@phdthesis{Kurz2020,
  author    = {Kurz, Andreas},
  title     = {Correlative live and fixed cell superresolution microscopy},
  doi       = {10.25972/OPUS-19945},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-199455},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Over the last decade life sciences have made an enormous leap forward. The development of complex analytical instruments, in particular in fluorescence microscopy, has played a decisive role in this. Scientist can now rely on a wide range of imaging techniques that offer different advantages in terms of optical resolution, recording speed or living cell compatibility. With the help of these modern microscopy techniques, multi-protein complexes can be resolved, membrane receptors can be counted, cellular pathways analysed or the internalisation of receptors can be tracked. However, there is currently no universal technique for comprehensive experiment execution that includes dynamic process capture and super resolution imaging on the same target object. In this work, I built a microscope that combines two complementary imaging techniques and enables correlative experiments in living and fixed cells. With an image scanning based laser spot confocal microscope, fast dynamics in several colors with low photodamage of the cells can be recorded. This novel system also has an improved resolution of 170 nm and was thoroughly characterized in this work. The complementary technique is based on single molecule localization microscopy, which can achieve a structural resolution down to 20-30 nm. Furthermore I implemented a microfluidic pump that allows direct interaction with the sample placed on the microscope. Numerous processes such as living cell staining, living cell fixation, immunostaining and buffer exchange can be observed and performed directly on the same cell. Thus, dynamic processes of a cell can be frozen and the structures of interest can be stained and analysed with high-resolution microscopy. Furthermore, I have equipped the detection path of the single molecule technique with an adaptive optical element. With the help of a deformable mirror, imaging functions can be shaped and information on the 3D position of the individual molecules can be extracted.},
  subject      = {Einzelmolek{\"u}lmikroskopie},
  language  = {en}
}
@phdthesis{Beliu2020,
  author    = {Beliu, Gerti},
  title     = {Bioorthogonale Tetrazin-Farbstoffe f{\"u}r die Lebendzell-Markierung und hochaufgel{\"o}ste Fluoreszenzmikroskopie},
  doi       = {10.25972/OPUS-18962},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-189628},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Der genetische Code beschreibt die Ver- und Entschl{\"u}sselung der Erb-information f{\"u}r das universelle Prinzip der Proteinbiosynthese aus einzelnen Aminos{\"a}uren. Durch Erweiterung des genetischen Codes lassen sich unna-t{\"u}rliche Aminos{\"a}uren (uAA) mit einzigartigen biophysikalischen Eigenschaf-ten ortsspezifisch in Proteine einf{\"u}hren und erm{\"o}glichen die spezifische Ma-nipulation von Proteinen. Die Click-Reaktion zwischen der unnat{\"u}rlichen Aminos{\"a}ure TCO*-Lysin und Tetrazin besitzt eine außergew{\"o}hnliche Reaktionskinetik (≥800 M-1s-1) und erm{\"o}glicht eine spezifische und bioorthogonale Markierung von Bio- ¬molek{\"u}len unter physiologischen Bedingungen. Im Fokus dieser Arbeit stand zun{\"a}chst die Markierung von Membran- ¬rezeptoren durch Click-Chemie in lebenden Zellen sowie die Untersuchung der Wechselwirkung 22 bekannter und neuartiger Tetrazin-Farbstoff- Konjugate. Dar{\"u}ber hinaus wurde die Anwendbarkeit von bioorthogonalen Click-Reaktionen f{\"u}r die hochaufl{\"o}sende Fluoreszenzmikroskopie untersucht. Durch Erweiterung des genetischen Codes in Proteine aus der Klasse der ionotropen Glutamatrezeptoren (iGluR), TNF-Rezeptoren oder Mikrotubu-li-assoziierten Proteinen (MAP) wurde ortspezifisch die unnat{\"u}rliche Amino-s{\"a}ure TCO*-Lysin eingef{\"u}hrt und dadurch die Fluoreszenzmarkierung durch Tetrazin-Farbstoffe erm{\"o}glicht. Die direkte chemische Kopplung von TCO an Liganden wie Phalloidin und Docetaxel, welche spezifisch das Aktin-Zytoskelett bzw. Mikrotubuli-Filamente binden k{\"o}nnen, erm{\"o}glichte zudem die Click-F{\"a}rbungen von fixierten und lebenden Zellen ohne genetische Ver-{\"a}nderungen der Zielproteine. Des Weiteren wurden die spektroskopischen Eigenschaften von 22 Tetrazin-Farbstoffen, verteilt {\"u}ber den gesamten sichtbaren Wellenl{\"a}ngenbereich, untersucht. Ein charakteristisches Kennzeichen der Click-Reaktion mit Tet-razin-Farbstoffen ist dabei ihre Fluorogenit{\"a}t. Das Tetrazin fungiert nicht nur als reaktive Gruppe w{\"a}hrend der Click-Reaktion mit Alkenen, sondern f{\"u}hrt in vielen Tetrazin-Farbstoff-Konjugaten zur Fluoreszenzl{\"o}schung. W{\"a}hrend bei gr{\"u}n-absorbierenden Farbstoffe vor allem FRET-basierte L{\"o}schprozesse dominieren, konnte photoinduzierter Elektronentransfer (PET) vom angeregten Farbstoff zum Tetrazin als Hauptl{\"o}schmechanismus bei rot-absorbierenden Oxazin- und Rhodamin-Derivaten identifiziert werden. Die effiziente und spezifische Markierung aller untersuchten Tetrazin- Farbstoffe erm{\"o}glichte die Visualisierung von Aktin-Filamenten, Mikrotubuli und Membranrezeptoren sowohl durch konventionelle Fluoreszenzmikrosko-pie als auch durch hochaufl{\"o}sende Verfahren, wie z.B. dSTORM, auf Ein-zelmolek{\"u}lebene. Die unterschiedliche Zellpermeabilit{\"a}t von Tetrazin-Farbstoffen kann dabei vorteilhaft f{\"u}r die spezifische intra- und extrazellul{\"a}re Markierung von Proteinen in fixierten und lebenden Zellen genutzt werden.},
  subject      = {Hochaufgel{\"o}ste Fluoreszenzmikroskopie},
  language  = {de}
}
@phdthesis{Becker2020,
  author    = {Becker, Mira Caroline},
  title     = {Principles of olfactory-visual integration to form a common percept in honeybees},
  doi       = {10.25972/OPUS-19919},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-199190},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {The honeybee is a well studied and important organism in neuroethology. The possibility to train them with a classical conditioning paradigm and their miniature brain provide a perfect requisite to investigate the neuronal principles of learning and memory. Honeybees use visual and olfactory cues to detect flowers during their foraging trips. Hence, the reward association of a nectar source is a multi-modal construct, which has at least two major components - olfactory and visual cues. It is still an open question, how both sensory components are converged in the mushroom body, which represent the multi-modal integration centre of the honeybee brain. The main goal of this study, is to investigate the processing of multiple modalities and how a reward association is formed. This includes, how and wether both sensory modalities interfere during learning. Thus, in this study stimulation with UV, blue and green light was used to evoke distinct photoreceptor activities in the compound eye. Furthermore, three different odours (Geraniol, Citronellol and Farnesol) were used. These stimuli were tested in three different experimental series. The first experiment involved classical differential conditioning of the single modalities - odour and colour. Honeybees showed high learning performances in differentiating olfactory stimuli and also reliable responses for visual conditioning. Furthermore, a temporal discrepancy in the stimulus length for best learning in the olfatcoty and visual cues was found. In the second series, it was tested how multi-modal compounds are perceived. This includes, unique cues (configural processing) or the sum of the single components of a compound (elemen- tal processing). This was tested by combining single odour components with monochromatic light in a positive (PP) and negative patterning (NP) experiment. During PP, the olfactory- visual compound was rewarded, whereas the single components were unrewarded. In contrast, during NP the single components were reinforced, but the compound was not. In addition, the ability to distinguish between two different light stimuli presented as a part of an olfactory-visual compound with the same odour component during acquisition was tested. In a memory test, the light stimuli were presented again as a compound and in addition as the single components. The results revealed that bees used elemental processing with compounds containing green and blue light. In contrast, when UV light was presented the bees used configural processing. Finally, a third experiment was conducted at the neuronal level. Multi-unit recordings were established to provide a suitable method to analyse extrinsic neurons at the mushroom body output region, the so called ventral lobe of the pedunculus. Here, three different odours (Geran- iol, Farnesol and Citronellol), two colours (green and blue) and two combined stimuli (colour + odour) were chosen as stimuli, to search for possible variations in processing stimuli with different modalities. Two units could be detected that responded mainly to visual stimuli.},
  language  = {en}
}
@phdthesis{Lu2020,
  author    = {Lu, Yunzhi},
  title     = {Kinetics of mouse and human muscle type nicotinic receptor channels},
  doi       = {10.25972/OPUS-19268},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-192688},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Acetylcholine (ACh) mediates transmission at vertebrate neuromuscular junctions and many other synapses. The postsynaptic ACh receptors at neuromuscular junctions are of the nicotinic subtype (nAChRs). They are among the best studied receptor channels and often serve as models or receptor prototypes. Despite a wealth of information on muscle type nAChRs so far little is known about species specific functional differences. In this work, mouse and human adult muscle type nAChRs are investigated. Cell attached recordings in the HEK293T heterologous expression system provided evidence that the ACh affinity of recombinant mouse and human adult muscle type nAChRs are different. To clarify this, I compared these receptors in outside-out patches employing a system for fast agonist application. Thus, the individual membrane patches with receptors can be exposed to various ligand concentrations. In response to 10 and 30 µM ACh normalized peak currents ({\^i}) were significantly larger and current rise-time (tr) shorter in human than in mouse receptors. Analyzing dose-response curves of {\^i} and tr and fitting them with a two-step equivalent binding-site kinetic mechanism revealed a two-fold higher ACh association rate constant in human compared to mouse receptors. Furthermore, human nAChRs were blocked faster in outside-out patches by superfusion of 300 nM α-Bungarotoxin (α-Bgtx) than mouse nAChRs. Finally, human nAChRs in outside-out patches showed higher affinity at 3 µM ACh than chimeric receptors consisting of mouse α- and human β-, γ- and ε-subunits. The higher affinity of human than mouse receptors for ACh and α-Bgtx is thus at least in part due to sequence difference in their α-subunits.},
  subject      = {Nicotinischer Acetylcholinrezeptor},
  language  = {en}
}
@phdthesis{Neubert2019,
  author    = {Neubert, Franziska},
  title     = {Markierung postsynaptischer Proteine f{\"u}r die hochaufl{\"o}sende Fluoreszenzmikroskopie},
  doi       = {10.25972/OPUS-19239},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-192394},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Das menschliche Gehirn ist ein Organ, das aufgrund seiner Komplexit{\"a}t und zellul{\"a}ren Diversit{\"a}t noch am wenigsten verstanden ist. Eine der Ursachen daf{\"u}r sind zahlreiche Herausforderungen in diversen neurobiologischen Bild-gebungsverfahren. Erst seit der Erfindung der hochaufl{\"o}senden Fluoreszenz-mikroskopie ist es m{\"o}glich, Strukturen unterhalb der Beugungsgrenze zu visua-lisieren und somit eine maximale Aufl{\"o}sung von bis zu 20 nm zu erreichen. Zus{\"a}tzlich h{\"a}ngt die F{\"a}higkeit, biologische Strukturen aufzul{\"o}sen, von der Markierungs-gr{\"o}ße und -dichte ab. Derzeit ist die h{\"a}ufigste Methode zur Proteinf{\"a}rbung die indirekte Antik{\"o}rperf{\"a}rbung, bei der ein Fluorophor-markierter Sekund{\"a}rantik{\"o}rper an einen Epitop-spezifischen Prim{\"a}rantik{\"o}rper bindet. Dabei kann der Abstand von Zielstruktur und Fluorophor bis zu 30 nm betragen, was eine Aufl{\"o}sungs-verminderung zur Folge haben kann. Aufgrund dessen wurden in dieser Arbeit alternative Markierungsmethoden getestet, um postsynaptische Proteine sicht-bar zu machen. Zun{\"a}chst wurde der postsynaptische N-Methyl-D-Aspartat (NMDA)-Rezeptor mit Hilfe konventioneller indirekter Antik{\"o}rperf{\"a}rbung markiert. Hier war die NR1-Untereinheit des NMDA-Rezeptors von besonderem Interesse, da diese in der Autoimmunerkrankung Anti-NMDA-Rezeptor-Enzephalitis invol-viert ist. Patienten dieser seltenen Krankheit bilden Autoantik{\"o}rper gegen die NR1-Untereinheit, wodurch ein schneller reversibler Verlust der NMDA-Rezeptoren auf der Postsynapse induziert wird. Wichtige Informationen k{\"o}nnen nicht mehr ausreichend weitergegeben werden, was psychiatrische und neurologi-sche St{\"o}rungen zur Folge hat. In dieser Arbeit wurden sowohl kommerzielle NR1-Antik{\"o}rper, als auch rekombinante monoklonale NR1-Antik{\"o}rper von Patien-ten mit Anti-NMDA-Rezeptor-Enzephalitis getestet. In konfokalen und in hochaufgel{\"o}sten SIM- (engl. structured illumination microscopy) und dSTORM- (engl. direct stochastic optical reconstruction microscopy) Messun-gen konnten kommerzielle NR1-Antik{\"o}rper keine erfolgreichen F{\"a}rbungen erzielen. Dagegen erwiesen sich die rekombinanten monoklonalen NR1-Patientenantik{\"o}rper als sehr spezifisch, sowohl in prim{\"a}ren Neuronen als auch im Hippocampus von murinen Gehirnschnitten und lieferten gute Kolokalisati-onen mit dem postsynaptischen Markerprotein Homer. Um die optische Aufl{\"o}sung zu verbessern, wurde eine neue Markierungs-methode mit sog. „Super-Binde-Peptiden" (SBPs) getestet. SBPs sind modifi-zierte Peptide, die erh{\"o}hte Affinit{\"a}ten und Spezifit{\"a}ten aufweisen und mit ei-ner Gr{\"o}ße von ~ 2,5 nm wesentlich kleiner als Antik{\"o}rper sind. In dieser Arbeit best{\"a}tigte sich ein kleines hochspezifisches SPB, das an den Fluoreszenzfarb-stoff Tetra- methylrhodamin (TMR) gekoppelt ist, als effektiver Marker f{\"u}r das Ankerpro-tein Gephyrin. Gephyrin ist f{\"u}r die Lokalisation und Verankerung einiger post-synaptischer Rezeptoren zust{\"a}ndig, indem es sie mit dem Cytoskelett der Zelle verbindet. SIM-Messungen in prim{\"a}ren Neuronen zeigten eine bessere Clus-terrepr{\"a}sentation bei der F{\"a}rbung von Gephyrin mit SBPs, als mit Antik{\"o}rper-f{\"a}rbung. Zus{\"a}tzlich wurden Kolokalisationsanalysen von Gephyrin zusammen mit dem inhibito-rischen pr{\"a}synaptischen vesikul{\"a}ren GABA-Transporter VGAT durchgef{\"u}hrt. Eine weitere F{\"a}rbemethode stellte die bioorthogonale Click-F{\"a}rbung durch die Erweiterung des eukaryotischen genetischen Codes (engl. genetic code ex-pansion, GCE) dar. Dabei wurde eine unnat{\"u}rliche, nicht-kanonische Amino-s{\"a}ure (engl. non-canonical amino acid, ncAA) ins Zielprotein eingebaut und in Kombination mit der Click-Chemie ortsspezifisch mit organischen Tetrazin-Farbstoff-Konjugaten angef{\"a}rbt. Organische Fluorophore haben den Vorteil, dass sie mit einer Gr{\"o}ße von 0,5 - 2 nm sehr klein sind und damit die nat{\"u}rli-chen Funktionen der Proteine in der Zelle kaum beeinflussen. In dieser Arbeit wurde zum ersten Mal gezeigt, dass der tetramere postsynaptische NMDA-Rezeptor durch die Amber-Supres-sionsmethode bioorthogonal angef{\"a}rbt werden konnte. Aus sieben verschiede-nen Amber-Mutanten der NR1-Untereinheit stellte sich die Y392TAG-NR1-Mutante als diejenige mit der besten Proteinexpression, F{\"a}rbeeffizienz und rezeptorfunktionalit{\"a}t heraus. Dies konnte durch Fluoreszenzmikroskopie- und Whole-Cell Patch-Clamp-Experimenten gezeigt werden. Die bioorthogo-nale Click-F{\"a}rbung durch GCE eignete sich f{\"u}r die F{\"a}rbung des NMDA-Rezeptors in verschiedenen Zelllinien, mit unterschiedlichen Tetrazin-Farbstoff-Konjugaten und f{\"u}r Lebendzellexperimente. In dSTORM-Messungen erwies sich das Tetrazin-Cy5-Farbstoff-Konjugat als ideal aufgrund seiner Gr{\"o}-ße, Photostabilit{\"a}t, Helligkeit und seines geeigneten Blinkverhaltens, sodass eine homogene NMDA-Rezeptorverteilung auf der Zellmembran gezeigt wer-den konnte. NR1-Antik{\"o}rperf{\"a}rbungen wiesen dagegen starke Clusterbildun-gen auf. Die Ergebnisse konnten belegen, dass kleinere Farbstoffe eine deut-lich bessere Zug{\"a}nglichkeit zu ihrem Zielprotein haben und somit besser f{\"u}r die hochaufl{\"o}sende Fluoreszenzmikroskopie geeignet sind.},
