@phdthesis{Peindl2024,
  author    = {Peindl, Matthias},
  title     = {Refinement of 3D lung cancer models for automation and patient stratification with mode-of-action studies},
  doi       = {10.25972/OPUS-31069},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-310693},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {Lung cancer is the main cause of cancer-related deaths worldwide. Despite the availability of several targeted therapies and immunotherapies in the clinics, the prognosis for lung cancer remains poor. A major problem for the low benefit of these therapies is intrinsic and acquired resistance, asking for pre-clinical models for closer investigation of predictive biomarkers for refined personalized medicine and testing of possible combination therapies as well as novel therapeutic approaches to break resistances. One third of all lung adenocarcinoma harbor mutations in the KRAS gene, of which 39 \% are transitions from glycine to cysteine in codon 12 (KRASG12C). Being considered "undruggable" in previous decades, KRASG12C-inhibitors now paved the way into the standard-of-care for lung adenocarcinoma treatment in the clinics. Still, the overall response rates as well as overall survival of patients treated with KRASG12C-inhibitors are sobering. Therefore, 3D KRASG12C-biomarker in vitro models were developed based on a decellularized porcine jejunum (SISmuc) using commercial and PDX-derived cell lines and characterized in regards of epithelial-mesenchymal-transition (EMT), stemness, proliferation, invasion and c-MYC expression as well as the sensitivity towards KRASG12C-inhibiton. The phenotype of lung tumors harboring KRAS mutations together with a c-MYC overexpression described in the literature regarding invasion and proliferation for in vivo models was well represented in the SISmuc models. A higher resistance towards targeted therapies was validated in the 3D models compared to 2D cultures, while reduced viability after treatment with combination therapies were exclusively observed in the 3D models. In the test system neither EMT, stemness nor the c-MYC expression were directly predictive for drug sensitivity. Testing of a panel of combination therapies, a sensitizing effect of the aurora kinase A (AURKA) inhibitor alisertib for the KRASG12C-inhibitor ARS-1620 directly correlating with the level of c-MYC expression in the corresponding 3D models was observed. Thereby, the capability of SISmuc tumor models as an in vitro test system for patient stratification was demonstrated, holding the possibility to reduce animal experiments. Besides targeted therapies the treatment of NSCLC with oncolytic viruses (OVs) is a promising approach. However, a lack of in vitro models to test novel OVs limits the transfer from bench to bedside. In this study, 3D NSCLC models based on the SISmuc were evaluated for their capability to perform efficacy and risk assessment of oncolytic viruses (OVs) in a pre-clinical setting. Hereby, the infection of cocultures of tumor cells and fibroblasts on the SISmuc with provided viruses demonstrated that in contrast to a wildtype herpes simplex virus 1 (HSV-1) based OV, the attenuated version of the OV exhibited specificity for NSCLC cells with a more advanced and highly proliferative phenotype, while fibroblasts were no longer permissive for infection. This approach introduced SISmuc tumor models as novel test system for in vitro validation of OVs. Finally, a workflow for validating the efficacy of anti-cancer therapies in 3D tumor spheroids was established for the transfer to an automated platform based on a two-arm-robot system. In a proof-of-concept process, H358 spheroids were characterized and treated with the KRASG12C-inhibitor ARS-1620. A time- and dose-dependent reduction of the spheroid area after treatment was defined together with a live/dead-staining as easy-to-perform and cost-effective assays for automated drug testing that can be readily performed in situ in an automated system.},
  subject      = {Krebs <Medizin>},
  language  = {en}
}
@phdthesis{Doll2024,
  author    = {Doll, Julia},
  title     = {Identifizierung und Charakterisierung neuer, mit H{\"o}rst{\"o}rungen assoziierter Gene und Varianten mittels Exom-Sequenzierung},
  doi       = {10.25972/OPUS-26109},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-261097},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {Laut des aktuellen Reports der Weltgesundheitsorganisation sind ca. 466 Millionen Menschen weltweit von einer H{\"o}rst{\"o}rung (HS) betroffen. Durch die enorme Heterogenit{\"a}t und die klinische Variabilit{\"a}t, die diese Erkrankung ausmacht, und viele bisher nicht mit HS assoziierte Gene, bleibt ein großer Teil der erblich bedingten HS in vielen Familien unaufgekl{\"a}rt. Die Entwicklung moderner Techniken, wie die Next-Generation Sequenzierung (NGS) und der Fortschritt bei der Untersuchung von Modellorganismen trugen jedoch in den letzten Jahren immens dazu bei, neue Gene zu identifizieren, die innerhalb des auditorischen Signalwegs oder damit assoziierten Strukturen beteiligt sind. Die vorliegende Arbeit umfasst Ergebnisse dreier Ver{\"o}ffentlichungen, in denen iranische und pakistanische Familien und eine deutsche Familie mit erblich bedingter HS untersucht und neue, krankheitsverursachende Varianten identifiziert und funktionell charakterisiert wurden. Im ersten Abschnitt konnten zwei neue rezessive Varianten im CDC14A-Gen als krankheitsverursachend identifiziert werden, die zu einem potentiellen Funktionsverlust des kodierten Proteins in einer iranischen und einer pakistanischen Familie f{\"u}hren. Mit Hilfe einer funktionellen Charakterisierung auf RNA-Ebene (Spleiß-Assay und RT-qPCR) konnte der Funktionsverlust beider Varianten best{\"a}tigt werden. Der zweite Abschnitt umfasst eine deutsche Familie mit sieben von einer HS betroffenen Familienmitgliedern, in der eine heterozygote missense Variante in MYO3A identifiziert wurde. In der vorliegenden Arbeit konnte somit die erste autosomal dominante Variante in einer europ{\"a}ischen Familie mit einer bilingualen, sensorineuralen Hochtonschwerh{\"o}rigkeit beschrieben werden und der dominante Charakter von MYO3A best{\"a}tigt werden. Im dritten Abschnitt konnten die krankheitsverursachenden Varianten in 13 Familien aus einer Kohorte mit 21 pakistanischen Familien mit einer syndromalen und nicht-syndromalen HS ausfindig gemacht werden. Hierbei wurden sowohl bekannte, als auch bisher nicht beschriebene Varianten detektiert. Die Aufkl{\"a}rungsrate innerhalb dieser Kohorte betrug 61,9\% und es konnte somit das Spektrum syndromaler und nicht-syndromaler HS erweitert werden. Der letzte Abschnitt dieser Arbeit beschreibt eine iranische Familie mit einer milden HS und milden Intelligenzminderung, in der eine homozygote missense Variante im Kandidatengen DBN1 ausfindig gemacht wurde. Um die Funktion und die Auswirkungen eines potentiellen Verlusts des codierten Proteins Drebrin zu untersuchen, wurden immunhistochemische F{\"a}rbungen und auditorische Messungen an Dbn1 Knockout (KO)-M{\"a}usen durchgef{\"u}hrt. Hierbei konnte eine Expression innerhalb der Nervenfasern, die innere Haarzellen innervieren, nachgewiesen werden. Eine leicht verl{\"a}ngerte Latenz f{\"u}r die ABR-Welle IV in KO-M{\"a}usen im Vergleich zum Wildtyp ergab den Hinweis auf einen Defekt innerhalb des zentralen auditorischen Signalwegs, der m{\"o}glicherweise mit einer Sprachverarbeitungsst{\"o}rung im Menschen korreliert.},
  subject      = {H{\"o}rst{\"o}rung},
  language  = {de}
}
@phdthesis{Steinfatt2023,
  author    = {Steinfatt, Tim Alexander},
  title     = {Modulation of regulatory T cells for the immunotherapy of inflammatory diseases and cancer},
  doi       = {10.25972/OPUS-19260},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-192600},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {Regulatory T cells (Tregs) are the masters of immune regulation controlling inflammation and tolerance, tissue repair and homeostasis. Multiple immunological diseases result from altered Treg frequencies and Treg dysfunction. We hypothesized that augmenting Treg function and numbers would prevent inflammatory disease whereas inhibiting or depleting Tregs would improve cancer immunotherapy. In the first part of this thesis, we explored whether in vivo activation and expansion of Tregs would impair acute graft-versus-host disease (aGvHD). In this inflammatory disease, Tregs are highly pathophysiological relevant and their adoptive transfer proved beneficial on disease outcome in preclinical models and clinical studies. IL-2 has been recognized as a key cytokine for Treg function. Yet, attempts in translating Treg expansion via IL-2 have remained challenging, due to IL-2s extremely broad action on other cell types including effector T cells, NK cells, eosinophils and vascular leakage syndrome, and importantly, due to poor pharmacokinetics in vivo. We addressed the latter issue using an IL-2-IgG-fusion protein (irrIgG-IL-2) with improved serum retention and demonstrated profound Treg expansion in vivo in FoxP3-luciferase reporter mice. Further, we augmented Treg numbers and function via the selective-TNF based agonists of TNFR2 (STAR2). Subsequently, we tested a next-generation TNFR2 agonist, termed NewSTAR, which proved even more effective. TNFR2 stimulation augmented Treg numbers and function and was as good as or even superior to the IL-2 strategy. Finally, in a mouse model of aGvHD we proved the clinical relevance of Treg expansion and activation with irrIgG-IL-2, STAR2 and NewSTAR. Notably, the TNFR2 stimulating constructs were outstanding as we observed not the IL-2 prototypic effects on other cell populations and no severe side effects. In the second part of this thesis, we explored Tregs in pancreatic ductal adenocarcinoma (PDAC) and developed targeting strategies. Among several tumor entities in which Tregs impact survival, preclinical and clinical data demonstrated their negative role on PDAC. In our studies we employed the orthotopic syngeneic Panc02 model in immunocompetent mice. Based on flow cytometric analysis of the tumor microenvironment we propose TIGIT and TNFRSF members as novel therapeutic targets. Surprisingly, we found that blocking TNFR2 did not interfere with intratumoral Treg accumulation. However, we decreased the highly abundant intratumoral Tregs when we disrupted the tumor extracellular matrix. In PDAC, Treg manipulation alone did not lead to tumor regression and we propose that an additional immune boost may be necessary for efficient tumor immune surveillance and cancer clearance. This contrasts with aGvHD, in which Treg manipulation alone was sufficient to improve disease outcome. Conclusively, we demonstrated the enormous medical benefit of Treg manipulation. Our promising data obtained with our newly developed powerful tools highlight the potential to translate our findings into clinical practice to therapeutically target human Tregs in patients. With novel TNFR2 agonists (STAR2, NewSTAR) we augmented Treg numbers and function as (or even more) effectively than with IL-2, without causing adverse side effects. Importantly, exogenous in vivo Treg expansion protected mice from aGvHD. For the therapy of PDAC, we identified novel targets on Tregs, notably TIGIT and members of the TNFRSF. We demonstrated that altering the extracellular tumor matrix can efficiently disrupt the Treg abundance in tumors. These novel targeting strategies appear as attractive new treatment options and they may benefit patients suffering from inflammatory disease and cancer in the future.},
  language  = {en}
}
@phdthesis{Riemens2023,
  author    = {Riemens, Renzo J. M.},
  title     = {Neuroepigenomics in Alzheimer's disease: The single cell ADds},
  isbn      = {978-94-6423-524-1},
  doi       = {10.25972/OPUS-25457},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-254574},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {Die Forschung, die in dieser Arbeit zusammengestellt wird, kann in zwei Teile geteilt werden. Der erste Teil, bestehend aus vier Kapiteln, konzentriert sich auf die Rolle der epigenetischen Dysregulation in der {\"A}tiopathophysiologie der sporadischen Alzheimer-Krankheit (sAD). Neben Einblicken in die neuesten Entwicklungen in neuroepigenomischen Studien zu dieser Krankheit geht der erste Teil der Arbeit auch auf verbleibende Herausforderungen ein und gibt einen Ausblick auf m{\"o}gliche Entwicklungen auf diesem Gebiet. Der zweite Teil, der drei weitere Kapitel umfasst, konzentriert sich auf die Anwendung von auf induzierten pluripotenten Stammzellen (iPSC) basierenden Krankheitsmodellen f{\"u}r das Studium der AD, einschließlich, aber nicht beschr{\"a}nkt auf mechanistische Studien zur epigenetischen Dysregulation unter Verwendung dieser Plattform. Neben der Skizzierung der bisherigen Forschung mit iPSC-basierten Modellen f{\"u}r sAD gibt der zweite Teil der Arbeit auch Einblicke in die Gewinnung krankheitsrelevanter Nervenkulturen auf Basis der gezielten Differenzierung von iPSCs und beinhaltet dar{\"u}ber hinaus einen experimentellen Ansatz f{\"u}r den Aufbau eines solchen Modellsystems.},
  subject      = {Epigenetik},
  language  = {en}
}
@phdthesis{Schulz2023,
  author    = {Schulz, Ellina},
  title     = {Lokale Ultraschall-vermittelte Zytostatika-Applikation zur Behandlung von Hirntumoren},
  doi       = {10.25972/OPUS-32016},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-320168},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {Glioblastoma (GBM) sind b{\"o}sartige hirneigene Tumore, deren schlechte Prognose einer innovativen Therapie bedarf. Aus diesem Grund wurde ein neuer Therapieansatz entwickelt, der auf einer lokalen Ultraschall-vermittelten Zytostatika Applikation beruht. Hierf{\"u}r wurden stabile Microbubbles (MB) bestehend aus Phospholipiden synthetisiert. Es konnte gezeigt werden, dass MB als auch fokussierter Ultraschall niedriger Intensit{\"a}t (LIFU) keinen negativen Einfluss auf GBM-Zellen hat. MB hingegen konnten mittels LIFU destruiert werden, wodurch das in den MB eingeschlossene Chemotherapeutikum freigesetzt werden kann. Es wurden verschiedene Platin(II)- und Palladium(II)-Komplexe auf GBM Zellen getestet. Zur Beladung der MB wurde Doxorubicin (Dox) verwendet. Es konnte eine Beladungseffizienz der MB mit Dox von 52 \% erreicht werden, auch eine Aufreinigung dieser mittel Ionenaustausch-Chromatographie und Dialyse war erfolgreich. Die Austestung der mit Dox beladenen MB (MBDox) erfolgte auf GBM-Zellen in 2D- und 3D-Zelkulturmodellen. Dabei zeigte sich, dass die Behandlung mit MBDox und LIFU f{\"u}r 48 h eine zytotoxische Wirkung hatte, die sich signifikant von der Behandlung mit MBDox ohne LIFU unterschied. Zur Austestung der MBDox in 3D-Zellkulturmodellen wurden zwei Scaffold-Systeme eingesetzt. Es zeigte sich in den Versuchen, dass MBDox mit LIFU im Vergleich zu MBDox ohne LIFU Applikation einen zytotoxischen Effekt auf GBM-Zellen haben. Somit konnte die Wirksamkeit der Zytostatika Applikation mittels MB und LIFU in 2D- und 3D-Zellkulturmodellen erfolgreich etabliert werden. Als weiterer Schritt wurden zwei 3D in vitro Modelle erarbeitet. Dabei wurden zun{\"a}chst organotypische hippocampale Slice Kulturen (organotypic hippocampal brain slice cultures, OHSC) aus der Maus hergestellt und anschließend mit fluoreszent-markierten Mikrotumoren aus GBM-Zelllinien, Prim{\"a}rzellen (PZ) und aus Patienten generierten GBM-Organoiden hergestellt. Diese GBM-Modelle wurden mit Tumor Treating Fields (TTFields) behandelt. Dabei war eine Abnahme der Tumorgr{\"o}ße von Mikrotumoren aus GBM-Zellen und PZ unter TTFields-Behandlung f{\"u}r 72 h messbar. Als weiteres in vitro Modell wurden humane Tumorschnitte aus intraoperativ entferntem GBM-Patientenmaterial hergestellt. Die Schnitte wiesen ein heterogenes Ansprechen nach 72 h TTFields-Applikation auf. Dies spiegelt die Heterogenit{\"a}t des GBM sehr gut wider und best{\"a}rkt die Eignung des Modelles zur Untersuchung von neuen Therapieans{\"a}tzen zur Behandlung von GBM.},
  subject      = {Glioblastom},
  language  = {de}
}
@phdthesis{Eckert2023,
  author    = {Eckert, Ina-Nathalie},
  title     = {Molecular markers of myeloid-derived suppressor cells and their functional role for homing and in disease models in mice},
  doi       = {10.25972/OPUS-31997},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-319974},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {MDSCs are suppressive immune cells with a high relevance in various pathologies including cancer, autoimmunity, and chronic infections. Surface marker expression of MDSCs resembles monocytes and neutrophils which have immunostimulatory functions instead of suppressing T cells. Therefore, finding specific surface markers for MDSCs is important for MDSC research and therapeutic MDSC manipulation. In this study, we analyzed if the integrin VLA-1 has the potential as a novel MDSC marker. VLA-1 was expressed by M-MDSCs but not by G-MDSCs as well as by Teff cells. VLA-1 deficiency did not impact iNOS expression, the distribution of M-MDSC and G-MDSC subsets, and the suppressive capacity of MDSCs towards na{\"i}ve and Teff cells in vitro. In mice, VLA-1 had no effect on the homing capability of MDSCs to the spleen, which is a major reservoir for MDSCs. Since the splenic red pulp contains collagen IV and VLA-1 binds collagen IV with a high affinity, we found MDSCs and Teff cells in this area as expected. We showed that T cell suppression in the spleen, indicated by reduced T cell recovery and proliferation as well as increased apoptosis and cell death, partially depended on VLA-1 expression by the MDSCs. In a mouse model of multiple sclerosis, MDSC injection prior to disease onset led to a decrease of the disease score, and this effect was significantly reduced when MDSCs were VLA-1 deficient. The expression of Sema7A by Teff cells, a ligand for VLA-1 which is implicated in negative T cell regulation, resulted in a slightly stronger Teff cell suppression by MDSCs compared to Sema7A deficient T cells. Live cell imaging and intravital 2-photon microscopy showed that the interaction time of MDSCs and Teff cells was shorter when MDSCs lacked VLA 1 expression, however VLA-1 expression had no impact on MDSC mobility. Therefore, the VLA-1-dependent interaction of MDSC and Teff cells on collagen IV in the splenic red pulp is implicated MDSC-mediated Teff cell suppression.},
  subject      = {Immunologie},
  language  = {en}
}
@article{LibreSeisslerGuerreroetal.2021,
  author    = {Libre, Camille and Seissler, Tanja and Guerrero, Santiago and Batisse, Julien and Verriez, C{\´e}dric and Stupfler, Benjamin and Gilmer, Orian and Cabrera-Rodriguez, Romina and Weber, Melanie M. and Valenzuela-Fernandez, Agustin and Cimarelli, Andrea and Etienne, Lucie and Marquet, Roland and Paillart, Jean-Christophe},
  title     = {A conserved uORF regulates APOBEC3G translation and is targeted by HIV-1 Vif protein to repress the antiviral factor},
  series = {Biomedicines},
  volume    = {10},
  journal   = {Biomedicines},
  number    = {1},
  issn      = {2227-9059},
  doi       = {10.3390/biomedicines10010013},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-252147},
  year      = {2021},
