@phdthesis{Kappenberger2024,
  author    = {Kappenberger, Jeannette Sarah},
  title     = {Biochemical characterization of the TFIIH translocase XPB from \(Chaetomium\) \(thermophilum\)},
  doi       = {10.25972/OPUS-24409},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-244096},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {DNA repair and gene expression are two major cellular processes that are fundamental for the maintenance of biological life. Both processes require the enzymatic activity of the super family 2 helicase XBP, which is an integral subunit of the general transcription factor TFIIH. During transcription initiation, XPB catalyzes the initial melting of promoter DNA enabling RNA polymerase II to engage with the coding DNA strand and start gene transcription. In nucleotide excision repair, XPB acts in concert with the other TFIIH helicase XPD causing strand separation around a lesion site. Mutations within the genes encoding XPB or other TFIIH subunits are associated with different cancer types as well as with the autosomal recessive disorders Xeroderma Pigmentosum and trichothiodystrophy and rarely combined features of Xeroderma Pigmentosum and Cockayne syndrome. In the last few years, great progress has been made towards unraveling the structure of TFIIH and its individual subunits including XPB. These structural insights tremendously improved our understandings with respect to the molecular interactions within this intriguing protein complex. However, the underlying regulation mechanisms that functionally control XPB during transcription and repair remained largely elusive. We thus executed the biochemical characterization of this protein to investigate the functional network that regulates XPB within the scaffold of TFIIH. Due to their enhanced stability compared to the human proteins, we utilized the proteins that originate from the thermophilic fungus Chaetomium thermophilum for this purpose as a model organism for eukaryotic TFIIH. The present work provides novel insights into the enzymatic function and regulation of XPB. We could show that both, DNA and the TFIIH subunit p52 stimulate XPB's ATPase activity and that the p52-mediated activity is further boosted by p8, another subunit within TFIIH. Surprisingly, DNA can activate XPB's ATPase activity to a greater extent than its TFIIH interaction partners p52/p8, but when both, i.e. p52/p8 and DNA are present at the same time, p52 dominates the activation and the enzymatic speed is maintained at the level observed through the sole activation of p52/p8. We thus defined p52 as the master regulator of XPB that simultaneously activates and represses XPB's enzymatic activity. Based on a correlative mutagenesis study of the main interface between p52 and XPB that was set into context with recent structural data, a model for the p52-mediated activation and speed limitation of XPB's ATPase was proposed. The research on XPB's ATPase was expanded with the investigation of the inhibition mechanism of XPB's ATPase via the natural compound Triptolide. Furthermore, we investigated XPB's DNA translocase function and could observe that XPB can only perform its translocase movement when it is fully incorporated into core TFIIH and this translocase movement is further enhanced by the nucleotide excision repair factor XPA. Fluorescence polarization measurements with nucleotide analogues revealed that XPB displays the highest affinity towards DNA in the ADP + Pi bound state and its binding is weakened when ADP is bound or the nucleotide is dissociated from the enzyme, suggesting a movement on the DNA during the distinct states of the ATPase cycle. Finally, the well-known and highly conserved RED motif was found to be the crucial element in XPB to enable this translocase movement. Combined, the data presented in this work provide novel insights into the intricate regulation network that controls XPB's enzymatic activity within TFIIH and furthermore show that XPB's enzymatic activity is tightly controlled by various factors.},
  subject      = {DNS-Reparatur},
  language  = {en}
}
@phdthesis{Lechermeier2024,
  author    = {Lechermeier, Carina},
  title     = {Neuroanatomical and functional evaluation of ADHD candidate genes in the model organism zebrafish (\(Danio\) \(rerio\))},