  subject      = {hochaufl{\"o}sende Fluoreszenzmikroskopie},
  language  = {de}
}
@phdthesis{Griffoni2019,
  author    = {Griffoni, Chiara},
  title     = {Towards advanced immunocompetent skin wound models for in vitro drug evaluation},
  doi       = {10.25972/OPUS-19212},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-192125},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Current preclinical models used to evaluate novel therapies for improved healing include both in vitro and in vivo methods. However, ethical concerns related to the use of animals as well as the poor physiological translation between animal and human skin wound healing designate in vitro models as a highly relevant and promising platforms for healing investigation. While current in vitro 3D skin models recapitulate a mature tissue with healing properties, they still represent a simplification of the in vivo conditions, where for example the inflammatory response originating after wound formation involves the contribution of immune cells. Macrophages are among the main contributors to the inflammatory response and regulate its course thanks to their plasticity. Therefore, their implementation into in vitro skin could greatly increase the physiological relevance of the models. As no full-thickness immunocompetent skin model containing macrophages has been reported so far, the parameters necessary for a successful triple co-culture of fibroblasts, keratinocytes and macrophages were here investigated. At first, cell source and culture timed but also an implementation strategy for macrophages were deter-mined. The implementation of macrophages into the skin model focused on the minimization of the culture time to preserve immune cell viability and phenotype, as the environment has a major influence on cell polarization and cytokine production. To this end, incorporation of macrophages in 3D gels prior to the combination with skin models was selected to better mimic the in vivo environment. Em-bedded in collagen hydrogels, macrophages displayed a homogeneous cell distribution within the gel, preserving cell viability, their ability to respond to stimuli and their capability to migrate through the matrix, which are all needed during the involvement of macrophages in the inflammatory response. Once established how to introduce macrophages into skin models, different culture media were evaluated for their effects on primary fibroblasts, keratinocytes and macrophages, to identify a suitable medium composition for the culture of immunocompetent skin. The present work confirmed that each cell type requires a different supplement combination for maintaining functional features and showed for the first time that media that promote and maintain a mature skin structure have negative effects on primary macrophages. Skin differentiation media negatively affected macrophages in terms of viability, morphology, ability to respond to pro- and anti-inflammatory stimuli and to migrate through a collagen gel. The combination of wounded skin equivalents and macrophage-containing gels con-firmed that culture medium inhibits macrophage participation in the inflammatory response that oc-curs after wounding. The described macrophage inclusion method for immunocompetent skin creation is a promising approach for generating more relevant skin models. Further optimization of the co-cul-ture medium will potentially allow mimicking a physiological inflammatory response, enabling to eval-uate the effects novel drugs designed for improved healing on improved in vitro models.},
  subject      = {Haut},
  language  = {en}
}
@phdthesis{Njovu2019,
  author    = {Njovu, Henry Kenneth},
  title     = {Patterns and drivers of herbivore diversity and invertebrate herbivory along elevational and land use gradients at Mt. Kilimanjaro, Tanzania},
  doi       = {10.25972/OPUS-17254},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-172544},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {This thesis elucidates patterns and drivers of invertebrate herbivory, herbivore diversity, and community-level biomass along elevational and land use gradients at Mt. Kilimanjaro, Tanzania. Chapter I provides background information on the response and predictor variables, study system, and the study design. First, I give an overview of the elevational patterns of species diversity/richness and herbivory published in the literature. The overview illuminates existing debates on elevational patterns of species diversity/richness and herbivory. In connection to these patterns, I also introduce several hypotheses and mechanisms put forward to explain macroecological patterns of species richness. Furthermore, I explain the main variables used to test hypotheses. Finally, I describe the study system and the study design used. Chapter II explores the patterns of invertebrate herbivory and their underlying drivers along extensive elevational and land use gradients on the southern slopes of Mt. Kilimanjaro. I recorded standing leaf herbivory from leaf chewers, leaf miners and gall-inducing insects on 55 study sites located in natural and anthropogenic habitats distributed from 866 to 3060 meters above sea level (m asl) on Mt. Kilimanjaro. Standing leaf herbivory was related to climatic variables [mean annual temperature - (MAT) and mean annual precipitation - (MAP)], net primary productivity (NPP) and plant functional traits (leaf traits) [specific leaf area (SLA), carbon to nitrogen ratio (CN), and nitrogen to phosphorous ratio (NP)]. Results revealed an unimodal pattern of total leaf herbivory along the elevation gradient in natural habitats. Findings also revealed differences in the levels and patterns of herbivory among feeding guilds and between anthropogenic and natural habitats. Changes in NP and CN ratios which were closely linked to NPP were the strongest predictors of leaf herbivory. Our study uncovers the role of leaf nutrient stoichiometry and its linkages to climate in explaining the variation in leaf herbivory along climatic gradients. Chapter III presents patterns and unravels direct and indirect effects of resource (food) abundance (NPP), resource (food) diversity [Functional Dispersion (FDis)], resource quality (SLA, NP, and CN rations), and climate variables (MAT and MAP) on species diversity of phytophagous beetles. Data were collected from 65 study sites located in natural and anthropogenic habitats distributed from 866 to 4550 m asl on the southern slopes of Mt. Kilimanjaro. Sweep net and beating methods were used to collect a total of 3,186 phytophagous beetles representing 21 families and 304 morphospecies. Two groups, weevils (Curculionidae) and leaf beetles (Chrysomelidae) were the largest and most diverse families represented with 898 and 1566 individuals, respectively. Results revealed complex (bimodal) and dissimilar patterns of Chao1-estimated species richness (hereafter referred to as species diversity) along elevation and land use gradients. Results from path analysis showed that temperature and climate-mediated changes in NPP had a significant positive direct and indirect effect on species diversity of phytophagous beetles, respectively. The results also revealed that the effect of NPP (via beetles abundance and diversity of food resources) on species diversity is stronger than that of temperature. Since we found that factors affecting species diversity were intimately linked to climate, I concluded that predicted climatic changes over the coming decades will likely alter the species diversity patterns which we observe today. Chapter IV presents patterns and unravels the direct and indirect effects of climate, NPP and anthropogenic disturbances on species richness and community-level biomass of wild large mammals which represent endothermic organisms and the most important group of vertebrate herbivores. Data were collected from 66 study sites located in natural and anthropogenic habitats distributed from 870 to 4550 m asl on the southern slopes of Mt. Kilimanjaro. Mammals were collected using camera traps and used path analysis to disentangle the direct and indirect effects of climatic variables, NPP, land use, land area, levels of habitat protection and occurrence of domesticated mammals on the patterns of richness and community-level biomass of wild mammals, respectively. Results showed unimodal patterns for species richness and community-level biomass of wild mammals along elevation gradients and that the patterns differed depending on the type of feeding guild. Findings from path analysis showed that net primary productivity and levels of habitat protection had a strong direct effect on species richness and community-level biomass of wild mammals whereas temperature had an insignificant direct effect. Findings show the importance of climate-mediated food resources in determining patterns of species richness of large mammals. While temperature is among key predictors of species richness in several ectotherms, its direct influence in determining species richness of wild mammals was insignificant. Findings show the sensitivity of wild mammals to anthropogenic influences and underscore the importance of protected areas in conserving biodiversity. In conclusion, despite a multitude of data sets on species diversity and ecosystem functions along broad climatic gradients, there is little mechanistic understanding of the underlying causes. Findings obtained in the three studies illustrate their contribution to the scientific debates on the mechanisms underlying patterns of herbivory and diversity along elevation gradients. Results present strong evidence that plant functional traits play a key role in determining invertebrate herbivory and species diversity along elevation gradients and that, their strong interdependence with climate and anthropogenic activities will shape these patterns in future. Additionally, findings from path analysis demonstrated that herbivore diversity, community-level biomass, and herbivory are strongly influenced by climate (either directly or indirectly). Therefore, the predicted climatic changes are expected to dictate ecological patterns, biotic interactions, and energy and nutrient fluxes in terrestrial ecosystems in the coming decades with stronger impacts probably occurring in natural ecosystems. Furthermore, findings demonstrated the significance of land use effects in shaping ecological patterns. As anthropogenic pressure is advancing towards more pristine higher elevations, I advocate conservation measures which are responsive to and incorporate human dimensions to curb the situation. Although our findings emanate from observational studies which have to take several confounding factors into account, we have managed to demonstrate global change responses in real ecosystems and fully established organisms with a wide range of interactions which are unlikely to be captured in artificial experiments. Nonetheless, I recommend additional experimental studies addressing the effect of top-down control by natural enemies on herbivore diversity and invertebrate herbivory in order to deepen our understanding of the mechanisms driving macroecological patterns along elevation gradients.  },
  subject      = {Species richness},
  language  = {en}
}
@phdthesis{Franke2019,
  author    = {Franke, Christian},
  title     = {Advancing Single-Molecule Localization Microscopy: Quantitative Analyses and Photometric Three-Dimensional Imaging},
