@article{DeaneBrunkCurranetal.2020,
  author    = {Deane, Katrina E. and Brunk, Michael G. K. and Curran, Andrew W. and Zempeltzi, Marina M. and Ma, Jing and Lin, Xiao and Abela, Francesca and Aksit, S{\"u}meyra and Deliano, Matthias and Ohl, Frank W. and Happel, Max F. K.},
  title     = {Ketamine anaesthesia induces gain enhancement via recurrent excitation in granular input layers of the auditory cortex},
  series = {The Journal of Physiology},
  volume    = {598},
  journal   = {The Journal of Physiology},
  number    = {13},
  doi       = {10.1113/JP279705},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-216123},
  pages     = {2741 -- 2755},
  year      = {2020},
  abstract  = {Ketamine is commonly used as an anaesthetic agent and has more recently gained attention as an antidepressant. It has been linked to increased stimulus-locked excitability, inhibition of interneurons and modulation of intrinsic neuronal oscillations. However, the functional network mechanisms are still elusive. A better understanding of these anaesthetic network effects may improve upon previous interpretations of seminal studies conducted under anaesthesia and have widespread relevance for neuroscience with awake and anaesthetized subjects as well as in medicine. Here, we investigated the effects of anaesthetic doses of ketamine (15 mg kg\(^{-1}\) h\(^{-1}\)i.p.) on the network activity after pure-tone stimulation within the auditory cortex of male Mongolian gerbils (Meriones unguiculatus). We used laminar current source density (CSD) analysis and subsequent layer-specific continuous wavelet analysis to investigate spatiotemporal response dynamics on cortical columnar processing in awake and ketamine-anaesthetized animals. We found thalamocortical input processing within granular layers III/IV to be significantly increased under ketamine. This layer-dependent gain enhancement under ketamine was not due to changes in cross-trial phase coherence but was rather attributed to a broadband increase in magnitude reflecting an increase in recurrent excitation. A time-frequency analysis was indicative of a prolonged period of stimulus-induced excitation possibly due to a reduced coupling of excitation and inhibition in granular input circuits - in line with the common hypothesis of cortical disinhibition via suppression of GABAergic interneurons.},
  language  = {en}
}
@phdthesis{Bauriedl2020,
  author    = {Bauriedl, Saskia Corinna},
  title     = {The influence of riboregulation on fitness and virulence in Neisseria meningitidis},
  doi       = {10.25972/OPUS-19297},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-192978},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Neisseria meningitidis (N. meningitidis) is a human commensal that occasionally causes life-threatening infections such as bacterial meningitis and septicemia. Despite experi-mental evidence that the expression of small non-coding RNAs (sRNAs) as well as the RNA chaperone Hfq affect meningococcal physiology, the impact of RNA-based regula-tion (riboregulation) on fitness and virulence in N. meningitidis is only poorly understood. Therefore, this study addressed these issues using a combination of high-throughput tech-nologies. A differential RNA-sequencing (dRNA-seq) approach was applied to produce a single-nucleotide resolution map of the primary transcriptome of N. meningitidis strain 8013. The dRNA-seq analysis predicted 1,625 transcriptional start sites including 65 putative sRNAs, of which 20 were further validated by northern blot analysis. By Hfq RNA im-munopreci-pitation sequencing a large Hfq-centered post-transcriptional regulatory net-work comprising 23 sRNAs and 401 potential mRNA targets was identified. Rifampicin stability assays demonstrated that Hfq binding confers enhanced stability on its associat-ed sRNAs. Based on these data, the interactions of two paralogous sRNAs and their cog-nate target mRNA prpB were validated in vivo as well as in vitro. Both sRNAs directly repress prpB encoding a methylisocitrate lyse which was previously shown to be involved in meningococcal colonization of the human nasopharynx. Besides the well-described RNA chaperone Hfq, FinO-domain proteins have recently been recognized as a widespread family of RNA-binding proteins (RBPs) with regulatory roles in diverse bacteria. They display an intriguing bandwidth of target sites, ranging from a single RNA pair as recognized by plasmid-encoded FinO to the global RNA regu-lons of enterobacterial ProQ proteins. To better understand the intrinsic targeting mode of