@phdthesis{Saal2017,
  author    = {Saal, Lena},
  title     = {Whole transcriptome profiling of compartmentalized motoneurons},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-140006},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {Spinal muscular atrophy and amyotrophic lateral sclerosis are the two most common devastating motoneuron diseases. The mechanisms leading to motoneuron degeneration are not resolved so far, although different hypotheses have been built on existing data. One possible mechanism is disturbed axonal transport of RNAs in the affected motoneurons. The underlying question of this study was therefore to characterize changes in transcript levels of distinct RNAs in cell culture models of spinal muscular atrophy and amyotrophic lateral sclerosis, especially in the axonal compartment of primary motoneurons. To investigate this in detail we first established compartmentalized cultures of Primary mouse motoneurons. Subsequently, total RNA of both compartments was extracted separately and either linearly amplified and subjected to microarray profiling or whole transcriptome amplification followed by RNA-Sequencing was performed. To make the whole transcriptome amplification method suitable for compartmentalized cultures, we adapted a double-random priming strategy. First, we applied this method for initial optimization onto serial dilutions of spinal cord RNA and later on to the compartmentalized motoneurons. Analysis of the data obtained from wildtype cultures already revealed interesting results. First, the RNA composition of axons turned out to be highly similar to the somatodendritic compartment. Second, axons seem to be particularly enriched for transcripts related to protein synthesis and energy production. In a next step we repeated the experiments by using knockdown cultures. The proteins depleted hereby are Smn, Tdp-43 and hnRNP R. Another experiment was performed by knocking down the non-coding RNA 7SK, the main interacting RNA of hnRNP R. Depletion of Smn led to a vast number of deregulated transcripts in the axonal and somatodendritic compartment. Transcripts downregulated in the axons upon Smn depletion were especially enriched for GOterms related to RNA processing and encode proteins located in neuron projections including axons and growth cones. Strinkingly, among the upregulated transcripts in the somatodendritic compartment we mainly found MHC class I transcripts suggesting a potential neuroprotective role. In contrast, although knockdown of Tdp-43 also revealed a large number of downregulated transcripts in the axonal compartment, these transcripts were mainly associated with functions in transcriptional regulation and RNA splicing. For the hnRNP R knockdown our results were again different. Here, we observed downregulated transcripts in the axonal compartment mainly associated with regulation of synaptic transmission and nerve impulses. Interestingly, a comparison between deregulated transcripts in the axonal compartment of both hnRNP R and 7SK knockdown presented a significant overlap of several transcripts suggesting some common mechanism for both knockdowns. Thus, our data indicate that a loss of disease-associated proteins involved in axonal RNA transport causes distinct transcriptome alterations in motor axons.},
  subject      = {Axon},
  language  = {en}
}
@phdthesis{Flohr2017,
  author    = {Flohr, Elena Leonie Ruth},
  title     = {The Scents of Interpersonality - On the Influence of Smells on the Evaluation and Processing of Social Stimuli},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-153352},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {In daily life, olfactory stimuli are potential generators of affective states, but also have a strong influence on social interaction. Pleasant odors have been shown to increase perceived attractiveness and pro-social behavior, whereas unpleasant body odors are often associated with negative personality traits. Since both pleasant odors and positive affective state facilitate pro-social behavior, it is conceivable that the influence of the odors on social interaction is mediated by the induced affective state elicited by the odor itself. The present thesis aims at exploring the impact of hedonic, i.e., pleasant or unpleasant, odors on the processing and evaluation of social stimuli as assessed by verbal, physiological, and behavioral indices. First, I investigate the effects of initially neutral odors which gained threatening value through an aversive conditioning procedure on social stimuli (Study 1). Second, I study the influence of naturally hedonic odors on social interaction. Third, this thesis aims at disentangling differences in the effects of an odor attributed to either a social interaction partner or the environment where the social encounter takes place (Study 