@phdthesis{Agnetta2019,
  author    = {Agnetta, Luca},
  title     = {Novel Photoswitchable and Dualsteric Ligands Acting on Muscarinic Acetylcholine Receptors for Receptor Function Investigation},
  doi       = {10.25972/OPUS-18717},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-187170},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {G protein-coupled receptor research looks out for new technologies to elucidate the complex processes of receptor activation, function and downstream signaling with spatiotemporal resolution, preferably in living cells and organisms. A thriving approach consists in making use of the unsurpassed properties of light, including its high precision in space and time, noninvasiveness and high degree of orthogonality regarding biological processes. This is realized by the incorporation of molecular photoswitches, which are able to effectively respond to light, such as azobenzene, into the structure of a ligand of a given receptor. The muscarinic acetylcholine receptors belong to class A GPCRs and have received special attention in this regard due to their role as a prototypic pharmacological system and their therapeutic potential. They mediate the excitatory and inhibitory effects of the neurotransmitter acetylcholine and thus regulate diverse important biological processes, especially many neurological functions in our brain. In this work, the application of photopharmacological tool compounds to muscarinic receptors is presented, consisting of pharmacophores extended with azobenzene as light-responsive motif. Making use of the dualsteric concept, such photochromic ligands can be designed to bind concomitantly to the orthosteric and allosteric binding site of the receptor, which is demonstrated for BQCAAI (M1) and PAI (M2) and may lead to subtype- and functionalselective photoswitchable ligands, suitable for further ex vivo and in vivo studies. Moreover, photoswitchable ligands based on the synthetic agonist iperoxo were investigated extensively with regard to their photochemical behavior and pharmacological profile, outlining the advantages and challenges of using red-shifted molecular photoswitches, such as tetraortho- fluoro azobenzene. For the first time on a GPCR it was examined, which impact the different substitution pattern has on both the binding and the activity on the M1 receptor. Results show that substituted azobenzenes in photopharmacological compounds (F4-photoiperoxo and F4-iper-azo-iper) not just represent analogs with other photophysical properties but can exhibit a considerably different biological profile that has to be investigated carefully. The achievements gained in this study can give important new insights into the binding mode and time course of activation processes, enabling precise spatial and temporal resolution of the complex signaling pathway of muscarinic receptors. Due to their role as exemplary model system, these findings may be useful for the investigation into other therapeutically relevant GPCRs.},
  subject      = {Muscarinrezeptor},
  language  = {en}
}
@phdthesis{RiechelmannverhSteinbacher2021,
  author    = {Riechelmann [verh. Steinbacher], Eva Katharina},
  title     = {Gaze interaction: Cognitive mechanisms of oculomotor action control},
  doi       = {10.25972/OPUS-21527},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-215279},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Humans use their eyes not only as visual input devices to perceive the environment, but also as an action tool in order to generate intended effects in their environment. For instance, glances are used to direct someone else's attention to a place of interest, indicating that gaze control is an important part of social communication. Previous research on gaze control in a social context mainly focused on the gaze recipient by asking how humans respond to perceived gaze (gaze cueing). So far, this perspective has hardly considered the actor's point of view by neglecting to investigate what mental processes are involved when actors decide to perform an eye movement to trigger a gaze response in another person. Furthermore, eye movements are also used to affect the non-social environment, for instance when unlocking the smartphone with the help of the eyes. This and other observations demonstrate the necessity to consider gaze control in contexts other than social communication whilst at the same time focusing on commonalities and differences inherent to the nature of a social (vs. non-social) action context. Thus, the present work explores the cognitive mechanisms that control such goal-oriented eye movements in both social and non-social contexts. The experiments presented throughout this work are built on pre-established paradigms from both the oculomotor research domain and from basic cognitive psychology. These paradigms are based on the principle of ideomotor action control, which provides an explanatory framework for understanding how goal-oriented, intentional actions come into being. The ideomotor idea suggests that humans acquire associations between their actions and the resulting effects, which can be accessed in a bi-directional manner: Actions can trigger anticipations of their effects, but the anticipated resulting effects can also trigger the associated actions. According to ideomotor theory, action generation involves the mental anticipation of the intended effect (i.e., the action goal) to activate the associated motor pattern. The present experiments involve situations where participants control the gaze of a virtual face via their eye movements. The triggered gaze responses of the virtual face are consistent to the participant's eye movements, representing visual action effects. Experimental situations are varied with respect to determinants of action-effect learning (e.g., contingency, contiguity, action mode during acquisition) in order to unravel the underlying dynamics of oculomotor control in these situations. In addition to faces, conditions involving changes in non-social objects were included to address the question of whether mechanisms underlying gaze control differ for social versus non-social context situations. The results of the present work can be summarized into three major findings. 1. My data suggest that humans indeed acquire bi-directional associations between their eye movements and the subsequently perceived gaze response of another person, which in turn affect oculomotor action control via the anticipation of the intended effects. The observed results show for the first time that eye movements in a gaze-interaction scenario are represented in terms of their gaze response in others. This observation is in line with the ideomotor theory of action control. 2. The present series of experiments confirms and extends pioneering results of Huestegge and Kreutzfeldt (2012) with respect to the significant influence of action effects in gaze control. I have shown that the results of Huestegge and Kreutzfeldt (2012) can be replicated across different contexts with different stimulus material given that the perceived action effects were sufficiently salient. 3. Furthermore, I could show that mechanisms of gaze control in a social gaze-interaction context do not appear to be qualitatively different from those in a non-social context. All in all, the results support recent theoretical claims emphasizing the role of anticipation-based action control in social interaction. Moreover, my results suggest that anticipation-based gaze control in a social context is based on the same general psychological mechanisms as ideomotor gaze control, and thus should be considered as an integral part rather than as a special form of ideomotor gaze control.},
  subject      = {Verhaltenskontrolle},
  language  = {en}
}
@phdthesis{Kaymak2019,
  author    = {Kaymak, Irem},
  title     = {Identification of metabolic liabilities in 3D models of cancer},
  doi       = {10.25972/OPUS-18154},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-181544},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Inefficient vascularisation of solid tumours leads to the formation of oxygen and nutrient gradients. In order to mimic this specific feature of the tumour microenvironment, a multicellular tumour spheroid (SPH) culture system was used. These experiments were implemented in p53 isogenic colon cancer cell lines (HCT116 p53 +/+ and HCT116 p53-/-) since Tp53 has important regulatory functions in tumour metabolism. First, the characteristics of the cells cultured as monolayers and as spheroids were investigated by using RNA sequencing and metabolomics to compare gene expression and metabolic features of cells grown in different conditions. This analysis showed that certain features of gene expression found in tumours are also present in spheroids but not in monolayer cultures, including reduced proliferation and induction of hypoxia related genes. Moreover, comparison between the different genotypes revealed that the expression of genes involved in cholesterol homeostasis is induced in p53 deficient cells compared to p53 wild type cells and this difference was only detected in spheroids and tumour samples but not in monolayer cultures. In addition, it was established that loss of p53 leads to the induction of enzymes of the mevalonate pathway via activation of the transcription factor SREBP2, resulting in a metabolic rewiring that supports the generation of ubiquinone (coenzyme Q10). An adequate supply of ubiquinone was essential to support mitochondrial electron transport and pyrimidine biosynthesis in p53 deficient cancer cells under conditions of metabolic stress. Moreover, inhibition of the mevalonate pathway using statins selectively induced oxidative stress and apoptosis in p53 deficient colon cancer cells exposed to oxygen and nutrient deprivation. This was caused by ubiquinone being required for electron transfer by dihydroorotate dehydrogenase, an essential enzyme of the pyrimidine nucleotide biosynthesis pathway. Supplementation with exogenous nucleosides relieved the demand for electron transfer and restored viability of p53 deficient cancer cells under metabolic stress. Moreover, the mevalonate pathway was also essential for the synthesis of ubiquinone for nucleotide biosynthesis to support growth of intestinal tumour organoids. Together, these findings highlight the importance of the mevalonate pathway in cancer cells and provide molecular evidence for an enhanced sensitivity towards the inhibition of mitochondrial electron transfer in tumour-like metabolic environments.},