  doi       = {10.25972/OPUS-15635},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-156355},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Since its first experimental implementation in 2005, single-molecule localization microscopy (SMLM) emerged as a versatile and powerful imaging tool for biological structures with nanometer resolution. By now, SMLM has compiled an extensive track-record of novel insights in sub- and inter- cellular organization.\\ Moreover, since all SMLM techniques rely on the analysis of emission patterns from isolated fluorophores, they inherently allocate molecular information \$per\$ \$definitionem\$.\\ Consequently, SMLM transitioned from its origin as pure high-resolution imaging instrument towards quantitative microscopy, where the key information medium is no longer the highly resolved image itself, but the raw localization data set.\\ The work presented in this thesis is part of the ongoing effort to translate those \$per\$ \$se\$ molecular information gained by SMLM imaging to insights into the structural organization of the targeted protein or even beyond. Although largely consistent in their objectives, the general distinction between global or segmentation clustering approaches on one side and particle averaging or meta-analyses techniques on the other is usually made.\\ During the course of my thesis, I designed, implemented and employed numerous quantitative approaches with varying degrees of complexity and fields of application.\\ \\ In my first major project, I analyzed the localization distribution of the integral protein gp210 of the nuclear pore complex (NPC) with an iterative \textit{k}-means algorithm. Relating the distinct localization statistics of separated gp210 domains to isolated fluorescent signals led, among others, to the conclusion that the anchoring ring of the NPC consists of 8 homo-dimers of gp210.\\ This is of particular significance, both because it answered a decades long standing question about the nature of the gp210 ring and it showcased the possibility to gain structural information well beyond the resolution capabilities of SMLM by crafty quantification approaches.\\ \\ The second major project reported comprises an extensive study of the synaptonemal complex (SNC) and linked cohesin complexes. Here, I employed a multi-level meta-analysis of the localization sets of various SNC proteins to facilitate the compilation of a novel model of the molecular organization of the major SNC components with so far unmatched extend and detail with isotropic three-dimensional resolution.\\ In a second venture, the two murine cohesin components SMC3 and STAG3 connected to the SNC were analyzed. Applying an adapted algorithm, considering the disperse nature of cohesins, led to the realization that there is an apparent polarization of those cohesin complexes in the SNC, as well as a possible sub-structure of STAG3 beyond the resolution capabilities of SMLM.\\ \\ Other minor projects connected to localization quantification included the study of plasma membrane glycans regarding their overall localization distribution and particular homogeneity as well as the investigation of two flotillin proteins in the membrane of bacteria, forming clusters of distinct shapes and sizes.\\ \\ Finally, a novel approach to three-dimensional SMLM is presented, employing the precise quantification of single molecule emitter intensities. This method, named TRABI, relies on the principles of aperture photometry which were improved for SMLM.\\ With TRABI it was shown, that widely used Gaussian fitting based localization software underestimates photon counts significantly. This mismatch was utilized as a \$z\$-dependent parameter, enabling the conversion of 2D SMLM data to a virtual 3D space. Furthermore it was demonstrated, that TRABI can be combined beneficially with a multi-plane detection scheme, resulting in superior performance regarding axial localization precision and resolution.\\ Additionally, TRABI has been subsequently employed to photometrically characterize a novel dye for SMLM, revealing superior photo-physical properties at the single-molecule level.\\ Following the conclusion of this thesis, the TRABI method and its applications remains subject of diverse ongoing research.},
  subject      = {Einzelmolek{\"u}lmikroskopie},
  language  = {en}
}
@phdthesis{KaltdorfgebSchuch2019,
  author    = {Kaltdorf [geb. Schuch], Kristin Verena},
  title     = {Mikroskopie, Bildverarbeitung und Automatisierung der Analyse von Vesikeln in \(C.\) \(elegans\) und anderen biologischen Strukturen},
  doi       = {10.25972/OPUS-16062},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-160621},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Thema dieser Thesis ist die Analyse sekretorischer Vesikelpools auf Ultrastrukturebene in unterschiedlichen biologischen Systemen. Der erste und zweite Teil dieser Arbeit fokussiert sich auf die Analyse synaptischer Vesikelpools in neuromuskul{\"a}ren Endplatten (NME) im Modellorganismus Caenorhabditis elegans. Dazu wurde Hochdruckgefrierung und Gefriersubstitution angewandt, um eine unverz{\"u}gliche Immobilisation der Nematoden und somit eine Fixierung im nahezu nativen Zustand zu gew{\"a}hrleisten. Anschließend wurden dreidimensionale Aufnahmen der NME mittels Elektronentomographie erstellt. Im ersten Teil dieser Arbeit wurden junge adulte, wildtypische C. elegans Hermaphroditen mit Septin-Mutanten verglichen. Um eine umfassende Analyse mit hoher Stichprobenzahl zu erm{\"o}glichen und eine automatisierte L{\"o}sung f{\"u}r {\"a}hnliche Untersuchungen von Vesikelpools bereit zu stellen wurde eine Software namens 3D ART VeSElecT zur automatisierten Vesikelpoolanalyse entwickelt. Die Software besteht aus zwei Makros f{\"u}r ImageJ, eines f{\"u}r die Registrierung der Vesikel und eines zur Charakterisierung. Diese Trennung in zwei separate Schritte erm{\"o}glicht einen manuellen Verbesserungsschritt zum Entfernen falsch positiver Vesikel. Durch einen Vergleich mit manuell ausgewerteten Daten neuromuskul{\"a}rer Endplatten von larvalen Stadien des Modellorganismus Zebrafisch (Danio rerio) konnte erfolgreich die Funktionalit{\"a}t der Software bewiesen werden. Die Analyse der neuromuskul{\"a}ren Endplatten in C. elegans ergab kleinere synaptische Vesikel und dichtere Vesikelpools in den Septin-Mutanten verglichen mit Wildtypen. Im zweiten Teil der Arbeit wurden neuromuskul{\"a}rer Endplatten junger adulter C. elegans Hermaphroditen mit Dauerlarven verglichen. Das Dauerlarvenstadium ist ein spezielles Stadium, welches durch widrige Umweltbedingungen induziert wird und in dem C. elegans {\"u}ber mehrere Monate ohne Nahrungsaufnahme {\"u}berleben kann. Da hier der Vergleich der Abundanz zweier Vesikelarten, der „clear-core"-Vesikel (CCV) und der „dense-core"-Vesikel (DCV), im Fokus stand wurde eine Erweiterung von 3D ART VeSElecT entwickelt, die einen „Machine-Learning"-Algorithmus zur automatisierten Klassifikation der Vesikel integriert. Durch die Analyse konnten kleinere Vesikel, eine erh{\"o}hte Anzahl von „dense-core"-Vesikeln, sowie eine ver{\"a}nderte Lokalisation der DCV in Dauerlarven festgestellt werden. Im dritten Teil dieser Arbeit wurde untersucht ob die f{\"u}r synaptische Vesikelpools konzipierte Software auch zur Analyse sekretorischer Vesikel in Thrombozyten geeignet ist. Dazu wurden zweidimensionale und dreidimensionale Aufnahmen am Transmissionselektronenmikroskop erstellt und verglichen. Die Untersuchung ergab, dass hierf{\"u}r eine neue Methodik entwickelt werden muss, die zwar auf den vorherigen Arbeiten prinzipiell aufbauen kann, aber den besonderen Herausforderungen der Bilderkennung sekretorischer Vesikel aus Thrombozyten gerecht werden muss.},
  subject      = {Mikroskopie},
  language  = {de}
}
@phdthesis{Pahlavan2019,
  author    = {Pahlavan, Pirasteh},
  title     = {Integrated Systems Biology Analysis; Exemplified on Potyvirus and Geminivirus interaction with \(Nicotiana\) \(benthamiana\)},
  doi       = {10.25972/OPUS-15341},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-153412},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Viral infections induce a significant impact on various functional categories of biological processes in the host. The understanding of this complex modification of the infected host immune system requires a global and detailed overview on the infection process. Therefore it is essential to apply a powerful approach which identifies the involved components conferring the capacity to recognize and respond to specific pathogens, which in general are defeated in so-called compatible virus-plant infections. Comparative and integrated systems biology of plant-virus interaction progression may open a novel framework for a systemic picture on the modulation of plant immunity during different infections and understanding pathogenesis mechanisms. In this thesis these approaches were applied to study plant-virus infections during two main viral pathogens of cassava: Cassava brown streak virus and African cassava mosaic virus. Here, the infection process was reconstructed by a combination of omics data-based analyses and metabolic network modelling, to understand the major metabolic pathways and elements underlying viral infection responses in different time series, as well as the flux activity distribution to gain more insights into the metabolic flow and mechanism of regulation; this resulted in simultaneous investigations on a broad spectrum of changes in several levels including the gene expression, primary metabolites, and enzymatic flux associated with the characteristic disease development process induced in Nicotiana benthamiana plants due to infection with CBSV or ACMV. Firstly, the transcriptome dynamics of the infected plant was analysed by using mRNA-sequencing, in order to investigate the differential expression profile according the symptom developmental stage. The spreading pattern and different levels of biological functions of these genes were analysed associated with the infection stage and virus entity. A next step was the Real-Time expression modification of selected key pathway genes followed by their linear regression model. Subsequently, the functional loss of regulatory genes which trigger R-mediated resistance was observed. Substantial differences were observed between infected mutants/transgenic lines and wild-types and characterized in detail. In addition, we detected a massive localized accumulation of ROS and quantified the scavenging genes expression in the infected wild-type plants relative to mock infected controls. Moreover, we found coordinated regulated metabolites in response to viral infection measured by using LC-MS/MS and HPLC-UV-MS. This includes the profile of the phytohormones, carbohydrates, amino acids, and phenolics at different time points of infection with the RNA and DNA viruses. This was influenced by differentially regulated enzymatic activities along the salicylate, jasmonate, and chorismate biosynthesis, glycolysis, tricarboxylic acid cycle, and pentose phosphate pathways, as well as photosynthesis, photorespiration, transporting, amino acid and fatty acid biosynthesis. We calculated the flux redistribution considering a gradient of modulation for enzymes along different infection stages, ranging from pre-symptoms towards infection stability. Collectively, our reverse-engineering study consisting of the generation of experimental data and modelling supports the general insight with comparative and integrated systems biology into a model plant-virus interaction system. We refine the cross talk between transcriptome modification, metabolites modulation and enzymatic flux redistribution during compatible infection progression. The results highlight the global alteration in a susceptible host, correlation between symptoms severity and the alteration level. In addition we identify the detailed corresponding general and specific responses to RNA and DNA viruses at different stages of infection. To sum up, all the findings in this study strengthen the necessity of considering the timing of treatment, which greatly affects plant defence against viral infection, and might result in more efficient or combined targeting of a wider range of plant pathogens.},
  language  = {en}
}
@phdthesis{Romanov2019,
  author    = {Romanov, Natalie},
  title     = {Characterizing Variation of Protein Complexes and Functional Modules on a Temporal Scale and across Individuals},
  doi       = {10.25972/OPUS-16813},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-168139},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {A fundamental question in current biology concerns the translational mechanisms leading from genetic variability to phenotypes. Technologies have evolved to the extent that they can efficiently and economically determine an individual's genomic composition, while at the same time big data on clinical profiles and diagnostics have substantially accumulated. Genome-wide association studies linking genomic loci to certain traits, however, remain limited in their capacity to explain the cellular mechanisms that underlie the given association. For most associations, gene expression has been blamed; yet given that transcript and protein abundance oftentimes do not correlate, that finding does not necessarily decrypt the underlying mechanism. Thus, the integration of further information is crucial to establish a model that could prove more accurate in predicting genotypic effects on the human organism. In this work we describe the so-called proteotype as a feature of the cell that could provide a substantial link between genotype and phenotype. Rather than looking at the proteome as a set of independent molecules, we demonstrate a consistent modular architecture of the proteome that is driven by molecular cooperativity. Functional modules, especially protein complexes, can be further interrogated for differences between individuals and tackled as imprints of genetic and environmental variability. We also show that subtle stoichiometric changes of protein modules could have broader effects on the cellular system, such as the transport of specific molecular cargos. The presented work also delineates to what extent temporal events and processes influence the stoichiometry of protein complexes and functional modules. The re-wiring of the glycolytic pathway for example is illustrated as a potential cause for an increased Warburg effect during the ageing of the human bone marrow. On top of analyzing protein abundances we also interrogate proteome dynamics in terms of stability and solubility transitions during the short temporal progression of the cell cycle. One of our main observations in the thesis encompass the delineation of protein complexes into respective sub-complexes according to distinct stability patterns during the cell cycle. This has never been demonstrated before, and is functionally relevant for our understanding of the dis- and assembly of large protein modules. The insights presented in this work imply that the proteome is more than the sum of its parts, and primarily driven by variability in entire protein ensembles and their cooperative nature. Analyzing protein complexes and functional modules as molecular reflections of genetic and environmental variations could indeed prove to be a stepping stone in closing the gap between genotype and phenotype and customizing clinical treatments in the future.},
  subject      = {Proteotype},
  language  = {en}
}
@phdthesis{Dirks2019,
  author    = {Dirks, Johannes},
  title     = {Charakterisierung der Wechselwirkung zwischen N-Myc und Aurora-A im MYCN-amplifizierten Neuroblastom},
