{\"o}ffnungen in der Epidermis der Bl{\"a}tter werden von zwei Schließzellen ums{\"a}umt. Um bei Trockenheit einen exzessiven Verlust von Wasser zu verhindern, synthetisieren/importieren Schließzellen das Stresshormon ABA (Abszisins{\"a}ure), das {\"u}ber eine schnelle ABA-Signalkaskade plasmamembrangebundene Ionenkan{\"a}le steuert. Dabei wird der Stomaschluss durch die Aktivit{\"a}t von R-(rapid) und S-(slow)Typ Anionenkan{\"a}len initiiert. Obwohl die R- und S-Typ Anionenstr{\"o}me in Schließzellen seit Jahrzehnten bekannt waren, konnte erst k{\"u}rzlich das Gen identifiziert werden, das f{\"u}r den S-Typ Anionenkanal (SLAC1, Slow activating Anion Channel 1) kodiert. Daraufhin wurde schnell der Zusammenhang zwischen dem Stresshormon ABA, der ABA-Signalkette und der Aktivit{\"a}t des SLAC1 Anionenkanals im heterologen Expressionssystem der X. laevis Oozyten als auch in Schließzellprotoplasten aufgekl{\"a}rt. Es konnte gezeigt werden, dass ABA durch einen zytosolischen Rezeptor/Phosphatasekomplex (RCAR1/ABI1) erkannt wird und die Aktivit{\"a}t von kalziumabh{\"a}ngigen Kinasen (CPK-Familie) sowie kalziumunabh{\"a}ngigen Kinasen der SnRK2-Familie (OST1) steuert. In Anwesenheit von ABA phosphorylieren diese Kinasen SLAC1 und sorgen so f{\"u}r die Aktivierung von Anionenstr{\"o}men und damit f{\"u}r die Initiierung des Stomaschlusses. Die genetische Herkunft der ABA-induzierten R-Typ Str{\"o}me in Schließzellen war zu Beginn der vorliegenden Arbeit noch nicht bekannt. R-Typ Str{\"o}me zeichnen sich durch eine strikte Spannungsabh{\"a}ngigkeit und sehr schnellen Aktivierungs- sowie Deaktivierungskinetiken aus. Die Charakterisierung von Verlustmutanten des Schließzell-exprimierten Gens ALMT12 (Aluminium-aktivierter Malattransporter 12) konnte in Zusammenarbeit mit der Arbeitsgruppe Martinoia (Z{\"u}rich) erste Hinweise auf die Beteiligung dieses Gens an der Stomabewegung demonstrieren. Anschließende Patch-Clamp Untersuchungen an Schließzellprotoplasten aus Wildtyppflanzen und ALMT12-Verlustmutanten zeigten, dass ALMT12 f{\"u}r die Malat-aktivierte R-Typ Anionenstromkomponente verantwortlich ist. Deshalb wurde der Anionenkanal QUAC1 (Quickly activating Anion Channel 1) benannt - in Anlehnung an die Benennung des Anionenkanals SLAC1. Mit der Identifizierung von QUAC1 in planta war es nun meine Aufgabe, die elektrischen Eigenschaften von ALMT12/QUAC1 und dessen Aktivit{\"a}tskontrolle durch die ABA-Signalkaskade im heterologen Expressionssystem der Xenopus Oozyten zu untersuchen. Protein-Protein Interaktionsstudien mit der Hilfe der Bimolekularen Fluoreszenz-Technik, sowie die Beobachtung von markant erh{\"o}hten QUAC1 Anionenstr{\"o}men in Anwesenheit der SnRK2 Kinase OST1 und den Calcium-abh{\"a}ngigen Kinasen CPK2 und CPK20, ließen den Schluss zu, dass QUAC1, ebenso wie SLAC1, unter der Kontrolle des schnellen ABA-Signalwegs steht. Eine zus{\"a}tzliche Expression des negativen Regulators ABI1 unterdr{\"u}ckte die aktivierenden Eigenschaften der QUAC1-aktivierenden Kinasen, was die Hypothese der Koregulation von S- und R-Typ Anionenkan{\"a}len durch die gleiche ABA-Signalkaskade weiter unterst{\"u}tzt. Zur weiteren Aufkl{\"a}rung der elektrischen Eigenschaften von QUAC1 wurden tiefgreifende elektrophysiologische Untersuchungen mit der Zwei-Elektroden-Spannungsklemmen Technik durchgef{\"u}hrt. Durch die Wahl von geschickten Spannungsprotokollen konnte sowohl die schnelle Aktivierungskinetik als auch die schnelle Deaktivierungskinetik von QUAC1 bestimmt und quantifiziert werden. Diese Stromantworten waren sehr {\"a}hnlich zu den R-Typ Str{\"o}men, die man von Patch-Clamp Untersuchungen an Schließzellprotoplasten kannte, was ein weiteres Indiz daf{\"u}r war, dass es sich bei QUAC1 tats{\"a}chlich um eine Komponente des R-Typ Kanals aus Schließzellen handelt. Weiterf{\"u}hrende Untersuchungen bez{\"u}glich der Spannungsabh{\"a}ngigkeit und der Selektivit{\"a}t von QUAC1 charakterisierten das Protein als einen Depolarisations-aktivierten Anionenkanal mit einer starken Pr{\"a}ferenz f{\"u}r Dicarbons{\"a}uren wie Malat und Fumarat. Zudem konnte auch eine Leitf{\"a}higkeit f{\"u}r Sulfat und Chlorid nachgewiesen werden. Interessanterweise erwies sich Malat nicht nur als ein permeierendes Ion, sondern auch als ein regulierendes Ion, welches das spannungsabh{\"a}ngige Schalten von QUAC1 maßgeblich beeinflusst. Extrazellul{\"a}res Malat verschob die Offenwahrscheinlichkeit von QUAC1 sehr stark zu negativeren Membranspannungen, so dass der Anionenkanal bereits bei typischen Ruhespannungen von Schließzellen (ca. -150 mV) aktiviert werden konnte. Eine Beladung von QUAC1-exprimierender Oozyten mit Malat bewirkte zum einen h{\"o}here Anioneneffluxstr{\"o}me, aber auch eine Verschiebung der spannungsabh{\"a}ngigen Offenwahrscheinlichkeit zu negativeren Membranpotentialen. Struktur-Funktionsanalysen sollten die umstrittene Topologie von ALMT-{\"a}hnlichen Proteinen beleuchten und die molekulare Herkunft der Phosphorylierungsaktivierung aufzeigen, sowie die Malatabh{\"a}ngigkeit und die starke Spannungsabh{\"a}ngigkeit von QUAC1 aufkl{\"a}ren. Es zeigte sich jedoch schnell, dass Punktmutationen und Deletionen im C-Terminus von QUAC1 sehr h{\"a}ufig zu nicht-funktionellen Mutanten f{\"u}hrten. Diese Tatsache weist darauf hin, dass es sich um einen hoch-strukturierten und funktionell sehr wichtigen Bereich des Anionenkanals handelt. Auch die Topologie des Anionenkanalproteins wird in der Literatur kontrovers diskutiert. Sowohl die Lage des N- und C-Terminus (extrazellul{\"a}r oder intrazellul{\"a}r), als auch die Anzahl der membrandurchspannenden Dom{\"a}nen war nicht abschließend gekl{\"a}rt. Deshalb wurde in einem Fluoreszenz-basiertem Ansatz die Lage der Termini bestimmt. Im Rahmen meiner Arbeit konnte somit eindeutig gezeigt werden, dass sich beide Termini im Zytosol der Zelle befinden. Auf Grundlage von Modellen aus der Literatur und meiner Topologiebestimmungen konnte schließlich ein erweitertes Modell zur Struktur von QUAC1 entwickelt werden. Dieses Modell kann in Zukunft als Ausgangspunkt f{\"u}r weiterf{\"u}hrende Struktur-Funktionsanalysen dienen. Diese Arbeit hat somit gezeigt, dass das Gen QUAC1 tats{\"a}chlich eine Komponente der R-Typ Str{\"o}me in Schließzellen kodiert. Ebenso wie SLAC1 steht der Malat-induzierte Anionenkanal QUAC1 unter der Kontrolle der schnellen ABA-Signalkaskade. In Zukunft bleibt zu kl{\"a}ren, welche weiteren Gene f{\"u}r die R-Typ Kanalproteine in Schließzellen kodieren und welche strukturelle Grundlage f{\"u}r die besonderen Eigenschaften von QUAC1 hinsichtlich seiner schnellen Kinetiken, seiner Selektivit{\"a}t und Aktivierbarkeit durch Malat.},
  subject      = {Ackerschmalwand},
  language  = {de}
}
@phdthesis{Loeschberger2014,
  author    = {L{\"o}schberger, Anna},
  title     = {Biologische Referenzstrukturen und Protokolloptimierung in der hochaufl{\"o}senden Fluoreszenzmikroskopie mit dSTORM},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-102630},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {Die Lokalisationsmikroskopie ist eine neue, vielversprechende Methode der hochaufl{\"o}senden Fluoreszenzmikroskopie. Sie erm{\"o}glicht detaillierte Einblicke in die Organisation und den strukturellen Aufbau von Zellen. Da die Vorbereitung der Proben und das Aufnehmen der Bilder im Vergleich zu herk{\"o}mmlichen Methoden h{\"o}here Anforderungen stellt, mussten ihr Potential und ihre Zuverl{\"a}ssigkeit erst noch {\"u}berzeugend gezeigt werden. Bis vor kurzem wurde das Aufl{\"o}sungsverm{\"o}gen vor allem an Mikrotubuli gezeigt, deren filament{\"o}se Struktur allerdings schon in konfokalen Bildern zu erkennen ist. Deswegen wurde in dieser Dissertation der Kernporenkomplex (NPC), dessen Struktur in der konventionellen Fluoreszenzmikroskopie nicht aufl{\"o}sbar ist, als Modellstruktur f{\"u}r die hochaufl{\"o}sende Fluoreszenzmikroskopie eingef{\"u}hrt. Dazu wurden Kernporenkomplexe aus Kernh{\"u}llen von Xenopus laevis Oocyten mit dSTORM (direct stochastic optical reconstruction microscopy), einer Methode der Lokalisationsmikroskopie, hochaufgel{\"o}st. Damit konnte nun erstmals die Achtfachsymmetrie dieses Proteinkomplexes lichtmikroskopisch dargestellt werden. Desweiteren konnte der Zentralkanal mit einem Durchmesser von ca. 40 nm aufgel{\"o}st werden. Die Daten eigneten sich außerdem f{\"u}r eine automatisierte Bildanalyse nach dem sogenannten "particle averaging" - einer aus der Elektronenmikroskopie bekannten Methode, um eine Durchschnittsstruktur zu ermitteln. Dar{\"u}ber hinaus wurden Zweifach-F{\"a}rbungen von NPCs benutzt, um verschiedene Ans{\"a}tze f{\"u}r Zweifarben-Aufnahmen mit dSTORM zu testen. Neben dem mittlerweile standardm{\"a}ßig benutzten, sequentiellen Ansatz mit zwei spektral getrennten Farbstoffen, wurde auch ein simultaner Ansatz mit zwei spektral {\"u}berlappenden Farbstoffen erfolgreich angewandt. Auch f{\"u}r 3D-Messungen mit den Ans{\"a}tzen Biplane und Astigmatismus eignete sich die Markierung der Kernh{\"u}lle. Hier wurden jedoch A6-Zellen benutzt und die Kr{\"u}mmung des Zellkerns {\"u}ber die gef{\"a}rbten Kernporen dargestellt. dSTORM-Messungen k{\"o}nnen nicht nur an fixierten, sondern auch in lebenden Zellen durchgef{\"u}hrt werden. Hierzu eignen sich vor allem sehr immobile Proteine, wie H2B oder Lamin C. Anhand von SNAP-Tag- und Halo-Tag-Konstrukten konnte gezeigt werden, dass sich kommerziell erh{\"a}ltliche, organische Farbstoffe auch in endogener zellul{\"a}rer Umgebung schalten lassen, wodurch Lebendzell-Aufnahmen mit dSTORM m{\"o}glich sind. Ein weiterer Teil dieser Arbeit befasst sich mit korrelativen Aufnahmen aus dSTORM und Rasterelektronenmikroskopie (SEM). Hierzu wurden Xenopus laevis Kernh{\"u}llen zuerst mit dSTORM hochaufgel{\"o}st und danach f{\"u}r die EM pr{\"a}pariert. Anschließend wurden zugeh{\"o}rige Bereiche am Rasterelektronenmikroskop aufgenommen. Mit den erhaltenen korrelativen Bildern konnte gezeigt werden, dass sich dSTORM und SEM bei geeigneten Proben durchaus kombinieren lassen. Proteine k{\"o}nnen somit spezifisch markiert und im Rahmen ihrer strukturellen Umgebung mit nahezu molekularer Aufl{\"o}sung dargestellt werden. Da hochwertige Aufnahmen eine ausgereifte Probenpr{\"a}paration voraussetzen, darf deren Etablierung nicht zu kurz kommen. Unter dieser Pr{\"a}misse wurde ein optimiertes Markierungsprotokoll mit dem Namen ClickOx entwickelt. Mit ClickOx bleibt bei der kupferkatalysierten Azid-Alkin-Cycloaddition die Feinstruktur von Aktinfilamenten, sowie die Fluoreszenz fluoreszierender Proteine, deutlich sichtbar erhalten. W{\"a}hrend bei den klassischen Click-Protokollen auf Grund der Entstehung von reaktiven Sauerstoff-Spezies (ROS) feine zellul{\"a}re Strukturen, wie Aktinfilamente, angegriffen oder zerst{\"o}rt werden, sch{\"u}tzt das neue Protokoll mit enzymatischem Sauerstoffentzug Proteine und somit Strukturen vor Reaktionen mit ROS. Das unterstreicht, wie wichtig es ist auch sogenannte "etablierte" Protokolle weiterzuentwickeln, denn bestimmte Nebeneffekte in Pr{\"a}parationen werden unter Umst{\"a}nden erstmals in der Hochaufl{\"o}sung sichtbar. Ein weiterer Aspekt war die Untersuchung des Einflusses von D1 auf die Chromatinorganisation. Mit verschiedenen mikroskopischen Methoden konnten Hinweise auf eine m{\"o}gliche DNA-Cross-Linking-F{\"a}higkeit dieses Proteins gesammelt werden. Hier wurde die Einzelmolek{\"u}linformation der dSTORM-Filme genutzt, um unterschiedliche Grade von DNA- bzw. Chromatin-Akkumulation zu vergleichen. Die Ergebnisse deuten darauf hin, dass wildtypisches D1 DNA vernetzen kann. Dies erfolgt {\"u}ber die sogenannten AT-Haken-Motive. Sobald diese alle durch Mutation funktionsunf{\"a}hig gemacht werden - wie bei der verwendeten R10xG-Mutante - l{\"a}sst sich keine Akkumulation der DNA mehr beobachten. Neben der Chromatinaggregation durch D1-Expression konnte in FRAP-Experimenten gezeigt werden, dass nur die "echten" AT-Haken eine hohe Affinit{\"a}t zum Chromatin aufweisen, die sogenannten "potentiellen" hingegen nicht.},
  subject      = {Fluoreszenzmikroskopie},
  language  = {de}
}
@phdthesis{Eck2016,
  author    = {Eck, Saskia},
  title     = {The impact of thermogenetic depolarizations of specific clock neurons on Drosophila melanogaster's circadian clock},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-137118},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {The rotation of the earth around its own axis determines periodically changing environmental conditions, like alterations in light and temperature. For the purpose of adapting all organisms' behavior, physiology and metabolism to recurring changes, endogenous clocks have evolved, which allow the organisms to anticipate environmental changes. In chronobiology, the scientific field dealing with the investigation of the underlying mechanisms of the endogenous clock, the fruit fly Drosophila melanogaster serves as a beneficial model organism. The fruit fly's circadian clock exhibits a rather simple anatomical organization, but nevertheless constitutes homologies to the mammalian system. Thus also in this PhD-thesis the fruit fly was used to decipher general features of the circadian clock's interneuronal communication. Drosophila melanogaster's circadian clock consists of about 150 clock neurons, which are located in the central nervous system of the fly. These clock neurons can be subdivided regarding to their anatomical position in the brain into the dorsal neurons (DN1s, DN2s, DN3s), as well as into the lateral neurons (LPNs, LNds, s-LNvs, l-LNvs). Functionally these clock neuron clusters can be classified as Morning- and Evening oscillators (M- and E- oscillators), driving different parts of the fly's locomotor activity in light-dark conditions (LD). The Morning-oscillators are represented by the s-LNvs and are known to be the main pacemakers, driving the pace of the clock in constant conditions (constant darkness; DD). The group of Evening-oscillators consists of the LNds, the DN1s and the 5th s-LNv and is important for the proper timing of the evening activity in LD. All of these clock neurons are not functionally independent, but form complex neuronal connections, which are highly plastic in their response to different environmental stimuli (Zeitgebers), like light or temperature. Even though a lot is known about the function and the importance of some clock neuron clusters, the exact interplay between the neurons is not fully known yet. To investigate the mechanisms, which are involved in communication processes among different clock neurons, we depolarized specific clock cells in a temporally and cell-type restricted manner using dTrpA1, a thermosensitive cation channel, which allows the depolarization of neurons by application of temperature pulses (TP) above 29°C to the intact and freely moving fly. Using different clock specific GAL4-driver lines and applying TPs at different time points within the circadian cycle in DD enabled us with the help of phase shift experiments to draw conclusions on the properties of the endogenous clock. The obtained phase shifts in locomotor behavior elicited by specific clock neuronal activation were plotted as phase response curves (PRCs). The depolarization of all clock neurons shifted the phase of activity the strongest, especially in the delay zone of the PRC. The exclusive depolarization of the M oscillators together with the l-LNvs (PDF+ neurons: s-LNvs \& l-LNvs) caused shifts in the delay and in the advance zone as well, however the advances were severely enhanced in their temporal occurrence ranging into the subjective day. We concluded that light might have inhibitory effects on the PDF+ cells in that particular part of the PRC, as typical light PRCs do not exhibit that kind of distinctive advances. By completely excluding light in the PRC-experiments of this PhD-thesis, this photic inhibitory input to the PDF+ neurons is missing, probably causing the broadened advance zone. These findings suggest the existence of an inhibitory light-input pathway to the PDF+ cells from the photoreceptive organs (Hofbauer-Buchner eyelet, photoreceptor cells of compound eyes, ocelli) or from other clock neurons, which might inhibit phase advances during the subjective day. To get an impression of the molecular state of the clock in the delay and advance zone, staining experiments against Period (PER), one of the most important core clock components, and against the neuropeptide Pigment Dispersing Factor (PDF) were performed. The cycling of PER levels mirrored the behavioral phase shifts in experimental flies, whereas the controls were widely unaffected. As just those neurons, which had been depolarized, exhibited immediate shifted PER oscillations, this effect has to be rapidly regulated in a cell-autonomous manner. However, the molecular link between clock neuron depolarization and shifts in the molecular clock's cycling is still missing. This issue was addressed by CREB (cAMP responsive element binding protein) quantification in the large ventrolateral neurons (l-LNvs), as these neurons responded unexpectedly and strongest to the artificial depolarization exhibiting a huge increase in PER levels. It had been previously suggested that CREB is involved in circadian rhythms by binding to regulatory sequences of the period gene (Belvin et al., 1999), thus activating its transcription. We were able to show, that CREB levels in the l-LNvs are under circadian regulation, as they exhibit higher CREB levels at the end of the subjective night relative to the end of the subjective day. That effect was further reinforced by artificial depolarization, independently of the time point of depolarization. Furthermore the data indicate that rises in CREB levels are coinciding with the time point of increases of PER levels in the l-LNvs, suggesting CREB being the molecular link between the neuronal electrical state and the molecular clock. Taking together, the results indicate that a temporal depolarization using dTrpA1 is able to significantly phase shift the clock on the behavioral and protein level. An artificial depolarization at the beginning of the subjective night caused phase delays, whereas a depolarization at the end of the subjective night resulted in advances. The activation of all clock neurons caused a PRC that roughly resembled a light-PRC. However, the depolarization of the PDF+ neurons led to a PRC exhibiting a shape that did not resemble that of a light-mediated PRC, indicating the complex processing ability of excitatory and inhibitory input by the circadian clock. Even though this experimental approach is highly artificial, just the exclusion of light-inputs enabled us to draw novel conclusions on the network communication and its light input pathways.},
  subject      = {Chronobiologie},
  language  = {en}
}
@phdthesis{Ullrich2014,
  author    = {Ullrich, Melanie},