this RBP family, in vivo targets of the minimal ProQ protein of N. meningitidis were de-termined. In vivo UV crosslinking with RNA deep sequencing (UV-CLIP) identified as-sociations of ProQ with 16 sRNAs and 166 mRNAs encoding a variety of biological functions and thus revealed ProQ as another global RBP in meningococci. It could be shown that meningococcal ProQ predominantly binds to highly structured RNA regions including DNA uptake sequences (DUS) and rho-independent transcription terminators and stabilizes many of its RNA targets as proved by rifampicin stability experiments. As expected from the large suite of ProQ-bound RNAs, proQ deletion globally affects both gene and protein expression in N. meningitidis, changing the expression levels of at least 244 mRNAs and 80 proteins. Phenotypic analyses suggested that ProQ promotes oxida-tive stress tolerance and UV damage repair capacity, both of which are required for full virulence of N. meningitidis. Together, this work uncovers the co-existence of two major post-transcriptional regulons, one governed by ProQ, the other by Hfq, in N. meningitidis. It further highlights the role of these distinct RBPs and its associated sRNAs to bacterial virulence and indicates that riboregulation is likely to contribute to the way how meningococci adapt to different host niches.},
  subject      = {Neisseria meningitidis},
  language  = {en}
}
@phdthesis{Eltschkner2020,
  author    = {Eltschkner, Sandra},
  title     = {Targeting the Bacterial Fatty-Acid Synthesis Pathway: Towards the Development of Slow-Onset Inhibitors and the Characterisation of Protein-Protein Interactions},
  doi       = {10.25972/OPUS-15664},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-156643},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {A continuous arms race between the development of novel antibiotics and the evolution of corresponding resistance mechanisms in bacteria has been observed, since antibiotic agents like arsphenamines (e.g. Salvarsan, developed by Paul Ehrlich [1]), sulphonamides (e.g. Prontosil, Gerhard Domagk [2]) and penicillin (Alexander Fleming [3]) were first applied to effectively cure bacterial infections in the early 20th century. The rapid emergence of resistances in contrast to the currently lagging discovery of antibiotics displays a severe threat to human health. Some serious infectious diseases, such as tuberculosis or melioidosis, which were either thought to be an issue only in Third-World countries in case of tuberculosis, or regionally restricted with respect to melioidosis, are now on the rise to expand to other areas. In contrast, methicillin-resistant Staphylococcus aureus (MRSA) is already present in clinical setups all over the world and causes severe infections in immunocompromised patients. Thus, there is an urgent need for new and effective antimicrobial agents, which impair vital functions of the pathogen's metabolism. One central metabolic pathway is represented by the bacterial fatty-acid synthesis pathway (FAS II), which is essential for the synthesis of long and branched-chain fatty acids, as well as mycolic acids. These substances play a major role as modulating components of the properties of the most important protective barrier - the cell envelope. The integrity of the bacterial cell wall and the associated membrane(s) is crucial for cell growth and for protection against physical strain, intrusion of antibiotic agents and regulation of uptake of ions and other small molecules. Thus, this central pathway represents a promising target for antibiotic action against pathogens to combat infectious diseases. The last and rate-limiting step is catalysed by the trans-2-enoyl-ACP reductase (ENR) FabI or InhA (in mycobacteria), which has been demonstrated to be a valuable target for drug design and can be addressed, amongst others, by diphenyl ether (DPE) compounds, derived from triclosan (TCL) - the first one of this class which was discovered to bind to ENR enzymes [4, 5]. Based on this scaffold, inhibitors containing different combinations of substituents at crucial positions, as well as a novel type of substituent at position five were investigated regarding their binding behaviour towards the Burkholderia pseudomallei and Mycobacterium tuberculosis ENR enzymes bpFabI and InhA, respectively, by structural, kinetic and in-vivo experiments. Generally, substitution patterns modulate the association and dissociation velocities of the different ENR inhibitors in the context of the two-step slow-onset binding mechanism, which is observed