@phdthesis{Yang2021,
  author    = {Yang, Tao},
  title     = {Functional insights into the role of a bacterial virulence factor and a host factor in Neisseria gonorrhoeae infection},
  doi       = {10.25972/OPUS-20895},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-208959},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Neisseria gonorrhoeae (GC) is a human specific pathogenic bacterium. Currently, N. gonorrhoeae developed resistance to virtually all the available antibiotics used for treatment. N. gonorrhoeae starts infection by colonizing the cell surface, followed by invasion of the host cell, intracellular persistence, transcytosis and exit into the subepithelial space. Subepithelial bacteria can reach the bloodstream and disseminate to other tissues causing systemic infections, which leads to serious conditions such as arthritis and pneumonia. A number of studies have well established the host-pathogen interactions during the initial adherence and invasion steps. However, the mechanism of intracellular survival and traversal is poorly understood so far. Hence, identification of novel bacterial virulence factors and host factors involved in the host-pathogen interaction is a crucial step in understanding disease development and uncovering novel therapeutic approaches. Besides, most of the previous studies about N. gonorrhoeae were performed in the conventional cell culture. Although they have provided insights into host-pathogen interactions, much information about the native infection microenvironment, such as cell polarization and barrier function, is still missing. This work focused on determining the function of novel bacterial virulence factor NGFG_01605 and host factor (FLCN) in gonococcal infection. NGFG_01605 was identified by Tn5 transposon library screening. It is a putative U32 protease. Unlike other proteins in this family, it is not secreted and has no ex vivo protease activity. NGFG_01605 knockout decreases gonococcal survival in the epithelial cell. 3D models based on T84 cell was developed for the bacterial transmigration assay. NGFG_01605 knockout does not affect gonococcal transmigration. The novel host factor FLCN was identified by shRNA library screening in search for factors that affected gonococcal adherence and/or internalization. We discovered that FLCN did not affect N. gonorrhoeae adherence and invasion but was essential for bacterial survival. Since programmed cell death is a host defence mechanism against intracellular pathogens, we further explored apoptosis and autophagy upon gonococcal infection and determined that FLCN did not affect apoptosis but inhibited autophagy. Moreover, we found that FLCN inhibited the expression of E-cadherin. Knockdown of E- cadherin decreased the autophagy flux and supported N. gonorrhoeae survival. Both non-polarized and polarized cells are present in the cervix, and additionally, E-cadherin represents different polarization properties on these different cells. Therefore, we established 3-D models to better understand the functions of FLCN. We discovered that FLCN was critical for N. gonorrhoeae survival in the 3-D environment as well, but not through inhibiting autophagy. Furthermore, FLCN inhibits the E-cadherin expression and disturbs its polarization in the 3-D models. Since N. gonorrhoeae can cross the epithelial cell barriers through both cell-cell junctions and transcellular migration, we further explored the roles FLCN and E-cadherin played in transmigration. FLCN delayed N. gonorrhoeae transmigration, whereas the knockdown of E-cadherin increased N. gonorrhoeae transmigration. In summary, we revealed roles of the NGFG_01605 and FLCN-E-cadherin axis play in N. gonorrhoeae infection, particularly in relation to intracellular survival and transmigration. This is also the first study that connects FLCN and human-specific pathogen infection.},
  language  = {en}
}
@phdthesis{Klein2021,
  author    = {Klein, Thomas},
  title     = {Establishing an in vitro disease model for Fabry Disease using patient specific induced pluripotent stem cell-derived sensory neurons},
  doi       = {10.25972/OPUS-19970},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-199705},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Fabry disease (FD) is an X-linked lysosomal storage disorder caused by deficiency of the α-galactosidase A (GLA), leading to intracellular accumulations of globotriaosylceramide (Gb3). Acral burning pain, which can be triggered by heat, fever or physical activity is an early hallmark of FD and greatly reduces patients' quality of life. The pathophysiology of FD pain is unknown and research is hindered by the limited in vivo availability of suitable human biomaterial. To overcome this obstacle, we generated induced pluripotent stem cells (iPSC) from one female and two male patients with a differing pain phenotype, and developed a refined differentiation protocol for sensory neurons to increase reliability and survival of these neurons, serving as an in vitro disease model. Neurons were characterized for the correct neuronal subtype using immunocytochemistry, gene expression analysis, and for their functionality using electrophysiological measurements. iPSC and sensory neurons from the male patients showed Gb3 accumulations mimicking the disease phenotype, whereas no Gb3 depositions were detected in sensory neurons derived from the female cell line, likely caused by a skewed X-chromosomal inactivation in favor of healthy GLA. Using super-resolution imaging techniques we showed that Gb3 is localized in neuronal lysosomes of male patients and in a first experiment using dSTORM microscopy we were able to visualize the neuronal membrane in great detail. To test our disease model, we treated the neurons with enzyme replacement therapy (ERT) and analyzed its effect on the cellular Gb3 load, which was reduced in the male FD-lines, compared to non-treated cells. We also identified time-dependent differences of Gb3 accumulations, of which some seemed to be resistant to ERT. We also used confocal Ca2+ imaging to investigate spontaneous neuronal network activity, but analysis of the dataset proofed to be difficult, nonetheless showing a high potential for further investigations. We revealed that neurons from a patient with pain pain are more easily excitable, compared to cells from a patient without pain and a healthy control. We provide evidence for the potential of patient-specific iPSC to generate a neuronal in vitro disease model, showing the typical molecular FD phenotype, responding to treatment, and pointing towards underlying electrophysiological mechanisms causing different pain phenotypes. Our sensory neurons are suitable for state-of-the-art microscopy techniques, opening new possibilities for an in-depth analysis of cellular changes, caused by pathological Gb3 accumulations. Taken together, our system can easily be used to investigate the effect of the different mutations of GLA on a functional and a molecular level in affected neurons.},
  subject      = {Induzierte pluripotente Stammzelle},
  language  = {en}
}
@phdthesis{Maimari2020,
  author    = {Maimari, Theopisti},
  title     = {The influence of N-terminal peptides of G-protein coupled receptor kinase (GRK) 2, 3 and 5 on β-adrenergic signaling},
  doi       = {10.25972/OPUS-19932},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-199322},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {G protein coupled receptor kinases (GRK) phosphorylate and thereby desensitize G protein coupled receptors (GPCR) including β-adrenergic receptors (βAR), which are critical regulators of cardiac function. We identified the Raf kinase inhibitor protein (RKIP) as an endogenous inhibitor of GRK2 that leads to increased cardiac contractility via βAR activation. RKIP binds to the N-terminus (aa1-185) of GRK2, which is important for the GRK2/receptor interaction. Thereby it interferes with the GRK2/receptor interaction without interference with cytosolic GRK2 target activation. In this project, the RKIP/GRK interface was investigated to develop strategies that simulate the effects of RKIP on βAR. RKIP binding to different isoforms of GRK expressed in the heart was analyzed by protein interaction assays using full-length and N-termini of GRK2, GRK3 and GRK5: 1-53, 54-185 and 1-185. Co-immunoprecipitation (Co-IPs) and pull-down assays revealed that RKIP binds to the peptides of GRK2 and GRK3 but not to the ones of GRK5, which suggests the existence of several binding sites of RKIP within the N-termini of GRK2 and GRK3. To analyze whether the peptides of GRK2 and GRK3 are able to simulate the RKIP mediated interference of the GRK2/receptor interaction, we analyzed the β2-AR phosphorylation in the absence and presence of the peptides. Interestingly, N-termini (aa1-185) of GRK2 and GRK3 reduced β2AR phosphorylation to a comparable extent as RKIP. In line with reduced receptor phosphorylation, the peptides also reduced isoproterenol-stimulated receptor internalization as shown by [3H] CGP-12177 radioligand binding assay and fluorescence microscopy compared to control cells. Subsequently, these peptides increased downstream signaling of β2AR, i.e. the phosphorylation of the PKA substrate phosducin. In an attempt to elucidate the mechanism behind the observed effects, Co-IPs were performed in order to investigate whether the peptides bind directly to the β2-AR and block its phosphorylation by GRK2. Indeed, GRK2 1-185 and GRK3 1-185 could bind the receptor, suggesting that this way GRK2 is prevented from inhibiting the receptor. To investigate the physiological effect of GRK2 1-185, GRK3 1-185 and GRK5 1-185, their effect on neonatal mouse cardiomyocyte contractility and hypertrophy was analyzed. After long-term isoproterenol stimulation, in the presence of GRK2 1 185 and GRK3 1-185 the cross-sectional area of the cardiomyocytes showed no significant increase in comparison to the unstimulated control cells. In addition, upon isoproterenol stimulation, GRK2 1-185 and GRK3 1-185 increased the beat rate in cardiomyocytes, mimicking RKIP while the base impedance, an indicator of viability, remained stable. The N-termini (1-185) of GRK2 and GRK3 simulated RKIP's function and had a significant influence on β2AR phosphorylation, on its downstream signaling and internalization, could bind β2-AR, increased beat rate and did not significantly induce hypertrophy, suggesting that they may serve as a model for the generation of new and more specific targeting strategies for GRK mediated receptor regulation.},
  language  = {en}
}
@phdthesis{Hagmann2020,
  author    = {Hagmann, Hanns Antony},
  title     = {The impact of the CRISPR/Cas system on the interaction of Neisseria meningitidis with human host cells},
  doi       = {10.25972/OPUS-19949},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-199490},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Neisseria meningitidis, a commensal β-proteobacterium residing exclusively in the human nasopharynx, is a leading cause of sepsis and epidemic meningitis worldwide. While comparative genome analysis was able to define hyperinvasive lineages that are responsible for most of the cases of invasive meningococcal disease (IMD), the genetic basis of their virulence remains unclear. Recent studies demonstrate that the type II C CRISPR/Cas system of meningococci is associated with carriage and less invasive lineages. CRISPR/Cas, an adaptive defence system against foreign DNA, was shown to be involved in gene regulation in Francisella novicida. This study shows that knockout strains of N. meningitidis lacking the Cas9 protein are impaired in the adhesion to human nasopharyngeal cells in a strain-dependant manner, which constitutes a central step in the pathogenesis of IMD. Consequently, this study indicates that the meningococcal CRISPR/Cas system fulfils functions beyond the defence of foreign DNA and is involved in the regulation of meningococcal virulence.},
  subject      = {CRISPR/Cas-Methode},
  language  = {en}
}
@phdthesis{Simons2020,
  author    = {Simons, Bibiane Stephanie Elisabeth},
  title     = {Modulation von emotionaler Anspannung mittels transkranieller Gleichstromstimulation (tDCS) des rechten inferioren pr{\"a}frontalen Kortex},
  doi       = {10.25972/OPUS-19928},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-199289},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Emotionale Kontrolle ist f{\"u}r unsere Zusammenleben unerl{\"a}sslich. Zum neuronalen Netzwerk der Emotionsverarbeitung und Emotionskontrolle geh{\"o}rt auch der rechte inferiore pr{\"a}frontale Kortex, wobei seine Funktion h{\"a}ufig mit der einer Bremse verglichen wird. Die Antizipationsangst, die bei manchen Angstst{\"o}rungen eine Rolle spielt und das daraus resultierende Vermeidungsverhalten, bieten einen relevanten Zusammenhang, den man in der Therapie von Angsterkrankungen beeinflussen k{\"o}nnte. Hierbei bieten nichtinvasive Hirnstimulationsverfahren einen m{\"o}glichen Ansatzpunkt und der rechte IFG ein m{\"o}gliches Ziel. In dieser Studie stimulierten wir den rechten inferioren frontalen Gyrus (rIFG) mittels anodaler transkranieller Gleichstromstimulation (tDCS) um zu pr{\"u}fen, ob dadurch die emotionale Anspannung moduliert werden kann. Zu diesem Zwecke wurde der rIFG bei gesunden Probanden (N = 80), aufgeteilt in eine tDCS Gruppe und eine Sham Gruppe, {\"u}ber einen Zeitraum von 20 Minuten mit einer Stromst{\"a}rke von 2 mA und einer Elektrodengr{\"o}ße von 35 cm² elektrisch stimuliert. W{\"a}hrenddessen wurde die Hautleitf{\"a}higkeiten (SCL) als psychophysiologischer Parameter in Antizipation eines akustischen neutralen bzw. aversiven Reizes gemessen. Die Art des akustischen Reizes war dabei f{\"u}r die Probanden durch einen visuellen Hinweisstimulus vorhersehbar, jedoch war der Zeitpunkt der Pr{\"a}sentation des akustischen Reizes nicht vorhersehbar. Dadurch konnte emotionale Anspannung in Antizipation des aversiven Stimulus induziert werden, was wir durch ein insgesamt h{\"o}heres SCL w{\"a}hrend der aversiven Bedingung nachweisen konnten. Wir konnten einen signifikanten Effekt der tDCS des rIFG auf die psychophysiologischen Parameter der Antizipationsangst nachweisen. Der Effekt beruhte dabei auf einem geringeren Anstieg des Hautleitf{\"a}higkeitslevels der tDCS Gruppe von neutraler zu aversiver Bedingung im Vergleich zu Sham Gruppe. Wir k{\"o}nnen daher best{\"a}tigen, dass es m{\"o}glich ist die physiologische Reaktion bei emotionaler Anspannung durch tDCS des rIFG zu regulieren. Dar{\"u}ber hinaus k{\"o}nnen wir dadurch die angenommene Rolle des rIFG in der Emotionsregulation best{\"a}tigen. Dieser scheint daher ein vielversprechender Stimulationsort f{\"u}r tDCS zur Verst{\"a}rkung der emotionalen Kontrolle zu sein. Auf Basis unserer Ergebnisse, k{\"o}nnte in zuk{\"u}nftigen Studien tDCS des rIFG in Kombination mit Verhaltenstherapie bei Angsterkrankungen oder zur Modulation von Vermeidungsverhalten eingesetzt werden. Durch unseren Versuch konnte damit ein grundlegender Beitrag f{\"u}r zuk{\"u}nftige Therapiestudien im Zusammenhang mit tDCS geleistet werden.},
  language  = {de}
}
@phdthesis{Stich2020,
  author    = {Stich, Manuel},
  title     = {Kompatibilit{\"a}t in der medizinischen Bildgebung: Beeinflussung von Gradientenfeldern durch das Magnetsystem und Beeinflussung elektronischer Bauteile durch ionisierende Strahlung},
  doi       = {10.25972/OPUS-20347},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-203474},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Diese Arbeit besch{\"a}ftigt sich mit der Kompatibilit{\"a}t in der medizinischen Bildgebung unter zwei verschiedenen Aspekten: (A) Beeinflussung von Gradientenfeldern durch das Magnetsystem eines Magnetresonanztomographen. (B) Beeinflussung elektronischer Bauteile durch ionisierende Strahlung. Imperfektionen in der Gradientenhardware (7-13) f{\"u}hren dazu, dass nicht die ideale zeitliche Gradientenform ausgespielt wird, sondern eine verzerrte Version der Gradienten (6,14). In der nicht-kartesischen Bildgebung f{\"u}hren diese resultierenden Abweichungen in den k-Raum Trajektorien zu Bildartefakten, die sich negativ auf die Diagnosestellung auswirken k{\"o}nnen. Die linearen und zeitinvarianten Eigenschaften des Gradientensystems erm{\"o}glichen die Bestimmung der {\"U}bertragungsfunktion (GSTF) (20). Diese {\"U}bertragungsfunktion kann innerhalb der Bildrekonstruktion zur Trajektorienkorrektur verwendet werden (14,15,70). In dieser Arbeit wurden mit der Feldkamera (Skope Magnetic Resonance Technologies, Z{\"u}rich, Schweiz) (22,23) und der schichtselektiven Phantommethode (5,6) zwei etablierte GSTF-Messverfahren verglichen. Dabei wurde die Notwendigkeit einer Abtastzeitkompensation festgestellt, um die GSTF-Informationen entsprechend der gew{\"a}hlten Abtastzeit zu korrigieren (s. Abbildung 16) und die Trajektorien hinreichend zu korrigieren und damit Bildartefakte zu reduzieren. Die Langzeit- und Temperaturanalyse der GSTF zeigte f{\"u}r zwei verschiedene Siemens-Tomographen (Siemens Healthcare, Erlangen, Germany) eine Langzeit und Temperaturstabilit{\"a}t, auch bei extensiven Duty-Cyclen. Damit l{\"a}sst sich auch einfach eine Pre-emphasis-Korrektur der Gradienten realisieren, was exemplarisch mit einer Zig-Zag- und einer Spiral-Sequenz gezeigt werden konnte. Die GSTF-Pre-emphasis-Korrektur lieferte dabei {\"a}hnliche Ergebnisse wie die GSTF-Post-Processing-Technik (s. Abbildung 44 und 47). In Bezug auf die Kompatibilit{\"a}t in der medizinischen Bildgebung wurde in dieser Arbeit auch die Beeinflussung von medizinischen Implantaten durch ionisierende Strahlung untersucht. Herzschrittmacher, Kardioverter-Defibrillatoren oder andere aktive medizini- sche Implantate k{\"o}nnen in ihrer Funktion durch ionisierende Strahlung, die bei verschiedenen diagnostischen und therapeutischen Anwendungen appliziert wird, beeintr{\"a}chtigt werden (28,97,111). In dieser Studie wurden verschiedene elektronische Bauteile, wie Kondensatoren, Transistoren, Batterien und Speicherkarten in einer gewebe{\"a}quivalenten Messumgebung bestrahlt und dabei auf ihre Funktionalit{\"a}t {\"u}berpr{\"u}ft. Die Messumgebung simuliert dabei die Wechselwirkungseigenschaften von menschlichem Gewebe mit ionisierender Strahlung in einem Energiebereich von 10 keV - 6 MeV. Zudem erm{\"o}glicht sie mit der Einschubeinheit die Integration von Implantaten/elektronischen Bauteilen, sowie eine realistische Bestrahlungsplanung und Dosisverifikation (35,77). Bei den Kondensatoren zeigten sich w{\"a}hrend der Bestrahlung ein ver{\"a}ndertes Funktionsverhalten, mit signifikant abweichenden Spannungen und Zeitkonstanten gegen{\"u}ber dem unbestrahlten Zustand. Auch die Batterien haben sich w{\"a}hrend der Bestrahlung signifikant schneller entladen, als ohne Strahlungsapplikation. Nach der Bestrahlung konnten bei den untersuchten SD-Speicherkarten auch Ver{\"a}nderungen in den Speicherzellen festgestellt werden. Bei den Transistoren war aufgrund von Fehlern im Messsetup und dem Schaltungsdesign keine genauere teststatistische Auswertung m{\"o}glich. Zusammenfassend l{\"a}sst sich sagen, dass sich charakteristische Kenngr{\"o}ßen der untersuchten Bauteile bei Strahlungsapplikation signifikant ver{\"a}nderten.},
  subject      = {Magnetresonanztomographie},
  language  = {de}
}
@phdthesis{Vollmuth2021,
  author    = {Vollmuth, Nadine},
  title     = {Role of the proto-oncogene c-Myc in the development of Chlamydia trachomatis},
  doi       = {10.25972/OPUS-20365},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-203655},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Chlamydia trachomatis, an obligate intracellular human pathogen, is the world's leading cause of infection related blindness and the most common, bacterial sexually transmitted disease. In order to establish an optimal replicative niche, the pathogen extensively interferes with the physiology of the host cell. Chlamydia switches in its complex developmental cycle between the infectious non-replicative elementary bodies (EBs) and the non-infectious replicative reticulate bodies (RBs). The transformation to RBs, shortly after entering a host cell, is a crucial process in infection to start chlamydial replication. Currently it is unknown how the transition from EBs to RBs is initiated. In this thesis, we could show that, in an axenic media approach, L glutamine uptake by the pathogen is crucial to initiate the EB to RB transition. L-glutamine is converted to amino acids which are used by the bacteria to synthesize peptidoglycan. Peptidoglycan inturn is believed to function in separating dividing Chlamydia. The glutamine metabolism is reprogrammed in infected cells in a c-Myc-dependent manner, in order to accomplish the increased requirement for L-glutamine. Upon a chlamydial infection, the proto-oncogene c-Myc gets upregulated to promote host cell glutaminolysis via glutaminase GLS1 and the L-glutamine transporter SLC1A5/ASCT2. Interference with this metabolic reprogramming leads to limited growth of C. trachomatis. Besides the active infection, Chlamydia can persist over a long period of time within the host cell whereby chronic and recurrent infections establish. C. trachomatis acquire a persistent state during an immune attack in response to elevated interferon-γ (IFN-γ) levels. It has been shown that IFN-γ activates the catabolic depletion of L-tryptophan via indoleamine 2,3-dioxygenase (IDO), resulting in the formation of non-infectious atypical chlamydial forms. In this thesis, we could show that IFN-γ depletes the key metabolic regulator c-Myc, which has been demonstrated to be a prerequisite for chlamydial development and growth, in a STAT1-dependent manner. Moreover, metabolic analyses revealed that the pathogen de routs the host cell TCA cycle to enrich pyrimidine biosynthesis. Supplementing pyrimidines or a-ketoglutarate helps the bacteria to partially overcome the persistent state. Together, the results indicate a central role of c-Myc induced host glutamine metabolism reprogramming and L-glutamine for the development of C. trachomatis, which may provide a basis for anti-infectious strategies. Furthermore, they challenge the longstanding hypothesis of L-tryptophan shortage as the sole reason for IFN-γ induced persistence and suggest a pivotal role of c-Myc in the control of the C. trachomatis dormancy.},
  language  = {en}
}
@phdthesis{Konrad2021,
  author    = {Konrad, Charlotte},
  title     = {Biochemische Charakterisierung von cAMP-Gradienten - Einfluss von Phosphodiesterasen},
  doi       = {10.25972/OPUS-20572},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-205728},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Cyclisches Adenosinmonophosphat ist ein ubiquit{\"a}rer zweiter Botenstoff zahlreicher Signalwege im menschlichen K{\"o}rper. Auf eine Vielzahl verschiedenster extrazellul{\"a}rer Signale folgt jedoch eine Erh{\"o}hung desselben intrazellul{\"a}ren Botenstoffs - cAMP. Nichtsdestotrotz schafft es die Zelle, Signalspezifit{\"a}t aufrecht zu erhalten. Ein anerkanntes, wenn auch bisher unverstandenes Modell, um dieses zu erm{\"o}glichen, ist das Prinzip der Kompartimentierung. Die Zelle besitzt demnach Areale verschieden hoher cAMP-Konzentrationen, welche lokal begrenzt einzelne Signalkaskaden beeinflussen und somit eine differenzierte Signal{\"u}bertragung erm{\"o}glichen. Eine m{\"o}gliche Ursache f{\"u}r die Ausbildung solcher Bereiche geringerer cAMP- Konzentrationen (hier als Dom{\"a}nen bezeichnet), ist die hydrolytische Aktivit{\"a}t von Phosphodiesterasen (PDEs), welche als einzige Enzyme die F{\"a}higkeiten besitzen, cAMP zu degradieren. In dieser Arbeit wird der Einfluss der cAMP-Hydrolyse verschiedener PDEs auf die Gr{\"o}ße dieser Dom{\"a}nen evaluiert und mit denen der PDE4A1 verglichen, welche bereits durch unsere Arbeitsgruppe aufgrund ihrer Gr{\"o}ße als Nanodom{\"a}nen definiert wurden. Der Fokus wird dabei auf den Einfluss von kinetischen Eigenschaften der Phosphodiesterasen gelegt. So werden eine PDE mit hoher Umsatzgeschwindigkeit (PDE2A3) und eine PDE mit hoher Substrataffinit{\"a}t (PDE8A1) verglichen. Mithilfe sogenannter Linker, Abstandshaltern definierter L{\"a}nge, werden zus{\"a}tzlich die Nanodom{\"a}nen ausgemessen, um einen direkten Zusammenhang zwischen Gr{\"o}ße und kinetischer Eigenschaft anzugeben. Die Zusammenschau der Ergebnisse zeigt, dass die maximale Umsatzgeschwindigkeit der Phosphodiesterasen direkt mit der Gr{\"o}ße der Nanodom{\"a}nen korreliert. Durch den unmittelbaren Vergleich der gesamten PDE mit ihrer katalytischen Dom{\"a}ne wird zus{\"a}tzlich der Einfluss von regulatorischen Dom{\"a}nen evaluiert. Es wird gezeigt, dass diese cAMP-Gradienten modulieren k{\"o}nnen. Bei der PDE2A3 geschieht die Modulation u.a. durch Stimulation mit cGMP, welche h{\"o}chstwahrscheinlich dosisabh{\"a}ngig ist und somit graduell verl{\"a}uft. Hiermit pr{\"a}sentieren sich die Dom{\"a}nen als dynamische Bereiche, d.h. sie k{\"o}nnen in ihrer Auspr{\"a}gung reguliert werden. In dieser Arbeit wird die Hypothese best{\"a}tigt, dass Phosphodiesterasen eine wichtige Rolle in der Kompartimentierung von cAMP spielen, die Gruppe jedoch inhomogener ist, als bislang angenommen. Die Gradienten-Bildung l{\"a}sst sich nicht bei jeder Phosphodiesterase darstellen (PDE8A1). Einige Phosphodiesterasen (PDE2A3) jedoch bilden Kompartimente, die durch externe Stimuli in ihrer Gr{\"o}ße reguliert werden k{\"o}nnen. Die Arbeit legt den Grundstein zur breiteren Charakterisierung des spezifischen Einflusses weiterer PDEs auf cAMP-Kompartimentierung, welches nicht nur das Verst{\"a}ndnis der Kompartimentierungs-Strategien voranbringt, sondern auch essentiell f{\"u}r das Verst{\"a}ndnis der Pathophysiologie zahlreicher Krankheitsbilder, aber auch f{\"u}r das Verst{\"a}ndnis bereits angewandter aber auch potentiell neuer Medikamente ist.},
  subject      = {Cyclo-AMP},
  language  = {de}
}
@phdthesis{Dennstaedt2020,
  author    = {Dennst{\"a}dt, Fabio Stefan},
  title     = {Modulation CD4+ humaner Treg- und Tconv-Zellen durch Inhibition der sauren Sphingomyelinase in vitro},
  doi       = {10.25972/OPUS-20542},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-205420},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Die saure Sphingomyelinase (ASM) stellt durch die Umwandlung von Sphingomyelin in Ceramid und Phosphorylcholin ein zentrales, fein reguliertes Enzym im Sphingolipidmetabolismus dar. Dadurch nimmt es Einfluss auf verschiedene zellul{\"a}re Mechanismen wie Signalvermittlung, Endo- und Exozytose und Zellaktivierung. Dementsprechend weitreichend ist auch die Bedeutung der ASM bei verschiedenen Krankheiten wie Arteriosklerose, Depression oder Neoplasien. Auch auf das Immunsystem, insbesondere auf die Signalvermittlung durch T-Zellen innerhalb des adaptiven Immunsystems, nimmt die saure Sphingomyelinase Einfluss. Aufbauend auf fr{\"u}heren Forschungsarbeiten zur pharmakologischen und genetischen Hemmung der ASM im Mausmodell untersuchten wir, welche Auswirkungen die Hemmung dieses Enzyms in humanen Zellkulturen auf die Population regulatorischer und konventioneller T-Zellen haben. Hierzu verwendeten wir die beiden selektiven Serotonin-Wiederaufnahmehemmer Sertralin und Citalopram; zwei antidepressiv wirksame Medikamente, die durch eine Verdr{\"a}ngung der ASM von der lysosomalen Membran eine hemmende Wirkung aus{\"u}ben. Wir konnten zeigen, dass diese beiden Substanzen sowohl in Maus-T-Zellen, als auch in humanen T-Zellen, in der Lage sind, die Aktivit{\"a}t der sauren Sphingomyelinase zu inhibieren. Durch Kultivierung von Immunzellen der Maus zusammen mit den Inhibitoren konnte dar{\"u}ber hinaus eine Erh{\"o}hung der Treg-Zellfrequenz erreicht werden. Verschiedene Zellkulturexperimente mit humanen PBMCs zeigten weiterhin, dass unter gewissen Umst{\"a}nden so auch eine Vermehrung regulatorischer T-Zellen im Menschen m{\"o}glich ist, und dass dies mutmaßlich durch Einbindung der ASM im CD3/CD28-Signalweg bedingt ist. In mit AntiCD3-Antik{\"o}rper stimulierten experimentellen Ans{\"a}tzen kam es jedoch nur bei einzelnen Individuen, die als Responder identifiziert werden konnten, zu einer Treg-Zellvermehrung. Umgekehrt kam es durch externe Zugabe von C6-Ceramid zu einer Verringerung des Anteils an regulatorischen T-Zellen. Des Weiteren wurden verschiedene Ver{\"a}nderungen im Expressionsverhalten von Treg- und Tconv-Zellen bez{\"u}glich CD25, CD69 und CTLA-4 in Anwesenheit der ASMInhibitoren beobachtet. Weiterhin best{\"a}tigte sich, dass die pharmakologische Hemmung der sauren Sphingomyelinase auch Auswirkungen auf die Effektorfunktion von T-Zellen hat. W{\"a}hrend die Proliferation der Zellen weitgehend unbeeintr{\"a}chtigt blieb, kam es zu einer verringerten Sekretion der Zytokine IFN-gamma, TNF, IL-5 und IL-10. In ihrer Gesamtheit sprechen diese Ergebnisse daf{\"u}r, dass Inhibitoren der sauren Sphingomyelinase beg{\"u}nstigend auf Krankheitsgeschehen mit {\"u}berschießender oder dysregulierter Aktivit{\"a}t des Immunsystems einwirken k{\"o}nnten. Immunmodulatorischen Wirkungen durch Inhibition der ASM erkl{\"a}ren m{\"o}glicherweise auch Einfl{\"u}sse auf das Immunsystem, die f{\"u}r verschiedene Antidepressiva beschrieben wurden. Insgesamt ist die Bedeutung der sauren Sphingomyelinase innerhalb der Regulation des adaptiven Immunsystems jedoch noch ein weitgehend ungekl{\"a}rtes Thema mit vielen offenen Fragen. Daher ist auch in Zukunft weitere klinische und experimentelle Forschung erforderlich, um zu kl{\"a}ren, welchen Einfluss dieses Enzyms auf Immunzellen hat und wie sich dieser auch klinisch anwenden l{\"a}sst.},
  subject      = {T-Lymphozyt},
  language  = {de}
}
@phdthesis{Hausmann2020,
  author    = {Hausmann, Michael},
  title     = {Analyse der Genexpression verschiedener Kandidatengene und der Methylierung im Xiphophorus Melanom},
  doi       = {10.25972/OPUS-20525},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-205258},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Das Melanom ist eine der aggressivsten Formen von malignen Tumoren beim Menschen. Bei Fischen der Gattung Xiphophorus kommt es zur spontanen Tumorformation, welche auch durch zwischenartliche Kreuzung herbeif{\"u}hrbar ist. Hybride mit angeborenem Melanom stellen ein n{\"u}tzliches Tiermodell zur Untersuchung der genetischen Grundlage der Tumorentwicklung dar. Ihre Tumorigenese hängt mit der pigmentzellspezifischen Überexpression der durch eine Mutation aktivierten Rezeptortyrosinkinase Xmrk zusammen. In reinrassigen Fischen wird die onkogene Funktion des xmrk durch den Genlocus R, welcher molekular noch nicht identifiziert wurde, unterdr{\"u}ckt. Zusammen mit der Überexpression von xmrk konnten mittels einer RNA-Seq Analyse weitere Gene gefunden werden, welche differenziell in den Proben von malignen und benignen Geweben des Xiphophorus exprimiert werden. Des Weiteren ist bekannt, dass die Methylierung des xmrk Promotors Einfluss auf die Expression des Genes hat. Um die Daten der durch RNA-Seq gefundenen Kandidatengene zu validieren, wurde deren Expression in malignen und benignen Geweben der Flossen und des Rumpfes mittels qPCR quantifiziert. Zusätzlich dazu wurde die Expression einiger humaner Orthologe dieser Gene in Proben aus humanen Melanomzelllinien gemessen. Mir war es möglich zu zeigen, dass mit Ausnahme von cdkn2ab, mitfb und xirp2b alle Kandidatengene signifikant unterschiedlich in mindestens einem Vergleich von benignem und malignem Gewebe exprimiert waren. Das mit xmrk verglichen gegensätzliche Expressionsmuster von pdcd4a macht es zu einem vielversprechenden Kandidaten als vom R-Locus codierten Tumorsuppressorgen. In den humanen Melanomzelllinien konnte ausschließlich von PDGFRB keine erhöhte Expression in irgendeiner Probe nachgewiesen werden. Während die Expression von PDCD4, C-MYC und MITF in mindestens drei der vier Zelllinien mittelstark erhöht war, ließ sich bei KIT eine enorm gesteigerte Überexpression in Zellen der Linie Hermes3a nachweisen. Da drei der f{\"u}nf analysierten Gene und ihre Orthologen ähnliche Expressionsmuster in Proben des Xiphophorus und der humanen Melanomzelllinien zeigen, deuten diese Ergebnisse auf die N{\"u}tzlichkeit des Tiermodells zur Identifizierung entscheidender Gene und Signalwege im malignen Melanom hin. Ein zweites Ziel der Arbeit war das Erlangen tieferer Einblicke in die Methylierung des Xiphophorus Melanoms auf einer globalen und promotor- spezifischen Ebene. Um die Hypothese einer Reduzierung der globalen Methylierung zu testen, f{\"u}hrte ich eine kolorimetrische Quantifizierung der 5-mC DNA in Kontroll- und Tumorgeweben aus. Diese Vorgehensweise zeigte zum ersten Mal eine signifikante Verminderung der methylierten globalen DNA in den benignen Läsionen und malignen Melanomen der Flossen verglichen mit dem Kontrollgewebe. Um herauszufinden, on diese Demethylierung direkt mit der Überexpression des xmrk verbunden ist, analysierte ich als nächstes die Methylierung eines CpG Dinukleotids des xmrk Promotors mithilfe von methylierungssensitiven Restriktionsendonukleasen. Obwohl nur in den Proben des exophytischen Tumorwachstums als Krebsgewebe eine verringerte Methylierung des CpG Dinukleotids verglichen mit den Kontrollen nachgewiesen werden konnte, zeigte sich die Stelle in Zellen der Xiphophorus Melanomzelllinie PSM komplett unmethyliert. Diese Ergebnisse deuten stark daraufhin, dass eine differenzierte Methylierung das onkogene Potential dieser Zellen bewirkt. Um die Effekte veränderter globaler und promotor-spezifischer Methylierung auf die Tumorigenese besser zu verstehen, sind weitere Untersuchungen nötig.},
  subject      = {Xiphophorus Melanom},
  language  = {de}
}
@phdthesis{Heitmann2020,
  author    = {Heitmann, Johanna Friederike},
  title     = {Signaltransduktionsweg nach rtPA-Behandlung im peripheren Nerven zur Barrieren{\"o}ffnung f{\"u}r hydrophile Analgetika in der Regionalan{\"a}sthesie},
  doi       = {10.25972/OPUS-20517},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-205177},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Zur Durchf{\"u}hrung peripherer Nervenblockaden werden im klinischen Alltag nichtselektive Lokalan{\"a}sthetika verwendet, die neben sensorischen auch motorische Nervenfasern blockieren. Diese Arbeit untersucht und beschreibt Grundlagen f{\"u}r die Verwendung selektiv wirksamer Co-Analgetika. Ziel dieser Arbeit war in diesem Kontext die Analyse der intrazellul{\"a}ren Signalwege, welche nach Applikation von rtPA am peripheren Nerven zur {\"O}ffnung der perineuralen Barriere und so zu einer opiat- vermittelten Analgesie f{\"u}hren. Gem{\"a}ß unserer Hypothese bindet rtPA an den LRP-1- Rezeptor und l{\"o}st eine intrazellul{\"a}re Signalkaskade aus: Erk wird phosphoryliert und inhibiert {\"u}ber bislang unklare Mechanismen die Claudin-1-Transkription. Claudin-1 wird weniger in die Zellmembran eingebaut und/oder verl{\"a}sst durch Endozytose/ Internalisierung die Zellmembran, was zur {\"O}ffnung der perineuralen Barriere f{\"u}hrt und den Durchtritt selektiv wirksamer Analgetika erlaubt. In der sp{\"a}teren Phase steht die Analyse der Wiederherstellung der Barrierefunktion der Zellmembran im Vordergrund. Die ist von zentraler Bedeutung um eine Sch{\"a}digung des Nervens durch das Umgebungsmilieu zu verhindern. Vermutlich wird die Wiederherstellung der Barrierefunktion {\"u}ber den Wnt-Signalweg gesteuert. Die Akkumulation von b-Catenin und Cdx2 f{\"u}hrt zu einem erneuten Anstieg der Claudin-1-Transkription. Der Claudin-1- Gehalt steigt in Western Blot-Untersuchungen jedoch bereits zu einem fr{\"u}heren Zeitpunkt in der Zellmembran wieder an. Dies legt nahe, dass weitere von der Transkription unabh{\"a}ngige Mechanismen zur Wiederherstellung der Barrierefunktion beitragen. Eine m{\"o}gliche Alternative zu rtPA stellt katalytisch inaktives rtPAi dar, welches in Untersuchungen {\"a}hnliche Ergebnisse wie rtPA zeigte. Dabei k{\"o}nnte die Verwendung von rtPAi anstatt rtPA pathophysiologisch denkbare Komplikationen wie beispielsweise Blutungen verhindern. In Versuchen anderer Mitglieder der Arbeitsgruppe wurde die {\"O}ffnung der perineuralen Barriere mittels immunhistochemischer und funktioneller Untersuchungen best{\"a}tigt. Auch konnten keine akute Neurotoxizit{\"a}t oder Blutungsgefahr beobachtet werden. Somit stellt rtPA in Kombination mit Opioiden eine m{\"o}gliche Alternative zur Verbesserung der postoperativen Analgesie dar, die jedoch weiterer Untersuchungen hinsichtlich von Nutzen, Risiken und Nebenwirkungen bedarf.},
  subject      = {Schmerz},
  language  = {de}
}
@phdthesis{John2020,
  author    = {John, Vini},
  title     = {Interaction of mycobacteria with myeloid-derived suppressor cells},
  doi       = {10.25972/OPUS-18350},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-183501},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Myeloid-derived suppressor cells (MDSCs) constitute of monocytic (M-MDSCs) and granulocytic cell subsets (G-MDSCs)and were initially described as suppressors of T-cell function in tumor microenvironments. Recent studies have shown the involvement of MDSCs in a number of infectious diseases including Mycobacterium tuberculosis (Mtb) infection. MDSCs are tremendously accumulated in patients with Mtb infection and exert a suppressive effect on T cell responses against mycobacteria. Mycobacterium bovis BCG, the only available vaccine against Mtb fails to protect against the adult pulmonary tuberculosis (TB). Understanding the mechanisms of MDSC suppression for immunity against mycobacterial infection will provide a rational basis to improve anti- TB vaccination and host-directed therapies against TB. In this study, we investigated the role of three lipid-rich components of the plasma membrane, Caveolin-1(Cav-1), Acid Sphingomyelinase (ASM) and asialo-GM1 on BCG-activated MDSCs. Cav-1 is one of the vital components of caveolae (plasma membrane invaginations) which regulates apoptosis and lipid metabolism. In this work, we found that MDSCs upregulated Cav-1, TLR4 and TLR2 expression after BCG infection on the cell surface. However, Cav-1 deficiency resulted in a selective defect in the intracellular TLR2 accumulation in the M-MDSC, but not G-MDSC subset. Further analysis indicated no difference in the phagocytosis of BCG by M-MDSCs from WT and Cav1-/- mice but a reduced capacity to up-regulate surface markers, to secrete various cytokines, induce iNOS and NO production. These defects correlated with deficits of Cav1-/- MDSCs in the suppression of T cell proliferation. Among the signaling pathways that were affected by Cav-1 deficiency, we found lower phosphorylation of NF-kB and p38 mitogen-activated protein kinase (MAPK) in BCG - activated MDSCs. ASM is an enzyme present in lysosomes and is translocated to the cell surface where it hydrolyzes sphingomyelin into ceramide. Flow cytometric studies revealed that MDSCs phagocytosed BCG independent of inhibiting ASMase using pharmacological inhibitors (amitryptiline or desipramine) or MDSCs from WT and ASM-/-. Suppression of ASMase or using ASM-/- MDSCs resulted in reduced NO production and decreased cytokine secretion by MDSCs in response to BCG. Furthermore, MDSCs inhibited by amitryptiline had impaired AKT phosphorylation upon BCG infection. Asialo-GM1 is a ganglioside expressed on the cell surface of MDSCs reported to cooperate with TLR2 for activating ERK signaling. Here, in this study, we found that asialo-GM1 expression was upregulated specifically upon mycobacterial infection and not upon any other stimulus. We noted that the soluble form of asialo-GM1 bound to BCG. Flow cytometric studies revealed that blocking 81 asialo-GM1 did not affect the phagocytosis of BCG into MDSCs. Furthermore, blocking of asialo- GM1 had no effect on the cytokine and NO secretion or AKT signaling. Collectively, the data presented in this work implicated that Cav-1, ASM, asialo-GM1 are dispensable for the internalization of BCG. Rather, Cav-1 and ASM are required for the functional activation of MDSCs. Although asialo-GM1 binds to BCG, we did not find any difference in the functional activation of MDSCs after blocking asialo-GM1. This study provides insights into the role of lipid raft components of the MDSC cell membrane during mycobacterial infection.},
  subject      = {MDSCs},
  language  = {en}
}
@phdthesis{Martens2020,
  author    = {Martens, Johannes},
  title     = {Development of an In-Silico Model of the Arterial Epicardial Vasculature},
  doi       = {10.25972/OPUS-18247},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-182478},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {In dynamic CE MR perfusion imaging the passage of an intravenously injected CA bolus through tissue is monitored to assess the myocardial pefusion state. To enable this, knowledge of the shape of CA wash-in through upstream epicardial vessels is required, the so-called AIF. For technical reasons this cannot be quantified directly in the supplying vessels and is thus measured in the left ventricle, which introduces the risk of systematic errors in quantification of MBF due to bolus dispersion in coronary vessels. This means occuring CA dispersion must be accounted in the quantification process in order to produce reliable and reproducible results. In order to do this, CFD simulations are performed to analyze and approximate these errors and deepen insights and knowledge gained from previous CFD analyses on both idealized as well as realistic and pathologically altered 3D geometries. In a first step, several different procedures and approaches are undertaken in order to accelerate the performed workflow, however, maintaining a sufficient degree of numerical accuracy. In the end, the implementation of these steps makes the analysis of the cardiovascular 3D model of unprecedented detail including vessels at pre-arteriolar level feasible at all. The findings of the Navier-Stokes simulations are thus validated with regard to different aspects of cardiac blood flow. These include the distribution of VBF into the different myocardial regions, the areals, which can be associated to the large coronary arteries as well as the fragmentation of VBF into vessels of different diameters. The subsequently performed CA transport simulations yield results on the one hand confirming previous studies. On the other hand, interesting additional knowledge about the behavior of CA dispersion in coronary arteries is obtained both regarding travelled distance as well as vessel diameters. The relative dispersion of the so-called vascular transport function, a characterizing feature of vascular networks, shows a linear decrease with vessel diameter. This results in asymptotically decreased additional dispersion of the CA time curve towards smaller and more distal vessels. Nonetheless, perfusion quantification errors are subject to strong regional variability and reach an average value of \$(-28\pm16)\$ \\% at rest across the whole myocardium. Depending on the distance from the inlet and the considered coronary tree, MBF errors up to 62 \\% are observed.},
  subject      = {Computerunterst{\"u}tztes Verfahren},
  language  = {en}
}
@phdthesis{Mayer2021,
  author    = {Mayer, Alexander E.},
  title     = {Protein kinase D3 signaling in the regulation of liver metabolism},