  title     = {Identification of SPRED2 as a Novel Regulator of Hypothalamic-Pituitary-Adrenal Axis Activity and of Body Homeostasis},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-107355},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {SPRED proteins are inhibitors of the Ras/ERK/MAPK signaling pathway, an evolutionary highly conserved and very widespread signaling cascade regulating cell proliferation, differentiation, and growth. To elucidate physiological consequences of SPRED2 deficiency, SPRED2 KO mice were generated by a gene trap approach. An initial phenotypical characterization of KO mice aged up to five months identified SPRED2 as a regulator of chondrocyte differentiation and bone growth. Here, the loss of SPRED2 leads to an augmented FGFR-dependent ERK activity, which in turn causes hypochondroplasia-like dwarfism. However, long term observations of older KO mice revealed a generally bad state of health and manifold further symptoms, including excessive grooming associated with severe self-inflicted wounds, an abnormally high water uptake, clear morphological signs of kidney deterioration, and a reduced survival due to sudden death. Based on these observations, the aim of this study was to discover an elicitor of this complex and versatile phenotype. The observed kidney degeneration in our SPRED2 KO mice was ascribed to hydronephrosis characterized by severe kidney atrophy and apoptosis of renal tubular cells. Kidney damage prompted us to analyze drinking behavior and routine serum parameters. Despite polydipsia, which was characterized by a nearly doubled daily water uptake, the significantly elevated Na+ and Cl- levels and the resulting serum hyperosmolality could not be compensated in SPRED2 KOs. Since salt and water balance is primarily under hormonal control of aldosterone and AVP, we analyzed both hormone levels. While serum AVP was similar in WTs and KOs, even after experimental water deprivation and an extreme loss of body fluid, serum aldosterone was doubled in SPRED2 KO mice. Systematic investigation of contributing upstream hormone axes demonstrated that hyperaldosteronism developed independently of an overactivated Renin-Angiotensin system as indicated by halved serum Ang II levels in KO mice. However, aldosterone synthase expression in the adrenal gland was substantially augmented. Serum corticosterone, which is like aldosterone released from the adrenal cortex, was more than doubled in SPRED2 KOs, too. Similar to corticosterone, the production of aldosterone is at least in part under control of pituitary ACTH, which is further regulated by upstream hypothalamic CRH release. In fact, stress hormone secretion from this complete hypothalamic-pituitary-adrenal axis was upregulated because serum ACTH, the mid acting pituitary hormone, and hypothalamic CRH, the upstream hormonal inductor of HPA axis activity, were also elevated by 30\% in SPRED2 KO mice. This was accompanied by an upregulated ERK activity in paraventricular nucleus-containing hypothalamic brain regions and by augmented hypothalamic CRH mRNA levels in our SPRED2 KO mice. In vitro studies using the hypothalamic cell line mHypoE-44 further demonstrated that both SPRED1 and SPRED2 were able to downregulate CRH promoter activity, CRH secretion, and Ets factor-dependent CRH transcription. This was in line with the presence of various Ets factor binding sites in the CRH promoter region, especially for Ets1. Thus, this study shows for the first time that SPRED2-dependent inhibition of Ras/ERK/MAPK signaling by suppression of ERK activity leads to a downregulation of Ets1 factor-dependent transcription, which further results in inhibition of CRH promoter activity, CRH transcription, and CRH release from the hypothalamus. The consecutive hyperactivity of the complete HPA axis in our SPRED2 KO mice reflects an elevated endogenous stress response becoming manifest by excessive grooming behavior and self-inflicted skin lesions on the one hand; on the other hand, in combination with elevated aldosterone synthase expression, this upregulated HPA hormone release explains hyperaldosteronism and the associated salt and water imbalances. Both hyperaldosteronism and polydipsia very likely contribute further to the observed kidney damage. Taken together, this study initially demonstrates that SPRED2 is essential for the appropriate regulation of HPA axis activity and of body homeostasis. To further enlighten and compare consequences of SPRED2 deficiency in mice and particularly in humans, two follow-up studies investigating SPRED2 function especially in heart and brain, and a genetic screen to identify human SPRED2 loss-of-function mutations are already in progress.},
  subject      = {Renin-Angiotensin-System},
  language  = {en}
}
@phdthesis{AppeltMenzel2016,
  author    = {Appelt-Menzel, Antje},
  title     = {Etablierung und Qualifizierung eines humanen Blut-Hirn-Schranken-Modells unter Verwendung von induziert pluripotenten und multipotenten Stammzellen},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-134646},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {Die Blut-Hirn-Schranke (BHS) stellt eine der dichtesten und wichtigsten Barrieren zwischen Blutzirkulation und Zentralnervensystem (ZNS) dar. Sie besteht aus spezialisierten Endothelzellen, welche die zerebralen Kapillaren auskleiden und durch sehr dichte Tight Junctions (TJs) miteinander verbunden sind. Weitere Komponenten der dynamischen Blut-Hirn-Schrankenbarriere stellen Perizyten, Astrozyten, Neurone und Mikrogliazellen dar, welche zusammen mit der extrazellul{\"a}ren Matrix der Basalmembran der Gehirnkapillaren und den zuvor genannten Endothelzellen ein komplexes regulatorisches System, die so genannte neurovaskul{\"a}re Einheit bilden (Hawkins und Davis 2005). Die Hauptfunktionen der BHS lassen sich in drei Untergruppen untergliedern, die physikalische, metabolische und Transport-Barriere (Neuhaus und Noe 2010). Haupts{\"a}chlich dient die BHS der Aufrechterhaltung der Hom{\"o}ostase des ZNS und dem Schutz vor neurotoxischen Substanzen sowie Pathogenen, wie Bakterien und Viren. Zudem ist sie auch f{\"u}r die Versorgung der Neuronen mit N{\"a}hrstoffen und regulierenden Substanzen sowie den Efflux von Stoffwechselendprodukten des ZNS zur{\"u}ck ins Blut verantwortlich. F{\"u}r die Entwicklung von Medikamenten zur Behandlung von neurodegenerativen Erkrankungen, wie Morbus Alzheimer, Morbus Parkinson und Multiple Sklerose oder Gehirntumoren, stellt die Dichtigkeit der BHS gegen{\"u}ber Substanzen und die hohe metabolische Aktivit{\"a}t der Endothelzellen aber ein großes Problem dar. Viele Medikamente sind nicht in der Lage in ausreichender Konzentration die BHS zu {\"u}berwinden, um an ihren Wirkort zu gelangen oder werden vor dem Transport metabolisiert und die Wirksamkeit dadurch eingeschr{\"a}nkt. Weiterhin spielen auch Defekte der BHS eine entscheidende Rolle in der Beeinflussung der Pathogenese vieler ZNS-Erkrankungen. Aufgrund des hohen Bedarfs an geeigneten Testsystemen in der Grundlagen- sowie pr{\"a}klinischen Forschung f{\"u}r Medikamentenentwicklung und Infektionsstudien wurden eine Vielzahl unterschiedlicher BHS-Modelle entwickelt. Neben in silico-, azellul{\"a}ren in vitro- und in vivo-Modellen sind auch zahlreiche zellbasierte Modelle der BHS entwickelt worden. Standardisierte Modelle auf Basis immortalisierter Zelllinien jedoch weisen nur eine inhomogene TJ-Expression auf und verf{\"u}gen meist {\"u}ber eine geringe Barriereintegrit{\"a}t, erfasst {\"u}ber transendotheliale elektrische Widerst{\"a}nde (TEER) unter 150 · cm2 (Deli et al. 2005). Im Vergleich dazu wurden in Tierexperimenten TEER-Werte von mehr als 1500 · cm2 an der BHS gemessen (Butt et al. 1990; Crone und Olesen 1982). Die Verf{\"u}gbarkeit humaner prim{\"a}rer BHS-Zellen ist sehr limitiert und ihr Einsatz nicht nur im Hinblick auf ethische Aspekte bedenklich. Humane Gehirnzellen k{\"o}nnen z. B. aus Biopsie- oder Autopsiematerial von Patienten mit Epilepsie oder Gehirntumoren isoliert werden. Allerdings besteht hier das Risiko, dass die isolierten Zellen krankheitsbedingt ver{\"a}ndert sind, was die Eigenschaften der BHS-Modelle erheblich beeinflussen kann. Eine Alternative, die diese Probleme umgeht, ist die Verwendung von humanen induziert pluripotenten Stammzellen (hiPSCs), um standardisierte humane BHS-Modelle unter reproduzierbaren Bedingungen bereitzustellen. Im Rahmen dieser Arbeit ist es gelungen, hiPSCs in vitro nach etablierten und standardisierten Methoden in Endothelzellen der BHS, neurale Stammzellen (hiPS-NSCs) sowie Astrozyten (hiPS-A) zu differenzieren (Lippmann et al. 2012; Lippmann et al. 2014; Wilson et al. 2015; Yan et al. 2013;Reinhardt et al. 2013) und zum Aufbau der Modelle einzusetzen. Die Endothelzellen wurden mit Hilfe protein- und genbasierter Nachweismethoden auf das Vorhandensein von endothelzellspezifischen TJ-Markern sowie spezifischen Transportern untersucht und funktionell charakterisiert. Die Kryokonservierung der hiPS-EC-Progenitoren, die im Rahmen der vorliegenden Arbeit entwickelt wurde, erm{\"o}glicht eine gr{\"o}ßere r{\"a}umliche und zeitliche Flexibilit{\"a}t beim Arbeiten mit den stammzellbasierten Modellen sowie das Anlegen standardisierter Zellbanken. Weiterhin wurden multipotente NSCs aus fetalen Gehirnbiopsien isoliert (fNSCs) und als Kontrollkulturen zu den hiPS-NSCs f{\"u}r den Aufbau von BHS-Modellen eingesetzt. Mit dem Ziel die in vivo-BHS bestm{\"o}glich zu imitieren und die Modelleigenschaften zu optimieren, wurde ein Set aus zehn unterschiedlichen BHS-Modellen basierend auf prim{\"a}ren Zellen, hiPSCs und fNSCs analysiert. Der Aufbau der BHS-Modelle erfolgte unter Verwendung von Transwellsystemen. Durch die systematische Untersuchung des Einflusses der unterschiedlichen Zelltypen der neurovaskul{\"a}ren Einheit auf die Barriereintegrit{\"a}t und Genexpression des BHS-Endothels, konnten die Quadrupel-Kulturen mit Perizyten, Astrozyten und hiPS-NSCs als die Kultur mit den physiologischsten Eigenschaften identifiziert werden. Auf Grund der signifikant erh{\"o}hten TEER-Werte von bis zu 2500 · cm2 und einer um mindestens 1,5-fachen Steigerung der Genexpression BHSrelevanter Transporter und TJ-Molek{\"u}le gegen{\"u}ber den Monokulturen, wurden diese Modelle f{\"u}r weiterf{\"u}hrende Studien ausgew{\"a}hlt. Das Vorhandensein eines komplexen, in vivo-{\"a}hnlichen TJ-Netzwerkes, bestehend aus Occludin, Claudin 1, 3, 4 und 5, konnte mittels quantitativer Realtime-PCR, Western Blot sowie ultrastruktureller Analyse in der Gefrierbruch- und Raster-Elektronenmikroskopie nachgewiesen werden. Neben der Begrenzung der parazellul{\"a}ren Permeabilit{\"a}t, welche {\"u}ber die geringe Permeation von FITC-Dextran (4 kDa und 40 kDa), Fluoreszein und Lucifer Yellow nachgewiesen wurde, stellt die BHS ebenfalls eine Barriere f{\"u}r den transzellul{\"a}ren Transport von Substanzen dar. Eine Beurteilung der Modelle hinsichtlich der Qualifikation f{\"u}r die Nutzung im Wirkstoffscreening wurde mit Hilfe von Transportversuchen unter dem Einsatz von BHS-relevanten Referenzsubstanzen durchgef{\"u}hrt. Die Klassifikation der Testsubstanzen erfolgte analog ihrer Permeationsgeschwindigkeiten: Diazepam und Koffein gelten als schnell transportierte Wirkstoffe, Ibuprofen, Celecoxib und Diclofenac werden mit einer mittleren Geschwindigkeit {\"u}ber die BHS transportiert und Loratadin sowie Rhodamin 123 sind langsam permeierende Substanzen. Innerhalb der Versuche mit den Quadrupelkulturen wurde diese Reihenfolge best{\"a}tigt, lediglich f{\"u}r Koffein wurde ein signifikant niedrigerer Permeationskoeffizient verglichen mit der Monokultur erzielt. Der Einsatz der hiPSC-Technologie erm{\"o}glicht es zudem, aus einer Stammzelllinie große Mengen an humanen somatischen Zelltypen zu generieren und f{\"u}r gezielte Anwendungen bereitzustellen. Es konnte im Rahmen dieser Arbeit gezeigt werden, dass mit Hilfe eines eigens f{\"u}r diese Zwecke konstruierten R{\"u}hrreaktorsystems eine reproduzierbare Expansion der hiPSCs unter definierten Bedingungen erm{\"o}glicht wurde. Basierend auf dieser Grundlage ist nun ein Hochdurchsatz-Screening von Medikamenten denkbar. Die in dieser Arbeit pr{\"a}sentierten Daten belegen die Etablierung eines stammzellbasierten in vitro- Quadrupelmodels der humanen BHS, welches {\"u}ber in vivo-{\"a}hnliche Eigenschaften verf{\"u}gt. Die Anforderungen, die an humane BHS-Modelle gestellt werden, wie die Reproduzierbarkeit der Ergebnisse, eine angemessene Charakterisierung, welche die Untersuchung der Permeabilit{\"a}t von Referenzsubstanzen einschließt, die Analyse der Expression von BHS-relevanten Transportermolek{\"u}len sowie die solide und physiologische Morphologie der Zellen, wurden erf{\"u}llt. Das etablierte BHS-Modell kann in der Pharmaindustrie f{\"u}r die Entwicklung von Medikamenten eingesetzt werden. Ausreichend qualifizierte Modelle k{\"o}nnen hier in der pr{\"a}klinischen Forschung genutzt werden, um Toxizit{\"a}ts- und Transportstudien an neu entwickelten Substanzen durchzuf{\"u}hren und eine bessere in vitro-in vivo-Korrelation der Ergebnisse zu erm{\"o}glichen oder Mechanismen zu entwickeln, um die BHS-Barriere gezielt zu {\"u}berwinden.},
  subject      = {Blut-Hirn-Schranke},
  language  = {de}
}
@phdthesis{Koenig2016,
  author    = {K{\"o}nig, Sebastian},
  title     = {Spatially selective visual attention in Drosophila melanogaster},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-134452},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {Finding the right behavior at the right time is one of the major tasks of brains. In a natural scenery there is often an abundance of stimuli present and the brain has to separate the relevant from the irrelevant ones. Selective visual attention (SVA) is a property of higher visual systems that achieves this separation, as it allows to '[…] focus on one source of sensory input to the exclusion of others' (Luck and Mangun, 1996). There are probably several forms of SVA depending upon the criteria used for the separation, such as salience, color, location in space, novelty, or motion. Many studies have investigated SVA in humans and non-human primates. However, complex functions like attention were initially not expected to be already implemented in the brains of simple organisms like Drosophila. After a first demonstration of selective attention in the fly (Wolf and Heisenberg, 1980), it took some time until other studies included attentional mechanisms in their argumentation to explain certain behaviors of Drosophila. However, their definition and characterization of attention differed and often was ambiguous. Here, one particular form, spatially selective visual attention in the fly Drosophila is investigated. It has been shown earlier that the fly spontaneously may restrict its behavioral responses in stationary flight to the visual stimuli on one side of the visual field. On the basis of experiments of Sareen et al., (2011) it has been conjectured that the fly has a focus of attention (FoA) and that the fly responds to the visual stimuli within this area of the visual field. Whether the FoA is the adequate concept for this spatial property of SVA in the fly needs to be further discussed and is a subject also of the present study. At this stage, the concept will be used in the description of the new results expanding the characterization of SVA. This study continued the investigation of SVA during tethered flight with variable but controlled visual input and an automated primary data evaluation. This standardized paradigm allowed for analysis of wild-type behavior as well as for a comparison of several mutant and pharmacologically manipulated strains to the wild-type. Some properties of human SVA like the occurrence of externally as well as internally caused shifts of attention were found in Drosophila and it could be shown, that SVA in the fly can be externally guided and has an attention span. Additionally, a neurotransmitter and proteins, which play a significant role in SVA were discovered. Based on this, the genetic tools available for Drosophila provided the means to a first examination of cells and circuits involved in SVA. Finally, the free walk behavior of flies that had been shown to have compromised SVA was characterized. The results suggested that the observed phenotypes of SVA were not behavior specific. Covert shifts of the FoA were investigated. The FoA can be externally guided by visual cues to one or the other side of the visual field and even after the cue has disappeared it remains there for <4s. An intriguing finding of this study is the fact, that the quality of the cue determines whether it is attractive or repellent. For example a cue can be changed from being repellent (negative) to being attractive (positive) by changing its oscillation amplitude from 4° to 2°. Testing the effectiveness of cues in the upper and lower visual field separately, revealed that the perception of a cue by the fly is not exclusively based on a sum of its specifications. Because positive cueing did not have an after-effect in each of the two half-fields alone, but did so if the cue was shown in both, the fly seems to evaluate the cue for each combination of parameters specifically. Whether this evaluation of the cue changed on a trial-to-trial basis or if the cue in some cases failed to shift the FoA can at this point not be determined. Looking at the responses of the fly to the displacement of a black vertical stripe showed that they can be categorized as no responses, syn-directional responses (following the direction of motion of the stripe) and anti-directional responses (in the opposite direction of the motion of the stripe). The yaw-torque patterns of the latter bared similarities with spontaneous body saccades and they most likely represented escape attempts of the fly. Syn-directional responses, however, were genuine object responses, distinguishable by a longer latency until they were elicited and a larger amplitude. These properties as well as the distribution of response polarities were not influenced by the presence or absence of a cue. When two stripes were displaced simultaneously in opposite directions the rate of no responses increased in comparison to the displacement of a single stripe. If one of the stripes was cued, both, the responses towards and away from the side of cue resembled the syn-directional responses. Significant progress was made with the elucidation of the neuronal underpinnings of SVA. Ablation of the mushroom bodies (MB) demonstrated their requirement for SVA. Furthermore, it was shown that dopamine signaling has to be balanced between too much and too little. Either inhibiting the synthesis of dopamine or its re-uptake at the synapse via the dDAT impaired the flies' susceptibility to cueing. Using the Gal4/UAS system, cell specific expression or knockdown of the dDAT was used to scrutinize the role of MB sub-compartments in SVA. The αβ-lobes turned out to be necessary and sufficient to maintain SVA. The Gal4-line c708a labels only a subset of Kenyon cells (KC) within the αβ-lobes, αβposterior. These cells stand out, because of (A) the mesh-like arrangement of their fibers within the lobes and (B) the fact that unlike the other KCs they bypass the calyx and thereby the main source of olfactory input to the MBs, forming connections only in the posterior accessory calyx (Tanaka et al., 2008). This structure receives no or only marginal olfactory input, suggesting for it a role in tasks other than olfaction. This study shows their requirement in a visual task by demonstrating that they are necessary to uphold SVA. Restoring dDAT function in these approximately only 90 cells was probably insufficient to lower the dopamine concentration at the relevant synapses and hence a rescue failed. Alternatively, the processes mediating SVA at the αβ-lobes might require an interplay between all of their KCs. In conclusion, the results provide an initial point for future research to fully understand the localization of and circuitry required for SVA in the brain. In the experiments described so far, attention has been externally guided. However, flies are also able to internally shift their FoA without any cues from the outside world. In a set of 60 consecutive simultaneous displacements of two stripes, they were more likely to produce a response with the same polarity as the preceding one than a random polarity selection predicted. This suggested a dwelling of the FoA on one side of the visual field. Assuming that each response was influenced by the previous one in a way that the probability to repeat the response polarity was increased by a certain factor (dwelling factor, df), a random selection of response type including a df was computed. Implementation of the df removed the difference between observed probability of polarity repetition and the one suggested by random selection. When the interval between displacements was iteratively increased to 5s, no significant df could be detected anymore for pauses longer than 4s. In conclusion, Drosophila has an attention span of approximately 4s. Flies with a mutation in the radish gene expressed no after-effect of cueing and had a shortened attention span of about 1s. The dDAT inhibitor methylphenidate is able to rescue the first, but does not affect the latter phenotype. Probably, radish is differently involved in the two mechanisms. This study showed, that endogenous (covert) shifts of spatially selective visual attention in the fly Drosophila can be internally and externally guided. The variables determining the quality of a cue turned out to be multifaceted and a more systematic approach is needed for a better understanding of what property or feature of the cue changes the way it is evaluated by the fly. A first step has been made to demonstrate that SVA is a fundamental process and compromising it can influence the characteristics of other behaviors like walking. The existence of an attention span, the dependence of SVA on dopamine as well as the susceptibility to pharmacological manipulations, which in humans are used to treat respective diseases, point towards striking similarities between SVA in humans and Drosophila.},
  subject      = {Taufliege},
  language  = {en}
}
@phdthesis{KuhngebBach2015,
  author    = {Kuhn [geb. Bach], Julia Elisa},