for both enzymes. These alterations in the rapidity of complex formation and decomposition have a crucial impact on the residence time of a compound and hence, on the pharmacokinetic properties of potential drug candidates. For example, the substituents at the 2'-position of the DPE scaffold influence the ground- and transition state stability during the binding process to bpFabI, whereas 4'-substituents primarily alter the transition state [6]. The novel triazole group attached to the 5-position of the scaffold, targeting the hydrophobic part of the substrate-binding pocket in InhA, significantly enhances the energy barrier of the transition state of inhibitor binding [7] and decelerates the association- as well as the dissociation processes. Combinations with different substituents at the 2'-position can enhance or diminish this effect, e.g. by ground-state stabilisation, which will result in an increased residence time of the respective inhibitor on InhA. Further structural investigations carried out in this work, confirm the proposed binding mode of a customised saFabI inhibitor [8], carrying a pyridone moiety on the DPE scaffold to expand interactions with the protein environment. Structural and preliminary kinetic data confirm the binding of the same inhibitor to InhA in a related fashion. Comparisons with structures of the ENR inhibitor AFN-1252 [9] bound to ENR enzymes from other organisms, addressing a similar region as the pyridone-moiety of the DPE inhibitor, suggest that also the DPE inhibitor bears the potential to display binding to homologues of saFabI and InhA and may be optimised accordingly. Both of the newly investigated substituents, the pyridone moiety at the 4'-position as well as the 5-triazole substituent, provide a good starting point to modify the DPE scaffold also towards improved kinetic properties against ENR enzymes other than the herein studied and combining both groups on the DPE scaffold may have beneficial effects. The understanding of the underlying binding mechanism is a crucial factor to promote the dedicated design of inhibitors with superior pharmacokinetic characteristics. A second target for a structure-based drug-design approach is the interaction surface between ENR enzymes and the acyl-carrier protein (ACP), which delivers the growing acyl chain to each distinct enzyme of the dissociated FAS-II system and presumably recognises its respective interaction partner via electrostatic contacts. The interface between saACP and saFabI was investigated using different approaches including crosslinking experiments and the design of fusion constructs connecting the ACP and the FabI subunits via a flexible linker region of varying lengths and compositions. The crosslinking studies confirmed a set of residues to be part of the contact interface of a previously proposed complex model [10] and displayed high crosslinking efficiency of saACP to saFabI when mutated to cysteine residues. However, crystals of the complex obtained from either the single components, or of the fusion constructs usually displayed weak diffraction, which supports the assumption that complex formation is highly transient. To obtain ordered crystals for structural characterisation of the complex it is necessary to trap the complex in a fixed state, e.g. by a high-affinity substrate attached to ACP [11], which abolishes rapid complex dissociation. For this purpose, acyl-coupled long-residence time inhibitors might be a valuable tool to elucidate the detailed architecture of the ACP-FabI interface. This may provide a novel basis for the development of inhibitors that specifically target the FAS-II biosynthesis pathway.},
  subject      = {Fetts{\"a}urestoffwechsel},
  language  = {en}
}
@phdthesis{Eckstein2020,
  author    = {Eckstein, Marie-Therese},
  title     = {Exploring the biology of the fungus Candida albicans in the gut of gnotobiotic mice},