  abstract  = {The HIV-1 Vif protein is essential for viral fitness and pathogenicity. Vif decreases expression of cellular restriction factors APOBEC3G (A3G), A3F, A3D and A3H, which inhibit HIV-1 replication by inducing hypermutation during reverse transcription. Vif counteracts A3G at several levels (transcription, translation, and protein degradation) that altogether reduce the levels of A3G in cells and prevent its incorporation into viral particles. How Vif affects A3G translation remains unclear. Here, we uncovered the importance of a short conserved uORF (upstream ORF) located within two critical stem-loop structures of the 5′ untranslated region (5′-UTR) of A3G mRNA for this process. A3G translation occurs through a combination of leaky scanning and translation re-initiation and the presence of an intact uORF decreases the extent of global A3G translation under normal conditions. Interestingly, the uORF is also absolutely required for Vif-mediated translation inhibition and redirection of A3G mRNA into stress granules. Overall, we discovered that A3G translation is regulated by a small uORF conserved in the human population and that Vif uses this specific feature to repress its translation.},
  language  = {en}
}
@phdthesis{Somody2023,
  author    = {Somody, Joseph Christian Campbell},
  title     = {Leveraging deep learning for identification and structural determination of novel protein complexes from \(in\) \(situ\) electron cryotomography of \(Mycoplasma\) \(pneumoniae\)},
  doi       = {10.25972/OPUS-31344},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-313447},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {The holy grail of structural biology is to study a protein in situ, and this goal has been fast approaching since the resolution revolution and the achievement of atomic resolution. A cell's interior is not a dilute environment, and proteins have evolved to fold and function as needed in that environment; as such, an investigation of a cellular component should ideally include the full complexity of the cellular environment. Imaging whole cells in three dimensions using electron cryotomography is the best method to accomplish this goal, but it comes with a limitation on sample thickness and produces noisy data unamenable to direct analysis. This thesis establishes a novel workflow to systematically analyse whole-cell electron cryotomography data in three dimensions and to find and identify instances of protein complexes in the data to set up a determination of their structure and identity for success. Mycoplasma pneumoniae is a very small parasitic bacterium with fewer than 700 protein-coding genes, is thin enough and small enough to be imaged in large quantities by electron cryotomography, and can grow directly on the grids used for imaging, making it ideal for exploratory studies in structural proteomics. As part of the workflow, a methodology for training deep-learning-based particle-picking models is established. As a proof of principle, a dataset of whole-cell Mycoplasma pneumoniae tomograms is used with this workflow to characterize a novel membrane-associated complex observed in the data. Ultimately, 25431 such particles are picked from 353 tomograms and refined to a density map with a resolution of 11 {\AA}. Making good use of orthogonal datasets to filter search space and verify results, structures were predicted for candidate proteins and checked for suitable fit in the density map. In the end, with this approach, nine proteins were found to be part of the complex, which appears to be associated with chaperone activity and interact with translocon machinery. Visual proteomics refers to the ultimate potential of in situ electron cryotomography: the comprehensive interpretation of tomograms. The workflow presented here is demonstrated to help in reaching that potential.},
  subject      = {Kryoelektronenmikroskopie},
  language  = {en}
}
@phdthesis{Lueffe2023,
  author    = {L{\"u}ffe, Teresa Magdalena},
  title     = {Behavioral and pharmacological validation of genetic zebrafish models for ADHD},
  doi       = {10.25972/OPUS-25716},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-257168},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {Attention-deficit/hyperactivity disorder (ADHD) is the most prevalent neurodevelopmental disorder described in psychiatry today. ADHD arises during early childhood and is characterized by an age-inappropriate level of inattention, hyperactivity, impulsivity, and partially emotional dysregulation. Besides, substantial psychiatric comorbidity further broadens the symptomatic spectrum. Despite advances in ADHD research by genetic- and imaging studies, the etiopathogenesis of ADHD remains largely unclear. Twin studies suggest a heritability of 70-80 \% that, based on genome-wide investigations, is assumed to be polygenic and a mixed composite of small and large, common and rare genetic variants. In recent years the number of genetic risk candidates is continuously increased. However, for most, a biological link to neuropathology and symptomatology of the patient is still missing. Uncovering this link is vital for a better understanding of the disorder, the identification of new treatment targets, and therefore the development of a more targeted and possibly personalized therapy. The present thesis addresses the issue for the ADHD risk candidates GRM8, FOXP2, and GAD1. By establishing loss of function zebrafish models, using CRISPR/Cas9 derived mutagenesis and antisense oligonucleotides, and studying them for morphological, functional, and behavioral alterations, it provides novel insights into the candidate's contribution to neuropathology and ADHD associated phenotypes. Using locomotor activity as behavioral read-out, the present work identified a genetic and functional implication of Grm8a, Grm8b, Foxp2, and Gad1b in ADHD associated hyperactivity. Further, it provides substantial evidence that the function of Grm8a, Grm8b, Foxp2, and Gad1b in activity regulation involves GABAergic signaling. Preliminary indications suggest that the three candidates interfere with GABAergic signaling in the ventral forebrain/striatum. However, according to present and previous data, via different biological mechanisms such as GABA synthesis, transmitter release regulation, synapse formation and/or transcriptional regulation of synaptic components. Intriguingly, this work further demonstrates that the activity regulating circuit, affected upon Foxp2 and Gad1b loss of function, is involved in the therapeutic effect mechanism of methylphenidate. Altogether, the present thesis identified altered GABAergic signaling in activity regulating circuits in, presumably, the ventral forebrain as neuropathological underpinning of ADHD associated hyperactivity. Further, it demonstrates altered GABAergic signaling as mechanistic link between the genetic disruption of Grm8a, Grm8b, Foxp2, and Gad1b and ADHD symptomatology like hyperactivity. Thus, this thesis highlights GABAergic signaling in activity regulating circuits and, in this context, Grm8a, Grm8b, Foxp2, and Gad1b as exciting targets for future investigations on ADHD etiopathogenesis and the development of novel therapeutic interventions for ADHD related hyperactivity. Additionally, thigmotaxis measurements suggest Grm8a, Grm8b, and Gad1b as interesting candidates for prospective studies on comorbid anxiety in ADHD. Furthermore, expression analysis in foxp2 mutants demonstrates Foxp2 as regulator of ADHD associated gene sets and neurodevelopmental disorder (NDD) overarching genetic and functional networks with possible implications for ADHD polygenicity and comorbidity. Finally, with the characterization of gene expression patterns and the generation and validation of genetic zebrafish models for Grm8a, Grm8b, Foxp2, and Gad1b, the present thesis laid the groundwork for future research efforts, for instance, the identification of the functional circuit(s) and biological mechanism(s) by which Grm8a, Grm8b, Foxp2, and Gad1b loss of function interfere with GABAergic signaling and ultimately induce hyperactivity.},
  language  = {en}
}
@phdthesis{Ferretti2022,
  author    = {Ferretti, Pamela},
  title     = {\(Clostridioides\) \(difficile\) beyond the disease-centred perspective: Beneficial properties in healthy infants and over-diagnosis in diseased adults identified by species- and SNV-based metagenomic analysis},
  doi       = {10.25972/OPUS-25417},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-254170},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {Clostridioides difficile is a bacterial species well known for its ability to cause C. difficile infection (also known as CDI). The investigation of the role of this species in the human gut has been so far dominated by a disease-centred perspective, focused on studying C. difficile in relation to its associated disease. In this context, the first aim of this thesis was to combine publicly available metagenomic data to analyse the microbial composition of stool samples from patients diagnosed with CDI, with a particular focus on identifying a CDI-specific microbial signature. However, similarly to many other bacterial species inhabiting the human gut, C. difficile association with disease is not valid in absolute terms, as C. difficile can be found also among healthy subjects. Further aims of this thesis were to 1) identify potential C. difficile reservoirs by screening a wide range of habitats, hosts, body sites and age groups, and characterize the biotic context associated with C. difficile presence, and 2) investigate C. difficile within-species diversity and its toxigenic potential across different age groups. The first part of the thesis starts with the description of the concepts and definitions used to identify bacterial species and within-species diversity, and then proceeds to provide an overview of the bacterial species at the centre of my investigation, C. difficile. The first Chapter includes a detailed description of the discovery, biology and physiology of this clinically relevant species, followed by an overview of the diagnostic protocols used in the clinical setting to diagnose CDI. The second part of the thesis describes the methodology used to investigate the questions mentioned above, while the third part presents the results of such investigative effort. I first show that C. difficile could be found in only a fraction of the CDI samples and that simultaneous colonization of multiple enteropathogenic species able to cause CDI-like clinical manifestations is more common than previously thought, raising concerns about CDI overdiagnosis. I then show that the CDIassociated gut microbiome is characterized by a specific microbial signature, distinguishable from the community composition associated with non-CDI diarrhea. Beyond the nosocomial and CDI context, I show that while rarely found in adults, C. difficile is a common member of the infant gut microbiome, where its presence is associated with multiple indicators typical of a desirable healthy microbiome development. In addition, I describe C. difficile extensive carriage among asymptomatic subjects, of all age groups and a potentially novel clade of C. difficile identified exclusively among infants. Finally, I discuss the limitations, challenges and future perspectives of my investigation.},
  language  = {en}
}
@phdthesis{Amelingmeier2022,
  author    = {Amelingmeier, Florian},
  title     = {Identifizierung und Untersuchung TOP-mRNA - bindender Faktoren},
  doi       = {10.25972/OPUS-28923},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-289231},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {Im Zellkern eukaryotischer Zellen werden Gene in mRNAs transkribiert, welche umfangreich prozessiert und aus dem Zellkern exportiert werden. Im Zytoplasma erfolgt die Translation der mRNAs in Proteine, ein Prozess, welcher viel Energie ben{\"o}tigt und daher mittels vielf{\"a}ltiger Mechanismen streng reguliert wird. Ein Beispiel hierf{\"u}r stellt die Klasse der TOP-mRNAs dar, eine RNA-Spezies, welche haupts{\"a}chlich Transkripte von Genen umfasst, die selbst in die Translation involviert sind. Die prominentesten Vertreter dieser Klasse sind die Proteine der kleinen und großen ribosomalen Untereinheiten. TOP-mRNAs zeichnen sich durch ein gemeinsames Sequenz-Motiv am Anfang Ihrer 5'-UTR aus, welches aus einem Pyrimidinstrang besteht und unmittelbar nach dem Cap mit einem Cytosin beginnt. Dieses allen TOP-RNAs gemeinsame Motiv erm{\"o}glicht die zeitgleiche Translationskontrolle dieser RNA-Klasse. So kann die Translation der TOP-mRNAs unter Stressbedingungen wie z.B. N{\"a}hrstoffmangel koordiniert inhibiert werden, wodurch Energie eingespart wird. Bereits lange wird nach einem Regulator gesucht, der an dieses TOP-Motiv bindet und die koordinierte Regulation erm{\"o}glicht. Man kann sich hier einen Inhibitor oder auch einen Aktivator vorstellen. Verschiedene Proteine wurden bereits in Erw{\"a}gung gezogen. In dieser Arbeit wurde das Protein TIAR mittels Massenspektrometrie als TOP-interagierender Faktor identifiziert und dessen Bindungseigenschaften mit dem TOP-Motiv durch Shift Assays untersucht. Hierbei konnten Minimalkonstrukte verschiedener Organismen sowie RNA-TOP - Sequenzen identifiziert werden, welche sich f{\"u}r Strukturanalysen eignen w{\"u}rden. Als weiterer TOP-interagierender Faktor wurde {\"u}ber verschiedene sequenzielle Reinigungsschritte das Protein 14-3-3ε identifiziert. Weiterhin wurden die TOP-Motiv-bindenden Proteine LARP1 und LARP7 auf Ihre Bindungseigenschaften mit Ihren Zielsequenzen untersucht. W{\"a}hrend gezeigt werden konnte, dass LARP1 einen inhibierenden Einfluss auf TOP-RNAs hat, wurde in weiteren Shift-Assays die Bindungseigenschaften von LARP7 mit 7SK untersucht, wobei ebenfalls ein minimales LARP7-Konstrukt sowie 7SK-Konstrukte f{\"u}r Strukturanalysen identifiziert werden konnten. Weiterhin konnte gezeigt werden, dass verschiedene Substanzen wie tRNA und Arginin einen starken Einfluss auf die LARP7-7SK - Interaktion aus{\"u}ben, welcher in weiteren Studien ber{\"u}cksichtigt werden sollte.},
  subject      = {Proteinbiosynthese},
  language  = {de}
}
@phdthesis{JimenezMartin2022,
  author    = {Jim{\´e}nez Mart{\´i}n, Ovidio Manuel},
  title     = {Analysis of MYCN and MAX alterations in Wilms Tumor},
  doi       = {10.25972/OPUS-24291},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-242919},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {Wilms tumor (WT) is the most common renal tumor in childhood. Among others, MYCN copy number gain and MYCN P44L and MAX R60Q mutations have been identified in WT. The proto-oncogene MYCN encodes a transcription factor that requires dimerization with MAX to activate transcription of numerous target genes. MYCN gain has been associated with adverse prognosis. The MYCN P44L and MAX R60Q mutations, located in either the transactivating or basic helix-loop-helix domain, respectively, are predicted to be damaging by different pathogenicity prediction tools. These mutations have been reported in several other cancers and remain to be functionally characterized. In order to further describe these events in WT, we screened both mutations in a large cohort of unselected WT patients, to check for an association of the mutation status with certain histological or clinical features. MYCN P44L and MAX R60Q revealed frequencies of 3 \% and 0.9 \% and also were significantly associated to higher risk of relapse and metastasis, respectively. Furthermore, to get a better understanding of the MAX mutational landscape in WT, over 100 WT cases were analyzed by Sanger sequencing to identify other eventual MAX alterations in its coding sequence. R60Q remained the only MAX CDS alteration described in WT to date. To analyze the potential functional consequences of these mutations, we used a doxycycline-inducible system to overexpress each mutant in HEK293 cells. This biochemical characterization identified a reduced transcriptional activation potential for MAX R60Q, while the MYCN P44L mutation did not change activation potential or protein stability. The protein interactome of N-MYC-P44L was likewise not altered as shown by mass spectrometric analyses of purified N-MYC complexes. However, we could identify a number of novel N-MYC partner proteins, several of these known for their oncogenic potential. Their correlated expression in WT samples suggested a role in WT oncogenesis and they expand the range of potential biomarkers for WT stratification and targeting, especially for high-risk WT.},
  subject      = {Nephroblastom},
  language  = {en}
}
@phdthesis{Christmann2022,
  author    = {Christmann, Gabriel},
  title     = {USP 10 als Deubiquitinase f{\"u}r β-Catenin},
  doi       = {10.25972/OPUS-27299},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-272998},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {Der WNT-Signalweg ist ein hochkonservierter Signalweg, dessen zentraler intrazellul{\"a}rer Regulationsschritt die Proteinstabilit{\"a}t des Proteins β-Catenin ist. Deregulierende Mutationen in diesem sind fr{\"u}he Ereignisse bei der Entstehung von Darmtumoren. Ist der Abbau von β-Catenin gest{\"o}rt, so ist unabh{\"a}ngig von {\"a}ußerer Kontrolle der Signalweg konstitutiv aktiviert und liefert ein Wachstumssignal. Untersuchungen haben aber gezeigt, dass beim Vorliegen solcher Mutationen immer noch eine - unzureichende - Ubiquitinylierung und ein Abbau von β-Catenin stattfindet. Ziel dieser Studie war Deubiquitinasen (DUBs) zu finden, die durch ihre Aktivit{\"a}t den Abbau von β-Catenin verhindern. Mithilfe eines siRNA Screens in der Vorarbeit konnten DUBs als Kandidaten f{\"u}r einen CRISPR Ansatz ausgew{\"a}hlt werden. APC Wildtyp HEK293T Zellen und Darmkrebszellen wurden mit lentiviralen CRISPR/Cas9 Vektoren infiziert, in welche sgRNAs gegen exonische Sequenzen von DUBs geklont waren. Einzelne Zellklone von USP10 CRISPR Zellen wurden weiter untersucht. In Western Blots und Immunofluoreszenz zeigte sich bei den USP10 CRISPR Zellen eine verminderte Expression von USP10 und damit einhergehend β-Catenin. Proteinstabilit{\"a}tsversuche mit MG132 und Cycloheximid zeigten einen erh{\"o}hten Abbau von β-Catenin in HEK293T USP10 CRISPR Zellen, vor allem nach Stimulierung des WNT-Signalwegs durch LiCl. In Aktivierungsassays (Luciferase und TOP-GFP FACS) des WNT-Signalwegs zeigte sich in HEK293T Zellen nach Behandlung mit LiCl eine geringere Aktivierung in den USP10 CRISPR Zellen. In einem Wachstumsassay zeigten HT29 USP10 CRISPR ein geringeres Wachstum als Kontrollzellen. W{\"a}hrend in einer histologischen F{\"a}rbung von Mausgewebe eine erh{\"o}hte Expression von USP10 nachweisbar war, zeigten sich in einer TMA F{\"a}rbung kein eindeutiger Unterschied zwischen gesundem Gewebe und Tumorgewebe. Die Studie identifiziert USP10 als eine m{\"o}gliche DUB f{\"u}r β-Catenin und potenzielles Ziel f{\"u}r eine Beeinflussung des mutierten WNT-Signalwegs in Darmkrebszellen.},
  subject      = {Darmkrebs},
  language  = {de}
}
@phdthesis{Amatobi2022,
  author    = {Amatobi, Kelechi Michael},
  title     = {Circadian clocks determine transport and membrane lipid oscillation in \(Drosophila\) hemolymph in complex interactions between nutrient-type, photic conditions and feeding behaviour},