  doi       = {10.25972/OPUS-37108},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-371084},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {Attention deficit hyperactivity disorder (ADHD) is one of the most prevalent developmental disorders, affecting 5.9\% children and adolescents and 2.5\% adults worldwide. The core characteristics are age-inappropriate levels of hyperactivity, impulsivity and inattention, often accompanied by co-morbidities such as mood and conduct disorders as wells as learning deficits. In the majority of cases, ADHD is caused by an interplay of accumulated genetic and environmental risk factors. Twin studies report a very high heritability of 70-80\%, however, common genetic variants in the population only explain a third of the heritability. The rest of the genetic predisposition is composed of rare copy number variations (CNVs) and gene x environment interactions including epigenetic alterations. Through genome wide association (GWAS) and linkage studies a number of likely candidate genes were identified. A handful of them play a role in dopamine or noradrenaline neurotransmitter systems, simultaneously those systems are the main targets of common drug treatment approaches. However, for the majority of candidates the biological function in relation to ADHD is unknown. It is crucial to identify those functions in order to gain a deeper understanding of the pathomechanism and genetic networks potentially responsible for the disorder. This work focuses on the three candidate genes GFOD1, SLC2A3 and LBX1 and their role in the healthy organism as well as in case of ADHD. The neuroanatomy was regarded through expression analysis and various behavioural assays of activity were performed to link alterations on the transcript level to phenotypes associated with the neurodevelopmental disorder. Zebrafish orthologues of the human risk genes were identified and extensive temporal and spacial expression characterisation performed via RNA in situ hybridisation. Through morpholino derived knock-down and mRNA overexpression zebrafish models with subsequent behavioural analysis, both hyper- and hypoactive phenotypes were discovered. Additional expression analysis through double in situ hybridisation revealed a co-localisation during zebrafish neurodevelopment of each gfod1 and slc2a3a together with gad1b, a marker for GABAergic neurons. Interestingly, both risk genes have previously been associated with glucose homeostasis and energy metabolism, which when disrupted could lead to alterations in signal transduction and neuron survival. Likewise, Lbx1 plays a pivotal role in GABAergic versus glutamatergic neuron specification during spinal cord and hindbrain development in mice and chicken. Preliminary results of this work suggest a similar role in zebrafish. Taken together, those findings on the one hand represent a sturdy basis to con- tinue studies of the function of the genes and on the other hand open up the opportunity to investigate novel aspects of ADHD research by exploring the role of the GABAergic neurotransmitter system or the connection between energy metabolism and psychiatric disorders.},
  subject      = {Aufmerksamkeitsdefizit-Syndrom},
  language  = {en}
}
@phdthesis{Zhu2024,
  author    = {Zhu, Yan},
  title     = {Small RNA-associated RNA-binding proteins in \(Fusobacterium\) \(nucleatum\)},
  doi       = {10.25972/OPUS-37073},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-370731},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {Fusobacterium nucleatum is an emerging cancer-associated bacterium belonging to the Fusobacteriota phylum, which is evolutionary distant from all model bacteria. Recent analysis generated global fusobacterial RNA maps, which enabled the discovery of 24 small noncoding RNAs (sRNAs) in F. nucleatum. Notably, the σE-dependent sRNA FoxI and FoxJ act as a posttranscriptional regulator of several cell envelope proteins. The σE-dependent sRNAs in Escherichia coli and Salmonella require the RNA chaperone Hfq for their functions. Intriguingly, F. nucleatum seems to have no homologs of the three common RNA-binding proteins (RBPs) CsrA, Hfq and ProQ. However, it remains unclear if other families of RBPs act in concert with FoxI, FoxJ and other fusobacterial sRNAs. This work has successfully established a 14-mer capture tagged-sRNA affinity purification procedure initially using 6S RNA as a proof-of-concept. Applying this method to 19 different F. nucleatum sRNAs led to a comprehensive mapping of sRNA-binding proteins in this bacterium. This screen identified a total of 75 proteins significantly enriched across all sRNAs and prominent in ribosomal proteins, uncharacterized proteins and enzymes associated with metabolism. This work further focused on the homologs of two KH domain proteins KhpA and KhpB, which were recently recognized as global RBPs in various Gram-positive bacteria such as Streptococcus pneumoniae, Clostridioides difficile, and Enterococcus faecalis. Comparative analyses revealed conserved domain composition and gene synteny of KhpA and KhpB across F. nucleatum, S. pneumoniae, C. difficle and E. faecalis, indicating conserved roles of these proteins in bacteria. Further protein-protein interaction assays and global RNA targets profiling demonstrated that KhpA and KhpB form dimers and act together as broad RBPs, binding to sRNAs, mRNAs and tRNAs in F. nucleatum. Further functional characterizations unveiled that KhpA/B are required for the growth of F. nucleatum under nutrient limitation conditions and impact cell morphology. Additionally, the two RBPs also influence global gene expression in F. nucleatum affecting various bacterial physiological processes, including ethanolamine utilization. In summary, this work established a sRNA-centric approach for screening sRNA-binding proteins in F. nucleatum. Further, the assay could be applied in other non-model organisms and is feasible to screen multiple sRNA baits in parallel for sRNA-interactors. By applying this procedure to nearly all known fusobacterial sRNAs, this work generated an extensive map of sRNA-interacting proteins in F. nucleatum. Molecular and genetic studies identified that KhpA/B act as major RBPs and gene regulators in F. nucleatum, representing important first steps in elucidating key players of post-transcriptional control at the root of the bacterial phylogenetic tree.},