  title     = {Design und Etablierung von Next Generation Sequencing-Methoden zur Diagnostik verschiedener Erbkrankheiten},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-116854},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {Innerhalb des letzten Jahrzehnts entstanden zahlreiche neue Anreicherungs- und Sequenzier-technologien der zweiten (und dritten) Generation, die in rasantem Tempo weiterentwickelt und schon jetzt in vielen Bereichen als neuer Goldstandard f{\"u}r molekulargenetische For-schung und Diagnostik angesehen werden. Als Hochdurchsatz-Verfahren erm{\"o}glichen diese Next Generation Sequencing-Methoden (NGS) in immer k{\"u}rzerer Zeit die parallele Analyse zahlreicher Proben und immer gr{\"o}ßerer Zielregionen bis hin zum ganzen Genom und f{\"u}hrten in der Humangenetik dadurch zu Forschungsans{\"a}tzen in neuen Dimensionen. In dieser Doktorarbeit, die im molekulargenetischen Diagnostik-Labor der Humangenetik W{\"u}rzburg durchgef{\"u}hrt wurde, wurden in f{\"u}nf Projekten NGS-Ans{\"a}tze unterschiedlicher Stufen bzw. Gr{\"o}ßenordnungen f{\"u}r verschiedene erblich bedingte Erkrankungen konzipiert und etabliert und in Forschungsprojekten sowie der Routinediagnostik eingesetzt. Dabei wurden verschiedene Methoden zur Anreicherung der Zielsequenzen und zur NGS-Sequenzierung erprobt und auf ihre Effizienz beurteilt. Die Ergebnisse des NGS und darauf basierender Nachweis-Experimente wurden in sieben Ver{\"o}ffentlichungen dokumentiert, auf denen diese Dissertation aufbaut. In den drei ersten Projekten wurden das Access Array-System (Fluidigm) zur Anreicherung der Zielsequenzen und der GS Junior (Roche) zur Erzeugung der Sequenzen verwendet. In Projekt 1 wurde COL4A6 als neues Kandidatengen f{\"u}r nicht-syndromale H{\"o}rst{\"o}rungen identifiziert. Um m{\"o}gliche weitere Mutationstr{\"a}ger zu detektieren, wurde erfolgreich ein kleiner NGS-Ansatz f{\"u}r das z{\"u}gige Screening dieses Gens bei knapp 100 weiteren Patienten etabliert. Diese und weitere Ergebnisse best{\"a}tigten die Kausalit{\"a}t der COL4A6-Mutation eines Index-Patienten mit schwerer, X-chromosomal-rezessiver H{\"o}rst{\"o}rung. Ein geeigneter NGS-Ansatz f{\"u}r die Analyse des großen RYR1-Gens wurde in Projekt 2 ge-sucht. Der erste Ansatz mit Access Array-System und GS Junior f{\"u}hrte zwar bei 39 von 87 Patienten mit Maligner Hyperthermie und/oder Central Core Disease zu dem Auffinden einer (potentiell) pathogenen Variante, allerdings mit hohen Ausfallquoten. Mit der zweiten Methode (Anreicherung: SureSelect-System custom design, Agilent; Sequenzierung: HiSeq, Illumina) wurden neben RYR1 noch 63 weitere Gene analysiert, was zu deutlich besseren Ergebnissen und vier Mutationsfunden f{\"u}hrte. Projekt 3 beinhaltete die Etablierung zwei kleiner Panels f{\"u}r Muskelkrankheiten. Ein Panel f{\"u}r drei Gene f{\"u}r Gliederg{\"u}rteldystrophien wurde sogar erfolgreich in die akkreditierte Rou-tinediagnostik {\"u}bernommen. Mit dem zweiten Panel f{\"u}r acht Kandidatengene myofibrill{\"a}rer Myopathien (MFM) wurde u.a. eine neue Mutation im BAG3-Gen identifiziert. Das Exom eines MFM-Patienten wurde in Projekt 4 nach Anreicherung mit dem SureSelect-System (Agilent) auf dem HiSeq (Illumina) sequenziert. Nach Auswertung und Beurteilung der identifizierten Varianten wurde ein neuer Erbgang f{\"u}r Myotilinopathien entdeckt. Verschiedene Nachweisexperimente best{\"a}tigten die Kausalit{\"a}t der Mutation im Myotilin-Gen. In Projekt 5 wurde die komplette genomische Sequenz des F8-Gens nach tiefen intronischen Mutationen bei H{\"a}mophilie-Patienten abgesucht (Anreicherung SureSelect custom design, Agilent; Sequenzierung MiSeq, Illumina). Bei jedem der analysierten Patienten konnte min-destens eine verd{\"a}chtige Variante identifiziert werden, die zu ver{\"a}ndertem Spleißverhalten f{\"u}hren k{\"o}nnte. Drei Mutationen waren schon durch Publikationen bekannt, bei einer weite-ren konnten in vitro-Spleißanalysen die Kausalit{\"a}t best{\"a}tigen. Die Ergebnisse dieser Arbeit zeigen, dass die zur Verf{\"u}gung stehenden Methoden zur An-reicherung von Zielsequenzen aus dem menschlichen Genom und zu deren Sequenzierung je nach Komplexit{\"a}t der Fragestellung, d.h. der Anzahl und Gr{\"o}ße der Gene sowie der Anzahl der zu untersuchenden Proben, sinnvoll und effizient kombiniert werden k{\"o}nnen. Im Verlauf der Arbeit haben sich die NGS-Techniken rasant weiterentwickelt. So sind PCR-basierte Ans{\"a}tze zur Anreicherung der Zielsequenzen f{\"u}r die meisten Anwendungen von hybridisierungs-basierten Methoden verdr{\"a}ngt worden. Von den urspr{\"u}nglich drei konkur-rierenden Verfahren zur Hochdurchsatzsequenzierung hat sich die Methode des „sequen-cing-by-synthesis" (Illumina) weitgehend durchgesetzt. Diese Entwicklung spiegelt sich auch in den w{\"a}hrend dieser Arbeit erhobenen Daten wider.},
  subject      = {Diagnostik},
  language  = {de}
}
@phdthesis{Schneider2015,
  author    = {Schneider, Gudrun},
  title     = {Effects of adjacent habitats and landscape composition on biodiversity in semi-natural grasslands and biological pest control in oilseed rape fields},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-113549},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {1) Modern European agricultural landscapes form a patchy mosaic of highly fragmented natural and semi-natural habitat remnants embedded in a matrix of intensively managed agricultural land. In those landscapes many organism frequently cross habitat borders including the crop - non-crop boundary, hereby connecting the biotic interactions of multiple habitat types. Therefore biodiversity and ecosystem functions within habitats are expected to depend on adjacent habitat types and the surrounding landscape matrix. In this thesis the biodiversity of non-crop habitats, and ecosystem services and disservices in crop habitats were studied in the human-dominated agricultural landscape in the district Lower Franconia, Bavaria, Germany. First we examined the effect of adjacent habitat type on species composition, diversity and ecosystem functions in semi-natural calcareous grasslands, a biodiversity-rich habitat of high conservation value (chapter 2 and 3). Second we studied the effect of habitat composition in the landscape on herbivory, biological pest control and yield in oilseed rape fields (chapter 4). 2) We examined the effect of adjacent habitat type on the diversity of carabid beetles in 20 calcareous grasslands using pitfall traps. Half of the grasslands were adjacent to a coniferous forest and half to a cereal crop field. We found different species compositions of carabid beetles depending on adjacent habitat type. In addition calcareous grasslands adjacent to crop fields harboured a higher species richness and activity density but a lower evenness of carabid beetles than calcareous grasslands adjacent to forests. These differences can be explained by the spillover of carabid beetles from the adjacent habitats. After crop harvest carabid beetle activity density in crop fields decreased while in parallel the activity density in the calcareous grasslands adjacent to the crop fields increased, indicating an unidirectional carabid beetle spillover. Our results underline that type and management of adjacent habitats affect community composition and diversity in calcareous grasslands. Therefore nature conservation measures, which focused on the improvement of local habitat quality so far, additionally need to consider adjacent habitat type. 3) In addition to carabid beetle communities we also surveyed predation rates of ground-dwelling predators on the same calcareous grasslands in two study periods (June and late August). As ground-dwelling predators of forests or crop fields can move into adjacent calcareous grasslands we expected different predation rates depending on adjacent habitat type. We exposed in total 32.000 lady bird eggs as prey items on the calcareous grasslands in distances of 5 and 20m from the habitat border. We found higher predation rates on calcareous grasslands adjacent to forests than on calcareous grasslands adjacent to crop fields, but only on cool days. On warm days a very high extent (often 100\%) of the exposed prey items were consumed adjacent to both habitat types, which did not allow the detection of possible differences between the adjacent habitat types. Predation rates differed not between the two study periods or the two distances to the habitat edge. The higher predation rates adjacent to forests can be explained by the spillover of ground-dwelling predators from forests into calcareous grasslands. Our results show, that spillover into semi-natural habitats affects ecosystem functioning in addition to species composition and diversity. 4) In chapter 4 of this thesis we examined the effect of spatiotemporal changes in crop cover on pest - natural enemy interactions and crop yields. During two study years we surveyed the abundance of adult and larval pollen beetles, parasitism of pollen beetle larvae by a hymenopteran parasitoid and oilseed rape yields of 36 oilseed rape fields. The surrounding landscape of the fields (1 km radius) differed in the oilseed rape proportion and in the inter-annual change in the oilseed rape proportion since the previous year. We found a dilution effect, i.e. a decreasing abundance with increasing oilseed rape proportions, for pollen beetle larvae and parasitoids in both study years and for adult pollen beetles in one study year. Oilseed rape yields increased with increasing oilseed rape proportions. Inter-annual changes in oilseed rape proportions led to inter-annual crowding and dilution effects for pollen beetles, but had no effect on parasitism or yield. Our results indicate the potential to reduce pest loads and increase yields in intensively managed oilseed rape fields by a coordinated management of the spatiotemporal oilseed rape cover in the landscape. 5) In summary, we showed in this thesis that the biodiversity and functioning of crop and non-crop habitats within agricultural landscapes is affected by the spillover of organisms and thus by the habitat composition in the close surrounding and in the broader landscape context. Spillover affects also ecosystem services and disservices and therefore crop productivity. Thereby the spatial and temporal variation of specific crop types in the landscape can be of particular importance for crop yields. Thus a coordinated landscape wide management can help to optimize both biodiversity conservation and the delivery of ecosystem services and thus crop yields. Future studies integrating landscape effects across several ecosystem functions, multiple taxonomic groups and different crop types are necessary to develop definite landscape management schemes.},
  subject      = {Landschafts{\"o}kologie},
  language  = {en}
}
@phdthesis{Brockmann2015,
  author    = {Brockmann, Markus},
  title     = {Inhibition von Aurora-A als neue Therapiestrategie gegen MYCN-amplifizierte Neuroblastome},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-135951},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {Im Neuroblastom ist die Amplifikation des MYCN-Gens, das f{\"u}r den Transkriptionsfaktor N-Myc kodiert, der klinisch bedeutendste Faktor f{\"u}r eine schlechte Prognose. Als Mitglied der onkogenen Myc-Familie induziert N-Myc die Expression von Genen, die in vielen biologischen Prozessen wie Metabolismus, Zellzyklusprogression, Zellwachstum und Apoptose eine wichtige Rolle spielen. Die Deregulation der MYCN-Expression f{\"u}hrt zu einem charakteristischen Genexpressionsprofil und einem aggressiven Phenotyp in den Tumorzellen. In normalen neuronalen Vorl{\"a}uferzellen wird N-Myc gew{\"o}hnlich sehr schnell proteasomal abgebaut. W{\"a}hrend der Mitose wird N-Myc an Serin 62 phosphoryliert. Diese Phosphorylierung dient als Erkennungssignal f{\"u}r die Kinase GSK3β, die die Phosphorylierung an Threonin 58 katalysiert. Das Phosphodegron wird von Fbxw7, einer Komponente des E3-Ubiquitinligase-Komplex SCFFbxw7, erkannt. Die anschließende Ubiquitinierung induziert den proteasomalen Abbau des Proteins. Die Reduktion der N-Myc-Proteinlevel erm{\"o}glicht den neuronalen Vorl{\"a}uferzellen den Austritt aus dem Zellzyklus und f{\"u}hrt zu einer terminalen Differenzierung. In einem shRNA Screen konnte AURKA als essentielles Gen f{\"u}r die Proliferation MYCN-amplifizierter Neuroblastomzellen identifiziert werden. Eine Aurora-A-Depletion hatte jedoch keinen Einfluss auf das Wachstum nicht-amplifizierter Zellen. W{\"a}hrend dieser Doktorarbeit konnte gezeigt werden, dass Aurora-A speziell den Fbxw7-vermittelten Abbau verhindert und dadurch N-Myc stabilisiert. F{\"u}r die Stabilisierung ist zwar die Interaktion der beiden Proteine von entscheidender Bedeutung, {\"u}berraschenderweise spielt die Kinaseaktivit{\"a}t von Aurora-A jedoch keine Rolle. Zwei spezifische Aurora-A-Inhibitoren, MLN8054 und MLN8237, sind allerdings in der Lage, nicht nur die Kinaseaktivit{\"a}t zu hemmen, sondern auch die N-Myc-Proteinlevel zu reduzieren. Beide Molek{\"u}le induzieren eine Konformations{\"a}nderung in der Kinasedom{\"a}ne von Aurora-A. Diese ungew{\"o}hnliche strukturelle Ver{\"a}nderung hat zur Folge, dass der N-Myc/Aurora-A-Komplex dissoziiert und N-Myc mit Hilfe von Fbxw7 proteasomal abgebaut werden kann. In MYCN-amplifizierten Zellen f{\"u}hrt diese Reduktion an N-Myc zu einem Zellzyklusarrest in der G1-Phase. Die in vitro Daten konnten in einem transgenen Maus-Modell f{\"u}r das MYCN-amplifizierte Neuroblastom best{\"a}tigt werden. Die Behandlung mit MLN8054 und MLN8237 f{\"u}hrte in den Tumoren ebenfalls zu einer N-Myc-Reduktion. Dar{\"u}ber hinaus konnte ein prozentualer Anstieg an differenzierten Zellen, die vollst{\"a}ndige Tumorregression in der Mehrzahl der Neuroblastome und eine gesteigerte Lebenserwartung beobachtet werden. Insgesamt zeigen die in vitro und in vivo Daten, dass die spezifischen Aurora-A-Inhibitoren ein hohes therapeutisches Potential gegen das MYCN-amplifizierte Neuroblastom besitzen.},
  subject      = {N-Myc},
  language  = {de}
}
@phdthesis{Scholl2015,
  author    = {Scholl, Christina},
  title     = {Cellular and molecular mechanisms contributing to behavioral transitions and learning in the honeybee},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-115527},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {The honeybee Apis mellifera is a social insect well known for its complex behavior and the ability to learn tasks associated with central place foraging, such as visual navigation or to learn and remember odor-reward associations. Although its brain is smaller than 1mm² with only 8.2 x 105 neurons compared to ~ 20 x 109 in humans, bees still show amazing social, cognitive and learning skills. They express an age - related division of labor with nurse bees staying inside the hive and performing tasks like caring for the brood or cleaning, and foragers who collect food and water outside the hive. This challenges foragers with new responsibilities like sophisticated navigation skills to find and remember food sources, drastic changes in the sensory environment and to communicate new information to other bees. Associated with this plasticity of the behavior, the brain and especially the mushroom bodies (MBs) - sensory integration and association centers involved in learning and memory formation - undergo massive structural and functional neuronal alterations. Related to this background my thesis on one hand focuses on neuronal plasticity and underlying molecular mechanisms in the MBs that accompany the nurse - forager transition. In the first part I investigated an endogenous and an internal factor that may contribute to the nurse - forager phenotype plasticity and the correlating changes in neuronal network in the MBs: sensory exposure (light) and juvenile hormone (JH). Young bees were precociously exposed to light and subsequently synaptic complexes (microglomeruli, MG) in the MBs or respectively hemolymph juvenile hormone (JH) levels were quantified. The results show that light input indeed triggered a significant decrease in MG density, and mass spectrometry JH detection revealed an increase in JH titer. Interestingly light stimulation in young bees (presumably nurse bees) triggered changes in MG density and JH levels comparable to natural foragers. This indicates that both sensory stimuli as well as the endocrine system may play a part in preparing bees for the behavioral transition to foraging. Considering a connection between the JH levels and synaptic remodeling I used gene knockdown to disturb JH pathways and artificially increase the JH level. Even though the knockdown was successful, the results show that MG densities remained unchanged, showing no direct effect of JH on synaptic restructuring. To find a potential mediator of structural synaptic plasticity I focused on the calcium-calmodulin-dependent protein kinase II (CaMKII) in the second part of my thesis. CaMKII is a protein known to be involved in neuronal and behavioral plasticity and also plays an important part in structural plasticity reorganizing synapses. Therefore it is an interesting candidate for molecular mechanisms underlying MG reorganization in the MBs in the honeybee. Corresponding to the high abundance of CaMKII in the learning center in vertebrates (hippocampus), CaMKII was shown to be enriched in the MBs of the honeybee. Here I first investigated the function of CaMKII in learning and memory formation as from vertebrate work CaMKII is known to be associated with the strengthening of synaptic connections inducing long term potentiation and memory formation. The experimental approach included manipulating CaMKII function using 2 different inhibitors and a specific siRNA to create a CaMKII knockdown phenotype. Afterwards bees were subjected to classical olfactory conditioning which is known to induce stable long-term memory. All bees showed normal learning curves and an intact memory acquisition, short-term and mid-term memory (1 hour retention). However, in all cases long-term memory formation was significantly disrupted (24 and 72 hour retention). These results suggests the necessity of functional CaMKII in the MBs for the induction of both early and late phases of long-term memory in honeybees. The neuronal and molecular bases underlying long-term memory and the resulting plasticity in behavior is key to understanding higher brain function and phenotype plasticity. In this context CaMKII may be an important mediator inducing structural synaptic and neuronal changes in the MB synaptic network.},
  subject      = {Biene},
  language  = {en}
}
@phdthesis{Konrad2015,
  author    = {Konrad, Tillmann},
  title     = {Governance of Protected Areas in West Africa - The case of the W-Arly-Pendjari (WAP) Complex in Benin and Burkina Faso},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-115331},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {Protected areas are the central strategy for preserving biodiversity in the face of overexploitation and global change. To ensure their long-term survival, however, these areas may not be regarded as last havens of wilderness, but as complex social-ecological systems. Modern approaches of protected area (PA) management support this view by balancing conservation and development issues in a sustainable way and adapted to the local context. However, success of these strategies in achieving their aims so far remains limited. This study therefore aimed at analysing processes and outcomes of PA co-management approaches implemented in a large transfrontier conservation area in West Africa. The W-Arly-Pendjari (WAP) complex spans over more than 30.000 square km in Benin, Burkina Faso and Niger and is composed of approximately 20 subunits. Due to national legal and administrative variety as well as a high diversity of local (project) implementation approaches, the general setting resembled a quasi-experimental design facilitating comparative studies. A mix of quantitative (e.g. survey of 549 households) and qualitative (e.g. expert interviews, literature review) methods was used to evaluate the institutional and organisational differences of PA management approaches implemented in the different parts of WAP belonging to Benin and Burkina Faso. I included an analysis of contextual factors (e.g. land-cover-change) and ecological data, but concentrated on the role of local resource users within the co-management arrangements and the effectiveness of governance regimes to deliver positive socio-economic outputs. Exploring the question whether promotion of development in PA surroundings indeed stipulates conservation success (and vice versa) remained challenging: the lack of sound ecological data, a general mismatch of spatial scale in existing data sets, as well as the high complexity of realities on the ground made me refrain from using simplified proxy indicators and (statistical) modelling approaches. I found that the Sudano-Sahelian context is a very difficult one for the implementation of effective participation approaches in the short-term. Political, demographic, socio-economic as well as ecological factors generated a very dynamic situation characterized by limited financial and natural resources as well as weak institutional and organisational settings. Arenas of interaction were often marked rather by a high degree of distrust and competition than by cooperation among actors. Amid all rhetoric, participation in most cases was hence limited to the transfer of (sparse) information, regulated resource access and financial funds. Options for participation of local resource users in decision-making arenas were generally scarce. Underlying processes were dominated by opacity and often low accountability of actors on all levels. Negative, but also positive affection of local residents by PA existence and management hence was high. Governance regimes of the complex performed very differently with regard to their ability of effectively empowering local village participatory bodies (vpb), generating and distributing benefits to individuals and village communities as well as providing mechanisms of conflict resolution. People around Pendjari enjoyed a relative wealth of high value benefits, while negative impacts caused by human-wildlife conflicts were widespread around the complex. Autochthonous farmers usually were better integrated in incentive schemes than were newcomers or herders. While there was functional separation of actors' roles in all parts of WAP, these roles differed significantly between blocks. Existence and functioning of village participatory bodies ameliorated the situation for local resource users fundamentally, as they acted as cut-points between different networks (governmental hierarchies, private concessionaires and local resource users). Vpbs in the Pendjari region proved to be most advanced in their capacity to push resource users' claims in action arenas on the micro-level. Via their union, these associations also managed to impact arenas on the meso- and the macro scale. Project interventions often had catalyst functions to empower local resource users and their vbps. However, they also contributed to social imbalance and intra-organisational competition. My results represent a snapshot of an ongoing process to establish effective co-governance regimes in the WAP-area. Though I identified a large scope of shortcomings, there were also very promising initiatives underway. This work is therefore meant to foster future research and further positive development by giving guidance scholars and decision-makers form the local to the global level alike.},