  doi       = {10.25972/OPUS-24446},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-244462},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {The interaction between circadian clocks and metabolism is of increasing interest, since clock dysfunction often correlates with metabolic pathologies. Many research articles have been published analysing the impact of factors such as circadian clock, light, feeding time and diet-type on energy homeostasis in various tissues/organs of organisms with most of the findings done in mammals. Little is known about the impact of circadian clock and the above-mentioned factors on circulating lipids, especially the transport form of lipids - diacylglycerol (DG) and membrane lipids such as phosphatidylethanolamine (PE) and phosphatidylcholine (PC) in the Drosophila hemolymph. The fruit fly Drosophila is a prime model organism in circadian, behaviour and metabolism research. To study the role of circadian clock and behaviour in metabolism, we performed an extensive comparative hemolymph lipid (diacylglycerol: DG, phosphatidylethanolamine: PE, phosphatidylcholine: PC) analysis using ultra performance liquid chromatography coupled to time-of-flight mass spectrometry (UPLC-MS) between wild-type flies (WTCS) and clock disrupted mutants (per01). In addition, clock controlled food intake- feeding behaviour was investigated. Time-dependent variation of transport (DG) and membrane lipids (PE and PC) were not rhythmic in WTCS under constant darkness and in per01 under LD, suggesting an impact of light and clock genes on daily lipid oscillations. Day-time and night-time restriction of food led to comparable lipid profiles, suggesting that lipid oscillations are not exclusively entrained by feeding but rather are endogenously regulated. Ultradian oscillations in lipid levels in WTCS under LD were masked by digested fatty acids since lipid levels peaked more robustly at the beginning and end of light phase when flies were fed a lipid- and protein-free diet. These results suggest that metabolite (DG, PE and PC) oscillation is influenced by complex interactions between nutrient-type, photic conditions, circadian clock and feeding time. In conclusion, the results of this thesis suggest that circadian clocks determine transport and membrane lipid oscillation in Drosophila hemolymph in complex interactions between nutrient-type, photic conditions and feeding behaviour.},
  subject      = {Pharmaceutische Biologie},
  language  = {en}
}
@article{HicklHeintzBuschartTrautweinSchultetal.2019,
  author    = {Hickl, Oskar and Heintz-Buschart, Anna and Trautwein-Schult, Anke and Hercog, Rajna and Bork, Peer and Wilmes, Paul and Becher, D{\"o}rte},
  title     = {Sample preservation and storage significantly impact taxonomic and functional profiles in metaproteomics studies of the human gut microbiome},
  series = {Microorganisms},
  volume    = {7},
  journal   = {Microorganisms},
  number    = {9},
  issn      = {2076-2607},
  doi       = {10.3390/microorganisms7090367},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-195976},
  year      = {2019},
  abstract  = {With the technological advances of the last decade, it is now feasible to analyze microbiome samples, such as human stool specimens, using multi-omic techniques. Given the inherent sample complexity, there exists a need for sample methods which preserve as much information as possible about the biological system at the time of sampling. Here, we analyzed human stool samples preserved and stored using different methods, applying metagenomics as well as metaproteomics. Our results demonstrate that sample preservation and storage have a significant effect on the taxonomic composition of identified proteins. The overall identification rates, as well as the proportion of proteins from Actinobacteria were much higher when samples were flash frozen. Preservation in RNAlater overall led to fewer protein identifications and a considerable increase in the share of Bacteroidetes, as well as Proteobacteria. Additionally, a decrease in the share of metabolism-related proteins and an increase of the relative amount of proteins involved in the processing of genetic information was observed for RNAlater-stored samples. This suggests that great care should be taken in choosing methods for the preservation and storage of microbiome samples, as well as in comparing the results of analyses using different sampling and storage methods. Flash freezing and subsequent storage at -80 °C should be chosen wherever possible.},
  language  = {en}
}
@phdthesis{Strack2022,
  author    = {Strack, Stefanie},
  title     = {Rolle der Polycomb Faktoren PCGF6 und E2F6 in undifferenzierten und differenzierenden embryonalen Stammzellen der Maus},
  doi       = {10.25972/OPUS-25560},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-255600},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {To investigate the role of PCGF6 and E2F6 in murine embryonic stem cells (mESCs) and at the beginning of differentiation, knockout cell lines of both proteins and in combination were generated by the CRISPR/Cas9n system. Characterization of these knockout cell lines (KO) was performed by growth analysis in mESCs and differentiating murine stem cells (EBs). It was found that Pcgf6 KO cells formed smaller EBs that also could not be maintained in culture for an extended period. To resolve this specific phenotype, further molecular analyses were performed by flow cytometry (FACS). Cells of the Pcgf6 KO exhibited an increased proportion of cells in G1 phase during differentiation as well as an increased apoptotic frequency. Supporting the assumption of a cell cycle defect, RNASeq data were analysed. It could be shown that cells of the Pcgf6 KO differentiated in a temporally uncontrolled manner. Evaluation of differentially expressed genes revealed that expression of E2f6, a regulator of the cell cycle and another component of the non-canonical PRC1.6, was downregulated in mESC and EB cultures, whereas cell cycle-specific targets of E2F6-dependent gene regulation were upregulated at day 2 of differentiation. These results indicated that deletion of Pcgf6 at the beginning of differentiation must have effects on E2F6-dependent cell cycle regulation. Due to mycoplasma contamination in the cell culture at this time point, the Pcgf6 KO cell line had to be re-established. In addition, KO cell lines of E2f6 in Wt and in Pcgf6 KO mESCs were established. The replication of cellular characterization of the phenotype revealed that EB cultures of the Pcgf6 KO and the double knockout of Pcgf6 and E2f6 (dKOPcgf6/E2f6) exhibited reduced cell numbers during differentiation. Molecular characterizations of the phenotype revealed that the increased proportion of cells in the G1 phase of the Pcgf6 KO, which was detected before mycoplasma contamination, could not be reproduced. However, an increased frequency of cells in the G2 phase of dKOPcgf6/E2f6 was detected in mESC and EB culture. Analysis of apoptotic frequency in all KO cell lines indicated an increase during differentiation. RNASeq data from two publications of PCGF6 and E2F6 were used to support the analyses performed to this point (Qui et al, 2021; Dahlet et al, 2021). Gene Ontology Enrichment analyses of these data revealed that germline genes were independently upregulated in both KO cell lines in mESCs. However, both KO cell lines also showed an overlap of commonly upregulated germline genes. Following these publications, gene expression analysis of individual germline genes revealed that loss of E2f6 leads to de-repression of genes that have a binding site for E2F6. In contrast, loss of Pcgf6 had no effect on expression of these targets. These results, as well as previously published data, support the assumption that there are distinct subcomplexes that regulate the expression of germline genes in mESC and EB cultures.},
  subject      = {Murine embryonale Stammzellen},
  language  = {de}
}
@misc{Gamaleldin2021,
  type      = {Master Thesis},
  author    = {Gamaleldin, Mariam},
  title     = {Non-canonical Signaling of μ-opioid Receptors},
  doi       = {10.25972/OPUS-24032},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-240327},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {According to the "canonical" paradigm of GPCR signaling, agonist-bound GPCRs only signal to the downstream adenylyl cyclase enzyme when they are seated at the plasma membrane. Upon prolonged binding of an agonist, receptor internalization usually takes place, leading to the termination of this downstream signaling pathway and activation of alternative ones. However, a set of recent studies have shown that at least some GPCRs (e.g. thyroid stimulating hormone receptor) continue signaling to adenylyl cyclase after internalization. In this study, I aimed to investigate canonical signaling by internalized μ opioid receptors (MORs), which are Gi-coupled receptors, using a fluorescence resonance energy transfer (FRET) sensor for cyclic AMP (cAMP) known as Epac1-camps. My results show that the cyclic AMP inhibition signal induced by the binding of DAMGO, a MOR agonist, persists after agonist washout. We hypothesized that this persistent signal might come from internalized DAMGO-bound receptors located in the endosomal compartment. To test this hypothesis, I used dynasore and Dyngo 4a, two dynamin inhibitors that are known to prevent clathrin-mediated endocytosis. Interestingly, dynasore but not Dyngo 4a pretreatment largely blunted the response to MOR activation as well as to adenylyl cyclase activation with Forskolin (FSK). In addition, DAMGO-induced cAMP signal remained persistent even in the presence of 30 M Dyngo 4a. These results might point to a complex interplay between clathrin-mediated internalization and MOR signaling. Further experiments are required to elucidate the mechanisms underlying the persistent MOR signaling and to fully clarify whether MORs are capable of Gi signaling in the endosomal compartment.},
  language  = {en}
}
@phdthesis{Bluemel2021,
  author    = {Bl{\"u}mel, Rabea},
  title     = {Der Zebrab{\"a}rbling (Danio rerio) als in vivo Modell zur Untersuchung der Entstehung von Kraniosynostosen},
  doi       = {10.25972/OPUS-20743},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-207436},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Die Entwicklung des Sch{\"a}deldachs beginnt beim Menschen bereits in der fr{\"u}hen Embryogenese und ist erst im Erwachsenenalter abgeschlossen. Das Wachstum der Sch{\"a}delknochen muss sich w{\"a}hrend der Entwicklung fortw{\"a}hrend dem Gehirnwachstum anpassen. An den Stellen, wo zwei Sch{\"a}delknochen aufeinandertreffen, formen sich Sch{\"a}deln{\"a}hte, die aus mesenchymalem Bindegewebe bestehen und als Wachstumsfugen des Sch{\"a}dels dienen. Tritt eine fr{\"u}hzeitige Verkn{\"o}cherung innerhalb einer oder mehrerer Sch{\"a}deln{\"a}hte auf, spricht man von einer Kraniosynostose. Als Konsequenz wird ein weiteres Knochenwachstum verhindert, sodass sich das Neurokranium in dieser Region nicht dem expansiven Wachstum des Gehirns anpassen kann. Dies geht in der Regel mit einem kompensatorischen Wachstum des Sch{\"a}dels und infolgedessen mit kraniofazialen Dysmorphien und einem erh{\"o}hten intrakraniellen Druck einher. Klinische Studien und Forschungen an Modellorganismen konnten bereits eine Vielzahl an Genen mit der Entstehung von Kraniosynostosen assoziieren, darunter die Transkriptionsfaktoren TCF12 und TWIST1. Beim Menschen sind heterozygote Mutationen in TCF12 und TWIST1 mit Kraniosynostosen der Koronarnaht assoziiert. Bei M{\"a}usen hingegen f{\"u}hrt eine heterozygote Tcf12 Mutation nur in Kombination mit einer heterozygoten Twist1 Mutation zu Fusionen der Koronarnaht. Der Zebrab{\"a}rbling (Danio rerio, {\"u}berwiegend auch Zebrafisch genannt) weist eine bemerkenswerte {\"A}hnlichkeit bez{\"u}glich der Anatomie und Morphologie des Sch{\"a}deldachs zum Menschen auf. Um die genaue Funktion von TCF12 bei der Ausbildung der Sch{\"a}deln{\"a}hte zu untersuchen, wurde im Rahmen dieser Arbeit der Zebrafisch als in vivo Modell f{\"u}r die Entstehung tcf12-induzierter Kraniosynostosen etabliert. Zu Beginn der Arbeit wurde das Expressionsmuster von tcf12 {\"u}ber die Entwicklung hinweg analysiert. Ein besonderer Fokus lag dabei auf einem Expressionsnachweis w{\"a}hrend der Entwicklung der Sch{\"a}delplatten und der Sch{\"a}deln{\"a}hte. Ein erster Expressionsnachweis von tcf12 mittels PCR-Analysen und Whole-mount RNA in-situ Hybridisierungen zeigte eine breite Expression von tcf12 ab dem 1-3 Somiten Stadium an. F{\"u}r tiefergehende in vivo Analysen wurden im Zuge dieser Arbeit tcf12:EGFP Reportergenlinien generiert. Mit diesen gelang ein Nachweis der tcf12 Expression entlang der Wachstumsfronten der Sch{\"a}delplatten, innerhalb der Sch{\"a}deln{\"a}hte sowie im Periost und der Dura mater. Mit den tcf12:EGFP Fischen als Referenz wurde in weiterf{\"u}hrenden Experimenten die Aktivit{\"a}t drei hochkonservierter CNEs (engl. conserved non-coding elements) in vivo im Zebrafisch untersucht. Zwei der CNEs konnten als tcf12 Enhancer verifiziert werden, die eine Genexpression w{\"a}hrend der Neurogenese des zentralen Nervensystems (ZNS) steuern. Die beiden Enhancer-Elemente zeichnen sich durch eine hohe Konservierung vom Menschen bis hin zum Zebrafisch aus. Aufgrund der unterschiedlichen Sensitivit{\"a}t gegen{\"u}ber einem Funktionsverlust von TCF12 und TWIST1 in Mensch und Maus sollte die Auswirkung eines Knockouts der orthologen Gene auf die Entwicklung der Sch{\"a}deln{\"a}hte des Zebrafisches untersucht werden. Mittels CRISPR/Cas9 wurden verschiedene Knockout-Linien f{\"u}r die Gene tcf12, twist1a und twist1b generiert. Analysen der Knockoutmutanten zeigten, dass ein heterozygoter Verlust von tcf12 und twist1b in seltenen F{\"a}llen zu partiellen Fusionen der Koronarn{\"a}hte im Zebrafisch f{\"u}hrt. Des Weiteren konnte bei tcf12 und twist1b Einzel- und Doppelmutanten ein abnormes Wachstum der Sch{\"a}delplatten im Bereich der Suturen beobachtet werden. Die Expressionsstudien und die Analysen der Knockoutmutanten deuten auf eine Regulation von TCF12 bei der Differenzierung der Stammzellen sowie der Proliferation der Osteoblasten innerhalb der Sch{\"a}deln{\"a}hte hin. Um die Auswirkung von TCF12 Mutationen auf funktioneller Ebene zu untersuchen wurden im Verlauf dieser Arbeit Luciferase-Reporter Assays durchgef{\"u}hrt. Anhand dieser konnte nachgewiesen werden, dass Mutationen, die die basic helix-loop-helix (bHLH)-Dom{\"a}ne beeintr{\"a}chtigen, die Transaktivierungsf{\"a}higkeit von TCF12 aufheben. Co-Transfektions-Experimente mit TWIST1 offenbarten eine Regulation der Transaktivierung von TCF12 durch TWIST1, sowohl im Menschen, als auch im Zebrafisch. Im Rahmen dieser Arbeit konnten die genauen Expressionsorte von TCF12 w{\"a}hrend der Morphogenese des Sch{\"a}deldachs nachgwiesen und die Funktion von TCF12 und seinem Interaktionspartner TWIST1 bei der Entstehung von Kraniosynostosen weiter aufgekl{\"a}rt werden.},
  subject      = {Kraniosynostose},
  language  = {de}
}
@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{Figueiredo2021,
  author    = {Figueiredo, Ludmilla},
  title     = {Extinction debt of plants, insects and biotic interactions: interactive effects of habitat fragmentation and climate change},
  doi       = {10.25972/OPUS-23873},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-238738},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {The importance of understanding species extinctions and its consequences for ecosystems and human life has been getting increasing public attention. Nonetheless, regardless of how pressing the current biodiversity loss is, with rare exceptions, extinctions are actually not immediate. Rather, they happen many generations after the disturbance that caused them. This means that, at any point in time after a given disturbance, there is a number of extinctions that are expected to happen. This number is the extinction debt. As long as all the extinctions triggered by the disturbance have not happened, there is a debt to be paid. This delay in extinctions can be interpreted as a window of opportunity, when conservation measures can be implemented. In this thesis, I investigated the relative importance of ecological and evolutionary processes unfolding after different disturbances scenarios, to understand how this knowledge can be used to improve conservation practices aiming at controlling extinctions. In the Introduction (chapter 1), I present the concept of extinction debts and the complicating factors behind its understanding. Namely, I start by presenting i) the theoretical basis behind the definition of extinction debts, and how each theory informed different methodologies of study, ii) the complexity of understanding and predicting eco-evolutionary dynamics, and iii) the challenges to studying extinctions under a regime of widespread and varied disturbance of natural habitats. I start the main body of the thesis (chapter 2) by summarizing the current state of empirical, theoretical, and methodological research on extinction debts. In the last 10 years, extinction debts were detected all over the globe, for a variety of ecosystems and taxonomic groups. When estimated - a rare occurrence, since quantifying debts requires often unavailable data - the sizes of these debts range from 9 to 90\\% of current species richness and they have been sustained for periods ranging from 5 to 570 yr. I identified two processes whose contributions to extinction debts have been studied more often, namely 1) life-history traits that prolong individual survival, and 2) population and metapopulation dynamics that maintain populations under deteriorated conditions. Less studied are the microevolutionary dynamics happening during the payment of a debt, the delayed conjoint extinctions of interaction partners, and the extinction dynamics under different regimes of disturbances (e.g. habitat loss vs. climate change). Based on these observations, I proposed a roadmap for future research to focus on these less studies aspects. In chapters 3 and 4, I started to follow this roadmap. In chapter 3, I used a genomically-explicit, individual-based model of a plant community to study the microevolutionary processes happening after habitat loss and climate change, and potentially contributing to the settlement of a debt. I showed that population demographic recovery through trait adaptation, i.e. evolutionary rescue, is possible. In these cases, rather than directional selection, trait change involved increase in trait variation, which I interpreted as a sign of disruptive selection. Moreover, I disentangled evolutionary rescue from demographic rescue and show that the two types of rescue were equally important for community resistance, indicating that community re-assembly plays an important role in maintaining diversity following disturbance. The results demonstrated the importance of accounting for eco-evolutionary processes at the community level to understand and predict biodiversity change. Furthermore, they indicate that evolutionary rescue has a limited potential to avoid extinctions under scenarios of habitat loss and climate change. In chapter 4, I analysed the effects of habitat loss and disruption of pollination function on the extinction dynamics of plant communities. To do it, I used an individual, trait-based eco-evolutionary model (Extinction Dynamics Model, EDM) parameterized according to real-world species of calcareous grasslands. Specifically, I compared the effects of these disturbances on the magnitude of extinction debts and species extinction times, as well as how species functional traits affect species survival. I showed that the loss of habitat area generates higher number of immediate extinctions, but the loss of pollination generates higher extinction debt, as species take longer to go extinct. Moreover, reproductive traits (clonal ability, absence of selfing and insect pollination) were the traits that most influenced the occurrence of species extinction as payment of the debt. Thus, the disruption of pollination functions arose as a major factor in the creation of extinction debts. Thus, restoration policies should aim at monitoring the status of this and other ecological processes and functions in undisturbed systems, to inform its re-establishment in disturbed areas. Finally, I discuss the implications of these findings to i) the theoretical understanding of extinction debts, notably via the niche, coexistence, and metabolic theories, ii) the planning conservation measures, including communicating the very notion of extinction debts to improve understanding of the dimension of the current biodiversity crisis, and iii) future research, which must improve the understanding of the interplay between extinction cascades and extinction debts.},