  subject      = {Tumor},
  language  = {en}
}
@phdthesis{MonyNair2021,
  author    = {Mony Nair, Rahul},
  title     = {Elucidating ubiquitin recognition by the HECT-type ubiquitin ligase HUWE1},
  doi       = {10.25972/OPUS-22103},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-221030},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {The small protein modifier ubiquitin is at the heart of an immensely versatile posttranslational modification system that orchestrates countless physiological and disease-associated cellular processes. Key to this versatility are the manifold modifications that can be assembled from ubiquitin "building blocks" and are associated with specific functional outcomes for the modified substrates. In particular, ubiquitin molecules can form polymeric chains of distinct lengths and linkage types that give rise to distinct chain conformations, thereby providing recognition sites for specific signaling receptors/effectors. The class of E3 enzymes (ubiquitin ligases) provides critical specificity determinants in ubiquitin linkage formation; it is therefore crucial to unravel precisely how E3 enzymes operate in order to understand the structural basis of ubiquitin signaling and exploit these insights for therapeutic benefit. Overexpression and deregulation of the HECT-type ubiquitin ligase HUWE1 is implicated in several different cancer types and neurodegenerative disorders. It is largely unknown which factors control the ubiquitin modifications formed by HUWE1, how the catalytic HECT domain interacts with functionally distinct ubiquitin molecules (donor, acceptor and regulatory ubiquitin molecules) and which conformational transitions enable these interactions during ubiquitin chain formation. One aim of this study was to structurally elucidate the recognition of donor ubiquitin by the HECT domain of HUWE1. To this end I utilized a ubiquitin activity-based probe to reconstitute a proxy for a donor ubiquitin-linked conjugate of the HECT domain of HUWE1 and determined its structure by X-ray crystallography. This structure reveals that the donor ubiquitin binds to the C-lobe of HUWE1 in the same way as NEDD4-type ligases, corroborating the idea that HECT ligases utilize a conserved mode of donor ubiquitin recognition. independent of their linkage and substrate specificities. With the help of biochemical analyses, I also validated specific features of the structure, in particular the positioning of the C-terminal tail of the ligase, which was known to be critical for activity. In the newly determined structure, which reflects an "L-shaped", active state of the HECT domain, this tail is fully resolved and coordinated at the N-lobe-C-lobe interface. I defined residues that are critical for this coordination and showed that they are also essential for the activity of HUWE1, including auto-ubiquitination, free ubiquitin chain formation, and substrate ubiquitination. Furthermore, I discovered that the N-lobe of HUWE1 harbors a ubiquitin-binding exosite similar to NEDD4-type ligases and E6AP. My in-vitro activity and binding assays show that HUWE1 uses the exosite for isopeptide bond formation, but that it is dispensable for thioester bond formation. The binding assays further show that the donor ubiquitin loaded HECT domain binds an additional ubiquitin molecule at the exosite more tightly than the apo HECT domain, which possibly suggests allosteric communication between the two sites. Finally, I showed that the ubiquitin activity-based probe (ubiquitin-propargylamine) can label the catalytic cysteine of HUWE1 and NEDD4-type with close to quantitative turn- over, while it does not react with the HECT domain of the evolutionarily more divergent E6AP. The determinants underlying these differential reactivities remain to be explored. Taken, together my results significantly enhance our mechanistic understanding of the catalytic domain of HUWE1 and pinpoint linchpins for therapeutic interventions with the activity of this disease-relevant enzyme.},
  language  = {en}
}
@phdthesis{Gorelashvili2019,
  author    = {Gorelashvili, Maximilian Georg},
  title     = {Investigation of megakaryopoiesis and the acute phase of ischemic stroke by advanced fluorescence microscopy},
  doi       = {10.25972/OPUS-18600},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-186002},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {In mammals, anucleate platelets circulate in the blood flow and are primarily responsible for maintaining functional hemostasis. Platelets are generated in the bone marrow (BM) by megakaryocytes (MKs), which mainly reside directly next to the BM sinusoids to release proplatelets into the blood. MKs originate from hematopoietic stem cells and are thought to migrate from the endosteal to the vascular niche during their maturation, a process, which is, despite being intensively investigated, still not fully understood. Long-term intravital two photon microscopy (2PM) of MKs and vasculature in murine bone marrow was performed and mean squared displacement analysis of cell migration was performed. The MKs exhibited no migration, but wobbling-like movement on time scales of 3 h. Directed cell migration always results in non-random spatial distribution. Thus, a computational modelling algorithm simulating random MK distribution using real 3D light-sheet fluorescence microscopy data sets was developed. Direct comparison of real and simulated random MK distributions showed, that MKs exhibit a strong bias to vessel-contact. However, this bias is not caused by cell migration, as non-vessel-associated MKs were randomly distributed in the intervascular space. Furthermore, simulation studies revealed that MKs strongly impair migration of other cells in the bone marrow by acting as large-sized obstacles. MKs are thought to migrate from the regions close to the endosteum towards the vasculature during their maturation process. MK distribution as a function of their localization relative to the endosteal regions of the bones was investigated by light sheet fluorescence microscopy (LSFM). The results show no bone-region dependent distribution of MKs. Taken together, the newly established methods and obtained results refute the model of MK migration during their maturation. Ischemia reperfusion (I/R) injury is a frequent complication of cerebral ischemic stroke, where brain tissue damage occurs despite successful recanalization. Platelets, endothelial cells and immune cells have been demonstrated to affect the progression of I/R injury in experimental mouse models 24 h after recanalization. However, the underlying Pathomechanisms, especially in the first hours after recanalization, are poorly understood. Here, LSFM, 2PM and complemental advanced image analysis workflows were established for investigation of platelets, the vasculature and neutrophils in ischemic brains. Quantitative analysis of thrombus formation in the ipsilateral and contralateral hemispheres at different time points revealed that platelet aggregate formation is minimal during the first 8 h after recanalization and occurs in both hemispheres. Considering that maximal tissue damage already is present at this time point, it can be concluded that infarct progression and neurological damage do not result from platelet aggregated formation. Furthermore, LSFM allowed to confirm neutrophil infiltration into the infarcted hemisphere and, here, the levels of endothelial cell marker PECAM1 were strongly reduced. However, further investigations must be carried out to clearly identify the role of neutrophils and the endothelial cells in I/R injury.},
  subject      = {Fluoreszenzmikroskopie},
  language  = {en}
}
@phdthesis{Flechsenhar2019,
  author    = {Flechsenhar, Aleya Felicia},
  title     = {The Ubiquity of Social Attention - a Detailed Investigation of the Underlying Mechanisms},