  subject      = {Gesch{\"u}tzte Natur},
  language  = {en}
}
@phdthesis{Riedinger2014,
  author    = {Riedinger, Verena},
  title     = {Landscape-scale spillover of pollinators from oil-seed rape to crop and semi-natural habitats on different temporal scales},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-96844},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {Organisms use different resources in different habitat types during their life cycle. Thereby, they connect habitats and provide ecosystem services or disservices in several habitat types. In agricultural landscapes, the spillover of organisms, i.e. movement of an organism and its function from one habitat to another, especially from semi-natural to managed habitats, is one of the most important processes that influence population dynamics and community composition. Importantly, spillover connects habitats not only spatially, but also on different temporal scales, because availability of resources changes over time in agricultural landscapes, e.g. by mass-flowering events of crops, harvesting or crop rotation. Most often, semi-natural habitats are seen as beneficial source of organisms, but also managed habitats can provide valuable resources, and thereby initiate spillover to other habitats. Mass-flowering crops, like oil-seed rape, are such valuable feeding resources for pollinators, and pollinators might spillover from oil-seed rape to other habitats which provide alternative foraging resources. The focus of this dissertation was to evaluate the influence of oil-seed rape on pollinators in agricultural landscapes by studying effects (1) on different temporal scales (from effects during the flowering period of oil-seed rape, Chapter II \& IV, to intermediate effects on a second mass-flowering crop, Chapter III, to spillover effects to the flowering period in the next year, Chapter IV), (2) semi-natural (Chapter II) and crop (Chapter III, IV) habitats, and (3) on various pollinator groups which differ in their life cycle (Chapter II, III, IV). In this dissertation effects from oil-seed rape on all temporal scales - in the short term during mass-flowering and in the long term on a late-flowering crop and even in the next year on oil-seed rape fields ─ were found. These effects might be important for crop and wild plant pollination, and pollinator conservation. Importantly, the effects on different temporal scales depend on the considered habitat (managed or different semi-natural habitats) and on the investigated pollinator group. The more pollinators match the flowering period of oil-seed rape in their activity period and the more dependent they are on flowering resources in their life cycle, the more pronounced are their responses. Effects were found for wild bees, but not for hoverflies and honey bees. Moreover, the availability of semi-natural habitats in the landscape is important and may modulate effects from oil-seed rape. The longevity of effects of oil-seed rape shows the importance of including several temporal scales into ecosystem-service studies, not only for pollinators, but also for other ecosystem-service providing species groups.},
  subject      = {Raps},
  language  = {en}
}
@phdthesis{Heidinger2015,
  author    = {Heidinger, Ina M. M.},
  title     = {Beyond metapopulation theory: Determinants of the dispersal capacity of bush crickets and grasshoppers},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-135068},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {Habitat fragmentation and destruction due to anthropogenic land use are the major causes of the increasing extinction risk of many species and have a detrimental impact on animal populations in numerous ways. The long-term survival and stability of spatially structured populations in fragmented landscapes largely depends on the colonisation of habitat patches and the exchange of individuals and genes between patches. The degree of inter-patch dispersal, in turn, depends on the dispersal ability of a species (i.e. the combination of physiological and morphological factors that facilitate dispersal) and the landscape structure (i.e. the nature of the landscape matrix or the spatial configuration of habitat patches). As fragmentation of landscapes is increasing and the number of species is continuously declining, a thorough understanding of the causes and consequences of dispersal is essential for managing natural populations and developing effective conservation strategies. In the context of animal dispersal, movement behaviour is intensively investigated with capture-mark-recapture studies. For the analysis of such experiments, the influence of marking technique, handling and translocation of marked animals on movement pattern is of crucial importance since it may mask the effects of the main research question. Chapter 2 of this thesis presents a capture-mark-recapture study investigating the effect of translocation on the movement behaviour of the blue-winged grasshopper Oedipoda caerulescens. Transferring individuals of this grasshopper species to suitable but unfamilliar sites has a significant influence on their movement behaviour. Translocated individuals moved longer distances, showed smaller daily turning angles, and thus their movements were more directed than those of resident individuals. The effect of translocation was most pronounced on the first day of the experiment, but may persist for longer. On average, daily moved distances of translocated individuals were about 50 \% longer than that of resident individuals because they have been transferred to an unfamiliar habitat patch. Depending on experiment duration, this leads to considerable differences in net displacement between translocated and resident individuals. In summary, the results presented in chapter 2 clearly point out that translocation effects should not be disregarded in future studies on arthropod movement, respectively dispersal. Studies not controlling for possible translocation effects may result in false predictions of dispersal behaviour, habitat detection capability or habitat preferences. Beside direct field observations via capture-mark-recapture methods, genetic markers can be used to investigate animal dispersal. Chapter 3 presents data on the genetic structure of populations of Metrioptera bicolor, a wing-dimorphic bush cricket, in a spatially structured landscape with patches of suitable habitat distributed within a diverse matrix of different habitat types. Using microsatellite markers, the effects of geographic distance and different matrix types on the genetic differentiation among 24 local populations was assessed. The results of this study clearly indicate that for M. bicolor the isolation of local populations severely depends on the type of surrounding matrix. The presence of forest and a river running through the study area was positively correlated with the extent of genetic differentiation between populations. This indicates that both matrix types severely impede gene flow and the exchange of individuals between local populations of this bush cricket. In addition, for a subsample of populations which were separated only by arable land or settlements, a significant positive correlation between pairwise genetic and geographic distances exists. For the complete data set, this correlation could not be found. This is most probably due to the adverse effect of forest and river on gene flow which dominates the effect of geographic distance in the limited set of patches investigated in this study. The analyses in chapter 3 clearly emphasize the differential resistance of different habitat types on dispersal and the importance of a more detailed view on matrix 'quality' in metapopulation studies. Studies that focus on the specific dispersal resistance of different matrix types may provide much more detailed information on the dispersal capacity of species than a mere analysis of isolation by distance. Such information is needed to improve landscape oriented models for species conservation. In addition to direct effects on realised dispersal (see chapter 3), landscape structure on its own is known to act as an evolutionary selection agent because it determines the costs and benefits of dispersal. Both morphological and behavioural traits of individuals and the degree to which a certain genotype responds to environmental variation have heritable components, and are therefore expected to be able to respond to selection pressures. Chapter 4 analyses the influence of patch size, patch connectivity (isolation of populations) and sand dynamics (stability of habitat) on thorax- and wing length as proxies for dispersal ability of O. caerulescens in coastal grey dunes. This study revealed clear and sex-specific effects of landscape dynamics and patch configuration on dispersal-related morphology. Males of this grasshopper species were smaller and had shorter wings if patches were larger and less connected. In addition, both sexes were larger in habitat patches with high sand dynamics compared to those in patches with lower dynamics. The investments in wing length were only larger in connected populations when sand dynamics were low, indicating that both landscape and patch-related environmental factors are of importance. These results are congruent with theoretical predictions on the evolution of dispersal in metapopulations. They add to the evidence that dispersal-related morphology varies and is selected upon in recently structured populations even at small spatial scales. Dispersal involves different individual fitness costs like increased predation risk, energy expenditure, costs of developing dispersal-related traits, failure to find new suitable habitat as well as reproductive costs. Therefore, the decision to disperse should not be random but depend on the developmental stage or the physiological condition of an individual just as on actual environmental conditions (context-dependent dispersal, e.g. sex- and wing morph-biased dispersal). Biased dispersal is often investigated by comparing the morphology, physiology and behaviour of females and males or sedentary and dispersive individuals. Studies of biased dispersal in terms of capture-mark-recapture experiments, investigating real dispersal and not routine movements, and genetic proofs of biased dispersal are still rare for certain taxa, especially for orthopterans. However, information on biased dispersal is of great importance as for example, undetected biased dispersal may lead to false conclusions from genetic data. In chapter 5 of this thesis, a combined approach of morphological and genetic analyses was used to investigate biased dispersal of M. bicolor. The presented results not only show that macropterous individuals are predestined for dispersal due to their morphology, the genetic data also indicate that macropters are more dispersive than micropters. Furthermore, even within the group of macropterous individuals, males are supposed to be more dispersive than females. To get an idea of the flight ability of M. bicolor, the morphological data were compared with that of Locusta migratoria and Schistocerca gregaria, which are proved to be very good flyers. Based on the morphological data presented here, one can assume a good flight ability for macropters of M. bicolor, although flying individuals of this species are seldom observed in natural populations.},
  subject      = {Heuschrecken <{\"U}berfamilie>},
  language  = {en}
}
@phdthesis{Hackl2016,
  author    = {Hackl, Thomas},
  title     = {A draft genome for the Venus flytrap, Dionaea muscipula : Evaluation of assembly strategies for a complex Genome - Development of novel approaches and bioinformatics solutions},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-133149},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {The Venus flytrap, \textit{Dionaea muscipula}, with its carnivorous life-style and its highly specialized snap-traps has fascinated biologist since the days of Charles Darwin. The goal of the \textit{D. muscipula} genome project is to gain comprehensive insights into the genomic landscape of this remarkable plant. The genome of the diploid Venus flytrap with an estimated size between 2.6 Gbp to 3.0 Gbp is comparatively large and comprises more than 70 \% of repetitive regions. Sequencing and assembly of genomes of this scale are even with state-of-the-art technology and software challenging. Initial sequencing and assembly of the genome was performed by the BGI (Beijing Genomics Institute) in 2011 resulting in a 3.7 Gbp draft assembly. I started my work with thorough assessment of the delivered assembly and data. My analysis showed that the BGI assembly is highly fragmented and at the same time artificially inflated due to overassembly of repetitive sequences. Furthermore, it only comprises about on third of the expected genes in full-length, rendering it inadequate for downstream analysis. In the following I sought to optimize the sequencing and assembly strategy to obtain an assembly of higher completeness and contiguity by improving data quality and assembly procedure and by developing tailored bioinformatics tools. Issues with technical biases and high levels of heterogeneity in the original data set were solved by sequencing additional short read libraries from high quality non-polymorphic DNA samples. To address contiguity and heterozygosity I examined numerous alternative assembly software packages and strategies and eventually identified ALLPATHS-LG as the most suited program for assembling the data at hand. Moreover, by utilizing digital normalization to reduce repetitive reads, I was able to substantially reduce computational demands while at the same time significantly increasing contiguity of the assembly. To improve repeat resolution and scaffolding, I started to explore the novel PacBio long read sequencing technology. Raw PacBio reads exhibit high error rates of 15 \% impeding their use for assembly. To overcome this issue, I developed the PacBio hybrid correction pipeline proovread (Hackl et al., 2014). proovread uses high coverage Illumina read data in an iterative mapping-based consensus procedure to identify and remove errors present in raw PacBio reads. In terms of sensitivity and accuracy, proovread outperforms existing software. In contrast to other correction programs, which are incapable of handling data sets of the size of D. muscipula project, proovread's flexible design allows for the efficient distribution of work load on high-performance computing clusters, thus enabling the correction of the Venus flytrap PacBio data set. Next to the assembly process itself, also the assessment of the large de novo draft assemblies, particularly with respect to coverage by available sequencing data, is difficult. While typical evaluation procedures rely on computationally extensive mapping approaches, I developed and implemented a set of tools that utilize k-mer coverage and derived values to efficiently compute coverage landscapes of large-scale assemblies and in addition allow for automated visualization of the of the obtained information in comprehensive plots. Using the developed tools to analyze preliminary assemblies and by combining my findings regarding optimizations of the assembly process, I was ultimately able to generate a high quality draft assembly for D. muscipula. I further refined the assembly by removal of redundant contigs resulting from separate assembly of heterozygous regions and additional scaffolding and gapclosing using corrected PacBio data. The final draft assembly comprises 86 × 10 3 scaffolds and has a total size of 1.45 Gbp. The difference to the estimated genomes size is well explained by collapsed repeats. At the same time, the assembly exhibits high fractions full-length gene models, corroborating the interpretation that the obtained draft assembly provides a complete and comprehensive reference for further exploration of the fascinating biology of the Venus flytrap.},
  subject      = {Venusfliegenfalle},
  language  = {en}
}
@phdthesis{MontalbandelBarrio2015,
  author    = {Montalb{\´a}n del Barrio, Itsaso},
  title     = {Immunosuppressive role of adenosine produced by ectonucleotidases CD39 and CD73 in ovarian cancer, tumor associated macrophages and the host immune system},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-133268},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {Eierstockkrebs ist der Tumor mit der schlechtesten Heilungsprognose unter allen gyn{\"a}kologischen Malignomen. Allein in Deutschland verursacht er {\"u}ber 6000 Tote pro Jahr. Patienten mit Ovarialkarzinom zeigen erst in einem sehr fortgeschrittenen Stadium charakteristische Symptome. Die einzig m{\"o}glichen Behandlungsmethoden sind dann die operative Tumorentfernung und die Verabreichung von platinbasierter Chemotherapien sowie von Anthrazyklinen. Da die aktuelle 5-Jahres-{\"U}berlebensrate lediglich 20-40\% betr{\"a}gt, besteht ein dringender Bedarf an neuen therapeutischen Optionen. Seit herausgefunden wurde, dass immunologische Parameter das {\"U}berleben der Patienten beeinflussen, ist Immuntherapie zu einer der vielversprechendsten Behandlungsarten des Eierstockkrebs geworden. Das Ziel unserer Forschung ist die {\"U}berwindung der Immunevasion des Tumors durch ein Verhindern der immun-unterdr{\"u}ckenden Mechanismen des Tumors. Im Speziellen befasst sich diese Arbeit mit dem Einfluss von Adenosin, das durch die Ectonukleotidasen CD39 und CD73 in der Mikroumgebung des Tumors gebildet wird. Die CD39- und CD73-Expression der Zellen f{\"u}hrt zu Immunosuppression da diese Ectonukleotidasen immun-stimulierendes, extrazellul{\"a}res ATP in immunsuppressives Adenosin umwandeln. Dies wurde zuerst als Effektormechanismus f{\"u}r regulatorische T-Zellen beschrieben, kann aber auch im Tumormikromilieu von Bedeutung sein. Mit dem Wissen, dass Tumorzellen von Eierstockkrebs-Patientinnen große Mengen der ATP-unterdr{\"u}ckenden Ectonukleotidasen CD39 und CD73 bilden, analysierten wir die adenosinvermittelte Unterdr{\"u}ckendung von Immunantwortenin der Mikroumgebung der Tumorzellen. Im Vergleich zu regulatorischen T Zellen konnten wir bei Eierstockkrebs-Zelllinien und bei aus Aszites gewonnenen Krebszellen eine 30- bis 60-fache Adenosinproduktion messen. Um diesen mutmaßlichen Immunevasions-Mechanismus zu best{\"a}tigen, untersuchten wir seine Auswirkungen auf mehrere Immunzellenpopulationen. CSFE-basierte Experimente zeigten zum Beispiel eine Hemmung der CD4+ T-Zell-Proliferation durch Adenosin, welches von Eierstockkrebs-Zellen produziert wurde. In diesem Zusammenhang haben wir auch eine in-vitro Methode entwickelt, mit der wir die Beeinflussung von Makrophagen durch Eierstockkrebszellen analysieren und modulieren konnten. Neben seiner suppressiven Wirkung {\"u}bt Adenosin auch chemotaktische Effekte auf menschliche Monozyten aus und lockt wahrscheinlich myeloide Vorl{\"a}uferzellen zum Tumorgewebe. Anschließend differenzieren sich menschliche Monozyten in einer von Eierstockkrebszellen geformten Mikroumgebung zu M2 Makrophagen oder tumor-assoziierten Makrophagen (TAMs), die ihrerseits erhebliche Mengen der Adenosin-produzierenden Ectonukleotidasen CD39 und CD73 bilden. W{\"a}hrend wir die Regulierung der Ectonukleotidasen-Expression untersuchten, entdeckten wir auch, dass klinisch genutzte Techniken zur Behandlung von Eierstockkrebs (zum Beispiel die Anwendung von Doxorubicin oder Bestrahlung) in vitro das CD73- und CD39-Level von Eierstockkrebs- und Immunzellen beeinflussen. In dieser Studie zeigen wir, wie dieser behandlungsbedingte Wechsel des ATP/Adenosine-Verh{\"a}ltnisses die Effektorfunktion verschiedener Immunzellen moduliert. Dar{\"u}ber hinaus untersuchen wir den potentiellen Vorteil von klinisch verf{\"u}gbaren, niedermolekularen Inhibitoren f{\"u}r CD39 und CD73, die die Immunsuppression in der Mikroumgebung des Tumors partiell aufheben k{\"o}nnten, und die vor allem in Kombination mit g{\"a}ngigen Behandlungsschemata von großem Interesse sein k{\"o}nnten.},