  doi       = {10.25972/OPUS-18660},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-186600},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Im Neuroblastom ist die Amplifikation des MYCN-Gens, eines Mitglieds der MYC-Onkogenfamilie, mit einer ung{\"u}nstigen Prognose assoziiert. Der von dem Gen kodierte Transkriptionsfaktor N-Myc ist f{\"u}r die Proliferation der MYCN-amplifizierten Neuroblastomzelllinien notwendig und seine Depletion oder Destabilisierung f{\"u}hren zum Proliferationsarrest (Otto et al., 2009). Da N-Myc auf Proteinebene durch die Interaktion mit der mitotischen Kinase Aurora-A stabilisiert wird, bewirkt deren Depletion oder die Hemmung der Interaktion der beiden Proteine mittels spezieller Aurora- A-Inhibitoren (z.B. MLN8054 und MLN8237) ebenso eine Hemmung der Proliferation - in vitro und in vivo (Brockmann et al., 2013). Bisher ist jedoch unklar, {\"u}ber welchen Mechanismus Aurora-A die Stabilisierung von N-Myc erreicht, die Kinaseaktivit{\"a}t spielt hierbei jedoch keine Rolle (Otto et al., 2009). Eine M{\"o}glichkeit stellt die Rekrutierung von Usps dar, die das angeh{\"a}ngte Ubiquitinsignal so modifizieren, dass die Erkennung und der Abbau des Proteins durch das Proteasom verringert werden. In der vorliegenden Arbeit wurde die Wirkung von Usp7 und Usp11 auf die Stabilit{\"a}t von N-Myc untersucht. F{\"u}r beide konnte in Immunpr{\"a}zipitationen die Interaktion mit N-Myc gezeigt werden. Ebenso erh{\"o}hten beide Proteasen in {\"U}berexpressionsexperimenten die vorhandene Menge an NMyc. Die Depletion von Usp7 mittels shRNAs f{\"u}hrte in IMR-32 zu einem Arrest in der G1-Phase und zur Differenzierung der Zellen. Gleichzeitig wurden stark erniedrigte mRNA- und Proteinmengen von N-Myc und Aurora-A nachgewiesen. Es konnte jedoch nicht eindeutig gezeigt werden, ob die beobachteten zellul{\"a}ren Effekte durch eine vermehrte proteasomale Degradation von N-Myc begr{\"u}ndet sind oder ob dabei die ver{\"a}nderte Regulation weiterer Zielproteine von Usp7 eine Rolle spielt. Die Depletion von Usp11 mit shRNAs bewirkte eine Abnahme der N-Myc-Mengen auf posttranslationaler Ebene. Somit stellen beide Usps vielversprechende Angriffspunkte einer gezielten Therapie in MYCN-amplifizierten Neuroblastomen dar und sollten deshalb Gegenstand weiterf{\"u}hrender Untersuchungen sein. {\"U}ber welche Proteindom{\"a}ne in N-Myc die Interaktion mit Aurora-A stattfindet ist nicht bekannt. Eine m{\"o}gliche Pseudosubstratbindungssequenz in Myc-Box I (Idee Richard Bayliss, University of Leicester) wurde in der vorliegenden Arbeit untersucht. Durch Mutation dieser Sequenz sollte die Bindung von Aurora-A unm{\"o}glich gemacht werden. Allerdings wurde die erwartete Abnahme der St{\"a}rke der Interaktion von Aurora-A und N-Myc durch die Mutation ebensowenig beobachtet wie eine verringerte Stabilit{\"a}t. Die Regulation der Phosphorylierung von N-Myc im Verlauf des Zellzyklus wurde durch die Mutation beeintr{\"a}chtigt. Wie diese Ver{\"a}nderung exakt zu begr{\"u}nden ist bedarf weiterer Experimente},
  subject      = {Neuroblastom},
  language  = {de}
}
@phdthesis{Memmel2019,
  author    = {Memmel, Simon},
  title     = {Automatisierte Algorithmen zur Analyse der Migration und der strahleninduzierten DNA-Sch{\"a}den humaner Glioblastomzellen nach kombinierter PI3K/mTOR/Hsp90-Inhibierung},
  doi       = {10.25972/OPUS-18571},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-185710},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Das hohe invasive Potential und die starke Resistenz gegen Radio-/Chemotherapie von Glioblastoma multiforme (GBM) Zellen machen sie zu dem t{\"o}dlichsten Tumor ihrer Art. Es ist deshalb von großem Interesse die Grundlagen, welche der Migrationsf{\"a}higkeit und DNA Reparatur zu Grunde liegen, besser zu verstehen. Im ersten Teil dieser Arbeit wurden zwei Algorithmen zur automatischen Analyse der Migration in der Einzelzellverfolgung und im Wundheilungsassay modifiziert. Die Auswertung der Daten konnte automatisch und somit schnell, effektiv und mit geringerem Arbeitsaufwand durchgef{\"u}hrt werden. Mit Hilfe dieser automatischen Algorithmen wurde die Migrationsf{\"a}higkeit von zwei GBM-Zelllinien (DK-MG und SNB19) untersucht. Zus{\"a}tzlich wurde die konfokale Laserscanning- sowie die hochaufl{\"o}sende dSTORM-Fluoreszenzmikroskopie verwendet um die, der Zellbewegung zu Grunde liegende, Struktur des F Aktin und der fokalen Adh{\"a}sionskinase (FAK) aufzul{\"o}sen und darzustellen. Unter Anwendung dieser genannten Methoden sind die Effekte des dualen PI3K/mTOR Inhibitors PI-103 alleine und in Kombination mit dem Hsp90 Inhibitor NVP AUY922 mit und ohne Bestrahlung auf die Bewegung untersucht worden. Es konnte festgestellt werden, dass sich beide Zelllinien deutlich in ihrem migratorischem Potential in vitro unterscheiden und zudem auch markante Unterschiede in ihrer Morphologie aufweisen. Die weniger invasiven DK MG-Zellen besitzen eine polarisierte Zellstruktur, wohingegen SNB19-Zellen sich durch multipolare ungerichtete Bewegung auszeichneten. Zudem wurde die Migration, durch PI3K/mTOR Inhibition mit PI-103 bei den DK-MG-Zellen (p53 wt, PTEN wt), sehr effektiv unterdr{\"u}ckt. Wohingegen sich die SNB19-Zellen (p53 mut, PTEN mut) resistent gegen diesen Inhibitor zeigten. Hsp90 Inhibition offenbarte in beiden Zelllinien einen starken inhibitorischen Effekt auf die Migration der Zellen sowie die Reorganisierung des F Aktinskelettes. In der zweiten H{\"a}lfte dieser Arbeit wurde ein Augenmerk auf die DNA-DSB-Reparatur der GBM Zellen nach ionisierender Strahlung gelegt. Zun{\"a}chst wurde eine automatische Analysesoftware „FocAn-3D" entwickelt, mit dessen Hilfe die DNA Doppelstrangbruchreparaturkinetik untersucht werden sollte. Diese Software erm{\"o}glicht es die gesamten Zellkerne mit ihren γH2AX-Foci in 3D-cLSM-Aufnahmen zu untersuchen. Es konnte somit eine Verbesserung der Genauigkeit in der Ausz{\"a}hlung der γH2AX-Foci erreicht werden, welche 2D beschr{\"a}nkter Software verwehrt bleibt. Mit FocAn-3D konnte der gesamte Verlauf der Induktions- und Abbauphase der γH2AX-Foci in DK MG- und SNB19-Zellen mit einem mathematischen Modell ausgewertet und dargestellt werden. Des Weiteren wurde die Nanometerstruktur von γH2AX- und pDNA-PKcs-Foci mittels hochaufl{\"o}sender dSTORM-Mikroskopie untersucht. Konventionelle Mikroskopiemethoden, begrenzt durch das Beugungslimit und einer Aufl{\"o}sung von ~200 nm, konnten die Nanometerstruktur (<100 nm) der Reparaturfoci bisher nicht darstellen. Mit Hilfe der beugungsunbegrenzten dSTORM-Mikroskopie war es m{\"o}glich in DK MG- und SNB19-Zellen die Nanometerstruktur genannten Reparaturproteine in den Foci mit einer Aufl{\"o}sung von bis zu ~20 nm darzustellen. γH2AX-Foci zeigten sich als eine Verteilung aus einzelnen Untereinheiten („Nanofoci") mit einem Durchmesser von ~45 nm. Dies l{\"a}sst die Vermutung zu, dass es sich hier um die elementare Substruktur der Foci und somit der γH2AX enthaltenen Nukleosome handelt. DNA-PK-Foci wiesen hingegen eine diffusere Verteilung auf. Die in dieser Arbeit ermittelten Unterschiede im Migrationsverhalten der Zellen rechtfertigen eine weitere pr{\"a}klinische Untersuchung der verwendeten Inhibitoren als potentielle Zelltherapeutika f{\"u}r die Behandlung von GBM. Zudem konnte sich dSTORM als machtvolles Hilfsmittel, sowohl zur Analyse der Migration zugrundeliegenden Zytoskelettstruktur und der Effekte der Hsp90 Inhibierung, als auch, der Nanostruktur der DNA-DSB-Reparaturfoci herausstellen. Es ist anzunehmen, dass beugungsunbegrenzte Mikroskopiemethoden sich als bedeutende Werkzeuge in der medizinischen und biologischen Erforschung der DNA-Reparaturmechanismen herausstellen werden. Das in dieser Arbeit entwickelte ImageJ Plugin „FocAn-3D" bewies sich ebenfalls als ein vielversprechendes Werkzeug f{\"u}r die Analyse der Reparaturkinetik. Mit Hilfe von „FocAn-3D" sollte es somit m{\"o}glich sein u.a. den Einfluss gezielter Inhibition auf den zeitlichen Verlauf der Induktion und des Abbaus der DNA-Reparaturmaschinerie genauer zu studieren.},
  subject      = {Glioblastom},
  language  = {de}
}
@phdthesis{Sauer2019,
  author    = {Sauer, Mark},
  title     = {Die microRNA-26 Familie kontrolliert {\"u}ber den REST-Komplex ein f{\"u}r die Neurogenese essentielles regulatorisches RNA Netzwerk},
  doi       = {10.25972/OPUS-18400},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-184008},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {In einem sich entwickelnden multizellul{\"a}ren Organismus ist die r{\"a}umlich-zeitliche Regulation der Genexpression von entscheidender Bedeutung f{\"u}r die Bildung, Identit{\"a}t und Funktion von Zellen. Der REST (repressor element silencing transcription factor) Komplex spielt bei der neuronalen Differenzierung und bei der Aufrechterhaltung des neuronalen Status eine essentielle Rolle, indem er in nicht neuronalen Zellen und neuralen Vorl{\"a}ufern die Expression neuronaler Gene unterdr{\"u}ckt, in deren Promotorregion eine RE1 (repressor element 1) Erkennungssequenz vorhanden ist. W{\"a}hrend der neuronalen Differenzierung wird der REST-Komplex schrittweise inaktiviert, was zur Einleitung eines neuronalen Genexpression-Programms f{\"u}hrt. Es wird daher angenommen, dass die Inhibierung des REST-Komplexes ein essentieller Vorgang der Neurogenese ist. Wichtige Bestandteile f{\"u}r die transkriptionell repressive Funktion des REST-Komplexes sind kleine Phosphatasen (CTDSP = C-terminal domain small phosphatases), welche die Polymerase-II-Aktivit{\"a}t an Zielgenen inhibieren. Im Zebrafisch wurde gezeigt, dass ctdsp2 durch die miR-26b negativ reguliert wird. Alle miR-26 Familienmitglieder sind in Vertebraten evolution{\"a}r konserviert und in Introns von Ctdsp Genen kodiert. Sie sind in der Lage, die Expression ihres eigenen Wirtsgens mittels einer autoregulatorischen R{\"u}ckkopplungsschleife zu regulieren. Im Rahmen dieser Dissertation wurde als Modellsystem f{\"u}r die Neurogenese ein neurales Differenzierungssystem, welches auf murinen, embryonalen Stammzellen (ESCs) aufbaut, eingesetzt. Zur funktionellen Analyse der miR-26 Familie wurden mit Hilfe der CRISPR/Cas9-Methode verschiedene miR-26 Knockout (KO) ESC-Linien hergestellt. Hierbei wurden die Sequenzen der einzelnen Familienmitglieder und der gesamten miR-26 Familie im Genom von Wildtyp (Wt) ESCs deletiert. Diese miR-26-defizienten ESCLinien behielten ihre Pluripotenz und zeigten keinen Ph{\"a}notyp hinsichtlich Proliferation, Morphologie und Identit{\"a}t der Zellen w{\"a}hrend der Differenzierung bis zum neuralen Vorl{\"a}uferzellstadium (NPCs, engl.: neural progenitor cells). Jedoch f{\"u}hrte die Deletion sowohl der gesamten miR-26 Familie als auch einzelner Mitglieder bei der terminalen Differenzierung zu einem spezifischen Entwicklungsstillstand im NPC Stadium und infolgedessen zu einer starken Reduktion der Anzahl von Neuronen und Astroglia. Die Transkriptom-Analyse der differenzierten miR-26-KO ESCs mittels RNA-Seq zeigte, dass die Expression von Genen die mit der Neurogenese und der neuronalen Differenzierung, aber auch der Gliogenese assoziert sind, herunterreguliert war. Die Abwesenheit der miR-26 Familie f{\"u}hrte außerdem zu einer selektiven Reduzierung bestimmter miRNAs (REST-miRs), die einerseits die Expression von REST-Komplex Komponenten unterdr{\"u}cken k{\"o}nnen, und andererseits selbst unter dessen transkriptioneller Kontrolle stehen. Zu diesem REST-miR Netzwerk geh{\"o}ren einige miRNAs (miR-9, miR-124, miR-132 und miR-218), die wichtige Funktionen bei verschiedenen Prozessen der neuronalen Entwicklung haben. Weiterhin f{\"u}hrte der miR-26-KO zu einer Derepression der Proteinlevel von REST und CTDSP2 w{\"a}hrend der terminalen Differenzierung. Funktionelle Analysen mit miRNA mimics zeigten, dass erh{\"o}hte miR-26 Level zu einer Hochregulation von REST-miRs f{\"u}hren. Weitere Experimente, die darauf zielten, die Hierarchie des REST-miR Netwerks aufzukl{\"a}ren zeigten, dass die miR-26 Familie stromaufw{\"a}rts die REST-miR Expression reguliert. Zusammengefasst weisen die in dieser Arbeit gezeigten Daten darauf hin, dass die miR-26 Familie als Initiator der schrittweisen Inaktivierung des REST-Komplexes eine zentrale Rolle bei der Differenzierung von neuralen Vorl{\"a}uferzellen zu postmitotischen Neuronen spielt.},
  language  = {de}
}
@phdthesis{Breitenbach2019,
  author    = {Breitenbach, Tim},
  title     = {A mathematical optimal control based approach to pharmacological modulation with regulatory networks and external stimuli},
  doi       = {10.25972/OPUS-17436},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-174368},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {In this work models for molecular networks consisting of ordinary differential equations are extended by terms that include the interaction of the corresponding molecular network with the environment that the molecular network is embedded in. These terms model the effects of the external stimuli on the molecular network. The usability of this extension is demonstrated with a model of a circadian clock that is extended with certain terms and reproduces data from several experiments at the same time. Once the model including external stimuli is set up, a framework is developed in order to calculate external stimuli that have a predefined desired effect on the molecular network. For this purpose the task of finding appropriate external stimuli is formulated as a mathematical optimal control problem for which in order to solve it a lot of mathematical methods are available. Several methods are discussed and worked out in order to calculate a solution for the corresponding optimal control problem. The application of the framework to find pharmacological intervention points or effective drug combinations is pointed out and discussed. Furthermore the framework is related to existing network analysis tools and their combination for network analysis in order to find dedicated external stimuli is discussed. The total framework is verified with biological examples by comparing the calculated results with data from literature. For this purpose platelet aggregation is investigated based on a corresponding gene regulatory network and associated receptors are detected. Furthermore a transition from one to another type of T-helper cell is analyzed in a tumor setting where missing agents are calculated to induce the corresponding switch in vitro. Next a gene regulatory network of a myocardiocyte is investigated where it is shown how the presented framework can be used to compare different treatment strategies with respect to their beneficial effects and side effects quantitatively. Moreover a constitutively activated signaling pathway, which thus causes maleficent effects, is modeled and intervention points with corresponding treatment strategies are determined that steer the gene regulatory network from a pathological expression pattern to physiological one again.},
  subject      = {Bioinformatik},
  language  = {en}
}
@phdthesis{Wilde2019,
  author    = {Wilde, Sabrina},
  title     = {Einsatz von mechanistischen Biomarkern zur Charakterisierung und Bewertung von \(in\) \(vitro\) Genotoxinen},