  doi       = {10.25972/OPUS-21870},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-218705},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {The human body is colonized by trillions of microbes from all three domains of life - eukaryotes, bacteria and archaea. The lower gastrointestinal tract is the most densely colonized part of the body, harbouring a diverse and dynamic community of microbes. While the importance of bacteria in this so-called microbiota is well acknowledged, the role of commensal fungi remains underexplored. The most prominent fungus of the human gastrointestinal microbiota is Candida albicans. This fungus occasionally causes life-threatening disseminated infections in individuals with debilitated immune defences. It is this "pathogenic" facet that has received the most attention from researchers in the past, leaving many aspects of its "commensal" lifestyle understudied. Using gnotobiotic mice as a model system to explore the biology of C. albicans in the mammalian gut, in this dissertation I establish the global response of the host to C. albicans monocolonization as well as the spatial distribution of the fungus in the intestine in the context of co-colonization with single gut bacterial species. The fungus elicited transcriptome changes in murine intestinal tissue, which included the activation of a reactive oxygen species-related defence mechanism and the induction of regulators of the circadian clock circuitry. Both responses have previously been described in the context of a complete bacterial microbiota. Imaging the intestine of animals monocolonized with the fungus or co-colonized with C. albicans and the gut bacteria Bacteroides thetaiotaomicron or Lactobacillus reuteri revealed that the fungus was embedded in a B. thetaiotaomicron-promoted outer mucus layer in the murine colon. The gel-like outer mucus constitutes a unique microhabitat, distinct in microbial composition from the adjacent intestinal lumen. This finding indicates that bacteria can shape the specific microhabitat occupied by the fungus in the intestine. Overall, the results described in this dissertation suggest that gnotobiotic mice constitute a valuable tool to dissect multiple aspects of the interactions among host, commensal fungi and cohabiting bacteria.},
  subject      = {Candida albicans},
  language  = {en}
}
@phdthesis{Yurdadogan2020,
  author    = {Yurdadogan, Tino},
  title     = {Endorganschaden und Gef{\"a}ßalter bei Patienten mit koronarer Herzkrankheit},
  doi       = {10.25972/OPUS-21846},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-218469},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Die nicht-invasive Gef{\"a}ßdiagnostik stellt einen wichtigen Pfeiler in der Pr{\"a}vention von Herz-Kreislauferkrankungen dar. W{\"a}hrend lange Zeit die sonographische Messung der cIMT, als morphologisches Korrelat der Gef{\"a}ßalterung, als Goldstandard galt, ist in den letzten Jahren in Gestalt der Pulswellenanalyse/PWV-Messung eine Technik weiterentwickelt worden, die, als funktionelles Korrelat der Gef{\"a}ßalterung, aufgrund der leichteren Durchf{\"u}hrbarkeit und geringerer Untersucherabh{\"a}ngigkeit und Kosten vielversprechend ist. So erlaubt die Messung der Pulswelle mittels gew{\"o}hnlicher Blutdruckmanschetten, genau wie die cIMT, die Berechnung des individuellen Gef{\"a}ßalters und die Diagnostik f{\"u}r das Vorliegen eines Endorganschadens der Blutgef{\"a}ße. Um die Messergebnisse der beiden Untersuchungen miteinander zu vergleichen, wurden beide in der EUROASPIRE-IV Studie an Patienten mit koronarer Herzkrankheit durchgef{\"u}hrt. Die Auswertung der Messergebnisse der mit dem Vascular Explorer durchgef{\"u}hrten Pulswellenanalyse/PWV-Messung ergab {\"u}berraschenderweise, dass die Mehrheit der herzkranken Patienten weder eine vaskul{\"a}re Voralterung noch einen Endorganschaden der Blutgef{\"a}ße aufweisen. Im Falle der cIMT-Messung war Gegenteiliges der Fall, was trotz der medikament{\"o}sen Therapie der Patienten so zu erwarten war. Weiterhin zeigte sich lediglich eine geringe Korrelation zwischen den Messergebnissen beider Untersuchungen. Die Determinanten der einzelnen Messwerte aus cIMT und Pulswellenanalyse/PWV-Messung waren deckungsgleich mit den in der Literatur beschriebenen Faktoren, wenn auch viele der sonst signifikanten Regressoren das Signifikanzniveau in unserer Auswertung nicht unterschritten. Eine Limitation der funktionellen Gef{\"a}ßdiagnostik liegt derzeit darin, dass die Messergebnisse stark von dem verwendeten Messger{\"a}t abh{\"a}ngen. Es liegen noch zu wenig Vergleichsstudien vor, um die Messergebnisse, speziell von neueren Ger{\"a}ten wie dem Vascular Explorer, auf andere zu {\"u}bertragen. Bei der Berechnung des Gef{\"a}ßalters sollten daher optimalerweise ger{\"a}tespezifische Normwerte vorliegen, was beim Vascular Explorer nicht der Fall ist. Gleiches gilt f{\"u}r die Verwendung des PWVcf-Grenzwerts f{\"u}r die Diagnose eines Endorganschadens der Blutgef{\"a}ße. Analog hat auch die Messung der cIMT gewisse Einschr{\"a}nkungen. So w{\"a}re eine weitere Standardisierung der Messorte (A. carotis communis vs Bulbus vs A. carotis interna), zwischen denen sich die durchschnittliche cIMT erheblich