  subject      = {Eierstockkrebs},
  language  = {en}
}
@phdthesis{Wiese2015,
  author    = {Wiese, Katrin Evelyn},
  title     = {Sensing supraphysiological levels of MYC : mechanisms of MIZ1-dependent MYC-induced Apoptosis in Mammary Epithelial Cells},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-132532},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {Deregulated MYC expression contributes to cellular transformation as well as progression and maintenance of human tumours. Interestingly, in the absence of additional genetic alterations, potentially oncogenic levels of MYC sensitise cells to a variety of apoptotic stimuli. Hence, MYC-induced apoptosis has long been recognised as a major barrier against cancer development. However, it is largely unknown how cells discriminate physiological from supraphysiological levels of MYC in order to execute an appropriate biological response. The experiments described in this thesis demonstrate that induction of apoptosis in mammary epithelial cells depends on the repressive actions of MYC/MIZ1 complexes. Analysis of gene expression profiles and ChIP-sequencing experiments reveals that high levels of MYC are required to invade low-affinity binding sites and repress target genes of the serum response factor SRF. These genes are involved in cytoskeletal dynamics as well as cell adhesion processes and are likely needed to transmit survival signals to the AKT kinase. Restoration of SRF activity rescues MIZ1- dependent gene repression and increases AKT phosphorylation and downstream function. Collectively, these results indicate that association with MIZ1 leads to an expansion of MYC's transcriptional response that allows sensing of oncogenic levels, which points towards a tumour-suppressive role for the MYC/MIZ1 complex in epithelial cells.},
  subject      = {Myc},
  language  = {en}
}
@phdthesis{Hoecherl2015,
  author    = {H{\"o}cherl, Nicole},
  title     = {Nesting behaviour of the paper wasp Polistes dominula - with special focus on thermoregulatory mechanisms},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-132681},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {Wasps of the genus Polistes comprise over 200 species and are nearly cosmopolitan. They show a lack of physiological caste differentiation and are therefore considered as primitively eusocial. Furthermore, paper wasps are placed between the solitary living Eumenidae and the highly social organized Vespinae. Hence, they are often called a "key genus" for understanding the evolution of sociality. Particularly, Polistes dominula, with its small easy manageable nests and its frequent occurrence and wide distribution range is often the subject of studies. In Europe, the invasion of this species into northern regions is on the rise. Since little was known about the nesting behaviour of P. dominula in Central Europe, the basic principles about nesting were investigated in W{\"u}rzburg, Germany (latitude 49°) by conducting a comprehensive field-study spanning three consecutive years. Furthermore, the thermoregulation of individual wasps in their natural habitat had not yet been investigated in detail. Therefore, their ability to respond to external hazards with elevated thorax temperatures was tested. In addition, different types of nest thermoregulation were investigated using modern methods such as infrared thermography and temperature data logger. In the present work, the investigation of basic nesting principles revealed that foundress groups (1-4 foundresses) and nests are smaller and that the nesting season is shorter in the W{\"u}rzburg area than in other regions. The mean size of newly founded nests was 83 cells and the average nesting season was around 4.6 months. The queens neither preferred single (54\%) nor multiple founding (46\%) in this study. The major benefit of multiple founding is an increased rate of survival. During the three years of observation, only 47\% of single-foundress colonies survived, whereas 100\% of colonies that were built by more than two queens, survived. However, an influence of the number of foundresses on the productivity of colonies in terms of number of cells and pupae per nest has not shown up. However, the length of the nesting season as well as the nest sizes varied strongly depending on the climatic conditions of the preceding winter during the three consecutive years. In order to investigate the thermoregulatory mechanisms of individual adult P. dominula wasps, I presented artificial threats by applying smoke or carbon dioxide simulating fire and predator attacks, respectively, and monitored the thorax temperature of wasps on the nest using infrared thermography. The results clearly revealed that P. dominula workers recognized smoke and CO2 and reacted almost instantaneously and simultaneously with an increase of their thorax temperature. The maximal thorax temperature was reached about 65 s after the application of both stressors, but subsequently the wasps showed a different behaviour pattern. They responded to a longer application of smoke with moving to the exit and fled, whereas in case of CO2 the wasps started flying and circling the nest without trying to escape. No rise of the thorax temperature was detectable after an air blast was applied or in wasps resting on the nest. Additionally, the thorax temperatures of queens were investigated during dominance battles. I found that the thorax temperature of the dominant queens rose up to 5°C compared to that of subordinate queens that attacked the former. The study of active mechanisms for nest thermoregulation revealed no brood incubation or clustering behaviour of P. dominula. Furthermore, I found out that wing fanning for cooling the nest was almost undetectable (4 documented cases). However, I could convincingly record that water evaporation is most effective for nest cooling. By the direct comparison of active (with brood and adults) and non-active (without brood and adults) nests, the start of cooling by water evaporation was detected above maximum outside temperatures of 25°C or at nest temperatures above 35°C. The powerful role of water in nest cooling was manifested by an average decrease of temperature of a single cell of about 8°C and a mean duration of 7 min until the cell reached again its initial temperature. The investigation of passive thermoregulatory mechanisms revealed that the nest site choice as well as nest orientation appears to be essential for P. dominula wasps. Furthermore, I was able to show that the architecture of the nests plays an important role. Based on the presented results, it can be assumed that the vertical orientation of cells helps maintaining the warmth of nests during the night, whereas the pedicel assists in cooling the nest during the day.},
  subject      = {Franz{\"o}sische Feldwespe},
  language  = {en}
}
@phdthesis{Wende2014,
  author    = {Wende, Beate},
  title     = {Diversity of saproxylic beetles and host tree specialisation in differently managed forests across Germany},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-107049},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {Chapter I The gradual turnover of dead organic material into mineral nutrients is a key ecological function, linking decomposition and primary production, the essential parts of the nutrient-energy cycle. However, disturbances in terms of species or resource losses might impair the equilibrium between production and decomposition. Humanity has converted large proportions of natural landscapes and intensified land-use activity for food production. Globally, only very few areas are totally unaffected by human activity today. To ensure the maintenance of both essential ecosystem services, knowledge about the interplay of biodiversity and ecosystem functioning as well as effects of intensified management on both is crucial. The vast majority of terrestrial biomass production as well as decomposition take place in forest ecosystems. Though forestry has a long sustainable history in Europe, its intensification during the last century has caused severe impacts on forest features and, consequently, on the associated biota, especially deadwood dependent organisms. Among these, saproxylic beetles are the most diverse group in terms of species numbers and functional diversity, but also most endangered due to habitat loss. These features classify them as ideal research organisms to study effects of intensified forestry on ecosystem services. The BELONGDEAD project located in Germany aimed to investigate deadwood decay and functional consequences of diversity changes in the associated fauna on the decomposition process from the initialisation of deadwood decay to complete degradation. As part of the BeLongDead project, this dissertation focussed on saproxylic beetle species, thereby evaluating (1) regionally effects of tree species identity of fresh deadwood and (2) forest management of varying intensities on the diversity, abundance and community composition of saproxylic beetles (chapter II); (3) the specialisation degree of different trophic guilds of saproxylic beetles, and thus the stability and robustness of their interaction networks against disturbances (chapter III); (4) the impact of environmental features of local to regional spatial scales on species richness of saproxylic beetles differing in their habitat niche in terms of deadwood decay stages (chapter IV). Chapter II The vast majority of European forest ecosystems have been anthropogenically affected, leaving less than 1\% of the about 1 milliard hectare as natural forests. A long history of forestry and especially the technological progress during the last century have caused massive habitat fragmentation as well as substantial loss of essential resources in European forest ecosystems. Due to this, the substrate-dependent group of saproxylic beetles has experienced severe species losses. Thus, investigations concerning saproxylic diversity and deadwood volume were badly needed. However, the importance of different deadwood in terms of tree species identity for the colonization by saproxylic beetles under different local and regional management regimes is poorly understood. Therefore, we studied possible regional differences in colonization patterns of saproxylic beetle species in a total of 688 fresh deadwood logs of 13 tree species in 9 sites of managed conifer and beech forests, and unmanaged beech forests, respectively. We found that tree species identity was an important driver in determining saproxylic species composition and abundance within fresh deadwood. However, saproxylic species showed different colonization patterns of deadwood items of the same tree species among the study regions. Regionally consistent, conifer forests were most diverse. We attribute the latter result to the historically adaption of saproxylic beetle species to semi-open forests, which conditions are actually best reflected by conifer forests. To preserve a diverse local species pool of early successional saproxylic beetles, we suggest an equal high degree of deadwood diversity in a tree species context in due consideration of regional differences. Chapter III The extinction risk of a particular species corresponds with its species-specific requirements on resources and habitat conditions, in other words with the width of the species` ecological niche. Species with a narrow ecological niche are defined as specialists. Members of this group experience higher extinction risk by resource limitation than generalists, which are able to utilize a variety of resources. For the classification of species as specialists or generalists, thus evaluating possible extinction risks, ecologists use the concept of interaction networks. This method has often been applied for mutualistic or antagonistic plant-animal interactions, but information for networks of detritivores is scarce. Therefore, saproxylic beetle species sampled as described in chapter II were categorised according to their larval diet; additionally their interaction networks (N=108) with 13 dead host tree species were analysed. Specialisation degree was highest for wood-digesting beetles and decreased with increasing trophic level. Also the network indices evaluating robustness and generality indicated a higher susceptibility to species extinctions for xylophagous than for mycetophagous and predatory beetles. The specialisation of xylophagous species on specific tree species might be an adaption to tree species specific ingredients stored for defence against pathogens and pests. However, we conclude that the high specialisation degree of xylophages and thus their higher extinction risk by resource loss harbours certain dangers for ecosystem function and stability as species diversity is positively linked to both. Chapter IV Populations depend on individual emigration and immigration events to ensure genetic exchange. For successful migration it is of utmost importance that spatially separated populations are obtainable by specimen. Migratory success depends on the one hand on the species dispersal abilities and on the other on the availability of suitable habitats in the surrounding landscape in which the distinct host populations exist. However, consequences of intensive forest management correspond not only to severe reduction of local deadwood amount, but, among others, also a change in tree species composition and high levels of fragmentation in the surrounding forest area. Saproxylic beetle species differ in their dispersal behaviour according to the temporal availability of their preferred habitat. Generally, early successional saproxylic beetles are able to disperse over large distances, whereas beetles inhabiting advanced decayed wood often remain close to their larval habitat. Due to this, environmental factors might affect saproxylic beetle guilds differently. We classified the saproxylic beetles sampled as described in chapter II according to their calculated habitat niche as early, intermediate or late successional saproxylic beetles. For the different guilds the effects of 14 environmental factors on different spatial scales (stand factors at 0.1 km radius, landscape composition at 2 km radius, and regionally differing abiotic factors in 400 km to 700 km distance) were investigated. Consistently for all guilds, species richness decreased with fragmentation at local and landscape scale, and increased in warmer climate. However, we found contradictory results between the guilds to some extent. We relate this to guild specific habitat requirements of the saproxylic beetles. Therefore, for the development of appropriate conservation practices guild-specific requirements saproxylic beetles have to be considered not only locally but on larger spatial scales. Chapter V In conclusion, this dissertation identified main drivers of early successional saproxylic beetle species richness on various spatial scales. Our results emphasize the importance to develop management schemes meeting species-specific and guild-specific habitat requirements of the saproxylic beetle fauna at relevant spatial and temporal scales. Therefore, short-term actions suggested for sustainable forest management should be the focus on a diverse tree species composition consisting of indigenous tree species with respect to regional differences. Moreover, senescent trees, fallen and standing deadwood should remain in the forests, and some tree individuals should be allowed to grow old. Long-term actions should involve the reduction of forest fragmentation and the connection of spatial widely separated forest fragments. Furthermore, to fully understand the effects of forest management long-term research should be conducted to compare habitat requirements of intermediate and late successional beetles with the results presented in this dissertation.},
  subject      = {Saproxylophage},
  language  = {en}
}
@phdthesis{Pusch2015,
  author    = {Pusch, Tobias},
  title     = {The transcription factor NFATc1 mediates cytotoxic T cell function in vitro and in vivo},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-123690},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {While numerous experiments on NFAT were already performed with CD4+ T cells showing defective cytokine release and a reduced T helper cell development, no detailed studies existed for CD8+ T cells. From this point, we wanted to examine the impact of NFATc1 and c2 on the physiological functions of CD8+ T cells in vitro and in vivo. Therefore, we used a murine infection model with the bacteria Listeria monocytogenes and mice in which NFATc1 was specifically depleted in the T cell compartment. Our first in vitro studies showed a typical NFATc1 and c2 nuclear translocation and changes on mRNA levels upon T cell activation similarly in CD4+ as well as in CD8+ T cells extracted from wild type mice. NFAT nuclear translocation is important for target gene activation and generation of effector functions. Stimulated T cell populations lacking NFATc1 and/or NFATc2 showed a markedly decreased expression of Th1/Tc1 cytokines, as e.g. IL 2 and IFNγ being important for the clearance of intracellular pathogens. From our in vitro model for the generation of allogenically reactive cytotoxic CD8+ T cells, we revealed a decreased killing and lytic granule-release capacity in Nfatc1 inactivated CD8+ T cells whereas NFATc2-/- cytotoxic T cells did not show an altered cytotoxic response compared to wild type cells. Interestingly, we found lytic granules accumulated and mitochondria not getting translocated to the immunological synapse upon re-stimulation in NFATc1-deficient CD8+ T cells. Together with results showing the CsA insensitivity of the CTL killing/degranulation capacities, we assume that some major cellular processes are affected by NFATc1 which are not directly linked to the TCR-induced signal transduction cascade. We also showed the importance of NFATc1 in T cells during intracellular infections with the bacteria Listeria monocytogenes in an in vivo mouse model. After five days, only few bacteria were detected in wt mice whereas high amounts of Listeria particles were extracted from livers of Nfatc1fl/fl x Cd4 cre mice. Although the reactivity towards the pathogen was similar in both groups, a decreased cytokine expression in NFATc1-/- CD8+ T cells was observed together with an altered memory cell generation. Our results show the importance of NFATc1 in CD8+ T cells and give some clue for a possible connection to other basal cellular functions, as e.g. the formation of an immunological synapse.},
  subject      = {Transkriptionsfaktor},
  language  = {en}
}
@phdthesis{Gnamlin2015,
  author    = {Gnamlin, Prisca},