  doi       = {10.25972/OPUS-18278},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-182782},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Die verf{\"u}gbaren in vitro Genotoxizit{\"a}tstests weisen hinsichtlich ihrer Spezifit{\"a}t und ihres Informationsgehalts zum vorliegenden Wirkmechanismus (Mode of Action, MoA) Einschr{\"a}nkungen auf. Um diese M{\"a}ngel zu {\"u}berwinden, wurden in dieser Arbeit zwei Ziele verfolgt, die zu der Entwicklung und Etablierung neuer in vitro Methoden zur Pr{\"u}fung auf Genotoxizit{\"a}t in der Arzneimittelentwicklung beitragen. 1. Etablierung und Bewertung einer neuen in vitro Genotoxizit{\"a}tsmethode (MultiFlow Methode) Die MultiFlow Methode basiert auf DNA-schadensassoziierten Proteinantworten von γH2AX (DNA-Doppelstrangbr{\"u}che), phosphorylierten H3 (S10) (mitotische Zellen), nukle{\"a}ren Protein p53 (Genotoxizit{\"a}t) und cleaved PARP1 (Apoptose) in TK6-Zellen. Insgesamt wurden 31 Modellsubstanzen mit dem MultiFlow Assay und erg{\"a}nzend mit dem etablierten Mikrokerntest (MicroFlow MNT), auf ihre F{\"a}higkeit verschiedene MoA-Gruppen (Aneugene/Klastogene/Nicht-Genotoxine) zu differenzieren, untersucht. Die Performance der „neuen" gegen{\"u}ber der „alten" Methode f{\"u}hrte zu einer verbesserten Sensitivit{\"a}t von 95\% gegen{\"u}ber 90\%, Spezifit{\"a}t von 90\% gegen{\"u}ber 72\% und einer MoA-Klassifizierungsrate von 85\% gegen{\"u}ber 45\% (Aneugen vs. Klastogen). 2. Identifizierung mechanistischer Biomarker zur Klassifizierung genotoxischer Substanzen Die Analyse 67 ausgew{\"a}hlter DNA-schadensassoziierter Gene in der QuantiGene Plex Methode zeigte, dass mehrere Gene gleichzeitig zur MoA-Klassifizierung beitragen k{\"o}nnen. Die Kombination der h{\"o}chstrangierten Marker BIK, KIF20A, TP53I3, DDB2 und OGG1 erm{\"o}glichte die beste Identifizierungsrate der Modellsubstanzen. Das synergetische Modell kategorisierte 16 von 16 Substanzen korrekt in Aneugene, Klastogene und Nicht-Genotoxine. Unter Verwendung der Leave-One-Out-Kreuzvalidierung wurde das Modell evaluiert und erreichte eine Sensitivit{\"a}t, Spezifit{\"a}t und Pr{\"a}diktivit{\"a}t von 86\%, 83\% und 85\%. Ergebnisse der traditionellen qPCR Methode zeigten, dass Genotoxizit{\"a}t mit TP53I3, Klastogenit{\"a}t mit ATR und RAD17 und oxidativer Stress mit NFE2L2 detektiert werden kann. Durch die Untersuchungen von posttranslationalen Modifikationen unter Verwendung der High-Content-Imaging-Technologie wurden mechanistische Assoziationen f{\"u}r BubR1 (S670) und pH3 (S28) mit Aneugenit{\"a}t, 53BP1 (S1778) und FANCD2 (S1404) mit Klastogenit{\"a}t, p53 (K373) mit Genotoxizit{\"a}t und Nrf2 (S40) mit oxidativem Stress identifiziert. Diese Arbeit zeigt, dass (Geno)toxine unterschiedliche Gen- und Proteinver{\"a}nderungen in TK6-Zellen induzieren, die zur Erfassung mechanistischer Aktivit{\"a}ten und Einteilung (geno)toxischer MoA-Gruppen (Aneugen/Klastogen/ Reaktive Sauerstoffspezies) eingesetzt werden k{\"o}nnen und daher eine bessere Risikobewertung von Wirkstoffkandidaten erm{\"o}glichen.},
  subject      = {Genotoxizit{\"a}t},
  language  = {de}
}
@phdthesis{DiegmanngebWeissbach2019,
  author    = {Diegmann [geb. Weißbach], Susann},
  title     = {Identifizierung des Mutationsspektrums und Charakterisierung relevanter Mutationen im Multiplen Myelom},
  doi       = {10.25972/OPUS-11480},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-114800},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Das Multiple Myelom (MM) ist eine maligne B-Zell-Erkrankung, welche von einer großen Heterogenit{\"a}t auf der biologischen und klinischen Ebene sowie in der Therapieantwort gepr{\"a}gt ist. Durch die biologische Interpretation von whole exome sequencing (WES)-Daten der Tumor- und Normalproben von f{\"u}nf MM-Patienten und sechs MM-Zelllinien (ZL) sowie dem Einbezug von publizierten next generation sequencing (NGS)-Daten von 38 MM-Patienten konnten in dieser Dissertation sowohl somatische tumorrelevante Mutationen identifiziert als auch ein MM-spezifisches Signaltransduktionsnetzwerk definiert werden. Interessanterweise wurde in fast 100 \% der MM-Patienten mindestens eine Mutation und in ~50 \% der MM-Patienten sogar mehr als eine Mutation innerhalb dieses Netzwerkes beobachtet, was auf eine inter- und intra-individuelle Signalweg-Redundanz hinweist, die f{\"u}r die individuelle Therapieentscheidung m{\"o}glicherweise von Bedeutung sein k{\"o}nnte. Außerdem konnte best{\"a}tigt werden, dass identische, positionsspezifische und genspezifische Mutationen im MM selten wiederholt auftreten. Als h{\"a}ufig mutierte Gene im MM konnten KRAS, NRAS, LRP1B, FAM46C, WHSC1, ALOX12B, DIS3 und PKHD1 identifiziert werden. Interessanterweise wurde die DIS3-Mutation in der MM-ZL OPM2 gemeinsam mit einer copy neutral loss of heterozygosity (CNLOH) im DIS3-Lokus detektiert, und in der MM-ZL AMO1 wurde eine noch nicht n{\"a}her charakterisierte KRAS-Mutation in Exon 4 in Verbindung mit einem copy number (CN)-Zugewinn und einer erh{\"o}hten KRAS-Genexpression gefunden. DIS3 ist ein enzymatisch aktiver Teil des humanen RNA-Exosom-Komplexes und KRAS ein zentrales Protein im RTK-Signalweg, wodurch genetische Aberrationen in diesen Genen m{\"o}glicherweise in der Entstehung oder Progression des MMs eine zentrale Rolle spielen. Daher wurde die gesamte coding sequence (CDS) der Gene DIS3 und KRAS an Tumorproben eines einheitlich behandelten Patientensets der DSMM-XI-Studie mit einem Amplikon-Tiefen-Sequenzierungsansatz untersucht. Das Patientenset bestand aus 81 MM-Patienten mit verf{\"u}gbaren zytogenetischen und klinischen Daten. Dies ergab Aufschluss {\"u}ber die Verteilung der Mutationen innerhalb der Gene und dem Vorkommen der Mutationen in Haupt- und Nebenklonen des Tumors. Des Weiteren wurde die Assoziation der Mutationen mit weiteren klassischen zytogenetischen Alterationen (z.B. Deletion von Chr 13q14, t(4;14)-Translokation) untersucht und der Einfluss der Mutationen in Haupt- und Nebenklonen auf den klinischen Verlauf und die Therapieantwort bestimmt. Besonders hervorzuheben war dabei die Entdeckung von sieben neuen Mutationen sowie drei zuvor unbeschriebenen hot spot-Mutationen an den Aminos{\"a}ure (AS)-Positionen p.D488, p.E665 und p.R780 in DIS3. Es wurde des Weiteren die Assoziation von DIS3-Mutationen mit einer Chr 13q14-Deletion und mit IGH-Translokationen best{\"a}tigt. Interessanterweise wurde ein niedrigeres medianes overall survival (OS) f{\"u}r MM-Patienten mit einer DIS3-Mutation sowie auch eine schlechtere Therapieantwort f{\"u}r MM-Patienten mit einer DIS3-Mutation im Nebenklon im Vergleich zum Hauptklon beobachtet. In KRAS konnten die bereits publizierten Mutationen best{\"a}tigt und keine Auswirkungen der KRAS-Mutationen in Haupt- oder Nebenklon auf den klinischen Verlauf oder die Therapieantwort erkannt werden. Erste siRNA vermittelte knockdown-Experimente von KRAS und {\"U}berexpressionsexperimente von KRAS-Wildtyp (WT) und der KRAS-Mutationen p.G12A, p.A146T und p.A146V mittels lentiviraler Transfektion zeigten eine Abh{\"a}ngigkeit der Phosphorylierung von MEK1/2 und ERK1/2 von dem KRAS-Mutationsstatus. Zusammenfassend liefert die vorliegende Dissertation einen detaillierten Einblick in die molekularen Strukturen des MMs, vor allem im Hinblick auf die Rolle von DIS3 und KRAS bei der Tumorentwicklung und dem klinischen Verlauf.},
  subject      = {Plasmozytom},
  language  = {de}
}
@phdthesis{Kaymak2019,
  author    = {Kaymak, Irem},
  title     = {Identification of metabolic liabilities in 3D models of cancer},
  doi       = {10.25972/OPUS-18154},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-181544},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Inefficient vascularisation of solid tumours leads to the formation of oxygen and nutrient gradients. In order to mimic this specific feature of the tumour microenvironment, a multicellular tumour spheroid (SPH) culture system was used. These experiments were implemented in p53 isogenic colon cancer cell lines (HCT116 p53 +/+ and HCT116 p53-/-) since Tp53 has important regulatory functions in tumour metabolism. First, the characteristics of the cells cultured as monolayers and as spheroids were investigated by using RNA sequencing and metabolomics to compare gene expression and metabolic features of cells grown in different conditions. This analysis showed that certain features of gene expression found in tumours are also present in spheroids but not in monolayer cultures, including reduced proliferation and induction of hypoxia related genes. Moreover, comparison between the different genotypes revealed that the expression of genes involved in cholesterol homeostasis is induced in p53 deficient cells compared to p53 wild type cells and this difference was only detected in spheroids and tumour samples but not in monolayer cultures. In addition, it was established that loss of p53 leads to the induction of enzymes of the mevalonate pathway via activation of the transcription factor SREBP2, resulting in a metabolic rewiring that supports the generation of ubiquinone (coenzyme Q10). An adequate supply of ubiquinone was essential to support mitochondrial electron transport and pyrimidine biosynthesis in p53 deficient cancer cells under conditions of metabolic stress. Moreover, inhibition of the mevalonate pathway using statins selectively induced oxidative stress and apoptosis in p53 deficient colon cancer cells exposed to oxygen and nutrient deprivation. This was caused by ubiquinone being required for electron transfer by dihydroorotate dehydrogenase, an essential enzyme of the pyrimidine nucleotide biosynthesis pathway. Supplementation with exogenous nucleosides relieved the demand for electron transfer and restored viability of p53 deficient cancer cells under metabolic stress. Moreover, the mevalonate pathway was also essential for the synthesis of ubiquinone for nucleotide biosynthesis to support growth of intestinal tumour organoids. Together, these findings highlight the importance of the mevalonate pathway in cancer cells and provide molecular evidence for an enhanced sensitivity towards the inhibition of mitochondrial electron transfer in tumour-like metabolic environments.},
  subject      = {Tumor},
  language  = {en}
}
@phdthesis{Grimm2019,
  author    = {Grimm, Johannes},
  title     = {Autocrine and paracrine effects of BRAF inhibitor induced senescence in melanoma},
  doi       = {10.25972/OPUS-18116},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-181161},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {The FDA approval of targeted therapy with BRAFV600E inhibitors like vemurafenib and dabrafenib in 2011 has been the first major breakthrough in the treatment of metastatic melanoma since almost three decades. Despite increased progression free survival and elevated overall survival rates, complete responses are scarce due to resistance development approximately six months after the initial drug treatment. It was previously shown in our group that melanoma cells under vemurafenib pressure in vitro and in vivo exhibit features of drug-induced senescence. It is known that some cell types, which undergo this cell cycle arrest, develop a so-called senescence associated secretome and it has been reported that melanoma cell lines also upregulate the expression of different factors after senescence induction. This work describes the effect of the vemurafenib-induced secretome on cells. Conditioned supernatants of vemurafenib-treated cells increased the viability of naive fibroblast and melanoma cell lines. RNA analysis of donor melanoma cells revealed elevated transcriptional levels of FGF1, MMP2 and CCL2 in the majority of tested cell lines under vemurafenib pressure, and I could confirm the secretion of functional proteins. Similar observations were also done after MEK inhibition as well as in a combined BRAF and MEK inhibitor treatment situation. Interestingly, the transcription of other FGF ligands (FGF7, FGF17) was also elevated after MEK/ERK1/2 inhibition. As FGF receptors are therapeutically relevant, I focused on the analysis of FGFR-dependent processes in response to BRAF inhibition. Recombinant FGF1 increased the survival rate of melanoma cells under vemurafenib pressure, while inhibition of the FGFR pathway diminished the viability of melanoma cells in combination with vemurafenib and blocked the stimulatory effect of vemurafenib conditioned medium. The BRAF inhibitor induced secretome is regulated by active PI3K/AKT signaling, and the joint inhibition of mTor and BRAFV600E led to decreased senescence induction and to a diminished induction of the secretome-associated genes. In parallel, combined inhibition of MEK and PI3K also drastically decreased mRNA levels of the relevant secretome components back to basal levels. In summary, I could demonstrate that BRAF inhibitor treated melanoma cell lines acquire a specific PI3K/AKT dependent secretome, which is characterized by FGF1, CCL2 and MMP2. This secretome is able to stimulate other cells such as naive melanoma cells and fibroblasts and contributes to a better survival under drug pressure. These data are therapeutically highly relevant, as they imply the usage of novel drug combinations, especially specific FGFR inhibitors, with BRAF inhibitors in the clinic.},
  subject      = {Inhibitor},
  language  = {en}
}
@phdthesis{Letschert2019,
  author    = {Letschert, Sebastian},
  title     = {Quantitative Analysis of Membrane Components using Super-Resolution Microscopy},