  doi       = {10.25972/OPUS-20797},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-207978},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {The liver plays a pivotal role in maintaining energy homeostasis. Hepatic carbohydrate and lipid metabolism are tightly regulated in order to adapt quickly to changes in nutrient availability. Postprandially, the liver lowers the blood glucose levels and stores nutrients in form of glycogen and triglycerides (TG). In contrast, upon fasting, the liver provides glucose, TG, and ketone bodies. However, obesity resulting from a discrepancy in food intake and energy expenditure leads to abnormal fat accumulation in the liver, which is associated with the development of hepatic insulin resistance, non-alcoholic fatty liver disease, and diabetes. In this context, hepatic insulin resistance is directly linked to the accumulation of diacylglycerol (DAG) in the liver. Besides being an intermediate product of TG synthesis, DAG serves as second messenger in response to G-protein coupled receptor signaling. Protein kinase D (PKD) family members are DAG effectors that integrate multiple metabolic inputs. However, the impact of PKD signaling on liver physiology has not been studied so far. In this thesis, PKD3 was identified as the predominantly expressed isoform in liver. Stimulation of primary hepatocytes with DAG as well as high-fat diet (HFD) feeding of mice led to an activation of PKD3, indicating its relevance during obesity. HFD-fed mice lacking PKD3 specifically in hepatocytes displayed significantly improved glucose tolerance and insulin sensitivity. However, at the same time, hepatic deletion of PKD3 in mice resulted in elevated liver weight as a consequence of increased hepatic lipid accumulation. Lack of PKD3 in hepatocytes promoted sterol regulatory element-binding protein (SREBP)-mediated de novo lipogenesis in vitro and in vivo, and thus increased hepatic triglyceride and cholesterol content. Furthermore, PKD3 suppressed the activation of SREBP by impairing the activity of the insulin effectors protein kinase B (AKT) and mechanistic target of rapamycin complexes (mTORC) 1 and 2. In contrast, liver-specific overexpression of constitutive active PKD3 promoted glucose intolerance and insulin resistance. Taken together, lack of PKD3 improves hepatic insulin sensitivity but promotes hepatic lipid accumulation. For this reason, manipulating PKD3 signaling might be a valid strategy to improve hepatic lipid content or insulin sensitivity. However, the exact molecular mechanism by which PKD3 regulates hepatocytes metabolism remains unclear. Unbiased proteomic approaches were performed in order to identify PKD3 phosphorylation targets. In this process, numerous potential targets of PKD3 were detected, which are implicated in different aspects of cellular metabolism. Among other hits, phenylalanine hydroxylase (PAH) was identified as a target of PKD3 in hepatocytes. PAH is the enzyme that is responsible for the conversion of phenylalanine to tyrosine. In fact, manipulation of PKD3 activity using genetic tools confirmed that PKD3 promotes PAH-dependent conversion of phenylalanine to tyrosine. Therefore, the data in this thesis suggests that PKD3 coordinates lipid and amino acid metabolism in the liver and contributes to the development of hepatic dysfunction.},
  subject      = {Metabolismus},
  language  = {en}
}
@phdthesis{Hoefner2020,
  author    = {H{\"o}fner, Christiane},
  title     = {Human Adipose-derived Mesenchymal Stem Cells in a 3D Spheroid Culture System - Extracellular Matrix Development, Adipogenic Differentiation, and Secretory Properties},
  doi       = {10.25972/OPUS-20424},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-204249},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {The ability to differentiate into mesenchymal lineages, as well as immunomodulatory, anti-inflammatory, anti-apoptotic, and angiogenic properties give ASCs great therapeutic potential. Through their culture as multicellular, three-dimensional spheroids this potential can even be enhanced. Accordingly, 3D spheroids are not only promising candidates for the application in regenerative medicine and inflammatory disease therapy, but also for the use as building blocks in tissue engineering approaches. Due to the resemblance to physiological cell-cell and cell-matrix interactions, 3D spheroids gain higher similarity to real tissues, what makes them a valuable tool in the development of bioactive constructs equivalent to native tissues in terms of its cellular and extracellular structure. Especially, to overcome the still tremendous clinical need for adequate implants to repair soft tissue defects, 3D spheroids consisting of ASCs are a promising approach in adipose tissue engineering. Nevertheless, studies on the use of ASC-based spheroids as building blocks for fat tissue reconstruction have so far been very rare. In order to optimally exploit their therapeutic potential to further their use in regenerative medicine, including adipose tissue engineering approaches, a 3D spheroid model consisting of ASCs was characterized extensively in this work. This included not only the elucidation of the structural features, but also the differentiation capacity, gene expression, and secretory properties. In addition, the elucidation of underlying mechanisms contributing to the improved therapeutic efficiency was addressed.},
  subject      = {adipose},
  language  = {en}
}
@phdthesis{Herz2021,
  author    = {Herz, Michaela},
  title     = {Genome wide expression profiling of Echinococcus multilocularis},
  doi       = {10.25972/OPUS-20380},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-203802},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Alveolar echinococcosis, which is caused by the metacestode stage of the small fox tapeworm Echinococcus multilocularis, is a severe zoonotic disease with limited treatment options. For a better understanding of cestode biology the genome of E. multilocularis, together with other cestode genomes, was sequenced previously. While a few studies were undertaken to explore the E. multilocularis transcriptome, a comprehensive exploration of global transcription profiles throughout life cycle stages is lacking. This work represents the so far most comprehensive analysis of the E. multilocularis transcriptome. Using RNA-Seq information from different life cycle stages and experimental conditions in three biological replicates, transcriptional differences were qualitatively and quantitatively explored. The analyzed datasets are based on samples of metacestodes cultivated under aerobic and anaerobic conditions as well as metacestodes obtained directly from infected jirds. Other samples are stem cell cultures at three different time points of development as well as non-activated and activated protoscoleces, the larval stage that can develop into adult worms. In addition, two datasets of metacestodes under experimental conditions suitable for the detection of genes that are expressed in stem cells, the so-called germinative cells, and one dataset from a siRNA experiment were analyzed. Analysis of these datasets led to expression profiles for all annotated genes, including genes that are expressed in the tegument of metacestodes and play a role in host-parasite interactions and modulation of the host's immune response. Gene expression profiles provide also further information about genes that might be responsible for the infiltrative growth of the parasite in the liver. Furthermore, germinative cell-specific genes were identified. Germinative cells are the only proliferating cells in E. multilocularis and therefore of utmost importance for the development and growth of the parasite. Using a combination of germinative cell depletion and enrichment methods, genes with specific expression in germinative cells were identified. As expected, many of these genes are involved in translation, cell cycle regulation or DNA replication and repair. Also identified were transcription factors, many of which are involved in cell fate commitment. As an example, the gene encoding the telomerase reverse transcriptase (TERT) was studied further. Expression of E. multilocularis tert in germinative cells was confirmed experimentally. Cell culture experiments indicate that TERT is required for proliferation and development of the parasite, which makes TERT a potentially interesting drug target for chemotherapy of alveolar echinococcosis. Germinative cell specific genes in E. multilocularis also include genes of densoviral origin. More than 20 individual densovirus loci with information for non-structural and structural densovirus proteins were identified in the E. multilocularis genome. Densoviral elements were also detected in many other cestode genomes. Genomic integration of these elements suggests that densovirus-based vectors might be suitable tools for genetic manipulation of tapeworms. Interestingly, only three of more than 20 densovirus loci in the E. multilocularis genome are expressed. Since the canonical piRNA pathway is lacking in cestodes, this raises the question about potential silencing mechanisms. Exploration of RNA-Seq information indicated natural antisense transcripts as a potential gene regulation mechanism in E. multilocularis. Preliminary experiments further suggest DNA-methylation, which was previously shown to occur in platyhelminthes, as an interesting avenue to explore in future. The transcriptome datasets also contain information about genes that are expressed in differentiated cells, for example the serotonin transporter gene that is expressed in nerve cells. Cell culture experiments indicate that serotonin and serotonin transport play an important role in E. multilocularis proliferation, development and survival. Overall, this work provides a comprehensive transcription data atlas throughout the E. multilocularis life cycle. Identification of germinative cell-specific genes and genes important for host-parasite interactions will greatly facilitate future research. A global overview of gene expression profiles will also aide in the detection of suitable drug targets and the development of new chemotherapeutics against alveolar echinococcosis.},
  subject      = {Fuchsbandwurm},
  language  = {en}
}
@phdthesis{LiessneeEller2021,
  author    = {Liess [n{\´e}e Eller], Anna Katharina Luise},
  title     = {Understanding the regulation of the ubiquitin-conjugating enzyme UBE2S},
  doi       = {10.25972/OPUS-20419},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-204190},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {The ubiquitination of proteins serves as molecular signal to control an enormous number of physiological processes and its dysregulation is connected to human diseases like cancer. The versatility of this signal stems from the diverse ways by which ubiquitin can be attached to its targets. Thus, specificity and tight regulation of the ubiquitination are pivotal requirements of ubiquitin signaling. Ubiquitin-conjugating enzymes (E2s) act at the heart of the ubiquitination cascade, transferring ubiquitin from a ubiquitin-activating enzyme (E1) to a ubiquitin ligase (E3) or substrate. When cooperating with a RING-type E3, ubiquitin-conjugating enzymes can determine linkage specificity in ubiquitin chain formation. Our understanding of the regulation of E2 activities is still limited at a structural level. The work described here identifies two regulation mechanisms in UBE2S, a cognate E2 of the human RING-type E3 anaphase-promoting complex/cyclosome (APC/C). UBE2S elongates ubiquitin chains on APC/C substrates in a Lys11 linkage-specific manner, thereby targeting these substrates for degradation and driving mitotic progression. In addition, UBE2S was found to have a role in DNA repair by enhancing non-homologous end-joining (NHEJ) and causing transcriptional arrest at DNA damage sites in homologous recombination (HR). Furthermore, UBE2S overexpression is a characteristic feature of many cancer types and is connected to poor prognosis and diminished response to therapy. The first regulatory mechanism uncovered in this thesis involves the intramolecular auto-ubiquitination of a particular lysine residue (Lys+5) close to the active site cysteine, presumably through conformational flexibility of the active site region. The Lys+5-linked ubiquitin molecule adopts a donor-like, 'closed' orientation towards UBE2S, thereby conferring auto-inhibition. Notably, Lys+5 is a major physiological ubiquitination site in ~25\% of the human E2 enzymes, thus providing regulatory opportunities beyond UBE2S. Besides the active, monomeric state and the auto-inhibited state caused by auto-ubiquitination, I discovered that UBE2S can adopt a dimeric state. The latter also provides an auto-inhibited state, in which ubiquitin transfer is blocked via the obstruction of donor binding. UBE2S dimerization is promoted by its unique C-terminal extension, suppresses auto-ubiquitination and thereby the proteasomal degradation of UBE2S. Taken together, the data provided in this thesis illustrate the intricate ways by which UBE2S activity is fine-tuned and the notion that structurally diverse mechanisms have evolved to restrict the first step in the catalytic cycle of E2 enzymes.},
  subject      = {E2},
  language  = {en}
}
@phdthesis{Kleefeldt2020,
  author    = {Kleefeldt, Florian},
  title     = {Einfluss von CEACAM1 auf die endotheliale Funktion},
  doi       = {10.25972/OPUS-20172},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-201726},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Dem Endothel, welches die luminale Oberfl{\"a}che aller Blutgef{\"a}ße auskleidet, kommt eine wichtige Barrierefunktion zwischen Blut und Gewebe zu. Nur durch eine bedarfsgerechte Justierung dieser Barriere, die den Durchtritt von Molek{\"u}len und Zellen reguliert, kann die Gewebehom{\"o}ostase aufrechterhalten werden. Dabei ist das Endothel nicht nur passive Barriere, sondern auch an dieser dynamischen Regulation aktiv beteiligt. St{\"o}rungen oder Fehlregulationen dieser Prozesse f{\"u}hren zu Pathologien, z.B. Arteriosklerose. Es ist seit l{\"a}ngerem bekannt, dass Carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1), ein Mitglied der Immunglobulin-Superfamilie, die Bildung und Morphogenese neuer Blutgef{\"a}ße beeinflusst. Die spontane Entwicklung kleiner Arteriosklerose-{\"a}hnlicher L{\"a}sionen in CEACAM1 knockout (Cc1-/-) M{\"a}usen zeigt, dass CEACAM1 auch f{\"u}r die Hom{\"o}ostase ausgereifter Blutgef{\"a}ße von Bedeutung ist. Ziel dieser Dissertationsarbeit war daher, den Einfluss von CEACAM1 auf wesentliche Aspekte der Endothelfunktion in Aorten in situ bzw. in Endothelzellkulturen in vitro zu analysieren. Es konnte zun{\"a}chst gezeigt werden, dass CEACAM1-defiziente Endothelzellen im Vergleich zu Wildtyp (WT) Endothelzellen eine rundlichere Zellmorphologie mit meanderf{\"o}rmigen Zellgrenzen und interzellul{\"a}ren L{\"u}cken aufweisen. Diese morphologischen Unterschiede stimmen mit Befunden in situ an Aorten von WT und Cc1-/- M{\"a}usen {\"u}berein. Weiterhin wurde eine Translokation der endothelialen NO-Synthase (eNOS) von der Zellmembran in den peri-nukle{\"a}ren Bereich bei CEACAM1-Defizienz festgestellt. Die erhobenen Daten bieten zwei m{\"o}gliche Erkl{\"a}rungen daf{\"u}r. Einerseits k{\"o}nnte CEACAM1 durch Interaktion mit eNOS als Membrananker fungieren. Daneben wiesen CEACAM1-defiziente Endothelzellen eine erh{\"o}hte Expression des Enzyms APT1 auf, welches eNOS depalmitoyliert. Die daraus resultierende, ebenfalls nachgewiesene geringere Palmitoylierung k{\"o}nnte auch zur verminderten Membran-lokalisation von eNOS beitragen. Zur endothelialen Funktion geh{\"o}rt, die Adh{\"a}sion von Blutzellen an die Gef{\"a}ßwand weitestgehend zu beschr{\"a}nken. CEACAM1-defiziente Endothelzellen zeigten im Vergleich zu WT Endothelzellen eine verst{\"a}rkte Adh{\"a}sivit{\"a}t gegen{\"u}ber murinen und humanen Monozyten. {\"A}hnliche Unterschiede wurden f{\"u}r Aortenexplantate aus WT und Cc1-/- M{\"a}usen festgestellt. Dies ist einerseits mit einer verst{\"a}rkten Expression des Zelladh{\"a}sionsmolek{\"u}ls ICAM-1 bei CEACAM1-Defizienz erkl{\"a}rbar. Dar{\"u}ber hinaus vermittelt die Glykokalyx anti-adh{\"a}sive Eigenschaften. Aus Vorbefunden war bekannt, dass die endotheliale Glykokalyx in der Aorta von Cc1-/- M{\"a}use reduziert ist. Im Rahmen dieser Arbeit konnte dies auf eine verst{\"a}rkte Expression der Glykokalyx-degradierenden Enzyme MMP9, Chondroitinase sowie Hyaluronidase-2 in Cc1-/- Endothelzellen zur{\"u}ckgef{\"u}hrt werden. Eine erh{\"o}hte Permeabilit{\"a}t stellt einen Indikator f{\"u}r ein dysfunktionales Endothel, eines der initialen Schritte in der Pathogenese der Arteriosklerose, dar. Zur Analyse der aortalen Permeabilit{\"a}t wurde ein modifizierter Miles-Assay etabliert. Unter Verwendung etablierter muriner Arteriosklerosemodelle konnte gezeigt werden, dass dieser Assay eine St{\"o}rung der vaskul{\"a}ren Permeabilit{\"a}t bereits vor Auftreten makroskopischer Ver{\"a}nderungen zuverl{\"a}ssig detektiert. Im Rahmen der folgenden Analysen an WT und Cc1-/- M{\"a}usen zeigte sich ein altersabh{\"a}ngiger Effekt von CEACAM1 auf die Gef{\"a}ßpermeabilit{\"a}t: Aorten von 3 Monate alten Cc1-/- M{\"a}use wiesen eine im Vergleich zum WT erh{\"o}hte Gef{\"a}ßpermeabilit{\"a}t auf, welche wahrscheinlich Folge einer verz{\"o}gerten Gef{\"a}ßreifung ist. Im Alter von 9 Monaten zeigte sich dagegen ein entgegengesetztes Bild. Dies wurde auf eine verst{\"a}rkte Expression des die Barriere sch{\"a}digenden Inflammationsmediators TNF-α in 9 Monate alten WT M{\"a}usen zur{\"u}ckgef{\"u}hrt. Außerdem modulierte CEACAM1 die TNF-α-vermittelte Lockerung der endothelialen Barriere, indem es die Phosphorylierung von Adherens Junction Proteinen beeinflusste. Basal stabilisierte CEACAM1 die endotheliale Barriere durch Hemmung der Phosphorylierung von Caveolin-1, welches Adherens Junctions destabilisiert. Unter Einfluss von TNF-α war CEACAM1 verst{\"a}rkt im Bereich von Adherens Junctions lokalisiert und rekrutierte dort Src-Kinase. Src-Kinase wiederum destabilisierte Adherens Junctions durch Phosphorylierung von β-Catenin, was in verst{\"a}rkter Gef{\"a}ßpermeabilit{\"a}t resultierte. Dagegen f{\"u}hrte TNF-α in CEACAM1-defizienten Endothelzellen zu einer Dephosphorylierung von Caveolin-1 und β-Catenin, wodurch Adherens Junctions und damit die endotheliale Barriere stabilisiert wurden. Diese CEACAM1-abh{\"a}ngige differenzielle Regulation der Stabilit{\"a}t von Adherens Junctions unter TNF-α tr{\"a}gt wahrscheinlich maßgeblich zu den Unterschieden der vaskul{\"a}ren Permeabilit{\"a}t in 3 bzw. 9 Monate alten WT und Cc1-/- M{\"a}usen bei. Zusammenfassend konnte im Rahmen dieser Arbeit nachgewiesen werden, dass CEACAM1 zentrale Funktionen des Endothels und hier{\"u}ber die Hom{\"o}ostase reifer Gef{\"a}ße beeinflusst. Da eine Expression von CEACAM1 auch in arteriosklerotischen Plaques nachgewiesen werden konnte, soll in weiteren Untersuchungen auch der Beitrag von CEACAM1 zur arteriosklerotischen Plaquebildung analysiert werden.},
  subject      = {Endothel},
  language  = {de}
}
@phdthesis{Dannhaeuser2021,
  author    = {Dannh{\"a}user, Sven},
  title     = {Function of the Drosophila adhesion-GPCR Latrophilin/CIRL in nociception and neuropathy},
  doi       = {10.25972/OPUS-20158},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-201580},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Touch sensation is the ability to perceive mechanical cues which is required for essential behaviors. These encompass the avoidance of tissue damage, environmental perception, and social interaction but also proprioception and hearing. Therefore research on receptors that convert mechanical stimuli into electrical signals in sensory neurons remains a topical research focus. However, the underlying molecular mechanisms for mechano-metabotropic signal transduction are largely unknown, despite the vital role of mechanosensation in all corners of physiology. Being a large family with over 30 mammalian members, adhesion-type G protein-coupled receptors (aGPCRs) operate in a vast range of physiological processes. Correspondingly, diverse human diseases, such as developmental disorders, defects of the nervous system, allergies and cancer are associated with these receptor family. Several aGPCRs have recently been linked to mechanosensitive functions suggesting, that processing of mechanical stimuli may be a common feature of this receptor family - not only in classical mechanosensory structures. This project employed Drosophila melanogaster as the candidate to analyze the aGPCR Latrophilin/dCIRL function in mechanical nociception in vivo. To this end, we focused on larval sensory neurons and investigated molecular mechanisms of dCIRL activity using noxious mechanical stimuli in combination with optogenetic tools to manipulate second messenger pathways. In addition, we made use of a neuropathy model to test for an involvement of aGPCR signaling in the malfunctioning peripheral nervous system. To do so, this study investigated and characterized nocifensive behavior in dCirl null mutants (dCirlKO) and employed genetically targeted RNA-interference (RNAi) to cell-specifically manipulate nociceptive function. The results revealed that dCirl is transcribed in type II class IV peripheral sensory neurons - a cell type that is structurally similar to mammalian nociceptors and detects different nociceptive sensory modalities. Furthermore, dCirlKO larvae showed increased nocifensive behavior which can be rescued in cell specific reexpression experiments. Expression of bPAC (bacterial photoactivatable adenylate cyclase) in these nociceptive neurons enabled us to investigate an intracellular signaling cascade of dCIRL function provoked by light-induced elevation of cAMP. Here, the findings demonstrated that dCIRL operates as a down-regulator of nocifensive behavior by modulating nociceptive neurons. Given the clinical relevance of this results, dCirl function was tested in a chemically induced neuropathy model where it was shown that cell specific overexpression of dCirl rescued nocifensive behavior but not nociceptor morphology.},
  subject      = {Drosophila},
  language  = {en}
}
@phdthesis{Spindler2020,
  author    = {Spindler, Markus},
  title     = {The role of the adhesion and degranulation promoting adapter protein (ADAP) in platelet production},
  doi       = {10.25972/OPUS-20097},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-200977},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Bone marrow (BM) megakaryocytes (MKs) produce platelets by extending proplatelets into sinusoidal blood vessels. Although this process is fundamental to maintain normal platelet counts in circulation only little is known about the regulation of directed proplatelet formation. As revealed in this thesis, ADAP (adhesion and degranulation promoting adapter protein) deficiency (constitutive as well as MK and platelet-specific) resulted in a microthrombocytopenia in mice, recapitulating the clinical hallmark of patients with mutations in the ADAP gene. The thrombocytopenia was caused by a combination of an enhanced removal of platelets from the circulation by macrophages and a platelet production defect. This defect led to an ectopic release of (pro)platelet-like particles into the bone marrow compartment, with a massive accumulation of such fragments around sinusoids. In vitro studies of cultured BM cell-derived MKs revealed a polarization defect of the demarcation membrane system, which is dependent on F-actin dynamics. ADAP-deficient MKs spread on collagen and fibronectin displayed a reduced F-actin content and podosome density in the lowest confocal plane. In addition, ADAP-deficient MKs exhibited a reduced capacity to adhere on Horm collagen and in line with that the activation of beta1-integrins in the lowest confocal plane of spread MKs was diminished. These results point to ADAP as a novel regulator of terminal platelet formation. Beside ADAP-deficient mice, three other knockout mouse models (deficiency for profilin1 (PFN1), Wiskott-Aldrich-syndrome protein (WASP) and Actin-related protein 2/3 complex subunit 2 (ARPC2)) exist, which display ectopic release of (pro)platelet-like particles. As shown in the final part of the thesis, the pattern of the ectopic release of (pro)platelet-like particles in these genetically modified mice (PFN1 and WASP) was comparable to ADAP-deficient mice. Furthermore, all tested mutant MKs displayed an adhesion defect as well as a reduced podosome density on Horm collagen. These results indicate that similar mechanisms might apply for ectopic release.},
  language  = {en}
}
@phdthesis{Geiger2021,
  author    = {Geiger, Ute},
  title     = {Erfassung der intraoperativen Ankopplungseffizienz mittels evozierten Potentialen bei mit Mittelohrimplantat versorgten Patienten},
  doi       = {10.25972/OPUS-20106},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-201068},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Patienten mit leicht bis hochgradigen Schallleitungs-, Schallempfindungs- und kombinierten Schwerh{\"o}rigkeiten werden routinem{\"a}ßig nach erfolglosem H{\"o}rger{\"a}tetrageversuch mit aktiven Mittelohrimplantaten versorgt. Aktive Mittelohrimplantate k{\"o}nnen an verschiedene Strukturen des Mittelohrs angekoppelt werden. Der Ort der Ankopplung ist abh{\"a}ngig vom H{\"o}rverlust und der individuellen Physiologie des Mittelohres. Die H{\"o}rverbesserung ist dabei stark von der Kopplungseffizienz des Implantatwandlers an die Mittelohrstruktur abh{\"a}ngig. Aktuell gibt es keine zufriedenstellende M{\"o}glichkeit die Kopplungseffizienz intraoperativ zu bestimmen. Daher wird eine objektive Methode eingef{\"u}hrt, um intraoperativ auditorische Hirnstammantworten (BERAs) bei Stimulation {\"u}ber das Implantat abzuleiten. Die Vibrant Soundbrigde® (VSB) wird dabei mit einem Drahtlos{\"u}bertr{\"a}ger (miniTEK, Signia GmbH, Erlangen) und der Carina®-Aktuator {\"u}ber ein Audiokabel mit der BERA-Anlage verbunden. Die BERA-Anlage {\"u}bertr{\"a}gt die Stimuli direkt an das Implantat, welches an die Mittelohrstruktur angekoppelt ist. Die BERA-Antworten werden bei der VSB durch einen optimierten VSB-CE-Chirp und beim Carina®-System durch den Standard CE-Chirp evoziert, beginnend bei Pegeln oberhalb der Knochenleitungsh{\"o}rschwelle bis unter die Registrierungsschwelle. Diese Methode kann die intraoperative Integrit{\"a}t des Implantats sowie die Kopplungseffizienz bestimmen, um eine Aussage {\"u}ber den zu erwartenden H{\"o}rerfolg treffen zu k{\"o}nnen. Dar{\"u}ber hinaus kann die versorgte H{\"o}rschwelle verwendet werden, um die Anpassung bei Kindern oder schwierigen F{\"a}llen zu unterst{\"u}tzen und um eine H{\"o}rverschlechterung {\"u}ber die Zeit zu erfassen. Zusammenfassend, konnte eine Methode zur Bestimmung der intraoperativen Kopplungseffizienz w{\"a}hrend der Implantation von VSBs und Carinas® etabliert werden. Dar{\"u}ber hinaus werden intraoperative BERA-Daten von 30 VSB- und 10-Carina®-Patienten sowie deren H{\"o}rergebnisse gezeigt.},
  subject      = {Mittelohrimplantat},
  language  = {de}
}
@phdthesis{Gruendl2021,
  author    = {Gr{\"u}ndl, Marco},
  title     = {Biochemical characterization of the MMB-Hippo crosstalk and its physiological relevance for heart development},
  doi       = {10.25972/OPUS-21332},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-213328},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {The Myb-MuvB (MMB) complex plays an essential role in the time-dependent transcriptional activation of mitotic genes. Recently, our laboratory identified a novel crosstalk between the MMB-complex and YAP, the transcriptional coactivator of the Hippo pathway, to coregulate a subset of mitotic genes (Pattschull et al., 2019). Several genetic studies have shown that the Hippo-YAP pathway is essential to drive cardiomyocyte proliferation during cardiac development (von Gise et al., 2012; Heallen et al., 2011; Xin et al., 2011). However, the exact mechanisms of how YAP activates proliferation of cardiomyocytes is not known. This doctoral thesis addresses the physiological role of the MMB-Hippo crosstalk within the heart and characterizes the YAP-B-MYB interaction with the overall aim to identify a potent inhibitor of YAP. The results reported in this thesis indicate that complete loss of the MMB scaffold protein LIN9 in heart progenitor cells results in thinning of ventricular walls, reduced cardiomyocyte proliferation and early embryonic lethality. Moreover, genetic experiments using mice deficient in SAV1, a core component of the Hippo pathway, and LIN9-deficient mice revealed that the correct function of the MMB complex is critical for proliferation of cardiomyocytes due to Hippo-deficiency. Whole genome transcriptome profiling as well as genome wide binding studies identified a subset of Hippo-regulated cell cycle genes as direct targets of MMB. By proximity ligation assay (PLA), YAP and B-MYB were discovered to interact in embryonal cardiomyocytes. Biochemical approaches, such as co-immunoprecipitation assays, GST-pulldown assays, and µSPOT-based peptide arrays were employed to characterize the YAP-B-MYB interaction. Here, a PY motif within the N-terminus of B-MYB was found to directly interact with the YAP WW-domains. Consequently, the YAP WW-domains were important for the ability of YAP to drive proliferation in cardiomyocytes and to activate MMB target genes in differentiated C2C12 cells. The biochemical information obtained from the interaction studies was utilized to develop a novel competitive inhibitor of YAP called MY-COMP (Myb-YAP competition). In MY-COMP, the protein fragment of B-MYB containing the YAP binding domain is fused to a nuclear localization signal. Co-immunoprecipitation studies as well as PLA revealed that the YAP-B-MYB interaction is robustly blocked by expression of MY-COMP. Adenoviral overexpression of MY-COMP in embryonal cardiomyocytes suppressed entry into mitosis and blocked the pro-proliferative function of YAP. Strikingly, characterization of the cellular phenotype showed that ectopic expression of MY-COMP led to growth defects, nuclear abnormalities and polyploidization in HeLa cells. Taken together, the results of this thesis reveal the mechanism of the crosstalk between the Hippo signaling pathway and the MMB complex in the heart and form the basis for interference with the oncogenic activity of the Hippo coactivator YAP.},
  subject      = {Zellzyklus},
  language  = {en}
}
@phdthesis{Boerner2020,
  author    = {B{\"o}rner, Kevin},
  title     = {How CLEC16A modifies the function of thymic epithelial cells},
  doi       = {10.25972/OPUS-20023},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-200230},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Genomweite Assoziationsstudien haben CLEC16A als ein Suszeptibilit{\"a}tsgen f{\"u}r Typ 1 Diabetes und weitere Autoimmunerkrankungen identifiziert. Die genaue Funktion von CLEC16A bleibt jedoch ungekl{\"a}rt. Studien zeigten, dass sowohl das Drosophila Ortholog ema als auch das murine Clec16a eine Rolle in Autophagie spielen. Autophagie tr{\"a}gt zur Beladung der MHC-Klasse-II Molek{\"u}le und somit der Antigenpr{\"a}sentation bei. Dar{\"u}ber hinaus konnten Studien belegen, dass Autophagie zur Antigenpr{\"a}sentation w{\"a}hrend der T-Zell Selektion in Thymus-Epithelzellen ben{\"o}tigt wird. Dies schl{\"a}gt eine m{\"o}gliche Funktion von CLEC16A in Thymus-Epithelzellen w{\"a}hrend der T-Zell Selektion vor. Außerdem berichteten Arbeiten, dass CLEC16A als quantitativer Trait Locus f{\"u}r seine Nachbargene fungiert und dass Clec16a KD in Langerhans Inseln im Pankreas die Insulinsekretion und den Glukosestoffwechsel beeintr{\"a}chtigt. Dieser Arbeit vorausgehend hatten Schuster et al. eine Clec16a KD NOD Maus generiert, welche vor spontanem autoimmunem Diabetes gesch{\"u}tzt war. F{\"u}r diese Arbeit wurde vermutet, dass CLEC16A als Suszeptibilit{\"a}tsgen f{\"u}r Typ 1 Diabetes den Prozess der Autophagie in Thymus-Epithelzellen beeintr{\"a}chtigt und somit Antigenpr{\"a}sentation und das T-Zell Repertoire beeinflusst. Um auf der Vorarbeit von Schuster et al. aufzubauen und diese zu erg{\"a}nzen, zielte diese Arbeit darauf ab, den Einfluss von CLEC16A auf Thymus-Epithelzellen zu untersuchen. Hierf{\"u}r wurde ein CLEC16A KD in menschlichen Zellen mittels RNA Interferenz erzeugt und Autophagie durch Immunoblotting untersucht. Zus{\"a}tzlich wurde die Entz{\"u}ndung im Pankreasgewebe von Clec16a KD NOD M{\"a}usen mittels H.E. F{\"a}rbung beurteilt und bewertet. Thymus-Transplanationen wurden durchgef{\"u}hrt, um zu sehen, ob der Einfluss von Clec16a KD T-Zell intrinsisch ist. Außerdem wurden intraperitoneale Glukosetoleranztests durchgef{\"u}hrt, um den Blutzuckerstoffwechsel in Clec16a KD M{\"a}usen zu beurteilen. Schließlich wurden mittels qPCR Expressionslevel der benachbarten Gene, wie zum Beispiel Dexi und Socs1, erhoben, um die Eigenschaften von CLEC16A als quantitativer Trait Locus einzuordnen. Gemeinsam mit den Ergebnissen von Schuster et al. kann diese Arbeit aufzeigen, dass Clec16a KD die Auspr{\"a}gung von Insulitis im Pankreas reduziert und Clec16a KD NOD M{\"a}use vor spontanem Autoimmundiabetes sch{\"u}tzt. Dieser Schutz vor Erkrankung wird durch beeintr{\"a}chtigte Autophagie in Thymus-Epithelzellen hervorgerufen, welche die T-Zell Selektion beeinflusst und die Reaktivit{\"a}t von T-Zellen reduziert. Der Einfluss des Clec16a KD ist innerhalb des Thymus wirksam. Der Blutzuckerstoffwechsel in Clec16a KD NOD M{\"a}usen bleibt unver{\"a}ndert und kann deshalb als Ursache f{\"u}r den Schutz vor Type 1 Diabetes ausgeschlossen werden. Clec16a und Dexi zeigen {\"a}hnliche Expressionslevel auf, dennoch ben{\"o}tigt es weitere detaillierte Studien, um eine Beziehung zwischen den beiden Genen etablieren zu k{\"o}nnen. Letztlich konnte die Beeintr{\"a}chtigung von Autophagie in menschlichen CLEC16A KD Zellen nachgewiesen werden, was bedeutet, dass die Funktion von CLEC16A evolution{\"a}r konserviert ist und ein m{\"o}glicher Zusammenhang zwischen CLEC16A Polymorphismen und einem erh{\"o}hten Risiko f{\"u}r Typ 1 Diabetes im Menschen besteht.},
  subject      = {Thymus},
  language  = {en}
}
@phdthesis{Boehm2020,
  author    = {B{\"o}hm, Lena},
  title     = {Dissecting Mechanisms of Host Colonization by C. albicans},
  doi       = {10.25972/OPUS-19230},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-192303},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {The human body is laden with trillions of microorganisms that belong to all three domains of life. Some species of this microbiota subsist as harmless commensals in healthy adults, but under certain circumstances, they can cause mucosal disease or even systemic, life-threatening infections. While the bacterial members of our microbiota are heavily studied today, much less attention is afforded to eukaryotic species that colonize different mucocutaneous surfaces of the human body. This dissertation focuses on identifying regulatory circuits that enable a prominent member of these eukaryotes, C. albicans, to, on the one hand, live on a specific mammalian mucosal surface as a harmless commensal and, on the other hand, proliferate as a pathogen. Since the ultimate source of many fatal Candida infections is the gastrointestinal (GI) tract of the infected individual, this organism is particularly suited to distinguishing traits essential for the gut colonization of commensal fungi and their ability to cause disease. Sequence-specific DNA-binding proteins that regulate transcription are important to most biological processes; I thus used these proteins as starting points to gain insights into 1) how a specific transcription regulator promotes virulence in C. albicans; 2) which traits C. albicans requires to inhabit the GI tract of a specific, well-defined mouse model as a harmless commensal; and 3) how three previously undescribed transcriptional regulators contribute to the commensal colonization of the digestive tract of this mouse model. Altogether, this work advances the knowledge concerning the biology of commensal fungi in the mammalian gut and genetic determinants of fungal commensalism, as well as pathogenicity.},
  subject      = {Candida albicans},
  language  = {en}
}
@phdthesis{Dufner2020,
  author    = {Dufner, Vera Christine},
  title     = {Effektivit{\"a}t und Sicherheit von Blinatumomab im Long-term Follow-up bei Non-Hodgkin-Lymphom-Patienten},
  doi       = {10.25972/OPUS-18476},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-184762},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Das Non-Hodgkin-Lymphom (NHL) steht an siebter Stelle der Inzidenzen aller Krebserkrankungen, mit j{\"a}hrlich steigender Tendenz. Wie kann einer so gef{\"a}hrlichen und heterogenen Krankheitsentit{\"a}t in der heutigen Medizin angemessen begegnet werden? Neben etablierten Therapien, die geraden bei rezidivierten oder refrakt{\"a}ren NHL an ihre Grenzen stoßen, bieten experimentelle Therapieans{\"a}tze neue Hoffnung: Blinatumomab ist ein bispezifischer Antik{\"o}rper, der durch seine beiden Dom{\"a}nen als Adapter f{\"u}r die T-Zelle und die Tumor-Zelle fungiert und eine Zytolyse der malignen B-Zelle induziert. Bei der ALL fand Blinatumomab schon Anwendung in mehreren klinischen Studien und wurde im Dezember 2014 von der FDA in den USA zur Behandlung von Philadelphia-Chromosom-negativer rezidivierten/ refrakt{\"a}ren B-Zell Vorl{\"a}ufer-ALL zugelassen. Als erste klinische Studie an NHL-Patienten wurde von 2004-2011 die MT103/104-Studie veranlasst. Im Zuge dieser unverblindeten, multizentrischen Phase I/II Studie wurden 76 Patienten mit refrakt{\"a}rem und rezidiviertem NHL vier bis acht Wochen mit Blinatumomab als Dauerinfusion behandelt und hierbei Informationen zu Toxizit{\"a}t und Tolerabilit{\"a}t gesammelt. Mit der Langzeitbeobachtung der W{\"u}rzburger Kohorte aus dieser Studie befasst sich die vorliegende Arbeit. Ziel ist es zun{\"a}chst, festzustellen, wie lange die Patienten nach Blinatumomab-Therapie im Zuge der MT103/104 Studie gesamt, rezidiv- oder therapiefrei {\"u}berlebten und ob bei einem bestimmten Patientensubkollektiv ein besonders vorteilhaftes Langzeit{\"u}berleben gezeigt werden kann. Die Frage nach der Sicherheit von Blinatumomab beantwortet die Erfassung des Langzeitnebenwirkungsspektrums: Somit werden als zweiter Endpunkt die h{\"a}ufigsten Gr{\"u}nde f{\"u}r Krankenhausaufenthalte nach Blinatumomabtherapie, eventuelle H{\"a}ufungen einer spezifischen Nebenwirkungsentit{\"a}t und die Reversibilit{\"a}t der unter der Therapie aufgetretenen Nebenwirkungen mit einem selbst entwickelten Fragebogen erfasst. Der MoCA-Test soll neurokognitive Langzeittoxizit{\"a}ten ausschließen. Die Arbeit konnte nicht nur zeigen, dass Patienten, die auf Blinatumomab ansprachen gegen{\"u}ber den Patienten ohne Ansprechen ein deutlich l{\"a}ngeres {\"U}berleben zeigten, sie best{\"a}tigte die Wichtigkeit des Erhalts der effektiven Dosis von 60 µg/m²/24h f{\"u}r das Erreichen und den Erhalt der Progressionsfreiheit. Sechs Patienten waren bei Beobachtungsende noch in Remission. Die unterschiedlichen Eindosierungsmodi hatten keinen Effekt auf das Langzeit{\"u}berleben, k{\"o}nnen aber nebenwirkungsbedingte Therapieabbr{\"u}che w{\"a}hrend der Therapie minimieren. Alle w{\"a}hrend der Therapie aufgetretenen Nebenwirkungen waren in der Langzeitnachbeobachtung vollst{\"a}ndig reversibel. Am h{\"a}ufigsten mussten Patienten auf Grund von Infektionen im Verlauf hospitalisiert werden, bei zwei Patienten traten zus{\"a}tzliche Tumorerkrankungen auf, die allerdings nicht mit der Blinatumomab-Therapie assoziiert waren. Die Rate der Transformationen von indolenten in aggressive NHL war nicht erh{\"o}ht. Im MoCA-Test lassen sich keine H{\"a}ufungen von neurokognitiven Defiziten finden. Blinatumomab zeigt sich auch in der Langzeitbeobachtung als ein f{\"u}r die Behandlung von rezidivierten und refrakt{\"a}ren NHLs effektives und sicheres Medikament.},
  subject      = {Non-Hodgkin-Lymphom},
  language  = {de}
}
@phdthesis{Friedrich2019,
  author    = {Friedrich, Maximilian Uwe},
  title     = {Funktionelle Charakterisierung einer Tripletdeletion in SLC5A4 (SGLT3) als Kandidatengen f{\"u}r das Aufmerksamkeitsdefizit-/ Hyperaktivit{\"a}tssyndrom (ADHS)},
  doi       = {10.25972/OPUS-18479},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-184791},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Natrium-Glukose Transporter (SGLT) geh{\"o}ren zur „solute carrier 5" (SLC5) Familie, die sich durch einen sekund{\"a}r aktiven, natriumabh{\"a}ngigen Transport von Zuckern und an-deren Molek{\"u}len nach intrazellul{\"a}r auszeichnen. Die durch das Gen SLC5A4 kodierte Isoform SGLT3 transportiert dagegen keinen Zucker, sondern verh{\"a}lt sich als Glukosesensor, der nach Bindung seiner Liganden eine Membrandepolarisation induziert. In genomweiten Exomsequenzierungsstudien (whole exome sequencing, WES) mehrerer erweiterter Stammb{\"a}ume mit hoher Pr{\"a}valenz des Aufmerksamkeitsdefizit-/Hyperaktivit{\"a}tssyndroms (ADHS) wurde im Vorfeld eine ATG-Tripletdeletion in SLC5A4 identifiziert, die zum Verlust einer Aminos{\"a}ure (ΔM500) in SGLT3 f{\"u}hrt und zumindest partiell mit dem klinischen Ph{\"a}notyp kosegregiert. In der vorliegenden Arbeit wurde die zentralnerv{\"o}se Expression von SGLT3 auf RNA- Ebene mittels Reverse-Transkriptase PCR sowie real-time PCR aus humanen Gesamt-RNAs nachgewiesen. Dabei konnte eine ubiquit{\"a}re Expression im Gehirn mit relativ erh{\"o}hter Expression unter anderem in Striatum und Hypothalamus, deren Dysfunktion in der Pathogenese des ADHS impliziert wurde, gezeigt werden. Da Mutationen in homologen Dom{\"a}nen der eng strukturverwandten Isoformen SGLT1 und SGLT2 sowohl intestinale als auch renale Funktionen schwer beeintr{\"a}chtigen, wurden in dieser Arbeit funktionelle Charakteristika sowohl des wildtypischen als auch der ΔM500 und der benachbarten ΔI501 Deletionsvariante von SGLT3 mittels Zwei-Elektroden Spannungs- und Stromklemme in entsprechend cRNA-injizierten Xenopus laevis Oozyten untersucht. Der hochpotente SGLT3-spezifische Iminozuckeragonist 1-Desoxynojirimycin (DNJ) induzierte an SGLT3-exprimierenden Oozyten in sauren Bedingungen etwa dreifach gr{\"o}ßere Kationeneinstr{\"o}me als D-Glukose, was sowohl im Spannungsklemmen-, und anhand einer entsprechenden Membrandepolarisation im Stromklemmenmodus gezeigt wurde. Die mit der ΔM500 bzw. ΔI501 Variante injizierten Oozyten dagegen zeigten in den maximalen Aktivierungsbedingungen um 92\% bzw. 96\% (p<0,01) reduzierte Kationeneinstr{\"o}me, sodass diese als hochgradig sch{\"a}dliche „Loss of Function" Mutationen in SGLT3 charakterisiert wurden. Dieser Befund wurde mittels bioinformatischer in-silico Effektvorhersage validiert. Um Konsequenzen der Sequenzalteration auf den Membraneinbau der Transporter zu untersuchen, wurden die mit einem gelb fluoreszierenden Farbstoff (YFP) markierten Transporter in Oozytenmembranen mittels Laser-Scanning Mikroskop nachgewiesen und die jeweiligen Mengen der Konstrukte anhand der Fluoreszenzintensit{\"a}ten quantifiziert. Dabei zeigte sich eine um 53\% bzw. 42\% (p<0,01) reduzierte Menge der mutierten Konstrukte ΔM500 bzw. ΔI501 in der Membran, was zus{\"a}tzliche sch{\"a}dliche Effekte der Mutationen auf das sogenannte Membrantargeting der Transporter belegt. Zusammenfassend demonstrieren die Ergebnisse dieser Arbeit, dass die ΔM500 Variante von SGLT3, welcher in ADHS-relevanten Hirnarealen exprimiert wird, dessen sub-stratinduzierte Natriumleitf{\"a}higkeit aufhebt und den Membraneinbau beeintr{\"a}chtigen k{\"o}nnte, was in Wechselwirkung mit anderen genetischen ADHS Risikovarianten das Risiko f{\"u}r ADHS in Mutationstr{\"a}gern beeinflussen kann.},
  subject      = {ADHS},
  language  = {de}
}
@phdthesis{Sasi2020,
  author    = {Sasi, Manju},
  title     = {A mouse model for genetic deletion of presynaptic BDNF from adult hippocampal mossy fiber terminals},