unterscheidet, sowie der Messparameter (Minimal- vs Maximal- vs Mittelwert) w{\"u}nschenswert. Die universelle Anwendung eines cIMT-Grenzwerts zur Diagnose eines Endorganschadens der Blutgef{\"a}ße ist daher kritisch zu sehen. Dies zeigt sich auch darin, dass in den neuesten Leitlinien der bislang geltende Grenzwert angezweifelt und kein aktuell g{\"u}ltiger Grenzwert mehr genannt wird. Wir interpretieren unsere Ergebnisse dahingehend, dass unsere Messung der cIMT die zu erwartende pathologische Gef{\"a}ßalterung bei Patienten mit koronarer Herzkrankheit besser widerspiegelt als die Messung der Pulswelle mit dem Vascular Explorer. Welche der beiden Untersuchungen hinsichtlich der prognostischen Wertigkeit {\"u}berlegen ist, muss im Rahmen von L{\"a}ngsschnittstudien gekl{\"a}rt werden.},
  subject      = {Arteriosklerose},
  language  = {de}
}
@phdthesis{Ehebauer2020,
  author    = {Ehebauer, Franziska},
  title     = {Regulation of Nicotinamide N-methyltransferase Expression in Adipocytes},
  doi       = {10.25972/OPUS-21764},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-217645},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Nicotinamide N-methyltransferase (NNMT) is a new regulator of energy homeostasis. Its expression is increased in models of obesity and diabetes. An enhanced NNMT level is also caused by an adipose tissue-specific knockout of glucose transporter type 4 (GLUT4) in mice, whereas the overexpression of this glucose transporter reduced the NNMT expression. Furthermore, the knockdown of the enzyme prevents mice from diet-induced obesity (DIO) and the recently developed small molecule inhibitors for NNMT reverses the DIO. These previous findings demonstrated the exclusive role of NNMT in adipose tissue and further make it to a promising target in obesity treatment. However, the regulation mechanism of this methyltransferase is not yet clarified. The first part of the thesis focus on the investigation whether pro-inflammatory signals are responsible for the enhanced NNMT expression in obese adipose tissue because a hallmark of this tissue is a low-level chronic inflammation. Indeed, the NNMT mRNA in our study was elevated in obese patients compared with the control group, whereas the GLUT4 mRNA expression does not differ between lean and obese humans. To analyze whether pro inflammatory signals, like interleukin (IL 6) and tumor necrosis factor α (TNF-α), regulate NNMT expression 3T3-L1 adipocytes were treated with these cytokines. However, IL 6, TNF α, and leptin, which is an alternative activator of the JAK/STAT pathway, did not affect the NNMT protein or mRNA level in differentiated 3T3-L1 adipocytes. The mRNA and protein levels were measured by quantitative polymerase chain reaction (qPCR) and western blotting. In the second part of this study, 3T3-L1 adipocytes were cultivated with varying glucose concentrations to show whether NNMT expression depends on glucose availability. Further studies with activators and inhibitors of AMP-activated protein kinase (AMPK) and mechanistic target of rapamycin (mTOR) signaling pathways were used to elucidate the regulation mechanism of the enzyme. The glucose deprivation of differentiated 3T3-L1 adipocytes led to a 2-fold increase in NNMT expression. This effect was confirmed by the inhibition of the glucose transports with phloretin as well as the inhibition of glycolysis with 2-deoxyglucose (2-DG). AMPK serves as an intracellular energy sensor and the pharmacological activation of it enhanced the NNMT expression. This increase was also caused by the inhibition of mTOR. Conversely, the activation of mTOR using MHY1485 prevented the effect of glucose deprivation on NNMT. Furthermore, the NNMT up-regulation was also blocked by the different autophagy inhibitors. Taken together, NNMT plays a critical role in autophagy in adipocytes, because an inhibition of this process prevented the augmented NNMT expression during glucose starvation. Moreover, the effect on NNMT protein and mRNA level depends on AMPK and mTOR. However, pro-inflammatory signals did not affect the expression. Further in vivo studies have to clarify whether AMPK activation and mTOR inhibition as well as autophagy are responsible for the increased NNMT levels in obese adipose tissue. In future this methyltransferase emerges as an awesome therapeutic target for obesity.},
  subject      = {Fettzelle},
  language  = {en}
}
@phdthesis{Gotru2020,
  author    = {Gotru, Sanjeev Kiran},
  title     = {Cation Homeostasis in Platelets},