  subject      = {Proteine},
  language  = {en}
}
@phdthesis{Drakopoulos2024,
  author    = {Drakopoulos, Antonios},
  title     = {Opioid receptor oligomerization study through fluorescent selective ligands},
  doi       = {10.25972/OPUS-20717},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-207179},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {Opioid receptors (ORs) are among the most intensively studied members of the G protein-coupled receptor (GPCR) family due to their important role in pain management and their involvement in psychological and neurological disorders. However, currently available opioid drugs exhibit both serious drawbacks, such as addiction, and life-threatening side effects, such as respiratory depression. Contrary to the classic monomeric model, indirect evidence suggests that ORs might form dimers, which could be endowed with a distinct pharmacological profile, and, thus, be exploited to develop innovative drugs. However, direct evidence for the spontaneous formation of OR dimers in living cells under physiological condition are missing. The focus of this thesis was the design, synthesis and characterization of new, highly subtype-selective OR fluorescent ligands to be used as tools for state-of-the-art microscopy methods, such as single molecule microscopy (SMM), in heterologous cells and potentially in native tissue, in order to investigate OR organization and mobility on the surface of intact, living cells, at low/physiological expression levels. The μOR is the OR subtype which plays the most critical role in pain modulation, while mediating the effects of the most powerful analgesic drugs. Also, it is the OR subtype which is mostly responsible for the major adverse effects of the currently marketed opioid drugs. We aimed to develop a new μOR-selective fluorescent ligand with a potential irreversible binding mode. Although the approach was in principle successful, i.e. the labelled cells were visible and distinguishable; this initial attempt was not suitable for SMM due to the ligands' poor selectivity and affinity as well as due to its high background noise. A second generation of the fluorescent ligand was designed; however the synthesis and characterization are part of another doctoral thesis. Lately, δOR has received attention as a promising drug target, due to its distinct pharmacological profile which features low abuse liability and lack of physical dependence. In addition, δOR expression has been associated with cancer regulation in the periphery, thus further highlighting the interest of imaging tools for this receptor. In this thesis, the development and characterization of two new δOR-selective fluorescent probes with excellent optical properties, based on the well-studied ligand naltrindole (NTI) is presented. Their application in SMM studies is currently underway at the group of Prof. Dr. Davide Calebiro at the University of Birmingham. The κOR is a subtype which has also emerged as a drug target due to its low abuse potential. Despite a growing interest in this receptor, κOR-selective fluorescent probes have been particularly scarce in literature. Herein, the design, synthesis and characterization of the first reported set of fluorescent κOR-selective probes with antagonistic properties, based on the established ligand 5'-guanidinonaltrindole (5'-GNTI) is presented. Two of these were employed for SMM experiments to investigate κOR homodimerization, localization and trafficking. Our findings do not support homodimerization of the κOR-bound probe complexes, while showing that the majority of them follow a normal Brownian diffusion on the cell surface.},
  subject      = {Opioidrezeptor},
  language  = {en}
}
@phdthesis{Steinmueller2024,
  author    = {Steinm{\"u}ller, Sophie Anna Maria},
  title     = {Benzimidazole-Based Photoswitches and Photoswitchable Cannabinoid 2 Receptor Ligands},