  subject      = {Aussterbedynamik},
  language  = {en}
}
@phdthesis{Kunz2021,
  author    = {Kunz, Tobias C.},
  title     = {Expansion Microscopy (ExM) as a tool to study organelles and intracellular pathogens},
  doi       = {10.25972/OPUS-22333},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-223330},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {The resolution of fluorescence light microscopy was long believed to be limited by the diffraction limit of light of around 200-250 nm described in 1873 by Ernst Abbe. Within the last decade, several approaches, such as structured illumination microscopy (SIM), stimulated emission depletion STED and (direct) stochastic optical reconstruction microscopy (d)STORM have been established to bypass the diffraction limit. However, such super-resolution techniques enabling a resolution <100 nm require specialized and expensive setups as well as expert knowledge in order to avoid artifacts. They are therefore limited to specialized laboratories. Recently, Boyden and colleagues introduced an alternate approach, termed expansion microscopy (ExM). The latter offers the possibility to perform superresolution microscopy on conventional confocal microscopes by embedding the sample into a swellable hydrogel that is isotropically expanded. Since its introduction in 2015, expansion microscopy has developed rapidly offering protocols for 4x, 10x and 20x expansion of proteins and RNA in cells, tissues and human clinical specimens. Mitochondria are double membrane-bound organelles and crucial to the cell by performing numerous tasks, from ATP production through oxidative phosphorylation, production of many important metabolites, cell signaling to the regulation of apoptosis. The inner mitochondrial membrane is strongly folded forming so-called cristae. Besides being the location of the oxidative phosphorylation and therefore energy conversion and ATP production, cristae have been of great interest because changes in morphology have been linked to a plethora of diseases from cancer, diabetes, neurodegenerative diseases, to aging and infection. However, cristae imaging remains challenging as the distance between two individual cristae is often below 100 nm. Within this work, we demonstrate that the mitochondrial creatine kinase MtCK linked to fluorescent protein GFP (MtCK-GFP) can be used as a cristae marker. Upon fourfold expansion, we illustrate that our novel marker enables visualization of cristae morphology and localization of mitochondrial proteins relative to cristae without the need for specialized setups. Furthermore, we show the applicability of expansion microscopy for several bacterial pathogens, such as Chlamydia trachomatis, Simkania negevensis, Neisseria gonorrhoeae and Staphylococcus aureus. Due to differences in bacterial cell walls, we reveal important aspects for the digestion of pathogens for isotropic expansion. We further show that expansion of the intracellular pathogens C. trachomatis and S. negevensis, enables the differentiation between the two distinct developmental forms, catabolic active reticulate bodies (RB) and infectious elementary bodies (EB), on a conventional confocal microscope. We demonstrate the possibility to precisely locate chlamydial effector proteins, such as CPAF or Cdu1, within and outside the chlamydial inclusion. Moreover, we show that expansion microscopy enables the investigation of bacteria, herein S. aureus, within LAMP1 and LC3-II vesicles. With the introduction of the unnatural α-NH2-ω-N3-C6-ceramide, we further present the first approach for the expansion of lipids that may also be suitable for far inaccessible molecule classes like carbohydrates. The efficient accumulation and high labeling density of our functionalized α-NH2-ω-N3-C6-ceramide in both cells and bacteria enables in combination with tenfold expansion nanoscale resolution (10-20 nm) of the interaction of proteins with the plasma membrane, membrane of organelles and bacteria. Ceramide is the central molecule of the sphingolipid metabolism, an important constituent of cellular membranes and regulates many important cellular processes such as differentiation, proliferation and apoptosis. Many studies report about the importance of sphingolipids during infection of various pathogens. While the transport of ceramide to Chlamydia has been reported earlier, one of the unanswered questions remaining was if ceramide forms parts of the outer or inner bacterial membrane. Expansion of α-NH2-ω-N3-C6-ceramide enabled the visualization of ceramide in the inner and outer membrane of C. trachomatis and their distance was determined to be 27.6 ± 7.7 nm.},
  subject      = {Fluoreszenzmikroskopie},
  language  = {en}
}
@phdthesis{Pickel2020,
  author    = {Pickel, Simone},
  title     = {Role of the β subunit of L-type calcium channels in cardiac hypertrophy},
  doi       = {10.25972/OPUS-19282},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-192829},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {L-type calcium channels (LTCCs) control crucial physiological processes in cardiomyocytes such as the duration and amplitude of action potentials, excitation-contraction coupling and gene expression, by regulating the entry of Ca2+ into the cells. Cardiac LTCCs consist of one pore-forming α1 subunit and the accessory subunits Cavβ, Cavα2δ and Cavγ. Of these auxiliary subunits, Cavβ is the most important regulator of the channel activity; however, it can also have LTCC-independent cellular regulatory functions. Therefore, changes in the expression of Cavβ can lead not only to a dysregulation of LTCC activity, but also to changes in other cellular functions. Cardiac hypertrophy is one of the most relevant risk factors for congestive heart failure and depends on the activation of calcium-dependent prohypertrophic signaling pathways. However, the role of LTCCs and especially Cavβ in this pathology is controversial and needs to be further elucidated. Of the four Cavβ isoforms, Cavβ2 is the predominant one in cardiomyocytes. Moreover, there are five different splice variants of Cavβ2 (Cavβ2a-e), differing only in the N-terminal region. We reported that Cavβ2b is the predominant variant expressed in the heart. We also revealed that a pool of Cavβ2 is targeted to the nucleus in cardiomyocytes. The expression of the nuclear Cavβ2 decreases during in vitro and in vivo induction of cardiomyocyte hypertrophy and overexpression of a nucleus-targeted Cavβ2 completely abolishes the in vitro induced hypertrophy. Additionally, we demonstrated by shRNA-mediated protein knockdown that downregulation of Cavβ2 enhances the hypertrophy induced by the α1-adrenergic agonist phenylephrine (PE) without involvement of LTCC activity. These results suggest that Cavβ2 can regulate cardiac hypertrophy through LTCC-independent pathways. To further validate the role of the nuclear Cavβ2, we performed quantitative proteome analyses of Cavβ2-deficient neonatal rat cardiomyocytes (NRCs). The results show that downregulation of Cavβ2 influences the expression of various proteins, including a decrease of calpastatin, an inhibitor of the calcium-dependent cysteine protease calpain. Moreover, downregulation of Cavβ2 during cardiomyocyte hypertrophy drastically increases calpain activity as compared to controls after treatment with PE. Finally, the inhibition of calpain by calpeptin abolishes the increase in PE-induced hypertrophy in Cavβ2-deficient cells. These results suggest that nuclear Cavβ2 has Ca2+- and LTCC-independent functions during the development of hypertrophy. Overall, our results indicate a new role for Cavβ2 in antihypertrophic signaling in cardiac hypertrophy.},
  subject      = {Herzhypertrophie},
  language  = {en}
}
@phdthesis{Sieger2020,
  author    = {Sieger, Charlotte Sophie},
  title     = {Potential evolutionary responses to landscape heterogeneity and systematic environmental trends},
  doi       = {10.25972/OPUS-21669},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-216690},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Over the course of the last century, humans have witnessed drastic levels of global environmental change that endangered both, the survival of single species as well as biodiversity itself. This includes climate change, in both environmental means and in variance and subsequently frequent extreme weather events, as well as land use change that species have to cope with. With increasing urbanization, increasing agricultural area and increasing intensification, natural habitat is not only lost, but also changes its shape and distribution in the landscape. Both aspects can heavily influence an individual's fitness and therefore act as a selective force promoting evolutionary change. This way climate change influences individuals' niches and dispersal. Local adaptation and dispersal are not independent of each other. Dispersal can have two opposite effects on local adaptation. It can oppose local adaptation, by promoting the immigration of maladapted indi- viduals or favor local adaptation by introducing better adapted genotypes. Which of those effects of dispersal on local adaptation emerges in a population depends on the dispersal strategies and the spatial structure of the landscape. In principle an adaptive response can include adjustment of the niche optimum as well as habitat tolerance (niche width) or (instead) ecological tracking of adequate conditions by dispersal and range shifting. So far, there has been no extensive modeling study of the evolution of the environmental niche optimum and tolerance along with dispersal probability in complex landscapes. Either only dispersal or (part of ) the environmental niche can evolve or the landscapes used are not realistic but rather a very abstract representation of spatial structures. I want to try and disentangle those different effects of both local adaptation and dispersal during global change, as well as their interaction, especially considering the separation between the effects of increasing mean and increasing variance. For this, I implemented an individual based model (IBM), with escalating complexity. I showed that both on a temporal as well as on a spatial scale, variation can be more influential then mean conditions. Indeed, the actual spatial configuration of this heterogeneity and the relationship between spatial and temporal heterogeneity affect the evolution of the niche and of dispersal probability more than temporal or spatial mean conditions. I could show that in isolated populations, an increase of an environmental attribute's mean or variance can lead to extinction, under certain conditions. In particular, increasing variance cannot be tracked forever, since increasing tolerance has distinct limits of feasibility. Increasing mean conditions can also occur too fast to be tracked, especially from generalist individuals. When expanding the model to the metapopulation level without a temporal environmental trend, the degree of spatial vs.temporal heterogeneity influenced the evolution of random dispersal heavily. With increasing spatial heterogeneity, individuals from extreme and rare patches evolve from being philopatric to dispersive, while individuals from average patches switch in the opposite direction. With the last expansion to a different set of landscapes with varying degrees of edge density, I could show that edge effects are strong in pseudo-agricultural landscapes, while in pseudo-natural habitats they were hardly found, regardless of emigration strategy. Sharp edges select against dispersal in the edge patches and could potentially further isolate populations in agricultural landscapes. The work I present here can also be expanded further and I present several suggestions on what to do next. These expansions could help the realism of the model and eventually shed light on its bearing on ecological global change predictions. For example species distribution models or extinction risk models would be more precise, if they included both spatial and temporal variation. The current modeling practices might not be suffcient to describe the possible outcomes of global change, because spatio-temporal heterogeneity and its influence on species' niches is too important to be ignored for longer.},
  language  = {en}
}
@phdthesis{Lekszas2020,
  author    = {Lekszas, Caroline},
  title     = {Erweiterung des genetischen Mutationsspektrums verschiedener Krankheitsbilder und Identifizierung neuer krankheitsrelevanter Gene im Menschen mittels Whole Exome Sequenzierung},
  doi       = {10.25972/OPUS-20880},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-208807},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Trotz der rasanten Entwicklung molekulargenetischer Analysemethoden sind die Ausl{\"o}ser vieler Erbrankheiten bislang ungekl{\"a}rt. Eine Identifikation der genetischen Ursache einer Erkrankung ist jedoch essenziell, um zus{\"a}tzliche invasive Tests vermeiden, ad{\"a}quate Therapiemaßnahmen in die Wege leiten, akkurate Prognosen stellen und eine entsprechende genetische Beratung anbieten zu k{\"o}nnen. Next Generation Sequencing (NGS)-basierte Techniken wie die Whole Exome Sequenzierung (WES) haben die humangenetische Forschung und Diagnostik in den letzten Jahren revolutioniert. Die WES erm{\"o}glicht die Sequenzierung der Exons aller proteincodierenden Gene von mehreren Individuen gleichzeitig und stellt ein hilfreiches Werkzeug bei der Suche nach neuen kranheitsrelevanten Genen im Menschen dar. Die vorliegende Arbeit besch{\"a}ftigt sich mit der Aufkl{\"a}rung genetischer Ursachen verschiedenster Erkrankungen in konsanguinen Familien aus dem nahen und mittleren Osten mittels WES. Insgesamt wurden 43 Patienten mit unterschiedlichen Krankheitsbildern untersucht, darunter viele mit Skelettdysplasien oder Neuropathien. In 22 F{\"a}llen (51\%) konnte die entsprechende krankheitsverursachende Mutation ausfindig gemacht werden. In 21\% der aufgekl{\"a}rten F{\"a}lle wurden Sequenzvarianten detektiert, die in der Literatur bereits als pathogen beschrieben wurden, w{\"a}hrend 63\% bisher noch unbekannte Mutationen in bereits als krankheitsrelevant beschriebenen Genen darstellten. Zudem konnten im Rahmen dieser Arbeit drei neue, f{\"u}r den Menschen krankheitsrelevante Gene identifiziert werden, solute carrier family 10 member 7 (SLC10A7), T-box 4 (TBX4) und MIA SH3 domain ER export factor 3 (MIA3). SLC10A7 codiert f{\"u}r einen Transporter aus der Familie der solute carrier, der in der Plasmamembran verankert ist. In dieser Arbeit geleistete Analyseergebnisse konnten zu der Erstbeschreibung von homozygoten pathogenen SLC10A7-Mutationen als Ursache f{\"u}r eine Skelettdysplasie mit Amelogenesis imperfecta beitragen. Bei TBX4 handelt es sich um einen hochkonservierten Transkriptionsfaktor, der w{\"a}hrend der embryonalen Entwicklung an der Ausbildung der unteren Extremit{\"a}ten beteiligt ist. Homozygote pathogene TBX4-Mutationen wurden im Kontext dieser Arbeit erstmalig mit einer posterioren Amelie mit Becken- und Lungenhypoplasie in Verbindung gebracht. MIA3 ist ein Transmembranprotein des endoplasmatischen Retikulums, das eine essenzielle Rolle bei der Proteinsekretion spielt. Die hier vorgestellten Patienten mit homozygoten pathogenen MIA3-Mutationen zeigen eine komplexe syndromale Erkrankung, die sich haupts{\"a}chlich in einer Kollagenopathie, Diabetes mellitus und milder mentaler Retardierung manifestiert und ein neues Krankheitsbild darstellt. Die im Rahmen dieser Arbeit erzielten Ergebnisse erweitern somit zum einen das Mutationsspektrum verschiedener bekannter Krankheitsbilder und offenbaren zum anderen neue krankheitsrelevante Gene im Menschen.},
  subject      = {Verwandtenehe},
  language  = {de}
}
@phdthesis{Bertolini2020,
  author    = {Bertolini, Enrico},
  title     = {Comparative analysis of insect circadian clocks: a behavioural, anatomical, and molecular study},
  doi       = {10.25972/OPUS-16465},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-164651},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Biological clocks are endogenous oscillators that give organisms the sense of time. Insects, as the largest taxonomic group, offer fascinating models to study the evolution of clocks and their adaptation to various environments. Although the laboratory fruit fly, Drosophila melanogaster, led the role in the field of circadian biology as it provides a powerful genetic experimental tool, new model insect species need to be established to understand photoperiodic responses and to enable comparative studies. This work reports the behavioural, anatomical, and molecular characterization of the circadian clock of five insect species. The malt fly Chymomyza costata carries a D. melanogaster-like clock network, which supports circadian rhythms under rhythmic environment but cannot self-sustain when isolated from external time cues. The olive fly Bactrocera oleae is the major pest of olive plantations and the characterization of its circadian clock will improve future pest management strategies. The linden bug Pyrrhocoris apterus, a well suited model for investigating circadian and photoperiodic timing interactions, shows high degree of homology of the clock network with D. melanogaster. The scuttle flies Megaselia scalaris and Megaselia abdita represent new fascinating models to study how the clock network controls circadian behaviour. Overall, this work highlights high degree of homology between different circadian clock systems, but at the same time also dramatic differences in terms of circadian behaviour and neuro-anatomical expression of clock components. These have been mainly discussed in regards to the evolution of clocks in Diptera, and the adaptation of clocks to high latitudes.},
  language  = {en}
}
@phdthesis{Kervarrec2020,
  author    = {Kervarrec, Thibault},
  title     = {Histogenesis of Merkel cell carcinoma},
  doi       = {10.25972/OPUS-19975},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-199750},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Merkel cell carcinoma (MCC) is a rare and aggressive skin cancer. In approximately 80\% of cases, genomic integration of the Merkel cell polyomavirus (MCPyV) is observed and overexpression of the two MCPyV T antigens (TAgs) is regarded as the main oncogenic determinant of MCPyV-positive MCC cases. However, the nature of the cells from which MCC arises is unknown. Therefore, the goal of the present work was to determine the cell of origin of MCC. First, we characterized MCC patients' tumors and demonstrated a high similarity of MCPyV- negative MCC with extracutaneous neuroendocrine carcinoma while MCPyV-positive MCC differs from these two groups with respect to morphology, immunohistochemical profile, genetics, origin and behavior. Based on the analysis of a trichoblastoma/MCC combined tumor, we demonstrated that a MCPyV-positive MCC can arise following MCPyV integration in an epithelial cell. In addition, the high similarity between trichoblastoma cells and Merkel cell (MC) progenitors of the hair follicle suggests that these hair follicle cells may represent a general start point for the development of MCPyV-positive MCC. A contribution of the viral TAgs to the development of the characteristic Merkel cell-like MCC phenotype is suggested by experiments demonstrating induction of Merkel cell markers upon TAg expression in human primary keratinocytes or hair follicle cells. As potential mechanisms mediating these phenotypic changes, we identified the capability of MCPyV LT to repress degradation of master regulator of MC development, i.e. the transcription factor ATOH1. To conclude, our work suggests that MCPyV integration in epithelial cells of the hair follicle may represent an important path for MCC development.},