  doi       = {10.25972/OPUS-17661},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-176616},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Divalent cations are important second messengers triggering various signal transduction events in platelets. Whereas calcium channel blockers have an established antithrombotic effect and the regulation of Ca2+ homeostasis has been elucidated in platelets, the molecular regulation of Mg2+ and Zn2+ homeostasis has not been investigated so far. In the first part of the thesis, the role of -type serine-threonine kinase linked to transient receptor potential cation channel, subfamily M, member 7 (TRPM7) in platelets was investigated. Using Trpm7R/R mice with a point mutation deleting the kinase activity, we showed that the TRPM7 kinase regulates platelet activation via immunoreceptor tyrosine-based activation motif (ITAM), hem(ITAM) and protease-activated receptor (PAR) signaling routes. Furthermore, Trpm7R/R mice were protected from in vivo thrombosis and stroke, thus establishing TRPM7 kinase as a promising anti-thrombotic target. In the second part of the thesis, the role of TRPM7 channel in a megakaryocyte (MK) and platelet-specific knockout mouse, Trpm7fl/fl-Pf4Cre, was investigated. Here, we observed that depending on the type of stimulation, Trpm7fl/fl-Pf4Cre platelets showed either enhanced or inhibited responses. Although Trpm7fl/fl-Pf4Cre mice were thrombocytopenic, no differences to wildtype mice were observed in models of in vivo thrombosis and stroke. The above two studies highlight that inhibition of TRPM7 kinase but not the channel itself (in MKs and platelets) may be a promising anti-thrombotic strategy. Besides TRPM7, we investigated the role of magnesium transporter 1 (MAGT1) in platelet Mg2+ homeostasis and found that MAGT1 primarily regulates receptor-operated calcium entry (ROCE) in platelets specifically upon GPVI activation. This physiological crosstalk is triggered by protein kinase C (PKC) isoforms. Platelets from Magt1-/y mice hyper-reacted to GPVI and thromboxane A2 (TXA2) receptor stimulation in vitro. Consequently, Magt1-/y platelets were found to be pro-thrombotic in disease models of thrombosis and stroke. To compare platelet ITAM-signaling to the immune system, we further investigated the role of MAGT1 in T and B cells. We described the primary role of MAGT1 in mice under pathogen-free conditions. Magt1-/y B cells showed dysregulated Mg2+ and Ca2+ homeostasis upon B-cell receptor activation, thereby altering Syk, LAT, phospholipase C (PLC)2 and PKC phosphorylation. In contrast to human MAGT1-deficient T cells, development and effector functions of mouse Magt1-/y T cells showed no alterations. Finally, in the last part of the thesis, we described methods to measure intracellular free zinc [Zn2+]i in human and mouse platelets with storage pool disease (SPD). We propose to measure the [Zn2+]i status in SPD platelets as a relatively easy diagnostic to screen platelet granule abnormalities.},
  subject      = {Thrombozyt},
  language  = {en}
}
@phdthesis{Volz2020,
  author    = {Volz, Julia},
  title     = {Studies on the influence of platelets on vascular integrity in primary tumors and the role of BIN2 in platelet calcium signaling},
  doi       = {10.25972/OPUS-21742},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-217427},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Maintenance of tumor vasculature integrity is indispensable for tumor growth and thus affects tumor progression. Previous studies have identified platelets as major regulators of tumor vascular integrity, as their depletion selectively renders tumor vessels highly permeable, causing massive intratumoral hemorrhage. While these results establish platelets as potential targets for anti-tumor therapy, depletion is not a treatment option due to the essential role of platelets for hemostasis. This thesis demonstrates for the first time that functional inhibition of glycoprotein (GP) VI on the platelet surface rapidly induces tumor hemorrhage and diminishes tumor growth similar to complete platelet depletion but without inducing systemic bleeding complications. Both, the intratumoral bleeding and tumor growth arrest could be reverted by depletion of Ly6G+ cells confirming them to be responsible for the induction of bleeding and necrosis within the tumor. In addition, GPVI inhibition increased intra-tumoral accumulation of co-administered chemotherapeutic agents, thereby resulting in a profound anti-tumor effect. In summary, this thesis manifests platelet GPVI as a key regulator of vascular integrity specifically