  doi       = {10.25972/OPUS-34894},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-348943},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {The field of photopharmacology has attracted considerable attention due to applying the spatial and temporal precision of light to pharmacological systems. Photoswitchable biologically active compounds have proven useful in the field of G protein-coupled receptors (GPCRs), which are of tremendous therapeutic relevance. Generally, the pharmacology of GPCRs is complex, perhaps even more complex than originally thought. Suitable tools are required to dissect the different signalling pathways and mechanisms and to unravel how they are connected in a holistic image. This is reflected in the enormous scientific interest in CB2R, as the neuroprotective and immunomodulatory effects attributed to CB2R agonists have not yet translated into effective therapeutics. This work focused on the development of a novel photoswitchable scaffold based on the privileged structure of benzimidazole and its application in photoswitchable CB2R ligands as photopharmacological tools for studying the CB2R. The visible-light photoswitchable ligand 10d enables the investigation of CB2R activation with regard to βarr2 bias, exhibiting a unique pharmacological profile as a "cis-on" affinity switch at receptor level and as a "trans-on" efficacy-switch in βarr2-mediated receptor internalization. The novel photoswitchable scaffold developed in this work further serves as a guide for the development of novel photoswitchable GPCR ligands based on the privileged structure of benzimidazole. To obtain a different tool compound for studying CB2R activation and signalling mechanisms, a previously reported putatively dualsteric CB2R ligand was rendered photoswitchable, by linking the orthosteric agonist to a CB2R-selective PAM via photoswitchable azobenzene. Compound 27-para exhibits a desirable "cis-on" behaviour across all investigated assays with >10-fold higher potency compared to its trans-isomer and can be used as an efficacy-switch employing specific concentrations.},
  subject      = {Cannabinoide},
  language  = {en}
}
@phdthesis{Hadi2024,
  author    = {Hadi, Naji Said Aboud},
  title     = {In vitro Studies on the Genotoxicity of Selected Pyrrolizidine Alkaloids},
  doi       = {10.25972/OPUS-37037},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-370376},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {Cancer is one of the leading causes of death worldwide. Toxic contaminants in human food or medicinal products, such as substances like pyrrolizidine alkaloids (PAs), have been thought to contribute to cancer incidence. PAs are found in many plant species as secondary metabolites, and they may affect humans through contaminated food sources, herbal medicines, and dietary supplements. Hundreds of compounds belonging to PAs have been identified, differing in their chemical structures, either in their necine base moiety or esterification at their necic acid moiety. PAs undergo hepatic metabolism, and after this process, they can induce hepatotoxicity, genotoxicity, and carcinogenicity. However, the mechanism of inducing genotoxicity and carcinogenicity is still unclear and warrants further investigation. Therefore, the present study aims to investigate the mechanism of genotoxicity induced by selected PAs with different chemical structures in in vitro systems. Primarily, human hepatoma HepG2 cells were utilized, and in co-culture, metabolically active HepG2 cells were combined with non-metabolically active human cervical HeLa H2B-GFP cells. First, the genotoxicity of the PAs europine, lycopsamine, retrorsine, riddelliine, seneciphylline, echimidine, and lasiocarpine was investigated in the cytokinesis-block micronucleus (CBMN) assay. All seven selected PAs caused the formation of micronuclei in a dose-dependent manner, with the maximal increase of micronucleus formation ranging from 1.64 to 2.0 fold. The lowest concentrations at which significant induction of micronuclei was found were 3.2 µM for lasiocarpine and riddelliine, 32 µM for retrorsine and echimidine, and 100 µM for seneciphylline, europine, and lycopsamine. These results confirmed previously published potency rankings in the micronucleus assay. The same PAs, with the exception of seneciphylline, were also investigated in a crosslink-modified comet assay, and reduced tail formation after hydrogen peroxide treatment was found in all diester-type PAs. Meanwhile, an equimolar concentration of the monoesters europine and lycopsamine did not significantly reduce DNA migration. Thus, the crosslinking activity was related to the ester type. Next, the role of metabolic enzymes and membrane transporters in PA-induced genotoxicity was assessed. Ketoconazole (CYP 450-3A4 inhibitor) prevented lasiocarpine-induced micronucleus formation completely, while furafylline (CYP 450-1A2 inhibitor) reduced lasiocarpine-induced micronucleus formation, but did not abolish it completely. This implies that the CYP 450 enzymes play an important role in PA-induced genotoxicity. Carboxylesterase 2 enzyme (CES 2) is commonly known to be involved in the detoxification of xenobiotics. Loperamide (CES 2 inhibitor) yielded an increased formation of lasiocarpine-induced micronuclei, revealing a possible role of CES-mediated detoxification in the genotoxicity of