2, 3, and 4). In the first study, a context conditioning procedure was applied, during which one out of two long-lasting neutral odors was paired with an unpredictable aversive unconditioned stimulus (US, i.e., white noise). This odor (CTX+) thereby gained threatening value, while another odor (CTX-) remained unpaired and therefore signaled safety. During a test session, facial stimuli were presented within both conditioned olfactory contexts. Results indicate that autonomic arousal was increased to faces when presented in the threatening odor context. Additionally, participants rated facial stimuli as more aversive when presented in the threatening odor as compared to the safety odor, indicating that faces acquire hedonic value from the odor they were presented in. Strikingly, angry facial expressions received additional processing resources when presented within a threatening olfactory context, as reflected on verbal reports and electrodermal activity (EDA). This latter finding suggests that threat-related stimuli, here angry faces, are preferentially processed within an olfactory context where a threat might happen. Considering that the hedonic value of an odor may be quite subjective, I conducted a pilot study in order to identify odors with pleasant vs. unpleasant properties for most participants. Seven odors (four pleasant and three unpleasant) were rated with respect to their valence (pleasant vs. unpleasant), arousal (arousing vs. calm), and intensity. Additionally, EDA was measured. Two pleasant (Citral and Eucalyptol) and two unpleasant ("Animalis" and Isobutyraldehyde) odors were chosen from the original seven. The unpleasant odors were rated as more negative, arousing, and intense than the positive ones, but no differences were found regarding EDA. These four odors were subsequently used in a virtual reality (VR) paradigm with two odor attribution groups. Participants of the social attribution group (n = 59) were always passively guided into the same room (an office) towards one out of two virtual agents who were either paired with the pleasant or the unpleasant odor. Participants of the contextual attribution group (n = 58) were guided into one out of two rooms which were either paired with the pleasant or the unpleasant odor and where they always met the same agent. For both groups, the agents smiled, frowned or remained with a neutral facial expression. This design allowed evaluating the influence of odor valence as a within-subjects factor and the influence of odor attribution as a between-subjects factor. Unpleasant odors facilitated the processing of social cues as reflected by increased verbal and physiological arousal as well as reduced active approach behavior. Specific influence of odor valence on emotional facial expressions was found for ratings, EDA, and facial mimicry, with the unpleasant odor causing a levelling effect on the differences between facial expressions. The social attribution group exhibited larger differences between odors than the contextual group with respect to some variables (i.e., ratings and EDA), but not to others (i.e., electrocortical potentials - ERPs - and approach behavior). In sum, unpleasant in comparison to pleasant odors diminished emotional responses during social interaction, while an additional enhancing effect of the social attribution was observed on some variables. Interestingly, the awareness that an interaction partner would smell (pleasantly or unpleasantly) boosted the emotional reactivity towards them. In Study 3, I adapted the VR paradigm to a within-subjects design, meaning that the different attribution conditions were now manipulated block-wise. Instead of an approach task, participants had to move away from the virtual agent (withdrawal task). Results on the ratings were replicated from Study 2. Specifically, the difference between pleasant and unpleasant odors on valence, arousal, and sympathy ratings was larger in the social as compared to the contextual attribution condition. No effects of odor or attribution were found on EDA, whereas heart rate (HR) showed a stronger acceleration to pleasant odors while participants were passively guided towards the agent. Instead of an approach task, I focused on withdrawal behavior in this study. Interestingly, independently of the attribution condition, participants spent more time withdrawing from virtual agents, when an unpleasant odor was presented. In sum, I demonstrated that the attribution of the odors to the social agent itself had an enhancing effect on their influence on social interaction. In the fourth and last study, I applied a similar within-subjects protocol as in Study 3 with an additional Ultimatum Game task as a measure of social interaction. Overall findings replicated the results of Study 3 with respect to HR and EDA. Strikingly, participants