  doi       = {10.25972/OPUS-18625},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-186250},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Brain-derived neurotrophic factor (BDNF) is a modulator and mediator of structural and functional plasticity at synapses in the central nervous system. Despite our profound knowledge about the synaptic function of BDNF at synapses, it is still controversially discussed whether synaptic BDNF acts primarily from pre- or postsynaptic sites. In the central nervous system, several studies show that mossy fiber (MF) projections formed by hippocampal granule neurons store the highest amount of BDNF. However, immunofluorescence and RNA labelling studies suggest that MF BDNF is primarily produced by granule neurons. Multiple other studies prefer the view that BDNF is primarily produced by postsynaptic neurons such as CA3 pyramidal neurons. Here, we question whether the BDNF, which is stored in the mossy fiber synapse, is primarily produced by granule neurons or whether by other cells in the MF-CA3 microcircuit. After standardization of immunolabelling of BDNF, confocal imaging confirmed the localization of BDNF in presynaptic MF terminals. This anterograde location of synaptic BDNF was also found in distinct regions of the fear and anxiety circuit, namely in the oval nucleus of the bed nucleus stria terminals (ovBNST) and in the central amygdala. To find out whether the presynaptic BDNF location is due to protein translation in the corresponding presynaptic dentate gyrus (DG) granule neuron, we developed and characterized a mouse model that exhibits BDNF deletion specifically from adult DG granule neurons. In this mouse model, loss of presynaptic BDNF immunoreactivity correlated with the specific Creactivity in granule neurons, thus confirming that MF BDNF is principally released by granule neurons. After BDNF deletion from granule neurons, we observed more immature neurons with widely arborized dendritic trees. This indicated that local BDNF deletion also affects the local adult neurogenesis, albeit Cre-mediated BDNF deletion only occur in adult granule neurons. Since BDNF is a master regulator of structural synaptic plasticity, it was questioned whether it is possible to visualize presynaptic, synapse-specific, structural plasticity in mossy fiber synapses. It was established that a combination of Cre-techniques together with targeting of GFP to membranes with the help of palmitoylation / myristoylation anchors was able to distinctly outline the synaptic structure of the BDNF-containing MF synapse. In summary, the mouse model characterized in here is suited to investigate the synaptic signalling function of presynaptic BDNF at the mossy fiber terminal, a model synapse to investigate microcircuit information processing from molecule to behaviour.},
  subject      = {Wachstumsfaktor},
  language  = {en}
}
@phdthesis{Seitz2020,
  author    = {Seitz, Nicola},
  title     = {Bee demise and bee rise: From honey bee colony losses to finding measures for advancing entire bee communities},
  doi       = {10.25972/OPUS-18418},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-184180},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {My dissertation comprises three studies: (1) an assessment of honey bee colony losses in the USA between 2014 and 2015, (2) an exploration of the potential of reclaimed sand mines as bee habitat, and (3) an evaluation of native and non-native pollinator friendly plants in regard to their attraction to bees. While the first study focuses on honey bees, the latter two studies primarily take wild bees or entire bee communities in focus. The study on honey bee colony losses was conducted within the framework of the Bee Informed Partnership (BIP, beeinformed.org) and aligns with the annual colony loss surveys which have been conducted in the USA since the winter of 2006/2007. It was the fourth year for which summer and annual losses were calculated in addition to winter losses. Among participants, backyard beekeepers were the largest group (n = 5690), although sideline (n = 169) and commercial (n = 78) beekeepers managed the majority (91.7 \%) of the 414 267 surveyed colonies. Overall, 15.1 \% of the estimated 2.74 million managed colonies in the USA were included in the study. Total honey bee colony losses (based on the entirety of included colonies) were higher in summer (25.3 \%) than in winter (22.3 \%) and amounted to 40.6 \% for the entire 2014/2015 beekeeping year. Average colony losses per beekeeper or operation were higher in winter (43.7 \%) than in summer (14.7 \%) and amounted to 49 \% for the entire 2014/2015 beekeeping year. Due to the dominance of backyard beekeepers among participants, average losses per operation (or unweighted loss) stronger reflected this smaller type of beekeeper. Backyard beekeepers mainly named colony management issues (e.g., starvation, weak colony in the fall) as causes for mortality, while sideline and commercial beekeepers stronger emphasized parasites or factors outside their control (e.g., varroa, nosema, queen failure). The second study took place at reclaimed sand mines. Sand mines represent anthropogenically impacted habitats found worldwide, which bear potential for bee conservation. Although floral resources can be limited at these habitats, vegetation free patches of open sandy soils and embankments may offer good nesting possibilities for sand restricted and other bees. We compared bee communities as found in three reclaimed sand mines and at adjacent roadside meadows in Maryland, USA, over two years. Both sand mines and roadsides hosted diverse bee communities with 111 and 88 bee species, respectively. Bee abundances as well as richness and Shannon diversity of bee species were higher in sand mines than at roadsides and negatively correlated with the percentage of vegetational ground cover. Species composition also differed significantly between habitats. Sand mines hosted a higher proportion of ground nesters, more uncommon and more 'sand loving' bees similar to natural sandy areas of Maryland. Despite the destruction of the original pre-mining habitat, sand mines thus appear to represent a unique habitat for wild bees, particularly when natural vegetation and open sand spots are encouraged. Considering habitat loss, the lack of natural disturbance regimes, and ongoing declines of wild bees, sand mines could add promising opportunities for bee conservation which has hitherto mainly focused on agricultural and urban habitats. The third study was an experimental field study on pollinator friendly plants. Bees rely on the pollen and nectar of plants as their food source. Therefore, pollinator friendly plantings are often used for habitat enhancements in bee conservation. Non-native pollinator friendly plants may aid in bee conservation efforts, but have not been tested and compared with native pollinator friendly plants in a common garden experiment. In this study, we seeded mixes of 20 native and 20 non-native pollinator friendly plants in two separate plots at three sites in Maryland, USA. For two years, we recorded flower visitors to the plants throughout the blooming period and additionally sampled bees with pan traps. A total of 3744 bees (120 species) were sampled in the study. Of these, 1708 bees (72 species) were hand netted directly from flowers for comparisons between native and non-native plants. Depending on the season, bee abundance and species richness was either similar or lower (early season and for richness also late season) at native plots compared to non-native plots. Additionally, the overall bee community composition differed significantly between native and non-native plots. Furthermore, native plants were associated with more specialized plant-bee visitation networks compared to non-native plants. In general, visitation networks were more specialized in the early season than the later seasons. Four species (Bombus impatiens, Halictus poeyi/ligatus, Lasioglossum pilosum, and Xylocopa virginica) out of the five most abundant bee species (also including Apis mellifera) foraged more specialized on native than non-native plants. Our study showed that non-native plants were well accepted by a diverse bee community and had a similar to higher attraction for bees compared to native plants. However, we also demonstrated alterations in foraging behavior, bee community assemblage, and visitation networks. As long as used with caution, non-native plants can be a useful addition to native pollinator friendly plantings. This study gives a first example of a direct comparison between native and non-native pollinator friendly plants.},
  subject      = {Biene},
  language  = {en}
}
@phdthesis{Fichtner2020,
  author    = {Fichtner, Alina Suzann},
  title     = {Alpaca, armadillo and cotton rat as new animal models for nonconventional T cells: Identification of cell populations and analysis of antigen receptors and ligands},
  doi       = {10.25972/OPUS-16910},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-169108},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {In this thesis, three species were investigated for the conservation of two non-conventional T cell systems, the CD1d/ iNKT cell system and the BTN3/ Vγ9Vδ2 T cell system. Non-conventional T cells are αβ or γδ T cells that do not fit into the classical mode of antigen recognition and adaptive responses. These T cells recognize antigens different from classical peptide antigens and are not restricted to the polymorphic MHC molecules but rather to non-polymorphic antigen-presenting molecules. The iNKT cell subset is restricted by the lipid antigen-presenting molecule CD1d and carries out immunomodulatory functions by rapid cytokine secretion. The molecular basis of this system, the semi-invariant iNKT TCR chains and CD1d were proven to be expressed and compared to homologs in human and rodents. Cotton rats possess multiple members of the AV14 and BV8 family and only one isoform of CD1d which is comparable to findings in the rat. Moreover, the reactivity of primary cells to glycolipid antigens could be shown, and an iNKT cell-like population was detected in primary cells using newly developed cotton rat CD1d oligomers. These were also applied to test the capacity of CD1d to present typical glycolipid antigens to iNKT TCR transductants. In addition, expression of cotton rat iNKT TCR α and β chains in TCR-negative cell lines was used to show successful pairing and detection of glycolipids in the context of CD1d. In summary, the conservation of a functional CD1d/iNKT cell system in the cotton rat could be shown, and tools were developed to study this cell subset in the course of infectious diseases. The Vγ9Vδ2 T cell subset is the major γδ T cell subset in human peripheral blood and has the unique ability to contribute to immune surveillance by detecting pyrophosphorylated metabolites of isoprenoid synthesis that indicate cell stress, transformation or infection. Up to this date, phosphoantigen-reactive γδ T cells have only been shown in primate species. However, evidence for the existence and functional conservation of the genes implied in the BTN3/Vγ9Vδ2 T cell system was found in several placental mammal species, and two candidate species were chosen for further investigation. The nine-banded armadillo, a valuable model for leprosy research, was shown to possess homologous genes to TRGV9, TRDV2 and BTN3. In this study, the expression of productive rearrangements of TRDV2 gene segments could be shown in peripheral blood samples, but no evidence was found for the expression of a functional TRGV9 rearrangement or BTN3 molecules. Moreover, determinants of phosphoantigen-reactive Vγ9Vδ2 T cells and functional BTN3 molecules were found to still be prevalent in armadillo genes. This makes the armadillo an interesting model to study the structural determinants that allow phosphoantigen recognition by a functional Vγ9Vδ2 T cell subset although this species is merely a witness for a functional system in a placental mammal ancestor. In contrast, alpacas were shown to express functional Vγ9Vδ2 T cells which conserved many features of the human counterpart. Expression of Vγ9Vδ2 pairings could be shown by single-cell PCR and functional phosphoantigenreactive pairings were observed. This phosphoantigen reactivity was also shown in PBMC cultures with a newly developed antibody specific for alpaca Vδ2Jδ4 chains. Moreover, a more detailed study of the alpaca TCR repertoire showed similarities to "γδ high" species like camelids and cattle which possess an extended family of TRDV genes. The γ and δ loci of alpaca TCR genes were drafted based on genomic information and cDNA studies and provide an overview for more detailed studies. Conservation of phosphoantigen recognition by the single BTN3 molecule of alpacas was shown in 293T knock out cell lines, and BTN3 detection on PBMCs was investigated with a newly developed alpaca BTN3-specific antibody. These findings prove the existence of a functional BTN3-dependent phosphoantigen-reactive Vγ9Vδ2 T cell subset and provide a basis for the future study of this cell system in a non-primate species. Moreover, as the first non-primate candidate species with the BTN3/Vγ9Vδ2 T cell system the alpaca is an important outgroup for research in this field. The use of a single BTN3 variant in contrast to three human isoforms that work together renders the alpaca a unique and to this date indispensable model for Vγ9Vδ2 T cells. In conclusion, this study provides an overview of the applicability of new animal models in the study of the non-conventional T cell subsets iNKT cells and Vγ9Vδ2 T cells and leads the way for a better understanding of structural and functional relationships.},
  subject      = {T-Lymphozyt},
  language  = {en}
}
@phdthesis{Dittert2020,
  author    = {Dittert, Natalie Christine},
  title     = {Modulation der Furchtextinktion durch transkranielle Gleichstromstimulation (tDCS)},
  doi       = {10.25972/OPUS-21095},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-210954},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Angsterkrankungen sowie die posttraumatische Belastungsst{\"o}rung sind weit verbreitete psychische Erkrankungen. Trotz gut evaluierter Therapiemethoden gibt es immer noch therapierefrakt{\"a}re oder rezidivierend erkrankende Patienten, f{\"u}r die nicht-invasive Hirnstimulationsverfahren wie die transkranielle Gleichstromstimulation (tDCS) eine zus{\"a}tzliche Option darstellen k{\"o}nnen. Diese Studie untersuchte daher die f{\"o}rderliche Wirkung der tDCS auf das Extinktionslernen, dem neuronalen Hintergrundmechanismus der Expositionstherapie. F{\"u}r die Untersuchung der Extinktionsprozesse wurde ein Ein-Tages-Furchtkonditionierungsparadigma mit weiblichen Gesichtern als konditionierte Stimuli (CS) und einem 95 dB lauten weiblichen Schrei als unkonditionierten Stimulus verwendet. Die tDCS zielte darauf ab den ventromedialen pr{\"a}frontalen Kortex (vmPFC), ein wichtiges Kontrollareal der Extinktion, zu aktivieren, wohingegen furchtgenerierende dorsomediale Hirnareale von der Stimulation ausgespart bleiben sollten. Hierf{\"u}r wurden zwei ca. 4 x 4 cm große Elektroden in bitemporaler Anordnung etwas unterhalb der EEG 10-20-Positionen F7 und F8 appliziert und ein Gleichstrom mit einer St{\"a}rke von 1.5 mA verwendet. Die 20- min{\"u}tige Stimulation startete w{\"a}hrend einer 10-min{\"u}tigen Pause zwischen Akquisition und Extinktion und lief bis zum Ende der Extinktion durch. Die gesunden Probanden wurden randomisiert und doppelt verblindet zwei sham- und zwei real-Stimulationsgruppen mit jeweils entgegengesetzten Stromflussrichtungen zugeordnet. Zur Messung der Furchtreaktion dienten die elektrodermale Reaktion sowie subjektive Arousal- und Valenzbewertungen. Zus{\"a}tzlich wurde die Kontingenzerwartung sowie verschiedene Frageb{\"o}gen zu Depressivit{\"a}t, Affekt, State- und Trait-Angst, Angstsensitivit{\"a}t und H{\"a}ndigkeit erhoben. Die Untersuchung der Effekte von tDCS und Stromflussrichtung erfolgte bei allen erfolgreich konditionierten Probanden (N = 84) mittels generalisierten Sch{\"a}tzgleichungen. Erwartet wurde insbesondere eine Verbesserung des fr{\"u}hen Extinktionslernens in den real-Stimulationsgruppen, wobei vermutetet wurde, dass rechts und links anodaler Stromfluss nicht zu identischen Resultaten f{\"u}hren w{\"u}rde. Die Ergebnisse wiesen auf eine Verbesserung der fr{\"u}hen Extinktion unter tDCS hin. Der Effekt spiegelte sich in den Maßen der elektrodermalen Aktivit{\"a}t in einer st{\"a}rkeren Reduktion der CS+/CS- Diskrimination und einem beschleunigten Reaktionsverlust auf CS+ wider. Der vermittelnde Mechanismus kann im intendierten Aktivit{\"a}tsanstieg des vmPFC liegen, eine Steigerung der dopaminergen Neurotransmission ist jedoch ebenso denkbar. Zus{\"a}tzlich ist auch die Verbesserung der Prozessierung von prediction errors durch die Ver{\"a}nderung der Dopaminsekretion bzw. Aktivit{\"a}tssteigerung im vmPFC, Orbitofrontalkortex und mittleren temporalen Gyrus m{\"o}glich. Die subjektiven Valenz- und Arousalbewertungen zeigten sich w{\"a}hrend des gesamten Experiments unbeeinflusst von der tDCS. Neben diesem Haupteffekt kam es zu weiteren nicht erwarteten Effekten. Einer dieser bedeutsamen Nebeneffekte war ein kurzer initialer Reaktionsanstieg auf den CS- zu Beginn des ersten und zweiten Extinktionsblocks in beiden real-Stimulationsgruppen, der u. a. mitverantwortlich f{\"u}r deren st{\"a}rkeren Verlust der CS+/CS- Diskrimination war. Auch negative Auswirkungen auf die stimulierten Personen - insbesondere in Kombination mit Angsterkrankungen - k{\"o}nnen eine denkbare Folge hiervon sein. Daher stellt dieser Nebeneffekt eine wichtige Limitation des Hauptergebnisses dar, dessen Ursachen dringend in weiteren Studien evaluiert werden sollten. Als m{\"o}gliche Gr{\"u}nde werden ein Verlust der Sicherheitsinformation des CS-, Angstgeneralisierungseffekte sowie ein erh{\"o}htes Maß an sustained fear vermutet. Dar{\"u}ber hinaus wurden unerwarteterweise auch keinerlei Unterschiede der Stromflussrichtung w{\"a}hrend der fr{\"u}hen Extinktion manifest, in der sp{\"a}ten bzw. gesamten Extinktion zeigten sich jedoch verschiedene Vor- und Nachteile. Vorteilhaft an der rechts anodalen im Vergleich zur links anodalen Stimulation war ein geringerer gemittelter Reaktionsanstieg auf CS+ und CS- zu Beginn des zweiten Extinktionsblocks. Dieser Effekt beruhte vermutlich auf einer Steigerung der Emotionsregulation durch Stimulation des rechten inferioren frontalen Gyrus. Als nachteilig erwies sich jedoch, dass die Reduktion der State-Angst w{\"a}hrend der Extinktion unter rechts anodaler tDCS geringer ausfiel. Bei Angstpatienten gibt es Hinweise auf eine Unteraktivierung des linken Frontalkortex, sodass angstreduzierende Effekte durch linksfrontale Aktivierung denkbar sind. Die Wahl der Stromflussrichtung sollte demnach je nach gew{\"u}nschten Effekten und Angstausmaß der stimulierten Probanden abgewogen werden. Aufgrund der experimentellen Anordnung ergeben sich einige Limitationen dieser Studie. Der gesamte Extinktionsvorgang war in allen Gruppen nur von sehr kurzer Dauer, dadurch hielten auch die positiven Effekte in den real-Stimulationsgruppen nicht lange an. Zudem fand keine Testung des Extinktionsrecalls statt, sodass keine Aussage {\"u}ber die langfristige Wirkung der tDCS gemacht werden kann. Da die Stimulation direkt nach der Akquisition gestartet wurde, kann es neben bzw. anstelle einer Verbesserung des Extinktionslernens auch zu einer St{\"o}rung der Furchtkonsolidierung und dadurch zu einer geringeren Furchtexpression gekommen sein. Zudem ist der vmPFC, das Hauptstimulationsziel dieser Studie, ebenso an der Suppression von Furchtreaktionen beteiligt, somit k{\"o}nnte auch dieser Mechanismus f{\"u}r die gefundenen Effekte verantwortlich sein. Eine Replikation der Studienergebnisse in einem mehrt{\"a}gigem Konditionierungsparadigma w{\"a}re damit sinnvoll, um die Dauer und Hintergr{\"u}nde der gefundenen Effekte besser zu verstehen. Insgesamt bilden die Ergebnisse dieser Studie eine gute Basis zur Anwendung der tDCS des vmPFC zur Verbesserung des Extinktionslernens. Die Schw{\"a}chen des hier getesteten Stimulationsprotokolls sollten jedoch in k{\"u}nftigen Studien weiter evaluiert und reduziert werden. Falls Testungen an Angstpatienten schließlich zu Erfolgen f{\"u}hren, k{\"o}nnte die tDCS des vmPFC als g{\"u}nstige und leicht anwendbare Erg{\"a}nzung zu Expositionstherapien bei Patienten mit bisher therapieresistenten oder rezidivierenden Angsterkrankungen eingesetzt werden.},
  subject      = {Hirnstimulation},
  language  = {de}
}
@phdthesis{Becker2021,
  author    = {Becker, Isabelle Carlotta},
  title     = {The role of megakaryocytes and platelets in vascular and osteogenic development},
  doi       = {10.25972/OPUS-21024},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-210241},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Platelets, small anucleate cell fragments in the blood stream, derive from large precursor cells, so-called megakaryocytes (MK) residing in the bone marrow (BM). In addition to their role in wound healing, platelets have been shown to play a significant role during inflammatory bleeding. Above all, the immunoreceptor tyrosine-based activation motif (ITAM) receptors GPVI as well as CLEC-2 have been identified as main regulators of vascular integrity. In addition to ITAM-bearing receptors, our group identified GPV as another potent regulator of hemostasis and thrombosis. Surprisingly, concomitant lack of GPV and CLEC-2 deteriorated blood-lymphatic misconnections observed in Clec2-/- mice resulting in severe edema formation and intestinal inflammation. Analysis of lymphatic and vascular development in embryonic mesenteries revealed severely defective blood-lymph-vessel separation, which translated into thrombocytopenia and increased vascular permeability due to reduced tight junction density in mesenteric blood vessels and consequent leakage of blood into the peritoneal cavity. Recently, platelet granule release has been proposed to ameliorate the progression of retinopathy of prematurity (ROP), a fatal disease in newborns leading to retinal degradation. The mechanisms governing platelet activation in this process remained elusive nonetheless, which prompted us to investigate a possible role of ITAM signaling. In the second part of this thesis, granule release during ROP was shown to be GPVI- and partly CLEC-2-triggered since blockade or loss of these receptors markedly deteriorated ROP progression. Proplatelet formation from MKs is highly dependent on a functional microtubule and actin cytoskeleton, the latter of which is regulated by several actin-monomer binding proteins including Cofilin1 and Twinfilin1 that have been associated with actin-severing at pointed ends. In the present study, a redundancy between both proteins especially important for the guided release of proplatelets into the bloodstream was identified, since deficiency in both proteins markedly impaired MK functionality mainly due to altered actin-microtubule crosstalk. Besides ITAM-triggered activation, platelets and MKs are dependent on inhibitory receptors, which prevent overshooting activation. We here identified macrothrombocytopenic mice with a mutation within Mpig6b encoding the ITIM-bearing receptor G6b-B. G6b-B-mutant mice developed a severe myelofibrosis associated with sex-specific bone remodeling defects resulting in osteosclerosis and -porosis in female mice. Moreover, G6b-B was shown to be indispensable for MK maturation as verified by a significant reduction in MK-specific gene expression in G6b-B-mutant MKs due to reduced GATA-1 activity.},
  subject      = {Megakaryozyt},
  language  = {en}
}
@phdthesis{Bandleon2020,
  author    = {Bandleon, Sandra},
  title     = {Der Einfluss von FKBP52 auf die Funktion von TRPC3 Kan{\"a}len im Herzen},
  doi       = {10.25972/OPUS-21008},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-210083},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Transient Receptor Potential (TRP; C-classical; TRPC) Kan{\"a}le sind Ionenkan{\"a}le in der Plasmamembran und erlauben einen nicht selektiven Ca2+-Einstrom in die Zelle. Durch die Stimulation von Gq-Protein-gekoppelten Rezeptoren (GqPCRs) wird dieser Ca2+-Einstrom erh{\"o}ht, wodurch {\"u}ber Calmodulin, die Phosphatase Calcineurin aktiviert wird. Der Transkriptionsfaktor nuclear factor of activated T-cells (NFAT) wird durch Calcineurin dephosphoryliert und wandert in den Nucleus, wo er mit anderen Transkriptionsfaktoren interagiert und Hypertrophie-induzierende Gene aktiviert. TRPC3 ist hierbei eine der relevantesten Isoformen f{\"u}r die Entwicklung einer Myokardhypertrophie, wie sie im Rahmen zahlreicher kardiovaskul{\"a}rer Erkrankungen zu finden ist. Eine kardiale Hypertrophie ist an der Pathogenese der Herzinsuffizienz beteiligt und stellt somit einen wichtigen Risikofaktor f{\"u}r den pl{\"o}tzlichen Herztod dar. Aus diesem Grund ist es von besonderer Bedeutung die Regulation von TRPC3 Kan{\"a}len und deren Einfluss auf hypertrophe Prozesse genauer zu untersuchen. In Vorversuchen wurde FK506 bindendes Protein 52 (FKBP52) als neuer Interaktionspartner von TRPC3 im Herzen gefunden. Die dabei gefundene FKBP52-Bindestelle von TRPC3 lag erstaunlicherweise außerhalb der zu erwartenden Bindestelle mit den vermeintlichen FKBP Bindemotiven. FKBP52 ist ein Immunophilin, das als cis/trans Isomerase fungiert und dadurch an der Regulation von verschiedenen Ionenkan{\"a}len beteiligt ist, darunter auch TRPC-Kan{\"a}le. Es zeigte sich, dass alle Dom{\"a}nen von FKBP52, bis auf die TPR3-Dom{\"a}ne und der C Terminus, in der Lage waren, mit TRPC3 zu interagieren. Aufgrund der Funktion der FKBPs und der Tatsache, dass TRPC3 eine Rolle in der Entwicklung einer kardialen Hypertrophie spielt, sollte in dieser Arbeit untersucht werden, ob FKBP52 die Aktivit{\"a}t und die nachgeschalteten hypertrophen Signalwege von TRPC3 beeinflusst. Die Downregulation von FKBP52 f{\"u}hrte zu einer verst{\"a}rkten TRPC3-abh{\"a}ngigen hypertrophen Antwort in neonatalen Rattenkardiomyozyten (engl. neonatal rat cardiomyocytes, NRCs). Der gleiche Effekt war sowohl in NRCs und in adulten Rattenkardiomyozyten (engl. adult rat cardiomyocytes, ARCs) zu sehen, wenn Peptidyl-Prolyl-cis-trans-Isomerase (PPIase) defiziente Mutanten von FKBP52 {\"u}berexprimiert wurde. Verk{\"u}rzte FKBP52 Mutanten erh{\"o}hten ebenfalls die TRPC3-abh{\"a}ngige Aktivit{\"a}t von Calcineurin, was durch eine verst{\"a}rkte Translokation von NFAT in den Nucleus von NRCs zu sehen war. Außerdem konnte in NRCs und in menschlichen embryonalen Nierenzellen (engl. human embryonic kidney cells, HEK 293 Zellen), die die PPIase defizienten Mutanten exprimierten, ein erh{\"o}hter Ca2+-Einstrom in die Zelle beobachtet werden. Das gleiche war nach Downregulation von FKBP52 in HEK 293 Zellen, die TRPC3 {\"u}berexprimieren (T3.9 Zellen), zu sehen. Eine funktionelle Interaktion von FKBP52 und TRPC3 konnte auch in elektrophysiologischen Messungen best{\"a}tigt werden. Nach der Interaktion von TRPC3 mit den FKBP52 Mutanten zeigte sich eine erh{\"o}hte TRPC3-Aktivit{\"a}t. Die Daten zeigen somit, dass TRPC3-Kan{\"a}le durch FKBP52 reguliert werden und diese Regulation abh{\"a}ngig von der PPIase Funktion ist. Eine Interaktion von TRPC3 mit vollfunktionsf{\"a}higem FKBP52 k{\"o}nnte vor einer Ca2+-{\"U}berlastung und einer damit einhergehenden pathologischen Hypertrophie des Herzens sch{\"u}tzen.},
  subject      = {Hypertrophie},
  language  = {de}
}
@phdthesis{Collenburg2018,
  author    = {Collenburg, Lena},
  title     = {The Role of Ceramides and Sphingomyelinases for Dynamic Membrane Processes in T Cells},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-151161},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Previous work of our group has established a role of sphingomyelinases in the regulation of T cell responses to TCR or pathogen stimulation, and this became particularly evident at the level of actin cytoskeletal dynamics. The formation of lipid membrane microdomains is crucial for receptor clustering and signal induction, and therefore, ceramide accumulation by membrane sphingomyelin breakdown is needed for signalling- complex-assembly. Pathogen-induced overshooting of SMase activation substantially impacted the formation of membrane protrusions, with T cell spreading as well as a front/rear polarisation upon CD3/CD28 co-stimulation [103]. On the other hand, NSM activation is part of the physiological TCR signal [67], indicating that a spatiotemporally balanced NSM activation is crucial for its physiological function. It involves actin cytoskeletal reorganisation and T cell polarisation. These two functions are also of central importance in directional T cell migration and motility in tissues. This thesis aims on defining the role of NSM in compartmentalisation of the T cell membrane in polarisation and migration. Therefore, functional studies on the impact of NSM activity in these processes had to be complemented by the development of tools to study ceramide compartmentalisation in living T cells.},
  subject      = {Ceramides},
  language  = {en}
}
@phdthesis{Candemir2018,
  author    = {Candemir, Esin},
  title     = {Involvement of neuronal nitric oxide synthase (NOS-I) PDZ interactions in neuropsychiatric disorders},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-151194},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Neuronal nitric oxide (NO) synthase (NOS-I) and its adaptor protein (NOS1AP) have been repeatedly and consistently associated with neuropsychiatric disorders in several genetic association and linkage studies, as well as functional studies. NOS-I has an extended PDZ domain which enables it to interact with postsynaptic density protein 95 (PSD-95) bringing NOS-I in close proximity to NMDA receptors. This interaction allows NMDA receptor activity dependent calcium-influx to activate NOS-I, linking NO synthesis to regulation of glutamatergic signaling pathways. NOS1AP is a PDZ-domain ligand of NOS-I and has been proposed to compete with PSD-95 for NOS-I interaction. Studies performed on post-mortem brain tissues have shown increased expression of NOS1AP in patients with schizophrenia and bipolar disorder, suggesting that increased NOS-I/NOS1AP interactions might be involved in neuropsychiatric disorders possibly through disruption of NOS-I PDZ interactions. Therefore, I have investigated the involvement of NOS-I in different endophenotypes of neuropsychiatric disorders by targeting its specific PDZ interactions in vitro and in vivo. To this end, I used recombinant adeno-associated virus (rAAV) vectors expressing NOS1AP isoforms/domains (NOS1AP-L: full length NOS1AP; NOS1AP-LC20: the last 20 amino acids of NOS1AP-L, containing the PDZ interaction motif suggested to stabilize interaction with NOS-I; NOS1AP-LΔC20: NOS1AP-L lacking the last 20 amino acids; NOS1AP-S: the short isoform of NOS1AP), residues 396-503 of NOS1AP-L (NOS1AP396-503) encoding the full NOS-I interaction domain, and N-terminal 133 amino acids of NOS-I (NOS-I1-133) encoding for the extended PDZ-domain. Neuropsychiatric disorders involve morphological brain changes including altered dendritic development and spine plasticity. Hence, I have examined dendritic morphology in primary cultured hippocampal and cortical neurons upon overexpression of constructed rAAV vectors. Sholl analysis revealed that overexpression of NOS1AP-L and NOS1AP-LΔC20 mildly reduced dendritic length/branching. Moreover, overexpression of all NOS1AP isoforms/domains resulted in highly altered spine plasticity including significant reduction in the number of mature spines and increased growth of filopodia. These findings suggest that NOS1AP affects dendritic growth and development of dendritic spines, which may involve both, increased NOS-I/NOS1AP interaction as well as interaction of NOS1AP with proteins other than NOS-I. Interestingly, the observed alterations in dendritic morphology were reminiscent of those observed in post-mortem brains of patients with neuropsychiatric disorders. Given the dendritic alterations in vitro, I have examined, whether disruption of NOS-I PDZ interaction would also result in behavioral deficits associated with neuropsychiatric disorders. To this end, rAAV vectors expressing NOS1AP-L, NOS1AP396-503, NOS-I1-133, and mCherry were stereotaxically delivered to the dorsal hippocampus of 6-week-old male C57Bl/6J mice. One week after surgery, mice were randomly separated into two groups. One of those groups underwent three weeks of chronic mild stress (CMS). Afterwards all mice were subjected to a comprehensive behavioral analysis. The findings revealed that overexpression of the constructs did not result in phenotypes related to anxiety or depression, though CMS had an anxiolytic effect independent of the injected construct. Mice overexpressing NOS-I1-133, previously shown to disrupt NOS-I/PSD-95 interaction, showed impaired spatial memory, sensorimotor gating, social interaction, and increased locomotor activity. NOS1AP overexpressing mice showed mild impairments in sensorimotor gating and spatial working memory and severely impaired social interaction. NOS1AP396-503 overexpressing mice also showed impaired social interaction but enhanced sensorimotor gating and reduced locomotor activity. Taken together, these behavioral findings indicate an involvement of NOS-I PDZ interactions in phenotypes associated with positive symptoms and cognitive deficits of psychotic disorders. In summary, this study revealed an important contribution of NOS-I protein interactions in the development of endophenotypic traits of neuropsychiatric disorders, in particular schizophrenia, at morphological and behavioral levels. These findings might eventually aid to a better understanding of NOS-I-dependent psychopathogenesis, and to develop pharmacologically relevant treatment strategies.},
  subject      = {Stickstoffmonoxid-Synthase},
  language  = {en}
}
@phdthesis{Kleffel2018,
  author    = {Kleffel, Sonja Beate},
  title     = {The role of cancer cell-expressed PD-1 in tumorigenesis and tumor immune evasion},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-151205},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Melanoma and Merkel cell carcinoma (MCC) are highly aggressive cancers of the skin that frequently escape immune recognition and acquire resistance to chemotherapeutic agents, which poses a major obstacle to successful cancer treatment. Recently, a new class of therapeutics targeting the programmed cell death-1 (PD-1) immune checkpoint receptor has shown remarkable efficacy in the treatment of both cancers. Blockade of PD-1 on T cells activates cancer-specific immune responses that can mediate tumor regression. The data presented in this Ph.D. thesis demonstrates that PD-1 is also expressed by subsets of cancer cells in melanoma and MCC. Moreover, this work identifies PD-1 as a novel tumor cell-intrinsic growth receptor, even in the absence of T cell immunity. PD-1 is expressed by tumorigenic cell subsets in melanoma patient samples and established human and murine cell lines that also co-express ABCB5, a marker of immunoregulatory tumor- initiating cells in melanoma. Consistently, melanoma-expressed PD-1 downmodulates T effector cell functions and increases the intratumoral frequency of tolerogenic myeloid- derived suppressor cells. PD-1 inhibition on melanoma cells by RNA interference, blocking antibodies, or mutagenesis of melanoma-PD-1 signaling motifs suppresses tumor growth in immunocompetent, immunocompromised, and PD-1-deficient tumor graft recipient mice. Conversely, melanoma-specific PD-1 overexpression enhances tumorigenicity, including in mice lacking adaptive immunity. Engagement of melanoma- PD-1 by its ligand PD-L1 promotes tumor growth, whereas melanoma-PD-L1 inhibition or knockout of host-PD-L1 attenuates growth of PD-1-positive melanomas. Mechanistically, the melanoma-PD-1 receptor activates mTOR signaling mediators, including ribosomal protein S6. In a proof-of-concept study, tumoral expression of phospho-S6 in pretreatment tumor biopsies correlated with clinical responses to anti-PD-1 therapy in melanoma patients. In MCC, PD-1 is similarly co-expressed by ABCB5+ cancer cell subsets in clinical tumor specimens and established human cell lines. ABCB5 renders MCC cells resistant to the standard-of-care chemotherapeutic agents, carboplatin and etoposide. Antibody-mediated ABCB5 blockade reverses chemotherapy resistance and inhibits tumor xenograft growth by enhancing chemotherapy-induced tumor cell killing. Furthermore, engagement of MCC-expressed PD-1 by its ligands, PD-L1 and PD-L2, promotes proliferation and activates MCC-intrinsic mTOR signaling. Consistently, antibody- mediated PD-1 blockade inhibits MCC tumor xenograft growth and phosphorylation of mTOR effectors in immunocompromised mice. In summary, these findings identify cancer cell-intrinsic functions of the PD-1 pathway in tumorigenesis and suggest that blocking melanoma- and MCC-expressed PD-1 might contribute to the striking clinical efficacy of anti-PD-1 therapy. Additionally, these results establish ABCB5 as a previously unrecognized chemoresistance mechanism in MCC.},
  subject      = {Melanom},
  language  = {en}
}
@phdthesis{Lerch2018,
  author    = {Lerch, Maike Franziska},
  title     = {Characterisation of a novel non-coding RNA and its involvement in polysaccharide intercellular adhesin (PIA)-mediated biofilm formation of \(Staphylococcus\) \(epidermidis\)},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-155777},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Coagulase-negative staphylococci, particularly Staphylococcus epidermidis, have been recognised as an important cause of health care-associated infections due to catheterisation, and livestock-associated infections. The colonisation of indwelling medical devices is achieved by the formation of biofilms, which are large cell-clusters surrounded by an extracellular matrix. This extracellular matrix consists mainly of PIA (polysaccharide intercellular adhesin), which is encoded by the icaADBC-operon. The importance of icaADBC in clinical strains provoking severe infections initiated numerous investigations of this operon and its regulation within the last two decades. The discovery of a long transcript being located next to icaADBC, downstream of the regulator gene icaR, led to the hypothesis of a possible involvement of this transcript in the regulation of biofilm formation (Eckart, 2006). Goal of this work was to characterise this transcript, named ncRNA IcaZ, in molecular detail and to uncover its functional role in S. epidermidis. The ~400 nt long IcaZ is specific for ica-positive S. epidermidis and is transcribed in early- and mid-exponential growth phase as primary transcript. The promotor sequence and the first nucleotides of icaZ overlap with the 3' UTR of the preceding icaR gene, whereas the terminator sequence is shared by tRNAThr-4, being located convergently to icaZ. Deletion of icaZ resulted in a macroscopic biofilm-negative phenotype with highly diminished PIA-biofilm. Biofilm composition was analysed in vitro by classical crystal violet assays and in vivo by confocal laser scanning microscopy under flow conditions to display biofilm formation in real-time. The mutant showed clear defects in initial adherence and decreased cell-cell adherence, and was therefore not able to form a proper biofilm under flow in contrast to the wildtype. Restoration of PIA upon providing icaZ complementation from plasmids revealed inconsistent results in the various mutant backgrounds. To uncover the functional role of IcaZ, transcriptomic and proteomic analysis was carried out, providing some hints on candidate targets, but the varying biofilm phenotypes of wildtype and icaZ mutants made it difficult to identify direct IcaZ mRNA targets. Pulse expression of icaZ was then used as direct fishing method and computational target predictions were executed with candidate mRNAs from aforesaid approaches. The combined data of these analyses suggested an involvement of icaR in IcaZ-mediated biofilm control. Therefore, RNA binding assays were established for IcaZ and icaR mRNA. A positive gel shift was maintained with icaR 3' UTR and with 5'/3' icaR mRNA fusion product, whereas no gel shift was obtained with icaA mRNA. From these assays, it was assumed that IcaZ regulates icaR mRNA expression in S. epidermidis. S. aureus instead lacks ncRNA IcaZ and its icaR mRNA was shown to undergo autoregulation under so far unknown circumstances by intra- or intermolecular binding of 5' UTR and 3' UTR (Ruiz de los Mozos et al., 2013). Here, the Shine-Dalgarno sequence is blocked through 5'/3' UTR base pairing and RNase III, an endoribonuclease, degrades icaR mRNA, leading to translational blockade. In this work, icaR mRNA autoregulation was therefore analysed experimentally in S. epidermidis and results showed that this specific autoregulation does not take place in this organism. An involvement of RNase III in the degradation process could not be verified here. GFP-reporter plasmids were generated to visualise the interaction, but have to be improved for further investigations. In conclusion, IcaZ was found to interact with icaR mRNA, thereby conceivably interfering with translation initiation of repressor IcaR, and thus to promote PIA synthesis and biofilm formation. In addition, the environmental factor ethanol was found to induce icaZ expression, while only weak or no effects were obtained with NaCl and glucose. Ethanol, actually is an ingredient of disinfectants in hospital settings and known as efficient effector for biofilm induction. As biofilm formation on medical devices is a critical factor hampering treatment of S. epidermidis infections in clinical care, the results of this thesis do not only contribute to better understanding of the complex network of biofilm regulation in staphylococci, but may also have practical relevance in the future.},
  subject      = {Biofilm},
  language  = {en}
}
@phdthesis{Semeniak2018,
  author    = {Semeniak, Daniela},
  title     = {Role of bone marrow extracellular matrix proteins on platelet biogenesis and function},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-155857},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Platelets, small anucleated blood cells responsible for hemostasis, interact at sights of injury with several exposed extracellular matrix (ECM) proteins through specific receptors. Ligand binding leads to activation, adhesion and aggregation of platelets. Already megakaryocytes (MKs), the immediate precursor cells in bone marrow (BM), are in constant contact to these ECM proteins (ECMP). The interaction of ECMP with MKs is, in contrast to platelets, less well understood. It is therefore important to study how MKs interact with sinusoids via the underlying ECMP. This thesis addresses three major topics to elucidate these interactions and their role in platelet biogenesis. First, we studied the topology of ECMP within BM and their impact on proplatelet formation (PPF) in vitro. By establishing a four-color immunofluorescence microscopy we localized collagens and other ECMP and determined their degree of contact towards vessels and megakaryocytes (MKs). In in vitro assays we could demonstrate that Col I mediates increased MK adhesion, but inhibits PPF by collagen receptor GPVI. By immunoblot analyses we identified that the signaling events underyling this inhibition are different from those in platelet activation at the Src family kinase level. Second, we determined the degree of MK-ECM interaction in situ using confocal laser scanning microscopy of four-color IF-stained femora and spleen sections. In transgenic mouse models lacking either of the two major collagen receptors we could show that these mice have an impaired association of MKs to collagens in the BM, while the MK count in spleen increased threefold. This might contribute to the overall unaltered platelet counts in collagen receptor-deficient mice. In a third approach, we studied how the equilibrium of ECMP within BM is altered after irradiation. Collagen type IV and laminin-α5 subunits were selectively degraded at the sinusoids, while the matrix degrading protease MMP9 was upregulated in MKs. Platelet numbers decreased and platelets became hyporesponsive towards agonists, especially those for GPVI activation. Taken together, the results indicate that MK-ECM interaction differs substantially from the well-known platelet-ECM signaling. Future work should further elucidate how ECMP can be targeted to ameliorate the platelet production and function defects, especially in patients after BM irradiation.},
  subject      = {Knochenmark},
  language  = {en}
}
@phdthesis{Mueller2017,
  author    = {M{\"u}ller, Stephanie},