  subject      = {Merkel-Zellkarzinom},
  language  = {en}
}
@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{Baur2019,
  author    = {Baur, Florentin Philipp},
  title     = {Establishment of a 3D tumour model and targeted therapy of BRAF-mutant colorectal cancer},
  doi       = {10.25972/OPUS-17412},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-174129},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Cancer remains after cardiovascular diseases the leading cause of death worldwide and an estimated 8.2 million people died of it in 2012. By 2030, 13 million cancer deaths are expected due to the growth and ageing of the population. Hereof, colorectal cancer (CRC) is the third most common cancer in men and the second in women with a wide geographical variation across the world. Usually, CRC begins as a non-cancerous growth leading to an adenomatous polyp, or adenoma, arising from glandular cells. Since research has brought about better understanding of the mechanisms of cancer development, novel treatments such as targeted therapy have emerged in the past decades. Despite that, up to 95\% of anticancer drugs tested in clinical phase I trials do not attain a market authorisation and hence these high attrition rates remain a key challenge for the pharmaceutical industry, making drug development processes enormously costly and inefficient. Therefore, new preclinical in vitro models which can predict drug responses in vivo more precisely are urgently needed. Tissue engineering not only provides the possibility of creating artificial three-dimensional (3D) in vitro tissues, such as functional organs, but also enables the investigation of drug responses in pathological tissue models, that is, in 3D cancer models which are superior to conventional two-dimensional (2D) cell cultures on petri dishes and can overcome the limitations of animal models, thereby reducing the need for preclinical in vivo models. In this thesis, novel 3D CRC models on the basis of a decellularised intestinal matrix were established. In the first part, it could be shown that the cell line SW480 exhibited different characteristics when grown in a 3D environment from those in conventional 2D culture. While the cells showed a mesenchymal phenotype in 2D culture, they displayed a more pronounced epithelial character in the 3D model. By adding stromal cells (fibroblasts), the cancer cells changed their growth pattern and built tumour-like structures together with the fibroblasts, thereby remodelling the natural mucosal structures of the scaffold. Additionally, the established 3D tumour model was used as a test system for treatment with standard chemotherapeutic 5-fluorouracil (5-FU). The second part of the thesis focused on the establishment of a 3D in vitro test system for targeted therapy. The US Food and Drug Administration has already approved of a number of drugs for targeted therapy of specific types of cancer. For instance, the small molecule vemurafenib (PLX4032, Zelboraf™) which demonstrated impressive response rates of 50-80\% in melanoma patients with a mutation of the rapidly accelerated fibrosarcoma oncogene type B (BRAF) kinase which belongs to the mitogen active protein kinase (MAPK) signalling pathway. However, only 5\% of CRC patients harbouring the same BRAF mutation respond to treatment with vemurafenib. An explanation for this unresponsiveness could be a feedback activation of the upstream EGFR, reactivating the MAPK pathway which sustains a proliferative signalling. To test this hypothesis, the two early passage cell lines HROC24 and HROC87, both presenting the mutation BRAF V600E but differing in other mutations, were used and their drug response to vemurafenib and/or gefitinib was assessed in conventional 2D cell culture and compared to the more advanced 3D model. Under 3D culture conditions, both cell lines showed a reduction of the proliferation rate only in the combination therapy approach. Furthermore, no significant differences between the various treatment approaches and the untreated control regarding apoptosis rate and viability for both cell lines could be found in the 3D tumour model which conferred an enhanced chemoresistance to the cancer cells. Because of the observed unresponsiveness to BRAF inhibition by vemurafenib as can be seen in the clinic for patients with BRAF mutations in CRC, the cell line HROC87 was used for further xenografting experiments and analysis of activation changes in the MAPK signalling pathway. It could be shown that the cells presented a reactivation of Akt in the 3D model when treated with both inhibitors, suggesting an escape mechanism for apoptosis which was not present in cells cultured under conventional 2D conditions. Moreover, the cells exhibited an activation of the hepatocyte growth factor receptor (HGFR, c-Met) in 2D and 3D culture, but this was not detectable in the xenograft model. This shows the limitations of in vivo models. The results suggest another feedback activation loop than that to the EGFR which might not primarily be involved in the resistance mechanism. This reflects the before mentioned high attrition rates in the preclinical drug testing.},
  subject      = {Dickdarmtumor},
  language  = {en}
}
@phdthesis{Yang2020,
  author    = {Yang, Manli},
  title     = {\(Chlamydia\) \(trachomatis\) metabolism during infection and metatranscriptome analysis in \(Neisseria\) \(gonorrhoeae\) coinfected STD patients},
  doi       = {10.25972/OPUS-18499},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-184993},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Chlamydia trachomatis (Ct) is an obligate intracellular human pathogen. It causes blinding trachoma and sexually transmitted disease such as chlamydia, pelvic inflammatory disease and lymphogranuloma venereum. Ct has a unique biphasic development cycle and replicates in an intracellular vacuole called inclusion. Normally it has two forms: the infectious form, elementary body (EB); and the non-infectious form, reticulate body (RB). Ct is not easily amenable to genetic manipulation. Hence, to understand the infection process, it is crucial to study how the metabolic activity of Ct exactly evolves in the host cell and what roles of EB and RB play differentially in Ct metabolism during infection. In addition, Ct was found regularly coinfected with other pathogens in patients who got sexually transmitted diseases (STDs). A lack of powerful methods to culture Ct outside of the host cell makes the detailed molecular mechanisms of coinfection difficult to study. In this work, a genome-scale metabolic model with 321 metabolites and 277 reactions was first reconstructed by me to study Ct metabolic adaptation in the host cell during infection. This model was calculated to yield 84 extreme pathways, and metabolic flux strength was then modelled regarding 20hpi, 40hpi and later based on a published proteomics dataset. Activities of key enzymes involved in target pathways were further validated by RT-qPCR in both HeLa229 and HUVEC cell lines. This study suggests that Ct's major active pathways involve glycolysis, gluconeogenesis, glycerolphospholipid biosynthesis and pentose phosphate pathway, while Ct's incomplete tricarboxylic acid cycle and fatty acid biosynthesis are less active. EB is more activated in almost all these carbohydrate pathways than RB. Result suggests the survival of Ct generally requires a lot of acetyl-CoA from the host. Besides, both EB and RB can utilize folate biosynthesis to generate NAD(P)H but may use different pathways depending on the demands of ATP. When more ATP is available from both host cell and Ct itself, RB is more activated by utilizing energy providing chemicals generated by enzymes associated in the nucleic acid metabolism. The forming of folate also suggests large glutamate consumption, which is supposed to be converted from glutamine by the glutamine-fructose-6-phosphate transaminase (glmS) and CTP synthase (pyrG). Then, RNA sequencing (RNA-seq) data analysis was performed by me in a coinfection study. Metatranscriptome from patient RNA-seq data provides a realistic overview. Thirteen patient samples were collected and sequenced by our collaborators. Six male samples were obtained by urethral swab, and seven female samples were collected by cervicovaginal lavage. All the samples were Neisseria gonorrhoeae (GC) positive, and half of them had coinfection with Ct. HISAT2 and Stringtie were used for transcriptomic mapping and assembly respectively, and differential expression analysis by DESeq2, Ballgown and Cuffdiff2 are parallelly processed for comparison. Although the measured transcripts were not sufficient to assemble Ct's transcriptome, the differential expression of genes in both the host and GC were analyzed by comparing Ct positive group (Ct+) against Ct-uninfected group. The results show that in the Ct+ group, the host MHC class II immune response was highly induced. Ct infection is associated with the regulation of DNA methylation, DNA double-strand damage and ubiquitination. The analysis also shows Ct infection enhances host fatty acid beta oxidation, thereby inducing mROS, and the host responds to reduce ceramide production and glycolysis. The coinfection upregulates GC's own ion transporters and amino acid uptake, while it downregulates GC's restriction and modification systems. Meanwhile, GC has the nitrosative and oxidative stress response and also increases the ability for ferric uptake especially in the Ct+ group compared to Ct-uninfected group. In conclusion, methods in bioinformatics were used here in analyzing the metabolism of Ct itself, and the responses of the host and GC respectively in a coinfection study with and without Ct. These methods provide metabolic and metatranscriptomic details to study Ct metabolism during infection and Ct associated coinfection in the human microbiota.},
  subject      = {chlamydia trachomatis},
  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{Ruecker2019,
  author    = {R{\"u}cker, Christoph},
  title     = {Development of a prevascularized bone implant},
  doi       = {10.25972/OPUS-17886},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-178869},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {The skeletal system forms the mechanical structure of the body and consists of bone, which is hard connective tissue. The tasks the skeleton and bones take over are of mechanical, metabolic and synthetic nature. Lastly, bones enable the production of blood cells by housing the bone marrow. Bone has a scarless self-healing capacity to a certain degree. Injuries exceeding this capacity caused by trauma, surgical removal of infected or tumoral bone or as a result from treatment-related osteonecrosis, will not heal. Critical size bone defects that will not heal by themselves are still object of comprehensive clinical investigation. The conventional treatments often result in therapies including burdening methods as for example the harvesting of autologous bone material. The aim of this thesis was the creation of a prevascularized bone implant employing minimally invasive methods in order to minimize inconvenience for patients and surgical site morbidity. The basis for the implant was a decellularized, naturally derived vascular scaffold (BioVaSc-TERM®) providing functional vessel structures after reseeding with autologous endothelial cells. The bone compartment was built by the combination of the aforementioned scaffold with synthetic β-tricalcium phosphate. In vitro culture for tissue maturation was performed using bioreactor technology before the testing of the regenerative potential of the implant in large animal experiments in sheep. A tibia defect was treated without the anastomosis of the implant's innate vasculature to the host's circulatory system and in a second study, with anastomosis of the vessel system in a mandibular defect. While the non-anastomosed implant revealed a mostly osteoconductive effect, the implants that were anastomosed achieved formation of bony islands evenly distributed over the defect. In order to prepare preconditions for a rapid approval of an implant making use of this vascularization strategy, the manufacturing of the BioVaSc-TERM® as vascularizing scaffold was adjusted to GMP requirements.},
  subject      = {Tissue Engineering},
  language  = {en}
}
@phdthesis{Aue2019,
  author    = {Aue, Annemarie},
  title     = {Lokalisation und Bedeutung der NO-sensitiven Guanylyl-Cyclase bei der Lungenfibrose in der Maus},
  doi       = {10.25972/OPUS-17671},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-176710},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Die im Rahmen dieser Arbeit behandelten Fragestellungen vermitteln neue Kenntnisse {\"u}ber die Pathogenese der Lungenfibrose auf zellul{\"a}rer Ebene. Bei der Lungenfibrose handelt es sich um eine chronische Erkrankung, die durch eine initiale Inflammation und das Auftreten von Myofibroblasten gekennzeichnet ist. Die Myofibroblasten f{\"u}hren zu einer vermehrten Produktion von EZM, was in einer Zerst{\"o}rung der Lungenarchitektur, Narbenbildung und folglich einem verminderten Gasaustausch resultiert. Eine modulatorische Rolle von Stickstoffmonoxid (NO) bei der Entwicklung der Lungenfibrose wird vermutet, dennoch sind die Effektorzellen in der Lunge noch nicht bekannt. Daher wurde im ersten Teil dieser Arbeit die Lokalisation des NO-Rezeptors, der NO-sensitiven Guanylyl-Cyclase (NO-GC), in der Lunge untersucht. Dazu wurden Knockout-M{\"a}use generiert, bei denen die NO-GC global (GCKO) oder Perizyten-spezifisch (PDGFRβ-GCKO, SMMHC-GCKO, NG2-GCKO und SMMHC/NG2-GCKO) deletiert ist. Zudem wurden tdTomato-Reporterm{\"a}use verwendet, die das Fluoreszenzprotein unter Kontrolle eines spezifischen Reporters exprimieren (PDGFRβ/tomato, SMMHC/tomato, NG2/tomato, FoxD1/tomato und Tie2/tomato). In der Lunge sind Perizyten der NO-GC-exprimierende Zelltyp. Durch Immunhistochemie konnten zudem zwei verschiedene Subpopulationen von NO-GC-exprimierenden Perizyten identifiziert werden: Eine große Population an SMMHC/PDGFRβ-positiven Perizyten und eine kleine Population an NG2/PDGFRβ-positiven Perizyten. Im zweiten Teil dieser Arbeit wurde die Funktion der NO-GC w{\"a}hrend der Bleomycin-induzierten Lungenfibrose untersucht. Bleomycin f{\"u}hrt zu einer fibrotischen Antwort in allen Genotypen, was durch ein erh{\"o}htes Lungengewicht und einen erh{\"o}hten Kollagengehalt deutlich wird. Der Schweregrad der Lungenverletzung ist in NO-GC-defizienten M{\"a}usen gr{\"o}ßer als in Anwesenheit der NO-GC. Dies deutet auf eine Rolle der NO-GC bei der Bleomycin-induzierten Lungenfibrose hin. W{\"a}hrend der Entstehung der Lungenfibrose kommt es zur Bildung von Myofibroblasten, die als die Schl{\"u}sselzellen der Wundheilung und fibrotischer Prozesse bezeichnet werden. Diese Zellen kommen unter physiologischen Bedingungen kaum vor und ihre Herkunft ist nach wie vor nicht eindeutig gekl{\"a}rt. Da Perizyten als m{\"o}gliche Vorl{\"a}uferzellen betrachtet werden, wurde Lineage Tracing von Perizyten durchgef{\"u}hrt. Erstmals wurden zwei verschiedene Myofibroblasten-Subtypen durch die Expression von NO-GC unterschieden: (1) NO-GC-positive Myofibroblasten, die in der Alveolarwand lokalisiert sind und von Perizyten abstammen und (2) NO-GC-negative Myofibroblasten, die sich innerhalb der Alveolen befinden, deren Ursprung jedoch nicht Perizyten sind. Diese Myofibroblasten zeigen jedoch eine de novo-Synthese von PDGFRβ. Durch Lineage Tracing-Versuche sowie immunhistochemische Analysen k{\"o}nnen Perizyten, Endothelzellen und Fibrozyten als Vorl{\"a}uferzellen ausgeschlossen werden. Die Ursprungszelle der intra-alveol{\"a}ren Myofibroblasten ist somit bislang nicht identifiziert. Im letzten Teil der Arbeit wurde die Rolle der an der Lungenfibrose beteiligten Zelltypen n{\"a}her untersucht. Dazu wurde die Aufl{\"o}sung der reversiblen Bleomycin-induzierten Lungensch{\"a}den betrachtet. Der Verlust der beiden Myofibroblasten-Subtypen weist darauf hin, dass sie zwar die Effektorzellen der Wundheilungsreaktion, jedoch nicht an der Entstehung der chronisch manifesten Fibrose beteiligt sind. Perizyten proliferieren in Folge der Gabe von Bleomycin und sind vermehrt im Lungenparenchym auch nach Aufl{\"o}sung der Bleomycin-induzierten Lungenverletzung vorzufinden. Diese Ergebnisse f{\"u}hren zu der Annahme, dass es sich hierbei um die Effektorzellen der chronisch manifesten Lungenfibrose handelt, die durch eine Verdickung der Alveolarwand gekennzeichnet ist. Um die zellul{\"a}ren Mechanismen der Lungenfibrose umfassend aufzukl{\"a}ren, m{\"u}ssen weitere Untersuchungen an irreversiblen Fibrosemodellen folgen, die auch die chronischen Charakteristiken der Erkrankung ber{\"u}cksichtigen.},
  subject      = {Lungenfibrose},
  language  = {de}
}
@phdthesis{Oerter2018,
  author    = {Oerter, Sabrina},