  doi       = {10.25972/OPUS-18452},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-184528},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {This dissertation highlights various aspects of basic social attention by choosing versatile approaches to disentangle the precise mechanisms underlying the preference to focus on other human beings. The progressive examination of different social processes contrasted with aspects of previously adopted principles of general attention. Recent research investigating eye movements during free exploration revealed a clear and robust social bias, especially for the faces of depicted human beings in a naturalistic scene. However, free viewing implies a combination of mechanisms, namely automatic attention (bottom-up), goal-driven allocation (top-down), or contextual cues and inquires consideration of overt (open exploration using the eyes) as well as covert orienting (peripheral attention without eye movement). Within the scope of this dissertation, all of these aspects have been disentangled in three studies to provide a thorough investigation of different influences on social attention mechanisms. In the first study (section 2.1), we implemented top-down manipulations targeting non-social features in a social scene to test competing resources. Interestingly, attention towards social aspects prevailed, even though this was detrimental to completing the requirements. Furthermore, the tendency of this bias was evident for overall fixation patterns, as well as fixations occurring directly after stimulus onset, suggesting sustained as well as early preferential processing of social features. Although the introduction of tasks generally changes gaze patterns, our results imply only subtle variance when stimuli are social. Concluding, this experiment indicates that attention towards social aspects remains preferential even in light of top-down demands. The second study (section 2.2) comprised of two separate experiments, one in which we investigated reflexive covert attention and another in which we tested reflexive as well as sustained overt attention for images in which a human being was unilaterally located on either the left or right half of the scene. The first experiment consisted of a modified dot-probe paradigm, in which peripheral probes were presented either congruently on the side of the social aspect, or incongruently on the non-social side. This was based on the assumption that social features would act similar to cues in traditional spatial cueing paradigms, thereby facilitating reaction times for probes presented on the social half as opposed to the non-social half. Indeed, results reflected such congruency effect. The second experiment investigated these reflexive mechanisms by monitoring eye movements and specifying the location of saccades and fixations for short as well as long presentation times. Again, we found the majority of initial saccades to be congruently directed to the social side of the stimulus. Furthermore, we replicated findings for sustained attention processes with highest fixation densities for the head region of the displayed human being. The third study (section 2.3), tackled the other mechanism proposed in the attention dichotomy, the bottom-up influence. Specifically, we reduced the available contextual information of a scene by using a gaze-contingent display, in which only the currently fixated regions would be visible to the viewer, while the remaining image would remain masked. Thereby, participants had to voluntarily change their gaze in order to explore the stimulus. First, results revealed a replication of a social bias in free-viewing displays. Second, the preference to select social features was also evident in gaze-contingent displays. Third, we find higher recurrent gaze patterns for social images compared to non-social ones for both viewing modalities. Taken together, these findings imply a top-down driven preference for social features largely independent of contextual information. Importantly, for all experiments, we took saliency predictions of different computational algorithms into consideration to ensure that the observed social bias was not a result of high physical saliency within these areas. For our second experiment, we even reduced the stimulus set to those images, which yielded lower mean and peak saliency for the side of the stimulus containing the social information, while considering algorithms based on low-level features, as well as pre-trained high-level features incorporated in deep learning algorithms. Our experiments offer new insights into single attentional mechanisms with regard to static social naturalistic scenes and enable a further understanding of basic social processing, contrasting from that of non-social attention. The replicability and consistency of our findings across experiments speaks for a robust effect, attributing social attention an exceptional role within the general attention construct, not only behaviorally, but potentially also on a neuronal level and further allowing implications for clinical populations with impaired social functioning.},
  subject      = {Aufmerksamkeit},
  language  = {en}
}
@phdthesis{Herrmann2019,
  author    = {Herrmann, Johannes Bernd},
  title     = {Rolle des Komplement C5a-Rezeptors 1 in der Pathophysiologie der Meningokokken-Sepsis},
  doi       = {10.25972/OPUS-18453},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-184533},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Das bekapselte, Gram-negative, diplokokkenf{\"o}rmige Bakterium Neisseria meningitidis (Nme) ist ein asymptomatischer Kommensale des oberen Nasenrachenraums im Men-schen. Gerade bei Kindern ist es dem humanspezifischen Pathogen in seltenen F{\"a}llen m{\"o}glich, in den Blutstrom einzuwandern und lebensbedrohliche Krankheitsbilder wie Meningoenzephalitis und Sepsis auszul{\"o}sen, welche als „Invasive Meningokokkener-krankung" (IMD) zusammengefasst werden. J{\"a}hrlich ereignen sich weltweit bis zu 1,2 Mio F{\"a}lle von IMD, welche aufgrund des fulminanten Verlaufs und der hohen Letalit{\"a}t gef{\"u}rchtet sind. In der Bek{\"a}mpfung der Nme-Sepsis ist das humane Komplementsystem von entscheidender Bedeutung. Vor diesem Hintergrund ist die protektive Rolle des lytischen (Membranangriffskomplex MAK) und opsonisierenden Arms (Opsonine iC3b und C1q) der Komplementkaskade gut dokumentiert. Dagegen ist der Beitrag des in-flammatorischen Arms (Anaphylatoxine C3a und C5a) in der Nme-Sepsis bisher unklar. Aus diesem Grunde wurde mit dieser Arbeit die Rolle des inflammatorischen Arms an-hand des Komplement C5a-Rezeptors 1 (C5aR1) in der Pathophysiologie der Nme-Sepsis am Mausmodell untersucht. Nach Etablierung des murinen, intraperitonealen In-fektionsmodells konnte ein sch{\"a}dlicher Effekt des C5aR1 in der Nme-Sepsis beobachtet werden. Aus der Abwesenheit des C5aR1 resultierte eine h{\"o}here {\"U}berlebensrate, ein besserer klinischer Zustand, eine niedrigere Bakteri{\"a}mie und niedrigere Konzentrationen der pro-inflammatorischen Mediatoren IL-6, CXCL-1 und TNF-α. Im Hinblick auf den zellul{\"a}ren Pathomechanismus sprechen Ergebnisse dieser Arbeit daf{\"u}r, dass der C5aR1 prim{\"a}r eine gesteigerte Freisetzung inflammatorischer Mediatoren durch verschiedene Zellpopulationen triggert (Zytokinsturm), wodurch sekund{\"a}r Zellparalyse, steigende Bakteri{\"a}mie und h{\"o}here Letalit{\"a}t bedingt sind. Durch Depletionsversuche und Immun-fluoreszenzf{\"a}rbungen konnte, unabh{\"a}ngig vom C5aR1, eine allgemein protektive Rolle von neutrophilen Granulozyten und Monozyten/Makrophagen in der Nme-Sepsis beo-bachtet werden. Dar{\"u}ber hinaus pr{\"a}sentierte sich der zyklische C5aR1-Antagonist PMX205 als erfolgsversprechende Therapieoption, um Parameter einer murinen Nme-Sepsis zu verbessern. Weitere Untersuchungen sind n{\"o}tig, um die Wirksamkeit dieser Substanz in der humanen Nme-Sepsis zu erforschen. Zudem k{\"o}nnte das murine, intrape-ritoneale Infektionsmodell zur Kl{\"a}rung der Rolle des C5aR2 in der Nme-Sepsis genutzt werden.},
  subject      = {Komplement C5a},
  language  = {de}
}
@phdthesis{Koenig2019,
  author    = {K{\"o}nig, Eva-Maria},
  title     = {Pathogenese von Kraniosynostosen},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-175181},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Das humane Sch{\"a}deldach besteht aus f{\"u}nf Sch{\"a}delplatten, die durch intramembran{\"o}se Ossifikation entstehen. Wenn diese in der Embryonalentwicklung aufeinandertreffen, bilden sich Sch{\"a}deln{\"a}hte aus, die eine Fusion der Sch{\"a}delplatten verhindern und damit ein Sch{\"a}delwachstum parallel zu Gehirnentwicklung erm{\"o}glichen. F{\"u}r diesen Prozess ist eine Balance aus Zellproliferation und Differenzierung n{\"o}tig, deren Aufrechterhaltung wiederum durch eine komplexe Regulation von verschiedenen Signalwegen gew{\"a}hrleistet wird. St{\"o}rungen in diesem regulatorischen System k{\"o}nnen zu einer vorzeitigen Fusion der Sch{\"a}delplatten, Kraniosynostose genannt, f{\"u}hren. Die Kraniosynostose ist eine der h{\"a}ufigsten kraniofazialen Fehlbildungen beim Menschen. Durch kompensatorisches Wachstum an den nicht fusionierten Suturen entstehen charakteristische Sch{\"a}deldeformationen, die sekund{\"a}r einen erh{\"o}hten intrakranialen Druck zur Folge haben k{\"o}nnen. Eine vorzeitige Fusion der Suturen kann sowohl isoliert als auch syndromal zusammen mit weiteren klinischen Auff{\"a}lligkeiten vorliegen. Bisher sind {\"u}ber 150 verschiedene Kraniosynostose Syndrome beschrieben und insgesamt 25-30\% aller Kraniosynostose Patienten sind von einer syndromalen Form