  doi       = {10.25972/OPUS-16213},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-162139},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {The plasma membrane is one of the most thoroughly studied and at the same time most complex, diverse, and least understood cellular structures. Its function is determined by the molecular composition as well as the spatial arrangement of its components. Even after decades of extensive membrane research and the proposal of dozens of models and theories, the structural organization of plasma membranes remains largely unknown. Modern imaging tools such as super-resolution fluorescence microscopy are one of the most efficient techniques in life sciences and are widely used to study the spatial arrangement and quantitative behavior of biomolecules in fixed and living cells. In this work, direct stochastic optical reconstruction microscopy (dSTORM) was used to investigate the structural distribution of mem-brane components with virtually molecular resolution. Key issues are different preparation and staining strategies for membrane imaging as well as localization-based quantitative analyses of membrane molecules. An essential precondition for the spatial and quantitative analysis of membrane components is the prevention of photoswitching artifacts in reconstructed localization microscopy images. Therefore, the impact of irradiation intensity, label density and photoswitching behavior on the distribution of plasma membrane and mitochondrial membrane proteins in dSTORM images was investigated. It is demonstrated that the combination of densely labeled plasma membranes and inappropriate photoswitching rates induces artificial membrane clusters. Moreover, inhomogeneous localization distributions induced by projections of three-dimensional membrane structures such as microvilli and vesicles are prone to generate artifacts in images of biological membranes. Alternative imaging techniques and ways to prevent artifacts in single-molecule localization microscopy are presented and extensively discussed. Another central topic addresses the spatial organization of glycosylated components covering the cell membrane. It is shown that a bioorthogonal chemical reporter system consisting of modified monosaccharide precursors and organic fluorophores can be used for specific labeling of membrane-associated glycoproteins and -lipids. The distribution of glycans was visualized by dSTORM showing a homogeneous molecule distribution on different mammalian cell lines without the presence of clusters. An absolute number of around five million glycans per cell was estimated and the results show that the combination of metabolic labeling, click chemistry, and single-molecule localization microscopy can be efficiently used to study cell surface glycoconjugates. In a third project, dSTORM was performed to investigate low-expressing receptors on cancer cells which can act as targets in personalized immunotherapy. Primary multiple myeloma cells derived from the bone marrow of several patients were analyzed for CD19 expression as potential target for chimeric antigen receptor (CAR)-modified T cells. Depending on the patient, 60-1,600 CD19 molecules per cell were quantified and functional in vitro tests demonstrate that the threshold for CD19 CAR T recognition is below 100 CD19 molecules per target cell. Results are compared with flow cytometry data, and the important roles of efficient labeling and appropriate control experiments are discussed.},
  subject      = {Fluoreszenzmikroskopie},
  language  = {en}
}
@phdthesis{Simon2019,
  author    = {Simon, Katja},
  title     = {Identifying the role of Myb-MuvB in gene expression and proliferation of lung cancer cells},
  doi       = {10.25972/OPUS-16181},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-161814},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {The evolutionary conserved Myb-MuvB (MMB) multiprotein complex is a transcriptional master regulator of mitotic gene expression. The MMB subunits B-MYB, FOXM1 as well as target genes of MMB are often overexpressed in different cancer types. Elevated expression of these genes correlates with an advanced tumor state and a poor prognosis for patients. Furthermore, it has been reported that pathways, which are involved in regulating the mitotic machinery are attractive for a potential treatment of cancers harbouring Ras mutations (Luo et al., 2009). This suggest that the MMB complex could be required for tumorigenesis by mediating overactivity of mitotic genes and that the MMB could be a useful target for lung cancer treatment. However, although MMB has been characterized biochemically, the contribution of MMB to tumorigenesis is largely unknown in particular in vivo. In this thesis, it was demonstrated that the MMB complex is required for lung tumorigenesis in vivo in a mouse model of non small cell lung cancer. Elevated levels of B-MYB, NUSAP1 or CENPF in advanced tumors as opposed to low levels of these proteins levels in grade 1 or 2 tumors support the possible contribution of MMB to lung tumorigenesis and the oncogenic potential of B-MYB.The tumor growth promoting function of B-MYB was illustrated by a lower fraction of KI-67 positive cells in vivo and a significantly high impairment in proliferation after loss of B-Myb in vitro. Defects in cytokinesis and an abnormal cell cycle profile after loss of B-Myb underscore the impact of B-MYB on proliferation of lung cancer cell lines. The incomplete recombination of B-Myb in murine lung tumors and in the tumor derived primary cell lines illustrates the selection pressure against the complete loss of B-Myb and further demonstrats that B-Myb is a tumor-essential gene. In the last part of this thesis, the contribution of MMB to the proliferation of human lung cancer cells was demonstrated by the RNAi-mediated depletion of B-Myb. Detection of elevated B-MYB levels in human adenocarcinoma and a reduced proliferation, cytokinesis defects and abnormal cell cycle profile after loss of B-MYB in human lung cancer cell lines underlines the potential of B-MYB to serve as a clinical marker.},
  subject      = {Lungenkrebs},
  language  = {en}
}
@phdthesis{Fleischmann2019,
  author    = {Fleischmann, Pauline Nikola},
  title     = {Starting foraging life: Early calibration and daily use of the navigational system in \(Cataglyphis\) ants},
  doi       = {10.25972/OPUS-15995},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-159951},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Cataglyphis ants are famous for their navigational abilities. They live in hostile habitats where they forage as solitary scavengers covering distances of more than hundred thousand times their body lengths. To return to their nest with a prey item - mainly other dead insects that did not survive the heat - Cataglyphis ants constantly keep track of their directions and distances travelled. The navigational strategy is called path integration, and it enables an ant to return to the nest in a straight line using its home vector. Cataglyphis ants mainly rely on celestial compass cues, like the position of the sun or the UV polarization pattern, to determine directions, and they use an idiothetic step counter and optic flow to measure distances. In addition, they acquire information about visual, olfactory and tactile landmarks, and the wind direction to increase their chances of returning to the nest safe and sound. Cataglyphis' navigational performance becomes even more impressive if one considers their life style. Most time of their lives, the ants stay underground and perform tasks within the colony. When they start their foraging careers outside the nest, they have to calibrate their compass systems and acquire all information necessary for navigation during subsequent foraging. This navigational toolkit is not instantaneously available, but has to be filled with experience. For that reason, Cataglyphis ants perform a striking behavior for up to three days before actually foraging. These so-called learning walks are crucial for the success as foragers later on. In the present thesis, both the ontogeny and the fine-structure of learning walks has been investigated. Here I show with displacement experiments that Cataglyphis ants need enough space and enough time to perform learning walks. Spatially restricted novices, i. e. na{\"i}ve ants, could not find back to the nest when tested as foragers later on. Furthermore, ants have to perform several learning walks over 1-3 days to gain landmark information for successful homing as foragers. An increasing number of feeder visits also increases the importance of landmark information, whereas in the beginning ants fully rely on their path-integration vector. Learning walks are well-structured. High-speed video analysis revealed that Cataglyphis ants include species-specific rotational elements in their learning walks. Greek Cataglyphis ants (C. noda and C. aenescens) inhabiting a cluttered pine forest perform voltes, small walked circles, and pirouettes, tight turns about the body axis with frequent stopping phases. During the longest stopping phases, the ants gaze back to their nest entrance. The Tunisian Cataglyphis fortis ants inhabiting featureless saltpans only perform voltes without directed gazes. The function of voltes has not yet been revealed. In contrast, the fine structure of pirouettes suggests that the ants take snapshots of the panorama towards their homing direction to memorize the nest's surroundings. The most likely hypothesis was that Cataglyphis ants align the gaze directions using their path integrator, which gets directional input from celestial cues during foraging. To test this hypothesis, a manipulation experiment was performed changing the celestial cues above the nest entrance (no sun, no natural polarization pattern, no UV light). The accurately directed gazes to the nest entrance offer an easily quantifiable readout suitable to ask the ants where they expect their nest entrance. Unexpectedly, all novices performing learning walks under artificial sky conditions looked back to the nest entrance. This was especially surprising, because neuronal changes in the mushroom bodies and the central complex receiving visual input could only be induced with the natural sky when comparing test animals with interior workers. The behavioral findings indicated that Cataglyphis ants use another directional reference system to align their gaze directions during the longest stopping phases of learning walk pirouettes. One possibility was the earth's magnetic field. Indeed, already disarraying the geomagnetic field at the nest entrance with an electromagnetic flat coil indicated that the ants use magnetic information to align their looks back to the nest entrance. To investigate this finding further, ants were confronted with a controlled magnetic field using a Helmholtz coil. Elimination of the horizontal field component led to undirected gaze directions like the disarray did. Rotating the magnetic field about 90°, 180° or -90° shifted the ants' gaze directions in a predictable manner. Therefore, the earth's magnetic field is a necessary and sufficient reference system for aligning nest-centered gazes during learning-walk pirouettes. Whether it is additionally used for other navigational purposes, e. g. for calibrating the solar ephemeris, remains to be tested. Maybe the voltes performed by all Cataglyphis ant species investigated so far can help to answer this question..},
  subject      = {Cataglyphis},
  language  = {en}
}
@phdthesis{Beck2019,
  author    = {Beck, Katharina},
  title     = {Die nitrerge Neurotransmission im Gastrointestinaltrakt der Maus},
  doi       = {10.25972/OPUS-15989},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-159896},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Die NO-sensitive Guanylyl-Cyclase (NO-GC) ist ein zentrales Enzym der NO/cGMP-Signalkaskade, das {\"u}ber die Aktivierung von NO zur Bildung des second messangers cGMP f{\"u}hrt. Die NO-GC setzt sich aus zwei Untereinheiten zusammen, sodass zwei Isoformen des Enzyms gebildet werden k{\"o}nnen (α1β1 und α2β1). Da die genaue Verteilung der beiden Isoformen im Colon nicht bekannt ist, wurde diese im ersten Teil dieser Arbeit charakterisiert. Immunhistochemie und In-situ-Hybridisierung zeigten die Expression beider Isoformen sowohl in der glatten Muskelschicht als auch in der Submukosa und Lamina propria. Dabei war die α1β1-Isoform ubiquit{\"a}r, die α2β1-Isoform dagegen haupts{\"a}chlich im Bereich des myenterischen Plexus vorzufinden. In der glatten Muskelschicht des Colons ist die NO-GC in glatten Muskelzellen (SMC), interstitiellen Zellen von Cajal (ICC) sowie Fibroblasten-{\"a}hnliche Zellen (FLC) exprimiert und haupts{\"a}chlich in die Modulation der gastrointestinalen Motilit{\"a}t involviert. Zur spezifischen Charakterisierung der Funktion der NO-GC in den einzelnen Zelltypen wurden Knockout-M{\"a}use generiert, denen die NO-GC global (GCKO) oder spezifisch in SMC (SMC-GCKO), ICC (ICC-GCKO) oder beiden Zelltypen (SMC/ICC-GCKO) fehlt. Anhand dieser Mausmodelle sollten im zweiten Teil dieser Arbeit die modulatorischen Effekte der NO-GC auf die spontanen Kontraktionen des Colons bestimmt werden. Zur Charakterisierung der spontanen Kontraktionen der zirkul{\"a}ren Muskelschicht wurden Myographiestudien mit 2,5 mm langen Colonringen durchgef{\"u}hrt. Hierbei konnten drei verschiedene Kontraktionen gemessen werden: Kleine, hochfrequente Ripples, mittlere Kontraktionen und große Kontraktionen. Die detaillierte Analyse der einzelnen Kontraktionen zeigte einerseits eine NO-unabh{\"a}ngige Regulation der Ripples, andererseits eine NO-abh{\"a}ngige Modulation der mittleren und großen Kontraktionen {\"u}ber die NO-GC in SMC und ICC. Die NO-GC in SMC beeinflusst die Kontraktionen vermutlich vor allem {\"u}ber die Regulation des Muskeltonus der zirkul{\"a}ren Muskelschicht. Die NO-GC in ICC dagegen modifiziert die spontanen Kontraktionen m{\"o}glicherweise {\"u}ber eine Ver{\"a}nderung der Schrittmacheraktivit{\"a}t. Allerdings f{\"u}hrt erst ein Funktionsverlust des NO/cGMP-Signalweges in beiden Zelltypen zu einem sichtbar ver{\"a}nderten Kontraktionsmuster, das dem von globalen Knockout-Tieren glich. Dies weist auf eine kompensatorische Wirkung der NO-GC im jeweils anderen Zelltyp hin. Zur Analyse der propulsiven Kontraktionen entlang des gesamten Colons wurden Videoaufnahmen der Darmbewegungen in Kontraktionsmusterkarten transformiert. Zudem wurde der Darm durchsp{\"u}lt und die Ausflusstropfen aufgezeichnet, um die Effektivit{\"a}t der Kontraktionen beurteilen zu k{\"o}nnen. Hierbei zeigte sich, dass eine Beeintr{\"a}chtigung des NO/cGMP-Signalweges eine verminderte Effektivit{\"a}t der Kontraktionen zur Folge hat und vermutlich durch eine beeintr{\"a}chtige Synchronisation der Kontraktionen erkl{\"a}rt werden kann. In diesem Regulationsmechanismus konnte vor allem der NO-GC in SMC eine {\"u}bergeordnete Rolle zugewiesen werden. Der dritte Teil der Arbeit thematisierte den Befund, dass SMC-GCKO-Tiere ca. 5 Monate nach Tamoxifen-Behandlung Entartungen der Mukosa entwickelten. Diese Entartung war lediglich in Tamoxifen-induzierten Knockout-Tieren vorzufinden. Histologische Analysen identifizierten die Entartungen als tubulovill{\"o}ses Adenom. Die Genexpressionsanalyse von Mukosafalten von SMC-GCKO- und heterozygoten Kontrolltieren zeigte eine Vielzahl von Genen, welche spezifisch bei colorectalem Karzinom differenziell exprimiert sind. Einer dieser Faktoren war der BMP-Antagonist Gremlin1. Dieser Faktor erschien von besonderem Interesse, da er in Zellen der Lamina muscularis mucosae und kryptennahen Myofibroblasten exprimiert wird. Immunhistochemische Analysen ließen vermuten, dass diese Zellen sowohl die NO-GC als auch die Cre-Rekombinase unter dem SMMHC-Promotor exprimieren. Diese Arbeit liefert demnach Hinweise darauf, dass die NO-GC einen wichtigen Regulator innerhalb der Stammzellnische bildet. Die Deletion der NO-GC f{\"u}hrt vermutlich zu einer verst{\"a}rkten Bildung bzw. Sekretion von Gremlin1, was die Hom{\"o}ostase der mukosalen Erneuerung st{\"o}rt und somit zur Entwicklung von Adenomen f{\"u}hrt.},
  subject      = {Gastrointestinaltrakt},
  language  = {de}
}
@phdthesis{Mekala2019,
  author    = {Mekala, SubbaRao},