  title     = {Use of Tumor Vasculature for Successful Treatment of Carcinomas by Oncolytic Vaccinia Virus},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-119019},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {Tumor-induced angiogenesis is of major interest for oncology research. Vascular endothelial growth factor (VEGF) is the most potent angiogenic factor characterized so far. VEGF blockade was shown to be sufficient for angiogenesis inhibition and subsequent tumor regression in several preclinical tumor models. Bevacizumab was the first treatment targeting specifically tumor-induced angiogenesis through VEGF blockade to be approved by the Food and Drugs Administration (FDA) for cancer treatment. However, after very promising results in preclinical evaluations, VEGF blockade did not show the expected success in patients. Some tumors became resistant to VEGF blockade. Several factors have been accounted responsible, the over-expression of other angiogenic factors, the noxious influence of VEFG blockade on normal tissues, the selection of hypoxia resistant neoplastic cells, the recruitment of hematopoietic progenitor cells and finally the transient nature of angiogenesis inhibition by VEGF blockade. The development of blocking agents against other angiogenic factors like placental growth factor (PlGF) and Angiopoietin-2 (Ang-2) allows the development of an anti-angiogenesis strategy adapted to the profile of the tumor. Oncolytic virotherapy uses the natural propensity of viruses to colonize tumors to treat cancer. The recombinant vaccinia virus GLV-1h68 was shown to infect, colonize and lyse several tumor types. Its descendant GLV-1h108, expressing an anti-VEGF antibody, was proved in previous studies to inhibit efficiently tumor induced angiogenesis. Additional VACVs expressing single chain antibodies (scAb) antibodies against PlGF and Ang-2 alone or in combination with anti VEGF scAb were designed. In this study, VACV-mediated anti-angiogenesis treatments have been evaluated in several preclinical tumor models. The efficiency of PlGF blockade, alone or in combination with VEGF, mediated by VACV has been established and confirmed. PlGF inhibition alone or with VEGF reduced tumor burden 5- and 2-folds more efficiently than the control virus, respectively. Ang-2 blockade efficiency for cancer treatment gave controversial results when tested in different laboratories. Here we demonstrated that unlike VEGF, the success of Ang-2 blockade is not only correlated to the strength of the blockade. A particular balance between Ang-2, VEGF and Ang-1 needs to be induced by the treatment to see a regression of the tumor and an improved survival. We saw that Ang-2 inhibition delayed tumor growth up to 3-folds compared to the control virus. These same viruses induced statistically significant tumor growth delays. This study unveiled the need to establish an angiogenic profile of the tumor to be treated as well as the necessity to better understand the synergic effects of VEGF and Ang-2. In addition angiogenesis inhibition by VACV-mediated PlGF and Ang-2 blockade was able to reduce the number of metastases and migrating tumor cells (even more efficiently than VEGF blockade). VACV colonization of tumor cells, in vitro, was limited by VEGF, when the use of the anti-VEGF VACV GLV-1h108 drastically improved the colonization efficiency up to 2-fold, 72 hours post-infection. These in vitro data were confirmed by in vivo analysis of tumors. Fourteen days post-treatment, the anti-VEGF virus GLV-1h108 was colonizing 78.8\% of the tumors when GLV-1h68 colonization rate was 49.6\%. These data confirmed the synergistic effect of VEGF blockade and VACV replication for tumor regression. Three of the tumor cell lines used to assess VACV-mediated angiogenesis inhibition were found, in certain conditions, to mimic either endothelial cell or pericyte functions, and participate directly to the vascular structure. The expression by these tumor cells of e-selectin, p-selectin, ICAM-1 and VCAM-1, normally expressed on activated endothelial cells, corroborates our findings. These proteins play an important role in immune cell recruitment, and there amount vary in presence of VEGF, PlGF and Ang-2, confirming the involvement of angiogenic factors in the immuno-modulatory abilities of tumors. In this study VACV-mediated angiogenesis blockade proved its potential as a therapeutic agent able to treat different tumor types and prevent resistance observed during bevacizumab treatment by acting on different factors. First, the expression of several antibodies by VACV would prevent another angiogenic factor to take over VEGF and stimulate angiogenesis. Then, the ability of VACV to infect tumor cells would prevent them to form blood vessel-like structures to sustain tumor growth, and the localized delivery of the antibody would decrease the risk of adverse effects. Next, the blockade of angiogenic factors would improve VACV replication and decrease the immune-modulatory effect of tumors. Finally the fact that angiogenesis blockade lasts until total regression of the tumor would prevent the recovery of the tumor-associated vasculature and the relapse of patients.},
  subject      = {Vaccinia-Virus},
  language  = {en}
}
@phdthesis{Kirscher2014,
  author    = {Kirscher, Lorenz},
  title     = {Melanogene rekombinante Vaccinia-Viren als diagnostisches und therapeutisches Agenz zur Tumorbehandlung},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-112074},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {Die g{\"a}ngigen therapeutischen Behandlungsmethoden f{\"u}r die verschiedensten Krebserkrankungen zeigen nach wie vor M{\"a}ngel bez{\"u}glich der Effizienz sowie zahlreiche Nebenwirkungen w{\"a}hrend und nach der Behandlung. Maßgeblich f{\"u}r diese Defizite ist die teilweise geringe Sensitivit{\"a}t der meisten konventionellen diagnostischen Systeme und damit einhergehend die oftmals zu sp{\"a}te Identifikation entarteter Gewebsbereiche. Zur L{\"o}sung dieser Problematik bieten onkolytische Vaccinia-Viren einen Ansatz, sowohl die Effizienz der Therapie wie auch die Diagnostik zu verbessern. In beiden F{\"a}llen sind die Tumorzell-spezifische Vermehrung der Viren und die M{\"o}glichkeit entscheidend, die Viren als Vektorsystem zur Expression therapeutischer oder diagnostischer Fremdgenkassetten zu nutzen. Um ein auf Vaccinia-Virus-basierendes Reportersystem zum diagnostischen Nachweis von Krebszellen mittels Tiefengewebs-Tomographie bereit zu stellen, wurden die f{\"u}r die murine Tyrosinase (mTyr) und das Tyrosinase-Helferprotein 1 (Tyrp1) kodierenden Gene in das Genom eines onkolytischen Vaccinia-Virus inseriert. Die Tyrosinase ist das Schl{\"u}sselenzym der Melaninsynthese. Bereits die solit{\"a}re Expression der Tyrosinase f{\"u}hrt in der transformierten Zelle zur Melaninproduktion. Das Tyrosinase-Helferprotein 1 ist an der Prozessierung und Stabilisierung der Tyrosinase beteiligt. Bereits in verschiedenen Studien konnte gezeigt werden, dass Melanin als Reportermolek{\"u}l f{\"u}r die Magnetresonanz sowie f{\"u}r die multispektrale optoakustische Tomographie einsetzbar ist. Es wurde deswegen angestrebt, die Kombination aus dem therapeutischen Potential des onkolytischen Vaccinia-Virus und der diagnostischen Anwendung des Melanins als Reporter auszunutzen. S{\"a}mtliche in dieser Arbeit aufgef{\"u}hrten rekombinanten Vaccinia-Viren (rVACV) wurden von der Firma Genelux Corporation zur Verf{\"u}gung gestellt und in dieser Arbeit hinsichtlich der therapeutischen Effizienz und des diagnostischen Potentials untersucht. In ersten Zellkultur-Versuchen wurde anhand verschiedener konstitutiv melanogener rVACV-Konstrukte festgestellt, dass die Kombination aus dem Vaccinia-Virus-spezifischen synthetic early/late Promotor und dem Enzym Tyrosinase (GLV-1h327) bzw. den Enzymen Tyrosinase und Tyrosinase-Helferprotein 1 (GLV-1h324) die h{\"o}chste Melaninsynthese-Rate zeigte. Anschließend wurde mittels der Bestimmung der spektralen Absorption und der Enzymaktivit{\"a}t der viral kodierten Melanin synthetisierenden Enzyme sowie mikroskopischer Analysen gezeigt, dass es mit diesen auf 8 Vaccinia-Virus-basierenden melanogenen Reportersystemen m{\"o}glich ist, die Melaninsynthese in nicht-melanogenen Zellen zu induzieren. Anhand elektronenmikroskopischer Untersuchungen in Zellkultur und ex vivo konnte gezeigt werden, dass die nach rVACV-Infektion stattfindende Melaninsynthese in den Lysosomen der Wirtszelle abl{\"a}uft. Eine Analyse der atomaren Zusammensetzung des viral vermittelten Melanins ergab, dass es sich um eine Mischform aus Eu- und Ph{\"a}omelanin handelt. Dieser Melanin-Mix {\"a}hnelte dem Melanin aus Haut und Augen, jedoch lagen an Melanin-gebundene Metallionen in erh{\"o}htem Maß vor...},
  subject      = {Melanin},
  language  = {de}
}
@phdthesis{LuiblneeHermann2014,
  author    = {Luibl [n{\´e}e Hermann], Christiane},
  title     = {The role of the neuropeptides NPF, sNPF, ITP and PDF in the circadian clock of Drosophila melanogaster},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-93796},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {Organisms have evolved endogenous clocks which allow them to organize their behavior, metabolism and physiology according to the periodically changing environmental conditions on earth. Biological rhythms that are synchronized to daily changes in environment are governed by the so-called circadian clock. Since decades, chronobiologists have been investigating circadian clocks in various model organisms including the fruitfly Drosophila melanogaster, which was used in the present thesis. Anatomically, the circadian clock of the fruitfly consists of about 150 neurons in the lateral and dorsal protocerebrum, which are characterized by their position, morphology and neurochemistry. Some of these neurons had been previously shown to contain either one or several neuropeptides, which are thought to be the main signaling molecules used by the clock. The best investigated of these neuropeptides is the Pigment Dispersing Factor (PDF), which had been shown to constitute a synchronizing signal between clock neurons as well as an output factor of the clock. In collaboration with various coworkers, I investigated the roles of three other clock expressed neuropeptides for the generation of behavioral rhythms and the partly published, partly unpublished data are presented in this thesis. Thereby, I focused on the Neuropeptide F (NPF), short Neuropeptide F (sNPF) and the Ion Transport Peptide (ITP). We show that part of the neuropeptide composition within the clock network seems to be conserved among different Drosophila species. However, the PDF expression pattern in certain neurons varied in species deriving from lower latitudes compared to higher latitudes. Together with findings on the behavioral level provided by other people, these data suggest that different species may have altered certain properties of their clocks - like the neuropeptide expression in certain neurons - in order to adapt their behavior to different habitats. We then investigated locomotor rhythms in Drosophila melanogaster flies, in which neuropeptide circuits were genetically manipulated either by cell ablation or RNA interference (RNAi). We found that none of the investigated neuropeptides seems to be of equal importance for circadian locomotor rhythms as PDF. PDF had been previously shown to be necessary for rhythm maintenance in constant darkness (DD) as well as for the generation of morning (M) activity and for the right phasing of the evening (E) activity in entrained conditions. We now demonstrate that NPF and ITP seem to promote E activity in entrained conditions, but are clearly not the only factors doing so. In addition, ITP seems to reduce nighttime activity. Further, ITP and possibly also sNPF constitute weak period shortening components in DD, thereby opposing the effect of PDF. However, neither NPF or ITP, nor sNPF seem to be necessary in the clock neurons for maintaining rhythmicity in DD. It had been previously suggested that PDF is released rhythmically from the dorsal projection terminals. Now we discovered a rhythm in ITP immunostaining in the dorsal projection terminals of the ITP+ clock neurons in LD, suggesting a rhythm in peptide release also in the case of ITP. Rhythmic release of both ITP and PDF seems to be important to maintain rhythmic behavior in DD, since constantly high levels of PDF and ITP in the dorsal protocerebrum lead to behavioral arrhythmicity. Applying live-imaging techniques we further demonstrate that sNPF acts in an inhibitory way on few clock neurons, including some that are also activated by PDF, suggesting that it acts as signaling molecule within the clock network and has opposing effects to PDF. NPF did only evoke very little inhibitory responses in very few clock neurons, suggesting that it might rather be used as a clock output factor. We were not able to apply the same live-imaging approach for the investigation of the clock neuron responsiveness to ITP, but overexpression of ITP with various driver lines showed that the peptide most likely acts mainly in clock output pathways rather than inter-clock neuron communication. Taking together, I conclude that all investigated peptides contribute to the control of locomotor rhythms in the fruitfly Drosophila melanogaster. However, this control is in most aspects dominated by the actions of PDF and rather only fine-tuned or complemented by the other peptides. I assume that there is a high complexity in spatial and temporal action of the different neuropeptides in order to ensure correct signal processing within the clock network as well as clock output.},
  subject      = {Taufliege},
  language  = {en}
}
@phdthesis{Schmitt2015,
  author    = {Schmitt, Alexandra},
  title     = {Role of Peroxiredoxin 6 in human melanoma},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-111465},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {Peroxiredoxin 6 (PRDX6) is a bifunctional enzyme comprising a peroxidase and a Ca2+-independent phospholipase (iPLA2) activity. This renders the enzyme capable of detoxifying reactive oxygen species (ROS) and of catalyzing the liberation of arachidonic acid (AA) from cellular membranes. Released AA can be further metabolized to bioactive lipids including eicosanoids, which are involved in inflammation, cell growth, differentiation, invasion and proliferation. Human melanoma cells are often characterized by imbalances in both ROS and lipid levels, which can be generated by oncogenic signaling, altered metabolism or UV irradiation. In previous studies, a comparative proteome analysis of the Xiphophorus fish melanoma model revealed a strong upregulation of Prdx6 in benign and malignant lesions compared to healthy skin. As the Xiphophorus melanoma model displays in many respects molecular characteristics that are similar to human melanoma, I investigated the functional role of PRDX6 in human melanoma cells. The first part of the study deals with the regulation of PRDX6 in melanocytes and human melanoma cells. I could demonstrate that the protein level of PRDX6 was strongly enhanced by the induction of the EGFR orthologue Xmrk from the Xiphophorus fish as well as the human EGFR. The upregulation of PRDX6 was further shown to be mediated in a PI3K-dependent and ROS-independent manner. The main part of the thesis comprises the investigation of the functional role of PRDX6 in human melanoma cells as well as the analysis of the underlying mechanism. I could show that knockdown of PRDX6 enhanced the oxidative stress response and led to decreased proliferation of melanoma cells. This cell growth effect was mainly mediated by the iPLA2 activity of PRDX6. Under conditions of strongly enhanced oxidative stress, the peroxidase activity became also important for cellular proliferation. Furthermore, the anti-proliferative effect in cells with lowered PRDX6 levels was the result of reduced cellular AA content and the decrease in the activation of SRC family proteins. Similarly, supplementation with AA led to regeneration of SRC family kinase activity and to an improvement in the reduced proliferation after knockdown of PRDX6. Since AA can be further processed into the prostaglandin PGE2, which has a pro-tumorigenic function in some cancer types, I further examined whether this eicosanoid is involved in the proliferative function of PRDX6. In contrast to AA, PGE2 was not consistently required for melanoma proliferation. In summary, I could demonstrate that PRDX6 plays a major role in AA-dependent lipid signaling in melanoma cells and thereby regulates proliferation. Interestingly, the proliferation relevant iPLA2 activity can be pharmacologically targeted, and melanoma cell growth was clearly blocked by the inhibitor BEL. Thus, I could identify the phospholipase activity of PRDX6 as a new therapeutically interesting target for melanoma treatment.},
  subject      = {Melanom},
  language  = {en}
}
@phdthesis{Endt2015,
  author    = {Endt, Daniela},
  title     = {Fanconi An{\"a}mie : Entwicklung von h{\"a}matopoetischen Mosaiken sowie funktionelle Studien von FANCO (RAD51C) und FANCN (PALB2)},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-127836},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {Zur Wahrung der Genomstabilit{\"a}t entwickelten sich verschiedene Reparaturmechanismen, deren Defekte zu diversen Erkrankungen f{\"u}hren. Der 1927 erstmals beschriebenen Fanconi An{\"a}mie (FA) (Fanconi 1927) liegt eine fehlerhafte Reparatur der DNA-Doppelstrang-Quervernetzung zugrunde. Als Ursache wurden Defekte innerhalb des FA/BRCA-Weges lokalisiert, welche zur Chromosomeninstabilit{\"a}t f{\"u}hren. Das Krankheitsbild der autosomal rezessiven oder X-chromosomalen Erkrankung wird meist von kongenitalen Fehlbildungen, progressivem Knochenmarkversagen sowie bereits im jugendlichen Alter erh{\"o}hten Tumor-raten und An{\"a}mien gepr{\"a}gt. Bisher wurden Defekte in 19 verschiedenen Genen als urs{\"a}chlich f{\"u}r diese Erkrankung diskutiert. Anhand des betroffenen Gens k{\"o}nnen nur begrenzt R{\"u}ckschl{\"u}sse auf die Auspr{\"a}-gung des Ph{\"a}notyps geschlossen werden, vielmehr scheinen die Art der Mutation und deren Position im Gen mit der Schwere der Erkrankung zu korrelieren. Im Laufe der Zeit wurden immer mehr Patienten mit mild ausgepr{\"a}gtem Erkrankungsbild beobachtet. Eine m{\"o}gliche Erkl{\"a}rung hierf{\"u}r liefern milde Mutationen, eine weitere das Vorhandensein von Mosaiken blutbildender Zellen. Zu letzterem f{\"u}hrt die Reversion einer der beiden Mutationen. Diese Art der „nat{\"u}rlichen Gentherapie" wurde bei 10-30\% der FA-Patienten beobachtet. Um die Entwicklung von Reversionen besser zu verstehen, erfolgte im Rahmen dieser Arbeit die Untersuchung verschiedener Zelllinien von 5 Patienten im Alter von 11 (Pat. 5) bis 33 (Pat. 4) Jahren. Die FA-A-Patienten 1 und 2 wurden bereits von Gross et al. 2002 als Mosaikpatienten beschrieben. F{\"u}r die weiteren Patienten f{\"u}hrten unterschiedliche Aspekte, wie normale Blutwerte, MMC-tolerante lympho-blastoide Zelllinien und gDNA-Analysen des Blutes zum Mosaikverdacht. N{\"a}here Analysen best{\"a}tigten f{\"u}r die FA-D2-Patienten (Pat. 4, 5) ebenfalls das Vorliegen einer Reversion in den Blutzellen. Allen Patienten gemein war die Reversion in Form einer R{\"u}ckmutation (Pat. 1: c.971T>G, Pat. 2: c.856 C>T, Pat. 4: c.3467-2A>G, Pat. 5: c.3707G>A), welche meist in einem oder in der N{\"a}he eines Mutationsmotives vorlag. Zur Einsch{\"a}tzung des Mosaikstatus in den Patientenblutzellen wurden, neben der meist mehrj{\"a}hrigen Be-obachtung der Blutwerte (Thrombo-, Mono-, Granulo-, Lymphozyten, H{\"a}moglobin), gDNA-, Chromoso-menbruch- und Zellzyklusanalysen durchgef{\"u}hrt. Chromosomenbruchanalysen von Metaphasen der T-Lymphozyten der Patienten 4 und 5 zeigten nach MMC-Behandlung die mosaik-typische bimodale Vertei-lung der Chromosomenbruchraten. Die nur moderat erh{\"o}hten Bruchraten in Metaphasen des Patienten 1 sprachen f{\"u}r eine starke Reversion. Zur besseren Absch{\"a}tzung des Mosaikstatus wurden Zellzyklusanaly-sen an Mischungsreihen aus FA- und nicht FA- Blut durchgef{\"u}hrt. Die Detektionsgrenze f{\"u}r FA-Mosaike lag bei einem Anteil von 30\% Zellen mit spontanem/MMC-induziertem G2-Phasen-Arrest. In Anlehnung an Mischungskurven wurden f{\"u}r die vier Patienten Reversionen von 0\% (Pat. 4) bis 90-95\% (Pat. 2) ange-nommen. Die gDNA-Analyse MACS-sortierter T-/B-Lympho-, Mono- und Granulozyten sowie von Fib-roblasten und lymphoblastoiden Zelllinien erm{\"o}glichte einen detaillierten Einblick in die Mosaikstatus auf molekularer Ebene. Wir fanden bei allen Patienten einen unterschiedlich stark ausgepr{\"a}gten Mosaikstatus ihrer Blutzellreihen. Tendenziell scheinen die Reversionsgrade mit der Zell-Lebensdauer korrelieren, hier-bei zeigen kurzlebige Zellen (Mono-, Granulo-, B-Lymphozyten) h{\"o}here Reversionsgrade als langlebige T-Lymphozyten. Das Auftreten von gleichen Reversionen in allen Zelllinien l{\"a}sst eine Reversion in einer gemeinsamen Vorl{\"a}uferzelle vermuten. Als Besonderheit fanden wir, unseren Erachtens erstmalig, eine komplette Reversion einer Knochenmark-Fibroblastenzelllinie (Pat. 1). H{\"a}ufig in Kultur stattfindende Re-versionen in lymphoblastoiden Zelllinien beobachteten wir f{\"u}r alle vier Patienten. Die Mosaikentstehung im Patientenblut konnte mit allen Methoden best{\"a}tigt werden. Jede Methode wies Vor- und Nachteile auf. Zur Absch{\"a}tzung der Mosaikstatus empfiehlt sich deshalb eine Kombination der Methoden. Ein weiteres Projekt besch{\"a}ftigte sich mit Interaktionen des FANCO (RAD51C) innerhalb der RAD51 Paraloge (RAD51B, -C, -D, XRCC2, XRCC3) und mit RAD51. Die Analysen erfolgten im Mammalian Two- und Three-Hybrid (M2H/M3H) System. Die Untersuchungen best{\"a}tigten die meisten der bisher detektierten Interaktionen, welche zur Ausbildung des RAD51C-XRCC3 Komplexes und des, aus den Subkomplexen RAD51B-RAD51C (BC) und RAD51D-XRCC2 (DX2) bestehenden, BCDX2-Komplex f{\"u}hren. Die M3H-Analysen weisen auf eine wichtige Rolle des RAD51B-Proteins bei der Auspr{\"a}gung dieses Komplexes hin. Es scheint die Ausbildung der RAD51C-RAD51D-Interaktion erst zu erm{\"o}glichen und zus{\"a}tzlich, anders als bisher beobachtet, auch mit XRCC2 zu interagieren. Diese Interaktion wiederum wird durch die Anwesenheit von RAD51D stark gef{\"o}rdert. Unsere M2H-/M3H-Beobachtungen weisen darauf hin, dass die Ausbildung der Subkomplexe f{\"u}r die Entstehung des BDCX2-Komplexes wichtig ist und dieser vermutlich als Ringstruktur vorliegt. Zus{\"a}tzlich fanden wir Hinweise auf m{\"o}gliche Wechselwir-kungen zwischen den BCDX2- und den XRCC3-Komplexproteinen. Aufgrund der Beteiligung der Protei-ne an der Doppelstrangl{\"a}sionsreparatur wurde die Auswirkung von MMC-induzierten DNA-Sch{\"a}den un-tersucht. Diese f{\"u}hrten innerhalb der Subkomplexe zu gegens{\"a}tzlichen {\"A}nderungen der Interaktionsinten-sit{\"a}t. W{\"a}hrend die Substanz im DX2-Komplex zum Sinken der Interaktionsst{\"a}rke f{\"u}hrte, erh{\"o}hte sich diese im BC-Komplex. Die in der Literatur beschriebene und charakterisierte RAD51C-FANCN-Interation war im M2H-Test nicht darstellbar. M{\"o}glicherweise w{\"u}rde diese jedoch durch die Anwesenheit eines drit-ten Proteins gef{\"o}rdert werden. Zus{\"a}tzlich wurde ein RAD51C-Protein, welches die Patientenmutation R258H enthielt, {\"u}berpr{\"u}ft. Es zeigte nur in der M3H-Analyse, mit pMRAD51D und nativem RAD51B, nach Behandlung mit MMC eine reduzierte Interaktionsst{\"a}rke im Vergleich zum Wildtyp. Dies unter-streicht einmal mehr die als hypomorph beschriebene Mutation des Proteins. Das dritte Projekt, die angestrebte Strukturaufkl{\"a}rung des RAD51C-Proteins erwies sich als schwierig. Eine f{\"u}r eine Kristallisation ausreichende Proteinmenge konnte, weder im E. coli-System noch in Insektenzellen oder in Co-Expression mit seinem Interaktionspartner XRCC3, isoliert und aufgereinigt werden. Elektro-phoretische Mobility Shift Assays des CX3-Proteinkomplexes mit DNA-Strukturen (ssDNA, Open Fork, 3'-/ 5'-{\"U}berhang-Struktur), zeigten eine Bevorzugung des 3'-{\"U}berhang-DNA-Substrates. Diese Art der Analyse k{\"o}nnte in weiterf{\"u}hrenden Analysen zur Absch{\"a}tzung der Auswirkung von Patientenmutationen herangezogen werden. bb},