betroffen. Da die klinischen Merkmale der Kraniosynostose Syndrome variabel sind und zum Teil {\"u}berlappen, ist eine klare klinische Diagnose h{\"a}ufig erschwert. Sowohl Umwelteinfl{\"u}sse als auch genetische Ver{\"a}nderungen k{\"o}nnen die Ursache f{\"u}r Kraniosynostosen sein. Vor allem bei syndromalen Kraniosynostosen wurden genetische Ver{\"a}nderungen, wie beispielsweise Mutationen in den Genen FGFR2, FGFR3, TWIST1 und EFNB1, identifiziert. Dar{\"u}ber hinaus wurden chromosomale Ver{\"a}nderungen wie partielle Monosomien von 7p, 9p oder 11p sowie partielle Trisomien von 5q, 13q oder 15q mit Kraniosynostose assoziiert. Trotzdem ist in {\"u}ber 50\% der F{\"a}lle die genetische Ursache unbekannt und die Pathogenese von Kraniosynostosen noch nicht vollst{\"a}ndig gekl{\"a}rt. Ziel dieser Arbeit war es neue genetische Ursachen bei Kraniosynostose Patienten zu identifizieren und so zur Aufkl{\"a}rung der Pathogenese beizutragen. Es wurde die genomische DNA von 83 Patienten molekulargenetisch durch Mikroarray basierte vergleichende Genomhybridisierung (Array-CGH) oder durch ein speziell entworfenes Next Generation Sequencing (NGS) Genpanel untersucht. Bei 30\% der Patienten konnte eine potentiell pathogene Ver{\"a}nderung identifiziert werden. Davon waren 23\% chromosomale Aberrationen wie unbalancierte Translokationen, isolierte interstitielle Verluste und ein Zugewinn an genomischen Material. Bei zwei Patienten wurden unbalancierte Translokationen mit partieller 5q Trisomie nachgewiesen. Das Gen MSX2 liegt innerhalb des duplizierten Bereichs, sodass m{\"o}glicherweise eine MSX2 {\"U}berexpression vorliegt. F{\"u}r ein normales Sch{\"a}delwachstum ist jedoch die richtige Menge an MSX2 kritisch. Des Weiteren wurde eine partielle Deletion von TCF12 detektiert, die in einer Haploinsuffizienz von TCF12 resultiert. TCF12 Mutationen sind mit Koronarnahtsynosten assoziiert. In einem anderen Fall lag das Gen FGF10 innerhalb der duplizierten 5p15.1-p12 Region. Das Gen kodiert f{\"u}r einen Liganden des FGF Signalwegs und wurde bisher noch nicht mit Kraniosynostose assoziiert. Aufgrund dessen wurden Analysen im Tiermodell Danio rerio durchgef{\"u}hrt. Eine simulierte {\"U}berexpression durch Injektion der fgf10a mRNA in das 1-Zell Stadium f{\"u}hrte zu schweren Gehirn-, Herz- und Augendefekten. Mittels NGS wurden 77\% der potentiell pathogenen genetischen Ver{\"a}nderungen identifiziert. Hierf{\"u}r wurde in dieser Arbeit ein Genpanel erstellt, das 68 Gene umfasst. Es wurden sowohl bekannte Kraniosynostose- als auch Kandidaten-Gene sowie Gene, die mit der Ossifikation assoziiert sind, in die Analyse eingeschlossen. Das Genpanel wurde durch die Sequenzierung von f{\"u}nf Kontrollproben mit bekannten Mutationen erfolgreich validiert. Anschließend wurde die genomische DNA von 66 Patienten analysiert. Es konnten 20 (potentiell) pathogene Varianten identifiziert werden. Neben bereits bekannten Mutationen in den Genen FGFR1, FGFR2, FGFR3 und TWIST1, konnten zus{\"a}tzlich 8 neue, potentiell pathogene Varianten in den Genen ERF, MEGF8, MSX2, PTCH1 und TCF12 identifiziert werden. Die Ergebnisse dieser Arbeit tragen dazu bei das Mutationsspektrum dieser Gene zu erweitern. Bei zwei der Varianten handelte es sich um potentielle Spleißvarianten. F{\"u}r diese konnte in einem in vitro Spleißsystem gezeigt werden, dass sie eine {\"A}nderung des Spleißmusters bewirken. Der Nachweis von zwei seltenen Varianten in den Genen FGFR2 und HUWE1 hat außerdem dazu beigetragen die Pathogenit{\"a}t dieser spezifischen Varianten zu bekr{\"a}ftigen. Eine Variante in POR, die aufgrund bioinformatischer Analysen als potentiell pathogen bewertet wurde, wurde nach der Segregationsanalyse als wahrscheinlich benigne eingestuft. Zusammenfassend konnten bei etwa einem Drittel der Patienten, die mit dem NGS Genpanel analysiert wurden, eine genetische Ursache identifiziert werden. Dieses Genpanel stellt somit ein effizientes diagnostisches Tool dar, das zuk{\"u}nftig in der genetischen Routine-Diagnostik von Kraniosynostose-Patienten eingesetzt werden kann. Die Ergebnisse dieser Arbeit zeigen, dass sowohl eine Untersuchung auf CNVs als auch auf Sequenz{\"a}nderungen bei Kraniosynostose Patienten sinnvoll ist.},
  subject      = {Kraniosynostose},
  language  = {de}
}
@phdthesis{Tiwarekar2019,
  author    = {Tiwarekar, Vishakha Rakesh},
  title     = {The APOBEC3G-regulated host factors REDD1 and KDELR2 restrict measles virus replication},
  doi       = {10.25972/OPUS-17952},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-179526},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Measles is an extremely contagious vaccine-preventable disease responsible for more than 90000 deaths worldwide annually. The number of deaths has declined from 8 million in the pre-vaccination era to few thousands every year due to the highly efficacious vaccine. However, this effective vaccine is still unreachable in many developing countries due to lack of infrastructure, while in developed countries too many people refuse vaccination. Specific antiviral compounds are not yet available. In the current situation, only an extensive vaccination approach along with effective antivirals could help to have a measles-free future. To develop an effective antiviral, detailed knowledge of viral-host interaction is required. This study was undertaken to understand the interaction between MV and the innate host restriction factor APOBEC3G (A3G), which is well-known for its activity against human immunodeficiency virus (HIV). Restriction of MV replication was not attributed to the cytidine deaminase function of A3G, instead, we identified a novel role of A3G in regulating cellular gene functions. Among two of the A3G regulated host factors, we found that REDD1 reduced MV replication, whereas, KDELR2 hampered MV haemagglutinin (H) surface transport thereby affecting viral release. REDD1, a negative regulator of mTORC1 signalling impaired MV replication by inhibiting mTORC1. A3G regulated REDD1 expression was demonstrated to inversely correlate with MV replication. siRNA mediated silencing of A3G in primary human blood lymphocytes (PBL) reduced REDD1 levels and simultaneously increased MV titres. Also, direct depletion of REDD1 improved MV replication in PBL, indicating its role in A3G mediated restriction of MV. Based on these finding, a new role of rapamycin, a pharmacological inhibitor of mTORC1, was uncovered in successfully diminishing MV replication in Vero as well as in human PBL. The ER and Golgi resident receptor KDELR2 indirectly affected MV by competing with MV-H for cellular chaperones. Due to the sequestering of chaperones by KDELR2, they can no longer assist in MV-H folding and subsequent surface expression. Taken together, the two A3G-regulated host factors REDD1 and KDELR2 are mainly responsible for mediating its antiviral activity against MV.},
  language  = {en}
}
@phdthesis{Reutter2019,
  author    = {Reutter, Mario},
  title     = {Biologische Marker f{\"u}r Aufmerksamkeitsverzerrungen bei sozialer {\"A}ngstlichkeit und deren Modifikation},
  doi       = {10.25972/OPUS-17870},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-178706},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Diese Dissertationsschrift besch{\"a}ftigt sich mit biologischen Korrelaten von Aufmerksamkeits-verzerrungen und eruiert deren Modifikation in einem l{\"a}ngsschnittlich angelegten Experiment. Hierf{\"u}r wurden {\"u}ber 100 sozial-{\"a}ngstliche Teilnehmer mit Hilfe einer Screening-Prozedur gewonnen und hinsichtlich der Auspr{\"a}gung einer ereigniskorrelierten Lateralisation namens „N2pc" untersucht. W{\"a}hrend der ersten Labormessung indizierte die N2pc bei der Bearbeitung eines Dot Probe Paradigmas einen mittelgroßen, statistisch hochbedeutsamen Attentional Bias hin zu w{\"u}tenden Gesichtern im Vergleich zu neutralen. Das hierf{\"u}r klassischerweise verwendete Maß von Reaktionszeitunterschieden hingegen konnte diese Verzerrung der Aufmerksamkeit nicht abbilden. Ferner zeigten weder die elektrophysiologische noch die behaviorale Messgr{\"o}ße einen Zusammenhang mit Frageb{\"o}gen sozialer Angst, was teilweise auf ein Fehlen interner Konsistenz zur{\"u}ckgef{\"u}hrt werden kann. Im weiteren Verlauf absolvierten die {\"u}berwiegend weiblichen Teilnehmer an acht unterschiedlichen Terminen {\"u}ber zwei bis vier Wochen fast 7000 Durchg{\"a}nge eines Aufmerksamkeitsverzerrungsmodifikationstrainings oder einer aktiven Kontrollprozedur. Daraufhin zeigte sich eine Ausl{\"o}schung der ereigniskorrelierten Lateralisation, allerdings in einem sp{\"a}teren Zeitfenster als erwartet. Dieses Verschwinden des Attentional Bias blieb bis elf Wochen nach Ende der Trainingsprozedur stabil. Außerdem trat dieselbe Modifikation ebenfalls f{\"u}r die Kontrollgruppe auf. Die selbstberichtete Schwere der Symptomauspr{\"a}gung ver{\"a}nderte sich zwar nicht, allerdings konnte eine Reduktion des Pers{\"o}nlichkeitsmerkmals Neurotizismus verzeichnet werden, welches konzeptuell mit dem Begriff der {\"A}ngstlichkeit eng verwoben ist. Durch explorative Folgeanalysen konnte eine st{\"a}rkere Modulation der rechten Großhirnh{\"a}lfte, also durch Reize im linken visuellen Halbfeld aufgedeckt werden. Eine Neuberechnung des Attentional Bias separat f{\"u}r jede Hemisph{\"a}re scheint daher auch f{\"u}r k{\"u}nftige Untersuchungen angebracht. Ferner wurde als Tr{\"a}ger der Modifikation {\"u}ber die Zeit eine Ver{\"a}nderung der Hyperpolarisation nach der N2-Komponente identifiziert. Ob durch eine Anpassung der Prozedur eine Modulation einer fr{\"u}heren ereigniskorrelierten Komponente erzielt werden kann, bleibt zum aktuellen Zeitpunkt unbeantwortet.},