lasiocarpine. Also, intracellular glutathione (GSH) plays an important role in the detoxification of xenobiotics or toxins in the cells. Cells which had been pretreated with L-buthionine sulfoximine (BSO) to reduce GSH content were significantly more sensitive for the induction of micronucleus formation by lasiocarpine revealing the importance of GSH in PA-induced genotoxicity. Quinidine (Q) and nelfinavir (NFR) are OCT1 and OATP1B1 influx transporter inhibitors, respectively, which reduced micronucleus induction by lasiocarpine (only quinidine significantly), but not completely, pointing to a relevance of OCT1 for PA uptake in HepG2 cells. Verapamil (V) and benzbromarone (Bz) are MDR1 and MRP2 efflux transporter inhibitors, respectively, and they caused a slightly increased micronucleus induction by lasiocarpine (significant only for benzbromarone) thus, revealing the role of efflux transporters in PA-induced genotoxicity. The mechanistic approach to PA-induced genotoxicity was further studied based on oxidative stress via the formation of reactive oxygen species (ROS) in HepG2 cells. Overproduction of ROS can cross-link cellular macromolecules such as DNA, leading to genomic damage. An equimolar concentration of 10 µM of lasiocarpine (open-diester PA), riddelliine (cyclic-diester PA), and europine (monoester) significantly induced ROS production, with the highest ROS generation observed after lasiocarpine treatment, followed by riddelliine and then europine. No significant increase in ROS production was found with lycopsamine (10 µM; monoester PA), even at a higher concentration (320 µM). The generation of ROS by these PAs was further analyzed for confirmation by using 5 mM of the thiol radical scavenger antioxidant N-acetyl cysteine (NAC) combined with lasiocarpine, riddelliine, or europine. This analysis yielded a significant decrease in ROS after combining NAC with lasiocarpine, riddelliine, and europine. In addition, lasiocarpine, riddelliine, and europine induced a loss of mitochondrial membrane potential, pointing to mitochondria as the source of ROS generation. In vivo, hepatic sinusoidal epithelial cells (HSECs) are known to be damaged first by PAs after hepatic metabolization, but HSECs themselves do not express the required metabolic enzymes for activation of PAs. To mimic this situation, HepG2 cells were used to metabolically activate PA in a co-culture with HeLa H2B-GFP cells as non-metabolically active neighbours. Due to the green fluorescent GFP label the HeLa cells could be identified easily based in the co-culture. The PAs europine, riddelliine and lasiocarpine induced micronucleus formation in HepG2 cells, and in HeLa H2B-GFP cells co-cultured with HepG2 cells, but not in HeLa H2B-GFP cells cultured alone. Metabolic inhibition of CYP 450 enzymes with ketoconazole abrogated micronucleus formation induced by the same PAs tested in the co-culture. The efflux transporter inhibitors verapamil and benzbromarone reduced the micronucleus formation in the co-culture. Furthermore, mitotic disturbances as an additional genotoxic mechanism of action were observed in HepG2 cells and in HeLa H2B-GFP cells co-cultured with HepG2 cells, but not in HeLa H2B-GFP cells cultured alone. Overall, we were able to show that PAs were activated by HepG2 cells and the metabolites induced genomic damage in co-cultured non-metabolically active green HeLa cells. Finally, in HepG2 cells as well as the co-culture, combinations of PAs lasiocarpine and riddelliine favoured an additive effect rather than synergism. Thus, this study therefore provides support that the assumption of dose-addition can be applied in the characterization of the genotoxicity risk of PAs present in a mixture.},
  subject      = {Pyrrolizidinalkaloide},
  language  = {en}
}
@phdthesis{Kawan2024,
  author    = {Kawan, Mona},
  title     = {The membrane trafficking protein myoferlin is a novel interactor of p97},
  doi       = {10.25972/OPUS-28121},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-281218},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {p97 uses the energy of ATP hydrolysis to unfold and thereby segregate proteins. It is involved in various cellular processes such as proteasomal degradation, DNA damage repair, autophagy, and endo-lysosomal trafficking. The specificity for these processes is controlled by more than 30 regulatory cofactors. Interactions of p97 with cofactors and target proteins are known to be highly dynamic and transient. To identify new interaction partners and to uncover novel cellular functions of p97, the interactome of endogenous p97 was determined by using in cellulo crosslinking followed by immunoprecipitation and mass spectrometry. Myoferlin (MYOF) was identified as a novel interactor of p97 and the interaction was validated in reciprocal immunoprecipitation experiments for different cell lines. The ferlin family member MYOF is a tail-anchored membrane protein containing multiple C2 domains. MYOF is involved in various