  title     = {Plant thermotolerance: The role of heat stress-induced triacylglycerols in \(Arabidopsis\) \(thaliana\)},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-152829},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {Plants are exposed to high temperature, especially during hot summer days. Temperatures are typically lowest in the morning and reach a maximum in the afternoon. Plants can tolerate and survive short-term heat stress even on hot summer days. A. thaliana seedlings have been reported to tolerate higher temperatures for different time periods, a phenomenon that has been termed basal thermotolerance. In addition, plants have the inherent capacity to acclimate to otherwise lethal temperatures. Arabidopsis thaliana seedlings acclimate at moderately elevated temperatures between 32-38° C. During heat acclimation, a genetically programmed heat shock response (HSR) is triggered that is characterized by a rapid activation of heat shock transcription factors (HSFs), which trigger a massive accumulation of heat shock proteins that are chiefly involved in protein folding and protection. Although the HSF-triggered heat-shock response is well characterized, little is known about the metabolic adjustments during heat stress. The aim of this work was to get more insight into heat-responsive metabolism and its importance for thermotolerance. In order to identify the response of metabolites to elevated temperatures, global metabolite profiles of heat-acclimated and control seedlings were compared. Untargeted metabolite analyses revealed that levels of polyunsaturated triacylglycerols (TG) rapidly increase during heat acclimation. TG accumulation was found to be temperature-dependent in a temperature range from 32-50° C (optimum at 42° C). Heat-induced TG accumulation was localized in extra-chloroplastic compartments by chloroplast isolation as well as by fluorescence microscopy of A. thaliana cell cultures. Analysis of mutants deficient in all four HSFA1 master regulator genes or the HSFA2 gene revealed that TG accumulation occurred independently to HSF. Moreover, the TG response was not limited to heat stress since drought and salt stress (but not short-term osmotic, cold and high light stress) also triggered an accumulation of TGs. In order to reveal the origin of TG synthesis, lipid analysis was carried out. Heat-induced accumulation of TGs does not derive from massive de novo fatty acid (FA) synthesis. On the other hand, lipidomic analyses of A. thaliana seedlings indicated that polyunsaturated FA from thylakoid galactolipids are incorporated into cytosolic TGs during heat stress. This was verified by lipidomic analyses of A. thaliana fad7/8 transgenic seedlings, which displayed altered FA compositions of plastidic lipids. In addition, wild type A. thaliana seedlings displayed a rapid conversion of plastidic monogalactosyldiacylglycerols (MGDGs) into oligogalactolipids, acylated MGDGs and diacylglycerols (DGs). For TG synthesis, DG requires a FA from the acyl CoA pool or phosphatidylcholine (PC). Seedlings deficient in phospholipid:diacylglycerol acyltransferase1 (PDAT1) were unable to accumulate TGs following heat stress; thus PC appears to be the major FA donor for TGs during heat treatment. These results suggest that TG and oligogalactolipid accumulation during heat stress is driven by post-translationally regulated plastid lipid metabolism. TG accumulation following heat stress was found to increase basal thermotolerance. Pdat1 mutant seedlings were more sensitive to severe heat stress without prior acclimatization, as revealed by a more dramatic decline of the maximum efficiency of PSII and lower survival rate compared to wild type seedlings. In contrast, tgd1 mutants over-accumulating TGs and oligogalactolipids displayed a higher basal thermotolerance compared to wild type seedlings. These results therefore suggest that accumulation of TGs increases thermotolerance in addition to the genetically encoded heat shock response.},
  subject      = {Triglyceride},
  language  = {en}
}
@phdthesis{Lagler2017,
  author    = {Lagler, Charlotte},
  title     = {Analyse von BMP2 und BMP2-Derivaten der TGF-β-Familie als potentielles Therapeutikum im Multiplen Myelom},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-155553},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {Diese Dissertation analysiert BMP2 und BMP2-Derivate als neue therapeutische Strategien f{\"u}r die Behandlung des Multiplen Myeloms (MM). Das MM ist eine maligne neoplastische Erkrankung des Knochenmarks mit Plasmazellvermehrung und erh{\"o}hten Leveln an Aktivin A im Blutserum, wobei eines der Hauptsymptome das Auftreten von schmerzvollen Osteolysen ist. In den letzten Jahren r{\"u}ckte Aktivin-A als interessantes Target zur Behandlung des Multiplen Myeloms in den Vordergrund. Die Reduzierung der Aktivin-A Level durch decoy-Rezeptoren f{\"u}hrte zu einer signifikanten Verbesserung der Osteolysen und einem reduzierten Proliferationsverhalten der neoplastischen B-Zellen, sowohl im Tierexperiment als auch in Studien der klinischen Phase II. Die Aktivin-A-Antagonisierung ist somit ein neuer und vielversprechender Ansatz in der Therapie des Multiplen Myeloms. Das Bone Morphogenetic Protein 2 ist aufgrund seiner molekularen und biologischen Eigenschaften ein interessantes Target f{\"u}r die Therapie des Multiplen Myeloms. Es ist auf molekularer Ebene ein Aktivin-A-Antagonist, besitzt aber auch osteoinduktives Potential und apoptotische bzw. anti-proliferative Eigenschaften auf neoplastische B-Zellen. Da die in der Literatur bereits beschriebenen, durch Mitglieder der TGF-β-Familie induzierten Apoptosemechanismen, noch nicht genauer untersucht waren, wurde in dieser Arbeit die BMP2-induzierte Apoptose in 10 unterschiedlichen humanen MM-Zellen analysiert. Erstens konnte dabei nachgewiesen werden, dass 7 von 10 Zelllinien nicht BMP2-responsiv waren. Eine genauere Untersuchung ergab, dass neben der Expression spezifischer BMP-Rezeptoren auch die Expression von inhibitorischen Smad-Proteinen {\"u}ber die BMP2-Responsivit{\"a}t entscheidet. Zweitens zeigte die genauere Analyse der Apoptosemechanismen, dass entgegen der in der Literatur publizierten Ergebnisse, BMP2 keine apoptotische Wirkung auf die von uns untersuchten Zelllinien hat. Mehrere verschieden durchgef{\"u}hrte Experimente, u.a. die Verwendung von spezifischen Inhibitoren des programmierten Zelltodes, unterst{\"u}tzen dieses Ergebnis und klassifizieren BMP2 als einen rein anti-proliferativen Faktor. Der letzte Teil der Arbeit befasst sich mit der Analyse von potentiellen Aktivin-A-Antagonisten in Form verschiedener BMP2- und GDF5-Derivate und inwiefern sie sich zum Einsatz in der Therapie des Multiplen Myeloms eignen. Die unterschiedlichen Eigenschaften der einzelnen Mutanten wurden in verschiedenen Zellsystemen getestet. So konnte aufgezeigt werden, dass neben einer erh{\"o}hten biologischen Aktivit{\"a}t in Form eines gesteigerten osteoinduktiven und anti-proliferativen Potentials auf neoplastische B-Zellen (Superagonisten), sich die verschiedenen Derivate als Super-Antagonisten zu Aktivin A eignen und damit unterschiedlichen Anspr{\"u}chen der adjuvanten Therapie im Multiplen Myelom gerecht werden.},
  subject      = {BMP},
  language  = {de}
}
@phdthesis{Schwab2017,
  author    = {Schwab, Andrea},
  title     = {Development of an osteochondral cartilage defect model},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-155617},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {The limited intrinsic self-healing capability of articular cartilage requires treatment of cartilage defects. Material assisted and cell based therapies are in clinical practice but tend to result in formation of mechanical inferior fibro-cartilage in long term follow up. If a lesion has not been properly restored degenerative diseases are diagnosed as late sequela causing pain and loss in morbidity. Complex three dimensional tissue models mimicking physiological situation allow investigation of cartilage metabolism and mechanisms involved in repair. A standardized and reproducible model cultured under controllable conditions ex vivo to maintain tissue properties is of relevance for comparable studies. Topic of this thesis was the establishment of an cartilage defect model that allows for testing novel biomaterials and investigate the effect of defined defect depths on formation of repair tissue. In part I an ex vivo osteochondral defect model was established based on isolation of porcine osteochondral explants (OCE) from medial condyles, 8 mm in diameter and 5 mm in height. Full thickness cartilage defects with 1 mm to 4 mm in diameter were created to define ex vivo cartilage critical size after 28 days culture with custom developed static culture device. In part II of this thesis hydrogel materials, namely collagen I isolated from rat tail, commercially available fibrin glue, matrix-metalloproteinase clevable poly(ethylene glycol) polymerized with heparin (starPEGh), methacrylated poly(N-(2-hydroxypropyl) methacrylamide mono-dilactate-poly(ethylene glycol) triblock copolymer/methacrylated hyaluronic acid (MP/HA), thiol functionalized HA/allyl functionalized poly(glycidol) (P(AGE/G)-HA-SH), were tested cell free and chondrocyte loaded (20 mio/ml) as implant in 4 mm cartilage defects to investigate cartilage regeneration. Reproducible chondral defects, 8 mm in diameter and 1 mm in height, were generated with an artificial tissue cutter (ARTcut®) to investigate effect of defect depth on defect regeneration in part III. In all approaches OCE were analyzed by Safranin-O staining to visualize proteoglycans in cartilage and/or hydrogels. Immuno-histological and -fluorescent stainings (aggrecan, collagen II, VI and X, proCollagen I, SOX9, RUNX2), gene expression analysis (aggrecan, collagen II and X, SOX9, RUNX2) of chondrocyte loaded hydrogels (part II) and proteoglycan and DNA content (Part I \& II) were performed for detailed analysis of cartilage regeneration. Part I: The development of custom made static culture device, consisting of inserts in which OCE is fixed and deep well plate, allowed tissue specific media supply without supplementation of TGF � . Critical size diameter was defined to be 4 mm. Part II: Biomaterials revealed differences in cartilage regeneration. Collagen I and fibrin glue showed presence of cells migrated from OCE into cell free hydrogels with indication of fibrous tissue formation by presence of proCollagen I. In chondrocyte loaded study cartilage matrix proteins aggrecan, collagen II and VI and transcription factor SOX9 were detected after ex vivo culture throughout the two natural hydrogels collagen I and fibrin glue whereas markers were localized in pericellular matrix in starPEGh. Weak stainings resulted for MP/HA and P(AGE/G)-HA-SH in some cell clusters. Gene expression data and proteoglycan quantification supported histological findings with tendency of hypertrophy indicated by upregulation of collagen X and RunX2 in MP/HA and P(AGE/G)-HA-SH. Part III: In life-dead stainings recruitment of cells from OCE into empty or cell free collagen I treated chondral defects was seen. Separated and tissue specific media supply is critical to maintain ECM composition in cartilage. Presence of OCE stimulates cartilage matrix synthesis in chondrocyte loaded collagen I hydrogel and reduces hypertrophy compared to free swelling conditions and pellet cultures. Differences in cartilage repair tissue formation resulted in preference of natural derived polymers compared to synthetic based materials. The ex vivo cartilage defect model represents a platform for testing novel hydrogels as cartilage materials, but also to investigate the effect of cell seeding densities, cell gradients, cell co-cultures on defect regeneration dependent on defect depth. The separated media compartments allow for systematic analysis of pharmaceutics, media components or inflammatory cytokines on bone and cartilage metabolism and matrix stability.},
  subject      = {Hyaliner Knorpel},
  language  = {en}
}
@phdthesis{Boeck2018,
  author    = {B{\"o}ck, Thomas},
  title     = {Multifunctional Hyaluronic Acid / Poly(glycidol) Hydrogels for Cartilage Regeneration Using Mesenchymal Stromal Cells},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-155345},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Improved treatment options for the degenerative joint disease osteoarthritis (OA) are of major interest, since OA is one of the main sources of disability, pain, and socioeconomic burden worldwide [202]. According to epidemiological data, already 27 million people suffer from OA in the US [23]. Moreover, the WHO expects OA to be the fourth most common cause of disability in 2020 [203], illustrating the need for effective and long-lasting therapy options of severe cartilage defects. Despite numerous clinically available products for the treatment of cartilage defects [62], the development of more cartilage-specific materials is still at the beginning. Hyaluronic acid (HA) is a major component of the cartilaginous extracellular matrix (ECM) and inherently creates a cell-friendly niche by providing cell attachment and migration sites. Furthermore, it is known that the functional groups of HA are well suited for chemical modification. These characteristics render HA an attractive material for hydrogel-based tissue engineering approaches. Poly(glycidol) (PG) as chemical crosslinker basically features similar chemical characteristics as the widely used poly(ethylene glycol) (PEG), but provides additional side groups at each repeating unit that can be further chemically functionalized. With the introduction of PG as multifunctional crosslinker for HA gels, a higher cross-linking density and, accordingly, a greater potential for biomimetic functionalization may be achieved. However, despite the mentioned potential benefits, PG has not been used for cartilage regeneration approaches so far. The initial aim of the study was to set up and optimize a HA-based hydrogel for the chondrogenic differentiation of mesenchymal stromal cells (MSCs), using different amounts and variations of cross-linkers. Therefore, the hydrogel composition was optimized by the utilization of different PEG diacrylate (PEGDA) concentrations to cross-link thiol-modified HA (Glycosil, HA-SH) via Michael addition. We aimed to generate volumestable scaffolds that simultaneously enable a maximum of ECM deposition. Histological and biochemical analysis showed 0.4\% PEGDA as the most suitable concentration for these requirements (Section 5.1.2). In order to evaluate the impact of a differently designed cross-linker on MSC chondrogenesis, HA-SH was cross-linked with PEGTA (0.6\%) and compared to PEGDA (0.4\%) in a next step. Following this, acrylated PG (PG-Acr) as multifunctional cross-linker alternative to acrylated PEG was evaluated. It provides around five times more functional groups when utilized in PG-Acr (0.6\%) HA-SH hydrogels compared to PEGTA (0.6\%) HA-SH hydrogels, thus enabling higher degrees of biomimetic functionalization. Determination of cartilage-specific ECM components showed no substantial differences between both cross-linkers while the deposition of cartilaginous matrix appeared more homogeneous in HA-SH PG-Acr gels. Taken together, we were able to successfully increase the possibilities for biomimetic functionalization in the developed HA-SH hydrogel system by the introduction of PG-Acr as cross-linker without negatively affecting MSC chondrogenesis (Section 5.1.3). The next part of this thesis focused extensively on the biomimetic functionalization of PG-Acr (0.6\%) cross-linked HA-SH hydrogels. Here, either biomimetic peptides or a chondrogenic growth factor were covalently bound into the hydrogels. Interestingly, the incorporation of a N-cadherin mimetic (HAV), a collagen type II binding (KLER), or a cell adhesion-mediating peptide (RGD) yielded no improvement of MSC chondrogenesis. For instance, the covalent binding of 2.5mM HAV changed morphology of cell nuclei and reduced GAG production while the incorporation of 1.0mM RGD impaired collagen production. These findings may be attributed to the already supportive conditions of the employed HA-based hydrogels for chondrogenic differentiation. Most of the previous studies reporting positive peptide effects on chondrogenesis have been carried out in less supportive PEG hydrogels or in significantly stiffer MeHA-based hydrogels [99, 101, 160]. Thus, the incorporation of peptides may be more important under unfavorable conditions while inert gel systems may be useful for studying single peptide effects (Section 5.2.1). The chondrogenic factor transforming growth factor beta 1 (TGF-b1) served as an example for growth factor binding to PG-Acr. The utilization of covalently bound TGF-b1 may thereby help overcome the need for repeated administration of TGF-b1 in in vivo applications, which may be an advantage for potential clinical application. Thus, the effect of covalently incorporated TGF-b1 was compared to the effect of the same amount of TGF-b1 without covalent binding (100nM TGF-b1) on MSC chondrogenesis. It was successfully demonstrated that covalent incorporation of TGF-b1 had a significant positive effect in a dose-dependent manner. Chondrogenesis of MSCs in hydrogels with covalently bound TGF-b1 showed enhanced levels of chondrogenesis compared to hydrogels into which TGF-b1 was merely mixed, as shown by stronger staining for GAGs, total collagen, aggrecan and collagen type II. Biochemical evaluation of GAG and collagen amounts, as well as Western blot analysis confirmed the histological results. Furthermore, the positive effect of covalently bound TGF-b1 was shown by increased expression of chondrogenic marker genes COL2A1, ACAN and SOX9. In summary, covalent growth factor incorporation utilizing PG-Acr as cross-linker demonstrated significant positive effects on chondrogenic differentiation of MSCs (Section 5.2.2). In general, PG-Acr cross-linked HA hydrogels generated by Michael addition represent a versatile hydrogel platform due to their high degree of acrylate functionality. These hydrogels may further offer the opportunity to combine several biological modifications, such as the incorporation of biomimetic peptides together with growth factors, within one cell carrier. A proof-of-principle experiment demonstrated the suitability of pure PG gels for studying single peptide effects. Here, the hydrogels were generated by the utilization of thiol-ene-click reaction. In this setting, without the supportive background of hyaluronic acid, MSCs showed enhanced chondrogenic differentiation in response to the incorporation of 1.0mM HAV. This was demonstrated by staining for GAGs, the cartilage-specific ECM molecules aggrecan and type II collagen, and by increased GAG and total collagen amounts shown by biochemical analysis. Thus, pure PG gels exhibit the potential to study the effects and interplay of peptides and growth factors in a highly modifiable, bioinert hydrogel environment. The last section of the thesis was carried out as part of the EU project HydroZONES that aims to develop and generate zonal constructs. The importance of zonal organization has attracted increased attention in the last years [127, 128], however, it is still underrepresented in tissue engineering approaches so far. Thus, the feasibility of zonal distribution of cells in a scaffold combining two differently composed hydrogels was investigated. A HA-SH(FMZ) containing bottom layer was generated and a pure PG top layer was subsequently cast on top of it, utilizing both times thiol-ene-click reaction. Indeed, stable, hierarchical constructs were generated that allowed encapsulated MSCs to differentiate chondrogenically in both zones as shown by staining for GAGs and collagen type II, and by quantification of GAG amount. Thus, the feasibility of differently composed zonal hydrogels utilizing PG as a main component was successfully demonstrated (Section 5.4). With the first-time utilization and evaluation of PG-Acr as versatile multifunctional cross-linker for the preparation of Michael addition-generated HA-SH hydrogels in the context of cartilage tissue engineering, a highly modifiable HA-based hydrogel system was introduced. It may be used in future studies as an easily applicable and versatile toolbox for the generation of biomimetically functionalized hydrogels for cell-based cartilage regeneration. The introduction of reinforcement structures to enhance mechanical resistance may thereby further increase the potential of this system for clinical applications. Additionally, it was also demonstrated that thiol-ene clickable hydrogels can be used for the generation of cell-laden, pure PG gels or for the generation of more complex, coherent zonal constructs. Furthermore, thiol-ene clickable PG hydrogels have already been further modified and successfully been used in 3D bioprinting experiments [204]. 3D bioprinting, as part of the evolving biofabrication field [205], offers the possibilities to generate complex and hierarchical structures, and to exactly position defined layers, yet at the same time alters the requirements for the utilized hydrogels [159, 206-209]. Since a robust chondrogenesis of MSCs was demonstrated in the thiol-ene clickable hydrogel systems, they may serve as a basis for the development of hydrogels as so called bioinks which may be utilized in more sophisticated biofabrication processes.},
  subject      = {Hyalurons{\"a}ure},
  language  = {en}
}
@phdthesis{Hollmann2017,
  author    = {Hollmann, Claudia Beate},
  title     = {Einfluss der sauren Sphingomyelinase auf anti-virale T-Zellantworten im Masernvirus-Infektionsmodell},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-153807},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {Die saure Sphingomyelinase (Asm), ein Enzym des Sphingolipidmetabolismus, spaltet Sphingomyelin zu Ceramid und Phosopocholin. Aktiviert wird die Asm unter anderem durch Stimulation des CD28 Rezeptors. CD28 Signale werden auch f{\"u}r die Aktivierung von konventionellen T-Zellen (Tconv) und f{\"u}r die Kostimulation ben{\"o}tigt und sind essentiell f{\"u}r die Differenzierung von regulatorischen T-Zellen (Treg) im Thymus und deren Erhalt in der Peripherie. Wir konnten zeigen, dass sich Tconv und Treg Zellen hinsichtlich der Asm unterscheiden. Treg haben eine h{\"o}here "basale" Asm Aktivit{\"a}t, widergespiegelt im h{\"o}heren Ceramidgehalt und haben eine niedrigere Lipidordnung als Tconv Zellen. Die Abwesenheit der Asm in defizienten M{\"a}usen bewirkt einen relativen Anstieg der Treg-Frequenz innerhalb der CD4+ T-Zellen. Außerdem f{\"u}hrt die Asm-Defizienz in Treg Zellen zu einer erh{\"o}hten Umsatzrate des immunsupprimierenden Molek{\"u}ls CTLA-4 und zu einer verst{\"a}rkten Suppressivit{\"a}t von Treg Zellen aus Asm-/- M{\"a}usen gegen{\"u}ber Wildtyp Zellen. Ein Anstieg in der Treg-Frequenz, {\"a}quivalent zur genetischen Defizienz, kann auch durch Inhibition der Asm, d. h. durch Wirkstoffe wie Amitriptylin und Desipramin erreicht werden. Es konnte gezeigt werden, dass die Inhibitorbehandlung die absolute Anzahl der Tconv Zellen selektiv verringert, da Treg Zellen gegen{\"u}ber dem Asm Inhibitor-induzierten Zelltod resistenter sind. Mechanistisch erkl{\"a}rbar sind die Unterschiede gegen{\"u}ber den proapoptotischen Inhibitoreffekten zwischen Tconv und Treg Zellen dadurch, dass Treg Zellen durch die Anwesenheit von IL-2 gesch{\"u}tzt sind. In Abwesenheit von IL-2 sterben die Treg Zellen ebenfalls. Die gezielte Ver{\"a}nderung des Verh{\"a}ltnisses von Treg zu Tconv durch den Einsatz von Asm-inhibitorischen Medikamenten kann hilfreich bei der therapeutischen Behandlung von inflammatorischen- und Autoimmunerkrankungen sein. Inwiefern die Asm f{\"u}r die Funktion von T-Zellen in der anti-viralen Immunantwort entscheidend ist, wurde im Masernvirus-Infektionsmodell n{\"a}her untersucht. In Asm-/- M{\"a}usen und Amitriptylin-behandelten M{\"a}usen konnte gezeigt werden, dass in Abwesenheit der Asm die Kontrolle der Masernvirusinfektion verschlechtert ist. Treg sind auch hier von entscheidender Bedeutung, da die Asm-abh{\"a}ngige, verst{\"a}rkte Masernvirusinfektion bei Fehlen der Asm nur in Gegenwart von Treg auftritt. In der akuten Phase gibt es in Asm-/- M{\"a}usen weniger masernvirusspezifische T-Zellen und dadurch eine verringerte Beseitigung der Viruslast. In der chronischen Phase ist die Anzahl masernvirusspezifischer T-Zellen zwischen WT und Asm-/- M{\"a}usen vergleichbar. In Letzteren ist allerdings die Anzahl und Frequenz von T-Zellen im Gehirn infizierter M{\"a}use noch deutlich erh{\"o}ht, was die verst{\"a}rkte Maserninfektion widerspiegelt. Zusammenfassend zeigt sich, dass die Asm die Funktion von Treg moduliert und einen Einfluss auf das Verh{\"a}ltnis von Tconv und Treg zueinander hat. Im Masernvirus-Infektionsmodell kann die Ver{\"a}nderung des Tconv zu Treg Verh{\"a}ltnisses in Abwesenheit der Asm urs{\"a}chlich f{\"u}r die verringerte Viruskontrolle sein. Die Asm Inhibitor-induzierte Treg-Aktivierung und die Beeinflussung des Treg zu Tconv Verh{\"a}ltnisses k{\"o}nnen wiederum f{\"u}r therapeutische Zwecke genutzt werden, wie beispielsweise bei Multipler Sklerose und Rheumatoider Arthritis.},
  subject      = {saure Sphingomyelinase},
  language  = {de}
}
@phdthesis{Karl2017,
  author    = {Karl, Franziska},
  title     = {The role of miR-21 in the pathophysiology of neuropathic pain using the model of B7-H1 knockout mice},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-156004},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {The impact of microRNA (miRNA) as key players in the regulation of immune and neuronal gene expression and their role as master switches in the pathophysiology of neuropathic pain is increasingly recognized. miR-21 is a promising candidate that could be linked to the immune and the nociceptive system. To further investigate the pathophysiological role of miR-21 in neuropathic pain, we assesed mice deficient of B7 homolog 1 (B7-H1 ko), a protein with suppressive effect on inflammatory responses. B7-H1 ko mice and wildtype littermates (WT) of three different age-groups, young (8 weeks), middle-aged (6 months), and old (12 months) received a spared nerve injury (SNI). Thermal withdrawal latencies and mechanical withdrawal thresholds were determined. Further, we investigated anxiety-, depression-like and cognitive behavior. Quantitative real time PCR was used to determine miR-21 relative expression in peripheral nerves, dorsal root ganglia and white blood cells (WBC) at distinct time points after SNI. Na{\"i}ve B7-H1 ko mice showed mechanical hyposensitivity with increasing age. Young and middle-aged B7-H1 ko mice displayed lower mechanical withdrawal thresholds compared to WT mice. From day three after SNI both genotypes developed mechanical and heat hypersensitivity, without intergroup differences. As supported by the results of three behavioral tests, no relevant differences were found for anxiety-like behavior after SNI in B7-H1 ko and WT mice. Also, there was no indication of depression-like behavior after SNI or any effect of SNI on cognition in both genotypes. The injured nerves of B7-H1 ko and WT mice showed higher miR-21 expression and invasion of macrophages and T cells 7 days after SNI without intergroup differences. Perineurial miR-21 inhibitor injection reversed SNI-induced mechanical and heat hypersensitivity in old B7-H1 ko and WT mice. This study reveals that reduced mechanical thresholds and heat withdrawal latencies are associated with miR-21 induction in the tibial and common peroneal nerve after SNI, which can be reversed by perineurial injection of a miR-21 inhibitor. Contrary to expectations, miR-21 expression levels were not higher in B7-H1 ko compared to WT mice. Thus, the B7-H1 ko mouse may be of minor importance for the study of miR-21 related pain. However, these results spot the contribution of miR-21 in the pathophysiology of neuropathic pain and emphasize the crucial role of miRNA in the regulation of neuronal and immune circuits that contribute to neuropathic pain.},
  subject      = {neuropathic pain},
  language  = {en}
}
@phdthesis{Quenzer2018,
  author    = {Quenzer, Anne},
  title     = {Der antiproliferative Effekt des Multidrug resistance-Protein 1 (MRP1)-Inhibitors Reversan und der Laktatdehydrogenase (LDH)-Inhibitoren Natriumoxamat und Galloflavin an kolorektalen Karzinomzellen bei tumorphysiologischen Sauerstoffkonzentrationen},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-156051},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Ziel der vorliegenden Arbeit waren pharmakologische Untersuchungen zum antiproliferativen Effekt der beiden Laktatdehydrogenase (LDH)-Inhibitoren Natriumoxamat und Galloflavin sowie des MRP1-Inhibitors Reversan einzeln und in Kombination bei verschiedenen Sauerstoffkonzentrationen in vitro zu untersuchen. Zus{\"a}tzlich wurde der antiproliferative Effekt der drei Inhibitoren mit dem antiproliferativen Effekt von 5-FU verglichen. Das Konzept zu dieser Arbeit basiert auf Gemeinsamkeiten zwischen LDH und MRP1 in malignen Zellen. Eine ist, dass beide Molek{\"u}le von zahlreichen Tumoren {\"u}berexprimiert werden. Weiter sind beide an der Ausbildung von Chemoresistenz beteiligt und beide werden auch in Hypoxie exprimiert. Zudem wird das f{\"u}r die Funktion von MRP1 notwendige ATP in malignen Zellen haupts{\"a}chlich mit der hyperaktiven Glykoloyse gebildet, deren Stoffumsatz auch von der LDH-Aktivit{\"a}t abh{\"a}ngig ist. Eine kombinierte Inhibition beider Zielstrukturen scheint somit geeignet zu sein, um die Proliferation maligner Zellen gezielt zu hemmen. Da in großen Teilen solider Tumoren hypoxische bzw. anoxische Bedingungen vorherrschen, wurde die Wirksamkeit der drei Inhibitoren auch bei 5 \% und 1 \% Sauerstoff, die als tumorphysiologisch gelten, untersucht. Die wichtigsten Ergebnisse aus dieser Arbeit sind, dass die beiden LDH-Inhibitoren Natriumoxamat und Galloflavin und der MRP1-Inhibitor Reversan einen antiproliferativen Effekt bei kolorektalen Karzinomzellen ausl{\"o}sen, der auch f{\"u}r tumorphysiologische Sauerstoffkonzentrationen nachzuweisen war. So verringerte sich durch Natriumoxamat bzw. Galloflavin der Anteil vitaler Zellen um bis zu 45 \% und durch Reversan um bis zu 60 \% bei 5 \% und 1 \% Sauerstoff im Vergleich zur unbehandelten Kontrolle. Auch unterschiedliche Kombination aus Natriumoxamat, Galloflavin und Reversan f{\"u}hrten zu einer Steigerung des antiproliferativen Effektes, der auch immer bei tumorphysiologischen Konzentrationen nachzuweisen war. Den st{\"a}rksten antiproli-ferativen Effekt wies die Dreifachkombination aus Galloflavin, Natriumoxamat und Reversan auf. So verringerte sich der Anteil vitaler Zellen bei 1 \% Sauerstoff durch diese Kombination auf bis zu 28 \% bei vier der f{\"u}nf kolorektalen Karzinomzelllinien. Die Dreifachkombination wies einen gleichstarken bzw. st{\"a}rkeren antiproliferativen Effekt auf als das Chemotherapeutikum 5-FU und zwar ebenfalls bei 5 \% und 1 \% Sauerstoff. Die Ergebnisse der vorliegenden Arbeit zum antiproliferativen Effekt von Natriumoxamat, Galloflavin (beides LDH-Inhibitoren) und Reversan (MRP1-Inhibitor) in vitro lassen den Schluss zu, dass das Konzept der Arbeit, einen antiproliferativen Effekt auch bei tumorphysiologischen Sauerstoffkonzentrationen zu induzieren, grunds{\"a}tzlich best{\"a}tigt wurde. Auch l{\"o}ste die gemeinsame Hemmung von LDH und MRP1 einen teilweise st{\"a}rkeren antiproliferativen Effekt aus als 5-FU. Weitere Untersuchungen sind aber ohne Frage n{\"o}tig, um die molekularen Interaktion zwischen LDH und MRP1 sowie ihrer Inhibition im Detail zu verstehen.},
  subject      = {Lactatdehydrogenase},
  language  = {de}
}
@phdthesis{Ziegenhals2018,
  author    = {Ziegenhals, Thomas},
  title     = {The role of the miR-26 family in neurogenesis},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-156395},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {For the differentiation of a embryonic stem cells (ESCs) to neuronal cells (NCs) a complex and coordinated gene regulation program is needed. One important control element for neuronal differentiation is the repressor element 1 silencing transcription factor (REST) complex, which represses neuronal gene expression in non-neuronal cells. Crucial effector proteins of the REST complex are small phosphatases such as the CTDSPs (C-terminal domain small phosphatases) that regulate polymerase II activity by dephosphorylating the C-terminal domain of the polymerase, thereby repressing target genes. The stepwise inactivation of REST, including the CTDSPs, leads to the induction of a neuron-specific gene program, which ultimately induces the formation of neurons. The spatio-temporal control of REST and its effector components is therefore a crucial step for neurogenesis. In zebrafish it was shown that the REST-associated CTDSP2 is negatively regulated by the micro RNA (miR) -26b. Interestingly, the miR-26b is encoded in an intron of the primary transcript of CTDSP2. This gives the fundament of an intrinsic regulatory negative feedback loop, which is essential for the proceeding of neurogenesis. This feedback loop is active during neurogenesis, but inactive in non-neuronal cells. The reason for this is that the maturation of the precursor miR (pre-miR) to the mature miR-26 is arrested in non neuronal cells, but not in neurons. As only mature miRs are actively repressing genes, the regulation of miR-26 processing is an essential step in neurogenesis. In this study, the molecular basis of miR-26 processing regulation in the context of neurogenesis was addressed. The mature miR is processed from two larger precursors: First the primary transcript is cleaved by the enzyme DROSHA in the nucleus to form the pre-miR. The pre-miR is exported from the nucleus and processed further through the enzyme DICER to yield the mature miR. The mature miR can regulate gene expression in association with the RNA-induced silencing complex (RISC). Multiple different scenarios in which miR processing was regulated were proposed and experimentally tested. Microinjection studies using Xenopus leavis oocytes showed that slowdown or blockage of the nucleo-cytoplasmic transport are not the reason for delayed pre-miR-26 processing. Moreover, in vitro and in vivo miR-processing assays showed that maturation is most likely regulated through a in trans acting factor, which blocks processing in non neuronal cells. Through RNA affinity chromatographic assays using zebrafish and murine lysates I was able to isolate and identify proteins that interact specifically with pre-miR-26 and could by this influence its biogenesis. Potential candidates are FMRP/FXR1/2, ZNF346 and Eral1, whose functional characterisation in the context of miR-biogenesis could now be addressed. The second part of my thesis was executed in close colaboration with the laboratory of Prof. Albrecht M{\"u}ller. The principal question was addressed how miR-26 influences neuronal gene expression and which genes are primarily affected. This research question could be addressed by using a cell culture model system, which mimics ex vivo the differentiation of ESCs to NCs via neuronal progenitor. For the functional analysis of miR-26 knock out cell lines were generated by the CRISPR/Cas9 technology. miR-26 deficient ESC keep their pluripotent state and are able to develop NPC, but show major impairment in differentiating to NCs. Through RNA deep sequencing the miR-26 induced transcriptome differences could be analysed. On the level of mRNAs it could be shown, that the expression of neuronal gene is downregulated in miR-26 deficient NCs. Interestingly, the deletion of miR-26 leads to selectively decreased levels of miRs, which on one hand regulate the REST complex and on the other hand are under transcriptional control by REST themself. This data and the discovery that induction of miR-26 leads to enrichment of other REST regulating miRs indicates that miR-26 initiates neurogenesis through stepwise inactivation of the REST complex.},
  subject      = {miRNS},
  language  = {en}
}
@phdthesis{Frank2019,
  author    = {Frank, Erik Thomas},
  title     = {Behavioral adaptations in the foraging behaviour of \(Megaponera\) \(analis\)},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-156544},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {An efficient foraging strategy is one of the most important traits for the fitness of animals. The theory of optimal foraging tries to predict foraging behaviour through the overarching question: how animals should forage so as to minimize costs while maximizing profits? Social insects, having occupied nearly every natural niche through widely different strategies, offer themselves as an ideal group to study how well optimal foraging theory can explain their behaviour and success. Specialization often leads to unique adaptations in morphology and behaviour. I therefore decided to investigate the behaviour of Megaponera analis. This ponerine ant species is specialized on hunting only termites of the subfamily Macrotermitinae at their foraging sites. Their foraging behaviour is regulated by a handful of individual scouts (10-20) that search for termite foraging sites before returning to the nest to recruit a large number of nestmates (200-500 ants). These ants then follow the scout in a column formation to the termites and after the hunt return together to the nest, these raids occur two to five times per day. Predators of highly defensive prey likely develop cost reducing adaptations. The evolutionary arms race between termites and ants led to various defensive mechanisms in termites, e.g. a caste specialized in fighting predators. As M. analis incurs high injury/mortality risks when preying on termites, some risk mitigating adaptations have evolved. I show that a unique rescue behaviour in M. analis, consisting of injured nestmates being carried back to the nest, reduces combat mortality. These injured ants "call for help" with pheromones present in their mandibular gland reservoirs. A model accounting for this rescue behaviour identifies the drivers favouring its evolution and estimates that rescuing allows for maintaining a 29\% larger colony size. Heavily injured ants that lost too many legs during the fight on the other hand are not helped. Interestingly, this was regulated not by the helper but by the uncooperativeness of the injured ant. I further observed treatment of the injury by nestmates inside the nest through intense allogrooming directly at the wound. Lack of treatment increased mortality from 10\% to 80\% within 24 hours, with the cause of death most likely being infections. Collective decision-making is one of the main mechanisms in social insects through which foraging is regulated. However, individual decision-making can also play an important role, depending on the type of foraging behaviour. In M. analis only a handful of individuals (the scouts) hold all the valuable information about foraging sites. I therefore looked at predictions made by optimal foraging theory to better understand the interplay between collective and individual decision-making in this obligate group-raiding predator. I found a clear positive relation between raid size and termite abundance at the foraging site. Furthermore, selectivity of the food source increased with distance. The confirmation of optimal foraging theory suggests that individual scouts must be the main driver behind raid size, choice and raiding behaviour. Therefore most central place foraging behaviours in M. analis were not achieved by collective decisions but rather by individual decisions of scout ants. Thus, 1\% of the colony (10-20 scouts) decided the fate and foraging efficiency of the remaining 99\%. Division of labour is one of the main reasons for the success of social insects. Worker polymorphism, age polyethism and work division in more primitive ants, like the ponerines, remain mostly unexplored though. Since M. analis specializes on a defensive prey, adaptations to reduce their foraging costs can be expected. I found that the work division, task allocation and column-formation during the hunt were much more sophisticated than was previously thought. The column-formation was remarkably stable, with the same ants resuming similar positions in subsequent raids and front ants even returning to their positions if displaced in the same raid. Most of the raid tasks were not executed by predetermined members of the raid but were filled out as need arose during the hunt, with a clear preference for larger ants to conduct most tasks. I show that specialization towards a highly defensive prey can lead to very unique adaptations in the foraging behaviour of a species. I explored experimentally the adaptive value of rescue behaviour focused on injured nestmates in social insects. This was not only limited to selective rescuing of lightly injured individuals by carrying them back (thus reducing predation risk) but moreover includes a differentiated treatment inside the nest. These observations will help to improve our understanding of the evolution of rescue behaviour in animals. I further show that most optimal foraging predictions are fulfilled and regulated by a handful of individuals in M. analis. Lastly, I propose that the continuous allometric size polymorphism in M. analis allows for greater flexibility in task allocation, necessary due to the unpredictability of task requirements in an irregular system such as hunting termites in groups. All of my observations help to further understand how a group-hunting predator should forage so as to minimize costs while maximizing profits.},
  subject      = {Stechameisen},
  language  = {en}
}
@phdthesis{Eltschkner2020,
  author    = {Eltschkner, Sandra},
  title     = {Targeting the Bacterial Fatty-Acid Synthesis Pathway: Towards the Development of Slow-Onset Inhibitors and the Characterisation of Protein-Protein Interactions},