  subject      = {Attention},
  language  = {de}
}
@phdthesis{Anany2019,
  author    = {Anany, Mohamed Ahmed Mohamed Mohamed},
  title     = {Enhancement of Toll-like receptor3 (TLR3)-induced death signaling by TNF-like weak inducer of apoptosis (TWEAK)},
  doi       = {10.25972/OPUS-18975},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-189757},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) is a member of the TNF superfamily (TNFSF) and is as such initially expressed as type II class transmembrane glycoprotein from which a soluble ligand form can be released by proteolytic processing. While the expression of TWEAK has been detected at the mRNA level in various cell lines and cell types, its cell surface expression has so far only been documented for dendritic cells, monocytes and interferon-γ stimulated NK cells. The fibroblast growth factor-inducible-14 (Fn14) is a TRAF2-interacting receptor of the TNF receptor superfamily (TNFRSF) and is the only receptor for TWEAK. The expression of Fn14 is strongly induced in a variety of non-hematopoietic cell types after tissue injury. The TWEAK/Fn14 system induces pleiotropic cellular activities such as induction of proinflammatory genes, stimulation of cellular angiogenesis, proliferation, differentiation, migration and in rare cases induction of apoptosis. On the other side, Toll-like receptor3 (TLR3) is one of DNA- and RNA-sensing pattern recognition receptors (PRRs), plays a crucial role in the first line of defense against virus and invading foreign pathogens and cancer cells. Polyinosinic-polycytidylic acid poly(I:C) is a synthetic analog of dsRNA, binds to TLR3 which acts through the adapter TRIF/TICAM1, leading to cytokine secretion, NF-B activation, IRF3 nuclear translocation, inflammatory response and may also elicit the cell death. TWEAK sensitizes cells for TNFR1-induced apoptosis and necroptosis by limiting the availability of protective TRAF2-cIAP1 and TRAF2-cIAP2 complexes, which interact with the TNFR1-binding proteins TRADD and RIPK1. In accordance with the fact that poly(I:C)-induced signaling also involves these proteins, we found enhanced necroptosis-induction in HaCaT and HeLa-RIPK3 by poly(I:C) in the presence of TWEAK (Figure 24). Analysis of a panel of TRADD, FADD, RIPK1 and caspase-8 knockout cells revealed furthermore similarities and differences in the way how these molecules act in cell death signaling by poly(I:C)/TWEAK and TNF and TRAIL. RIPK1 turned out to be essential for poly(I:C)/TWEAK-induced caspase-8-mediated apoptosis but was dispensable for these responses in TNF and TRAIL signaling. Lack of FADD protein abrogated TRAIL- but not TNF- and poly(I:C)-induced necroptosis. Moreover, we observed that both long and short FLIP rescued HaCaT and HeLa-RIPK3 cells from poly(I:C)-induced apoptosis or necroptosis. To sum up, our results demonstrate that TWEAK, which is produced by interferon stimulated myeloid cells, controls the induction of apoptosis and necroptosis by the TLR3 ligand poly(I:C) and may thus contribute to cancer or anti-viral immunity treatment.},
  subject      = {Immunologe},
  language  = {en}
}
@phdthesis{Ji2022,
  author    = {Ji, Changhe},
  title     = {The role of 7SK noncoding RNA in development and function of motoneurons},
  doi       = {10.25972/OPUS-22463},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-224638},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {In mammals, a major fraction of the genome is transcribed as non-coding RNAs. An increasing amount of evidence has accumulated showing that non-coding RNAs play important roles both for normal cell function and in disease processes such as cancer or neurodegeneration. Interpreting the functions of non-coding RNAs and the molecular mechanisms through which they act is one of the most important challenges facing RNA biology today. In my Ph.D. thesis, I have been investigating the role of 7SK, one of the most abundant non-coding RNAs, in the development and function of motoneurons. 7SK is a highly structured 331 nt RNA transcribed by RNA polymerase III. It forms four stem-loop (SL) structures that serve as binding sites for different proteins. Larp7 binds to SL4 and protects the 3' end from exonucleolytic degradation. SL1 serves as a binding site for HEXIM1, which recruits the pTEFb complex composed of CDK9 and cyclin T1. pTEFb has a stimulatory role for transcription and is regulated through sequestration by 7SK. More recently, a number of heterogeneous nuclear ribonucleoproteins (hnRNPs) have been identified as 7SK interactors. One of these is hnRNP R, which has been shown to have a role in motoneuron development by regulating axon growth. Taken together, 7SK's function involves interactions with RNA binding proteins, and different RNA binding proteins interact with different regions of 7SK, such that 7SK can be considered as a hub for recruitment and release of different proteins. The questions I have addressed during my Ph.D. are as follows: 1) which region of 7SK interacts with hnRNP R, a main interactor of 7SK? 2) What effects occur in motoneurons after the protein binding sites of 7SK are abolished? 3) Are there additional 7SK binding proteins that regulate the functions of the 7SK RNP? Using in vitro and in vivo experiments, I found that hnRNP R binds both the SL1 and SL3 region of 7SK, and also that pTEFb cannot be recruited after deleting the SL1 region but is able to bind to a 7SK mutant with deletion of SL3. In order to answer the question of how the 7SK mutations affect axon outgrowth and elongation in mouse primary motoneurons, we proceeded to conduct rescue experiments in motoneurons by using lentiviral vectors. The constructs were designed to express 7SK deletion mutants under the mouse U6 promoter and at the same time to drive expression of a 7SK shRNA from an H1 promoter for the depletion of endogenous 7SK. Using this system we found that 7SK mutants harboring deletions of either SL1 or SL3 could not rescue the axon growth defect of 7SK-depleted motoneurons suggesting that 7SK/hnRNP R complexes are integral for this process. In order to identify novel 7SK binding proteins and investigate their functions, I proceeded to conduct pull-down experiments by using a biotinylated RNA antisense oligonucleotide that targets the U17-C33 region of 7SK thereby purifying endogenous 7SK complexes. Following mass spectrometry of purified 7SK complexes, we identified a number of novel 7SK interactors. Among these is the Smn complex. Deficiency of the Smn complex causes the motoneuron disease spinal muscular atrophy (SMA) characterized by loss of lower motoneurons in the spinal cord. Smn has previously been shown to interact with hnRNP R. Accordingly, we found Smn as part of 7SK/hnRNP R complexes. These proteomics data suggest that 7SK potentially plays important roles in different signaling pathways in addition to transcription.},
  subject      = {Spliceosome},
  language  = {en}
}
@phdthesis{Turakhiya2019,
  author    = {Turakhiya, Ankit},
  title     = {Functional characterization of the role of ZFAND1 in stress granule turnover},
  doi       = {10.25972/OPUS-16375},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-163751},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Protein quality control systems are critical for cellular proteostasis and survival under stress conditions. The ubiquitin proteasome system (UPS) plays a pivotal role in proteostasis by eliminating misfolded and damaged proteins. However, exposure to the environmental toxin arsenite results in the accumulation of polyubiquitylated proteins, indicating an overload of the UPS. Arsenite stress induces the rapid formation of stress granules (SGs), which are cytoplasmic assemblies of mRNPs stalled in translation initiation. The mammalian proteins ZFAND2A/B (also known as AIRAP and AIRAPL, respectively) bind to the 26S proteasome, and ZFAND2A has been shown to adapt proteasome activity to arsenite stress. They belong to a small subfamily of AN1 type zinc finger containing proteins that also comprises the unexplored mammalian member ZFAND1 and its yeast homolog Cuz1. In this thesis, the cellular function of Cuz1 and ZFAND1 was investigated. Cuz1/ZFAND1 was found to interact with the ubiquitin-selective, chaperone-like ATPase Cdc48/p97 and with the 26S proteasome. The interaction between Cuz1/ZFAND1 and Cdc48/p97 requires a predicted ubiquitin-like domain of Cuz1/ZFAND1. In vivo, this interaction was strongly dependent on acute arsenite stress, suggesting that it is a part of the cellular arsenite stress response. Lack of Cuz1/ZFAND1 caused a defect in the clearance of arsenite induced SG clearance. ZFAND1 recruits both, the 26S proteasome and p97, to arsenite-induced SGs for their normal clearance. In the absence of ZFAND1, SGs lack the 26S proteasome and p97, accumulate defective ribosomal products and become aberrant. These aberrant SGs persist after arsenite removal and undergo degradation via autophagy. ZFAND1 depletion is epistatic to the expression of pathogenic mutant p97 with respect to SG clearance, suggesting that ZFAND1 function is relevant to the multisystem degenerative disorder, inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia and amyotrophic lateral sclerosis (IBMPFD/ALS).},