membrane repair and trafficking processes such as the endocytic recycling of cell surface receptors. The MYOF interactome was determined by mass spectrometry. Among others, the p97 cofactor PLAA, CD71 and Rab14 were identified as common interactors of p97 and MYOF. Immunoprecipitation experiments with PLAA KO cells revealed that the interaction between MYOF and p97 depends on PLAA. Immunofluorescence microscopy showed a co-localization of MYOF with Rab14 and Rab11, which are both involved in endocytic recycling pathways. Furthermore, immunofluoroscence experiments revealed that MYOF and the p97 cofactor PLAA are localized to Rab14- and Rab5-positive endosomal compartments. Using p97 inhibitors and p97 trapping mutants, the presence of p97 at MYOF-positive and Rab14-positive structures could be demonstrated. Consistent with this finding, the endocytic recycling of transferrin was delayed upon inhibition of p97. Taken together, this work identified MYOF as a novel interactor of p97 and suggests a role for p97 in the recycling of endocytic cargo.},
  subject      = {Endosom},
  language  = {en}
}
@article{JarickMokhtariSchelleretal.2018,
  author    = {Jarick, Katja J. and Mokhtari, Zeinab and Scheller, Lukas and Hartweg, Julia and Thusek, Sina and Le, Duc-Dung and Ranecky, Maria and Shaikh, Haroon and Qureischi, Musga and Heinze, Katrin G. and Beilhack, Andreas},
  title     = {Photoconversion of Alloreactive T Cells in Murine Peyer's Patches During Acute Graft-Versus-Host Disease: Tracking the Homing Route of Highly Proliferative Cells In Vivo},
  series = {Frontiers in Immunology},
  volume    = {9},
  journal   = {Frontiers in Immunology},
  doi       = {10.3389/fimmu.2018.01468},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-323309},
  year      = {2018},
  abstract  = {The regulation of immune cell migration throughout the body is essential to warrant immunosurveillance and to maintain immune homeostasis. Marking and tracking of these cells has proven important to study mechanisms of immune cell trafficking and cell interaction in vivo. Photoconversion is a well-suited technique for intravital application because it enables contactless time- and location-specific marking of cells in the tissue without surgically manipulating the microenvironment of the cells in question. However, in dividing cells the converted fluorescent protein may decline quickly. Here, we provide a detailed description of the photoconversion technique and its applicability to tracking highly proliferating T cells from the priming site of T cell activation to peripheral target organs of effector function in a preclinical model. Dendra2+ T cells were photoconverted in the Peyer's patches during the initiation phase of acute graft-versus-host disease (GvHD) and tracked through the mesenteric lymph nodes and the peripheral blood to the small intestine with flow cytometry and intravital two-photon microscopy. Photoconverted alloreactive T cells preserved the full proliferative capacity, homing, and migration of alloreactive T cells in the intestinal lamina propria. We conclusively proved that photoconversion of highly proliferative alloreactive T cells in the Peyer's patches is an effective tool to study trafficking of alloreactive T cells under physiologic conditions and to GvHD target tissues. This technique can also be applied to the study of immune cell tracking under inflammatory and non-inflammatory conditions.},
  language  = {en}
}
@phdthesis{Waltmann2024,
  author    = {Waltmann, Maria},
  title     = {Neurocognitive mechanisms of loss of control in Binge Eating Disorder},
  doi       = {10.25972/OPUS-36430},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-364300},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {Binge Eating Disorder (BED) is a common, early-onset mental health condition characterised by uncontrollable episodes of overeating followed by negative emotions such as guilt and shame. An improved understanding of the neurocognitive mechanisms underlying BED is central to the development of more targeted and effective treatments. This thesis comprises a systematic review and three empirical studies contributing to this endeavour. BED can be thought of as a disorder of cognitive-behavioural control. Indeed, self-report evidence points towards enhanced impulsivity and compulsivity in BED. However, retrospective self-reports do not capture the mechanisms underlying impulsive and compulsive lapses of control in the moment. The systematic review therefore focussed on the experimental literature on impulsivity and compulsivity in BED. The evidence was very mixed, although there was some indication of altered goal-directed control and behavioural flexibility in BED. We highlight poor reliability of experimental paradigms and the failure to properly account for weight status as potential reasons for inconsistencies between studies. Moreover, we propose that impulsivity and/or compulsivity may be selectively enhanced in negative