  title     = {Generation of cardiomyocytes from vessel wall-resident stem cells},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-146046},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Myocardial infarction (MI) is a major cause of health problems and is among the leading deadly ending diseases. Accordingly, regenerating functional myocardial tissue and/or cardiac repair by stem cells is one of the most desired aims worldwide. Indeed, the human heart serves as an ideal target for regenerative intervention, because the capacity of the adult myocardium to restore itself after injury or infarct is limited. Thus, identifying new sources of tissue resident adult stem or progenitor cells with cardiovascular potential would help to establish more sophisticated therapies in order to either prevent cardiac failure or to achieve a functional repair. Ongoing research worldwide in this field is focusing on a) induced pluripotent stem (iPS) cells, b) embryonic stem (ES) cells and c) adult stem cells (e. g. mesenchymal stem cells) as well as cardiac fibroblasts or myofibroblasts. However, thus far, these efforts did not result in therapeutic strategies that were transferable into the clinical management of MI and heart failure. Hence, identifying endogenous and more cardiac-related sources of stem cells capable of differentiating into mature cardiomyocytes would open promising new therapeutic opportunities. The working hypothesis of this thesis is that the vascular wall serves as a niche for cardiogenic stem cells. In recent years, various groups have identified different types of progenitors or mesenchymal stem cell-like cells in the adventitia and sub-endothelial zone of the adult vessel wall, the so called vessel wall-resident stem cells (VW-SCs). Considering the fact that heart muscle tissue contains blood vessels in very high density, the physiological relevance of VW-SCs for the myocardium can as yet only be assumed. The aim of the present work is to study whether a subset of VW-SCs might have the capacity to differentiate into cardiomyocyte-like cells. This assumption was challenged using adult mouse aorta-derived cells cultivated in different media and treated with selected factors. The presented results reveal the generation of spontaneously beating cardiomyocyte-like cells using specific media conditions without any genetic manipulation. The cells reproducibly started beating at culture days 8-10. Further analyses revealed that in contrast to several publications reporting the Sca-1+ cells as cardiac progenitors the Sca-1- fraction of aortic wall-derived VW-SCs reproducibly delivered beating cells in culture. Similar to mature cardiomyocytes the beating cells developed sarcomeric structures indicated by the typical cross striated staining pattern upon immunofluorescence analysis detecting α-sarcomeric actinin (α-SRA) and electron microscopic analysis. These analyses also showed the formation of sarcoplasmic reticulum which serves as calcium store. Correspondingly, the aortic wall-derived beating cardiomyocyte-like cells (Ao-bCMs) exhibited calcium oscillations. This differentiation seems to be dependent on an inflammatory microenvironment since depletion of VW-SC-derived macrophages by treatment with clodronate liposomes in vitro stopped the generation of Ao bCMs. These locally generated F4/80+ macrophages exhibit high levels of VEGF (vascular endothelial growth factor). To a great majority, VW-SCs were found to be positive for VEGFR-2 and blocking this receptor also stopped the generation VW-SC-derived beating cells in vitro. Furthermore, the treatment of aortic wall-derived cells with the ß-receptor agonist isoproterenol or the antagonist propranolol resulted in a significant increase or decrease of beating frequency. Finally, fluorescently labeled aortic wall-derived cells were implanted into the developing chick embryo heart field where they became positive for α-SRA two days after implantation. The current data strongly suggest that VW-SCs resident in the vascular adventitia deliver both progenitors for an inflammatory microenvironment and beating cells. The present study identifies that the Sca-1- rather than Sca-1+ fraction of mouse aortic wall-derived cells harbors VW-SCs differentiating into cardiomyocyte-like cells and reveals an essential role of VW-SCs-derived inflammatory macrophages and VEGF-signaling in this process. Furthermore, this study demonstrates the cardiogenic capacity of aortic VW-SCs in vivo using a chimeric chick embryonic model.},
  subject      = {Herzmuskelzelle},
  language  = {en}
}
@phdthesis{Hieke2019,
  author    = {Hieke, Marie},
  title     = {Synaptic arrangements and potential communication partners of \(Drosophila's\) PDF-containing clock neurons within the accessory medulla},
  doi       = {10.25972/OPUS-17598},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-175988},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Endogenous clocks regulate physiological as well as behavioral rhythms within all organisms. They are well investigated in D. melanogaster on a molecular as well as anatomical level. The neuronal clock network within the brain represents the center for rhythmic activity control. One neuronal clock subgroup, the pigment dispersing factor (PDF) neurons, stands out for its importance in regulating rhythmic behavior. These neurons express the neuropeptide PDF (pigment dispersing factor). A small neuropil at the medulla's edge, the accessory medulla (AME), is of special interest, as it has been determined as the main center for clock control. It is not only highly innervated by the PDF neurons but also by terminals of all other clock neuron subgroups. Furthermore, terminals of the photoreceptors provide light information to the AME. Many different types of neurons converge within the AME and afterward spread to their next target. Thereby the AME is supplied with information from a variety of brain regions. Among these neurons are the aminergic ones whose receptors' are expressed in the PDF neurons. The present study sheds light onto putative synaptic partners and anatomical arrangements within the neuronal clock network, especially within the AME, as such knowledge is a prerequisite to understand circadian behavior. The aminergic neurons' conspicuous vicinity to the PDF neurons suggests synaptic communication among them. Thus, based on former anatomical studies regarding this issue detailed light microscopic studies have been performed. Double immunolabellings, analyses of the spatial relation of pre- and postsynaptic sites of the individual neuron populations with respect to each other and the identification of putative synaptic partners using GRASP reenforce the hypothesis of synaptic interactions within the AME between dopaminergic/ serotonergic neurons and the PDF neurons. To shed light on the synaptic partners I performed first steps in array tomography, as it allows terrific informative analyses of fluorescent signals on an ultrastructural level. Therefore, I tested different ways of sample preparation in order to achieve and optimize fluorescent signals on 100 nm thin tissue sections and I made overlays with electron microscopic images. Furthermore, I made assumptions about synaptic modulations within the neuronal clock network via glial cells. I detected their cell bodies in close vicinity to the AME and PDFcontaining clock neurons. It has already been shown that glial cells modulate the release of PDF from s-LNvs' terminals within the dorsal brain. On an anatomical level this modulation appears to exist also within the AME, as synaptic contacts that involve PDF-positive dendritic terminals are embedded into glial fibers. Intriguingly, these postsynaptic PDF fibers are often VIIAbstract part of dyadic or even multiple-contact sites in opposite to prolonged presynaptic active zonesimplicating complex neuronal interactions within the AME. To unravel possible mechanisms of such synaptic arrangements, I tried to localize the ABC transporter White. Its presence within glial cells would indicate a recycling mechanism of transmitted amines which allows their fast re-provision. Taken together, synapses accompanied by glial cells appear to be a common arrangement within the AME to regulate circadian behavior. The complexity of mechanisms that contribute in modulation of circadian information is reflected by the complex diversity of synaptic arrangements that involves obviously several types of neuron populations},
  subject      = {Taufliege},
  language  = {en}
}
@phdthesis{Horn2019,
  author    = {Horn, Jessica},
  title     = {Molecular and functional characterization of the long non-coding RNA SSR42 in \(Staphylococcus\) \(aureus\)},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-175778},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Staphylococcus aureus asymptomatically colonizes the skin and anterior nares of 20-30\% of the healthy human population. As an opportunistic human pathogen it elicits a variety of infections ranging from skin and soft tissue infections to highly severe manifestations such as pneumonia, endocarditis and osteomyelitis. Due to the emergence of multi resistant strains, treatment of staphylococcal infections becomes more and more challenging and the WHO therefore classified S. aureus as a "superbug". The variety of diseases triggered by S. aureus is the result of a versatile expression of a large set of virulence factors. The most prominent virulence factor is the cytotoxic and haemolytic pore-forming α-toxin whose expression is mediated by a complex regulatory network involving two-component systems such as the agr quorum-sensing system, accessory transcriptional regulators and alternative sigma-factors. However, the intricate regulatory network is not yet understood in its entirety. Recently, a transposon mutation screen identified the AraC-family transcriptional regulator 'Repressor of surface proteins' (Rsp) to regulate haemolysis, cytotoxicity and the expression of various virulence associated factors. Deletion of rsp was accompanied by a complete loss of transcription of a 1232 nt long non-coding RNA, SSR42. This doctoral thesis focuses on the molecular and functional characterization of SSR42. By analysing the transcriptome and proteome of mutants in either SSR42 or both SSR42 and rsp, as well as by complementation of SSR42 in trans, the ncRNA was identified as the main effector of Rsp-mediated virulence. Mutants in SSR42 exhibited strong effects on transcriptional and translational level when compared to wild-type bacteria. These changes resulted in phenotypic alterations such as strongly reduced haemolytic activity and cytotoxicity towards epithelial cells as well as reduced virulence in a murine infection model. Deletion of SSR42 further promoted the formation of small colony variants (SCV) during long term infection of endothelial cells and demonstrated the importance of this molecule for intracellular bacteria. The impact of this ncRNA on staphylococcal haemolysis was revealed to be executed by modulation of sae mRNA stability and by applying mutational studies functional domains within SSR42 were identified. Moreover, various stressors modulated the transcription of SSR42 and antibiotic challenge resulted in SSR42-dependently increased haemolysis and cytotoxicity. Transcription of SSR42 itself was found under control of various important global regulators including AgrA, SaeS, CodY and σB, thereby illustrating a central position in S. aureus virulence gene regulation. The present study thus demonstrates SSR42 as a global virulence regulatory RNA which is important for haemolysis, disease progression and adaption of S. aureus to intracellular conditions via formation of SCVs.},
  subject      = {Staphylococcus aureus},
  language  = {en}
}
@phdthesis{Schubert2019,
  author    = {Schubert, Frank Klaus},
  title     = {The circadian clock network of \(Drosophila\) \(melanogaster\)},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-157136},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {All living organisms need timekeeping mechanisms to track and anticipate cyclic changes in their environment. The ability to prepare for and respond to daily and seasonal changes is endowed by circadian clocks. The systemic features and molecular mechanisms that drive circadian rhythmicity are highly conserved across kingdoms. Therefore, Drosophila melanogaster with its relatively small brain (ca. 135.000 neurons) and the outstanding genetic tools that are available, is a perfect model to investigate the properties and relevance of the circadian system in a complex, but yet comprehensible organism. The last 50 years of chronobiological research in the fruit fly resulted in a deep understanding of the molecular machinery that drives circadian rhythmicity, and various histological studies revealed the neural substrate of the circadian system. However, a detailed neuroanatomical and physiological description on the single-cell level has still to be acquired. Thus, I employed a multicolor labeling approach to characterize the clock network of Drosophila melanogaster with single-cell resolution and additionally investigated the putative in- and output sites of selected neurons. To further study the functional hierarchy within the clock network and to monitor the "ticking clock" over the course of several circadian cycles, I established a method, which allows us to follow the accumulation and degradation of the core clock genes in living brain explants by the means of bioluminescence imaging of single-cells.},
  subject      = {Taufliege},
  language  = {en}
}
@phdthesis{Maierhofer2018,
  author    = {Maierhofer, Anna},