  title     = {Expression von Natrium/Glukose-Cotransportern im menschlichen Gehirn bei Todesf{\"a}llen durch Sch{\"a}del-Hirn-Trauma und Todesf{\"a}llen durch Ersticken},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-164093},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Glukosetransporter spielen eine wichtige Rolle in der Versorgung des Gehirns mit N{\"a}hrstoffen und somit f{\"u}r den Erhalt der physiologischen Zellintegrit{\"a}t. Glukose wird {\"u}ber die Blut-Hirn-Schranke (BHS) mittels spezifischen transmembranen Transportproteinen der SLC-Genfamilie (GLUT, SGLT) bef{\"o}rdert. Dabei scheint w{\"a}hrend physiologischen Bedingungen haupts{\"a}chlich der Glukosetransporter GLUT1 (SLC2A1) f{\"u}r die Energieversorgung des Gehirns zust{\"a}ndig zu sein. Die Erforschung der SGLT-Expression ist in den letzten Jahren ein wichtiger Ansatzpunkt f{\"u}r neue Behandlungsstrategien vieler Erkrankungen, wie Diabetes Mellitus, maligne Neoplasien oder eines Herzinfarkts, geworden. Jedoch ist {\"u}ber deren Expression und Funktion im menschlichen Gehirn nur wenig bekannt. Besonders die Lokalisation entlang der BHS bleibt fraglich. Ein Großteil bisheriger Forschungsarbeiten besch{\"a}ftigt sich haupts{\"a}chlich mit der Expressionsanalyse des Transporters SGLT1 im tierischen Gehirn in vivo (Poppe et al. 1997; Balen et al. 2008; Yu et al. 2013). Es konnte aufgezeigt werden, dass SGLT1 mRNA exklusiv in Neuronen und nicht an der BHS exprimiert wird. Dies wird durch in vitro Analysen einer humanen Hirnendothelzelllinie best{\"a}tigt. Demnach kann kein SGLT1 unter physiologischen Bedingungen nachgewiesen werden (Sajja et al. 2014). Im menschlichen Hirngewebe besitzen SGLTs somit keine zentrale Funktion f{\"u}r den Glukosetransport an der BHS. Im Gegensatz dazu konnte eine Expression von SGLT sowohl in vivo als auch in vitro w{\"a}hrend hypoglyk{\"a}mischen Bedingungen belegt werden (Vemula et al. 2009; Sajja et al. 2014). Die Expression der SGLT-Transporter w{\"a}hrend einer isch{\"a}mischen Hypoglyk{\"a}mie f{\"u}hrt zu der Annahme, dass diese Transporter f{\"u}r die Aufrechterhaltung der Energieversorgung des gesch{\"a}digten Hirngewebes notwendig sind. Um die physiologischen Mechanismen nach einem Glukosemangel zu untersuchen, wurden SHT-Modelle etabliert (Salvador et al. 2013). In einem experimentellen Modell des Sch{\"a}del-Hirn-Traumas im Rahmen eines DFG-gef{\"o}rdertes Projekts ist ein Expressionsverlauf von Glukosetransportern im Maushirn und in Hirnendothelzellen erarbeitet worden (Wais 2012; Salvador et al. 2015). Somit k{\"o}nnten SGLTs als Ansatzpunkt f{\"u}r den Nachweis der {\"U}berlebenszeit nach einem SHT fungieren. Die vorliegende Arbeit fokussiert sich auf die Expression der Natrium-abh{\"a}ngigen Glukosetransporter SGLT1 und SGLT2 im menschlichen Gehirn. Hierbei liegt das Hauptaugenmerk auf der Lokalisation dieser Transporter an der menschlichen BHS von post mortalem Hirngewebe. Weiterhin wird untersucht ob die Expressionsst{\"a}rke von SGLT1 und SGLT2 eine Aussage {\"u}ber die {\"U}berlebenszeit von Verstorbenen nach einer traumatisch bedingten Hirnver{\"a}nderung zul{\"a}sst. Die Lokalisation von SGLT1 und SGLT2 an der menschlichen BHS konnte durch die Etablierung eines Protokolls zur Isolation von Hirnkapillaren erfolgen. Vorab wurden alle verwendeten Antik{\"o}rper auf ihre Spezifit{\"a}t mittels siRNA Transfektion und Blockierung der Immunfluoreszenzsignale mittels immunisierten Peptids getestet. Somit ist die Spezifit{\"a}t der detektierten SGLT1- und SGLT2-Expression in menschlichen Hirnkapillaren gew{\"a}hrleistet. Anschließend wird untersucht, in welchen zeitlichem Verlauf nach einer traumatisch bedingten Hirnver{\"a}nderung die verschiedenen Formen der Glukosetransporter exprimiert werden und ob ggf. der Umfang und die Verteilung von SGLT1, SGLT2 und GLUT1 sowie das Verh{\"a}ltnis zueinander Ausk{\"u}nfte {\"u}ber eine vitale bzw. postmortale Entstehung eines Traumas bzw. dessen {\"U}berlebenszeit zul{\"a}sst. Hierf{\"u}r wird ein Expressionsschema der Glukosetransporter generiert, abh{\"a}ngig von Todeszeitpunkt und Todesursache. Es konnte festgestellt werden, dass GLUT1 nicht als Target f{\"u}r die Ermittlung der {\"U}berlebenszeit nach einem Trauma geeignet ist. Dahingegen zeigen SGLT1 und SGLT2 eine signifikante {\"A}nderung der Expressionsst{\"a}rke im contusionalen Gewebe in Abh{\"a}ngigkeit von der {\"U}berlebenszeit. Obwohl diese vorl{\"a}ufigen Daten einen neuen Ansatzpunkt f{\"u}r die forensische Fragestellung aufzeigen, m{\"u}ssen weitere Experimente mit einem erh{\"o}hten Umfang der Probenanzahl und k{\"u}rzere Zeitspannen der {\"U}berlebenszeitr{\"a}ume durchgef{\"u}hrt werden.},
  subject      = {Sodium-Glucose Transporter 2},
  language  = {de}
}
@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{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{Lichtenstein2018,
  author    = {Lichtenstein, Leonie},
  title     = {Color vision and retinal development of the compound eye in bees},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-150997},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {The superfamiliy of bees, Apiformes, comprises more than 20,000 species. Within the group, the eusocial species like honeybees and bumblebees are receiving increased attention due to their outstanding importance for pollination of many crop and wild plants, their exceptional eusocial lifestyle and complex behavioral repertoire, which makes them an interesting invertebrate model to study mechanisms of sensory perception, learning and memory. In bees and most animals, vision is one of the major senses since almost every living organism and many biological processes depend on light energy. Bees show various forms of vision, e.g. color vision, achromatic vision or polarized vision in order to orientate in space, recognize mating partners, detect suitable nest sites and search for rewarding food sources. To catch photons and convert light energy into electric signals, bees possess compound eyes which consists of thousands of single ommatidia comprising a fixed number of photoreceptors; they are characterized by a specific opsin protein with distinct spectral sensitivity. Different visual demands, e.g. the detection of a single virgin queen by a drone, or the identification and discrimination of flowers during foraging bouts by workers, gave rise to the exceptional sex-specific morphology and physiology of male and female compound eyes in honeybees. Since Karl von Frisch first demonstrated color vision in honeybees more than 100 years ago, much effort has been devoted to gain insight into the molecular, morphological and physiological characteristics of (sex-specific) bee compound eyes and the corresponding photoreceptors. However, to date, almost nothing is known about the underlying mechanisms during pupal development which pattern the retina and give rise to the distinct photoreceptor distribution. Hence, in Chapter 2 and 3 I aimed to better understand the retinal development and photoreceptor determination in the honeybee eye. In a first step, the intrinsic temporal expression pattern of opsins within the retina was evaluated by quantifying opsin mRNA expression levels during the pupal phase of honeybee workers and drones. First results revealed that honeybee workers and drones express three different opsin genes, UVop, BLop and Lop1 during pupal development which give rise to an ultraviolet, blue, and green-light sensitive photoreceptor. Moreover, opsin expression patterns differed between both sexes and the onset of a particular opsin occurred at different time points during retinal development. Immunostainings of the developing honeybee retina in Chapter 2 showed that at the beginning of pupation the retina consist only of a thin hypodermis. However, at this stage all retinal structures are already present. From about mid of pupation, opsin expression levels increase and goes hand in hand with the differentiation of the rhabdoms, suggesting a two-step process in photoreceptor development and differentiation in the honeybee compound eye. In a first step the photoreceptor cells meet its fate during late pupation; in a second step, the quantity of opsin expression in each photoreceptor strongly increase up to the 25-fold shortly after eclosion. To date, the underlying mechanisms leading to different photoreceptor types have been intensively studied in the fruit fly, Drosophila melanogaster, and to some extend in butterflies. Interestingly, the molecular mechanisms seemed to be conserved within insects and e.g. the two transcription factors, spalt and spineless, which have been shown to be essential for photoreceptor determination in flies and butterflies, have been also identified in the honeybee. In chapter 3, I investigated the expression patterns of both transcription factors during pupal development of honeybee workers and showed that spalt is mainly expressed during the first few pupal stages which might correlate with the onset of BLop expression. Further, spineless showed a prominent peak at mid of pupation which might initiates the expression of Lop1. However, whether spalt and spineless are also essential for photoreceptor determination in the honeybee has still to be investigated, e.g. by a knockdown/out of the respective transcription factor during retinal development which leads to a spectral phenotype, e.g. a dichromatic eye. Such spectral phenotypes can then be tested in behavioral experiments in order to test the function of specific photoreceptors for color perception and the entrainment of the circadian clock. In order to evaluate the color discrimination capabilities of bees and the quality of color perception, a reliable behavioral experiment under controlled conditions is a prerequisite. Hence, in chapter 4, I aimed to establish the visual PER paradigm as a suitable method for behaviorally testing color vision in bees. Since PER color vision has considered to be difficult in bees and was not successful in Western honeybees without ablating the bee's antennae or presenting color stimuli in combination with other cues for several decades, the experimental setup was first established in bumblebees which have been shown to be robust and reliable, e.g. during electrophysiological recordings. Workers and drones of the bufftailed bumblebee, Bombus terrestris were able to associate different monochromatic light stimuli with a sugar reward and succeeded in discriminating a rewarded color stimulus from an unrewarded color stimulus. They were also able to retrieve the learned stimulus after two hours, and workers successfully transferred the learned information to a new behavioral context. In the next step, the experimental setup was adapted to honeybees. In chapter 5, I tested the setup in two medium-sized honeybees, the Eastern honeybee, Apis cerana and the Western honeybee, Apis mellifera. Both honeybee species were able to associate and discriminate between two monochromatic light stimuli, blue and green light, with peak sensitivities of 435 nm and 528 nm. Eastern and Western honeybees also successfully retrieve the learned stimulus after two hours, similar to the bumblebees. Visual conditioning setups and training protocols in my study significantly differed from previous studies using PER conditioning. A crucial feature found to be important for a successful visual PER conditioning is the duration of the conditioned stimulus presentation. In chapter 6, I systematically tested different length of stimuli presentations, since visual PER conditioning in earlier studies tended to be only successful when the conditioned stimulus is presented for more than 10 seconds. In this thesis, intact honeybee workers could successfully discriminate two monochromatic lights when the stimulus was presented 10 s before reward was offered, but failed, when the duration of stimulus presentation was shorter than 4 s. In order to allow a more comparable conditioning, I developed a new setup which includes a shutter, driven by a PC based software program. The revised setup allows a more precise and automatized visual PER conditioning, facilitating performance levels comparable to olfactory conditioning and providing now an excellent method to evaluate visual perception and cognition of bees under constant and controlled conditions in future studies.},
  subject      = {Biene},
  language  = {en}
}
@phdthesis{TawkTaouk2018,
  author    = {Tawk [Taouk], Caroline S.},
  title     = {The role of host-stress in the infection by the bacterial pathogen \(Shigella\) \(flexneri\)},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-151107},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {The human-bacterial pathogen interaction is a complex process that results from a prolonged evolutionary arms race in the struggle for survival. The pathogen employs virulence strategies to achieve host colonization, and the latter counteracts using defense programs. The encounter of both organisms results in drastic physiological changes leading to stress, which is an ancient response accompanying infection. Recent evidence suggests that the stress response in the host converges with the innate immune pathways and influences the outcome of infection. However, the contribution of stress and the exact mechanism(s) of its involvement in host defense remain to be elucidated. Using the model bacterial pathogen Shigella flexneri, and comparing it with the closely related pathogen Salmonella Typhimurium, this study investigated the role of host stress in the outcome of infection. Shigella infection is characterized by a pronounced pro-inflammatory response that causes intense stress in host tissues, particularly the intestinal epithelium, which constitutes the first barrier against Shigella colonization. In this study, inflammatory stress was simulated in epithelial cells by inducing oxidative stress, hypoxia, and cytokine stimulation. Shigella infection of epithelial cells exposed to such stresses was strongly inhibited at the adhesion/binding stage. This resulted from the depletion of sphingolipidrafts in the plasma membrane by the stress-activated sphingomyelinases. Interestingly, Salmonella adhesion was not affected, by virtue of its flagellar motility, which allowed the gathering of bacteria at remaining membrane rafts. Moreover, the intracellular replication of Shigella lead to a similar sphingolipid-raft depletion in the membrane across adjacent cells inhibiting extracellular bacterial invasion. Additionally, this study shows that Shigella infection interferes with the host stress granule-formation in response to stress. Interestingly, infected cells exhibited a nuclear depletion of the global RNA-binding stress-granule associated proteins TIAR and TIA-1 and their accumulation in the cytoplasm. Overall, this work investigated different aspects of the host stress-response in the defense against bacterial infection. The findings shed light on the importance of the host stress-pathways during infection, and improve the understanding of different strategies in host-pathogen interaction.},
  subject      = {Shigella flexneri},
  language  = {en}
}
@phdthesis{Kalleda2018,
  author    = {Kalleda, Nataraja Swamy},
  title     = {Spatiotemporal analysis of immune cell recruitment and Neutrophil defence functions in \(Aspergillus\) \(fumigatus\) lung infections},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-150931},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Humans are continuously exposed to airborne spores of the saprophytic fungus Aspergillus fumigatus. In healthy individuals, local pulmonary host defence mechanisms can efficiently eliminate the fungus without any overt symptoms. In contrast, A. fumigatus causes devastating infections in immunocompromised patients. However, local host immune responses against A. fumigatus lung infections in immunocompromised conditions have remained largely elusive. Given the dynamic changes in immune cell subsets within tissues upon immunosuppressive therapy, we dissected the spatiotemporal pulmonary immune response after A. fumigatus infection to reveal basic immunological events that fail to effectively control the invasive fungal disease. In different immunocompromised murine models, myeloid but not lymphoid cells were strongly recruited upon infection. Notably, neutrophils and macrophages were recruited to infected lungs in different immunosuppressed regimens. Other myeloid cells, particularly dendritic cells and monocytes were only recruited in the corticosteroid model after infection. Lymphoid cells, particularly CD4+ or CD8+ T-cells and NK cells were highly reduced upon immunosuppression and were not recruited after A. fumigatus infection. Importantly, adoptive CD11b+ myeloid cell transfer rescued immunosuppressed mice from lethal A. fumigatus infection. These findings illustrate that CD11b+ myeloid cells are critical for anti-A. fumigatus defence under immunocompromised conditions. Despite improved antifungal agents, invasive A. fumigatus lung infections cause a high rate morbidity and mortality in neutropenic patients. Granulocyte transfusions have been tested as an alternative therapy for the management of high-risk neutropenic patients with invasive A. fumigatus infections. To increase the granulocyte yield for transfusion, donors are treated with corticosteroids. Yet, the efficacy of granulocyte transfusion and the functional defence mechanisms of granulocytes collected from corticosteroid treated donors remain largely elusive. We aimed to assess the efficacy of granulocyte transfusion and functional defence mechanisms of corticosteroid treated granulocytes using mouse models. In this thesis, we show that transfusion of granulocytes from corticosteroid treated mice did not protect cyclophosphamide immunosuppressed mice against lethal A. fumigatus infection in contrast to granulocytes from untreated mice. Upon infection, increased levels of inflammatory cytokines helped to recruit granulocytes to the lungs without any recruitment defects in corticosteroid treated and infected mice or in cyclophosphamide immunosuppressed and infected mice that have received the granulocytes from corticosteroid treated mice. However, corticosteroid treated human or mouse neutrophils failed to form neutrophil extracellular traps (NETs) in in vitro and in vivo conditions. Further, corticosteroid treated granulocytes exhibited impaired ROS production against A. fumigatus. Notably, corticosteroids impaired the β-glucan receptor Dectin-1 (CLEC7A) on mouse and human granulocytes to efficiently recognize and phagocytize A. fumigatus, which markedly impaired fungal killing. We conclude that corticosteroid treatment of granulocyte donors for increasing neutrophil yields or patients with ongoing corticosteroid treatment could result in deleterious effects on granulocyte antifungal functions, thereby limiting the benefit of granulocyte transfusion therapies against invasive fungal infections.},
  subject      = {Aspergillus fumigatus},
  language  = {en}
}
@phdthesis{Knies2018,
  author    = {Knies, Kerstin},
  title     = {Neue Fanconi-An{\"a}mie-Gene als W{\"a}chter des Genoms},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-150669},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Fanconi An{\"a}mie (FA) geh{\"o}rt zu den seltenen Chromsomeninstabilit{\"a}ts-Syndromen. Urs{\"a}chlich f{\"u}r die Erkrankung sind biallelische Mutationen mit autosomal rezessiver Vererbung in einem der bisher bekannten 21 Genen (FANCA, -B, -C, -D1, -D2, -E, -F, -G, -I, -J, -L, -M, -N, -O, -P, -Q, -R, -S, -T, -U und -V). Eine Ausnahme stellen FANCB und FANCS dar, die X-chromosomal rezessiv bzw. mit einem dominant negativen Effekt vererbt werden. Die Genprodukte sind als Teil des FA/BRCA-DNA-Reparatur Netzwerks bei der Beseitigung von DNA-Interstrang-Quervernetzungen (ICL) involviert. ICLs f{\"u}hren zu einer Stagnation der Replikationsgabel und blockieren somit wichtige zellul{\"a}re Prozesse wie Replikation und Transkription, sodass eine Aufrechterhaltung der Genomstabilit{\"a}t nicht mehr gew{\"a}hrleistet ist. FA ist gekennzeichnet durch angeborene Fehlbildungen, fortschreitendes Knochenmarkversagen und eine erh{\"o}hte Pr{\"a}disposition gegen{\"u}ber Krebserkrankungen. Die Diagnose basiert auf ph{\"a}notypischen Auff{\"a}lligkeiten und wird auf zellul{\"a}rer Ebene durch die Hypersensititv{\"a}t gegen{\"u}ber DNA-quervernetzenden Substanzen wie Mitomycin C (MMC) best{\"a}tigt. Da nicht jeder Patient einer bisher bekannten Komplementationsgruppe zugeordnet werden kann und herk{\"o}mmliche molekulare Diagnostikverfahren mit der steigenden Anzahl an FA-Genen m{\"u}hsam, zeitaufw{\"a}ndig und teuer geworden sind, war es n{\"o}tig, neue molekulare Verfahren wie Whole Exome Sequencing (WES) zu etablieren. Im Rahmen dieser Arbeit wurde das Potential dieser Methode im Bezug auf die FA-Genotypisierung erforscht. Bei der Suche nach einer optimalen Anwendung des WES, untersuchten wir verschiedene Anreicherungs- und Sequenziertechniken. Dennoch f{\"u}hren Fehler in den Datenbanken sowie Pseudogene zu falschen Dateninterpretationen und -darstellungen und stellen somit eine Herausforderung dar. Trotzdem zeigen unserer Daten, dass WES eine wertvolle Methode in der Molekulardiagnostik von FA ist. Dies best{\"a}tigte sich durch die Zuordnung mehrerer, vorher unklassifizierter FA-Patienten zu den bekannten Komplementationsgruppen und der Erg{\"a}nzung eines siebten Patienten zum Subtyp FA-P, im Rahmen von zwei Next Generation Sequencing (NGS) Publikationen. Außerdem wurden mit Hilfe von WES zwei neue FA-Gene (FANCQ und FANCW) im Rahmen dieser Arbeit gefunden, wobei XPF (FANCQ) das erste Gen {\"u}berhaupt war, welches anhand von NGS detektiert wurde. ERCC4/XPF ist eine strukturspezifische Endonuklease, die durch ein Gen kodiert wird, welches bereits vorher mit den Krankheiten Xeroderma Pigmentosum (XP) und dem segmentalen XFE progeroid Syndrom in Verbindung gebracht wurde. Unsere Daten zeigen, dass abh{\"a}ngig von der Mutation in XPF, Patienten eine der drei unterschiedlichen Funktionsst{\"o}rungen aufweisen. Dies hebt die multifunktionale Stellung der XPF Endonuklease im Rahmen der Genomstabilit{\"a}t und von humanen Erkrankungen hervor. Das zweite Gen, das w{\"a}hrend dieser Arbeit entdeckt wurde, ist die WD40-Dom{\"a}ne tragende E3 Ubiquitin Ligase RFWD3, die k{\"u}rzlich mit DNA Reparatur und insbesondere HR verkn{\"u}pft wurde. Wir konnten zeigen, dass eine RFWD3 Mutation in der WD40-Dom{\"a}ne bei einem FA-Patienten mit der genetischen Erkrankung Fanconi An{\"a}mie assoziiert ist. Die HR ist in RFWD3 (FANCW) mutierten Zellen gest{\"o}rt, was auf einer verminderten Relokalisation von mutiertem RFWD3 an das Chromatin und einer defekten Interaktion mit RPA beruht. Des Weiteren weisen Rfwd3 defiziente M{\"a}use typische Merkmale anderer FA-Mausmodelle auf, wie verminderte Fertilit{\"a}t, ovarielle und testikul{\"a}re Atrophie sowie eine reduzierte Lebenserwartung. Insgesamt zeigt diese Arbeit, dass neue molekulare Ans{\"a}tze wie NGS ein wertvolles Hilfsmittel in der FA-Diagnostik sind um bisher unklassifizierte Patienten einer Komplementationsgruppe zuordnen zu k{\"o}nnen. Zudem konnten mit Hilfe dieser Technik zwei neue Gene identifiziert werden. Deren Charakterisierung tr{\"a}gt zu einer Vervollst{\"a}ndigung und weiteren Aufkl{\"a}rung des FA/BRCA-DNA-Reparatur-Netzwerks bei.},