  subject      = {ubiquitin},
  language  = {en}
}
@phdthesis{Nieberler2019,
  author    = {Nieberler, Matthias},
  title     = {The physiological role of autoproteolysis of the Adhesion GPCR Latrophilin/dCIRL},
  doi       = {10.25972/OPUS-16589},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-165894},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {G protein-coupled receptors of the Adhesion family (aGPCRs) comprise the second largest group within the GPCR realm with over 30 mammalian homologs. They contain a unique structure with unusually large extracellular domains (ECDs) holding many structural folds known to mediate cell-cell and cell-matrix interactions. Furthermore, aGPCRs undergo autoproteolytic cleavage at the GPCR proteolysis site (GPS), an integral portion of the GPCR autoproteolysis inducing (GAIN) domain. Thus far, it is largely unknown if and how self-cleavage affects aGPCR activation and signaling and how these signals may shape the physiological function of cells. Latrophilin, alternatively termed the calcium-independent receptor of α-latrotoxin (CIRL) constitutes a highly conserved, prototypic aGPCR and has been assigned roles in various biological processes such as synaptic development and maturation or the regulation of neurotransmitter release. The Drosophila melanogaster homolog dCIRL is found in numerous sensory neurons including the mechanosensory larval pentascolopidial chordotonal organs (CHOs), which rely on dCIRL function in order to sense mechanical cues and to modulate the mechanogating properties of present ionotropic receptors. This study reveals further insight into the broad distribution of dCirl expression throughout the larval central nervous system, at the neuromuscular junction (NMJ), as well as subcellular localization of dCIRL in distal dendrites and cilia of chordotonal neurons. Furthermore, targeted mutagenesis which disabled GPS cleavage of dCIRL left intracellular trafficking in larval CHOs unaffected and proved autoproteolysis is not required for dCIRL function in vivo. However, substitution of a threonine residue, intrinsic to a putative tethered agonist called Stachel that has previously been documented for several other aGPCRs, abrogated receptor function. Conclusively, while this uncovered the presence of Stachel in dCIRL, it leaves the question about the biological relevance of the predetermined breaking point at the GPS unanswered. In an independent approach, the structure of the "Inter-RBL-HRM" (IRH) region, the region linking the N-terminal Rhamnose-binding lectin-like (RBL) and the hormone receptor motif (HRM) domains of dCIRL, was analyzed. Results suggest random protein folding, excessive glycosylation, and a drastic expansion of the size of IRH. Therefore, the IRH might represent a molecular spacer ensuring a certain ECD dimension, which in turn may be a prerequisite for proper receptor function. Taken together, the results of this study are consistent with dCIRL's mechanoceptive faculty and its role as a molecular sensor that translates mechanical cues into metabotropic signals through a yet undefined Stachel-dependent mechanism.},
  subject      = {Latrophilin},
  language  = {en}
}
@phdthesis{İşbilir2022,
  author    = {İ{\c{s}}bilir, Ali},
  title     = {Localization and Trafficking of CXCR4 and CXCR7},
  doi       = {10.25972/OPUS-24937},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-249378},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {G protein-coupled receptors (GPCRs) constitute the largest class of membrane proteins, and are the master components that translate extracellular stimulus into intracellular signaling, which in turn modulates key physiological and pathophysiological processes. Research within the last three decades suggests that many GPCRs can form complexes with each other via mechanisms that are yet unexplored. Despite a number of functional evidence in favor of GPCR dimers and oligomers, the existence of such complexes remains controversial, as different methods suggest diverse quaternary organizations for individual receptors. Among various methods, high resolution fluorescence microscopy and imagebased fluorescence spectroscopy are state-of-the-art tools to quantify membrane protein oligomerization with high precision. This thesis work describes the use of single molecule fluorescence microscopy and implementation of two confocal microscopy based fluorescence fluctuation spectroscopy based methods for characterizing the quaternary organization of two class A GPCRs that are important clinical targets: the C-X-C type chemokine receptor 4 (CXCR4) and 7 (CXCR7), or recently named as the atypical chemokine receptor 3 (ACKR3). The first part of the results describe that CXCR4 protomers are mainly organized as monomeric entities that can form transient dimers at very low expression levels allowing single molecule resolution. The second part describes the establishment and use of spatial and temporal brightness methods that are based on fluorescence fluctuation spectroscopy. Results from this part suggests that ACKR3 forms clusters and surface localized monomers, while CXCR4 forms increasing amount of dimers as a function of receptor density in cells. Moreover, CXCR4 dimerization can be modulated by its ligands as well as receptor conformations in distinct manners. Further results suggest that antagonists of CXCR4 display distinct binding modes, and the binding mode influences the oligomerization and the basal activity of the receptor: While the ligands that bind to a "minor" subpocket suppress both dimerization and constitutive activity, ligands that bind to a distinct, "major" subpocket only act as neutral antagonists on the receptor, and do not modulate neither the quaternary organization nor the basal signaling of CXCR4. Together, these results link CXCR4 dimerization to its density and to its activity, which may represent a new strategy to target CXCR4.},
  subject      = {G-Protein gekoppelter Rezeptor},
  language  = {en}
}
@phdthesis{Kaiser2020,
  author    = {Kaiser, Sebastian},
  title     = {A RecQ helicase in disguise: Characterization of the unconventional Structure and Function of the human Genome Caretaker RecQ4},
  doi       = {10.25972/OPUS-16041},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-160414},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {From the simplest single-cellular organism to the most complex multicellular life forms, genetic information in form of DNA represents the universal basis for all biological processes and thus for life itself. Maintaining the structural and functional integrity of the genome is therefore of paramount importance for every single cell. DNA itself, as an active and complex macromolecular structure, is both substrate and product of many of these biochemical processes. A cornerstone of DNA maintenance is thus established by the tight regulation of the multitude of reactions in DNA metabolism, repressing adverse side reactions and ensuring the integrity of DNA in sequence and function. The family of RecQ helicases has emerged as a vital class of enzymes that facilitate genomic integrity by operating in a versatile spectrum of nucleic acid metabolism processes, such as DNA replication, repair, recombination, transcription and telomere stability. RecQ helicases are ubiquitously expressed and conserved in all kingdoms of life. Human cells express five different RecQ enzymes, RecQ1, BLM, WRN, RecQ4 and RecQ5, which all exhibit individual as well as overlapping functions in the maintenance of genomic integrity. Dysfunction of three human RecQ helicases, BLM, WRN and RecQ4, causes different heritable cancer susceptibility syndromes, supporting the theory that genomic instability is a molecular driving force for cancer development. However, based on their inherent DNA protective nature, RecQ helicases represent a double-edged sword in the maintenance of genomic integrity. While their activity in normal cells is essential to prevent cancerogenesis and cellular aging, cancer cells may exploit this DNA protective function by the overexpression of many RecQ helicases, aiding to overcome the disadvantageous results of unchecked DNA replication and simultaneously gaining resistance against chemotherapeutic drugs. Therefore, detailed knowledge how RecQ helicases warrant genomic integrity is required to understand their implication in cancerogenesis and aging, thus setting the stage to develop new strategies towards the treatment of cancer. The current study presents and discusses the first high-resolution X-ray structure of the human RecQ4 helicase. The structure encompasses the conserved RecQ4 helicase core, including a large fraction of its unique C- terminus. Our structural analysis of the RecQ4 model highlights distinctive differences and unexpected similarities to other, structurally conserved, RecQ helicases and permits to draw conclusions about the functional implications of the unique domains within the RecQ4 C-terminus. The biochemical characterization of various RecQ4 variants provides functional insights into the RecQ4 helicase mechanism, suggesting that RecQ4 might utilize an alternative DNA strand separation technique, compared to other human RecQ family members. Finally, the RecQ4 model permits for the first time the analysis of multiple documented RecQ4 patient mutations at the atomic level and thus provides the possibility for an advanced interpretation of particular structure-function relationships in RecQ4 pathogenesis.},