in growing tumors, serving as a potential basis for the development of anti-tumor strategies. In the second part of this thesis, light is shed on the modulating role of bridging integrator 2 (BIN2) in platelet Ca2+ signaling. Stromal interaction molecule 1 (STIM1) mediated store-operated calcium entry (SOCE) is the major route of Ca2+ influx in platelets, triggered by inositol trisphosphate receptor (IP3R)-dependent Ca2+ store release. In this thesis, the BAR domain superfamily member BIN2 was identified as the first Ca2+ signaling modulator, interacting with both, STIM1 and IP3R in platelets. Deletion of BIN2 resulted in reduced Ca2+ store release and Ca2+ influx in response to all tested platelet agonists. These defects were a consequence of impaired IP3R function in combination with defective STIM1-mediated SOC channel activation, while Ca2+ store content and agonist-induced IP3 production were unaltered. These results establish BIN2 as a central regulator of platelet Ca2+ signaling. The third part of this thesis focuses on the effect of the soluble neuronal guidance protein Sema7A on platelet function. Rosenberger et al. discovered that Sema7A cleavage from red blood cells increases the formation of platelet-neutrophil complexes, thereby reinforcing thrombo-inflammation in myocardial ischemia-reperfusion injury (MIRI). This thesis establishes soluble Sema7A as a stimulator of platelet thrombus formation via its interaction with platelet GPIbα, thereby reinforcing PNC formation. Thus, interfering with the GPIb-Sema7A interaction during MIRI represents a potential strategy to reduce cardiac damage and improve clinical outcome following MI.},
  subject      = {Thrombozyt},
  language  = {en}
}
@phdthesis{Misra2020,
  author    = {Misra, Mohit},
  title     = {Knowing then defeating: The Ubiquitin activating enzyme, a promising target for cancer therapy},
  doi       = {10.25972/OPUS-16722},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-167227},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Ubiquitin is a 76 amino acid long polypeptide, which is present throughout eukaryotes in a highly conserved fashion. Ubiquitin can modify proteins by becoming covalently attached to them. Eukaryotic cells employ ubiquitin to maintain and regulate fundamental cellular processes like protein degradation, the immune response and transcriptional and translational regulation. Transfer of ubiquitin to the substrate is achieved by the catalysis of three classes of enzymes namely E1, E2 and E3. Together these enzymes form a pyramidal hierarchy, where E1 stands at the apex and E3 enzymes form the base of the pathway. The ubiquitin activating enzyme 1 (UBA1) plays a major role in ubiquitylation being the ubiquitin-dedicated E1 enzyme. In addition, it is the only enzyme in this pathway to use ATP as an energy source to catalyze two important reactions. The products of these reactions, ubiquitin adenylate and ubiquitin thioester, are the essential intermediate states of ubiquitin, for being conjugated to the target protein. With the help of X-ray crystallography and biochemical approaches, snapshots of multiple catalytic states of UBA1, where it is bound to Mg-ATP, ubiquitin and the E2 Ubc13 as substrates could be captured. With the help of these high-resolution crystal structures, deeper insights into the enzymatic mechanism of UBA1 could be attained. The resulting insights into the catalytic cycle were further validated by biochemical assays. It could be shown that ATP acts as a molecular switch to induce the enzyme's open conformation. Ubiquitin-binding to the enzyme leads to domain rotations, which facilitate the recruitment of a cognate E2 enzyme. The interdomain communication as well as the cross-talk with the substrates and the products fuel the enzymatic cycle of UBA1. Due to the proven efficacy of proteasome inhibitors for cancer treatment, which block degradation of proteins labeled with ubiquitin, enzymes participating in the ubiquitylation cascade have been targeted by researchers for the development of novel anti-cancer therapeutics. UBA1 inhibition has been shown to preferentially induce cell death in malignant cells, and it can also be used as a strategy to overcome resistance against proteasome inhibitors. MLN7243, an adenosyl sulfamate inhibitor developed by Millenium Pharmaceutical to specifically target UBA1, is currently in Phase-I clinical trials for the treatment of solid tumors. UBA1 could be crystallized in complex with three adenosyl sulfamate inhibitors covalently linked to ubiquitin, which are promising drug candidates for cancer therapy. The inhibitors employed, MLN7243, MLN4924 and ABPA3, show distinct specificities towards different E1 enzymes. With the help of crystal structures the specificity determinants of these inhibitors could be deciphered, which were further confirmed by inhibition assays as well as molecular dynamics simulations. Together these crystal structures provide a starting point for developing E1-specific inhibitors, which, besides their potential for medicinal purposes, are important tools to better understand the function of the ubiquitin system as well as the action of ubiquitin-like proteins.},