offered less money to virtual agents in the bad smelling room than in the good smelling room. In contrast to Study 3, no effects of odor attribution were found in Study 4. In sum, again I demonstrated that unpleasant odor may lessen social interaction not only when the interaction partner smells badly, but also in more complex interaction situations. In conclusion, I demonstrated that hedonic odors in general influence social interaction. Thus, pleasant odors seem to facilitate, while unpleasant odors seem to reduce interpersonal exchanges. Therefore, the present thesis extends the body of literature on the influence of odors on the processing of social stimuli. Although I found a direct influence of odors on social preferences as well as on the physiological and behavioral responses to social stimuli, I did not disentangle impact of odor per se from the impact of the affective state. Interestingly, odor attribution might play an additional role as mediator of social interactions such as odor effects in social interactions might be boosted when the smell is attributed to an individual. However, the results in this regard were less straightforward, and therefore further investigations are needed. Future research should also take into account gender or other inter-individual differences like social anxiety.},
  subject      = {smell},
  language  = {en}
}
@phdthesis{Stritt2017,
  author    = {Stritt, Simon},
  title     = {The role of the cytoskeleton in platelet production and the pathogenesis of platelet disorders in humans and mice},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-122662},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {Platelets are continuously produced from megakaryocytes (MK) in the bone marrow by a cytoskeleton-driven process of which the molecular regulation is not fully understood. As revealed in this thesis, MK/ platelet-specific Profilin1 (Pfn1) deficiency results in micro- thrombocytopenia, a hallmark of the Wiskott-Aldrich syndrome (WAS) in humans, due to accelerated platelet turnover and premature platelet release into the bone marrow. Both Pfn1-deficient mouse platelets and platelets isolated from WAS patients contained abnormally organized and hyper-stable microtubules. These results reveal an unexpected function of Pfn1 as a regulator of microtubule organization and point to a previously unrecognized mechanism underlying the platelet formation defect in WAS patients. In contrast, Twinfilin2a (Twf2a) was established as a central regulator of platelet reactivity and turnover. Twf2a-deficient mice revealed an age-dependent macrothrombocytopenia that could be explained by a markedly decreased platelet half-life, likely due to the pronounced hyper-reactivity of \(Twf2a^{-/-}\) platelets. The latter was characterized by sustained integrin acti- vation and thrombin generation in vitro that translated into accelerated thrombus formation in vivo. To further elucidate mechanisms of integrin activation, Rap1-GTP-interacting adaptor molecule (RIAM)-null mice were generated. Despite the proposed critical role of RIAM for platelet integrin activation, no alterations in this process could be found and it was concluded that RIAM is dispensable for the activation of β1 and β3 integrins, at least in platelets. These findings change the current mechanistic understanding of platelet integrin activation. Outside-in signaling by integrins and other surface receptors was supposed to regulate MK migration, but also the temporal and spatial formation of proplatelet protrusions. In this the- sis, phospholipase D (PLD) was revealed as critical regulator of actin dynamics and podo- some formation in MKs. Hence, the unaltered platelet counts and production in \(Pld1/2^{-/-}\) mice and the absence of a premature platelet release in the bone marrow of \(Itga2^{-/-}\) mice question the role of podosomes in platelet production and raise the need to reconsider the proposed inhibitory signaling by α2β1 integrins on proplatelet formation. Non-muscle myosin IIA (NMMIIA) has been implicated as a downstream effector of the in- hibitory signals transmitted via α2β1 integrins. Besides Rho-GTPase signaling, also \(Mg^{2+}\) and transient receptor potential melastatin-like 7 (TRPM7) channel α-kinase are known regulators of NMMIIA activity. In this thesis, TRPM7 was identified as major regulator of \(Mg^{2+}\) homeostasis in MKs and platelets. Furthermore, decreased \([Mg^{2+}]_i\) led to deregulated NMMIIA activity and altered cytoskeletal dynamics that impaired thrombopoiesis and resulted in macrothrombocytopenia in humans and mice.},
  subject      = {Thrombozytopoese},
  language  = {en}
}
@phdthesis{Deppermann2017,
  author    = {Deppermann, Carsten},