  subject      = {Helikasen},
  language  = {en}
}
@phdthesis{Uthe2018,
  author    = {Uthe, Henriette},
  title     = {Analyse des Interaktoms des Mediatorkomplexes und seiner posttranslationalen Modifikationen in \(Saccharomyces\) \(Cerevisae\) mittels Massenspektrometrie},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-162619},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Eukaryotic messenger RNA (mRNA) synthesis catalyzed by the RNA Polymerase II is the central and critical process for the regulation of gene expression. Several decades of research unearthed many details about this essential process of high complexity and dynamic. The mediator complex turned out to be crucial for the regulation of Pol II mediated transcription, especially the process of initiation. It functions as an interface between the general transcription machinery and multiple DNA binding transcriptional regulators. Binding these regulators via its tail module and binding the polymerase II via its head module, the mediator forms a bridge between upstream activating sequences and the core promotor and initiates the assembling of the Pre-Initiation complex consisting of the polymerase II and the general transcription factors. However, particularly the last years of research suggest the mediator complex within many other functions including transcription elongation, gene looping and chromatin remodeling. Considering the facts, that the mediator (a) consist of 25 subunits, which are partially flexible associated, (b) shows a flexible intrinsic structure and (c) is highly and dynamically phosphorylated it becomes easy to imagineplausible that the mediator complex meets all this functions, by serving as a transcriptional platform. In context of this thesis, and it was possible to "illustrate" the mediator within its versatile tasks and functions by presenting the most comprehensive analysis of the Mediator complex interactome to date. By optimizing the conditions of cell lysis and co-immunoprecipitation it was possible to preserve even transient and labile protein-protein interactions. The use of metabolic labeling (15N) in the control experiment, allowed us to distinguish between specific and non-specific captured proteins. In combination with high performance mass spectrometry, more than 400 proteins and even complete protein complexes interacting with the mediator complex could be identified, naming RNA-Polymerase II, all general transcription factors the SAGA complex, chromatin remodeling complexes and highly acetylated histones. Furthermore, many candidates where identified playing a role in co-transcriptional processes of mRNA, such as splicing, mRNA-decapping, mRNA transport and decay. This analysis not only confirmed several interactions , already can be found in the literature, but furthermore provide clear evidence, that mediator complex interacts not only with the RNA-Polymerase II, but also with the RNA Polymerase I and III. Next to the high numbers of potential known and unknown interacting proteins, it could be shown, that the interactome is highly dynamic and sensitive to detergent.},
  subject      = {Mediator Komplex},
  language  = {de}
}
@phdthesis{Bach2019,
  author    = {Bach, Matthias},
  title     = {Massenspektrometrische Analyse der Interaktionen von Protein mit Proteinen und Proteinen mit niedermolekularen Verbindungen},
  doi       = {10.25972/OPUS-16046},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-160462},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Proteine k{\"o}nnen aufgrund ihrer biochemischen Vielfalt eine Vielzahl von Interaktionen mit anderen Proteinen oder chemischen Verbindungen eingehen. Im ersten Teil dieser Arbeit wurden Protein-Protein Interaktionen mittels chemischen Quervernetzens untersucht. Das Ziel war, neue und verbesserte Methoden zu entwickeln, um Interaktionsnetzwerke zu erstellen. Im zweiten Teil wurden die Interaktionen von Proteinen mit niedermolekularen Verbindungen untersucht, um Drug Targets zu identifizieren und zu validieren. Die Untersuchung von Protein-Protein Interaktionen mittels Massenspektrometrie (MS) ist eine leistungsf{\"a}hige Methode, um alle potentiellen Interaktionen eines Proteins nach einer Anreicherung (Co-IP) aus einem Zelllysat zu detektieren. Durch das zus{\"a}tzliche Quervernetzen dieser Proteine und anschließender MS kann ein Interaktionsnetzwerk erstellt werden, um direkte von indirekten Interaktionen unterscheiden zu k{\"o}nnen (Topology Mapping). Zur Methodenetablierung wurden kommerzielle Crosslinker und rekombinante Proteine von bekannten Interaktionspartnern mit niedriger Komplexit{\"a}t verwendet. Die beiden Interaktionspartner NPL4 und UFD1 konnten mit dem Crosslinker BS3 erfolgreich quervernetzt und anhand der vernetzten Peptide identifiziert werden. Im n{\"a}chsten Schritt wurde dieser Arbeitsablauf auf eine Co-IP des Mediatorkomplexes aus Hefe angewendet. Die Probenkomplexit{\"a}t ist hierbei 500 - 1000-fach h{\"o}her als bei der Verwendung von rekombinanten Proteinen. Nach der erfolgreichen Quervernetzung konnte innerhalb des Komplexes ein Interaktionsnetzwerk erstellt werden. Diese Daten passen zu dem bereits bekannten Modell des Mediatorkomplexes. Interaktionen zu bekannten Interaktionspartnern, wie der RNA-Pol II, konnten aufgrund deren subst{\"o}chiometrischen Anreicherung nicht identifiziert werden. Aufgrund der genannten Limitationen beim Quervernetzen von Proteinen wurden folgende neue und verbesserte Methoden entwickelt: 1. Verwendung des spaltbaren Crosslinkers (DSSO), der w{\"a}hrend der Messung selektiv durch niedrige Kollisionsenergie gespalten werden kann, um die Datenbanksuche zu vereinfachen. Die Funktionalit{\"a}t der DSSO-Strategie konnte erfolgreich am Protein Cytochrom C getestet werden. Bei der ersten Fragmentierung wird der Linker gespalten, anschließend k{\"o}nnen die getrennten Peptide separat fragmentiert werden. Die erzeugten Daten sind mit einer Standarddatenbanksuche kompatibel, was bei gemischten Spektren von zwei Peptiden nicht der Fall w{\"a}re. Beim Quervernetzen der rekombinanten Interaktionspartner UBX und p97N mit DSSO konnte der zu best{\"a}tigende Crosslink zwischen zwei Lysinen nicht identifziert werden. Grund hierf{\"u}r k{\"o}nnte eine zu kurze Linkerl{\"a}nge von DSSO sein. Diese Versuche brachten jedoch einige Limitationen des Ansatzes zum Vorschein, wie die Beschr{\"a}nkung auf die Protease Trypsin, aufgrund der positiven Ladung am C-Terminus und die Notwendigkeit von großen Proteinmengen, da das Spalten des Linkers einen zus{\"a}tzlichen Intensit{\"a}tsverlust f{\"u}r die folgende Identifizierung der Peptide mit sich bringt. 2. Da die niedrige Abundanz von quervernetzten Peptiden das Hauptproblem bei deren Identifizierung ist, wurde eine Methode entwickelt, um w{\"a}hrend der Messung direkt nach diesen niedrig abundanten Spezies zu suchen. Entscheidendes Kriterium hierf{\"u}r war, dass quervernetzte Peptide zwei C-Termini haben. Diese wurden zur H{\"a}lfte enzymatisch mit 18O bzw. 16O markiert und wieder vereinigt. Der resultierende Massenunterschied von 8 Da (4 x 18O) kommt ausschließlich bei zwei quervernetzten Peptiden vor und kann w{\"a}hrend der Messung direkt gesucht werden. Die vollst{\"a}ndige Markierung von Peptiden mit 18O wurde zun{\"a}chst am Protein Beta-Galaktosidase getestet. Bereits hier stellte sich heraus, dass der enzymatische R{\"u}cktausch von 18O zu 16O ein Problem darstellt und die Markierungseffizienz von Aminos{\"a}uren beeinflusst wird, die sich C-terminal nach der Spaltstelle befinden. Mit dieser Strategie ließ sich somit keine vollst{\"a}ndige Markierung f{\"u}r alle Peptide erreichen, was f{\"u}r diese Strategie essentiell gewesen w{\"a}re. 3. Um alle Probleme zu umgehen, die bei der Identifizierung von quervernetzten Peptiden auftreten, wurde eine Methode entwickelt, um quervernetzte Proteine anhand von Profilen nach einer Auftrennung im Polyacrylamidgel (SDS-PAGE) zu identifizieren. Durch das Quervernetzen von Proteinen entstehen zus{\"a}tzliche Proteinbanden nach einer SDSPAGE, die im Gel nach oben verschoben sind. Alle Proteine in diesen neu erzeugten Bereichen stellen somit potentielle Interaktionspartner dar. Als Modellsystem wurde der Mediatorkomplex verwendet. Er wurde aus einem Zelllysat mittels Co-IP angereichert und anschließend quervernetzt. Aus den mittels LC-MS/MS gemessenen Gelfraktionen wurden Proteinprofile erstellt und miteinander verglichen. Die Intensit{\"a}tsmaxima der Proteine des Mediatorkomplexes konnten in bestimmten zus{\"a}tzlichen Fraktionen gefunden werden, was den indirekten Nachweis f{\"u}r eine Interaktion darstellt. Die Funktionalit{\"a}t der Strategie konnte somit best{\"a}tigt werden. Ein verbleibender Nachteil ist jedoch die zu geringe Trennleistung von Polyacrylamidgelen. Befinden sich mehr als 50 Proteine in einer Fraktion, k{\"o}nnen potentielle Interaktionspartner nicht eindeutig zu einer Untereinheit eines Komplexes zugeordnet werden. Im zweiten Teil der Arbeit wurde im Rahmen der Klinischen