mood states in BED and may therefore not be consistently detected in lab-based studies. In the empirical studies, we explored the role of behavioural flexibility in BED using experimental and neuroimaging methods in concert with computational modelling. In the first empirical study, we assessed the reliability of a common measure of behavioural flexibility, the Probabilistic Reversal Learning Task (PRLT). We demonstrate that the behavioural and computational metrics of the PRLT have sufficient reliability to justify past and future applications if calculated using hierarchical modelling. This substantially improves reliability by reducing error variance. The results support the use of the PRLT in the second and third empirical studies on development and BED. Because a majority of patients develop BED as adolescents or young adults, we speculated that it may emerge as a consequence of disrupted or deficient maturation of behavioural flexibility. Little is known about typical development in this domain. We therefore investigated normative development of reversal learning from adolescence to adulthood in the second empirical study. Typically- developing adolescents exhibited less adaptive and more erratic and explorative behaviour than adults. This behaviour was accounted for by reduced sensitivity to positive feedback in a reinforcement learning model, and partially mediated by reduced activation reflecting uncertainty in the medial prefrontal cortex, a region known to mature substantially during adolescence. In the third empirical study, we investigated reversal learning in BED, paying special attention to potential biases associated with learning from wins vs learning from losses. We speculated that negative urgency could make it more difficult for BED patients to learn and make decisions under pressure to avoid losses. To dissociate between effects of excess weight and BED, we collected data from obese individuals with and without BED as well as normal-weight controls. As hypothesised, there were subtle neurocognitive differences between obese participants with and without BED with regard to learning to obtain rewards and to avoid losses. Obese individuals showed relatively impaired learning to obtain rewards, while BED patients showed relatively impaired learning to avoid losses. This was reflected in differential learning signals in the brain and associated with BED symptom severity. In sum, this thesis shows that the evidence on impulsivity and compulsivity in BED is inconsistent and offers potential explanations for this inconsistency. It highlights the need for reliability in interindividual difference research and indicates ways to improve it. Further, it charts the typical development of reversal learning from adolescence to adulthood and underscores the relevance of exploration in the context of learning and decision-making in adolescence. Finally, it demonstrates qualitative differences between BED and obesity, hinting at a pivotal role of aversive states in loss of control in BED.},
  subject      = {Binge-eating Disorder},
  language  = {en}
}
@phdthesis{Jorgacevic2024,
  author    = {Jorgacevic, Ivana},
  title     = {Elucidating the interconnection of GvHD and Western diet-induced atherosclerosis},
  doi       = {10.25972/OPUS-32579},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-325792},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {Allogeneic hematopoietic cell transplantation (Allo-HCT) is the main and only treatment for many malignant and non-malignant haematological disorders. Even though the treatment has improved through the years and patient life expectancy has increased, graft versus host disease (GvHD) is still considered the main obstacle and one of the main reasons for increased mortality. Furthermore, improved patient's survival and life expectancy brought into question the late post-HCT complications. The leading cause of late death after allo-HCT is the relapse of primary disease as well as chronic GvHD (cGvHD). However, a clear relationship was also described with pulmonary complications, endocrine dysfunction and infertility, and cataracts in post-HCT patients. In the last years big concern regarding a cumulative cardiovascular incidence in long-term survivors has been raised. Severe cardiovascular disease (CVD) is caused by atherosclerosis which is considered a chronic inflammatory disease of blood vessels. As such, it takes a long time from endothelial damage, as the onset event, and followed plaque formation to a manifestation of severe consequences, such as stroke, coronary heart disease, or peripheral arterial disease. Endothelial damage is well documented in patients post-HCT. In the context of allo-HCT, the endothelial damage is induced by the conditioning regimen with or without total body irradiation (TBI). Furthermore, endothelial cells (ECs) have been documented as a target of GvHD and increased concentration of circulating endothelial cells (CEC) coinciding with an increase