  subject      = {Fanconi An{\"a}mie},
  language  = {de}
}
@phdthesis{Czakai2015,
  author    = {Czakai, Kristin Bernadette},
  title     = {Interaktionen des humanpathogenen Pilzes Aspergillus fumigatus mit dem angeborenen Immunsystem und Thrombozyten},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-117496},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {Pilze sind in unserer Umwelt allgegenw{\"a}rtig und besiedeln im Fall von Candida albicans (C. albicans) sogar bei {\"u}ber 50\% der Menschen die Schleimh{\"a}ute, w{\"a}hrend Sporen von Aspergillus fumigatus (A. fumigatus) t{\"a}glich {\"u}ber die Atmung in die Lunge des Menschen gelangen. Dennoch sind Erkrankungen, die durch diese zwei Pilze ausgel{\"o}st werden, bei gesunden Menschen selten. Ist jedoch das Immunsystem beeintr{\"a}chtigt, k{\"o}nnen diese Pilze zu systemischen und damit lebensbedrohlichen Erkrankungen wie der invasiven Aspergillose und der systemischen Candidiasis f{\"u}hren. F{\"u}r eine Verbesserung der Behandlung solcher Infektionen ist das genaue Verst{\"a}ndnis der Immunabwehrmechanismen entscheidend. Da A. fumigatus {\"u}ber die Lunge in den K{\"o}rper gelangt, wurden in dieser Arbeit die h{\"a}ufigsten Immunzellen der Lunge, die Makrophagen, und deren Immunantwort auf A. fumigatus untersucht. Parallel hierzu wurden dendritische Zellen (DCs) verwendet, die als Br{\"u}cke zwischen dem angeborenen und adaptiven Immunsystem wirken. Ein besonderes Augenmerk wurde hierbei auf A. fumigatus induzierte Genexpressions{\"a}nderungen und deren Regulationsmechanismen gelegt. Dabei wurden kurze, regulatorische RNAs, die sogenannten miRNAs, untersucht, die eine wichtige Rolle in der post-transkriptionalen Genregulation spielen. Bislang ist nur wenig {\"u}ber die miRNA-abh{\"a}ngigen Genregulationen in DCs, die auf eine Infektion mit A. fumigatus oder C. albicans reagieren, bekannt. Um alle durch A. fumigatus und C. albicans regulierten miRNAs zu identifizieren, wurden DCs mit A. fumigatus und C. albicans ko-kultiviert und anschließend eine Komplettsequenzierung der kurzen RNAs durchgef{\"u}hrt. Die Pilz-spezifische Induktion der miRNA-Regulation wurde zudem mit der miRNA-Regulation durch den bakteriellen Zellwandbestandteil Lipopolysaccharid verglichen. Durch die Stimulation mit Keimschl{\"a}uchen von A. fumigatus wurden die miRNAs miR-132-3p/5p, miR-155-5p, miR129-2-3p, miR-129-5p, miR-212-3p/5p und miR-9-5p in DCs induziert. Diese wurden ebenfalls durch C. albicans induziert, zudem noch die miRNAs miR-147a und miR-147b. Spezifisch f{\"u}r A. fumigatus war die Regulation der miR-129-2-3p. Neben dem miRNA-Profiling wurde auch das mRNA-Transkriptom {\"u}ber Microarrays analysiert und dadurch 18 potentielle Zielgene der Pilz-induzierten miRNAs identifiziert. Neben den Elementen der Translationsregulation wurden auch die Transkriptionsfaktoren untersucht. Als einziger unter den 60 regulierten Transkriptionsfaktoren zeigte KLF4 eine ver{\"a}nderte Expressionsrichtung in DCs, die mit Pilzen oder LPS behandelt waren. W{\"a}hrend die Stimulation mit LPS die Expression von KLF4 induzierte, wurde es durch die Pilze A. fumigatus und C. albicans reprimiert. In einer Untersuchung der unterschiedlichen A. fumigatus-Rezeptoren, wurde deren Einfluss auf die KLF4-Regulation gezeigt. W{\"a}hrend TLR4-Liganden KLF4 induzierten, f{\"u}hrten Liganden, die an die Rezeptoren TLR2/TLR1 und Dectin-1 binden, zu einer Reduktion von KLF4. Nach einem erfolgreich etablierten KLF4-knock-down mittels RNA-Interferenz wurden KLF4-Zielgene untersucht. W{\"a}hrend kein bzw. nur ein geringer Effekt auf die Genexpression von CCL2, RANTES, CXCL10 und TNF beobachtet wurde, sorgte der KLF4 knock-down f{\"u}r eine hoch signifikante Reduktion der IL6-Genexpression in LPS-stimulierten DCs. Um die KLF4-Regulation weiter zu untersuchen, wurde zudem eine weitere Zellpopulation des angeborenen Immunsystems, die Makrophagen, verwendet. Auch hier wurde die Immunantwort gegen A. fumigatus analysiert. Zudem wurde die Rolle der Thrombozyten als Immunmediatoren betrachtet. Zuerst wurde ein Zytokinprofil des pl{\"a}ttchenreichen Plasmas (PRP), das mit A. fumigatus stimuliert wurde, erstellt. In diesem konnte nur RANTES in hoher Konzentration nachgewiesen werden. Daraufhin wurde der Einfluss von PRP auf die Reifung von DCs, die Phagozytosef{\"a}higkeit von Makrophagen und DCs sowie der Einfluss von DCs und Makrophagen auf die metabolische Aktivit{\"a}t von A. fumigatus in An- und Abwesenheit von pl{\"a}ttchenreichem Plasma untersucht. Es konnte eine gering verst{\"a}rkte Reifung der DCs durch PRP gezeigt werden. Isolierte Thrombozyten konnten die Phagozytose von DCs steigern, w{\"a}hrend Makrophagen durch PRP verst{\"a}rkt Konidien phagozytierten. In einem genomweiten Transkriptomprofiling wurde die Immunantwort von DCs und Makrophagen verglichen. Zudem wurde untersucht, wie PRP die Immunantwort dieser Immunzellen beeinflusst. Es wurden 2 bzw. 24 Gene identifiziert, die signifikant in A. fumigatus-stimulierten DCs und Makrophagen reguliert waren. Hierbei wurde gezeigt, dass KLF4 durch die Zugabe von PRP herabreguliert wurde. Das zuvor beschriebene Zielgen IL6 wurde durch PRP in A. fumigatus-stimulierten DCs gegen{\"u}ber stimulierten DCs ohne PRP deutlich reduziert, wodurch sich eine immunmodulatorische F{\"a}higkeit des PRP zeigte. Die Induktion von IL-6, weiteren Zytokinen und der Reifemarker durch A. fumigatus in DCs wurden zudem in einem Booleschen Modell simuliert. Dieses Modell soll in Zukunft Vorhersagen {\"u}ber experimentelle Ergebnisse und dadurch eine optimale Versuchsvorbereitung erm{\"o}glichen.},
  subject      = {Aspergillus fumigatus},
  language  = {de}
}
@phdthesis{Bruttel2015,
  author    = {Bruttel, Valentin Stefan},
  title     = {Soluble HLA-G binds to dendritic cells which likely suppresses anti-tumour immune responses in regional lymph nodes in ovarian carcinoma},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-127252},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {Zusammenfassung Einleitung HLA-G, ein nicht-klassisches HLA bzw. MHC Klasse Ib Molek{\"u}l, kann sowohl als membrangebundenes als auch als l{\"o}sliches Molek{\"u}l verschiedenste Immunzellpopulationen effektiv inhibieren. Unter physiologischen Bedingungen wird HLA-G vor allem in der Plazenta exprimiert, wo es dazu beitr{\"a}gt den semiallogenen Embryo vor einer Abstoßung durch das m{\"u}tterliche Immunsystem zu besch{\"u}tzen. Außerdem wird HLA-G in einer Vielzahl von Tumoren wie zum Beispiel in Ovarialkarzinomen {\"u}berexprimiert. Ziel dieser Arbeit war es besonders die Rolle von l{\"o}slichem HLA-G im Ovarialkarzinom und die Expression von HLA-G in verschiedenen Subtypen des Ovarialkarzinoms genauer zu untersuchen. Ergebnisse Anhand eines Tissue Microarrays wurde best{\"a}tigt dass HLA-G unter physiologischen Bedingungen nur in sehr wenigen Geweben wie Plazenta oder Testes exprimiert wird. Außerdem wurden erstmals auch im Nebennierenmark hohe Expressionslevel detektiert. Im Gegensatz zur physiologischen Expression wurde HLA-G in ser{\"o}sen, muzin{\"o}sen, endometrioiden und Klarzellkarzinomen und somit in Tumoren aller untersuchten Subtypen des Ovarialkarzinoms detektiert. Am h{\"a}ufigsten war HLA-G in hochgradigen ser{\"o}sen Karzinomen {\"u}berexprimiert. Hier konnte gezeigt werden dass auf Genexpressionslevel in Ovarialkarzinomen die Expression des immunsuppressiven HLA-G mit der Expression von klassischen MHC Molek{\"u}len wie HLA-A, -B oder -C hochsignifikant korreliert. Außerdem konnte in Aszitesproben von Patientinnen mit Ovarialkarzinomen hohe Konzentrationen von l{\"o}slichem HLA-G nachgewiesen werden. Auch auf metastasierten Tumorzellen in regionalen Lymphknoten war HLA-G nachweisbar. {\"U}berraschenderweise wurde aber besonders viel HLA-G auf Dendritischen Zellen in Lymphknoten detektiert. Da in Monozyten und Dendritischen Zellen von gesunden Spendern durch IL-4 oder IL-10 im Gegensatz zu Literatur keine Expression von HLA-G induzierbar war, untersuchten wir ob Dendritische Zellen l{\"o}sliches HLA-G binden. Es konnte gezeigt werden, dass besonders Dendritische Zellen die in Gegenwart von IL-4, IL-10 und GM-CSF aus Monozyten generiert wurden (DC-10) effektiv l{\"o}sliches HLA-G {\"u}ber ILT Rezeptoren binden. In Abh{\"a}ngigkeit von ihrer Beladung mit HLA-G hemmen auch fixierte DC-10 Zellen noch die Proliferation von zytotoxischen CD8+ T Zellen. Zudem wurden regulatorische T Zellen induziert. Schlussfolgerungen Besonders in den am h{\"a}ufigsten diagnostizierten hochgradigen ser{\"o}sen Ovarialkarzinomen ist HLA-G in den meisten F{\"a}llen {\"u}berexprimiert. Durch die Expression immunsuppressiver MHC Klasse Ib Molek{\"u}le wie HLA-G k{\"o}nnen wahrscheinlich auch Tumore wachsen, die noch klassische MHC Molek{\"u}le exprimieren und aufgrund ihrer Mutationslast eigentlich vom Immunsystem erkannt und eliminiert werden m{\"u}ssten. L{\"o}sliches HLA-G k{\"o}nnte zudem lokal Immunantworten gegen Tumorantigene unterdr{\"u}cken indem es an Dendritische Zellen in regionalen Lymphknoten bindet. Diese Zellen pr{\"a}sentieren nomalerweise zytotoxischen T Zellen Tumorantigene und spielen daher eine entscheidende Rolle in der Entstehung von protektiven Immunantworten. Mit l{\"o}slichem HLA-G beladene Dendritische Zellen hemmen jedoch die Proliferation von CD8+ T Zellen und induzieren regulatorische T Zellen. Dadurch k{\"o}nnten Ovarialkarzinome "aus der Ferne" auch in metastasenfreien Lymphknoten die Entstehung von gegen den Tumor gerichteten Immunantworten unterdr{\"u}cken. Dieser erstmals beschriebene Mechanismus k{\"o}nnte auch in anderen malignen Erkrankungen eine Rolle spielen, da l{\"o}sliches HLA-G in einer Vielzahl von Tumorindikationen nachgewiesen wurde.},
  subject      = {HLA-G},
  language  = {en}
}
@phdthesis{Kuger2015,
  author    = {Kuger, Sebastian},
  title     = {Radiosensibilisierung humaner Tumorzelllinien unterschiedlicher Entit{\"a}ten durch den dualen PI3K/mTOR-Inhibitor NVP-BEZ235 alleine oder in Kombination mit dem MEK-Inhibitor AZD6244: Einfluss des Behandlungsschemas und der Hypoxie},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-126715},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {Eine wichtige Standardtherapie in der modernen Behandlung von Krebserkrankungen ist die Strahlentherapie, in welcher Tumorzellen mittels ionisierender Strahlung gesch{\"a}digt und abget{\"o}tet werden. Dabei soll die Sch{\"a}digung des umgebenden Normalgewebes m{\"o}glichst gering gehalten und trotzdem eine maximale Sch{\"a}digung des Tumorgewebes erreicht werden. Deshalb sind neue Strategien zur Steigerung der Radiosensitivit{\"a}t des Tumorgewebes sehr wichtig, die es erlauben, bei gleicher Dosis eine verst{\"a}rkte Strahlenantwort im Tumorgewebe zu erreichen. Hier kommen zunehmend sog. Radiosensibilisatoren zum Einsatz, die unter anderem onkogene Signalwege in den Tumorzellen inhibieren. Der PI3K/Akt/mTOR Signalweg stellt hierbei einen wichtigen Ansatzpunkt dar, da er in vielen Tumorentit{\"a}ten dereguliert vorliegt und diese Signalkaskade bekanntermaßen einen Einfluss auf die zellul{\"a}re Strahlensensitivit{\"a}t hat. Obwohl es f{\"u}r diesen Signalweg schon eine Reihe von Inhibitoren gibt, f{\"u}r die bereits neben einer anti-proliferativen Wirkung auch ein radiosensibilisierender Effekt nachgewiesen wurde (z.B. Wortmannin und Rapamycin), machten eine geringe Spezifit{\"a}t, starke Nebenwirkungen und negative R{\"u}ckkopplungsmechanismen im Signalweg, die die Wirkung des Inhibitors kompensieren, die Entwicklung neuer Inhibitoren notwendig. Das Imidazoquinolinderivat NVP-BEZ235 inhibiert den PI3K/Akt/mTOR Signalweg an mehreren Stellen gleichzeitig, indem es kompetitiv zu ATP das katalytische Zentrum von PI3K und mTOR blockiert. F{\"u}r diesen kleinmolekularen, dualen Inhibitor gibt es bereits erste vielversprechende Forschungsergebnisse hinsichtlich einer radiosensibilisierenden Wirkung, allerdings sind die zugrunde liegenden molekularbiologischen Mechanismen noch nicht vollst{\"a}ndig gekl{\"a}rt. Deshalb war das Ziel der vorliegenden Dissertation, in drei Teilprojekten mehrere Aspekte der NVP-BEZ235-induzierten Radiosensibilisierung aufzukl{\"a}ren: a) Einfluss des Behandlungsschemas f{\"u}r NVP-BEZ235 in vier Glioblastomzelllinien mit unterschiedlichem PTEN und TP53 Mutationsstatus, b) Einfluss der Sauerstoffversorgung (Hypoxie, Normoxie, reoxygeniert nach Bestrahlung) auf die strahlensensibilisierende Wirkung von NVP-BEZ235 in zwei Mammakarzinomzelllinien, c) gleichzeitige Inhibierung des MAPK Signalwegs durch AZD6244 und der PI3K/Akt/mTOR Signalkaskade durch NVP-BEZ235 in zwei Zelllinien mit unter-schiedlichem Mutationsstatus aus verschiedenen Tumorentit{\"a}ten, um synergistische Effekte zu untersuchen. Um diese Fragestellungen zu beantworten, wurde im Rahmen - 142 - der Dissertation eine Auswahl an humanen Tumorzelllinien mit unterschiedlich deregulierten Signalwegen bearbeitet. Dabei wurde die Expression von Schl{\"u}sselproteinen der MAPK/Erk und der PI3K/Akt/mTOR Signalwege analysiert und mit zellbiologischen Daten verschiedener ph{\"a}notypischer Endpunkte nach Inhibitor Behandlung und Bestrahlung integriert (Proliferationsrate, klonogenes {\"U}berleben, Zellzyklusaberrationen, DNS-Sch{\"a}den und -Reparatur, Zelltod und Autophagie). Im Teilprojekt zum Behandlungsschema der NVP-BEZ235 Inhibierung und Bestrahlung konnte in vier Glioblastomzelllinien mit Behandlungsschema I (NVP-BEZ235 Behandlung 24 Stunden vor Bestrahlung) kein radiosensibilisierender Effekt hinsichtlich klonogenem {\"U}berleben nachgewiesen werden, wohingegen Behandlungsschema II (NVP-BEZ235 Behandlung 1 h vor und im Anschluss an die Bestrahlung) unabh{\"a}ngig vom Mutationsstatus in allen vier Zelllinien eine starke Radiosensibilisierung bewirkte. Auf molekularer Ebene war zwischen beiden Behandlungsschemata f{\"u}r das antiapoptotische Protein Akt ein großer Unterschied zu beobachten, welches bei Behandlung nach Schema I zum Zeitpunkt der Bestrahlung {\"u}beraktiviert, nach Behandlung mit Schema II hingegen inhibiert war. Weiterhin resultierte Behandlungsschema I in einem erh{\"o}hten Anteil der Zellen in der radioresistenteren G1-Phase des Zellzyklus zum Zeit-punkt der Bestrahlung. Behandlungsschema II f{\"u}hrte hingegen nach Bestrahlung zu einer verminderten Expression des Reparaturproteins Rad51 und damit zu verminderter DNS-Schadensreparatur und schließlich zu einem stabilen Arrest in der G2/M-Phase des Zellzyklus sowie zu verst{\"a}rkter Apoptose (erh{\"o}hte Spaltung von PARP, erh{\"o}hter Anteil hypodiploider Zellen). Somit zeigen diese Ergebnisse, dass unabh{\"a}ngig vom PTEN und TP53 Mutationsstatus eine Radiosensibilisierung nur durch das Behandlungsschema II erreicht werden konnte. Ferner deuten die Ergebnisse der Proteinexpression darauf hin, dass durch NVP-BEZ235 ein negativer R{\"u}ckkopplungsmechanismus ausgel{\"o}st wird, wodurch die PI3K/Akt/mTOR Signalkaskade 24h nach Zugabe des Inhibitors aktiviert und synergistische Effekte mit ionisierender Bestrahlung aufgehoben wurden. Im Teilprojekt zur Abh{\"a}ngigkeit der NVP-BEZ235 Inhibition vom Sauerstoffgehalt wurden in den beiden Brustkrebszelllinien MCF-7 (ER-positiv) und TN MDA-MB-231 (TP53 mutiert) normoxische, hypoxische und nach Bestrahlung reoxygenierte Kulturbedingungen im Hinblick auf die Koloniebildungsf{\"a}higkeit nach NVP-BEZ235 Behandlung und Bestrahlung untersucht. Die beobachtete Radiosensibilisierung war unter allen getesteten Bedingungen auf gleichem Niveau. In beiden Zelllinien bewirkte NVP-BEZ235 eine Inhibition des antiapoptotischen HIF-1α Proteins, eine stabile Inaktivierung des PI3K/Akt/mTOR Signalweges und eine Aktivierung der Autophagie. Nach Bestrahlung waren zudem erh{\"o}hte residuale DNS-Sch{\"a}den und ein stabiler Arrest in der G2/M-Phase des Zellzyklus unter allen Oxygenierungsbedingungen in beiden Zelllinien zu beobachten. Eine Apoptose Induktion (Spaltung von PARP, hypodiploide Zellen) trat nur in der TP53 wildtypischen MCF-7 Zelllinie nach NVP-BEZ235 Behandlung auf. Somit konnte in beiden Zelllinien in allen pathophysiologisch relevanten Oxygenierungszust{\"a}nden eine sauerstoffunabh{\"a}ngige Radiosensibilisierung durch NVP-BEZ235 gezeigt werden. Der bisher nicht erforschte Aspekt zur synergistischen Wirkung des MEK Inhibitors AZD6244 und des dualen PI3K/Akt/mTOR Inhibitors NVP-BEZ235 nach Bestrahlung wurde an der Glioblastomzelllinie SNB19 und der Lungenkarzinomzelllinie A549 anhand der Koloniebildungsf{\"a}higkeit der behandelten Zellen untersucht. Eine Behandlung mit dem MEK Inhibitor bewirkte lediglich eine moderate Radiosensibilisierung, wohin-gegen der duale PI3K/Akt/mTOR Inhibitor beide Zelllinien in st{\"a}rkerem Maße sensibilisierte. Eine Kombination beider Inhibitoren resultierte bei keiner Zelllinie in einer Verst{\"a}rkung der durch NVP-BEZ235 induzierten Radiosensibilisierung. Eine m{\"o}gliche Erkl{\"a}rung f{\"u}r die fehlende Synergie im Bezug auf die Radiosensibilisierung k{\"o}nnen die gegens{\"a}tzlichen Effekte der beiden Inhibitoren auf den Zellzyklus sein. Auf Proteinebene f{\"u}hrte eine simultane Behandlung mit beiden Substanzen zur Inhibition beider Signalwege. Dar{\"u}ber hinaus war in SNB19 Zellen eine verst{\"a}rkte Dephosphorylierung von Rb und ein erh{\"o}hter Anteil an G1-Phase Zellen bei kombinierter Gabe der Inhibitoren zu beobachten. Im Rahmen dieser Arbeit konnte somit die radiosensibilisierende Wirkung von NVP-BEZ235 in Abh{\"a}ngigkeit vom Behandlungsschema gezeigt werden. Ferner wurde nachgewiesen, dass die Radiosensibilisierung unabh{\"a}ngig von der Sauerstoffversorgung sowie von den PTEN und TP53 Mutationsstatus der Tumorzellen ist. Die kombinierte Inhibition der MAPK und PI3K/Akt/mTOR Signalwege resultierte zwar in einem verst{\"a}rkten zytostatischen, aber nicht in einem verst{\"a}rkten radiosensibilisierenden Effekt. Da allerdings eine große Anzahl verschiedener Inhibitoren der MAPK/Erk und der PI3K/Akt/mTOR Signalkaskade verf{\"u}gbar sind, sollte die kombinatorische Inhibition dieser Signalwege systematisch weiter verfolgt werden. Die vorliegende Arbeit liefert auch weitere grundlegende Erkenntnisse zu den molekularen Mechanismen der Radiosensibilisierung durch NVP-BEZ235, die auch auf Verkn{\"u}pfungen und Wechselwirkungen mit anderen als den bisher bekannten Proteinen hindeuten, die f{\"u}r jeden Inhibitor aufgekl{\"a}rt werden m{\"u}ssen, um eine effektive radiosensibilisierende Wirkung vorher-sagen zu k{\"o}nnen.},
  subject      = {Strahlensensibilisator},
  language  = {de}
}
@phdthesis{Bettaga2014,
  author    = {Bettaga, Noomen},