  doi       = {10.25972/OPUS-15664},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-156643},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {A continuous arms race between the development of novel antibiotics and the evolution of corresponding resistance mechanisms in bacteria has been observed, since antibiotic agents like arsphenamines (e.g. Salvarsan, developed by Paul Ehrlich [1]), sulphonamides (e.g. Prontosil, Gerhard Domagk [2]) and penicillin (Alexander Fleming [3]) were first applied to effectively cure bacterial infections in the early 20th century. The rapid emergence of resistances in contrast to the currently lagging discovery of antibiotics displays a severe threat to human health. Some serious infectious diseases, such as tuberculosis or melioidosis, which were either thought to be an issue only in Third-World countries in case of tuberculosis, or regionally restricted with respect to melioidosis, are now on the rise to expand to other areas. In contrast, methicillin-resistant Staphylococcus aureus (MRSA) is already present in clinical setups all over the world and causes severe infections in immunocompromised patients. Thus, there is an urgent need for new and effective antimicrobial agents, which impair vital functions of the pathogen's metabolism. One central metabolic pathway is represented by the bacterial fatty-acid synthesis pathway (FAS II), which is essential for the synthesis of long and branched-chain fatty acids, as well as mycolic acids. These substances play a major role as modulating components of the properties of the most important protective barrier - the cell envelope. The integrity of the bacterial cell wall and the associated membrane(s) is crucial for cell growth and for protection against physical strain, intrusion of antibiotic agents and regulation of uptake of ions and other small molecules. Thus, this central pathway represents a promising target for antibiotic action against pathogens to combat infectious diseases. The last and rate-limiting step is catalysed by the trans-2-enoyl-ACP reductase (ENR) FabI or InhA (in mycobacteria), which has been demonstrated to be a valuable target for drug design and can be addressed, amongst others, by diphenyl ether (DPE) compounds, derived from triclosan (TCL) - the first one of this class which was discovered to bind to ENR enzymes [4, 5]. Based on this scaffold, inhibitors containing different combinations of substituents at crucial positions, as well as a novel type of substituent at position five were investigated regarding their binding behaviour towards the Burkholderia pseudomallei and Mycobacterium tuberculosis ENR enzymes bpFabI and InhA, respectively, by structural, kinetic and in-vivo experiments. Generally, substitution patterns modulate the association and dissociation velocities of the different ENR inhibitors in the context of the two-step slow-onset binding mechanism, which is observed for both enzymes. These alterations in the rapidity of complex formation and decomposition have a crucial impact on the residence time of a compound and hence, on the pharmacokinetic properties of potential drug candidates. For example, the substituents at the 2'-position of the DPE scaffold influence the ground- and transition state stability during the binding process to bpFabI, whereas 4'-substituents primarily alter the transition state [6]. The novel triazole group attached to the 5-position of the scaffold, targeting the hydrophobic part of the substrate-binding pocket in InhA, significantly enhances the energy barrier of the transition state of inhibitor binding [7] and decelerates the association- as well as the dissociation processes. Combinations with different substituents at the 2'-position can enhance or diminish this effect, e.g. by ground-state stabilisation, which will result in an increased residence time of the respective inhibitor on InhA. Further structural investigations carried out in this work, confirm the proposed binding mode of a customised saFabI inhibitor [8], carrying a pyridone moiety on the DPE scaffold to expand interactions with the protein environment. Structural and preliminary kinetic data confirm the binding of the same inhibitor to InhA in a related fashion. Comparisons with structures of the ENR inhibitor AFN-1252 [9] bound to ENR enzymes from other organisms, addressing a similar region as the pyridone-moiety of the DPE inhibitor, suggest that also the DPE inhibitor bears the potential to display binding to homologues of saFabI and InhA and may be optimised accordingly. Both of the newly investigated substituents, the pyridone moiety at the 4'-position as well as the 5-triazole substituent, provide a good starting point to modify the DPE scaffold also towards improved kinetic properties against ENR enzymes other than the herein studied and combining both groups on the DPE scaffold may have beneficial effects. The understanding of the underlying binding mechanism is a crucial factor to promote the dedicated design of inhibitors with superior pharmacokinetic characteristics. A second target for a structure-based drug-design approach is the interaction surface between ENR enzymes and the acyl-carrier protein (ACP), which delivers the growing acyl chain to each distinct enzyme of the dissociated FAS-II system and presumably recognises its respective interaction partner via electrostatic contacts. The interface between saACP and saFabI was investigated using different approaches including crosslinking experiments and the design of fusion constructs connecting the ACP and the FabI subunits via a flexible linker region of varying lengths and compositions. The crosslinking studies confirmed a set of residues to be part of the contact interface of a previously proposed complex model [10] and displayed high crosslinking efficiency of saACP to saFabI when mutated to cysteine residues. However, crystals of the complex obtained from either the single components, or of the fusion constructs usually displayed weak diffraction, which supports the assumption that complex formation is highly transient. To obtain ordered crystals for structural characterisation of the complex it is necessary to trap the complex in a fixed state, e.g. by a high-affinity substrate attached to ACP [11], which abolishes rapid complex dissociation. For this purpose, acyl-coupled long-residence time inhibitors might be a valuable tool to elucidate the detailed architecture of the ACP-FabI interface. This may provide a novel basis for the development of inhibitors that specifically target the FAS-II biosynthesis pathway.},
  subject      = {Fetts{\"a}urestoffwechsel},
  language  = {en}
}
@phdthesis{PompergebMueller2019,
  author    = {Pomper [geb. M{\"u}ller], Laura Dorothea},
  title     = {Unterschiede in Frontaler Kortex Oxygenierung in zweierlei Risikogruppen der Alzheimer Demenz},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-156757},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Die verbesserte medizinische Versorgung f{\"u}hrt zu einer zunehmenden Lebenserwartung unserer Gesellschaft. Damit steigt auch die sozio{\"o}konomische Relevanz neurodegenerativer Erkrankungen kontinuierlich. F{\"u}r die Alzheimer Demenz (AD), die dabei die h{\"a}ufigste Ursache darstellt, stehen bisher keine krankheitsmodifizierenden Behandlungsoptionen zur Verf{\"u}gung. Die lange pr{\"a}klinische Phase der Erkrankung birgt jedoch großes Potential f{\"u}r die Entwicklung neuer Behandlungsoptionen. Das Untersuchen von Risikogruppen ist f{\"u}r die Identifikation von Pr{\"a}diktoren einer sp{\"a}teren AD Manifestation von besonderem Interesse. In diesem Zusammenhang werden insbesondere das Vorliegen genetischer Risikokonstellationen, wie dem Apolipoprotein E (APOE) Ɛ4-Allel, sowie kognitiver Risikofaktoren, wie der „leichten kognitiven Beeintr{\"a}chtigung" (MCI), diskutiert. Die Identifikation pr{\"a}klinischer Aktivierungsunterschiede in relevanten Gehirnregionen von Risikogruppen kann als Basis f{\"u}r die Entwicklung neurofunktioneller Fr{\"u}herkennungs-Marker dienen. Der pr{\"a}frontale Kortex (PFC), welcher mit der Steuerung von Exekutivfunktionen assoziiert wird, hat sich in diesem Zusammenhang in bisherigen Studien als eine relevante Schl{\"u}sselregion manifestiert. Aufgrund der aufwendigen und kostenintensiven bildgebenden Untersuchungsmethoden, sind die genauen Prozesse jedoch noch unklar. Ziel der vorliegenden Arbeit war es daher, Unterschiede in der PFC Oxygenierung in zweierlei Risikogruppen der AD mit einer kosteng{\"u}nstigeren Bildgebungsmethode, der funktionellen Nahinfrarot Spektroskopie (fNIRS), zu untersuchen. Daf{\"u}r wurde in einem ersten Schritt, der Trailmaking Test (TMT), ein weitverbreiteter neuropsychologischer Test zur Erfassung exekutiver Funktionen, f{\"u}r fNIRS implementiert. Als Grundlage f{\"u}r die Untersuchung fr{\"u}hpathologischer Prozesse, wurden zun{\"a}chst gesunde Alterungsprozesse betrachtet. Der Vergleich von jungen und {\"a}lteren Probanden (n = 20 pro Gruppe) wies neben der Eignung der Testimplementierung f{\"u}r fNIRS auf eine spezifische bilaterale PFC Oxygenierung hin, welche bei jungen Probanden rechtshemisph{\"a}risch lateralisiert war. {\"A}ltere Probanden hingegen zeigten bei vergleichbaren Verhaltensdaten insgesamt mehr signifikante Kan{\"a}le sowie eine Abnahme der Lateralisierung. Dies kann als zus{\"a}tzlicher Bedarf an Ressourcen in gesunden Alterungsprozessen interpretiert werden. Im Rahmen der Hauptstudie wurden anschließend insgesamt 604 {\"a}ltere Probanden im Alter von 70 bis 76 Jahren untersucht. Zun{\"a}chst wurde die genetische Risikogruppe der Ɛ4-Allel-Tr{\"a}ger (n = 78) mit den neutralen Ɛ3-Allel-Tr{\"a}gern (n = 216) und den Tr{\"a}gern des als protektiv geltenden Ɛ2-Allels (n = 50) verglichen. Hierbei zeigte sich eine geringere Oxygenierung der Risikogruppe bei geringer Aufgabenschwierigkeit, w{\"a}hrend sich ein erh{\"o}hter Oxygenierungsanstieg im medialen PFC mit steigender Aufgabenschwierigkeit zeigte. Dies deutet auf einen erh{\"o}hten Bedarf an neuronalen Kontrollmechanismen der Risikogruppe zur Bew{\"a}ltigung der steigenden Aufgabenschwierigkeit hin. Die protektive Gruppe zeigte hingegen eine erh{\"o}hte Oxygenierung im ventralen PFC mit steigender Aufgabenschwierigkeit, was m{\"o}glicherweise auf einen pr{\"a}ventiven Effekt hindeuten k{\"o}nnte. Weiterf{\"u}hrend wurden MCI-Patienten mit gesunden Probanden (n = 57 pro Gruppe) hinsichtlich des kognitiven Risikofaktors verglichen. Hierbei zeigte sich ein punktuell reduzierter Oxygenierunganstieg der MCI Patienten mit steigender Aufgabenschwierigkeit vor allem im ventralen PFC bei ebenfalls stabiler Verhaltensleistung. Die gefundene Reduktion k{\"o}nnte ein Zeichen f{\"u}r eine aufgebrauchte kognitive Reserve sein, welche Einbußen auf Verhaltensebene voranzugehen scheint. Diese charakteristischen Unterschiede in den frontalen Oxygenierungsmustern von Risikogruppen (APOE, MCI) k{\"o}nnten als Biomarker zur Fr{\"u}herkennung von AD noch vor dem Auftreten kognitiver Einbußen dienen. Die fNIRS-Untersuchung w{\"a}hrend der Durchf{\"u}hrung des TMT hat sich in diesem Zusammenhang als potentielles Instrument zur Fr{\"u}hdiagnose der pr{\"a}klinischen Phase der AD als geeignet erwiesen. Die Ergebnisse werden unter Einbezug des wissenschaftlichen Kontexts interpretiert und Implikationen f{\"u}r weitere notwendige Studien sowie die klinische Anwendbarkeit diskutiert.},
  subject      = {Alzheimerkrankheit},
  language  = {de}
}
@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{Wilhelm2018,
  author    = {Wilhelm, Christian},
  title     = {Die Rolle von Chronophin bei Schlaganfall-induziertem Funktionsverlust der Blut-Hirn-Schranke},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-163877},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Der isch{\"a}mische Schlaganfall ist mit einer j{\"a}hrlichen Inzidenz von 200/100 000 Einwohnern die h{\"a}ufigste Gef{\"a}ßerkrankung in Deutschland. Atherothrombose, arterielle Hypertonie und Embolien unterschiedlichen Ursprungs sind die wesentlichen Ursachen des isch{\"a}mischen Schlaganfalls. Die neurologischen Defizite nach einem Schlaganfall resultieren aus einem gest{\"o}rten zerebralen Blutfluss und somit einer insuffizienten Sauerstoffversorgung. Zus{\"a}tzlich ist die {\"O}dembildung, welche von einer gesteigerten Permeabilit{\"a}t der Blut-Hirn-Schranke verursacht wird, am neuronalen Zelltod beteiligt. Chronophin ist eine Aktinzytoskelett-regulierende Serin-Phosphatase. In einem isch{\"a}mischen Schlaganfall-Modell konnte im Rahmen dieser Arbeit gezeigt werden, dass der globale Verlust von Chronophin zu einer vermehrten {\"O}dembildung und einem aggravierten neurologischen Zustand der M{\"a}use im Vergleich zu wildtypischen Kontrollen f{\"u}hrte. Hirnlysate von wildtypischen M{\"a}usen zeigten verringerte Chronophin-Level in der vom Schlaganfall betroffenen Hemisph{\"a}re. Jedoch konnten initiale immunhistochemische und zellbiologische Untersuchungen weder Chronophin-abh{\"a}ngige Ver{\"a}nderungen der Blut-Hirn-Schranke feststellen noch einen zerebralen Zelltyp identifizieren, der f{\"u}r den sch{\"u}tzenden Effekt von Chronophin verantwortlich ist. Diese Ergebnisse weisen auf einen komplexen, vielzelligen Mechanismus hin, dem die sch{\"u}tzende Rolle von Chronophin im isch{\"a}mischen Schlaganfall unterliegt. Die Entschl{\"u}sselung dieses Mechanismus ist Aufgabe k{\"u}nftiger Untersuchungen.},
  subject      = {Schlaganfall},
  language  = {de}
}
@phdthesis{Balasubramanian2018,
  author    = {Balasubramanian, Srikkanth},
  title     = {Novel anti-infectives against pathogenic bacteria},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-163882},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Marine sponge-associated actinomycetes are reservoirs of diverse natural products with novel biological activities. Their antibiotic potential has been well explored against a range of Gram positive and negative bacteria. However, not much is known about their anti-infective or anti-virulence potential against human pathogens. This Ph.D. project aimed to investigate the anti-infective (anti-Shiga toxin and anti-biofilm) potential of sponge-derived actinobacteria through identification and isolation of their bioactive metabolites produced and characterizing their mechanism of action by transcriptomics. This thesis is divided into three studies with the overall objective of exploring the anti-infective efficacy of actinomycetes-derived extracts and compound(s) that could possibly be used as future therapeutics. The first study deals with investigation on the anti-Shiga toxin effects of sponge-associated actinomycetes. Diarrheal infections pose a huge burden in several developing and developed countries. Diarrheal outbreaks caused by Enterohemorrhagic Escherichia coli (EHEC) could lead to life-threatening complications like gastroenteritis and haemolytic uremic syndrome (HUS) if left untreated. Shiga toxin (Stx) produced by EHEC is a major virulence factor that negatively affects the human cells, leading them to death via apoptosis. Antibiotics are not prescribed against EHEC infections since they may enhance the risk of development of HUS by inducing the production and release of Stx from disintegrating bacteria and thereby, worsening the complications. Therefore, an effective drug that blocks the Stx production without affecting the growth needs to be urgently developed. In this study, the inhibitory effects of 194 extracts and several compounds originating from a collection of marine sponge-derived actinomycetes were evaluated against the Stx production in EHEC strain EDL933 with the aid of Ridascreen® Verotoxin ELISA assay kit. It was found that treatment with the extracts did not lead to significant reduction in Stx production. However, strepthonium A isolated from the culture of Streptomyces sp. SBT345 (previously cultivated from the Mediterranean sponge Agelas oroides) reduced the Stx production (at 80 μM concentration) in EHEC strain EDL933 without affecting the bacterial growth. The structure of strepthonium A was resolved by spectroscopic analyses including 1D and 2D-NMR, as well as ESI-HRMS and ESI-HRMS2 experiments. This demonstrated the possible application of strepthonium A in restraining EHEC infections. VI In the second study, the effect of marine sponge-associated actinomycetes on biofilm formation of staphylococci was assessed. Medical devices such as contact lenses, metallic implants, catheters, pacemakers etc. are ideal ecological niches for formation of bacterial biofilms, which thereby lead to device-related infections. Bacteria in biofilms are multiple fold more tolerant to the host immune responses and conventional antibiotics, and hence are hard-to-treat. Here, the anti-biofilm potential of an organic extract derived from liquid fermentation of Streptomyces sp. SBT343 (previously cultivated from the Mediterranean sponge Petrosia ficiformis) was reported. Results obtained in vitro demonstrated its anti-biofilm (against staphylococci) and non-toxic nature (against mouse macrophage (J774.1), fibroblast (NIH/3T3) and human corneal epithelial cell lines). Interestingly, SBT343 extract could inhibit staphylococcal biofilm formation on polystyrene, glass and contact lens surfaces without affecting the bacterial growth. High Resolution Fourier Transform Mass Spectrometry (HR-MS) analysis indicated the complexity and the chemical diversity of components present in the extract. Preliminary physio-chemical characterization unmasked the heat stable and non-proteinaceous nature of the active component(s) in the extract. Finally, fractionation experiments revealed that the biological activity was due to synergistic effects of multiple components present in the extract. In the third study, anti-biofilm screening of 50 organic extracts generated from solid and liquid fermentation of 25 different previously characterized sponge-derived actinomycetes was carried out. This led to identification of the anti-biofilm organic extract derived from the solid culture of Streptomyces sp. SBT348 (previously cultivated from the Mediterranean sponge Petrosia ficiformis). Bioassay-guided fractionation was employed to identify the active fraction Fr 7 in the SBT348 crude extract. Further purification with semi-preparative HPLC led to isolation of the bioactive SKC1, SKC2, SKC3, SKC4 and SKC5 sub-fractions. The most active sub-fraction SKC3 was found to be a pure compound having BIC90 and MIC values of 3.95 μg/ml and 31.25 μg/ml against S. epidermidis RP62A. SKC3 had no apparent toxicity in vitro on cell lines and in vivo on the greater wax moth Galleria melonella larvae. SKC3 was stable to heat and enzymatic treatments indicating its non-proteinaceous nature. HR-MS analysis revealed the mass of SKC3 to be 1258.3 Da. Structure elucidation of SKC3 with the aid of 1D and 2D-NMR data is currently under investigation. Further, to obtain insights into the mode of action of SKC3 on S. epidermidis RP62A, RNA sequencing was done. Transcriptome data revealed that SKC3 was recognized by RP62A at 20 min and SKC3 negatively interfered with the central metabolism of staphylococci at 3 h. Taken VII together, these findings suggest that SKC3 could be a lead structure for development of new anti-staphylococcal drugs. Overall, the results obtained from this work underscore the anti-infective attributes of actinomycetes consortia associated with marine sponges, and their applications in natural product drug discovery programs.},
  subject      = {Marine sponges},
  language  = {en}
}
@phdthesis{Bury2018,
  author    = {Bury, Susanne},
  title     = {Molekularbiologische Untersuchungen der antagonistischen Effekte des probiotischen \(Escherichia\) \(coli\) Stamms Nissle 1917 auf Shiga-Toxin produzierende \(Escherichia\) \(coli\) St{\"a}mme},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-163401},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Shiga toxin produzierende E. coli (STEC) stellen mit einer Infektionsdosis von gerade einmal 100 Bakterien ein großes Risiko f{\"u}r unsere Gesundheit dar. Betroffene Patienten k{\"o}nnen milde Krankheitssymptome wie w{\"a}ssrigen Durchfall aufweisen, welcher sich allerdings zu blutigem Durchfall oder dem h{\"a}molytisch ur{\"a}mischen Syndrom (HUS) weiterentwickeln kann. Die Ursache f{\"u}r das Krankheitsbild ist das zytotoxische Protein Shiga-Toxin (Stx), welches von STEC St{\"a}mmen produziert wird, eukaryotischen Zellen angreift und den apoptotischen Zelltod induziert. Es konnte gezeigt werden, dass infizierte Patienten in ihrem Krankheitsverlauf stark variieren, was unter anderem auf die Zusammensetzung ihrer Mikrobiota zur{\"u}ckzuf{\"u}hren sein k{\"o}nnte. Diesbez{\"u}glich k{\"o}nnen zum Beispiel einige Bakterien bereits die Darmbesiedlung von STEC St{\"a}mmen unterbinden, wohingegen andere die Toxin Produktion der pathogenen St{\"a}mme beeinflussen und wieder andere von den stx tragenden Phagen infiziert werden k{\"o}nnen und daraufhin selbst zu Toxin produzierenden St{\"a}mmen werden. Da die genetischen Informationen f{\"u}r das Toxin auf einem Prophagen im Genom der STEC St{\"a}mme kodiert ist, f{\"u}hrt eine Antibiotika Behandlung von infizierten Patienten zwar zum Tod der Bakterien, hat allerdings auch einen Wechsel vom lysogenen zum lytischen Phagen Zyklus und damit einen enormen Anstieg an freigesetztem Stx zur Folge. In den letzten Jahrzehnten kam es immer wieder zu Epidemien mit STEC St{\"a}mmen, welche auch einige Todesopfer forderten. Die Behandlung von Patienten erfolgt auf Grund von mangelnden Behandlungsm{\"o}glichkeiten meist nur symptomatisch, weswegen neue Strategien f{\"u}r die Behandlung einer STEC Infektion dringend ben{\"o}tigt werden. Der probiotische E. coli Stamm Nissle 1917 (EcN) z{\"a}hlt bereits seit mehr als 100 Jahren als Medikament f{\"u}r Behandlungen von Darmentz{\"u}ndungen. In vitro und in vivo Studien mit dem probiotischen Stamm und STEC St{\"a}mmen konnten zeigen, dass EcN die Produktion von Stx unterdr{\"u}ckt und gleichzeitig die STEC Zellzahl reduziert. Diese Ergebnisse waren der Anlass f{\"u}r diese Studie in der die Auswirkungen von EcN auf STEC St{\"a}mme genauer untersucht wurden, um eine m{\"o}gliche Behandlung von STEC Infektionen mit dem Probiotikum zu gew{\"a}hrleisten. Eines der Hauptziele dieser Studie war es, herauszufinden, ob EcN von stx-Phagen infiziert werden kann und damit selbst zu einem Toxin Produzenten wird. In diesem Falle w{\"a}re eine Behandlung mit dem E. coli Stamm ausgeschlossen, da es den Krankheitsverlauf verschlimmern k{\"o}nnte. Verschiedene experimentelle Ans{\"a}tze in denen versucht wurde den YaeT stx-Phagen Rezeptor tragenden Stamm zu infizieren schlugen fehl. Weder mittels PCR Analysen, Phagen Plaque Assays oder der Phagen Anreicherung konnte eine Lyse oder eine Prophagen Integration nachgewiesen werden. Transkriptom Analysen konnten zeigen, dass Gene eines lambdoiden Prophagen in EcN in Anwesenheit von stx-Phagen stark reguliert sind. Auch andere E. coli St{\"a}mme, welche sich ebenfalls durch eine Resistenz gegen{\"u}ber einer stx-Phagen Infektion auswiesen, wurden positiv auf lambdoide Prophagen untersucht. Einzig dem stx-Phagen sensitiven K-12 Stamm MG1655 fehlt ein kompletter lambdoider Prophage, weswegen die Vermutung nahe liegt, dass ein intakter lambdoider Prophage vor der Superinfektion mit stx-Phagen sch{\"u}tzten kann. In weiteren Experimenten wurde der Einfluss der Mikrozin-negativen EcN Mutante SK22D auf STEC St{\"a}mme untersucht. Es konnte gezeigt werden, dass SK22D nicht nur die Produktion des zytotoxischen Proteins unterdr{\"u}ckt, sondern auch mit der Produktion der stx-Phagen von allen getesteten STEC St{\"a}mmen interferiert (O157:H7, O26:H11, O145:H25, O103:H2, O111:H- und zwei O104:H4 Isolate vom STEC Ausbruch in Deutschland im Jahr 2011). Transwell Studien konnten zeigen, dass der Faktor, welcher die Transkription des Prophagen unterdr{\"u}ckt, von SK22D sekretiert wird. Die Ergebnisse lassen vermuten, dass die Pr{\"a}senz von SK22D den lysogenen Zustand des Prophagen st{\"u}tzt und somit den lytischen Zyklus unterdr{\"u}ckt. Da stx-Phagen eine große Gefahr darstellen andere E. coli St{\"a}mme zu infizieren, haben wir uns in weiteren Studien dem Einfluss von EcN auf isolierte Phagen gewidmet. Die Kultivierungsexperimente von EcN mit Phagen zeigten, dass der probiotische Stamm in der Lage war die stx-Phagen in ihrer Effizienz der Lyse des K 12 Stammes MG1655 von~ 1e7 pfus/ml auf 0 pfus/ml nach einer 44 st{\"u}ndigen Inkubation zu inaktivieren. Diese Inaktivierung konnte auf die Aktivit{\"a}t eines hitzestabilen Proteins, welches in der station{\"a}ren Wachstumsphase synthetisiert wird, zur{\"u}ckgef{\"u}hrt werden. Studien welche einen Anstieg der Biofilmmasse zur Folge hatten zeigten eine gesteigerte Effizienz in der Phagen Inaktivierung, weswegen Komponenten des Biofilms m{\"o}glicherweise die Phagen Inaktivierung herbeif{\"u}hren. Neben dem direkten Einfluss auf die Phagen wurde auch ein Schutzeffekt von SK22D gegen{\"u}ber dem stx-Phagen empf{\"a}nglichen K 12 St{\"a}mmen untersucht. Lysogene K 12 St{\"a}mme zeichneten sich durch eine enorme Stx und stx-Phagen Produktion aus. Die Pr{\"a}senz von SK22D konnte den K 12 vermittelten Anstieg der pathogenen Faktoren unterbinden. Transwell Ergebnisse und Kinetik Studien lassen vermuten, dass SK22D eher die Phagen Infektion von K-12 St{\"a}mmen unterbindet als die Lyse von lysogenen K-12 St{\"a}mmen zu st{\"o}ren. Eine m{\"o}gliche Erkl{\"a}rung f{\"u}r den Schutz der K-12 St{\"a}mme vor einer stx-Phagen Infektion k{\"o}nnte darin liegen, dass die K-12 St{\"a}mme innerhalb der SK22D Kultur wachsen und dadurch von den infekti{\"o}sen Phagen abgeschirmt werden. Zusammenfassend konnte in dieser Studie gezeigt werden, dass der probiotische Stamm EcN sowohl die Lyse von STEC St{\"a}mmen unterdr{\"u}ckt als auch die infekti{\"o}sen stx-Phagen inaktiviert und sensitive E. coli St{\"a}mme vor der Phagen Infektion sch{\"u}tzen kann. Diese Ergebnisse sollten als Grundlage f{\"u}r in vivo Studien herangezogen werden, um eine m{\"o}gliche Behandlung von STEC infizierten Patienten mit dem Probiotikum zu gew{\"a}hrleisten.},
  subject      = {EHEC},
  language  = {en}
}
@phdthesis{Pennington2018,
  author    = {Pennington, Laura Sophie},
  title     = {The role of Cadherin-13 in serotonergic neurons during different murine developmental stages},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-161331},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Abstract Background: Attention-deficit/ hyperactivity disorder (ADHD) ranges among the most common neurodevelopmental disorders worldwide with a prevalence of 3-12\% in childhood and 1-5\% for adults. Over the last decade extensive genetic research has been conducted in order to determine its causative genetic factors. None of the so far identified susceptibility genes, however, could explain the estimated ADHD heritability of 76\%. In this thesis one of the most promising candidates -Cadherin 13 (Cdh13) - was examined in terms of its influence on the central serotonergic (5-HT) system. In addition to that, the Cdh13 protein distribution pattern was analysed over time. Methods: The developing serotonergic system was compared over three embryonic and postnatal stages (E13.5, E17.5 and P7) in different Cdh13 genotypes (WT, HZ and KO) using immunohistochemistry and various double staining protocols. Results: The raphe nuclei of the 5-HT system develop in spite of Cdh13 absence and show a comparable mature constellation. The cells in the KO, however, are slightly more scattered than in the WT. Furthermore the dynamics of their formation is altered, with a transient delay in migration at E13.5. In early developmental stages the total amount of serotonergic cells is reduced in KO and HZ, though their proportional distribution to the raphe nuclei stays constant. Strikingly, at P7 the absolute numbers are comparable again. Concerning the Cdh13 protein, it shows high concentrations on fibres running through hindbrain and midbrain areas at E13.5. This, however, changes over time, and it becomes more evenly spread until P7. Furthermore, its presence in serotonergic cells could be visualised using confocal microscopy. Since the described pattern is only in parts congruent to the localisation of serotonergic neurons, it is most likely that Cdh13 is present in other developing neurotransmitter systems, such as the dopaminergic one, as well. Conclusion: It could be proven that Cdh13 is expressed in serotonergic cells and that its knockout does affect the developing serotonergic system to some degree. Its absence, however, only slightly and transiently affects the measured parameters of serotonergic system development, indicating a possible compensation of CDH13 function by other molecules in the case of Cdh13 deficiency. In addition further indicators could be found for an influence of Cdh13 on outgrowth and path finding of neuronal processes.},
  subject      = {Cadherine},
  language  = {en}
}
@phdthesis{Kruber2019,
  author    = {Kruber, Philip},
  title     = {Functional analysis of DROSHA and SIX1 mutations in kidney development and Wilms tumor},
  doi       = {10.25972/OPUS-16141},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-161418},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Wilms tumor (WT) is the most common kidney cancer in childhood. It is a genetically heterogeneous tumor and several genetic alterations have been identified in WT patients. Recurrent mutations were found in the homeo-domain of SIX1 and SIX2 in high proliferative tumors (18.1\% of the blastemal-type tumors) as well as in the microprocessor genes DROSHA and DGCR8 (18.2\% of the blastemal-type tumors), indicating a critical role of the SIX-SALL pathway and aberrant miRNA processing in WT formation. Underlined by the fact that a significant overlap between mutations in DROSHA and SIX1 was found, indicating a synergistic effect. To characterize the in vivo role of DROSHA and SIX mutations during kidney development and their oncogenic potential, I analyzed mouse lines with either a targeted deletion of Drosha or an inducible expression of human DROSHA or SIX1 carrying a tumor-specific E1147K or Q177R mutation, respectively. The DROSHA mutation E1147K was predicted to act in a dominant negative manner. Six2-cre mediated deletion of Drosha in nephron progenitors led to a lethal phenotype with apoptotic loss of progenitor cells and early termination of nephrogenesis. Mosaic deletions via Wt1-creERT2 resulted in a milder phenotype with viable offspring that developed proteinuria after 2-4 weeks, but no evidence of tumor formation. Activation of the DROSHA-E1147K transgene via Six2-cre, on the other hand, induced a more severe phenotype with apoptosis of progenitor cells, proteinuria and glomerular sclerosis. The severely growth-retarded mice died within the first two months. This strong phenotype was consistent with the predicted dominant-negative effect of DROSHA-E1147K. Analysis of the SIX1-Q177R mutation suggested that the mutation leads to a shift in DNA binding specificity instead of a complete loss of DNA binding. This may end up in subtle changes of the gene regulatory capacity of SIX1. Six2-cre mediated activation of SIX1-Q177R lead to a viable phenotype with no alterations or shortened life span. Yet a global activation of SIX1-Q177R mediated by Zp3-cre resulted in bilateral hydronephrosis and juvenile death of the mice. To mimic the synergistic effect of DROSHA and SIX1 mutations, I generated compound mutants in two combinations: A homozygous deletion of Drosha combined with an activation of SIX1-Q177R and a compound mutant with activation of DROSHA-E1147K and SIX1-Q177R. Each mouse model variant displayed new phenotypical alterations. Mice with Six2-cre mediated homozygous deletion of Drosha and activation of SIX1-Q177R were not viable, yet heterozygous deletion of Drosha and activation of SIX1-Q177R led to hydronephrosis, proteinuria and an early death around stage P28. Combined activation of DROSHA-E1147K and SIX1-Q177R under Six2-cre resulted in proteinuria, glomerulosclerosis and lesions inside the kidney. These mice also suffered from juvenile death. Both mouse models could confirm the predicted synergistic effect. While these results underscore the importance of a viable self-renewing progenitor pool for kidney development, there was no evidence of tumor formation. This suggests that either additional alterations in mitogenic or antiapoptotic pathways are needed for malignant transformation, or premature loss of a susceptible target cell population and early lethality prevent WT formation.},
  subject      = {Nephroblastom},
  language  = {en}
}
@phdthesis{Lennartz2018,
  author    = {Lennartz, Simon},
  title     = {Tissue Engineering der menschlichen Speicheldr{\"u}se unter Verwendung von Epithel- und mikrovaskul{\"a}ren Endothelzellen auf einer Matrix aus dezellularisiertem Schweinedarm},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-164116},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Eine ausgepr{\"a}gte Mundtrockenheit, Xerostomie, entsteht h{\"a}ufig durch eine irreversible Funktionseinschr{\"a}nkung der Speicheldr{\"u}sen. Diese ist unter anderem durch die Einnahme bestimmter Medikamente, Autoimmunerkrankungen, fortgeschrittenes Alter oder die Bestrahlungstherapie von Tumoren der Kopf-Hals-Region bedingt, wobei letztere eine der h{\"a}ufigsten Ursachen darstellt. Konsequenzen der eingeschr{\"a}nkten Dr{\"u}senfunktion sind herabgesetzte Speichelflussraten, eine Reduktion des Mund-pH-Werts, eine ver{\"a}nderte Elektrolyt- und Immunglobulin-Zusammensetzung des Speichels und somit eine Verringerung des Infektionsschutzes. Die resultierenden Komplikationen erstrecken sich von Karies und rezidivierenden Infektionen bis hin zu Pilzbesiedelungen der Mundschleimhaut. Diese schr{\"a}nken die Lebensqualit{\"a}t der Patienten stark ein und f{\"u}hren h{\"a}ufig zu Therapieunterbrechungen. Fast die H{\"a}lfte der Patienten leidet unter Depressionen oder psychischen Belastungszust{\"a}nden. Es gibt wenige Therapieans{\"a}tze zur Behandlung der postradiogenen Xerostomie: Pilocarpin erh{\"o}ht zwar die Speichelflussraten, hat jedoch keinen signifikanten Effekt auf die Lebensqualit{\"a}t. Die operative Translokation der Glandula submandibularis hat den Weg in die klinische Routine noch nicht gefunden, w{\"a}hrend die intensit{\"a}tsmodulierte Bestrahlung (IMRT) nicht f{\"u}r jeden Patienten geeignet ist; beide zeigen jedoch einen positiven Effekt auf die Lebensqualit{\"a}t. Gentechnische und stammzellbasierte Ans{\"a}tze zur Regeneration des Dr{\"u}sengewebes befinden sich im Experimentalstadium. Somit ergibt sich ein dringender Bedarf an innovativen Optionen zur Behandlung der postradiogenen Xerostomie. Das Tissue Engineering, die Erstellung einer k{\"u}nstlichen Speicheldr{\"u}se aus k{\"o}rpereigenen Zellen, b{\"o}te hier ein potentielles Behandlungskonzept. Diese Studie soll deshalb untersuchen, ob humane Speicheldr{\"u}senepithelzellen (hSEZ) auf einer Matrix aus dezellularisiertem, porzinem Jejunum, der sogenannten Small intestinal submucosa + mucosa (SIS-muc), kultiviert werden k{\"o}nnen. K{\"o}nnen die Zellen innerhalb der Wachstumsperiode wichtige physiologische Differenzierungsmarker beibehalten? Kann die Produktion von α-Amylase, einem der wichtigsten Enzyme des menschlichen Speichels, erhalten werden? Welchen Einfluss hat die Kokultur mit mikrovaskul{\"a}ren Endothelzellen (mvEZ)? Und zuletzt: Ist dezellularisierter Schweinedarm eine potentiell geeignete Matrix f{\"u}r das Tissue Engineering der menschlichen Speicheldr{\"u}se? Zun{\"a}chst erfolgte die Entnahme von humanem Speicheldr{\"u}sengewebe, woraus hSEZ isoliert wurden. Diese wurden dann sowohl in Mono- als auch in Kokultur mit mvEZ auf die SIS-muc aufgebracht und auf dieser kultiviert. Die SIS-muc wurde aus kurzen Schweinedarm-Segmenten gewonnen, die in einem mehrstufigen Verfahren dezellularisiert wurden. Die besiedelte SIS-muc wurde mittels konventioneller sowie Immunfluoreszenzf{\"a}rbungen, Raster- und Transmissionsektronenmikroskopie (REM/TEM) sowie quantitativer Polymerasekettenreaktion (qPCR) untersucht, dar{\"u}ber hinaus erfolgte die Messung der α-Amylase-Enzymaktivit{\"a}t. Histologisch sowie in der REM zeigte sich sowohl in der Mono- als auch in der Kokultur eine konfluente Besiedelung der SIS-muc mit hSEZ. In der Kokultur formten mvEZ einen Monolayer auf der serosalen Matrixseite. Bei der Charakterisierung der hSEZ zeigte sich in den Immunfluoreszenzaufnahmen eine starke Auspr{\"a}gung von Zytokeratin, α-Amylase und Aquaporin-5 und eine moderate Auspr{\"a}gung von Claudin-1. Bei der Untersuchung der Funktion der α-Amylase konnte in der Kokultur von hSEZ mit mvEZ eine im Gegensatz zur Mono- und 2D-Kultur signifikant erh{\"o}hte Enzymaktivit{\"a}t der α-Amylase nachgewiesen werden. In der qPCR-Analyse der α-Amylase-Genexpression war die 3D-Kultur der 2D-Kultur {\"u}berlegen. Die vorliegende Arbeit zeigt, dass die Kultur von hSEZ auf der SIS-muc m{\"o}glich ist. Es konnte nachgewiesen werden, dass die Zellen in 3D-Kultur spezifische Differenzierungsmerkmale beibehalten, die in der 2D-Kultur teils verloren gehen und dass hSEZ in Kokultur mit mvEZ eine gegen{\"u}ber der Monokultur signifikant erh{\"o}hte Produktion von α-Amylase aufweisen. Diese Arbeit liefert die Datengrundlage f{\"u}r zuk{\"u}nftige Studien im dynamischen Bioreaktor-Modell (BioVaSc), die auf dem Weg zur klinischen Translation notwendig sind. Somit stellt sie einen wichtigen Schritt in Richtung einer auf Tissue Engineering basierten Therapie der belastenden Xerostomie dar.},
  subject      = {Tissue Engineering},
  language  = {de}
}
@phdthesis{Goettlich2019,
  author    = {G{\"o}ttlich, Claudia},
  title     = {Etablierung eines humanen 3D Lungentumor-Testsystems zur Analyse von Behandlungseffekten},
  doi       = {10.25972/OPUS-16413},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-164132},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Lungenkrebs ist weltweit f{\"u}r die meisten krebsassoziierten Tode verantwortlich. Ursache daf{\"u}r ist unter anderem, dass viele Medikamente in der klinischen Anwendung, aufgrund nicht {\"u}bertragbarer Ergebnisse aus der Pr{\"a}klinik, scheitern. Zur Entwicklung neuer Therapiestrategien werden deshalb Modelle ben{\"o}tigt, welche die in vivo Situation besser widerspiegeln. Besonders wichtig ist es dabei, zu zeigen, f{\"u}r welche Fragestellungen ein neues Testsystem valide Ergebnisse liefert. In dieser Arbeit ist es mit Hilfe des Tissue Engineering gelungen, ein humanes 3D in vitro Lungentumor-Testsystem weiter zu entwickeln und f{\"u}r verschiedene Fragestellungen zu validieren. Zudem konnten sowohl f{\"u}r die Herstellung als auch f{\"u}r die Behandlung der Tumormodelle SOPs etabliert werden. Hier wurde zun{\"a}chst beobachtet, dass die Auswerteparameter f{\"u}r die Beurteilung von Behandlungseffekten eine geringe Varianz aufweisen und das 3D Modell deshalb als Testsystem geeignet ist. Ein Vergleich der Morphologie, des EMT-Status und der Differenzierung der Tumorzelllinien im 3D Modell mit Tumorbiopsaten von Adenokarzinompatienten verdeutlichte, dass die 3D Modelle tumorrelevante Merkmale besitzen. So sind die Zelllinien auf der biologischen Matrix, verglichen mit der jeweiligen 2D Kultur, durch eine reduzierte Proliferationsrate gekennzeichnet, welche eher der in vivo Situation entspricht. F{\"u}r die Etablierung und Validierung des 3D Modells als Testsystem war es notwendig, klinisch relevante Therapien in dem Modell anzuwenden und die Ergebnisse der Behandlung in vitro mit denen im Patienten zu vergleichen. Dabei konnte zun{\"a}chst best{\"a}tigt werden, dass eine zielgerichtete Therapie gegen den EGFR in dem 3D System zu einer verst{\"a}rkten Induktion der Apoptose im Vergleich zu 2D f{\"u}hrt. Dies entspricht klinischen Beobachtungen, bei denen EGFR-mutierte Patienten gut auf eine Therapie mit Tyrosin-Kinase-Inhibitoren (TKI) ansprechen. Anschließend wurde in dieser Arbeit erstmals in vitro gezeigt, dass die Behandlung mit einem HSP90-Inhibitor bei KRAS-Mutation wie in behandelten Patienten keine eindeutigen Vorteile bringt, diese jedoch in Experimenten der 2D Zellkultur mit den entsprechenden Zelllinien vorhergesagt werden. Die Ergebnisse aus dem in vitro Modell spiegeln damit verschiedene klinische Studien wider und unterstreichen das Potenzial des 3D Lungentumor-Testsystems die Wirkung zielgerichteter Therapien vorherzusagen. Durch die Messung von Signalwegsaktivierungen {\"u}ber Phospho-Arrays und Western Blot konnten in dieser Arbeit Unterschiede zwischen 2D und 3D nach Behandlung gezeigt werden. Diese lieferten die Grundlage f{\"u}r bioinformatische Vorhersagen f{\"u}r Medikamente. Mit fortschreitender Erkrankung und dem Entstehen invasiver Tumore, die m{\"o}glicherweise Metastasen bilden, verschlechtert sich die Prognose von Krebspatienten. Zudem entwickeln Patienten, die zun{\"a}chst auf eine Therapie mit TKI ansprechen, bereits nach kurzer Zeit Resistenzen, die ebenfalls zur Progression des Tumorwachstums f{\"u}hren. Zur Wirkungsuntersuchung von Substanzen in solchen fortgeschrittenen Erkrankungsstadien wurde das bestehende Testsystem erweitert. Zum einen wurde mit Hilfe des Wachstumsfaktors TGF-β1 eine EMT ausgel{\"o}st. Hier konnte beobachtet werden, dass sich die Expression verschiedener EMT- und invasionsassoziierter Gene und Proteine ver{\"a}nderte und die Zellen vor allem in dynamischer Kultur verst{\"a}rkt die Basalmembran der Matrix {\"u}berquerten. Zum anderen wurde die Ausbildung von Resistenzen gegen{\"u}ber TKI durch die Generierung von resistenten Subpopulationen aus einer urspr{\"u}nglich sensitiven Zelllinie und anschließender Kultivierung auf der Matrix abgebildet. Dabei zeigte sich keine der klinisch bekannten Mutationen als urs{\"a}chlich f{\"u}r die Resistenz, sodass weitere Mechanismen untersucht wurden. Hier konnten Ver{\"a}nderungen in der Signaltransduktion sowie der Expression EMT-assoziierter Proteine festgestellt werden. Im letzten Teil der Arbeit wurde eine neuartige Behandlung im Bereich der Immuntherapie erfolgreich in dem 3D Modell angewendet. Daf{\"u}r wurden T-Zellen, die einen chim{\"a}ren Antigen-Rezeptor (CAR) gegen ROR1 tragen, in statischer und dynamischer Kultur zu den Tumorzellen gegeben und der Therapieeffekt mittels histologischer F{\"a}rbung und der Bestimmung der Apoptose evaluiert. Zus{\"a}tzlich konnten Eigenschaften der T-Zellen, wie deren Proliferation sowie Zytokinaussch{\"u}ttung quantifiziert und damit eine spezifische Wirkung der CAR transduzierten T-Zellen gegen{\"u}ber Kontroll-T-Zellen nachgewiesen werden. Zusammenfassend ist es in dieser Arbeit gelungen, ein humanes 3D Lungentumor-Testsystem f{\"u}r die Anwendung in der pr{\"a}klinischen Entwicklung von Krebsmedikamenten sowie der Grundlagenforschung im Bereich der Tumorbiologie zu etablieren. Dieses Testsystem ist in der Lage relevante Daten zu Biomarker-geleiteten Therapien, zur Behandlung fortgeschrittener Tumorstadien und zur Verbesserung neuartiger Therapiestrategien zu liefern.},
  subject      = {Tissue Engineering},
  language  = {de}
}
@phdthesis{Horn2017,
  author    = {Horn, Hannes},
  title     = {Analysis and interpretation of (meta-)genomic data from host-associated microorganisms},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-152035},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {Host-microbe interactions are the key to understand why and how microbes inhabit specific environments. With the scientific fields of microbial genomics and metagenomics, evolving on an unprecedented scale, one is able to gain insights in these interactions on a molecular and ecological level. The goal of this PhD thesis was to make (meta-)genomic data accessible, integrate it in a comparative manner and to gain comprehensive taxonomic and functional insights into bacterial strains and communities derived from two different environments: the phyllosphere of Arabidopsis thaliana and the mesohyl interior of marine sponges. This thesis focused first on the de novo assembly of bacterial genomes. A 5-step protocol was developed, each step including a quality control. The examination of different assembly software in a comparative way identified SPAdes as most suitable. The protocol enables the user to chose the best tailored assembly. Contamination issues were solved by an initial filtering of the data and methods normally used for the binning of metagenomic datasets. This step is missed in many published assembly pipelines. The described protocol offers assemblies of high quality ready for downstream analysis. Subsequently, assemblies generated with the developed protocol were annotated and explored in terms of their function. In a first study, the genome of a phyllosphere bacterium, Williamsia sp. ARP1, was analyzed, offering many adaptions to the leaf habitat: it can deal with temperature shifts, react to oxygen species, produces mycosporins as protection against UV-light, and is able to uptake photosynthates. Further, its taxonomic position within the Actinomycetales was infered from 16S rRNA and comparative genomics showing the close relation between the genera Williamsia and Gordonia. In a second study, six sponge-derived actinomycete genomes were investigated for secondary metabolism. By use of state-of-the-art software, these strains exhibited numerous gene clusters, mostly linked to polykethide synthases, non-ribosomal peptide synthesis, terpenes, fatty acids and saccharides. Subsequent predictions on these clusters offered a great variety of possible produced compounds with antibiotic, antifungal or anti-cancer activity. These analysis highlight the potential for the synthesis of natural products and the use of genomic data as screening toolkit. In a last study, three sponge-derived and one seawater metagenomes were functionally compared. Different signatures regarding the microbial composition and GC-distribution were observed between the two environments. With a focus on bacerial defense systems, the data indicates a pronounced repertoire of sponge associated bacteria for bacterial defense systems, in particular, Clustered Regularly Interspaced Short Palindromic Repeats, restriction modification system, DNA phosphorothioation and phage growth limitation. In addition, characterizing genes for secondary metabolite cluster differed between sponge and seawater microbiomes. Moreover, a variety of Type I polyketide synthases were only found within the sponge microbiomes. With that, metagenomics are shown to be a useful tool for the screening of secondary metabolite genes. Furthermore, enriched defense systems are highlighted as feature of sponge-associated microbes and marks them as a selective trait.},
  subject      = {Bakterien},
  language  = {en}
}
@phdthesis{HockSiew2018,
  author    = {Hock Siew, Tan},