in the number of circulating alloreactive T cells. According to 2021 ESC Guidelines on CVD prevention, the main atherosclerotic CVD (ASCVD) risk factors are blood apolipoprotein B (ApoB)-containing lipoproteins (of which low-density lipoprotein (LDL) is the most abundant), high blood pressure, cigarette smoking and diabetes mellitus (DM). GvHD is considered a high-risk factor for the onset of dyslipidaemia, hypertension, and DM. Overall, the risk of premature cardiovascular death is 2.7 fold increased in comparison to the general population, while the cumulative incidence of cardiovascular complications was shown to be up to 47\% at ten years after reduced intensity conditioning (RIC), post-HCT. However, up to date, there are no available studies elucidating the interconnection between GvHD and atherosclerosis. The goal of this study was, therefore, to investigate the involvement of GvHD in the progression of atherosclerosis as well as to elucidate whether cytotoxic, CD8+ T cells that were shown to play a significant role in endothelial damage during the course of skin GvHD on one hand, and inducers of formation of unstable plaque on the other, are involved in this interconnection. For that purpose we established a novel minor histocompatibility anti gens (miHAg) allo-HCT Western diet (WD)-induced atherosclerosis mouse model. We were able to show that GvHD has a significant impact on atherosclerosis development in B6.Ldlr-/- recipient mice even in the absence of overt clinical disease activity. It seems that the impact is at least partly induced by CD8+ T cells, that showed significantly increased infiltration of aortic lesions in mice facing subclinical GvHD. As studies have shown in regular atherosclerotic mouse models as well as in humans, these CD8+ T cells exhibited not only increased expression of genes involved in activation, survival and differentiation to cytotoxic phenotype, but also some genes pointing out their exhaustion, that were absent in CD4+ T cell cluster. When anti-CD8β antibody was applied once per week along with WD feeding for eight weeks, the plaque formation was significantly reduced in aorta and aortic root pointing out the importance of these cells in an alloreactivity induced lesion formation. Furthermore, anti-CD8β treatment led to significantly decreased necrotic core formation followed by overall increase in plaque stability. Strikingly, bone marrow plus T cells (BMT) recipients fed WD showed significantly increased serum cholesterol levels in comparison to bone marrow (BM) (a group lacking alloreactive T cells that induce GvHD). This effect was reversed when anti-CD8β treatment was applied, suggesting, at least partly, an impact of alloreactive CD8+ T cells on cholesterol levels. Expression of genes responsible for lipid metabolism pointed out the tendency of the liver to regulate the increased cholesterol levels, however, the mechanism behind this phenotype still remains to be revealed. On the other hand, the impact of obesity, induced by chronic high-fat diet (HFD) feeding, has been shown to be an independent risk factor for gastrointestinal GvHD. Similarly, in major histocompatibility complex (MHC) disparate allo-HCT mouse model, we have noticed that even short-term WD intake leads to a significant decrease in survival of mice post-HCT. When the concentration of transplanted alloreactive T cells was reduced, the survival was improved, pointing out the involvement of these cells in the pathogenesis. Additionally, bioluminescence imaging (BLI) during initiation and effector phase of acute GvHD (aGvHD) revealed increased infiltration of alloreactive T cells in mice fed WD. Studies in an obesity model, we could confirm the involvement of specifically CD4+ T cells in WD induced impact, as the relative number of these cells was significantly increased in small intestine on day six post-HCT in mice fed WD. This increased intestinal infiltration was preceded by increase in the number of alloreactive T cells expressing intestine homing receptor (α4β7 integrin) in peripheral lymph nodes (LNs). Even though the number of T cells was not changed in the spleen of WD fed mice, the subset of CD4+ and CD8+ T cells that were highly secreting TNFα was increased as well as the expression of genes regulating pro-inflammatory cytokines such as IL-6 and interferon (IFN)γ pointing out significant WD-induced inflammation. Moreover, slight tendency towards increased intestinal permeability and load of translocated luminal bacteria, that we observed, could induce severe endotoxemia and dysregulated systemic immune response that could lead to detrimental induction of cell death. Justifying our speculations, we noted increased levels of transaminases and an increase in lactate dehydrogenase (LDH) levels (pointing out significant tissue damages). However, the exact mechanism behind this detrimental WD impact still remains to be elucidated.},
  subject      = {Periphere Stammzellentransplantation},
  language  = {en}
}