  subject      = {DNA Reparatur},
  language  = {de}
}
@phdthesis{Brendtke2018,
  author    = {Brendtke, Rico},
  title     = {Entwicklungsaspekte eines Medizinproduktes zur Pr{\"a}vention und {\"U}berwachung von Hydrierungszust{\"a}nden},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-157181},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Der demografische Wandel und das Populationswachstum stellen eine globale Herausforderung f{\"u}r die Gesundheitssysteme dar. Eine vielversprechende L{\"o}sungsstrategie liegt in der digitalen {\"U}berwachung, Pr{\"a}vention und Therapie akuter und chronischer Erkrankungen durch die Nutzung von innovativen Technologien aus dem Bereich der personalisierten Medizin. Die Digitalisierung in der {\"U}berwachung von Vitalparametern mittels Sensorik besitzt großes Potential f{\"u}r die l{\"a}ngere Gesunderhaltung der Patienten und somit die Entlastung der Gesundheitssyteme im Ganzen. Da Wassermangel f{\"u}r eine Vielfalt von Krankheiten einen Katalysator darstellt, ist die Hydratation ein wichtiger aber bislang nur invasiv zug{\"a}nglicher Vitalparameter. Zur Etablierung nicht invasiver Messungen des Wasserhaushaltes am Menschen wurde im Rahmen dieser Arbeit die Eignung der Mikrowellentechnologie untersucht. Dehydratation resultiert in der Ver{\"a}nderung des Osmolythaushaltes und beeinflusst biochemische Prozesse, was zur Entstehung von Morbidit{\"a}t f{\"u}hren kann. Im Rahmen der Arbeit werden Teilbereiche der Entwicklung eines Medizinproduktes abgebildet. Zu diesem Zweck wird die Machbarkeit der mikrowellenbasierten Analyse des Wasserhaushaltes in einer technischen Machbarkeitsstudie untersucht, um im zweiten Prozessschritt einen technischen Demonstrator in vitro und in vivo am Probanden erproben zu k{\"o}nnen. Hochfrequente elektromagnetische Wellen interagieren mit Molek{\"u}len, speziell Wasser. Enth{\"a}lt eine Probe freie Wassermolek{\"u}le, kann dies im reflektierten Signal detektiert werden. Zur {\"U}berpr{\"u}fung des Sensorsystems in vitro dienen humane 3D-Vollhautmodelle mit spezifischer Hydratation und Gewebedichte der Matrixkomponenten als standardisiertes Modell zur Untersuchung definierter Exsikkoseszenarien und des Einflusses verschiedener Modellkomplexit{\"a}ten. Die Eignungs{\"u}berpr{\"u}fung des Systems mit einem technischen Demonstrator des k{\"u}nftigen Medizinproduktes belegt die Anwendbarkeit des Messsystems zur Erfassung des relativen Wassergehaltes. Die Technologie zeichnet sich durch eine hohe Sensitivit{\"a}t bei der Destinktion von Proben mittels Frequenz- und Signalreflektionsdifferenzen aus. Neben den In-vitro-Testungen wird das entwickelte Sensorsystem aus regulatorischer Sicht zur klinischen Leistungs{\"u}berpr{\"u}fung vorbereitet und im Rahmen eines bewilligten Ethikvotums in vivo erprobt. Die Ergebnisse belegen die Machbarkeit der nichtinvasiven Erfassung des Wasserhaushaltes durch mikrowellenbasierte Messungen. Die Technologie birgt das Potential, in ein k{\"o}rpernahes Sensorsystem integriert zu werden, welches als Medizinprodukt zur pers{\"o}nlichen Gesundheits{\"u}berwachung zugelassen werden kann.},
  subject      = {Medizinprodukt},
  language  = {de}
}
@phdthesis{Haertle2018,
  author    = {Haertle, Larissa},
  title     = {Gestationsdiabetes und fetale Programmierung: Epigenetische Untersuchungen mit verschiedenen Next Generation Sequencing Techniken},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-156465},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Eine intrauterine Gestationsdiabetes (GDM) Exposition induziert in den betroffenen Nachkommen eine lebenslang erh{\"o}hte Pr{\"a}disposition f{\"u}r metabolische und komplexe Erkrankungen. Die Krankheitssuszeptibilit{\"a}t wird dabei durch epigenetische Ver{\"a}nderungen vermittelt, die sich {\"u}ber die Regulation der Genaktivit{\"a}t auch auf das Expressionsniveau und den Ph{\"a}notypen auswirken. Um neue Gene zu finden, die eine Rolle in der fetalen Programmierung spielen, wurden in dieser Arbeit genomweite Methylierungsmuster von Nabelschnurbluten (FCBs) aus GDM-Schwangerschaften und Kontrollen miteinander verglichen. Mit Illumina Infinium HumanMethylation 450K Arrays konnten signifikante Gruppenunterschiede f{\"u}r insgesamt 65 CpG-Stellen (52 davon genassoziiert) festgestellt werden, die multiplem Testen standhielten. Mittels Pyrosequenzierung wurden vier dieser Kandidaten-Loci (ATP5A1, MFAP4, PRKCH, SLC17A4), sowie ein Gen aus der Literatur (HIF3A) genauer untersucht und die Effekte erfolgreich validiert. F{\"u}r das zugrundeliegende multivariate Regressionsmodell wurden die potenziellen St{\"o}rfaktoren Gestationsalter, kindliches Geschlecht und m{\"u}tterlicher BMI ber{\"u}cksichtigt. Der GDM-Effekt zeigte sich st{\"a}rker in der insulinbehandelten Subgruppe (I-GDM) als in der di{\"a}tisch behandelten (D GDM) und scheint insgesamt multifaktoriell bedingt zu sein, da viele Gene betroffen waren, jedoch alle mit einer vergleichsweise niedrigen Effekt-Gr{\"o}ße. Zus{\"a}tzlich konnten f{\"u}r den MEG3 Promotor, MEST und PEG3, drei von vier gepr{\"a}gten Genen, die mittels Deep Bisulfite Sequencings (DBS) analysiert wurden, ebenfalls signifikante Methylierungs-unterschiede zwischen der GDM- und Kontroll-Gruppe detektiert werden. Die identifizierten Gene stellen labile Zielregionen f{\"u}r die GDM-induzierte intrauterine Programmierung dar und k{\"o}nnen zuk{\"u}nftig n{\"u}tzliche Biomarker f{\"u}r Krankheitsdiagnosen und Prognosen sein. Mittels DBS k{\"o}nnen dar{\"u}ber hinaus Einzelmolek{\"u}l-Analysen durchgef{\"u}hrt werden, f{\"u}r die in differentiell methylierten Regionen (DMRs) anhand eines informativen SNPs die parentale Allel-Herkunft bestimmt und bei der Berechnung von Epimutationsraten einbezogen werden kann. Epimutationen wurde als solche gewertet, wenn sie ein > 50 \% abnormal (de)methyliertes Methylierungsprofil aufwiesen. Die DBS-Daten wurden mit zwei verschiedenen Sequenzierplattformen generiert (Roche GS Junior und Illumina MiSeq). F{\"u}r Zweitere wurde ein eigenes, unabh{\"a}ngiges Library-Pr{\"a}parations-Protokoll entwickelt. In Nabelschnurblut, adultem Blut und Viszeralfett wurden f{\"u}r die paternal exprimierte MEST Promotor DMR und die maternal exprimierte MEG3 intergenic (IG) DMR hohe Epimutationsraten f{\"u}r das jeweils unmethylierte Allel detektiert. Die gepr{\"a}gten (methylierten) Allele wiesen dagegen nur niedrige Epimutationsraten auf. Da MEST und MEG3 invers gepr{\"a}gte Gene sind, war die Hypermethylierung des nicht gepr{\"a}gten Allels (HNA) demnach unabh{\"a}ngig von der parentalen Allel-Herkunft. Die HNA scheint außerdem erst nach der Fertilisation aufzutreten, da in Spermien nur sehr wenige Epimutationen gefunden wurden. F{\"u}r die sekund{\"a}re MEG3 Promotor DMR (deren Pr{\"a}gung von der prim{\"a}ren MEG3 IG-DMR reguliert wird) wurde ein deutlich schw{\"a}cherer, wenngleich signifikanter HNA-Effekt im FCB gemessen, f{\"u}r die paternal exprimierte PEG3 Promotor DMR konnte dagegen kein signifikanter Unterschied zwischen den beiden parentalen Epimutationsraten festgestellt werden. Der HNA-Effekt f{\"u}r die MEST DMR, MEG3 IG-DMR und MEG3 Promotor DMR war weder mit GDM noch mit Adipositas assoziiert und zeigte allgemein eine große interindividuelle Varianz. Die Aufrechterhaltung differenzieller Methylierungsmuster in Imprinting Kontrollregionen (ICRs) scheint in manchen Entwicklungs-Zeitspannen von großer Bedeutung und damit streng kontrolliert zu sein, sp{\"a}ter jedoch redundant zu werden, was sich in der Anreicherung von stochastischen sowie umweltinduzierten Fehlern auf dem nicht gepr{\"a}gten Allel {\"a}ußern kann. HNA-suszeptible gepr{\"a}gte Gene {\"a}hneln in mancherlei Hinsicht metastabilen Epiallelen. Diese Studie zeigt, dass sowohl stochastische Faktoren als auch Umweltstimuli w{\"a}hrend der fr{\"u}hen embryonalen Entwicklung u.a. {\"u}ber HNA-Effekte gepr{\"a}gte Gen-Netzwerke programmieren, die in Wachstumsprozesse involviert sind. Um tiefere Einblicke in allelspezifische Pr{\"a}gungsprofile zu erhalten, w{\"a}ren umfangreiche DBS HNA-L{\"a}ngsschnittstudien aller 50-100 human gepr{\"a}gten Gene in unterschiedlichen Gewebetypen und Differenzierungsstadien w{\"u}nschenswert.  },
  subject      = {Schwangerschaftsdiabetes},
  language  = {de}
}
@phdthesis{Schmitt2017,
  author    = {Schmitt, Franziska},
  title     = {Neuronal basis of temporal polyethism and sky-compass based navigation in \(Cataglyphis\) desert ants},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-142049},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {Desert ants of the genus Cataglyphis (Formicinae) are widely distributed in arid areas of the palearctic ecozone. Their habitats range from relatively cluttered environments in the Mediterranean area to almost landmark free deserts. Due to their sophisticated navigational toolkit, mainly based on the sky-compass, they were studied extensively for the last 4 decades and are an exceptional model organism for navigation. Cataglyphis ants exhibit a temporal polyethism: interior workers stay inside the dark nest and serve as repletes for the first ∼2 weeks of their adult life (interior I). They then switch to nursing and nest maintenance (interior II) until they transition to become day-active outdoor foragers after ∼4 weeks. The latter switch in tasks involves a transition phase of ∼2-3 days during which the ants perform learning and orientation walks. Only after this last phase do the ants start to scavenge for food as foragers. In this present thesis I address two main questions using Cataglyphis desert ants as a model organism: 1. What are the underlying mechanisms of temporal polyethism? 2. What is the neuronal basis of sky-compass based navigation in Cataglyphis ants? Neuropeptides are important regulators of insect physiology and behavior and as such are promising candidates regarding the regulation of temporal polyethism in Cataglyphis ants. Neuropeptides are processed from large precursor proteins and undergo substantial post-translational modifications. Therefore, it is crucial to biochemically identify annotated peptides. As hardly any peptide data are available for ants and no relevant genomic data has been recorded for Cataglyphis, I started out to identify the neuropeptidome of adult Camponotus floridanus (Formicinae) workers (manuscript 1). This resulted in the first neuropeptidome described in an ant species - 39 neuropeptides out of 18 peptide families. Employing a targeted approach, I identified allatostatin A (AstA), allatotropin (AT), short neuropeptide F (sNPF) and tachykinin (TK) using mass spectrometry and immunohistology to investigate the distribution of AstA, AT and TK in the brain (manuscript 2). All three peptides are localized in the central complex, a brain center for sensory integration and high-order control of locomotion behavior. In addition, AstA and TK were also found in visual and olfactory input regions and in the mushroom bodies, the centers for learning and memory formation. Comparing the TK immunostaining in the brain of 1, 7 and 14 days old dark kept animals revealed that the distribution in the central complex changes, most prominently in the 14 day old group. In the Drosophila central complex TK modulates locomotor activity levels. I therefore hypothesize that TK is involved in the internal regulation of the interior I-interior II transition which occurs after ∼2 weeks of age. I designed a behavioral setup to test the effect of neuropeptides on the two traits: 'locomotor activity level' and 'phototaxis' (manuscript 3). The test showed that interior I ants are less active than interior II ants, which again are less active than foragers. Furthermore, interior ants are negatively phototactic compared to a higher frequency of positive phototaxis in foragers. Testing the influence of AstA and AT on the ants' behavior revealed a stage-specific effect: while interior I behavior is not obviously influenced, foragers become positively phototactic and more active after AT injection and less active after AstA injection. I further tested the effect of light exposure on the two behavioral traits of interior workers and show that it rises locomotor activity and results in decreased negative phototaxis in interior ants. However, both interior stages are still more negatively phototactic than foragers and only the activity level of interior II ants is raised to the forager level. These results support the hypothesis that neuropeptides and light influence behavior in a stage-specific manner. The second objective of this thesis was to investigate the neuronal basis of skycompass navigation in Cataglyphis (manuscript 4). Anatomical localization of the sky-compass pathway revealed that its general organization is highly similar to other insect species. I further focused on giant synapses in the lateral complex, the last relay station before sky-compass information enters the central complex. A comparison of their numbers between newly eclosed ants and foragers discloses a rise in synapse numbers from indoor worker to forager, suggesting task-related synaptic plasticity in the sky-compass pathway. Subsequently I compared synapse numbers in light preexposed ants and in dark-kept, aged ants. This experiment showed that light as opposed to age is necessary and sufficient to trigger this rise in synapse number. The number of newly formed synapses further depends on the spectral properties of the light to which the ants were exposed to. Taken together, I described neuropeptides in C. floridanus and C. fortis, and provided first evidence that they influence temporal polyethism in Cataglyphis ants. I further showed that the extent to which neuropeptides and light can influence behavior depends on the animals' state, suggesting that the system is only responsive under certain circumstances. These results provided first insight into the neuronal regulation of temporal polyethism in Cataglyphis. Furthermore, I characterized the neuronal substrate for sky-compass navigation for the first time in Cataglyphis. The high level of structural synaptic plasticity in this pathway linked to the interior-forager transition might be particularly relevant for the initial calibration of the ants' compass system.},
  subject      = {Cataglyphis},
  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{GarciaGuerrero2017,
  author    = {Garcia Guerrero, Estefania},
  title     = {Strategies to Obtain Tumor-Reactive Cells for Cancer Immunotherapy by Cell Sorting and Genetic Modifications of T Lymphocytes},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-150547},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {Recent advances in the field of cancer immunotherapy have enabled this therapeutic approach to enter the mainstream of modern cancer treatment. In particular, adoptive T cell therapy (ACT) is a potentially powerful immunotherapy approach that relies on the administration of tumor-specific T cells into the patient. There are several strategies to obtain tumor-reactive cytotoxic T lymphocytes (CTLs), which have already been shown to induce remarkable responses in the clinical setting. However, there are concerns and limitations regarding the conventional approaches to obtain tumor-reactive T cells, such as accuracy of the procedure and reproducibility. Therefore, we aimed to develop two approaches to improve the precision and efficacy of tumor-reactive T cells therapy. These two techniques could constitute effective, safe and broadly applicable alternatives to the conventional methods for obtaining tumor-specific CTLs. The first approach of this study is the so called "Doublet Technology". Here, we demonstrate that peptide-human leukocyte antigen-T cell receptor (pHLA-TCR) interactions that involve immune reactive peptides are stable and strong. Therefore, the CTLs that are bound by their TCR to tumor cells can be selected and isolated through FACS-based cell sorting taking advantage of this stable interaction between the CTLs and the target cells. The CTLs from acute myeloid leukemia (AML) patients obtained with this technique show cytolytic activity against blast cells suggesting a potential clinical use of these CTLs. "Doublet Technology" offers a personalized therapy in which there is no need for a priori knowledge of the exact tumor antigen. The second approach of this study is the Chimeric Antigen Receptor (CAR) Technology. We design several CARs targeting the B-Cell Maturation Antigen (BCMA). BCMA CAR T cells show antigen-specific cytolytic activity, production of cytokines including IFN-γ and IL-2, as well as productive proliferation. Although we confirm the presence of soluble BCMA in serum of multiple myeloma (MM) patients, we demonstrate that the presence of soluble protein does not abrogate the efficacy of BCMA CAR T cells suggesting that BCMA CAR T cells can be used in the clinical setting to treat MM patients. The high antigen specificity of CAR T cells allows efficient tumor cell eradication and makes CAR Technology attractive for broadly applicable therapies.},
  subject      = {Immunotherapy},
  language  = {en}
}
@phdthesis{Karl2016,
  author    = {Karl, Stefan},