  title     = {Functional characterization of an acid-regulated sRNA in \(Helicobacter\) \(pylori\)},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-150671},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Low pH is the main environmental stress encountered by Helicobacter pylori in the human stomach. To ensure its survival under acidic conditions, this bacterium utilizes urease (encoded by the ureAB operon), a nickel-activated metalloenzyme, which cleaves urea into ammonia to buffer the periplasmic space. Expression of the ureAB operon is tightly regulated at the transcriptional level. Moreover, the urease activity is modulated post translationally via the activity of nickel-binding proteins such as HP1432 that act as nickel sponges to either sequester or release nickel depending on the pH. However, little is known how the levels of these nickel-binding proteins are regulated at the post-transcriptional level. Interestingly, more than 60 candidate small regulatory RNAs (sRNAs) have been identified in a differential RNA-seq approach in H. pylori strain 26695, suggesting an uncharacterized layer of post-transcriptional riboregulation in this pathogen. sRNAs control their trans- or cis- encoded targets by direct binding. Many of the characterized sRNAs are expressed in response to specific environmental cues and are ideal candidates to confer post-transcriptional regulation under different growth conditions. This study demonstrates that a small RNA termed ArsZ (Acid Responsive sRNA Z) and its target HP1432 constitute yet another level of urease regulation. In-vitro and in-vivo experiments show that ArsZ interacts with the ribosome binding site (RBS) of HP1432 mRNA, effectively repressing translation of HP1432. During acid adaptation, the acid-responsive ArsRS two-component system represses expression of ArsZ. ArsRS and ArsZ work in tandem to regulate expression of HP1432 via a coherent feedforward loop (FFL). ArsZ acts as a delay mechanism in this feedforward loop to ensure that HP1432 protein levels do not abruptly change upon transient pH drops encountered by the bacteria. ArsZ "fine-tunes" the dynamics of urease activity after pH shift presumably by altering nickel availability through post transcriptional control of HP1432 expression. Interestingly, after adaptation to acid stress, ArsZ indirectly activates the transcription of HP1432 and forms an incoherent FFL with ArsRS to regulate HP1432. This study identified a non-standard FFL in which ArsZ can participate directly or indirectly in two different network configurations depending on the state of acid stress adaptation. The importance of ArsZ in the acid response of H. pylori is further supported by bioinformatics analysis showing that the evolution of ArsZ is closely related to the emergence of modern H. pylori strains that globally infect humans. No homologs of arsZ were found in the non-pylori species of Helicobacter. Moreover, this study also demonstrates that the physiological role of a sRNA can be elucidated without the artificial overexpression of the respective sRNA, a method commonly used to characterize sRNAs. Coupled with time-course experiments, this approach allows the kinetics of ArsZ regulation to be studied under more native conditions. ArsZ is the first example of a trans-acting sRNA that regulates a nickel storage protein to modulate apo-urease maturation. These findings may have important implications in understanding the details of urease activation and hence the colonization capability of H. pylori, the only bacterial class I carcinogen to date (WHO, 1994).},
  subject      = {Small RNA},
  language  = {en}
}
@phdthesis{Lorenzin2016,
  author    = {Lorenzin, Francesca},
  title     = {Regulation of transcription by MYC - DNA binding and target genes},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-150766},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {MYC is a transcription factor, whose expression is elevated or deregulated in many human cancers (up to 70\%) and is often associated with aggressive and poorly differentiated tumors. Although MYC is extensively studied, discrepancies have emerged about how this transcription factor works. In primary lymphocytes, MYC promotes transcriptional amplification of virtually all genes with an open promoter, whereas in tumor cells MYC regulates specific sets of genes that have significant prognostic value. Furthermore, the set of target genes that distinguish MYC's physiological function from the pathological/oncogenic one, whether it exists or not, has not been fully understood yet. In this study, it could be shown that MYC protein levels within a cell and promoter affinity (determined by E-box presence or interaction with other proteins) of target genes toward MYC are important factors that influence MYC activity. At low levels, MYC can amplify a certain transcriptional program, which includes high affinity binding sites, whereas at high levels MYC leads to the specific up- and down regulation of genes with low affinity. Moreover, the promoter affinity characterizes different sets of target genes which can be distinguished in the physiological or oncogenic MYC signatures. MYC-mediated repression requires higher MYC levels than activation and formation of a complex with MIZ1 is necessary for inhibiting expression of a subset of MYC target genes.},
  subject      = {MYC},
  language  = {en}
}
@phdthesis{Monjezi2018,
  author    = {Monjezi, Razieh},
  title     = {Engineering of chimeric antigen receptor T cells with enhanced therapeutic index in cancer immunotherapy using non-viral gene transfer and genome editing},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-152521},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {The advances in genetic engineering have enabled us to confer T cells new desired functions or delete their specific undesired endogenous properties for improving their antitumor function. Due to their efficient gene delivery, viral vectors have been successfully used in T-cell engineering to provide gene transfer medicinal products for the treatment of human disease. One example is adoptive cell therapy with T cells that were genetically modified with gamma-retroviral and lentiviral (LV) delivery vectors to express a CD19-specific chimeric antigen receptor (CAR) for cancer treatment. This therapeutic approach has shown remarkable results against B-cell malignancies in pilot clinical trials. Consequently, there is a strong desire to make CAR T cell therapy scalable and globally available to patients. However, there are persistent concerns and limitations with the use of viral vectors for CAR T cell generation with regard to safety, cost and scale of vector production. In order to address these concerns, we aimed to improve non-viral gene transfer and genome editing tools as an effective, safe and broadly applicable alternative to viral delivery methods for T-cell engineering. In the first part of the study, we engineered CAR T cells through non-viral Sleeping Beauty (SB) transposition of CAR genes from minimalistic DNA vectors called minicircles rather than conventional SB plasmids. This novel approach dramatically increased stable gene transfer rate and cell viability and resulted in higher yield of CAR+ T cells without the need of long ex vivo expansion to generate therapeutic doses of CAR+ T cells. Importantly, CD19-CAR T cells modified by MC-based SB transposition were equally effective as LV transduced CD19-CAR T cells in vitro and in a murine xenograft model (NSG/Raji-ffLuc), where a single administration of CD8+ and CD4+ CAR T cells led to complete eradication of lymphoma and memory formation of CAR T cells after lymphoma clearance. To characterize the biosafety profile of the CAR T cell products, we did the most comprehensive genomic insertion site analysis performed so far in T cells modified with SB. The data showed a close-to-random integration profile of the SB transposon with a higher number of insertions in genomic safe harbors compared to LV integrants. We developed a droplet digital PCR assay that enables rapid determination of CAR copy numbers for clinical applications. In the second part of the study, we ablated expression of PD-1, a checkpoint and negative regulator of T cell function to improve the therapeutic index of CAR T cells. This was accomplished using non-viral CRISPR/Cas9 via pre-assemble Cas9 protein and in vitro-transcribed sgRNA (Cas9 RNP). Finally, we combined our developed Cas9 RNP tool with CAR transposition from MC vectors into a single-step protocol and successfully generated PD-1 knockout CAR+ T cells. Based on the promising results achieved from antibody-mediated PD-1 blockade in the treatment of hematological and solid tumors, we are confident that PD-1 knockout CAR T cells enhance the potency of CAR T cell therapies for treatment of cancers without the side effects of antibody-based therapies. In conclusion, we provide a novel platform for virus-free genetic engineering of CAR T cells that can be broadly applied in T-cell cancer therapy. The high level of gene transfer rate and efficient genome editing, superior safety profile as well as ease-of-handling and production of non-viral MC vectors and Cas9 RNP position our developed non-viral strategies to become preferred approaches in advanced cellular and gene-therapy.},
  subject      = {Krebs <Medizin>},
  language  = {en}
}
@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{Flohr2017,
  author    = {Flohr, Elena Leonie Ruth},
  title     = {The Scents of Interpersonality - On the Influence of Smells on the Evaluation and Processing of Social Stimuli},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-153352},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {In daily life, olfactory stimuli are potential generators of affective states, but also have a strong influence on social interaction. Pleasant odors have been shown to increase perceived attractiveness and pro-social behavior, whereas unpleasant body odors are often associated with negative personality traits. Since both pleasant odors and positive affective state facilitate pro-social behavior, it is conceivable that the influence of the odors on social interaction is mediated by the induced affective state elicited by the odor itself. The present thesis aims at exploring the impact of hedonic, i.e., pleasant or unpleasant, odors on the processing and evaluation of social stimuli as assessed by verbal, physiological, and behavioral indices. First, I investigate the effects of initially neutral odors which gained threatening value through an aversive conditioning procedure on social stimuli (Study 1). Second, I study the influence of naturally hedonic odors on social interaction. Third, this thesis aims at disentangling differences in the effects of an odor attributed to either a social interaction partner or the environment where the social encounter takes place (Study 2, 3, and 4). In the first study, a context conditioning procedure was applied, during which one out of two long-lasting neutral odors was paired with an unpredictable aversive unconditioned stimulus (US, i.e., white noise). This odor (CTX+) thereby gained threatening value, while another odor (CTX-) remained unpaired and therefore signaled safety. During a test session, facial stimuli were presented within both conditioned olfactory contexts. Results indicate that autonomic arousal was increased to faces when presented in the threatening odor context. Additionally, participants rated facial stimuli as more aversive when presented in the threatening odor as compared to the safety odor, indicating that faces acquire hedonic value from the odor they were presented in. Strikingly, angry facial expressions received additional processing resources when presented within a threatening olfactory context, as reflected on verbal reports and electrodermal activity (EDA). This latter finding suggests that threat-related stimuli, here angry faces, are preferentially processed within an olfactory context where a threat might happen. Considering that the hedonic value of an odor may be quite subjective, I conducted a pilot study in order to identify odors with pleasant vs. unpleasant properties for most participants. Seven odors (four pleasant and three unpleasant) were rated with respect to their valence (pleasant vs. unpleasant), arousal (arousing vs. calm), and intensity. Additionally, EDA was measured. Two pleasant (Citral and Eucalyptol) and two unpleasant ("Animalis" and Isobutyraldehyde) odors were chosen from the original seven. The unpleasant odors were rated as more negative, arousing, and intense than the positive ones, but no differences were found regarding EDA. These four odors were subsequently used in a virtual reality (VR) paradigm with two odor attribution groups. Participants of the social attribution group (n = 59) were always passively guided into the same room (an office) towards one out of two virtual agents who were either paired with the pleasant or the unpleasant odor. Participants of the contextual attribution group (n = 58) were guided into one out of two rooms which were either paired with the pleasant or the unpleasant odor and where they always met the same agent. For both groups, the agents smiled, frowned or remained with a neutral facial expression. This design allowed evaluating the influence of odor valence as a within-subjects factor and the influence of odor attribution as a between-subjects factor. Unpleasant odors facilitated the processing of social cues as reflected by increased verbal and physiological arousal as well as reduced active approach behavior. Specific influence of odor valence on emotional facial expressions was found for ratings, EDA, and facial mimicry, with the unpleasant odor causing a levelling effect on the differences between facial expressions. The social attribution group exhibited larger differences between odors than the contextual group with respect to some variables (i.e., ratings and EDA), but not to others (i.e., electrocortical potentials - ERPs - and approach behavior). In sum, unpleasant in comparison to pleasant odors diminished emotional responses during social interaction, while an additional enhancing effect of the social attribution was observed on some variables. Interestingly, the awareness that an interaction partner would smell (pleasantly or unpleasantly) boosted the emotional reactivity towards them. In Study 3, I adapted the VR paradigm to a within-subjects design, meaning that the different attribution conditions were now manipulated block-wise. Instead of an approach task, participants had to move away from the virtual agent (withdrawal task). Results on the ratings were replicated from Study 2. Specifically, the difference between pleasant and unpleasant odors on valence, arousal, and sympathy ratings was larger in the social as compared to the contextual attribution condition. No effects of odor or attribution were found on EDA, whereas heart rate (HR) showed a stronger acceleration to pleasant odors while participants were passively guided towards the agent. Instead of an approach task, I focused on withdrawal behavior in this study. Interestingly, independently of the attribution condition, participants spent more time withdrawing from virtual agents, when an unpleasant odor was presented. In sum, I demonstrated that the attribution of the odors to the social agent itself had an enhancing effect on their influence on social interaction. In the fourth and last study, I applied a similar within-subjects protocol as in Study 3 with an additional Ultimatum Game task as a measure of social interaction. Overall findings replicated the results of Study 3 with respect to HR and EDA. Strikingly, participants offered less money to virtual agents in the bad smelling room than in the good smelling room. In contrast to Study 3, no effects of odor attribution were found in Study 4. In sum, again I demonstrated that unpleasant odor may lessen social interaction not only when the interaction partner smells badly, but also in more complex interaction situations. In conclusion, I demonstrated that hedonic odors in general influence social interaction. Thus, pleasant odors seem to facilitate, while unpleasant odors seem to reduce interpersonal exchanges. Therefore, the present thesis extends the body of literature on the influence of odors on the processing of social stimuli. Although I found a direct influence of odors on social preferences as well as on the physiological and behavioral responses to social stimuli, I did not disentangle impact of odor per se from the impact of the affective state. Interestingly, odor attribution might play an additional role as mediator of social interactions such as odor effects in social interactions might be boosted when the smell is attributed to an individual. However, the results in this regard were less straightforward, and therefore further investigations are needed. Future research should also take into account gender or other inter-individual differences like social anxiety.},
  subject      = {smell},
  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{Awad2019,
  author    = {Awad, Eman Da'as},
  title     = {Modulation of insulin-induced genotoxicity in vitro and genomic damage in gestational diabetes},
  doi       = {10.25972/OPUS-16186},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-161866},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Diabetes mellitus is a global health problem, where the risk of diabetes increases rapidly due to the lifestyle changes. Patients with type II diabetes have many complications with increased risk of morbidity and mortality. High levels of insulin may lead to DNA oxidation and damage. Several studies proposed that hyperinsulinemia may be an important risk factor for various types of cancer. To investigate insulin signaling pathway inducing oxidative stress and genomic damage, pharmaceutical and natural compounds which can interfere with the insulin pathway including PI3K inhibitors, resveratrol, lovastatin, and RAD-001 were selected due to their beneficial effects against metabolic disorder. Thus, the anti-genotoxic potential of these compounds regarding insulin-mediated oxidative stress were investigated in normal rat kidney cells in vitro. Our compounds showed protective effect against genotoxic damage and significantly decreased reactive oxygen specious after treatment of cells with insulin with different mechanisms of protection between the compounds. Thus, these compounds may be attractive candidates for future support of diabetes mellitus therapy. Next, we explored the link between gestational diabetes mellitus and genomic damage in cells derived from human blood. Moreover, we investigated the influence of estradiol, progesterone, adrenaline and triiodothyronine on insulin-induced genomic damage in vitro. First, we studied the effect of these hormones in human promyelocytic leukemia cells and next ex vivo with non-stimulated and stimulated peripheral blood mononuclear cells. In parallel, we also measured the basal genomic damage using three conditions (whole blood, non-stimulated and stimulated peripheral blood mononuclear cells) in a small patient study including non-pregnant controls with/without hormonal contraceptives, with a subgroup of obese women, pregnant women, and gestational diabetes affected women. A second-time point after delivery was also applied for analysis of the blood samples. Our results showed that GDM subjects and obese individuals exhibited higher basal DNA damage compared to lower weight nonpregnant or healthy pregnant women in stimulated peripheral blood mononuclear cells in both comet and micronucleus assays. On the other hand, the DNA damage in GDM women had decreased at two months after birth. Moreover, the applied hormones also showed an influence in vitro in the enhancement of the genomic damage in cells of the control and pregnant groups but this damage did not exceed the damage which existed in obese and gestational diabetes mellitus patients with high level of genomic damage. In conclusion, insulin can induce genomic damage in cultured cells, which can be modulated by pharmaceutical and naturals substances. This may be for future use in the protection of diabetic patients, who suffer from hyperinsulinemia during certain disease stages. A particular form of diabetes, GDM, was shown to lead to elevated DNA damage in affected women, which is reduced again after delivery. Cells of affected women do not show an enhanced, but rather a reduced sensitivity for further DNA damage induction by hormonal treatment in vitro. A potential reason may be an existence of a maximally inducible damage by hormonal influences.},
  subject      = {Gestationsdiabetes},
  language  = {en}
}
@phdthesis{Schulze2020,
  author    = {Schulze, Andrea},
  title     = {Investigating the mechanism of the Hsp90 molecular chaperone using photoinduced electron transfer fluorescence quenching},
  doi       = {10.25972/OPUS-16215},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-162155},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {The molecular chaperone Hsp90 facilitates the folding and activation of a wide array of structurally and functionally diverse client proteins. Hsp90 presents a central node of protein homeostasis and is frequently involved in the development of many human diseases. Although Hsp90 is a promising target for disease treatment, the mechanism by which Hsp90 facilitates client recognition and maturation is poorly understood. The shape of the homodimeric protein resembles a molecular clamp that opens and closes in response to binding and hydrolysis of ATP. Structural studies reveal a network of distinct local conformational rearrangements that coordinate the slow transition into the hydrolysis-active, closed state configuration (time order of minutes). However, the kinetics of local conformational changes remain elusive because spectroscopic tools that can detect them have been missing so far. Fluorescence quenching of extrinsic fluorophores by the natural amino acid Tryptophan is based on a photoinduced electron transfer (PET) reaction, which requires sub-nanometer contact between fluorophore and Tryptophan. This quenching mechanism has been developed into a 1-nm spectroscopic tool for the detection of rapid protein folding dynamics. Within the scope of this doctoral thesis, PET-reporter systems were designed to investigate the kinetics of local conformational motions that are part of the mechanistic core of the Hsp90 chaperone cycle. ATP-triggered kinetics of closure of the ATP-lid as well as swapping of the N-terminal ß-strand across subunits and association of the N-terminal and middle-domain were estimated in solution. Bulk experiments revealed that local motions occur on similar timescales and are in good agreement with the ATP-hydrolysis rate. Functional mutations demonstrated that local motions act cooperatively. Furthermore, the lid was shown to close via a two-step process consisting of a rapid lid-reconfiguration in direct response to ATP-binding, followed by slow closure of the lid. The co-chaperone Aha1 seems to act early in the chaperone cycle by remodelling of the lid and by stabilization of apo Hsp90 in a NM-domain pre-associated conformation. A two-colour single-molecule PET microscopy method was developed to observe local motions at remote positions simultaneously and in real-time. Thus, directionality within the network of local conformational changes could be revealed. In a first attempt, the feasibility of detecting PET-complexes on the single-molecule surface was tested on Hsp90 constructs that report on only one motion (one-colour single-molecule PET microscopy). PET-quenched complexes could be distinguished from photobleached fluorophores through oxidation by molecular oxygen, resulting in fluorescence recovery. In two-colour experiments, a dimmed state was identified for PET-quenched complexes, but not for all of the used PET-reporter systems. Results suggest that local motions occur simultaneously within the time-resolution of the experiment (0.3 sec). Furthermore, bi-exponential kinetics of transition into the closed clamp configuration indicate a more complex mechanism of clamp-closure than of clamp-opening, which could be well described by a mono-exponential function.},
  subject      = {Hitzeschock-Proteine},
  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{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{Wermser2019,
  author    = {Wermser, Charlotte},
  title     = {Morphology, regulation and interstrain interactions in a new macrocolony biofilm model of the human pathogen \(Staphylococcus\) \(aureus\)},
  doi       = {10.25972/OPUS-16593},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-165931},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {The role of multicellularity as the predominant microbial lifestyle has been affirmed by studies on the genetic regulation of biofilms and the conditions driving their formation. Biofilms are of prime importance for the pathology of chronic infections of the opportunistic human pathogen Staphylococcus aureus. The recent development of a macrocolony biofilm model in S. aureus opened new opportunities to study evolution and physiological specialization in biofilm communities in this organism. In the macrocolony biofilm model, bacteria form complex aggregates with a sophisticated spatial organization on the micro- and macroscale. The central positive and negative regulators of this organization in S. aureus are the alternative sigma factor σB and the quorum sensing system Agr, respectively. Nevertheless, nothing is known on additional factors controlling the macrocolony morphogenesis. In this work, the genome of S. aureus was screened for novel factors that are required for the development of the macrocolony architecture. A central role for basic metabolic pathways was demonstrated in this context as the macrocolony architecture was strongly altered by the disruption of nucleotide and carbohydrate synthesis. Environmental signals further modulate macrocolony morphogenesis as illustrated by the role of an oxygen-sensitive gene regulator, which is required for the formation of complex surface structures. A further application of the macrocolony biofilm model was demonstrated in the study of interstrain interactions. The integrity of macrocolony communities was macroscopically visibly disturbed by competitive interactions between clinical isolates of S. aureus. The results of this work contribute to the characterization of the macrocolony biofilm model and improve our understanding of developmental processes relevant in staphylococcal infections. The identification of anti-biofilm effects exercised through competitive interactions could lead to the design of novel antimicrobial strategies targeting multicellular bacterial communities.},
  subject      = {Staphylococcus aureus},
  language  = {en}
}
@phdthesis{Uri2019,
  author    = {Uri, Anna},
  title     = {Differential requirement for CD28 co-stimulation on donor T cell subsets in mouse models of acute graft versus host disease and graft versus tumour effect},
  doi       = {10.25972/OPUS-16586},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-165863},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Hematopoietic stem cell transplantation is a curative therapy for malignant diseases of the haematopoietic system. The patients first undergo chemotherapy or irradiation therapy which depletes the majority of tumour cells before they receive the transplant, consisting of haematopoietic stem cells and mature T cells from a healthy donor. The donor T cells kill malignant cells that have not been eliminated by the conditioning therapy (graft versus leukaemia effect, GvL), and, therefore, are crucially required to prevent relapse of the tumour. However, the donor T cells may also severely damage the patient's organs causing acute graft versus host disease (aGvHD). In mice, aGvHD can be prevented by interfering with the co-stimulatory CD28 signal on donor T cells. However, experimental models using conventional CD28 knockout mice as T cell donors or αCD28 antibodies have some disadvantages, i.e. impaired T cell development in the thymus of CD28 knockout mice and systemic CD28 blockade with αCD28 antibodies. Thus, it remains unclear how CD28 co-stimulation on different donor T cell subsets contributes to the GvL effect and aGvHD, respectively. We developed mouse models of aGvHD and the GvL effect that allowed to selectively delete CD28 on certain donor T cell populations or on all donor T cells. CD4+ conventional T cells (Tconv cells), regulatory T cells (Treg cells) or CD8+ T cells were isolated from either Tamoxifen-inducible CD28 knockout (iCD28KO) mice or their wild type (wt) littermates. Allogeneic recipient mice were then transplanted with T cell depleted bone marrow cells and different combinations of iCD28KO and wt T cell subsets. Tamoxifen treatment of the recipients caused irreversible CD28 deletion on the iCD28KO donor T cell population. In order to study the GvL response, BCL-1 tumour cells were injected into the mice shortly before transfer of the T cells. CD4+ Tconv mediated aGvHD was efficiently inhibited when wt Treg cells were co-transplanted. In contrast, after selective CD28 deletion on donor Treg cells, the mice developed a late and lethal flare of aGvHD, i.e. late-onset aGvHD. This was associated with a decline in iCD28KO Treg cell numbers around day 20 after transplantation. CD28 ablation on either donor CD4+ Tconv cells or CD8+ T cells reduced but did not abrogate aGvHD. Moreover, iCD28KO and wt CD8+ T cells were equally capable of killing allogeneic target cells in vivo and in vitro. Due to this sufficient anti-tumour activity of iCD28KO CD8+ T cells, they had a therapeutic effect in our GvL model and 25\% of the mice survived until the end of the experiment (day 120) without any sign of the malignant disease. Similarly, CD28 deletion on all donor T cells induced long-term survival. This was not the case when all donor T cells were isolated from wt donor mice. In contrast to the beneficial outcome after CD28 deletion on all donor T cells or only CD8+ T cells, selective CD28 deletion on donor CD4+ Tconv cells completely abrogated the GvL effect due to insufficient CD4+ T cell help from iCD28KO CD4+ Tconv cells. This study demonstrates that therapeutic inhibition of the co-stimulatory CD28 signal in either all donor T cells or only in CD8+ T cells might protect patients from aGvHD without increasing the risk of relapse of the underlying disease. Moreover, deletion of CD28 on donor Treg cells constitutes a mouse model of late-onset aGvHD which can be a useful tool in aGvHD research.},
  subject      = {Antigen CD28},
  language  = {en}
}
@phdthesis{Goetz2018,
  author    = {G{\"o}tz, Silvia},
  title     = {Zuo1 - ein neues G-Quadruplex-bindendes Protein in \(Saccharomyces\) \(cerevisiae\)},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-152158},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {G-Quadruplex (G4)-Strukturen sind sehr stabile und polymorphe DNA und RNA Sekund{\"a}rstrukturen mit einem konservierten Guanin-reichen Sequenzmotiv (G4-Motiv). Sie bestehen aus {\"u}bereinander gestapelten planaren G-Quartetts, in denen je vier Guanine durch Wasserstoffbr{\"u}ckenbindungen zusammengehalten werden. Da G4-Motive in Eukaryoten an bestimmten Stellen im Genom angereichert vorkommen, wird angenommen, dass die Funktion von G4-Strukturen darin besteht, biologische Prozesse positiv oder negativ zu regulieren. Aufgrund der hohen thermodynamischen Stabilit{\"a}t von G4 Strukturen ist davon auszugehen, dass Proteine in die Faltung, Stabilisierung und Entfaltung dieser Nukleins{\"a}ure-Strukturen regulatorisch involviert sind. Bis heute wurden viele Proteine in der Literatur beschrieben, die G4-Strukturen entwinden k{\"o}nnen. Jedoch konnten bisher nur wenige Proteine identifiziert werden, die in vivo die Faltung f{\"o}rdern oder G4-Strukturen stabilisieren. Durch Yeast One-Hybrid (Y1H)-Screenings habe ich Zuo1 als neues G4 bindendes Protein identifiziert. In vitro Analysen best{\"a}tigten diese Interaktion und es stellte sich heraus, dass Zuo1 G4-Strukturen stabilisiert. {\"U}bereinstimmend mit den in vitro Daten konnte gezeigt werden, dass Zuo1 signifikant an G4-Motive im Genom von Saccharomyces ceresivisiae bindet. Genomweit {\"u}berlappen G4-Motive, an die Zuo1 bindet, mit Stellen, an denen die DNA Replikation zum Stillstand kommt und vermehrt DNA Sch{\"a}den vorkommen. Diese Ergebnisse legen nahe, dass Zuo1 eine Funktion w{\"a}hrend der DNA Reparatur oder in Zusammenhang mit dem Vorankommen der DNA Replikationsgabel hat, indem G4-Strukturen stabilisiert werden. Diese Hypothese wird außerdem durch genetische Experimente gest{\"u}tzt, wonach in Abwesenheit von Zuo1 die Genominstabilit{\"a}t zunimmt. Aufgrund dieser Daten war es m{\"o}glich ein Model zu entwickeln, bei dem Zuo1 w{\"a}hrend der S-Phase G4-Strukturen bindet und stabilisiert wodurch die DNA Replikation blockiert wird. Diese Interaktion findet neben Stellen schadhafter DNA statt und unterst{\"u}tzt somit DNA Reparatur-Prozesse wie beispielsweise die Nukleotidexzisionsreparatur. Als weiteres potentielles G4-bindendes Protein wurde Slx9 in Y1H-Screenings identifiziert. In vitro Experimente zeigten zwar, dass Slx9 mit h{\"o}herer Affinit{\"a}t an G4-Strukturen bindet im Vergleich zu anderen getesteten DNA Konformationen, jedoch wurde in S. cerevisiae genomweit keine signifikante Bindung an G4-Motive festgestellt.},
  subject      = {Saccharomyces cerevisiae},
  language  = {de}
}
@phdthesis{Weigand2021,
  author    = {Weigand, Isabel},
  title     = {Consequences of Protein Kinase A mutations in adrenocortical cells and tumours},
  doi       = {10.25972/OPUS-16064},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-160646},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Adrenal Cushing's Syndrome (CS) is a rare but life-threatening disease and therefore it is of great importance to understand the pathogenesis leading to adrenal CS. It is well accepted that Protein Kinase A (PKA) signalling mediates steroid secretion in adrenocortical cells. PKA is an inactive heterotetramer, consisting of two catalytic and two regulatory subunits. Upon cAMP binding to the regulatory subunits, the catalytic subunits are released and are able to phosphorylate their target proteins. Recently, activating somatic mutations affecting the catalytic subunit a of PKA have been identified in a sub-population of cortisol-producing adenomas (CPAs) associated with overt CS. Interestingly, the PKA regulatory subunit IIb has long been known to have significantly lower protein levels in a sub-group of CPAs compared to other adrenocortical tumours. Yet, it is unknown, why these CPAs lack the regulatory subunit IIb, neither are any functional consequences nor are the underlying regulation mechanisms leading to reduced RIIb levels known. The results obtained in this thesis show a clear connection between Ca mutations and reduced RIIb protein levels in CPAs but not in other adrenocortical tumours. Furthermore, a specific pattern of PKA subunit expression in the different zones of the normal adrenal gland is demonstrated. In addition, a Ca L206R mutation-mediated degradation of RIIb was observed in adrenocortical cells in vitro. RIIb degradation was found to be mediated by caspases and by performing mutagenesis experiments of the regulatory subunits IIb and Ia, S114 phosphorylation of RIIb was identified to make RIIb susceptible for degradation. LC-MS/MS revealed RIIb interaction partners to differ in the presence of either Ca WT and Ca L206R. These newly identified interaction partners are possibly involved in targeting RIIb to subcellular compartments or bringing it into spatial proximity of degrading enzymes. Furthermore, reducing RIIb protein levels in an in vitro system were shown to correlate with increased cortisol secretion also in the absence of PRKACA mutations. The inhibiting role of RIIb in cortisol secretion demonstrates a new function of this regulatory PKA subunit, improving the understanding of the complex regulation of PKA as key regulator in many cells.},
  subject      = {Cushing-Syndrom},
  language  = {en}
}
@phdthesis{Martin2018,
  author    = {Martin, Corinna},
  title     = {Oxidized phospholipids and their role in neuronal excitation of primary sensory neurons},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-160665},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Recently, our research group identified in a study novel proalgesic targets in acute and chronic inflammatory pain: oxidized phospholipids (OxPL). OxPL, endogenous chemical irritants, are generated in inflamed tissue and mediate their pain-inducing function by activating the transient receptor potential channels TRPA1 and TRPV1. Both channels are sensors for chemical stimuli on primary afferent nociceptors and are involved in nociception. Here, with the help of calcium imaging and whole cell patch clamp recording techniques, it was found that OxPL metabolites acutely activate TRPA1 and TRPV1 ion channels to excite DRG neurons. OxPL species act predominantly via TRPA1 ion channels and mediate long- lasting non-selective inward currents. Notably, one pure OxPL compound, PGPC, activated a TRPA1 mutant lacking the binding site for electrophilic agonists, suggesting that OxPL activate TRP ion channels by an indirect mechanical mechanism. Next, it was investigated how OxPL influence the excitability of primary sensory neurons. Acute stimulation and fast calcium imaging revealed that OxPL elicit repetitive, spike-like calcium transients in small- diameter DRG neurons, which were fully blocked by antagonists against TRPA1/V1 and N- type voltage-gated calcium channels. In search of a mechanism that drives repetitive spiking of DRG neurons, it was asked whether NaV1.9, a voltage-gated sodium channel involved in subthreshold excitability and nociception, is needed to trigger OxPL-induced calcium spikes and action potential firing. In electrophysiological recordings, both the combination of local application of OxPL and current injection were required to efficiently increase the action potential (AP) frequency of small-diameter sensory neurons. However, no difference was monitored in the resting membrane potential or OxPL-induced AP firing rate between wt and NaV1.9-deficient small diameter DRG neurons. To see whether NaV1.9 needs inflammatory conditions to be integrated in the OxPL-induced excitation cascade, sensory neurons were pretreated with a mixture of inflammatory mediators before OxPL application. Under inflammatory conditions both the AP and the calcium-spike frequency were drastically enhanced in response to an acute OxPL stimulus. Notably, this potentiation of OxPL stimuli was entirely lost in NaV1.9 deficient sensory neurons. Under inflammatory conditions, the resting membrane potential of NaV1.9-deficient neurons was more negative compared to wt neurons, suggesting that NaV1.9 shows resting activity only under inflammatory conditions. In conclusion, OxPL are endogenous irritants that induce excitability in small-diameter DRG neurons, a cellular model of nociceptors, via TRP activation. This effect is potentiated under inflammatory conditions. Under these conditions, NaV1.9 functions as essential mediator as it eases the initiation of excitability after OxPL stimulation. As mutants in the human NaV1.9 mediate an enhanced or painless perception, this study provides new insight into the mechanism on how NaV1.9 amplifies stimuli of endogenous irritants under inflammatory conditions.},
  subject      = {Entz{\"u}ndung},
  language  = {en}
}
@phdthesis{Gerner2019,
  author    = {Gerner, Frank},
  title     = {Functional analysis of polarization and podosome formation of murine and human megakaryocytes},
  doi       = {10.25972/OPUS-16050},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-160508},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {In mammals, blood platelets are produced by large bone marrow (BM) precursor cells, megakaryocytes (MK) that extend polarized cell protrusions (proplateles) into BM sinusoids. Proplatelet formation (PPF) requires substantial cytoskeletal rearrangements that have been shown to involve the formation of podosomes, filamentous actin (F-actin) and integrin-rich structures. However, the exact molecular mechanisms regulating MK podosome formation, polarization and migration within the BM are poorly defined. According to current knowledge obtained from studies with other cell types, these processes are regulated by Rho GTPase proteins like RhoA and Cdc42. In this thesis, polarization and podosome formation were investigated in MKs from genetically modified mice, as well as the cell lines K562 and Meg01 by pharmacological modulation of signaling pathways. The first part of this thesis describes establishment of the basic assays for investigation of MK polarization. Initial data on polarization of the MK-like erythroleukemia cell line K562 revealed first insights into actin and tubulin dynamics of wild type (WT) and RhoA knock-out (RhoA-/-) K562 cells. Phorbol 12-myristate 13-acetate (PMA)-induction of K562 cells led to the expected MK-receptor upregulation but also RhoA depletion and altered polarization patterns. The second part of this thesis focuses on podosome formation of MKs. RhoA is shown to be dispensable for podosome formation. Cdc42 is revealed as an important, but not essential regulator of MK spreading and podosome formation. Studies of signaling pathways of podosome formation reveal the importance of the tyrosine kinases Src, Syk, as well as glycoprotein (GP)VI in MK spreading and podosome formation. This thesis provides novel insights into the mechanisms underlying polarization and podosome formation of MKs and reveals new, important information about cytoskeletal dynamics of MKs and potentially also platelets.},
  subject      = {Megakaryozyt},
  language  = {en}
}
@phdthesis{Hofmann2018,
  author    = {Hofmann, Lukas},
  title     = {The α-galactosidase A deficient mouse as a model for Fabry disease and the effect of Gb3 depositions on peripheral nociceptive ion channel function},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-158513},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Fabry disease (FD) is an X-linked lysosomal storage disorder with intracellular accumulation of globotriaosylceramide (Gb3) due to α-galactosidase A deficiency. We studied α-galactosidase A knockout mice (GLA KO) as a model for sensory disturbance and pain in FD. Pain associated behavior of young (3 months) and old (≥18 months) GLA KO mice and wildtype (WT) littermates in an inflammatory and a neuropathic pain model was investigated. Furthermore, affective and cognitive behavior was assessed in the na{\"i}ve state and in an inflammatory pain model. Gene and protein expression of pain associated ion channels and Gb3 accumulation in dorsal root ganglion (DRG) neurons was determined. We also performed patch clamp analysis on cultivated DRG neurons and human embryonic kidney 293 (HEK) cells expressing voltage-gated-sodium channel 1.7 (Nav1.7) as an in vitro model of FD. Intracellular Gb3 deposits were modulated using shRNA silencing of α-galactosidase A. After intraplantar injection of complete Freund`s adjuvant (CFA) and chronic constriction injury (CCI) of the right sciatic nerve, old GLA KO mice did not develop heat and mechanical hypersensitivity in contrast to young GLA KO and old WT mice. Additionally, we found no relevant differences between genotypes and age-groups in affective and cognitive behavior in the na{\"i}ve state and after CFA injection. Gene and protein expression analysis provided no explanation for the observed sensory impairment. However, cultured DRG neurons of old GLA KO mice revealed a marked decrease of sodium and Ih-currents compared to young GLA KO and old WT mice. DRG neurons of old GLA KO mice displayed substantial intracellular accumulation of Gb3 compared to young GLA KO and old WT mice. Similar to cultured neurons, sodium currents were also decreased in HEK cells treated with shRNA and consecutively increased intracellular Gb3 deposits compared to the control condition, but could be rescued by treatment with agalsidase-alpha. Our study unveils that, similar to patients with FD, GLA KO mice display age-dependent sensory deficits. However, contrary to patients, GLA KO mice are also protected from hypersensitivity induced by inflammation and nerve lesion due to Gb3-dependent and reversible reduction of neuronal sodium- and Ih-currents. Our data provide evidence for direct Gb3-dependent ion channel impairment in sensory DRG neurons as a potential contributor to sensory dysfunction and pain in FD.},
