@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{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{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{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}
}
@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{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{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}
}
@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{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{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}
}