Forschergruppe 216 (CRU216) Interaktionen von Proteinen mit verschiedenen niedermolekularen Verbindungen massenspektrometrisch untersucht, um potentielle Drug Targets zu identifizieren. Diese Versuche sind vergleichbar mit Co-IP Experimenten, da sich der Arbeitsablauf nur durch die Anreicherung mittels chemischer Verbindung unterscheidet. Hierzu wurden biotinylierte Verbindungen immobilisiert und potentielle Drug Targets aus einem komplexen Zelllysat angereichert. Die Identifzierung der echten Bindungspartner wurde {\"u}ber quantitive Massenspektrometrie erreicht. Dabei wurden die angereicherten Proteine, die an die niedermolekularen Substanzen binden mit einer geeigneten Kontrollanreicherung verglichen. Mit den getesteten α-acyl Aminocarboxamiden konnten verschiedene Proteinkomplexe und interagierende Proteine spezifisch angereichert werden. Hierbei waren die vier Kinasen DNA-PK, ATM, ATR und mTOR besonders interessant, da sie mit onkogenem Signalling und {\"U}berlebensmechanismen wie der Hitzeschockantwort in Zellen des Multiplen Myeloms (MM) in Verbidnung stehen. Die Inhibition der DNA-PK, ATM, ATR und mTOR mit α- acyl Aminocarboxamiden stellt somit einen m{\"o}glichen Therapieansatz dar, wenn er zusammen mit hitzestressausl{\"o}senden Inhibitoren verwendet wird. Weiterhin konnte gezeigt werden, dass die Armadillodom{\"a}ne innerhalb der potentiellen Drug targets signifkant angereichert wurde. Sie stellt damit eine potentielle Bindestelle der α-acyl Aminocarboxamide dar. Abschließend wurden Proteine mit biotinylierten Naphtylisochinolinen aus einem MMZelllysat angereichert, deren Vorl{\"a}ufersubstanzen eine Wirkung auf Tumorzellen und den Malariaparasit Plasmodium falciparum gezeigt hatten. Hierbei konnten vor allem RNAbindende- und mRNA-Splicing Proteine identifiziert werden, die zum Teil essentiell f{\"u}r das Spleißen in-vivo sind. Hierzu geh{\"o}ren mehrere Untereinheiten der Splicing Factoren 3A und 3B. Die Ver{\"a}nderung der transkriptionellen Regulation und der resultierende Effekt auf Krebszellen konnte bereits in anderen Studien mit dem Inhibitor Spliceostatin A gezeigt werden, der das Spleißen beeinflusst.},
  subject      = {Massenspektrometrie},
  language  = {de}
}
@phdthesis{Wendlinger2023,
  author    = {Wendlinger, Simone Alice},
  title     = {Function of Peripheral Blood Eosinophils in Melanoma},
  publisher = {Cancers (Basel)},
  doi       = {10.25972/OPUS-30119},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-301194},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {Despite accounting for only a small proportion of all skin cancers, malignant melanoma displays a serious health risk with increasing incidence and high mortality rate. Fortunately, advances in the treatment of malignant melanoma now prolong survival and enhance response and treatment efficacy. Established biomarkers help evaluate disease progression and facilitate choosing appropriate and individual treatment options. However, the need for easily accessible and reliable biomarkers is rising to predict patient-specific clinical outcome. Eosinophil infiltration into the tumor and high peripheral eosinophil counts prior and during treatment have been associated with better response in patients for various cancer entities, including melanoma. An analysis of a heterogeneous study cohort reported high serum ECP levels in non-responders. Hence, eosinophil frequency and serum ECP as a soluble eosinophil-secreted mediator were suggested as prognostic biomarkers in melanoma. We examined whether melanoma patients treated with first-line targeted therapy could also benefit from the effects of eosinophils. In total, 243 blood and serum samples from patients with advanced melanoma were prospectively and retrospectively collected before and after drug initiation. To link eosinophil function to improved clinical outcome, soluble serum markers and peripheral blood counts were used for correlative studies using a homogeneous study cohort. In addition, functional and phenotypical characterizations provided insights into the expression profile and activity of freshly isolated eosinophils, including comparisons between patients and healthy donors. Our data showed a significant correlation between high pre-treatment blood eosinophil counts and improved response to targeted therapy and by trend to combinatorial immunotherapy in patients with metastatic melanoma. In accordance with previous studies our results links eosinophil blood counts to better response in melanoma patients. High pre-treatment ECP serum concentration correlated with response to immunotherapy but not to targeted therapy. Eosinophils from healthy donors and patients showed functional and phenotypical similarities. Functional assays revealed a strong cytotoxic potential of blood eosinophils towards melanoma cells in vitro, inducing apoptosis and necrosis. In addition, in vitro cytotoxicity was an active process of peripheral eosinophils and melanoma cells with bidirectional features and required close cell-cell interaction. The extent of cytotoxicity was dose-dependent and showed susceptibility to changes in physical factors like adherence. Importantly, we provide evidence of an additive tumoricidal function of eosinophils and combinatorial targeted therapy in vitro. In summary, we give valuable insights into the complex and treatment-dependent role of eosinophils in melanoma. As a result, our data support the suggestion of eosinophils and their secreted mediators as potential prognostic biomarkers. It will take additional studies to examine the molecular mechanisms that underlie our findings.},
  subject      = {Melanom},
  language  = {en}
}
@phdthesis{Gulve2019,
  author    = {Gulve, Nitish},
  title     = {Subversion of Host Genome Integrity by Human Herpesvirus 6 and \(Chlamydia\) \(trachomatis\)},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-162026},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2019},
  abstract  = {Ovarian cancer is one of the most common gynecological malignancies in the world. The prevalence of a microbial signature in ovarian cancer has been reported by several studies till date. In these microorganisms, Human herpesvirus 6 (HHV-6) and Chlamydia trachomatis (C.tr) are especially important as they have significantly high prevalence rate. Moreover, these pathogens are directly involved in causing DNA damage and thereby disrupting the integrity of host genome which is the underlying cause of any cancer. This study focuses on how the two pathogens, HHV-6 and C. trachomatis can affect the genome integrity in their individual capacities and thereby may drive ovarian epithelial cells towards transformation. HHV-6 has unique tendency to integrate its genome into the host genome at subtelomeric regions and achieve a state of latency. This latent virus may get reactivated during the course of life by stress, drugs such as steroids, during transplantation, pregnancy etc. The study presented here began with an interesting observation wherein the direct repeat (DR) sequences flanking the ends of double stranded viral genome were found in unusually high numbers in human blood samples as opposed to normal ratio of two DR copies per viral genome. This study was corroborated with in vitro data where cell lines were generated to mimic the HHV-6 status in human samples. The same observation of unusually high DR copies was found in these cell lines as well. Interestingly, fluorescence in situ hybridization (FISH) and inverse polymerase chain reaction followed by southern blotting showed that DR sequences were found to be integrated in nontelomeric regions as opposed to the usual sub-telomeric integration sites in both human samples and in cell lines. Sanger sequencing confirmed the non-telomeric integration of viral DR sequences in the host genome. Several studies have shown that C. trachomatis causes DNA damage and inhibits the signaling cascade of DNA damage response. However, the effect of C. trachomatis infection on process of DNA repair itself was not addressed. In this study, the effect of C. trachomatis infection on host base excision repair (BER) has been addressed. Base excision repair is a pathway which is responsible for replacing the oxidized bases with new undamaged ones. Interestingly, it was found that C. trachomatis infection downregulated polymerase β expression and attenuated polymerase β- mediated BER in vitro. The mechanism of the polymerase β downregulation was found to be associated with the changes in the host microRNAs and downregulation of tumor suppressor, p53. MicroRNA-499 which has a binding site in the polymerase β 3'UTR was shown to be upregulated during C. trachomatis infection. Inhibition of miR-499 using synthetic miR-499 inhibitor indeed improved the repair efficiency during C. trachomatis infection in the in vitro repair assay. Moreover, p53 transcriptionally regulates polymerase β and stabilizing p53 during C. trachomatis infection enhanced the repair efficiency. Previous studies have shown that C. trachomatis can reactivate latent HHV-6. Therefore, genomic instability due to insertions of unstable 'transposon-like' HHV-6 DR followed by compromised BER during C. trachomatis infection cumulatively support the hypothesis of pathogenic infections as a probable cause of ovarian cancer},
  subject      = {Chlamydia trachomatis},
  language  = {en}
}