  subject      = {Fabry-Krankheit},
  language  = {en}
}
@phdthesis{MontaggebKukielka2018,
  author    = {Montag [geb. Kukielka], Gracia Anna},
  title     = {Rolle des Differenzierungszustandes f{\"u}r die Empfindlichkeit von S{\"a}ugerzellen gegen{\"u}ber genotoxischen Agenzien},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-164670},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {In der vorliegenden Arbeit wurden die Einfl{\"u}sse verschiedener genotoxischer Substanzen auf S{\"a}ugertierzellen untersucht. Da ein Organismus der Ontogenese unterliegt und sich Zellen aus Stamm- und Vorl{\"a}uferzellen entwickelt, gilt es diese urspr{\"u}nglichen Zellen vor {\"a}ußeren Einfl{\"u}ssen zu sch{\"u}tzen. Da bisher kaum Untersuchungen von Zellen in verschiedenen Differenzierungsstadien durchgef{\"u}hrt wurden, wurden unter Verwendung vieler unterschiedlicher biologischer Endpunkte Effekte auf die Vitalit{\"a}t, Proliferation, Mitose und Apoptose dieser Zellen untersucht. Zudem erfolgte eine Interpretation der Ausbildung von Mikrokernen, Entstehung von DNS-Sch{\"a}den und der zugrundeliegenden Reparaturmechanismen. So konnte mit Hilfe der Untersuchungen der h{\"a}matopoetischen Stammzellen und der TK6-Zellen postuliert werden, dass h{\"a}matopoetische Stammzellen weitestgehend weniger empfindlich gegen{\"u}ber Zytostatika (Doxorubicin, Vinblastin, Methylmethansulfonat und Mitomycin C) sind als die lymphoblastoide Zelllinie TK6, welche in der Entwicklungshierarchie den Stammzellen folgt. Die Bef{\"u}rchtung, dass der Mikrokerntest in immortalisierten TK6-Zellen als Grundlage f{\"u}r Genotoxizit{\"a}tsuntersuchungen nicht gen{\"u}gen w{\"u}rden, konnte mit Hilfe der Versuchsergebnisse dieser Arbeit widerlegt werden. Die Ergebnisse belegen, dass der Mikrokerntest in TK6-Zellen relevant ist, da TK6-Zellen empfindlicher auf genotoxische Agentien im Vergleich zu h{\"a}matopoetischen Stammzellen reagieren. Bei der Untersuchung der Leuk{\"a}miezelllinie HL-60 wurden die Effekte klassischer (Vinblastin, Vincristin, Vinflunin und Vinorelbin) mit neu synthetisierten Vinca-Alkaloiden (4-Chlorochablastin, 4-Chlorochacristin, 16a, 17b und 18a) verglichen. Vinca-Alkaloide werden sehr h{\"a}ufig mit Nebenwirkungen, wie Neuropathien assoziiert, welche w{\"a}hrend einer Chemotherapie oftmals zu Therapieabbr{\"u}chen durch die Patienten f{\"u}hren. Aus diesem Grund war es erstrebenswert, neuartige Vinca-Alkaloide zu entwickeln, welche weniger Nebenwirkungen aber zugleich eine {\"a}hnliche Wirksamkeit aufweisen. Obwohl die Potenz der neuen Substanzen niedriger war als bei Vinblastin, Vincristin und Vinorelbin, zeigte ein Teil eine {\"a}hnliche Wirkung wie das Vinca-Alkaloid Vinflunin auf die Krebszelllinie HL-60 auf. Die Ergebnisse diese Arbeit k{\"o}nnen als erste Indikation in vitro genommen werden, dass sich diese Substanzen in der Krebstherapie als wirksam erweisen k{\"o}nnten und nach weiteren Ergebnissen in vivo als therapeutische Alternativen in Betracht gezogen werden. Auch bei der vergleichenden Untersuchung von exponentiell wachsenden mit differenzierten Zelllinien konnten Unterschiede detektiert werden. Die Zelllinie HT-22, welche selbst keine Krebszelllinie ist, zeigte nach Differenzierung zu nicht exponentiell wachsenden Zellen eine erh{\"o}hte Empfindlichkeit gegen{\"u}ber dem Alkylanz Methylmethansulfonat, was auf einer verminderten Basenexzisionsreparatur beruhen k{\"o}nnte. Auch die differenzierte Form der Adenokarzinom-Zelllinie CaCo2 zeigte eine gesteigerte Sensitivit{\"a}t gegen{\"u}ber dem Topoisomerase II-Inhibitor Etoposid auf, wohingegen der unselektive Topoisomerase II-Hemmer Doxorubicin keinen Effekt aufwies. Um den Sachverhalt zu kl{\"a}ren ob die festgestellten Unterschiede auf das Enzym Topoisomerase II zur{\"u}ckzuf{\"u}hren oder zellartspezifisch waren, wurden weitere Analysen der Zelllinien HL-60 und deren differenzierten Zellart durchgef{\"u}hrt. Auch hier konnten signifikante Unterschiede bei der Einzelzellgelelektrophorese nach Behandlung mit Doxorubicin und Etoposid festgestellt werden. Neben den in dieser Arbeit nachgewiesenen Unterschieden bei der Reparatur zwischen den Zelltypen, k{\"o}nnten aber auch weitere Faktoren zu Varianzen f{\"u}hren und die Mutagenit{\"a}tsforschung beeinflussen. Folglich ist davon auszugehen, dass zuk{\"u}nftige Testungen bei der pharmakologischen Substanzentwicklung in verschiedenen Zellsystemen von N{\"o}ten sind, bevor neue Substanzen zugelassen werden. Alles in allem konnte die Komplexit{\"a}t der Ergebnisse zwischen Zellen der verschiedenen Differenzierungsstadien in dieser Arbeit aufgezeigt werden. Deswegen sollte auch bei weiteren Forschungsvorhaben insbesondere ein Augenmerk auf den Differenzierungszustand der zu untersuchenden Zellpopulation geworfen werden.},
  subject      = {S{\"a}ugetiere},
  language  = {de}
}
@phdthesis{Heidenreich2018,
  author    = {Heidenreich, Julius Frederik},
  title     = {Characterization of the widely used Rac1-inhibitors NSC23766 and EHT1864 in mouse platelets},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-165453},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Platelet activation and aggregation at sites of vascular injury is critical to prevent excessive blood loss, but may also lead to life-threatening ischemic diseases, such as myocardial infarction and stroke. Extracellular agonists induce platelet activation by stimulation of platelet membrane receptors. Signal transduction results in reorganization of the cytoskeleton, shape change, platelet adhesion and aggregation, cumulating in thrombus formation. Several Rho GTPases, including Rac1, Cdc42 and RhoA, are essential mediators of subsequent intracellular transduction of ITAM- and GPCR-signaling. Therefore, inhibition or knockout can result in severely defective platelet signaling. Mice with platelet specific Rac1-deficiency are protected from arterial thrombosis. This benefit highlights further investigation of Rac1-specific functions and its potential as a new pharmacological target for prevention of cardiovascular diseases. Two newly developed synthetic compounds, NSC23766 and EHT1864, were proposed to provide highly specific inhibition of Rac1 activity, but both drugs have never been tested in Rac1-deficient cell systems to rule out potential Rac1-independent effects. This study revealed significant off-target effects of NSC23766 and EHT1864 that occurred in a dose-dependent fashion in both wild-type and Rac1-deficient platelets. Both inhibitors individually affected resting platelets after treatment, either by altering membrane protein expression (NSC23766) or by a marked decrease of platelet viability (EHT1864). Platelet apoptosis could be confirmed by enhanced levels of phosphatidylserine exposure and decreased mitochondrial membrane potential. Phosphorylation studies of the major effector proteins of Rac1 revealed that NSC23766 and EHT1864 abolish PAK1/PAK2 activation independently of Rac1 in wild-type and knockout platelets, which may contribute to the observed off-target effects. Additionally, this study demonstrated the involvement of Rac1 in G protein-coupled receptor-mediated platelet activation and GPIb-induced signaling. Furthermore, the data revealed that Rac1 is dispensable in the process of integrin IIb 3-mediated clot retraction. This study unveiled that new pharmacological approaches in antithrombotic therapy with Rac1 as molecular target have to be designed carefully in order to obtain high specificity and minimize potential off-target effects.},
  subject      = {Thrombozyt},
  language  = {en}
}
@phdthesis{Potabattula2019,
  author    = {Potabattula, Ramya Sri Krishna},
  title     = {Male aging and obesity effects on sperm methylome and consequences for the next generation},
  doi       = {10.25972/OPUS-16548},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-165481},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Besides a growing tendency for delayed parenthood, sedentary lifestyle coupled with overnutrition has dramatically increased worldwide over the last few decades. Epigenetic mechanisms can help us understand the epidemics and heritability of complex traits like obesity to a significant extent. Majority of the research till now has focused on determining the impact of maternal factors on health and disease risk in the offspring(s). This doctoral thesis is focused on deciphering the potential effects of male aging and obesity on sperm methylome, and consequences/transmission via germline to the next generation. In humans, this was assessed in a unique cohort of ~300 sperm samples, collected after in vitro fertilization/intracytoplasmic sperm injection, as well as in conceived fetal cord blood samples of the children. Furthermore, aging effect on sperm samples derived from a bovine cohort was analyzed. The study identified that human male aging significantly increased the DNA methylation levels of the promoter, the upstream core element, the 18S, and the 28S regions of ribosomal DNA (rDNA) in sperm. Prediction models were developed to anticipate an individual's age based on the methylation status of rDNA regions in his sperm. Hypermethylation of alpha satellite and LINE1 repeats in human sperm was also observed with aging. Epimutations, which are aberrantly methylated CpG sites, were significantly higher in sperm of older males compared to the younger ones. These effects on the male germline had a negative impact on embryo quality of the next generation. Consistent with these results, DNA methylation of rDNA regions, bovine alpha satellite, and testis satellite repeats displayed a significant positive correlation with aging sperm samples within the same individual and across different age-grouped bulls. A positive association between human male obesity/body mass index (BMI) and DNA methylation of the imprinted MEG3 gene and the obesity-related HIF3A gene was detected in sperm. These BMI-induced sperm DNA methylation signatures were transmitted to next generation fetal cord blood (FCB) samples in a gender-specific manner. Males, but not female offsprings exhibited a significant positive correlation between father's BMI and FCB DNA methylation in the two above-mentioned amplicons. Additionally, hypomethylation of IGF2 with increased paternal BMI was observed in female FCB samples. Parental allele-specific in-depth methylation analysis of imprinted genes using next generation sequencing technology also revealed significant correlations between paternal factors like age and BMI, and the corresponding father's allele DNA methylation in FCB samples. Deep bisulphite sequencing of imprinted genes in diploid somatic cord blood cells of offspring detected that the levels of DNA methylation signatures largely depended on the underlying genetic variant, i.e. sequence haplotypes. Allele-specific epimutations were observed in PEG1, PEG5, MEG3, H19, and IGF2 amplicons. For the former three genes, the non-imprinted unmethylated allele displayed more epimutations than the imprinted methylated allele. On the other hand, for the latter two genes, the imprinted allele exhibited higher epimutation rate than that of the non-imprinted allele. In summary, the present study proved that male aging and obesity impacts the DNA methylome of repetitive elements and imprinted genes respectively in sperm, and also has considerable consequences on the next generation. Nevertheless, longitudinal follow-up studies are highly encouraged to elucidate if these effects can influence the risk of developing abnormal phenotype in the offspring during adulthood.},
  language  = {en}
}
@phdthesis{Thomas2021,
  author    = {Thomas, Sarah Katharina},
  title     = {Design of novel IL-4 antagonists employing site-specific chemical and biosynthetic glycosylation},
  doi       = {10.25972/OPUS-17517},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-175172},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {The cytokines interleukin 4 (IL-4) and IL-13 are important mediators in the humoral immune response and play a crucial role in the pathogenesis of chronic inflammatory diseases, such as asthma, allergies, and atopic dermatitis. Hence, IL-4 and IL-13 are key targets for treatment of such atopic diseases. For cell signalling IL-4 can use two transmembrane receptor assemblies, the type I receptor consisting of receptors IL-4R and γc, and type II receptor consisting of receptors IL-4R and IL-13R1. The type II receptor is also the functional receptor of IL-13, receptor sharing being the molecular basis for the partially overlapping effects of IL-4 and IL-13. Since both cytokines require the IL-4R receptor for signal transduction, this allows the dual inhibition of both IL-4 and IL-13 by specifically blocking the receptor IL-4R. This study describes the design and synthesis of novel antagonistic variants of human IL-4. Chemical modification was used to target positions localized in IL-4 binding sites for γc and IL-13R1 but outside of the binding epitope for IL-4R. In contrast to existing studies, which used synthetic chemical compounds like polyethylene glycol for modification of IL-4, we employed glycan molecules as a natural alternative. Since glycosylation can improve important pharmacological parameters of protein therapeutics, such as immunogenicity and serum half-life, the introduced glycan molecules thus would not only confer a steric hindrance based inhibitory effect but simultaneously might improve the pharmacokinetic profile of the IL-4 antagonist. For chemical conjugation of glycan molecules, IL-4 variants containing additional cysteine residues were produced employing prokaryotic, as well as eukaryotic expression systems. The thiol-groups of the engineered cysteines thereby allow highly specific modification. Different strategies were developed enabling site-directed coupling of amine- or thiol- functionalized monosaccharides to introduced cysteine residues in IL-4. A linker-based coupling procedure and an approach requiring phenylselenyl bromide activation of IL-4 thiol-groups were hampered by several drawbacks, limiting their feasibility. Surprisingly, a third strategy, which involved refolding of IL-4 cysteine variants in the presence of thiol- glycans, readily allowed synthesis of IL-4 glycoconjugates in form of mixed disulphides in milligram amount. This approach, therefore, has the potential for large-scale synthesis of IL-4 antagonists with highly defined glycosylation. Obtaining a homogenous glycoconjugate with exactly defined glycan pattern would allow using the attached glycan structures for fine-tuning of pharmacokinetic properties of the IL-4 antagonist, such as absorption and metabolic stability. The IL-4 glycoconjugates generated in this work proved to be highly effective antagonists inhibiting IL-4 and/or IL-13 dependent responses in cell-based experiments and in in vitro binding studies. Glycoengineered IL-4 antagonists thus present valuable alternatives to IL-4 inhibitors used for treatment of atopic diseases such as the neutralizing anti-IL-4R antibody Dupilumab.},
  subject      = {Glykosylierung},
  language  = {en}
}
@phdthesis{Wunsch2019,
  author    = {Wunsch, Marie},
  title     = {Das enterische Nervensystem als m{\"o}gliche Zielstruktur der Autoimmunreaktion in der Multiplen Sklerose},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-175888},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Bei der Multiplen Sklerose (MS) handelt es sich um eine Autoimmunerkrankung des zentralen Nervensystems (ZNS). Abh{\"a}ngig von der betroffenen ZNS-Region kann es zu vielf{\"a}ltigen Symptomen kommen. Neben neurologischen Symptomen verursacht durch ZNS-L{\"a}sionen leidet ein Großteil der MS-Patienten auch unter gastrointestinalen Funktionsst{\"o}rungen. Diese gastrointestinalen Symptome wurden bisher eher auf L{\"a}sionen im R{\"u}ckenmark zur{\"u}ckgef{\"u}hrt und nicht direkt in Verbindung mit der autoimmunen {\"A}tiologie der Erkrankung gebracht. In dieser Studie wurde das enterische Nervensystem (ENS) in einem B-Zell- und Antik{\"o}rper-abh{\"a}ngigen Mausmodell der MS untersucht. Daf{\"u}r wurde der Autoimmunprozess durch Immunisierung mit MP4, einem Fusionsprotein aus dem Myelin-Basischen-Protein (MBP) und dem Proteolipid-Protein (PLP), ausgel{\"o}st. Das ZNS und ENS wurden in den unterschiedlichen Erkrankungsstadien immunhistochemisch und elektronenmikroskopisch analysiert. Neben der Immunpathologie des ZNS konnte dabei eine Degeneration des ENS schon vor dem Einsetzen der ersten neurologischen Defizite nachgewiesen werden. Die ENS-Pathologie war antik{\"o}rper-mediiert und ging einher mit einer verringerten gastrointestinalen Motilit{\"a}t sowie mit einer Gliose und Neurodegeneration des ENS. Mithilfe von Immunpr{\"a}zipitation und Massenspektrometrie konnten im ENS vier m{\"o}gliche Zielstrukturen des Autoimmunprozesses identifiziert werden, was auf sog. epitope spreading hindeutet. Auch im Plasma von MS-Patienten konnten Antik{\"o}rper gegen drei dieser Antigene nachgewiesen werden. Des Weiteren zeigten sich in Kolon-Resektaten von MS-Patienten erste Ans{\"a}tze einer Neurodegeneration und Gliose des ENS. In dieser Studie wurde zum ersten Mal ein direkter Zusammenhang zwischen der Autoimmunreaktion gegen das ZNS und einer simultanen Reaktion gegen das ENS gezeigt. Dies kann einen Paradigmenwechsel im Verst{\"a}ndnis der Immunpathogenese der MS anstoßen und neue therapeutische und diagnostische Ans{\"a}tze initiieren.},
  subject      = {Multiple Sklerose},
  language  = {de}
}
@phdthesis{Mayer2019,
  author    = {Mayer, Rafaela},
  title     = {OxPAPC as an endogenous agonist of TRPA1 channels on nociceptors},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-175890},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Non-steroidal antiinflammatory drugs are most commonly used for inflammatory and postoperative pain. But they lack effectiveness and specificity, leading to severe side effects, like gastric ulcers, asthma and severe bleeding. Oxidized 1-palmitoyl-2-arachinidonoyl-sn-glycero-3-phosphocholine (OxPAPC) plays an important role in inflammatory pain. PAPC is a common phosphatidylcholine of membranes, which can be oxidized by reactive oxygen species. In preliminary experiments, our group found that local injection of OxPAPC in rat paws induces hyperalgesia. In this study we examined the effect of OxPAPC on transient receptor potential A1 (TRPA1), an ion channel expressed in C-fiber neurons. Furthermore, we investigated if intracellular cysteine residues of TRPA1 were necessary for agonist-channel-interactions and if a subsequent TRPA1 activation could be prevented by OxPAPC scavengers. To answer these questions, we performed calcium imaging using HEK-293 cells stably expressing hTRPA1, or transiently expressing the triple mutant channel hTRPA1-3C and na{\"i}ve DRG neurons. Cells were incubated with the ratiometric, fluorescent dye Fura-2/AM and stimulated with OxPAPC. The change of light emission after excitation with 340 and 380 nm wavelengths allowed conclusions regarding changes of intracellular calcium concentrations after TRPA1 activation. In our investigation we proved evidence that OxPAPC activates TRPA1, which caused a flow of calcium ions into the cytoplasm. The TRPA1-specific channel blocker HC-030031 eliminated this agonist-induced response. TRPA1-3C was not completely sensitive to OxPAPC. The peptide D-4F and the monoclonal antibody E06 neutralized OxPAPC-induced TRPA1 activation. In this work, the importance of OxPAPC as a key mediator of inflammatory pain and as a promising target for drug design is highlighted. Our results indicate that TRPA1 activation by OxPAPC involves cysteine-dependent mechanisms, but there are other, cysteine-independent activation mechanisms as well. Potential pharmaceuticals for the treatment of inflammatory pain are D-4F and E06, whose efficiency has recently been confirmed in the animal model by our research group.},
  subject      = {Schmerzforschung},
  language  = {en}
}
@phdthesis{Ruedenauer2021,
  author    = {R{\"u}denauer, Fabian},
  title     = {Nutrition facts of pollen: nutritional quality and how it affects reception and perception in bees},
  doi       = {10.25972/OPUS-21254},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-212548},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Nutrients belong to the key elements enabling life and influencing an organism's fitness. The intake of nutrients in the right amounts and ratios can increase fitness; strong deviations from the optimal intake target can decrease fitness. Hence, the ability to assess the nutritional profile of food would benefit animals. To achieve this, they need the according nutrient receptors, the ability to interpret the receptor information via perceptive mechanisms, and the ability to adjust their foraging behavior accordingly. Additionally, eventually existing correlations between the nutrient groups and single nutrient compounds in food could help them to achieve this adjustment. A prominent interaction between food and consumer is the interaction between flowering plants (angiosperms) and animal pollinators. Usually both of the interacting partners benefit from this mutualistic interaction. Plants are pollinated while pollinators get a (most of the times) nutritional reward in form of nectar and/or pollen. As similar interactions between plants and animals seem to have existed even before the emergence of angiosperms, these interactions between insects and angiosperms very likely have co-evolved right from their evolutionary origin. Therefore, insect pollinators with the ability to assess the nutritional profile may have shaped the nutritional profile of plant species depending on them for their reproduction via selection pressure. In Chapter I of this thesis the pollen nutritional profile of many plant species was analyzed in the context of their phylogeny and their dependence on insect pollinators. In addition, correlations between the nutrients were investigated. While the impact of phylogeny on the pollen protein content was little, the mutual outcome of both of the studies included in this chapter is that protein content of pollen is mostly influenced by the plant's dependence on insect pollinators. Several correlations found between nutrients within and between the nutrient groups could additionally help the pollinators to assess the nutrient profile of pollen. An important prerequisite for this assessment would be that the pollinators are able to differentiate between pollen of different plant species. Therefore, in Chapter II it was investigated whether bees have this ability. Specifically, it was investigated whether honeybees are able to differentiate between pollen of two different, but closely related plant species and whether bumblebees prefer one out of three pollen mixes, when they were fed with only one of them as larvae. Honeybees indeed were able to differentiate between the pollen species and bumblebees preferred one of the pollen mixes to the pollen mix they were fed as larvae, possibly due to its nutritional content. Therefore, the basis for pollen nutrient assessment is given in bees. However, there also was a slight preference for the pollen fed as larvae compared to another non-preferred pollen mix, at least hinting at the retention of larval memory in adult bumblebees. Chapter III looks into nutrient perception of bumblebees more in detail. Here it was shown that they are principally able to perceive amino acids and differentiate between them as well as different concentrations of the same amino acid. However, they do not seem to be able to assess the amino acid content in pollen or do not focus on it, but instead seem to focus on fatty acids, for which they could not only perceive concentration differences, but also were able to differentiate between. These findings were supported by feeding experiments in which the bumblebees did not prefer any of the pollen diets containing less or more amino acids but preferred pollen with less fatty acids. In no choice feeding experiments, bumblebees receiving a diet with high fatty acid content accepted undereating other nutrients instead of overeating fat, leading to increased mortality and the inability to reproduce. Hence, the importance of fat in pollen needs to be looked into further. In conclusion, this thesis shows that the co-evolution of flowering plants and pollinating insects could be even more pronounced than thought before. Insects do not only pressure the plants to produce high quality nectar, but also pressure those plants depending on insect pollination to produce high quality pollen. The reason could be the insects' ability to receive and perceive certain nutrients, which enables them to forage selectively leading to a higher reproductive success of plants with a pollinator-suitable nutritional pollen profile.},
  subject      = {Pollen},
  language  = {en}
}
@phdthesis{Schmitt2017,
  author    = {Schmitt, Dominique},
  title     = {Initial characterization of mouse Syap1 in the nervous system: Search for interaction partners, effects of gene knockdown and knockout, and tissue distribution with focus on the adult brain},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-147319},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {The synapse-associated protein of 47 kDa (Sap47) in Drosophila melanogaster is the founding member of a phylogenetically conserved protein family of hitherto unknown molecular function. Sap47 is localized throughout the entire neuropil of adult and larval brains and closely associated with glutamatergic presynaptic vesicles of larval motoneurons. Flies lacking the protein are viable and fertile and do not exhibit gross structural or marked behavioral deficiencies indicating that Sap47 is dispensable for basic synaptic function, or that its function is compensated by other related proteins. Syap1 - the mammalian homologue of Sap47 - was reported to play an essential role in Akt1 phosphorylation in various non-neuronal cells by promoting the association of mTORC2 with Akt1 which is critical for the downstream signaling cascade for adipogenesis. The function of Syap1 in the vertebrate nervous system, however, is unknown so far. The present study provides a first description of the subcellular localization of mouse Syap1 in cultured motoneurons as well as in selected structures of the adult mouse nervous system and reports initial functional experiments. Preceding all descriptive experiments, commercially available Syap1 antibodies were tested for their specificity and suitability for this study. One antibody raised against the human protein was found to recognize specifically both the human and murine Syap1 protein, providing an indispensable tool for biochemical, immunocytochemical and immunohistochemical studies. In the course of this work, a Syap1 knockout mouse was established and investigated. These mice are viable and fertile and do not show obvious changes in morphology or phenotype. As observed for Sap47 in flies, Syap1 is widely distributed in the synaptic neuropil, particularly in regions rich in glutamatergic synapses but it was also detected at perinuclear Golgi-associated sites in certain groups of neuronal somata. In motoneurons the protein is especially observed in similar perinuclear structures, partially overlapping with Golgi markers and in axons, dendrites and axonal growth cones. Biochemical and immunohistochemical analyses showed widespread Syap1 expression in the central nervous system with regionally distinct distribution patterns in cerebellum, hippocampus or olfactory bulb. Besides its expression in neurons, Syap1 is also detected in non-neuronal tissue e.g. liver, kidney and muscle tissue. In contrast, non-neuronal cells in the brain lack the typical perinuclear accumulation. First functional studies with cultured primary motoneurons on developmental, structural and functional aspects reveal no influence of Syap1 depletion on survival and morphological features such as axon length or dendritic length. Contrary to expectations, in neuronal tissues or cultured motoneurons a reduction of Akt phosphorylation at Ser473 or Thr308 was not detected after Syap1 knockdown or knockout.},
  subject      = {Synapse},
  language  = {en}
}
@phdthesis{Godbole2018,
  author    = {Godbole, Amod Anand},
  title     = {A new paradigm in GPCR signaling at the trans-Golgi network of thyroid cells},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-147159},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Whereas G-protein coupled receptors (GPCRs) have been long believed to signal through cyclic AMP exclusively at cell surface, our group has previously shown that GPCRs not only signal at the cell surface but can also continue doing so once internalized together with their ligands, leading to persistent cAMP production. This phenomenon, which we originally described for the thyroid stimulating hormone receptor (TSHR) in thyroid cells, has been observed also for other GPCRs. However, the intracellular compartment(s) responsible for such persistent signaling and its consequences on downstream effectors were insufficiently characterized. The aim of this study was to follow by live-cell imaging the trafficking of internalized TSHRs and other involved signaling proteins as well as to understand the consequences of signaling by internalized TSHRs on the downstream activation of protein kinase A (PKA). cAMP and PKA activity was measured in real-time in living thyroid cells using FRET-based sensors Epac1-camp and AKAR2 respectively. The results suggest that TSH co-internalizes with its receptor and that the internalized TSH/TSHR complexes traffic retrogradely to the trans-Golgi network (TGN). This study also provides evidence that these internalized TSH/TSHR complexes meet an intracellular pool of Gs proteins in sorting endosomes and in TGN and activate it there, as visualized in real-time using a conformational biosensor nanobody, Nb37. Acute Brefeldin A-induced Golgi collapse hinders the retrograde trafficking of TSH/TSHR complexes, leading to reduced cAMP production and PKA signaling. BFA pretreatment was also able to attenuate CREB phosphorylation suggesting that an intact Golgi/TGN organisation is essential for an efficient cAMP/PKA signaling by internalized TSH/TSHR complexes. Taken together this data provides evidence that internalized TSH/TSHR complexes meet and activate Gs proteins in sorting endosomes and at the TGN, leading to a local activation of PKA and consequently increased CREB activation. These findings suggest unexpected functions for receptor internalization, with major pathophysiological and pharmacological implications.},
  subject      = {G-Protein gekoppelte Rezeptoren},
  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{LeBlancSoto2017,
  author    = {Le Blanc Soto, Solange},
  title     = {Role of FGF signaling in the adipogenic and osteogenic differentiation of human bone marrow stromal cells in a three-dimensional \(in\) \(vitro\) model},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-147659},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {Adult human skeletal stem cells are considered to give rise to the bone marrow stromal compartment, including bone-forming osteoblasts and marrow adipocytes. Reduced osteogenesis and enhanced adipogenesis of these skeletal progenitors may contribute to the bone loss and marrow fat accumulation observed during aging and osteoporosis, the main disorder of bone remodeling. Concordantly, in vitro evidence indicates that adipogenic and osteogenic differentiation of human bone marrow stromal cells (hBMSCs) display an inverse relationship under numerous conditions. Hence, the identification of factors modulating inversely both differentiation pathways is of great therapeutic interest. Based on mRNA expression analysis of inversely regulated genes after switching differentiation conditions, our group had previously proposed that fibroblast growth factor 1 (FGF1) might play such a modulator role in hBMSC differentiation. The main aim of this work was, therefore, to investigate the role of FGF1 signaling in the adipogenic and osteogenic differentiation of hBMSCs using a three-dimensional (3D) culture system based on collagen type I hydrogels in order to better mimic the natural microenvironment. Adipogenic and osteogenic differentiation of hBMSCs embedded in collagen gels was successfully established. Treatment with recombinant human FGF1 (rhFGF1), as well as rhFGF2, throughout differentiation induction was found to exert a dose-dependent inhibitory effect on adipogenesis in hBMSCs. This inhibitory effect was found to be reversible and dependent on FGF receptors (FGFR) signaling, given that simultaneous pharmacological blockage of FGFRs rescued adipogenic differentiation. Additionally, matrix mineralization under osteogenic induction was also inhibited by rhFGF1 and rhFGF2 in a dose-dependent manner. A transient treatment with rhFGF1 and rhFGF2 during an expansion phase, however, enhanced proliferation of hBMSCs without affecting the differentiation capacity, although matrix mineralization under osteogenic conditions was hindered. Additionally, rhFGF1 and rhFGF2 treatments affected the matrix remodeling ability of hBMSCs, which displayed alterations in the cytoskeletal phenotype and the expression patterns of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). On the other hand, inhibition of FGFR signaling throughout differentiation induction elicited a strong enhancement of matrix mineralization under osteogenic conditions but had no significant effect on adipocyte formation under adipogenic induction. IX In conclusion, FGF1 and FGF2 signaling was found to support the expansion of bone marrow stromal precursors with adipogenic and osteogenic capacities, to hinder adipogenic and osteogenic differentiation if continuously present during differentiation induction and to alter the matrix remodeling ability of hBMSCs within a 3D collagenous microenvironment.},
  subject      = {Fettzelle},
  language  = {en}
}
@phdthesis{Ziegler2016,
  author    = {Ziegler, Christiane},
  title     = {Epigenetic Mechanisms in the Pathogenesis and Therapy of Anxiety Disorders},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-146815},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {Anxiety disorders (AD) are common, disabling mental disorders, which constitute the most prevalent mental health condition conveying a high individual and socioeconomic burden. Social anxiety disorder (SAD), i.e. fear in social situations particularly when subjectively scrutinized by others, is the second most common anxiety disorder with a life time prevalence of 10\%. Panic disorder (PD) has a life time prevalence of 2-5\% and is characterized by recurrent and abrupt surges of intense fear and anticipatory anxiety, i.e. panic attacks, occurring suddenly and unexpected without an apparent cue. In recent years, psychiatric research increasingly focused on epigenetic mechanisms such as DNA methylation as a possible solution for the problem of the so-called "hidden heritability", which conceptualizes the fact that the genetic risk variants identified so far only explain a small part of the estimated heritability of mental disorders. In the first part of this thesis, oxytocin receptor (OXTR) gene methylation was investigated regarding its role in the pathogenesis of social anxiety disorder. In summary, OXTR methylation patterns were implicated in different phenotypes of social anxiety disorder on a categorical, neuropsychological, neuroendocrinological as well as on a neural network level. The results point towards a multilevel role of OXTR gene hypomethylation particularly at one CpG site (CpG3, Chr3: 8 809 437) within the protein coding region of the gene in SAD. The second part of the thesis investigated monoamine oxidase A (MAOA) gene methylation regarding its role in the pathogenesis of panic disorder as well as - applying a psychotherapy-epigenetic approach - its dynamic regulation during the course of cognitive behavioural therapy (CBT) in PD patients. First, MAOA hypomethylation was shown to be associated with panic disorder as well as with panic disorder severity. Second, in patients responding to treatment MAOA hypomethylation was shown to be reversible up to the level of methylation in healthy controls after the course of CBT. This increase in MAOA methylation along with successful psychotherapeutic treatment was furthermore shown to be associated with symptom improvement regarding agoraphobic avoidance in an independent replication sample of non-medicated patients with PD. Taken together, in the future the presently identified epigenetic patterns might contribute to establishing targeted preventive interventions and personalized treatment options for social anxiety disorder or panic disorder, respectively.},
  subject      = {Angst},
  language  = {en}
}
@phdthesis{Moradi2017,
  author    = {Moradi, Mehri},
  title     = {Differential roles of α-, β- and γ-actin isoforms in regulation of cytoskeletal dynamics and stability during axon elongation and collateral branch formation in motoneurons},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-147453},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {In highly polarized cells like neurons, cytoskeleton dynamics play a crucial role in establishing neuronal connections during development and are required for adult plasticity. Actin turnover is particularly important for neurite growth, axon path finding, branching and synaptogenesis. Motoneurons establish several thousand branches that innervate neuromuscular synapses (NMJs). Axonal branching and terminal arborization are fundamental events during the establishment of synapses in motor endplates. Branching process is triggered by the assembly of actin filaments along the axon shaft giving rise to filopodia formation. The unique contribution of the three actin isoforms, α-, β- and γ-actin, in filopodia stability and dynamics during this process is not well characterized. Here, we performed high resolution in situ hybridization and qRT-PCR and showed that in primary mouse motoneurons α-, β- and γ-actin isoforms are expressed and their transcripts are translocated into axons. Using FRAP experiments, we showed that transcripts for α-, β- and γ-actin become locally translated in axonal growth cones and translation hot spots of the axonal branch points. Using live cell imaging, we showed that shRNA depletion of α-actin reduces dynamics of axonal filopodia which correlates with reduced number of collateral branches and impairs axon elongation. Depletion of β-actin correlates with reduced dynamics of growth cone filopoida, disturbs axon elongation and impairs presynaptic differentiation. Also, depletion of γ-actin impairs axonal growth and decreases axonal filopodia dynamics. These findings implicate that actin isoforms accomplish unique functions during development of motor axons. Depletions of β- and γ-actin lead to compensatory upregulation of other two isoforms. Consistent with this, total actin levels remain unaltered and F-actin polymerization capacity is preserved. After the knockdown of either α- or γ-actin, the levels of β-actin increase in the G-actin pool indicating that polymerization and stability of β-actin filaments depend on α- or γ-actin. This study provides evidence both for unique and overlapping function of actin isoforms in motoneuron growth and differentiation. In the soma of developing motoneurons, actin isoforms act redundantly and thus could compensate for each other's loss. In the axon, α-, β- and γ-actin accomplish specific functions, i.e. β-actin regulates axon elongation and plasticity and α- and γ-actin regulate axonal branching. Furthermore, we show that both axonal transport and local translation of α-, β- and γ-actin isoforms are impaired in Smn knockout motoneurons, indicating a role for Smn protein in RNA granule assembly and local translation of these actin isoforms in primary mouse motoneurons.},
  subject      = {Motoneuron},
  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{Schuerlein2016,
  author    = {Sch{\"u}rlein, Sebastian},
  title     = {Entwicklung von Technologien zur Optimierung von Tissue Engineering Prozessen am Beispiel der Herstellung von kardialem Gewebe},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-142432},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {Kardiovaskul{\"a}re Erkrankungen, wie beispielsweise der Herzinfarkt, sind die h{\"a}ufigste Todesursache weltweit. Bei einem Herzinfarkt sterben Areale des Herzens aufgrund einer Unterversorgung mit Blut ab. Da das Herzmuskelgewebe ein sogenanntes terminal differenziertes Gewebe ist, kommt es zu keiner Regeneration des Gewebes, mit der Folge einer Herzinsuffizienz beziehungsweise dem Tod des Patienten. Eine alternative Behandlungsm{\"o}glichkeit zu einer Herztransplantation stellt das Tissue Engineering dar. Mit Hilfe des Tissue Engineerings k{\"o}nnen dreidimensionale Gewebe aufgebaut und kultiviert werden, um auf diese Weise ein funktionelles Gewebe zu erhalten, durch welches das abgestorbene Gewebeareal des Herzens zuk{\"u}nftig auch ersetzt werden k{\"o}nnte. In der vorliegenden Arbeit wurden notwendige Technologien f{\"u}r den Aufbau von Geweben entwickelt sowie erste Versuche f{\"u}r die Erzeugung eines funktionellen Herzmuskelgewebes durchgef{\"u}hrt. Beim Aufbau von dreidimensionalen Geweben finden Tr{\"a}gerstrukturen Anwendung, die mit Zellen besiedelt werden. Solche Tr{\"a}gerstrukturen k{\"o}nnen aus biologischen oder synthetischen Polymeren hergestellt sein oder aus der extrazellul{\"a}ren Matrix eines dezellularisierten Gewebes bestehen. F{\"u}r eine standardisierte Dezellularisierung von Geweben wurde eine computergesteuerte Pumpeneinheit, f{\"u}r die Herstellung von Nanofaserscaffolds eine Elektrospinninganlage entwickelt. Mit Hilfe der Dezellularisierungseinheit k{\"o}nnen komplexe Organe, wie ein Herz im Ganzen, reproduzierbar dezellularisiert werden. Untersuchungen der mittels Elektrospinning hergestellten Nanofaserscaffolds, welche als Alternative zu der dezellularisierten, nat{\"u}rlichen Matrix eingesetzt werden k{\"o}nnen, zeigten bei allen hergestellten Zusammensetzungen eine Orientierung der Zellen entlang der Fasern. Die Kultivierung von Zellmatrixkonstrukten erfolgt im Tissue Engineering h{\"a}ufig unter dynamischen Bedingungen. Hierf{\"u}r wurde ein mobiler Stand Alone Inkubator mit der erforderlichen Peripherie f{\"u}r eine Kultur unter Perfusion des Gewebes entwickelt. Als Weiterentwicklung des Stand Alone Inkubators ist eine modulare Bioreaktorplattform, bestehend aus W{\"a}rmetauscher, Beutelpumpe und Gasaustauscher, aufgebaut worden. In dieses System kann {\"u}ber Standard Anschl{\"u}sse jegliche Art von Bioreaktor in das System eingebunden werden. Durch die Kompaktheit des Systems ist es m{\"o}glich mehrere Ans{\"a}tze parallel auf engem Raum durchzuf{\"u}hren. Die Funktion der Plattform, wurde in der vorliegenden Arbeit durch die Gewebekultur einer nativen porzinen Karotis nachgewiesen. F{\"u}r den Aufbau des kardialen Gewebes dient die small intestinal submucosa ohne Serosa (SISser) als Tr{\"a}gerstruktur. Der Aufbau des Gewebekonstrukts erfolgte in verschiedenen Ans{\"a}tzen unter Einsatz verschiedener Zellarten. Native, aus Herzbiopsien generierte Cardiosphere derived cells (CDCs) verteilten sich gleichm{\"a}ßige {\"u}ber die Oberfl{\"a}che der Matrix, jedoch konnten immunhistologisch keine spezifischen kardialen Marker bei den artifiziellen Geweben nachgewiesen werden. Zellmatrixkonstrukte aus einer Mono Kultur von Kardiomyozyten, differenziert aus induzierten pluripotenten Stammzellen (iPS Zellen) sowie einer Co Kultur dieser Kardiomyozyten mit mesenchymalen Stammzellen und Zellen aus einer Herzbiopsie zeigten nach wenigen Tagen in Kultur ein kontraktiles Verhalten. Immunhistologische F{\"a}rbungen der beiden Gewebe best{\"a}tigten die Expression der spezifischen kardialen Marker, wie beispielsweise kardiales Troponin T, kardiales Troponin C und alpha Actinin. Die Kardiomyozyten der Mono Kultur sind jedoch nicht {\"u}ber die gesamte Matrixoberfl{\"a}che verteilt, sondern bilden Aggregate. Bei der Co Kultur kann eine gleichm{\"a}ßige Verteilung der Zellen auf der Matrix beobachtet werden. Der vielversprechendste Ansatz f{\"u}r den Aufbau eines Herzmuskelgewebes, welches als Implantat oder Testsystem eingesetzt werden kann, bildet nach den in dieser Arbeit erzielten Ergebnissen, ein Konstrukt aus der SISser und der Co Kultur der Zellen. Allerdings muss die Zusammensetzung der Co Kultur sowie das Verh{\"a}ltnis der Zellzahlen optimiert werden.},