  subject      = {Ubiquitin-aktivierende Enzym 1},
  language  = {en}
}
@phdthesis{PerpinaViciano2020,
  author    = {Perpi{\~n}{\´a} Viciano, Cristina},
  title     = {Study of the activation mechanisms of the CXC chemokine receptor 4 (CXCR4) and the atypical chemokine receptor 3 (ACKR3)},
  doi       = {10.25972/OPUS-19237},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-192371},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {The CXC chemokine receptor 4 (CXCR4) and the atypical chemokine receptor 3 (ACKR3) are seven transmembrane receptors that are involved in numerous pathologies, including several types of cancers. Both receptors bind the same chemokine, CXCL12, leading to significantly different outcomes. While CXCR4 activation generally leads to canonical GPCR signaling, involving Gi proteins and β-arrestins, ACKR3, which is predominantly found in intracellular vesicles, has been shown to signal via β-arrestin-dependent signaling pathways. Understanding the dynamics and kinetics of their activation in response to their ligands is of importance to understand how signaling proceeds via these two receptors. In this thesis, different F{\"o}rster resonance energy transfer (FRET)-based approaches have been combined to individually investigate the early events of their signaling cascades. In order to investigate receptor activation, intramolecular FRET sensors for CXCR4 and ACKR3 were developed by using the pair of fluorophores cyan fluorescence protein and fluorescence arsenical hairpin binder. The sensors, which exhibited similar functional properties to their wild-type counterparts, allowed to monitor their ligand-induced conformational changes and represent the first RET-based receptor sensors in the field of chemokine receptors. Additional FRET-based settings were also established to investigate the coupling of receptors with G proteins, rearrangements within dimers, as well as G protein activation. On one hand, CXCR4 showed a complex activation mechanism in response to CXCL12 that involved rearrangements in the transmembrane domain of the receptor followed by rearrangements between the receptor and the G protein as well as rearrangements between CXCR4 protomers, suggesting a role of homodimers in the activation course of this receptor. This was followed by a prolonged activation of Gi proteins, but not Gq activation, via the axis CXCL12/CXCR4. In contrast, the structural rearrangements at each step of the signaling cascade in response to macrophage migration inhibitory factor (MIF) were dynamically and kinetically different and no Gi protein activation via this axis was detected. These findings suggest distinct mechanisms of action of CXCL12 and MIF on CXCR4 and provide evidence for a new type of sequential signaling events of a GPCR. Importantly, evidence in this work revealed that CXCR4 exhibits some degree of constitutive activity, a potentially important feature for drug development. On the other hand, by cotransfecting the ACKR3 sensor with K44A dynamin, it was possible to increase its presence in the plasma membrane and measure the ligand-induced activation of this receptor. Different kinetics of ACKR3 activation were observed in response to CXCL12 and three other agonists by means of using the receptor sensor developed in this thesis, showing that it is a valuable tool to study the activation of this atypical receptor and pharmacologically characterize ligands. No CXCL12-induced G protein activation via ACKR3 was observed even when the receptor was re-localized to the plasma membrane by means of using the mutant dynamin. Altogether, this thesis work provides the temporal resolution of signaling patterns of two chemokine receptors for the first time as well as valuable tools that can be applied to characterize their activation in response to pharmacologically relevant ligands.},
  subject      = {G protein-coupled receptors},
  language  = {en}
}