  title     = {Bedeutung der NO-sensitiven Guanylyl Cyclase bei der Angiogenese und der Arteriogenese in der Maus},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-111284},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {Stickstoffmonoxid (NO) spielt eine wichtige Rolle bei Gef{\"a}ßremodelling-Prozessen wie Angiogenese und Arteriogenese. Die NO-Synthese im Gef{\"a}ßsystem wird haupts{\"a}chlich durch die endotheliale NO-Synthase (eNOS) gew{\"a}hrleistet. Sie kann durch verschiedene Faktoren wie Scherkr{\"a}fte und Zytokine wie der vaskul{\"a}re endotheliale Wachstumsfaktor (VEGF) reguliert werden. VEGF ist ein wichtiger Stimulator der Angiogenese und wird w{\"a}hrend dieses Prozesses hochreguliert. Die meisten physiologischen Effekte von NO werden durch die NO-sensitive Guanylyl-Cyclase (NO-GC) vermittelt. Als Hauptrezeptor f{\"u}r NO produziert die NO-GC den sekund{\"a}ren Botenstoff cyklisches Guanosinmonophosphat (cGMP) und f{\"u}hrt dadurch zur Stimulation der verschiedenen Effektoren wie z.B. der PKG. Ob die Wirkung von NO in Angiogenese und Arteriogenese ebenfalls durch NO-GC vermittelt wird, war bis zum Beginn dieser Arbeit noch unklar. Die NO-GC besteht aus zwei Untereinheiten (α und ß). Die Deletion der ß1-Untereinheit in M{\"a}usen resultiert in einer vollst{\"a}ndigen Knockout Maus (GCKO). Mithilfe des Cre-LoxP-Systems wurden zus{\"a}tzlich zellspezifische Knockout-M{\"a}use f{\"u}r glatte Muskelzellen (SMC-GCKO) und Endothelzellen (EC-GCKO) generiert. Um die Rolle der NO-GC in der Angiogenese und Arteriogenese zu untersuchen, wurden drei gut etablierte Methoden benutzt. Im ersten Teil des Projekts sollte die Expression der NO-GC in Endothelzellen untersucht werden. Zu diesem Zweck wurde die reverse Transkriptase-Polymerase-Kettenreaktion (RT-PCR) benutzt. Die Ergebnisse zeigen, dass die NO-GC in Endothelzellen der Lunge nur {\"a}ußerst gering wenig exprimiert ist. Durch den Aortenring-Assay wurde eine Rolle der NO-GC bei der VEGF-vermittelten Angiogenese festgestellt. Dabei zeigte sich eine st{\"a}rkere Angiogeneserate bei globaler Abwesenheit der NO-GC. Bei Fehlen der NO-GC ausschließlich in Endothelzellen zeigte sich kein Unterschied in den aussprossenden Aorten im Vergleich zu den Kontroll-Tieren. Dies zeigt, dass die NO-GC in Endothelzellen sehr wahrscheinlich keine Rolle bei der VEGF-vermittelten Angiogenese spielt. Im zweiten Teil wurde die Rolle der NO-GC bei der Angiogenese in einem in vivo-Modell untersucht. In dem Modell der Sauerstoff-induzierten-Retinopathie zeigten die GCKO-M{\"a}use eine verringerte Vaso-Obliteration, eine verlangsamte Angiogenese und eine erh{\"o}hte Tuft-Bildung. {\"A}hnliche Ergebnisse wurden bei den SMC-GCKO-Tieren beobachtet. EC-GCKO-M{\"a}use zeigten eine gegen{\"u}ber den Kontroll-Tieren unver{\"a}nderte Vaso-Obliteration, Angiogeneserate und Tuft-Bildung. Diese Ergebnisse lassen darauf schließen, dass die NO-GC in Endothelzellen keine Rolle spielt. Immunfluoreszenz-Aufnahmen zeigten die Expression von NO-GC in Perizyten der Gef{\"a}ßkapillaren der Mausretina. Daher k{\"o}nnte die NO-GC in diesem Zelltyp letztendlich f{\"u}r die Effekte bei den GCKO- und SMC-GCKO-Tieren verantwortlich sein. Im letzten Teil dieser Arbeit wurde eine Versuchsreihe unter Anwendung des Hinterlauf-Isch{\"a}mie-Modells durchgef{\"u}hrt. Hierbei entwickelten die Pfoten aller GCKO- und teilweise der SMC-GCKO-Tiere nach der Ligation der Femoralarterie eine Nekrose. Die Regeneration der Hinterl{\"a}ufe der EC-GCKO-Tiere nach der Operation verlief normal. Diese Ergebnisse schließen eine bedeutende Rolle der NO-GC in Endothelzellen aus, zeigen allerdings, dass die NO-GC in den glatten Muskelzellen essentiell f{\"u}r den Arteriogenese-Prozess ist. Zusammengefasst f{\"u}hrt die Deletion der NO-GC in glatten Muskelzellen und wahrscheinlich auch in Perizyten zur einer verlangsamten Angiogenese und Inhibierung der Arteriogenese.},
  subject      = {Guanylylcyclase},
  language  = {de}
}
@phdthesis{Grimmig2015,
  author    = {Grimmig, Tanja Maria},
  title     = {Immunity, Inflammation and Cancer: The role of Foxp3, TLR7 and TLR8 in gastrointestinal cancer},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-125248},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {Regulatory T cells (Treg) expressing the transcription factor forkhead box protein P3 (Foxp3) have been demonstrated to mediate evasion from anti-tumor immune responses during tumor progression. Moreover, Foxp3 expression by tumor cells themselves may allow them to counteract effector T cell responses, resulting in a survival benefit of the tumor. For gastrointestinal cancers, in particular pancreatic and colorectal cancer (CRC), the clinical relevance of Foxp3 is not clear to date. Therefore the aim of this study was to analyze its impact in CRC and pancreatic cancer. To determine the relevance of Foxp3 for tumor progression and patient survival, gene and protein analysis of human pancreatic and colon cancer cell lines as well as tumor tissues from patients with CRC was performed. The results derived from the patients with CRC were correlated with clinicopathological parameters and patients' overall survival. Cancer cell mediated Foxp3 expression in vitro was demonstrated in human pancreatic cancer cell lines PANC1, PaCa DD 135, PaCa DD 159 and PaCa DD 185 as well as in human colon cancer cell lines SW480 and SW620. Additionally, Foxp3 expressing cancer cells were found in ex vivo tumor tissue samples of patients with CRC. The percentage of Foxp3+ cancer cells increased from stages UICC I/II to UICC III/IV compared to normal tissue. Moreover, high tumor cell mediated Foxp3 expression was associated with poor prognosis compared to patients with low Foxp3 expression. In contrast, low and high Foxp3 level in tumor infiltrating Treg cells demonstrated no significant differences in patients' overall survival. Correlation analysis demonstrated a significant association of Foxp3 cancer cell expression with the expression of immunosuppressive cytokines IL-10 and TGF-β. These findings suggest that Immunosuppressive cytokines such as IL-10 and TGF-β released by rather Foxp3+ cancer cells than Foxp3+ Treg cells may inhibit the activation of naive T cells, hence limiting antitumor immune responses and favoring tumorigenesis and progression. Chronic inflammation has been shown to be an important epigenetic and environmental factor in numerous tumor entities. Recent data suggest that tumorigenesis and tumor progression may be associated with inflammation-triggered activation of Toll-like receptors (TLR). In this study, the specific impact of both TLR7 and TLR8 expression and signaling on tumor cell proliferation and chemoresistance is analyzed in inflammation linked CRC and pancreatic cancer. By gene and protein expression analysis of human pancreatic and colon cancer cell lines TLR7 and TLR8 expression was determined in vitro. Additionally, expression of TLR7/TLR8 in UICC stage I-IV pancreatic cancer, chronic pancreatitis and normal pancreatic tissue was examined. For in vitro/in vivo studies TLR7/TLR8 overexpressing PANC1 cell lines were generated and analyzed for effects of TLR expression and stimulation on tumor cell proliferation and chemoresistance. Cancer cell mediated TLR7 and TLR8 expression in vitro was demonstrated in human colon cancer cell lines SW480, SW620 and HT-29 as well as in primary pancreatic cancer cell lines PaCa DD 135, PaCa DD 159 and PaCa DD 185. Additionally, TLR7 and TLR8 expressing tumor cells were found in ex vivo tissue samples of patients with pancreatic cancer and chronic pancreatitis. Significantly elevated expression levels of TLR7 and TLR8 were found in advanced tumor stages (UICC III) compared to early tumor stages (UICC II) and chronic pancreatitis. No or occasionally low expression was detected in normal pancreatic tissue. In contrast to the tissues from patients with pancreatic cancer or chronic pancreatitis, established pancreatic tumor cell lines express only very low levels of TLR7 and TLR8. Therefore, for in vitro and xenograft studies TLR7 or TLR8 overexpressing PANC1 cells were generated. Proliferation promoting effects of TLR7 and TLR8 expression and stimulation with R848 were detected in vitro. Additionally, increased tumor growth of TLR expressing PANC1 cells was demonstrated in subcutaneously injected Balb/c nude mice. Interestingly, activation of TLR7 or TLR8 induced not only an increase in tumor cell proliferation but also a strong chemoresistance of PANC1 cells against 5-fluorouracil (5-FU). Moreover, treatment with R848 resulted in elevated expression levels of NF-κB, COX-2 and inflammatory cytokines IL-1β, IL-8 and TNF-α, suggesting TLR7/8 signaling to contribute to an inflammatory, anti-apoptotic and proliferation promoting tumor microenvironment. These findings emphasize the particular role of TLR7 and TLR8 in inflammation related cancers and their relevance as potential targets for cancer therapy.  },
  subject      = {Bauchspeicheldr{\"u}senkrebs},
  language  = {en}
}
@phdthesis{Dusik2015,
  author    = {Dusik, Verena},
  title     = {Immunhistochemische und funktionelle Charakterisierung der Mitogen-aktivierten Proteinkinase p38 in der inneren Uhr von Drosophila melanogaster},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-124636},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {Circadianes und Stress-System sind zwei physiologische Systeme, die dem Organismus helfen sich an Ver{\"a}nderungen ihrer Umwelt anzupassen. W{\"a}hrend letzteres spontane und schnelle Antworten auf akute, unvorhersehbare Umweltreize liefert, sagt das circadiane System t{\"a}glich wiederkehrende Ereignisse vorher and bereitet den Organismus so vorzeitig auf diese nahende Umweltver{\"a}nderung vor. Dennoch, trotz dieser unterschiedlichen Reaktionsmechanismen agieren beide Systeme nicht komplett autonom. Studien der vergangen Jahre belegen vielmehr eine Interaktion beider Systeme. So postulieren sie zum einem Unterschiede in der Stressantwort in Abh{\"a}ngigkeit von der Tageszeit zu der der Reiz auftritt und weisen zugleich auf eine Zunahme von gest{\"o}rten biologischen Tagesrhythmen, wie zum Beispiel Schlafst{\"o}rungen, in Folge von unkontrollierten oder exzessiven Stress hin. Ebenso liefern k{\"u}rzlich durchgef{\"u}hrte Studien an Vertebraten und Pilzen Hinweise, dass mit p38, eine Stress-aktivierte Kinase, an der Signalweiterleitung zur inneren Uhr beteiligt ist (Hayashi et al., 2003), sogar durch dieses endogene Zeitmesssystem reguliert wird (Vitalini et al., 2007; Lamb et al., 2011) und deuten damit erstmals eine m{\"o}gliche Verbindung zwischen Stress-induzierten und regul{\"a}ren rhythmischen Anpassungen des Organismus an Umweltver{\"a}nderungen an. Molekulare und zellul{\"a}re Mechanismen dieser Verkn{\"u}pfung sind bisher noch nicht bekannt. W{\"a}hrend die Rolle von p38 MAPK bei der Stress- und Immunantwort in Drosophila melanogaster gut charakterisiert ist, wurden Expression und Funktion von p38 in der inneren Uhr hingegen bislang nicht untersucht. Die hier vorliegende Arbeit hatte daher zum Ziel mittels immunhistochemischer, verhaltensphysiologischer und molekularer Methoden eine m{\"o}gliche Rolle der Stress-aktivierten Kinase im circadianen System der Fliege aufzudecken. Antik{\"o}rperf{\"a}rbungen sowie Studien mit Reporterlinien zeigen deutliche F{\"a}rbesignale in den s-LNv, l-LNv und DN1a und erbringen erstmals einen Nachweis f{\"u}r p38 Expression in den Uhrneuronen der Fliege. Ebenso scheint die Aktivit{\"a}t von p38 MAPK in den DN1a uhrgesteuert zu sein. So liegt p38 vermehrt in seiner aktiven Form in der Dunkelphase vor und zeigt, neben seiner circadian regulierten Aktivierung, zus{\"a}tzlich auch eine Inaktivierung durch Licht. 15-Minuten-Lichtpulse in der subjektiven Nacht f{\"u}hren zu einer signifikanten Reduktion von aktivierter, phosphorylierter p38 MAPK in den DN1a von Canton S Wildtypfliegen im Vergleich zu Fliegen ohne Lichtpuls-Behandlung. Aufzeichnungen der Lokomotoraktivit{\"a}t offenbaren zus{\"a}tzlich die Notwendigkeit von p38 MAPK f{\"u}r wildtypisches Timing der Abendaktivit{\"a}t sowie zum Erhalt von 24-Stunden-Verhaltensrhythmen unter konstanten Dauerdunkel-Bedindungen. So zeigen Fliegen mit reduzierten p38 Level in Uhrneuronen einen verz{\"o}gerten Beginn der Abendaktivit{\"a}t und stark verl{\"a}ngerte Freilaufperioden. In {\"U}bereinstimmung mit Effekten auf das Laufverhalten scheint dar{\"u}ber hinaus die Expression einer dominant-negativen Form von p38b in Drosophila's wichtigsten Uhrneuronen eine versp{\"a}tete nukle{\"a}re Translokation von Period zur Folge zu haben. Westernblots legen zus{\"a}tzlich einen Einfluss von p38 auf den Phosphorylierungsgrad von Period nahe und liefern damit einen m{\"o}gliche Erkl{\"a}rung f{\"u}r den versp{\"a}teten Kerneintritt des Uhrproteins. Abschließende St{\"u}tzung der Westernblotergebnisse bringen in vitro Kinasenassays und deuten auf p38 als eine potentielle „Uhrkinase" hin, welche auch in vivo Period an Serin 661 sowie weiteren potentiellen Phosphorylierungsstellen phosphorylieren k{\"o}nnte. Zusammengenommen deuten die Ergebnisse der hier vorliegenden Arbeit eindeutig auf eine bedeutende Rolle von p38, neben dessen Funkion im Stress-System, auch im circadianen System der Fliege hin und offenbaren damit die M{\"o}glichkeit, dass p38 als Schnittstelle zwischen beider Systeme fungiert.},
  subject      = {Taufliege},
  language  = {de}
}
@phdthesis{Frank2015,
  author    = {Frank, Nicolas Clemens},
  title     = {Lokale axonale Wirkungen der CNTF-STAT3 Signalkaskade in Motoneuronen der pmn Maus - einem Mausmodel f{\"u}r die Amyotrophe Lateralsklerose},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-121065},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {1. Zusammenfassung W{\"a}hrend der Embryogenese und nach Verletzungen von Nerven regulieren neurotrophe Faktoren Signalwege f{\"u}r Apoptose, Differenzierung, Wachstum und Regeneration von Neuronen. In vivo Experimente an neugeborenen Nagern haben gezeigt, dass der Verlust von Motoneuronen nach peripherer Nervenl{\"a}sion durch die Behandlung mit GDNF, BDNF, und CNTF reduziert werden kann In der pmn-Mausmutante, einem Modell f{\"u}r die Amyotrophe Lateralsklerose, f{\"u}hrt die Gabe von CNTF, nicht aber von GDNF zu einem verz{\"o}gerten Krankheitsbeginn und einem verlangsamten Fortschreiten der Motoneuronendegeneration. Ausl{\"o}ser der Motoneuronendegeneration in der pmn-Maus ist eine Mutation im Tubulin spezifischen Chaperon E (Tbce) Gen, das f{\"u}r eines von f{\"u}nf Tubulin spezifischen Chaperonen (TBCA-TBCE) kodiert und an der Bildung von -Tubulinheterodimeren beteiligt ist. Diese Arbeit sollte dazu beitragen, die CNTF-induzierten Signalwege zu entschl{\"u}sseln, die sich lindernd auf den progredienten Verlauf der Motoneuronendegeneration in der pmn-Maus auswirken. Prim{\"a}re pmn mutierte Motoneurone zeigen ein reduziertes Axonwachstum und eine erh{\"o}hte Anzahl axonaler Schwellungen mit einer anomalen H{\"a}ufung von Mitochondrien - ein fr{\"u}hes Erkennungsmerkmal bei ALS-Patienten. Die Applikation von CNTF nicht aber von BDNF oder GDNF, kann in vitro die beobachteten Wachstumsdefekte und das bidirektionale axonale Transportdefizit in pmn mutierten Motoneurone verhindern. Aus {\"a}lteren Untersuchungen war bekannt, dass CNTF {\"u}ber den dreiteiligen transmembranen Rezeptorkomplex, bestehend aus CNTFR, LIFR und gp130, Januskinasen aktiviert, die STAT3 an Tyrosin 705 phosphorylieren (pSTAT3Y705). Ich konnte beobachten, dass axonales fluoreszenzmarkiertes pSTAT3Y705 nach CNTF-Gabe nicht retrograd in den Nukleus transportiert wird. Stattdessen f{\"u}hrt die CNTF-induzierte Phosphorylierung von STAT3 an Tyrosin 705 zu einer transkriptionsunabh{\"a}ngigen lokalen Reaktion im Axon. Diese pSTAT3Y705 abh{\"a}ngige Reaktion ist notwendig und ausreichend, um das reduzierte Axonwachstum pmn mutierter Motoneurone zu beheben. Wie die Kombination einer CNTF Behandlung mit dem shRNA vermittelten knock-down von Stathmin in pmn mutierten Motoneuronen zeigt, zielt die CNTF-STAT3 Signalkaskade auf die Stabilisierung axonaler Mikrotubuli ab und wirkt sich positiv auf die anterograde und retrograde Mobilit{\"a}t von axonalen Mitochondrien aus. Interessanter Weise konnte ich außerdem feststellen, dass eine akute Gabe von CNTF das mitochondriale Membranpotential in Axonen prim{\"a}rer pmn mutierter und wildtypischer Motoneurone erh{\"o}ht und einen Anstieg von ATP ausl{\"o}st. Meine Beobachtungen legen nahe, dass CNTF unerwarteter Weise auch eine transiente Phosphorylierung an STAT3 Serin 727 (pSTAT3S727) ausl{\"o}st, die zur anschließenden Translokation von pSTAT3S727 in Mitochondrien f{\"u}hrt. Diese Ergebnisse zeigen, dass STAT3 mehrere lokale Ziele im Axon besitzt, n{\"a}mlich axonale Mikrotubuli und Mitochondrien.},
  subject      = {Motoneuron},
  language  = {de}
}
@phdthesis{Siegl2014,
  author    = {Siegl, Christine},
  title     = {Degradation of Tumour Suppressor p53 during Chlamydia trachomatis Infections},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-108679},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {The intracellular pathogen Chlamydia is the causative agent of millions of new infections per year transmitting diseases like trachoma, pelvic inflammatory disease or lymphogranuloma venereum. Undetected or recurrent infections caused by chlamydial persistence are especially likely to provoke severe pathologies. To ensure host cell survival and to facilitate long term infections Chlamydia induces anti-apoptotic pathways, mainly at the level of mitochondria, and restrains activity of pro-apoptotic proteins. Additionally, the pathogen seizes host energy, carbohydrates, amino acids, lipids and nucleotides to facilitate propagation of bacterial progeny and growth of the chlamydial inclusion. At the beginning of this study, Chlamydia-mediated apoptosis resistance to DNA damage induced by the topoisomerase inhibitor etoposide was investigated. In the course of this, a central cellular protein crucial for etoposide-mediated apoptosis, the tumour suppressor p53, was found to be downregulated during Chlamydia infections. Subsequently, different chlamydial strains and serovars were examined and p53 downregulation was ascertained to be a general feature during Chlamydia infections of human cells. Reduction of p53 protein level was established to be mediated by the PI3K-Akt signalling pathway, activation of the E3-ubiquitin ligase HDM2 and final degradation by the proteasome. Additionally, an intriguing discrepancy between infections of human and mouse cells was detected. Both activation of the PI3K-Akt pathway as well as degradation of p53 could not be observed in Chlamydia-infected mouse cells. Recently, production of reactive oxygen species (ROS) and damage to host cell DNA was reported to occur during Chlamydia infection. Thus, degradation of p53 strongly contributes to the anti-apoptotic environment crucial for chlamydial infection. To verify the importance of p53 degradation for chlamydial growth and development, p53 was stabilised and activated by the HDM2-inhibiting drug nutlin-3 and the DNA damage-inducing compound etoposide. Unexpectedly, chlamydial development was severely impaired and inclusion formation was defective. Completion of the chlamydial developmental cycle was prevented resulting in loss of infectivity. Intriguingly, removal of the p53 activating stimulus allowed formation of the bacterial inclusion and recovery of infectivity. A similar observation of growth recovery was made in infected cell lines deficient for p53. As bacterial growth and inclusion formation was strongly delayed in the presence of activated p53, p53-mediated inhibitory regulation of cellular metabolism was suspected to contribute to chlamydial growth defects. To verify this, glycolytic and pentose phosphate pathways were analysed revealing the importance of a functioning PPP for chlamydial growth. In addition, increased expression of glucose-6-phosphate dehydrogenase rescued chlamydial growth inhibition induced by activated p53. The rescuing effect was even more pronounced in p53-deficient cells treated with etoposide or nutlin-3 revealing additional p53-independent aspects of Chlamydia inhibition. Removal of ROS by anti-oxidant compounds was not sufficient to rescue chlamydial infectivity. Apparently, not only the anti-oxidant capacities of the PPP but also provision of precursors for nucleotide synthesis as well as contribution to DNA repair are important for successful chlamydial growth. Modulation of host cell signalling was previously reported for a number of pathogens. As formation of ROS and DNA damage are likely to occur during infections of intracellular bacteria, several strategies to manipulate the host and to inhibit induction of apoptosis were invented. Downregulation of the tumour suppressor p53 is a crucial point during development of Chlamydia, ensuring both host cell survival and metabolic support conducive to chlamydial growth.},
  subject      = {Chlamydia-trachomatis-Infektion},
  language  = {en}
}