  subject      = {Tissue Engineering},
  language  = {de}
}
@phdthesis{Schmid2016,
  author    = {Schmid, Evelyn},
  title     = {Effekte des Raf Kinase Inhibitor Proteins (RKIP) auf β-adrenerge Signalwege, Herzfunktion und die Entwicklung der Herzinsuffizienz},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-142486},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {Das Raf kinase inhibitor protein (RKIP) ist ein Kinaseregulator, der im Herzen eine Pr{\"a}ferenz f{\"u}r die G-Protein-gekoppelte Rezeptorkinase 2 (GRK2) zeigt. Die Regulation erfolgt durch direkte Interaktion beider Proteine, wird durch eine PKC-Phosphorylierung an Serin 153 des RKIP induziert und inhibiert die GRK2-vermittelte Phosphorylierung von G-Protein-gekoppelten Rezeptoren (GPCR). Die GRK2 desensitiviert GPCR und eine Hemmung der GRK2-Aktivit{\"a}t wirkt sich so positiv auf die Ansprechbarkeit von GPCR aus. Die \textbeta-adrenergen Rezeptoren (\textbeta AR) sind im Herzen maßgeblich an der Regulation der kardialen Kontraktilit{\"a}t beteiligt. Erste Zusammenh{\"a}nge zwischen der RKIP-Expression und der kontraktilen Antwort von Kardiomyozyten wurden bereits in einer fr{\"u}heren Arbeit untersucht und best{\"a}tigt. Sie begr{\"u}nden die Fragestellung nach Effekten einer verst{\"a}rkten RKIP-Expression auf \textbeta-adrenerge Rezeptorsignale, Herzfunktion und die Entwicklung der Herzinsuffizienz. Im Rahmen dieses Projektes konnten die Effekte des RKIP auf \textbeta-adrenerge Signalwege detaillierter beschrieben werden. Dabei erwies sich die inhibitorische Funktion auf die GRK2 als rezeptorspezifisch ohne Einfluss auf zytosolische Angriffspunkte der GRK2 zu nehmen. Verst{\"a}rkte \textbeta-adrenerge Signale zeigten sich in neonatalen Kardiomyozyten an Hand der erh{\"o}hten cAMP-Level, PKA-Aktivit{\"a}t, sowie Kontraktionsrate und Relaxationsgeschwindigkeit nach \textbeta-adrenerger Stimulation. Im Einklang damit konnte eine erh{\"o}hte PKA- und CaMKII-Aktivit{\"a}t und eine positive Inotropie in transgenen Tieren, mit herzspezifischer {\"U}berexpression von RKIP, beobachtet werden. Durch Messung des Calcium-\textit{Cyclings} in Kardiomyozyten konnte der Ph{\"a}notyp auf eine verbesserte R{\"u}ckf{\"u}hrung des Calciums, einer daraus resultierenden erh{\"o}hten Calciumbeladung des sarkoplasmatischen Retikulums und einem gesteigerten systolischen Calciumspiegel, zur{\"u}ckgef{\"u}hrt werden. Die Untersuchung der Phosphorylierung von Calciumkan{\"a}len, L-Typ-Calciumkanal und Ryanodin-Rezeptor 2, die den einw{\"a}rtsgerichteten Calciumstrom vermitteln konnte ihre Beteiligung an der positiv inotropen Wirkung ausschließen. Neben dem kontraktilen Ph{\"a}notyp konnten zus{\"a}tzliche protektive Effekte beobachtet werden. In Modellen, die eine chronische \textbeta-adrenerge Stimulation imitieren, bzw. eine Nachlasterh{\"o}hung induzieren konnte eine Verringerung der interstitiellen Fibrose und der damit assoziierten Marker, gezeigt werden. Mit Hilfe von \textit{in vivo} EKG-Messungen konnte die Neigung zur Ausbildung von Arrhythmien untersucht werden. Auch im Hinblick auf die Anzahl der Extrasystolen waren RKIP-transgene Tiere gesch{\"u}tzt. Infolge der Untersuchung der Ph{\"a}notypen in Deletionshintergr{\"u}nden der einzelnen \textbeta AR-Subtypen (\textbeta\textsubscript{1}AR, \textbeta\textsubscript{2}AR) konnte die positive Inotropie mit den spezifischen Signalwegen des \textbeta\textsubscript{1}AR assoziiert und die protektiven Effekte gegen{\"u}ber den Umbauprozessen und der Arrhythmieneigung dem \textbeta\textsubscript{2}-adrenergen Signalen zugeschrieben werden. Zus{\"a}tzlich best{\"a}tigt sich eine besondere Rolle der G\textalpha\textsubscript{i}-Kopplung des \textbeta\textsubscript{2}AR, durch die er einen hemmenden Einfluss auf die \textbeta\textsubscript{1}AR-Singale nehmen kann. Die Untersuchung einiger Marker, die eine physiologische von einer pathologischen Hypertrophie unterscheiden, konnte das in den RKIP-transgenen M{\"a}usen auftretende Wachstum der Kardiomyozyten als kompensatorische und physiologische Hypertrophie charakterisieren. Zusammengenommen weisen diese Ergebnisse auf eine ausgeglichene Aktivierung der beiden Rezeptoren hin, die sich gegenseitig regulieren und durch die Inhibition der GRK2 in ihrer Anregbarkeit erhalten bleiben. Mittels einer AAV9-vermittelten Gentherapie konnte das therapeutische Potential dieses Prinzips weiter best{\"a}tigt werden, da es die prominentesten Ver{\"a}nderungen w{\"a}hrend der Herzinsuffizienzentwicklung, wie die Verschlechterung der linksventrikul{\"a}ren Funktion, die Dilatation des linken Ventrikels, die Ausbildung von Lungen{\"o}demen und interstitieller Fibrose sowie die Expression von Herzinsuffizienz-assoziierten Genen, verhindern konnte. Auch konnten die Auswirkungen der Deletion des RKIP, die sich durch eine beschleunigte und gravierendere Herzinsuffizienzentwicklung auszeichnet, durch Reexpression von RKIP verhindert werden. Diese Arbeit kann somit zeigen, dass das RKIP eine ausgeglichene Verst{\"a}rkung von \textbeta-adrenergen Signalwegen verursacht, die positiv inotrop und gleichzeitig protektiv wirkt. Dieses Wirkprinzip k{\"o}nnte ferner eine Strategie zur Erh{\"o}hung der Kontraktilit{\"a}t in der Herzinsuffizienz darstellen, die entgegen etablierter Theorien auf der Stimulation beider \textbeta AR basiert.},
  subject      = {Herzinsuffizienz},
  language  = {de}
}
@phdthesis{Wanzek2016,
  author    = {Wanzek, Katharina},
  title     = {The investigation of the function of repair proteins at G-quadruplex structures in \(Saccharomyces\) \(cerevisiae\) revealed that Mms1 promotes genome stability},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-142547},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {G-quadruplex structures are highly stable alternative DNA structures that can, when not properly regulated, impede replication fork progression and cause genome instability (Castillo Bosch et al, 2014; Crabbe et al, 2004; Koole et al, 2014; Kruisselbrink et al, 2008; London et al, 2008; Lopes et al, 2011; Paeschke et al, 2013; Paeschke et al, 2011; Piazza et al, 2015; Piazza et al, 2010; Piazza et al, 2012; Ribeyre et al, 2009; Sabouri et al, 2014; Sarkies et al, 2012; Sarkies et al, 2010; Schiavone et al, 2014; Wu \& Spies, 2016; Zimmer et al, 2016). The aim of this thesis was to identify novel G-quadruplex interacting proteins in Saccharomyces cerevisiae and to unravel their regulatory function at these structures to maintain genome integrity. Mms1 and Rtt101 were identified as G-quadruplex binding proteins in vitro via a pull-down experiment with subsequent mass spectrometry analysis. Rtt101, Mms1 and Mms22, which are all components of an ubiquitin ligase (Rtt101Mms1/Mms22), are important for the progression of the replication fork following fork stalling (Luke et al, 2006; Vaisica et al, 2011; Zaidi et al, 2008). The in vivo binding of endogenously tagged Mms1 to its target regions was analyzed genome-wide using chromatin-immunoprecipitation followed by deep-sequencing. Interestingly, Mms1 bound independently of Mms22 and Rtt101 to G-rich regions that have the potential to form G-quadruplex structures. In vitro, formation of G-quadruplex structures could be shown for the G-rich regions Mms1 bound to. This binding was observed throughout the cell cycle. Furthermore, the deletion of MMS1 caused replication fork stalling as evidenced by increased association of DNA Polymerase 2 at Mms1 dependent sites. A gross chromosomal rearrangement assay revealed that deletion of MMS1 results in a significantly increased genome instability at G-quadruplex motifs compared to G-rich or non-G-rich regions. Additionally, binding of the helicase Pif1, which unwinds G4 structures in vitro (Paeschke et al, 2013; Ribeyre et al, 2009; Sanders, 2010; Wallgren et al, 2016), to Mms1 binding sites was reduced in mms1 cells. The data presented in this thesis, together with published data, suggests a novel mechanistic model in which Mms1 binds to G-quadruplex structures and enables Pif1 association. This allows for replication fork progression and genome integrity.},
  subject      = {Quadruplex-DNS},
  language  = {en}
}
@phdthesis{Gupta2017,
  author    = {Gupta, Sanjay Kumar},
  title     = {The human CCHC-type Zinc Finger Nucleic Acid Binding Protein (CNBP) binds to the G-rich elements in target mRNA coding sequences and promotes translation},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-142917},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {The genetic information encoded with in the genes are transcribed and translated to give rise to the functional proteins, which are building block of a cell. At first, it was thought that the regulation of gene expression particularly occurs at the level of transcription by various transcription factors. Recent discoveries have shown the vital role of gene regulation at the level of RNA also known as post-transcriptional gene regulation (PTGR). Apart from non-coding RNAs e.g. micro RNAs, various RNA binding proteins (RBPs) play essential role in PTGR. RBPs have been implicated in different stages of mRNA life cycle ranging from splicing, processing, transport, localization and decay. In last 20 years studies have shown the presence of hundreds of RBPs across eukaryotic systems many of which are widely conserved. Given the rising number of RBPs and their link to human diseases it is quite evident that RBPs have major role in cellular processes and their regulation. The current study is aimed to describe the so far unknown molecular mechanism of CCHC-type Zinc Finger Nucleic Acid Binding Protein (CNBP/ZNF9) function in vivo. CNBP is ubiquitously expressed across various human tissues and is a highly conserved RBP in eukaryotes. It is required for embryonic development in mammals and has been implicated in transcriptional as well as post-transcriptional gene regulation; however, its molecular function and direct target genes remain elusive. Here, we use multiple systems-wide approaches to identify CNBP targets and document the consequences of CNBP binding. We established CNBP as a cytoplasmic RNA-binding-protein and used Photoactivatable Ribonucleoside Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) to identify direct interactions of CNBP with 4178 mRNAs. CNBP preferentially bound a G-rich motif in the target mRNA coding sequences. Functional analyses, including ribosome profiling, RNA sequencing, and luciferase assays revealed the CNBP mode of action on target transcripts. CNBP binding was found to increase the translational efficiency of its target genes. We hypothesize that this is consistent with an RNA chaperone function of CNBP helping to resolve secondary structures, thus promoting translation. Altogether this study provides a novel mechanism of CNBP function in vivo and acts as a step-stone to study the individual CNBP targets that will bring us closer to understand the disease onset.},
  subject      = {CNBP},
  language  = {en}
}
@phdthesis{vanEeuwijk2018,
  author    = {van Eeuwijk, Judith Martina Maria},
  title     = {Studies on thrombopoiesis and spleen tyrosine kinase-mediated signaling in platelets},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-142933},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {In mammals, anucleate blood platelets are constantly produced by their giant bone marrow (BM) progenitors, the megakaryocytes (MKs), which originate from hematopoietic stem cells. Megakaryopoiesis and thrombopoiesis have been studied intensively, but the exact mechanisms that control platelet generation from MKs remain poorly understood. Using multiphoton intravital microscopy (MP-IVM), thrombopoiesis and proplatelet formation were analyzed in the murine BM in real-time and in vivo, identifying an important role for several proteins, including Profilin1, TRPM7 and RhoA in thrombopoiesis. Currently, it is thought that blood cell precursors, such as MKs, migrate from the endosteal niche towards the vascular niche during maturation. In contrast to this paradigm, it was shown that MKs are homogeneously distributed within the dense BM blood vessel network, leaving no space for vessel-distant niches. By combining results from in vivo MP-IVM, in situ light-sheet fluorescence microscopy (LSFM) of the intact BM as well as computational simulations, surprisingly slow MK migration, limited intervascular space and a vessel-biased MK pool were revealed, contradicting the current concept of directed MK migration during thrombopoiesis. Platelets play an essential role in hemostasis and thrombosis, but also in the pathogenesis of ischemic stroke. Ischemic stroke, which is mainly caused by thromboembolic occlusion of brain arteries, is among the leading causes of death and disability worldwide with limited treatment options. The platelet collagen receptor glycoprotein (GP) VI is a key player in arterial thrombosis and a critical determinant of stroke outcome, making its signaling pathway an attractive target for pharmacological intervention. The spleen tyrosine kinase (Syk) is an essential signaling mediator downstream of GPVI, but also of other platelet and immune cell receptors. In this thesis, it was demonstrated that mice lacking Syk specifically in platelets are protected from arterial thrombus formation and ischemic stroke, but display unaltered hemostasis. Furthermore, it was shown that mice treated with the novel, selective and orally bioavailable Syk inhibitor BI1002494 were protected in a model of arterial thrombosis and had smaller infarct sizes and a significantly better neurological outcome 24 h after transient middle cerebral artery occlusion (tMCAO), also when BI1002494 was administered therapeutically, i.e. after ischemia. These results provide direct evidence that pharmacological Syk inhibition might become a safe therapeutic strategy. The T cell receptor  chain-associated protein kinase of 70 kDA (Zap-70) is also a spleen tyrosine kinase family member, but has a lower intrinsic activity compared to Syk and is expressed in T cells and natural killer (NK) cells, but not in platelets. Unexpectedly, arterial thrombus formation in vivo can occur independently of Syk kinase function as revealed by studies in Sykki mice, which express Zap-70 under the control of intrinsic Syk promoter elements.},
  subject      = {Thrombose},
  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{Rodrigues2016,
  author    = {Rodrigues, Johannes},
  title     = {Let me change your mind… Frontal brain activity in a virtual T-maze},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-143280},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {Frontal asymmetry, a construct invented by Richard Davidson, linking positive and negative valence as well as approach and withdrawal motivation to lateralized frontal brain activation has been investigated for over thirty years. The frontal activation patterns described as relevant were measured via alpha-band frequency activity (8-13 Hz) as a measurement of deactivation in electroencephalography (EEG) for homologous electrode pairs, especially for the electrode position F4/ F3 to account for the frontal relative lateralized brain activation. Three different theories about frontal activation patterns linked to motivational states were investigated in two studies. The valence theory of Davidson (1984; 1998a; 1998b) and its extension to the motivational direction theory by Harmon-Jones and Allen (1998) refers to the approach motivation with relative left frontal brain activity (indicated by relative right frontal alpha activity) and to withdrawal motivation with relative right frontal brain activation (indicated by relative left frontal alpha activity). The second theory proposed by Hewig and colleagues (2004; 2005; 2006) integrates the findings of Davidson and Harmon - Jones and Allen with the reinforcement sensitivity theory of Jeffrey A. Gray (1982, 1991). Hewig sees the lateralized frontal approach system and withdrawal system proposed by Davidson as subsystems of the behavioral activation system proposed by Gray and bilateral frontal activation as a biological marker for the behavioral activation system. The third theory investigated in the present studies is the theory from Wacker and colleagues (2003; 2008; 2010) where the frontal asymmetrical brain activation patterns are linked to the revised reinforcement sensitivity theory of Gray and McNaughton (2000). Here, right frontal brain activity (indicated by lower relative right frontal alpha activity) accounts for conflict, behavioral inhibition and activity of the revised behavioral inhibition system, while left frontal brain activation (indicated by lower relative left frontal alpha activity) stands for active behavior and the activity of the revised behavioral activation system as well as the activation of the revised flight fight freezing system. In order to investigate these three theories, a virtual reality T-maze paradigm was introduced to evoke motivational states in the participants, offering the opportunity to measure frontal brain activation patterns via EEG and behavior simultaneously in the first study. In the second study the virtual reality paradigm was additionally compared to mental imagery and a movie paradigm, two well-known state inducing paradigms in the research field of frontal asymmetry. In the two studies, there was confirming evidence for the theory of Hewig and colleages (2004; 2005; 2006), showing higher bilateral frontal activation for active behavior and lateralized frontal activation patterns for approach (left frontal brain activation) and avoidance (right frontal brain activation) behavior. Additionally a limitation for the capability model of anterior brain asymmetry proposed by Coan and colleagues (2006), where the frontal asymmetry should be dependent on the relevant traits driving the frontal asymmetry pattern if a relevant situation occurs, could be found. As the very intense virtual reality paradigm did not lead to a difference of frontal brain activation patterns compared to the mental imagery paradigm or the movie paradigm for the traits of the participants, the trait dependency of the frontal asymmetry in a relevant situation might not be given, if the intensity of the situation exceeds a certain level. Nevertheless there was an influence of the traits in the virtual reality T-maze paradigm, because the shown behavior in the maze was trait-dependent. The implications of the findings are multifarious, leading from possible objective personality testing via diversification of the virtual reality paradigm to even clinical implications for depression treatments based on changes in the lateralized frontal brain activation patterns for changes in the motivational aspects, but also for changes in bilateral frontal brain activation when it comes to the drive and preparedness for action in patients. Finally, with the limitation of the capability model, additional variance in the different findings about frontal asymmetry can be explained by taking the intensity of a state manipulation into account.},
  subject      = {Electroencephalographie},
  language  = {en}
}
@phdthesis{Hoffmann2017,
  author    = {Hoffmann, Helene},
  title     = {Identifying regulators of tumor vascular morphology},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-142348},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {In contrast to normal vessels, tumor vasculature is structurally and functionally abnormal. Tumor vessels are highly disorganized, tortuous and dilated, with uneven diameter and excessive branching. Consequently, tumor blood flow is chaotic, which leads to hypoxic and acidic regions in tumors. These conditions lower the therapeutic effectiveness and select for cancer cells that are more malignant and metastatic. The therapeutic outcome could be improved by increasing the functionality and density of the tumor vasculature. Tumor angiogenesis also shows parallels to epithelial to mesenchymal transition (EMT), a process enabling metastasis. Metastasis is a multi-step process, during which tumor cells have to invade the surrounding host tissue to reach the circulation and to be transported to distant sites. We hypothesize that the variability in the phenotype of the tumor vasculature is controlled by the differential expression of key transcription factors. Inhibiting these transcription factors might be a promising way for angiogenic intervention and vascular re-engineering. Therefore, we investigated the interdependence of tumor-, stroma- and immune cell-derived angiogenic factors, transcription factors and resulting vessel phenotypes. Additionally, we evaluated whether transcription factors that regulate EMT are promising targets for vascular remodeling. We used formalin fixed paraffin embedded samples from breast cancer patients, classified according to estrogen-, progesterone- and human epidermal growth factor receptor (HER) 2 status. Establishing various techniques (CD34 staining, laser microdissection, RNA isolation and expression profiling) we systematically analyzed tumor and stroma-derived growths factors. In addition, vascular parameters such as microvessel size, area, circularity and density were assessed. Finally the established expression profiles were correlated with the observed vessel phenotype. As the SNAI1 transcriptional repressor is a key regulator of EMT, we examined the effect of vascular knockdown of Snai1 in murine cancer models (E0771, B16-F10 and lewis lung carcinoma). Among individual mammary carcinomas, but not among subtypes, strong differences of vascular parameters were observed. Also, little difference between lobular carcinomas and ductal carcinomas was found. Vessel phenotype of Her2 enriched carcinomas was similar to that of lobular carcinomas. Vessel morphology of luminal A and B and basal-like tumors resembled each other. Expression of angiogenic factors was variable across subtypes. We discovered an inverse correlation of PDGF-B and VEGF-A with vessel area in luminal A tumors. In these tumors expression of IL12A, an inhibitor of angiogenesis, was also correlated with vessel size. Treatment of endothelial cells with growth factors revealed an increased expression of transcription factors involved in the regulation of EMT. Knockdown of Snai1 in endothelial cells of mice increased tumor growth and decreased hypoxia in the E0771 and the B16-F10 models. In the lewis lung carcinomas, tumor vascularity and biodistribution of doxorubicin were improved. Here, doxorubicin treatment in combination with the endothelial cell-specific knockdown did slow tumor growth. This shows that SNAI1 is important for a tumor's vascularization, with the significance of its role depending on the tumor model. The methods established in this work open the way for the analysis of the expression of key transcription factors in vessels of formalin fixed paraffin embedded tumors. This research enables us to find novel targets for vascular intervention and to eventually design novel targeted drugs to inhibit these targets.},
  subject      = {Antiangiogenese},
  language  = {en}
}
@phdthesis{Schweinlin2016,
  author    = {Schweinlin, Matthias Oliver},
  title     = {Development of advanced human intestinal in vitro models},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-142571},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {The main function of the small intestine is the absorption of essential nutrients, water and vitamins. Moreover, it constitutes a barrier protecting us from toxic xenobiotics and pathogens. For a better understanding of these processes, the development of intestinal in vitro models is of great interest to the study of pharmacological and pathological issues such as transport mechanisms and barrier function. Depending on the scientific questions, models of different complexity can be applied. In vitro Transwell® systems based on a porous PET-membrane enable the standardized study of transport mechanisms across the intestinal barrier as well as the investigation of the influence of target substances on barrier integrity. However, this artificial setup reflects only limited aspects of the physiology of the native small intestine and can pose an additional physical barrier. Hence, the applications of this model for tissue engineering are limited. Previously, tissue models based on a biological decellularized scaffold derived from porcine gut tissue were demonstrated to be a good alternative to the commonly used Transwell® system. This study showed that preserved biological extracellular matrix components like collagen and elastin provide a natural environment for the epithelial cells, promoting cell adhesion and growth. Intestinal epithelial cells such as Caco-2 cultured on such a scaffold showed a confluent, tight monolayer on the apical surface. Additionally, myofibroblasts were able to migrate into the scaffold supporting intestinal barrier formation. In this thesis, dendritic cells were additionally introduced to this model mimicking an important component of the immune system. This co-culture model was then successfully proven to be suitable for the screening of particle formulations developed as delivery system for cancer antigens in peroral vaccination studies. In particular, nanoparticles based on PLGA, PEG-PAGE-PLGA, Mannose-PEG-PAGE-PLGA and Chitosan were tested. Uptake studies revealed only slight differences in the transcellular transport rate among the different particles. Dendritic cells were shown to phagocytose the particles after they have passed the intestinal barrier. The particles demonstrated to be an effective carrier system to transport peptides across the intestinal barrier and therefore present a useful tool for the development of novel drugs. Furthermore, to mimic the complex structure and physiology of the gut including the presence of multiple different cell types, the Caco-2 cell line was replaced by primary intestinal cells to set up a de novo tissue model. To that end, intestinal crypts including undifferentiated stem cells and progenitor cells were isolated from human small intestinal tissue samples (jejunum) and expanded in vitro in organoid cultures. Cells were cultured on the decellularized porcine gut matrix in co-culture with intestinal myofibroblasts. These novel tissue models were maintained under either static or dynamic conditions. Primary intestinal epithelial cells formed a confluent monolayer including the major differentiated cell types positive for mucin (goblet cells), villin (enterocytes), chromogranin A (enteroendocrine cells) and lysozyme (paneth cells). Electron microscopy images depicted essential functional units of an intact epithelium, such as microvilli and tight junctions. FITC-dextran permeability and TEER measurements were used to assess tightness of the cell layer. Models showed characteristic transport activity for several reference substances. Mechanical stimulation of the cells by a dynamic culture system had a great impact on barrier integrity and transporter activity resulting in a tighter barrier and a higher efflux transporter activity. In Summary, the use of primary human intestinal cells combined with a biological decellularized scaffold offers a new and promising way to setup more physiological intestinal in vitro models. Maintenance of primary intestinal stem cells with their proliferation and differentiation potential together with adjusted culture protocols might help further improve the models. In particular, dynamic culture systems and co culture models proofed to be a first crucial steps towards a more physiological model. Such tissue models might be useful to improve the predictive power of in vitro models and in vitro in vivo correlation (IVIVC) studies. Moreover, these tissue models will be useful tools in preclinical studies to test pharmaceutical substances, probiotic active organisms, human pathogenic germs and could even be used to build up patient-specific tissue model for personalized medicine.},
  subject      = {Tissue Engineering},
  language  = {en}
}
@phdthesis{Kasaragod2022,
  author    = {Kasaragod, Vikram Babu},
  title     = {Biochemical and Structural Basis for the Moonlighting Function of Gephyrin},
  doi       = {10.25972/OPUS-14307},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-143077},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {Neurons are specialized cells dedicated to transmit the nerve impulses throughout the human body across specialized structures called synapses. At the synaptic terminals, a crosstalk between multiple macromolecules regulates the structure and function of the presynaptic nerve endings and the postsynaptic recipient sites. Gephyrin is the central organizer at inhibitory postsynaptic specializations and plays a crucial role in the organization of these structures by anchoring GABAA receptors (GABAAR) and glycine receptors (GlyR) to the postsynaptic membrane. This 93 kDa protein features an N-terminal G domain and a C-terminal E domain and the latter interacts directly with the intracellular loop between transmembrane helices 3 and 4 of certain subunits of the GlyRs and GABAARs. Biochemical and structural analyses have already provided valuable insights into the gephyrin-GlyR interaction. Interestingly, biochemical studies on the gephyrin-GABAAR interaction demonstrated that the GABAARs also depend on the same binding site as the GlyRs for the interaction with the gephyrin, but the molecular basis for this receptor specific interaction of gephyrin was still unknown. Co-crystal structures of GephE-GABAAR α3- derived peptides with supporting biochemical data presented in this study deciphered the receptor-specific interactions of gephyrin in atomic detail. In its moonlighting function, gephyrin also catalyzes the terminal step of the evolutionarily conserved molybdenum cofactor biosynthesis. Molybdenum, an essential transition element has to be complexed with a pterin-based cofactor resulting in the formation of the molybdenum cofactor (Moco). Moco is an essential component at the active site of all molybdenum-containing enzymes with the exception of nitrogenase. Mutations in enzymes involved in this pathway lead to a rare yet severe disease called Moco deficiency, which manifest itself in severe neurodevelopmental abnormalities and early childhood death. Moco biosynthesis follows a complex multistep pathway, where in the penultimate step, the N-terminal G domain of gephyrin activates the molybdopterin to form an adenylated molybdopterin intermediate. In the terminal step, this intermediate is then transferred to the C-terminal E domain of gephyrin, which catalyzes the metal insertion and deadenylation reaction to form active Moco. Previous biochemical and structural studies provided valuable insights into the penultimate step of the Moco biosynthesis but the terminal step remained elusive. Through the course of my dissertation, I crystallized the C-terminal E domain in the apo-form as well as in complex with ADP and AMP. These structures shed lightonto the deadenylation reaction and the formation of a ternary E-domain-ADP-Mo/W complex and thus provide structural insight into the metal insertion mechanism. Moreover, the structures also provided molecular insights into a mutation leading to Moco deficiency. Finally, ternary complexes of GephE, ADP and receptor-derived peptides provided first clues regarding the integration of gephyrin's dual functionality. In summary, during the course of the dissertation I was able to derive high resolution structural insights into the interactions between gephyrin and GABAARs, which explain the receptor-specific interaction of gephyrin and, furthermore, these studies can be extended in the future to understand GABAAR subunit-specific interactions of gephyrin. Finally, the understanding of Moco biosynthesis shed light on the molecular basis of the fatal Moco deficiency.},
  subject      = {Gephyrin},
  language  = {en}
}
@phdthesis{Meduri2017,
  author    = {Meduri, Rajyalakshmi},
  title     = {Elucidation of an intricate surveillance network for cellular U snRNP homeostasis},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-143173},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {Spliceosomal U-rich small ribonucleoprotein particles (U snRNPs) are the major building blocks of the nuclear pre-mRNA splicing machinery. The core composition of U snRNPs includes the name giving U snRNA and a set of seven common (Sm) proteins termed Sm B/B', D1, D2, D3, E, F and G. These Sm proteins are arranged in the form of a toroidal ring on the single stranded conserved sequence element in the snRNA to form the Sm core domain. Even though U snRNPs assemble spontaneously in vitro, their assembly in vivo requires an amazingly large number of trans-acting assembly factors united in the Protein Arginine Methyltransferase 5 (PRMT5) and the Survival Motor Neuron (SMN) complexes. The cytoplasmic assembly pathway of U snRNPs can be divided into the early and the late phase. The early phase is dominated by the assembly chaperone, pICln, a subunit of the PRMT5 complex. This factor binds to Sm proteins and delivers them in a pICln-bound form to the PRMT5 complex. The early assembly phase then segregates into two lines. In one assembly line, a stable hexameric ring intermediate (6S complex) composed of pICln and the five Sm proteins D1, D2, F, E and G, is formed. This intermediate forms at the PRMT5 complex but dissociates from the latter upon completion of its assembly. Within the 6S complex, these Sm proteins are pre-organized into respective spatial positions adopted in the assembled U snRNP. The other assembly line forms a protein trimer composed of pICln, Sm B/B' and D3, which unlike the 6S complex is not released from the PRMT5 complex. As a consequence of their association with pICln, Sm proteins are kinetically trapped and fail to proceed in the assembly pathway. The late phase of the U snRNP formation is dominated by the SMN complex, which resolves this kinetic trap by dissociating pICln from the pre-organized Sm proteins and, subsequently catalyzes the loading of the Sm proteins on the U snRNA. Even though basic principles of U snRNP assembly have been understood in some detail, the question arises as to why cells employ sophisticated assembly machinery for the assembly despite the reaction occurring spontaneously in vitro. A few studies have shown that the system works towards rendering specificity to the assembly reaction. However, Sm proteins in their free form expose hydrophobic surfaces to the cytosolic solvent. Hence, I reasoned that the assembly machinery of snRNPs might also prevent Sm protein aggregation. In this thesis, I describe the work that leads to the discovery of a multi-layered regulatory network for Sm proteins involving post-transcriptional and post-translational surveillance mechanisms. Here, I show that the reduced level of SMN (a key assembly factor of the late phase) leads to the initial tailback of Sm proteins over pICln followed by the transcriptional down regulation of Sm protein encoding mRNAs. In contrast, depletion of pICln, a key factor of the early phase, results in the retention of Sm proteins on the ribosomes followed by their degradation via autophagy. Furthermore, I show that exceeding levels of Sm proteins over pICln caused by overexpression results in aggregation and mis-localization of Sm proteins. Thus, my findings uncover a complex regulatory network that helps to maintain the cellular U snRNP homeostasis by either preventing or clearing the unassembled Sm protein aggregates when they are not faithfully incorporated into the U snRNPs.},
  language  = {en}
}
@phdthesis{HergovitsgebWalter2018,
  author    = {Hergovits [geb. Walter], Sabine},
  title     = {Relevanz der gp130 Endozytose bei der Maturierung und Differenzierung dendritischer Zellen sowie Charakterisierung des molekularen Mechanismus der OSM-vermittelten Induktion antiviraler Gene},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-150562},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Interleukin-6 (IL-6)-Typ Zytokine, allen voran IL-6 und Oncostatin M (OSM), besitzen pleiotrope Eigenschaften und spielen eine wichtige Rolle bei einer Vielzahl biologischer Prozesse. Als Akutphaseinduktoren sind IL-6 und OSM an der Initialisierung entz{\"u}ndlicher und immunologischer Prozesse beteiligt, k{\"o}nnen aber ebenso die Differenzierung und das Zellwachstum beeinflussen. Ihre biologische Wirkung vermitteln sie {\"u}ber die Bindung an einen multimeren Rezeptorkomplex. Dieser weist im Fall von IL-6 eine hexamere Struktur auf und besteht aus je zwei Molek{\"u}len des Glykoproteins 130 (gp130), des IL-6 α Rezeptors (IL-6R) und IL-6. Der Rezeptor von OSM hingegen ist ein Heterodimer und besteht aus gp130 und dem OSM Rezeptor (OSMR) oder aber aus gp130 und dem leukemia inhibitory factor (LIF) Rezeptor (LIFR). Die Zytokine der IL-6-Familie vermitteln die Induktion des Jak/STAT (Januskinase/signal transducer and activator of transcription), PI3K/Akt (Phosphoinositid-3-Kinasen/AKR thymoma oncogene homolog) und MAPK (mitogenaktivierte Proteinkinase) Signalwegs. Da eine unkontrollierte Aktivierung dieser Signalkaskaden jedoch zu chronisch entz{\"u}ndlichen Erkrankungen oder abnormen Zellwachstum f{\"u}hren kann, ist eine Regulation durch verschiedene R{\"u}ckkopplungsmechanismen essentiell. Neben der Rekrutierung inhibitorisch wirkender Proteine, wie den Mitgliedern der SOCS (suppressors of cytokine signaling) Familie und Tyrosinphosphatasen, gilt die Rezeptorinternalisierung als regulierender Mechanismus. Der erste Teil der vorliegenden Dissertation geht der Fragestellung nach, wie die Expression des IL-6-Rezeptors w{\"a}hrend der Differenzierung und Maturierung dendritischer Zellen (DZ) reguliert ist und welche Relevanz die gp130-Internalisierung bei diesen Entwicklungsprozessen spielt. DZ geh{\"o}ren zu den professionellen antigenpr{\"a}sentieren Zellen (APZ) und gelten als Bindeglied zwischen der angeborenen und adaptiven Immunantwort. Sie spielen insbesondere bei der Polarisation der T-Helferzellen (Th1, Th2, Th17, Treg) eine wichtige Rolle. DZ repr{\"a}sentieren eine heterogene Zellpopulation, die auf Basis ihrer Entstehung, ihres Vorkommens und/oder ihrer Funktionen in verschiedene Subtypen unterteilt werden und sich u.a. durch die Expression bestimmter Oberfl{\"a}chenmarker unterscheiden lassen. Es ist bekannt, dass DZ sowohl gp130 als auch den IL-6R auf ihrer Oberfl{\"a}che exprimieren, weshalb ihre Differenzierung und Maturierung durch IL-6 beeinflusst werden kann. Obwohl Studien der letzten Jahre bereits eindrucksvoll die Relevanz des IL-6-Signals f{\"u}r die DZ Entwicklung belegt haben, bleibt dessen Funktion f{\"u}r diese Prozesse bisher kontrovers diskutiert. Inwiefern eine ver{\"a}nderte Rezeptorexpression auf diesen Zellen Einfluss auf die biologische Wirkung von IL-6 nimmt, wurde bisher nicht untersucht und war daher Ziel der hier durchgef{\"u}hrten Experimente. Mit Hilfe GM-CSF-gereifter DZ aus murinem Knochenmark (KM-DZ) sowie steady state DZ aus peripheren lymphatischen Organen konnte gezeigt werden, dass die Expression von gp130 und IL-6R bereits w{\"a}hrend der DZ Differenzierung unterschiedlich reguliert ist. Konventionelle DZ (kDZ) aus Milz und Lymphknoten wiesen ein h{\"o}heres Expressionsniveau f{\"u}r den IL-6-Rezeptor auf als plasmazytoide DZ (pDZ). Es wurde nachgewiesen, dass die gp130 Expression im Verlauf der DZ Differenzierung stetig zunimmt, w{\"a}hrend nahezu keine Ver{\"a}nderung f{\"u}r die Expression des IL-6R festzustellen war. In weiteren Experimenten konnte dar{\"u}ber hinaus belegt werden, dass die Reifung der DZ, induziert durch Lipopolysaccharid (LPS), Tumornekrosefaktor-α (TNFα) oder Choleratoxin (Ctx), zu einer Cross-Regulation von gp130 f{\"u}hrt. Diese konnte im Fall von TNFα und Ctx auf eine vor{\"u}bergehende Internalisierung des Rezeptors in Folge der Aktivierung der Serin-/Threonin-Kinase MK2 (MAPK-aktivierte Proteinkinase 2) zur{\"u}ckgef{\"u}hrt werden. Untersuchungen von KM-DZ aus der gp130 knockin Mauslinie gp130LLAA, die sich durch eine Punktmutation des beschriebenen Endozytose-Motivs des Rezeptors auszeichnet, f{\"u}hrten zu dem Schluss, dass LPS gp130 {\"u}ber einen bisher noch nicht beschriebenen Mechanismus reguliert. Dieser vermittelt eine fr{\"u}he Clathrin-abh{\"a}ngige Internalisierung von gp130 und f{\"u}hrt schließlich zu einer langfristigen Inhibierung der gp130 Expression. Die Regulation der IL-6 Rezeptorexpression ist insofern von Bedeutung als dass vermutet werden kann, dass das IL-6/STAT3-Signal f{\"u}r die Aufrechterhaltung eines unreifen DZ Ph{\"a}notyps wichtig ist. Ferner kann vermutet werden, dass die Limitierung der Responsivit{\"a}t gegen{\"u}ber LIF (und vermutlich auch OSM) f{\"u}r die Differenzierung von DZ wichtig ist, da die Expression des LIFR {\"u}ber die Dauer der KM-DZ Differenzierung stark herunterreguliert wurde. Eine Expression des OSMR auf DZ wurde hingegen nicht nachgewiesen und steht demzufolge im Gegensatz zu bisher ver{\"o}ffentlichten Untersuchungen. Im Rahmen dieses ersten Projekts wurde ebenfalls untersucht, wie sich die ver{\"a}nderte gp130 Endozytose auf die Hom{\"o}ostase myeloider und lymphoider Zellen auswirkt. Bei den hierf{\"u}r analysierten gp130LLAA M{\"a}usen wurde eine geringf{\"u}gige Verminderung der Frequenz und absoluten Zellzahl von kDZ in der Milz sowie eine Zunahme der Frequenz und absoluten Zellzahl von pDZ und Makrophagen in den inguinalen Lymphknoten nachgewiesen. Dar{\"u}ber hinaus wurde ein Anstieg in der Frequenz und absoluten Zellzahl von T-Zellen, jedoch eine Abnahme der B-Zellen in der Milz beobachtet. Im zweiten Teil der Dissertation sollte die OSM-vermittelte Induktion antiviraler Gene in prim{\"a}ren humanen dermalen Fibroblasten (HDF) untersucht werden. Typ I Interferone (IFN) gelten als zentrale Zytokine der antiviralen Immunantwort. Studien der letzten Jahre haben gezeigt, dass eine Reihe proinflammatorischer Zytokine ebenso die antivirale Immunantwort beeinflussen k{\"o}nnen, indem sie u.a. die Expression der pattern recognition receptors (PRR) regulieren. PRR sind Teil des angeborenen Immunsystems und f{\"u}r die Erkennung von Pathogenen durch die Bindung an konservierte Pathogen-assoziierte molekulare Strukturen (PAMPs, Pathogen-associated molecular patterns) wichtig. Im zweiten Teilprojekt der vorliegenden Arbeit konnte gezeigt werden, dass OSM dazu in der Lage ist, die Induktion der im Zytoplasma lokalisierten PRR Retinoic acid inducible gene-I (RIG-I) und Melanoma differentiation-associated protein 5 (MDA5) zu vermitteln. Mit Hilfe von RNA Interferenzstudien konnte nachgewiesen werden, dass die Expression dieser beiden RNA-Helikasen von der OSM-vermittelten STAT1 Aktivierung abh{\"a}ngig ist und {\"u}ber einen STAT3/SOCS3-abh{\"a}ngigen Mechanismus reguliert wird. W{\"a}hrend die Blockade der STAT1 Expression zu einer Inhibierung der OSM-induzierten Expression der Helikasen f{\"u}hrte, wurde durch den Verlust von STAT3 oder SOCS3 die Expression von RIG-I und MDA5, aufgrund einer st{\"a}rkeren und l{\"a}nger anhaltenden STAT1-Phosphorylierung, signifikant erh{\"o}ht. Zus{\"a}tzlich konnte ein additiver Effekt zwischen der OSM- und IFNγ-vermittelten Helikasenexpression belegt werden. Dieses Resultat steht im Einklang mit vorhergehenden Ver{\"o}ffentlichungen, die einen Crosstalk zwischen OSM und Typ I IFN bei der antiviralen Abwehr gegen das Hepatitis C Virus beschreiben.},
  language  = {de}
}