  title     = {Control Centrality in Non-Linear Biological Networks},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-150838},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {Biological systems such as cells or whole organisms are governed by complex regulatory networks of transcription factors, hormones and other regulators which determine the behavior of the system depending on internal and external stimuli. In mathematical models of these networks, genes are represented by interacting "nodes" whose "value" represents the activity of the gene. Control processes in these regulatory networks are challenging to elucidate and quantify. Previous control centrality metrics, which aim to mathematically capture the ability of individual nodes to control biological systems, have been found to suffer from problems regarding biological plausibility. This thesis presents a new approach to control centrality in biological networks. Three types of network control are distinguished: Total control centrality quantifies the impact of gene mutations and identifies potential pharmacological targets such as genes involved in oncogenesis (e.g. zinc finger protein GLI2 or bone morphogenetic proteins in chondrocytes). Dynamic control centrality describes relaying functions as observed in signaling cascades (e.g control in mouse colon stem cells). Value control centrality measures the direct influence of the value of the node on the network (e.g. Indian hedgehog as an essential regulator of proliferation in chondrocytes). Well-defined network manipulations define all three centralities not only for nodes, but also for the interactions between them, enabling detailed insights into network pathways. The calculation of the new metrics is made possible by substantial computational improvements in the simulation algorithms for several widely used mathematical modeling paradigms for genetic regulatory networks, which are implemented in the regulatory network simulation framework Jimena created for this thesis. Applying the new metrics to biological networks and artificial random networks shows how these mathematical concepts correspond to experimentally verified gene functions and signaling pathways in immunity and cell differentiation. In contrast to controversial previous results even from the Barab{\´a}si group, all results indicate that the ability to control biological networks resides in only few driver nodes characterized by a high number of connections to the rest of the network. Autoregulatory loops strongly increase the controllability of the network, i.e. its ability to control itself, and biological networks are characterized by high controllability in conjunction with high robustness against mutations, a combination that can be achieved best in sparsely connected networks with densities (i.e. connections to nodes ratios) around 2.0 - 3.0. The new concepts are thus considerably narrowing the gap between network science and biology and can be used in various areas such as system modeling, plausibility trials and system analyses. Medical applications discussed in this thesis include the search for oncogenes and pharmacological targets, as well their functional characterization.},
  subject      = {Bioinformatik},
  language  = {en}
}
@phdthesis{Duennes2016,
  author    = {D{\"u}nnes, Sarah},
  title     = {Einfluss der NO-sensitiven Guanylyl-Cyclase auf den cGMP/cAMP-Crosstalk und die Steifigkeit der murinen Aorta},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-141479},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2016},
  abstract  = {Die NO/cGMP-vermittelte Signalkaskade ist im vaskul{\"a}ren System entscheidend an der Regulation des Blutdrucks beteiligt. Innerhalb der Kaskade nimmt die NO-sensitive Guanylyl-Cyclase (NO-GC) eine Schl{\"u}sselfunktion als wichtigster Rezeptor f{\"u}r das Signalmolek{\"u}l Stickstoffmonoxids (NO) ein. NO wird endogen von verschiedenen Isoformen der NO Synthase produziert. Die Bindung von NO an die NO GC f{\"u}hrt zur Produktion des sekund{\"a}ren Botenstoffs cyclisches Guanosinmonophosphat (cGMP). Dieser Botenstoff aktiviert verschiedene Effektor-Molek{\"u}le und bewirkt letztlich eine Relaxation der glatten Muskulatur. Ein weiterer sekund{\"a}rer Botenstoff, das Signalmolek{\"u}l cyclisches Adenosinmonophosphat (cAMP), ist ebenfalls an der Regulation des Tonus der glatten Muskulatur und dadurch an der Blutdruckregulation beteiligt. Unterschiedliche Phosphodiesterasen (PDE) bauen die sekund{\"a}ren Botenstoffe ab und beenden dadurch die Signalkaskaden. Die PDE3 spielt hierbei eine besondere Rolle, da sie eine gemischte Substratspezifit{\"a}t besitzt. Um den Einfluss der NO-GC auf das kardiovaskul{\"a}re System zu untersuchen, wurden NO-GC Knockout(KO)-M{\"a}use mit globaler (GCKO) oder Glattmuskel-spezifischer (SMC-GCKO) Deletion der NO-GC generiert. Um das Zusammenspiel von cAMP und cGMP n{\"a}her zu beleuchten, wurde im ersten Teil dieser Arbeit die PDE3 genauer untersucht. Im Gef{\"a}ßsystem wird lediglich die PDE3A und nicht die PDE3B exprimiert. Die Aorten von GCKO- und SMC-GCKO-Tieren reagieren sensitiver auf PDE3A-Blockade als die Kontroll-Tiere. Auch die akute Blockade der NO-GC f{\"u}hrt zu diesem Sensitivit{\"a}tseffekt. Die PDE3A ist in Folge der NO-GC-Deletion sowohl in ihrer Expression, als auch ihrer Aktivit{\"a}t um die H{\"a}lfte reduziert. Dies dient vermutlich kompensatorisch dazu, das cAMP-Signal weitgehend zu erhalten und so eine cAMP-induzierte Relaxation der Gef{\"a}ße zu gew{\"a}hrleisten. Ohne R{\"u}ckkopplung zwischen den beiden Signalwegen k{\"a}me es vermutlich zu weiteren negativen Konsequenzen f{\"u}r das Herz-Kreislaufsystem. Diese Daten weisen auf eine direkte Regulation der PDE3 in glatten Muskelzellen durch die NO/cGMP-Signalkaskade und einen PDE3-vermittelten cAMP/cGMP-Crosstalk hin. Der genaue Mechanismus dieser Expressionsregulation ist noch unklar. Denkbar w{\"a}re eine cGMP-vermittelte Transkriptionsregulation oder eine Modulation der Translation der PDE3A. Der Verlust der NO-GC f{\"u}hrt in GCKO- und SMC-GCKO-M{\"a}usen zu einem erh{\"o}hten systolischen Blutdruck von ~30 mmHg. Bei der Entwicklung der arteriellen Hypertonie k{\"o}nnte eine erh{\"o}hte Aortensteifigkeit beteiligt sein, die im zweiten Teil dieser Arbeit n{\"a}her untersucht wurde. In GCKO-M{\"a}usen ist die aortale Steifigkeit und daraus resultierend die Pulswellengeschwindigkeit (PWV) deutlich erh{\"o}ht. Die Steigerung der PWV wird in den GCKO-Tieren zus{\"a}tzlich durch den verminderten Aorten-Durchmesser bedingt. Außerdem weisen die Aorten dieser Tiere eine ver{\"a}nderte Wandstruktur auf, die zu einer Verminderung der aortalen Windkesselfunktion f{\"u}hrt. Diese Ver{\"a}nderungen k{\"o}nnten die Blutdruckerh{\"o}hung in GCKO-M{\"a}usen erkl{\"a}ren. In SMC-GCKO-Tieren tritt keine dieser Gef{\"a}ß-Modifikationen auf. Eine Aortensteifigkeit als m{\"o}gliche Ursache f{\"u}r den erh{\"o}hten systolischen Blutdruck in den SMC-GCKO-Tieren kann somit ausgeschlossen werden. Zur Aufkl{\"a}rung m{\"u}ssen weitere Versuche zum Aufbau der Gef{\"a}ßw{\"a}nde und zur Bestimmung des peripheren Widerstands gemacht werden. Auch der Einfluss anderer Zelltypen, wie z.B. Perizyten oder Fibroblasten, auf die Blutdruckregulation sollte untersucht werden.},
  subject      = {Knock-Out <Molekulargenetik>},
  language  = {de}
}
@phdthesis{Gmeiner2014,
  author    = {Gmeiner, Florian},
  title     = {Der Einfluss der Neurotransmitter Dopamin, Serotonin und GABA sowie ihrer Transporter auf das Schlafverhalten von Drosophila melanogaster},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-99152},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {In der vorliegenden Arbeit wurde der Einfluss von Dopamin, Serotonin und GABA auf das Schlafverhalten von Drosophila melanogaster genauer untersucht. Mit Hilfe von Mutanten in Wiederaufnahmetransportern f{\"u}r Dopamin und Serotonin konnte gezeigt werden, dass Dopamin und Serotonin entgegengesetzte Wirkungen auf die Schlafmenge der Fliegen haben. Dopamin hat eine schlafhemmende, Serotonin eine schlaff{\"o}rdernde Wirkung. Die Nutzung eines neuronal dopamindefizienten Fliegenstammes erweitert diese Erkenntnisse. Die Nutzung von RNAi zur Hinunterregulierung der Rezeptoren f{\"u}r Dopamin brachte keine weiteren Erkenntnisse, da sie zu keinem messbaren Effekt f{\"u}hren. Jedoch ergab eine parallel dazu durchgef{\"u}hrte Hinunterregulierung des GABABR2 Rezeptors, dass dieser maßgeblich f{\"u}r die Aufrechterhaltung des Schlafes in der zweiten H{\"a}lfte der Nacht verantwortlich ist. Es konnte gezeigt werden, dass f{\"u}r diese Aufgabe vor allem ihre Expression in den l-LNv Neuronen relevant ist. Dabei ist f{\"u}r die GABABR2 Rezeptoren kein Effekt, f{\"u}r Dopamin und Serotonin nur in geringen Ausmaß ein Effekt auf die Innere Uhr in Form von gering ver{\"a}nderter Periode zu beobachten. Durch eine Kombination der Transportermutanten f{\"u}r Dopamin und Serotonin mit dem intakten, als auch mutierten WHITE Transporter zeigte sich eine interessante Interaktion dieser drei Transporter bei der Regulation der Gesamtschlafmenge, wobei die white Mutation zu einer Reduzierung der Gesamtschlafmenge f{\"u}hrt. UPLC Messungen der St{\"a}mme ergaben, dass der Effekt von white vermutlich auf dessen Einfluss auf den beta-Alanyldopamingehalt der Fliegen basiert. beta-Alanyldopamin wird bei dem Transport von Dopamin {\"u}ber die Gliazellen durch das Enzym EBONY gebildet, dessen Mutation in der Kombination mit intaktem WHITE und mutiertem Dopamintransporter zu einer drastischen Reduktion des Schlafes w{\"a}hrend der Nacht f{\"u}hrt. Im Rahmen der Untersuchung konnte zudem gezeigt werden, dass entgegen des bisherigen Wissens aus Zellkulturstudien in Drosophila melanogaster kein beta-Alanylserotonin gebildet wird. M{\"o}glicherweise wird nur Dopamin, nicht jedoch Serotonin {\"u}ber die Gliazellen recycelt. Dies ist ein interessanter Unterschied, der sowohl eine zeitliche, als auch lokale Feinregulation der Gegenspieler Dopamin und Serotonin erm{\"o}glicht. Die Untersuchung der Dimerpartner BROWN und SCARLET zeigte, dass lediglich BROWN zu einer Reduktion des Schlafes f{\"u}hrt. Ein Effekt, der auch in einer Fliegenlinie mit spontaner white Mutation beobachtet werden konnte. Die genaue Funktion dieses Heterodimertransporters und seine neuronale Lokalisation wurden im Rahmen dieser Arbeit noch nicht gekl{\"a}rt. Dennoch liegt eine Funktion als Dopamin- oder beta-Alanyldopamintransporter in Gliazellen auf Grund der ermittelten Ergebnisse nahe. Zus{\"a}tzlich konnte zum ersten Mal in Drosophila melanogaster eine Funktion der Amintransporter bei der Anpassung der Inneren Uhr an extreme kurze bzw. lange Photoperioden gezeigt werden. Eine anatomische Lokalisierung des WHITE Transporters im Gehirn von Drosophila melanogaster, die weitere Charakterisierung der Rolle des WHITE/BROWN Dimers und die Zuordnung bestimmter dopaminerger und serotonerger Neurone bei der Modulation der Aktivit{\"a}tsmaxima stellen spannende Fragen f{\"u}r zuk{\"u}nftige Arbeiten dar.},
  subject      = {Taufliege},
  language  = {de}
}
@phdthesis{Groeber2014,
  author    = {Groeber, Florian},
  title     = {Etablierung eines vaskularisierten Haut{\"a}quivalentes},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-107453},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {Durch Methoden des Tissue Engineerings hergestellte dreidimensionale Haut{\"a}quivalente bilden die native humane Haut hinsichtlich ihrer histologischen Architektur, zellul{\"a}ren Zusammensetzung und metabolischen Aktivit{\"a}t ab. Diese Gewebe eignen sich daher als zellbasierte Wundauflagen f{\"u}r großfl{\"a}chige Hautdefekte oder als In-vitro-Testsysteme f{\"u}r den Ersatz von Tierversuchen. Bei bisherigen Haut{\"a}quivalenten fehlt jedoch ein funktionelles Blutgef{\"a}ßsystem. Wird solch ein Gewebe als Implantat eingesetzt, f{\"u}hrt das Fehlen von Blutgef{\"a}ßen zu einer unzureichenden Versorgung mit N{\"a}hrstoffen und zur Nekrose. Neben dieser klinischen Limitation ist auch das Anwendungsspektrum als In-vitro-Testsystem begrenzt. Bei nicht vaskularisierten Hautmodellen kann eine transdermale Penetration von Substanzen nicht akkurat abgesch{\"a}tzt werden, da die zus{\"a}tzliche Barriere, welche die gef{\"a}ßauskleidenden Endothelzellen bilden, nicht enthalten ist. In Studien zur Integration eines Gef{\"a}ßsystems in Haut{\"a}quivalente konnte bislang lediglich gezeigt werden, dass sich Endothelzellen zu gef{\"a}ßartigen Strukturen zusammenlagern. Die Bildung von funktionellen perfundierbaren Gef{\"a}ßen in einem in vitro generierten Haut{\"a}quivalent ist bisher jedoch noch nicht belegt. Entsprechend ist eine direkte Anastomose mit dem Blutkreislauf eines Patienten bei einem klinischen Einsatz als Hautimplantat nicht m{\"o}glich. Bei einer Anwendung in In-vitro-Studien ist zudem das Gef{\"a}ßsystem experimentell nicht zug{\"a}nglich. In der vorliegenden Arbeit kann durch die Kombination einer biologischen, vaskularisierten Tr{\"a}gerstruktur (BioVaSc) mit einem neu entwickelten Bioreaktorsystems, ein Haut{\"a}quivalent mit einem perfundierbaren Gef{\"a}ßsystem hergestellt werden. Die Generierung dieser sogenannten SkinVaSc erfolgt {\"u}ber die Besiedlung der BioVaSc mit humanen Keratinozyten (hEK) und Fibroblasten. Parallel dazu werden die eingebetteten Gef{\"a}ßstrukturen der BioVaSc mit humanen mikrovaskul{\"a}ren Endothelzellen (hDMEC) rebesiedelt. Durch eine Anastomose zwischen den Gef{\"a}ßen der BioVaSc und dem Bioreaktorsystem ist eine Perfusion mit physiologisch, gepulsten Dr{\"u}cken zwischen 80 und 120 mmHG m{\"o}glich. Optimale Kulturbedingungen f{\"u}r die Haut- zellen k{\"o}nnen ferner durch zwei Kulturmodi generiert werden. Zur optimalen Versorgung der hEK innerhalb einer Proliferationsphase, die sich an die Zellaussaat anschließt, erfolgt eine kontinuierliche Versorgung der Oberfl{\"a}che der SkinVaSc mit Medium. Der zweite Modus stimuliert die Differenzierung der hEK durch eine Kultivierung des Modells an der Grenzfl{\"a}che zwischen Luft und Medium. Nach einer vierzehnt{\"a}gigen Kultivierung der SkinVaSc an der Luft Medium Grenzfl{\"a}che l{\"a}sst sich die Bildung einer hautspezifischen histologischen Architektur durch H{\"a}malaun/Eosin und immunhistologische F{\"a}rbungen belegen. Eine nat{\"u}rlich differenzierte Epidermis wird durch eine Basalmembran, die Kollagen Typ IV und Laminin 5 enth{\"a}lt von einen dermalen Teil getrennt. Die Dermale-Epidermale-Verbindung erscheint durch die Mikrostrukturierung der BioVaSc wellenf{\"o}rmig. Damit bildet die SkinVaSc die papillare Struktur der nativen humanen Haut ab. Innerhalb des dermalen Anteils k{\"o}nnen zudem Gef{\"a}ßstrukturen ausgemacht werden. Die Innenseite der Gef{\"a}ße sind durch eine Schicht aus hDMEC ausgekleidet, die endothelzellspe- zifische Oberfl{\"a}chenmarker wie "platelet endothelial cell adhesion molecule 1" und "von Willebrand Faktor" aufweisen. Eine zerst{\"o}rungsfreie {\"U}berwachung der SkinVaSc hinsichtlich der epidermalen Differenzierung ist durch eine integrierte Sensortechnologie auf Basis der Impedanz-spektroskopie m{\"o}glich. Dabei erlaubt ein entwickeltes mathematisches Modell die Extraktion von biologisch relevanten Informationen aus Impedanzspektren in einem Frequenzbereich zwischen 1 Hz und 100 kHz. Innerhalb dieser Studien ließ sich zeigen, dass die epidermale Differenzierung zu einer signifikanten Steigerung des ohmschen Widerstandes von 245,3 Ohm*cm2 zu 1108,1 Ohm*cm2 f{\"u}hrt. Gleichzeitig sinkt die zellul{\"a}re Kapazit{\"a}t von 131,5µF/cm2 auf 5,4µF/cm2 ab. Durch diese Parameter ist es m{\"o}glich die epidermale Barriere zerst{\"o}rungsfrei {\"u}ber die Kultivierungszeit zu {\"u}berwachen. Das Gef{\"a}ßsystem der SkinVaSc erm{\"o}glicht es mehr dermatologische Fragestellungen in vitro zu untersuchen und damit Tierversuche zu ersetzen. Zudem kann auf Basis der SkinVaSc ein vaskularisiertes Hautimplantat entwickelt werden, das es erm{\"o}glicht tiefe Hautverletzungen zu behandeln.},
  subject      = {Tisuue Engineering},
  language  = {de}
}
@phdthesis{FiebeckgebApfel2014,
  author    = {Fiebeck [geb. Apfel], Johanna Natalie},
  title     = {Etablierung eines fluoreszenzbasierten Zellassays zum Screening potentieller Krebstherapeutika des Wnt-Signalwegs},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-93101},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {Der Wnt Signalweg spielt eine entscheidende Rolle in der Embryogenese durch Steuerung der Proliferation, Apoptose, Differenzierung und der Festlegung der K{\"o}rperachsen im fr{\"u}hen Embryo. Eine Fehlregulation des Signalwegs durch Mutationen in einem der Proteine und Gene dieser hochkomplexen Signalkaskade kann fatale Folgen haben, und ist ein erster Schritt auf dem Weg der Krebsentstehung. Dabei spielt das Protein β-Catenin eine Schl{\"u}sselrolle im kanonischen Zweig des Wnt Signalwegs. Durch Steuerung seiner Konzentration im Zytoplasma wird die Expression seiner direkten Zielgene reguliert, da β-Catenin im aktiven Signalweg als Co-Transkriptionsfaktor agiert. Durch Sichtbarmachung dieses Proteins durch fluoreszierende Reportergenkonstrukte kann der Aktivit{\"a}tsstatus des Wnt Signalwegs in der Zelle beobachtet werden. Das erm{\"o}glicht zum einen genaue Analysen des Signalwegs, wie zum Beispiel das Studium des Zusammenspiels mit anderen Signalwegen. Vor allem aber erlaubt es die gezielte Suche nach Wnt-Signalwegs-modulierenden Substanzen als potentielle Wirkstoffe in der Krebsmedikamentenentwicklung. In der vorliegenden Arbeit wurden mehrere Reportergenkonstrukte f{\"u}r die stabile Transfektion von Zelllinien entwickelt und hinsichtlich eines m{\"o}glichen Einsatzes sowohl in der Forschung, als auch in Wirkstoffscreenings validiert. Dies umfasst sowohl mehrere Reporter mit β-Catenin als Fusionsprotein, als auch Wnt-Promoter-regulierte eGFP-Reporter, die den Akitvit{\"a}tsstatus des Wnt-Signalwegs anzeigen. Mit Hilfe dieser Reporter konnten Untersuchungen zur Wirkung des Wnt-Signalwegs auf die Morphologie von transfizierten und nicht-transfizierten MDCK-Zellen durchgef{\"u}hrt werden. {\"U}berdies wurde ein promotorregulierter eGFP-Reporter konstruiert, mit welchem transfizierte Zellen mit aktiviertem Wnt-Signalweg aus einem Zellpool gefischt werden k{\"o}nnen. Diese Methode ist sowohl f{\"u}r den Einsatz in kultivierten Zelllinien, als auch in der Diagnostik nach der Transfektion prim{\"a}rer Zellen geeignet. Auf Grundlage der neuen Zelllinien wurde weiterhin ein neuer Screeningansatz f{\"u}r potentielle Wnt-Signalwegsinhibitoren entwickelt, der auf dem Ausbleichen der Fluoreszenz in einem Well einer Multiwell-Kulturplatte beruht.},
  subject      = {Wnt-Proteine},
  language  = {de}
}
@phdthesis{Haug2014,
  author    = {Haug, Daniela},
  title     = {Untersuchung von FOSL1 im humanen Melanom},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-91064},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {Bei Melanomen handelt es sich um die gef{\"a}hrlichste Form von Hautkrebs mit der h{\"o}chsten Mortalit{\"a}tsrate. Deshalb sind Untersuchungen dieser Hautkrebsart von immenser Bedeutung. Es ist bekannt, dass der AP-1-Transkriptionsfaktorkomplex eine große Rolle f{\"u}r Melanomentstehung und -progression spielt. In der vorliegenden Arbeit wurde die Funktion der AP-1 Komponente FOSL1 in Melanomen untersucht. Hierbei konnte zun{\"a}chst ermittelt werden, dass die FOSL1 Expression im humanen Melanom durch den MAPK-Signalweg vermittelt wird und von den Onkogenen BRAF und NRAS abh{\"a}ngig ist. Dies wird auch durch die Tatsache unterst{\"u}tzt, dass die Stabilit{\"a}t von FOSL1 durch MAPK reguliert wird. Des Weiteren konnte gezeigt werden, dass FOSL1 in vielen Melanomzellen die Proliferation verst{\"a}rkt und auch an Migration beteiligt ist. Da diese Prozesse zur Krebsprogression beitragen, deutet dies darauf hin, dass FOSL1 bei der Melanomentwicklung eine wichtige Funktion besitzt. Weiterhin konnten SLUG, SNAI3, IL6 und MMP14 als FOSL1-Zielgene identifiziert werden, deren Regulierbarkeit durch FOSL1, jedoch abh{\"a}ngig von der jeweiligen Zelllinie war. Somit konnte mit dieser Arbeit gezeigt werden, dass FOSL1 nicht nur, wie zuvor f{\"u}r Brustkrebszellen beschrieben, an Migration beteiligt ist, sondern auch zur Proliferation humaner Melanome beitr{\"a}gt. Zuk{\"u}nftige Arbeiten werden zeigen, ob die identifizierten Gene f{\"u}r die FOSL1-vermittelte Migration und Proliferation verantwortlich sind.},
  subject      = {Proliferation},
  language  = {de}
}