  title     = {Altersassoziierte und strahleninduzierte Ver{\"a}nderungen des genomweiten DNA-Methylierungs-Profils},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-174134},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Der Prozess des Alterns ist ein komplexer multifaktorieller Vorgang, der durch eine sukzessive Verschlechterung der physiologischen Funktionen charakterisiert ist. Ein hohes Alter ist der Hauptrisikofaktor f{\"u}r die meisten Krankheiten, einschließlich Krebs und Herz-Kreislauf-Erkrankungen. Das Verst{\"a}ndnis der epigenetischen Mechanismen, die in den Prozess des Alterns involviert sind, k{\"o}nnte zur Entwicklung pharmakologischer Interventionen beitragen, die nicht nur die Lebenserwartung erh{\"o}hen, sondern auch den Beginn des altersassoziierten funktionellen Abbaus verz{\"o}gern k{\"o}nnten. Durch die Langzeit-Kultivierung prim{\"a}rer humaner Fibroblasten wurde ein in vitro Modell f{\"u}r das Altern etabliert, das die Identifizierung altersassoziierter DNA-Methylierungs-Ver{\"a}nderungen erm{\"o}glichte. Die in vitro Alterung konnte mit einer globalen Hypomethylierung und einer erh{\"o}hten DNA-Methylierung der ribosomalen DNA assoziiert werden. Dar{\"u}ber hinaus konnten DNA-Methylierungs-Ver{\"a}nderungen in Genen und Signalwegen, die f{\"u}r das Altern relevant sind, und ein erh{\"o}htes epigenetisches Alter nachgewiesen werden. Das in vitro Modell f{\"u}r das Altern wurde verwendet, um neben den direkten Effekten ionisierender Strahlung auf die DNA-Methylierung auch deren Langzeit-Effekte zu untersuchen. Die Strahlentherapie ist ein entscheidendes Element der Krebstherapie, hat aber auch negative Auswirkungen und kann unter anderem das Risiko f{\"u}r die Entwicklung eines Zweittumors erh{\"o}hen. Bei externer Bestrahlung wird neben dem Tumor auch gesundes Gewebe ionisierender Strahlung ausgesetzt. Daher ist es wichtig zu untersuchen, wie Zellen mit intakten DNA-Reparatur-Mechanismen und funktionierenden Zellzyklus-Checkpoints durch diese beeinflusst werden. In der fr{\"u}hen Phase der DNA-Schadensantwort auf Bestrahlung wurden in normalen Zellen keine wesentlichen DNA-Methylierungs-Ver{\"a}nderungen beobachtet. Mehrere Populations-Verdoppelungen nach Strahlenexposition konnten dagegen eine globale Hypomethylierung, eine erh{\"o}hte DNA-Methylierung der ribosomalen DNA und ein erh{\"o}htes epigenetisches Alter detektiert werden. Des Weiteren zeigten Gene und Signalwege, die mit Krebs in Verbindung gebracht wurden, Ver{\"a}nderungen in der DNA-Methylierung. Als Langzeit-Effekte ionisierender Strahlung traten somit die mit der in vitro Alterung assoziierten DNA-Methylierungs-Ver{\"a}nderungen verst{\"a}rkt auf und ein epigenetisches Muster, das stark an das DNA-Methylierungs-Profil von Tumorzellen erinnert, entstand. Man geht davon aus, dass Ver{\"a}nderungen der DNA-Methylierung eine aktive Rolle in der Entwicklung eines Tumors spielen. Die durch ionisierende Strahlung induzierten DNA-Methylierungs-Ver{\"a}nderungen in normalen Zellen k{\"o}nnten demnach in die Krebsentstehung nach Strahlenexposition involviert sein und zu dem sekund{\"a}ren Krebsrisiko nach Strahlentherapie beitragen. Es ist bekannt, dass Patienten unterschiedlich auf therapeutische Bestrahlung reagieren. Die Ergebnisse dieser Arbeit weisen darauf hin, dass die individuelle Sensitivit{\"a}t gegen{\"u}ber ionisierender Strahlung auch auf epigenetischer Ebene beobachtet werden kann. In einem zweiten Projekt wurden Gesamtblutproben von Patienten mit Werner-Syndrom, einer segmental progeroiden Erkrankung, und gesunden Kontrollen analysiert, um mit dem vorzeitigen Altern in Verbindung stehende DNA-Methylierungs-Ver{\"a}nderungen zu identifizieren. Werner-Syndrom konnte nicht mit einer globalen Hypomethylierung, jedoch mit einer erh{\"o}hten DNA-Methylierung der ribosomalen DNA und einem erh{\"o}hten epigenetischen Alter assoziiert werden. Das vorzeitige Altern geht demzufolge mit spezifischen epigenetischen Ver{\"a}nderungen einher, die eine Beschleunigung der mit dem normalen Altern auftretenden DNA-Methylierungs-Ver{\"a}nderungen darstellen. Im Rahmen dieser Arbeit konnte die Bedeutung epigenetischer Mechanismen im Prozess des Alterns hervorgehoben werden und gezeigt werden, dass sowohl exogene Faktoren, wie ionisierende Strahlung, als auch endogene Faktoren, wie das in Werner-Syndrom-Patienten mutiert vorliegende WRN-Gen, altersassoziierte DNA-Methylierungs-Ver{\"a}nderungen beeinflussen k{\"o}nnen.},
  subject      = {Methylierung},
  language  = {de}
}
@phdthesis{Nuernberger2018,
  author    = {N{\"u}rnberger, Fabian},
  title     = {Timing of colony phenology and foraging activity in honey bees},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-155105},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {I. Timing is a crucial feature in organisms that live within a variable and changing environment. Complex mechanisms to measure time are wide-spread and were shown to exist in many taxa. These mechanisms are expected to provide fitness benefits by enabling organisms to anticipate environmental changes and adapt accordingly. However, very few studies have addressed the adaptive value of proper timing. The objective of this PhD-project was to investigate mechanisms and fitness consequences of timing decisions concerning colony phenology and foraging activity in the honey bee (Apis mellifera), a social insect species with a high degree of social organization and one of the most important pollinators of wild plants and crops. In chapter II, a study is presented that aimed to identify the consequences of disrupted synchrony between colony phenology and the local environment by manipulating the timing of brood onset after hibernation. In a follow-up experiment, the importance of environmental factors for the timing of brood onset was investigated to assess the potential of climate change to disrupt synchronization of colony phenology (Chapter III). Chapter IV aimed to prove for the first time that honey bees can use interval time-place learning to improve foraging activity in a variable environment. Chapter V investigates the fitness benefits of information exchange between nest mates via waggle dance communication about a resource environment that is heterogeneous in space and time. II. In the study presented in chapter II, the importance of the timing of brood onset after hibernation as critical point in honey bee colony phenology in temperate zones was investigated. Honey bee colonies were overwintered at two climatically different sites. By translocating colonies from each site to the other in late winter, timing of brood onset was manipulated and consequently colony phenology was desynchronized with the local environment. Delaying colony phenology in respect to the local environment decreased the capability of colonies to exploit the abundant spring bloom. Early brood onset, on the other hand, increased the loads of the brood parasite Varroa destructor later in the season with negative impact on colony worker population size. This indicates a timing related trade-off and illustrates the importance of investigating effects of climate change on complex multi-trophic systems. It can be concluded that timing of brood onset in honey bees is an important fitness relevant step for colony phenology that is highly sensitive to climatic conditions in late winter. Further, phenology shifts and mismatches driven by climate change can have severe fitness consequences. III. In chapter III, I assess the importance of the environmental factors ambient temperature and photoperiod as well as elapsed time on the timing of brood onset. Twenty-four hibernating honey bee colonies were placed into environmental chambers and allocated to different combinations of two temperature regimes and three different light regimes. Brood onset was identified non-invasively by tracking comb temperature within the winter cluster. The experiment revealed that ambient temperature plays a major role in the timing of brood onset, but the response of honey bee colonies to temperature increases is modified by photoperiod. Further, the data indicate the involvement of an internal clock. I conclude that the timing of brood onset is complex but probably highly susceptible to climate change and especially spells of warm weather in winter. IV. In chapter IV, it was examined if honey bees are capable of interval time-place learning and if this ability improves foraging efficiency in a dynamic resource environment. In a field experiment with artificial feeders, foragers were able to learn time intervals and use this ability to anticipate time periods during which feeders were active. Further, interval time-place learning enabled foragers to increase nectar uptake rates. It was concluded that interval time-place learning can help honey bee foragers to adapt to the complex and variable temporal patterns of floral resource environments. V. The study presented in chapter V identified the importance of the honey bee waggle dance communication for the spatiotemporal coordination of honey bee foraging activity in resource environments that can vary from day to day. Consequences of disrupting the instructional component of honey bee dance communication were investigated in eight temperate zone landscapes with different levels of spatiotemporal complexity. While nectar uptake of colonies was not affected, waggle dance communication significantly benefitted pollen harvest irrespective of landscape complexity. I suggest that this is explained by the fact that honey bees prefer to forage pollen in semi-natural habitats, which provide diverse resource species but are sparse and presumably hard to find in intensively managed agricultural landscapes. I conclude that waggle dance communication helps to ensure a sufficient and diverse pollen diet which is crucial for honey bee colony health. VI. In my PhD-project, I could show that honey bee colonies are able to adapt their activities to a seasonally and daily changing environment, which affects resource uptake, colony development, colony health and ultimately colony fitness. Ongoing global change, however, puts timing in honey bee colonies at risk. Climate change has the potential to cause mismatches with the local resource environment. Intensivation of agricultural management with decreased resource diversity and short resource peaks in spring followed by distinctive gaps increases the probability of mismatches. Even the highly efficient foraging system of honey bees might not ensure a sufficiently diverse and healthy diet in such an environment. The global introduction of the parasitic mite V. destructor and the increased exposure to pesticides in intensively managed landscapes further degrades honey bee colony health. This might lead to reduced cognitive capabilities in workers and impact the communication and social organization in colonies, thereby undermining the ability of honey bee colonies to adapt to their environment.},
  subject      = {Biene},
  language  = {en}
}
@phdthesis{Glogger2018,
  author    = {Glogger, Marius},
  title     = {Single-molecule fluorescence microscopy in live \(Trypanosoma\) \(brucei\) and model membranes},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-169222},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Der eukaryotische Parasit Trypanosoma brucei hat komplexe Strategien entwickelt um der Immunantwort eines Wirtes zu entkommen und eine persistente Infektion innerhalb dessen aufrechtzuerhalten. Ein zentrales Element seiner Verteidigungsstrategie st{\"u}tzt sich auf die Schutzfunktion seines Proteinmantels auf der Zelloberfl{\"a}che. Dieser Mantel besteht aus einer dichten Schicht aus identischen, Glykosylphosphatidylinositol (GPI)-verankerten variablen Oberfl{\"a}chenglykoproteinen (VSG). Der VSG Mantel verhindert die Erkennung der darunterliegenden, invarianten Epitope durch das Immunsystem. Obwohl es notwendig ist die Funktionsweise des VSG Mantels zu verstehen, vor allem um ihn als m{\"o}gliches Angriffsziel gegen den Parasiten zu verwenden, sind seine biophysikalischen Eigenschaften bisher nur unzureichend verstanden. Dies ist vor allem der Tatsache geschuldet, dass die hohe Motilit{\"a}t der Parasiten mikroskopische Studien in lebenden Zellen bisher weitestgehend verhinderten. In der vorliegenden Arbeit wird nun hochmoderne Einzelmolek{\"u}l-Fluoreszenzmikroskopie (EMFM) als M{\"o}glichkeit f{\"u}r mikroskopische Untersuchungen im Forschungsbereich der Trypanosomen vorgestellt. Die Arbeit umfasst Untersuchungen der VSG Dynamik unter definierten Bedingungen k{\"u}nstlicher Membransysteme. Es wurde zuerst der Einfluss der lateralen Proteindichte auf die VSG Diffusion untersucht. Experimente mittels Fluoreszenz- Wiederkehr nach irreversiblem Photobleichen und komplement{\"a}re Einzelmolek{\"u}l- Verfolgungs Experimente offenbarten, dass ein molekularer Diffusionsschwellenwert existiert. {\"U}ber diesem Schwellenwert wurde eine dichteabh{\"a}nige Reduzierung des Diffusionskoeffizienten gemessen. Eine relative Quantifizierung der rekonstituierten VSGs verdeutlichte, dass der Oberfl{\"a}chenmantel der Trypanosomen sehr nahe an diesem Schwellenwert agiert. Der VSG Mantel ist optimiert um eine hohe Proteindichte bei gleichzeitiger hoher Mobilit{\"a}t der VSGs zu gew{\"a}hrleisten. Des Weiteren wurde der Einfluss der VSG N-Glykosylierung auf die Diffusion des Proteins quantitativ untersucht. Die Messungen ergaben, dass die N-Glykosylierung dazu beitr{\"a}gt eine hohe Mobilit{\"a}t bei hohen Proteindichten aufrechtzuerhalten. Eine detaillierte Analyse von VSG Trajektorien offenbarte, dass zwei unterschiedliche Populationen frei diffundierender VSGs in der k{\"u}nstlichen Membran vorlagen. K{\"u}rzlich wurde entdeckt, dass VSGs zwei strukturell unterschiedliche Konformationen annehmen k{\"o}nnen. Die Messungen in der Arbeit stimmen mit diesen Beschreibungen {\"u}berein. Die Ergebnisse der EMFM in k{\"u}nstlichen Membranen wurden durch VSG Einzelmolek{\"u}l- Verfolgungs Experimente auf lebenden Zellen erg{\"a}nzt. Es wurde eine hohe Mobilit{\"a}t und Dynamik einzelner VSGs gemessen, was die allgemein dynamische Natur des VSG Mantels verdeutlicht. Dies f{\"u}hrte zu der Schlussfolgerung, dass der VSG Mantel auf lebenden Trypanosomen ein dichter und dennoch dynamischer Schutzmantel ist. Die F{\"a}higkeit der VSGs ihre Konformation flexibel anzupassen, unterst{\"u}tzt das Erhalten der Fluidit{\"a}t bei variablen Dichten. Diese Eigenschaften des VSG Mantels sind elementar f{\"u}r die Aufrechterhaltung einer presistenden Infektion eines Wirtes. In dieser Arbeit werden des Weiteren verschiedene, auf Hydrogel basierende Einbettungsmethoden vorgestellt. Diese erm{\"o}glichten die Zellimmobilisierung und erlaubten EMFM in lebenden Trypanosomen. Die Hydrogele wiesen eine hohe Zytokompatibilit{\"a}t auf. Die Zellen {\"u}berlebten in den Gelen f{\"u}r eine Stunde nach Beginn der Immobilisierung. Die Hydrogele erf{\"u}llten die Anforderungen der Superresolution Mikroskopie (SRM) da sie eine geringe Autofluoreszenz im Spektralbereich der verwendeten Fluorophore besaßen. Mittels SRM konnte nachgewiesen werden, dass die Hydrogele die Zellen effizient immobilisierten. Als erstes Anwendungsbeispiel der Methode wurde die Organisation der Plasmamembran in lebenden Trypanosomen untersucht. Die Untersuchung eines fluoreszenten Tracers in der inneren Membranschicht ergab, dass dessen Verteilung nicht homogen war. Es wurden spezifische Membrandom{\"a}nen gefunden, in denen das Molek{\"u}l entweder vermehrt oder vermindert auftrat. Dies f{\"u}hrte zu der Schlussfolgerung, dass diese Verteilung durch eine Interaktion des Tracers mit Proteinen des zellul{\"a}ren Zytoskeletts zustande kam. Die in dieser Arbeit pr{\"a}sentierten Ergebnisse zeigen, dass EMFM erfolgreich f{\"u}r verschiedene biologische Untersuchungen im Forschungsfeld der Trypanosomen angewendet werden kann. Dies gilt zum Beispiel f{\"u}r die Untersuchung von der VSG Dynamik in k{\"u}nstlichen Membransystemen, aber auch f{\"u}r Studien in lebenden Zellen unter Verwendung der auf Hydrogelen basierenden Zelleinbettung.},
  subject      = {Trypanosoma brucei},
  language  = {en}
}
@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}
}