  title     = {The role of platelet granules in thrombosis, hemostasis, stroke and inflammation},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-121010},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {Platelets are small anucleate cell fragments derived from bone marrow megakaryocytes (MKs) and are important players in hemostasis and thrombosis. Platelet granules store factors which are released upon activation. There are three major types of platelet granules: alpha-granules, dense granules and lysosomes. While dense granules contain non-proteinacious factors which support platelet aggregation and adhesion, platelet alpha-granules contain more than 300 different proteins involved in various functions such as inflammation, wound healing and the maintenanceof vascular integrity, however, their functional significance in vivo remains unknown. This thesis summarizes analyses using three mouse models generated to investigate the role of platelet granules in thrombosis, hemostasis, stroke and inflammation. Unc13d-/- mice displayed defective platelet dense granule secretion, which resulted in abrogated thrombosis and hemostasis. Remarkably, Munc13-4-deficient mice were profoundly protected from infarct progression following transient middle cerebral artery occlusion (tMCAO) and this was not associated with increased intracranial bleeding indicating an essential involvementof dense granule secretion in infarct progression but not intracranial hemostasis during acute stroke with obvious therapeutic implications. In the second part of this thesis, the role of platelet alpha-granules was investigated using the Nbeal2-/- mouse. Mutations in NBEAL2 have been linked to the gray platelet syndrome (GPS), a rare inherited bleeding disorder. Nbeal2-/- mice displayed the characteristics of human GPS, with defective alpha-granule biogenesis in MKs and their absence from platelets. Nbeal2-deficiency did not affect MK differentiation and proplatelet formation in vitro or platelet life span in vivo. Nbeal2-/- platelets displayed impaired adhesion, aggregation, and coagulant activity ex vivo that translated into defective arterial thrombus formation and protection from thrombo-inflammatory brain infarction in vivo. In a model of skin wound repair, Nbeal2-/- mice exhibited impaired development of functional granulation tissue due to severely reduced differentiation of myofibroblasts. In the third part, the effects of combined deficiency of alpha- and dense granule secretion were analyzed using Unc13d-/-/Nbeal2-/- mice. Platelets of these mice showed impaired aggregation and adhesion to collagen under flow ex vivo, which translated into infinite tail bleeding times and severely defective arterial thrombus formation in vivo. When subjected to in vivo models of skin or lung inflammation, the double mutant mice showed no signs of hemorrhage. In contrast, lack of platelet granule release resulted in impaired vascular integrity in the ischemic brain following tMCAO leading to increased mortality. This indicates that while defective dense granule secretion or the paucity of alpha-granules alone have no effect on vascular integrity after stroke, the combination of both impairs vascular integrity and causes an increase in mortality.},
  subject      = {Thrombozyten},
  language  = {en}
}
@phdthesis{Karl2017,
  author    = {Karl, Franziska},
  title     = {The role of miR-21 in the pathophysiology of neuropathic pain using the model of B7-H1 knockout mice},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-156004},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {The impact of microRNA (miRNA) as key players in the regulation of immune and neuronal gene expression and their role as master switches in the pathophysiology of neuropathic pain is increasingly recognized. miR-21 is a promising candidate that could be linked to the immune and the nociceptive system. To further investigate the pathophysiological role of miR-21 in neuropathic pain, we assesed mice deficient of B7 homolog 1 (B7-H1 ko), a protein with suppressive effect on inflammatory responses. B7-H1 ko mice and wildtype littermates (WT) of three different age-groups, young (8 weeks), middle-aged (6 months), and old (12 months) received a spared nerve injury (SNI). Thermal withdrawal latencies and mechanical withdrawal thresholds were determined. Further, we investigated anxiety-, depression-like and cognitive behavior. Quantitative real time PCR was used to determine miR-21 relative expression in peripheral nerves, dorsal root ganglia and white blood cells (WBC) at distinct time points after SNI. Na{\"i}ve B7-H1 ko mice showed mechanical hyposensitivity with increasing age. Young and middle-aged B7-H1 ko mice displayed lower mechanical withdrawal thresholds compared to WT mice. From day three after SNI both genotypes developed mechanical and heat hypersensitivity, without intergroup differences. As supported by the results of three behavioral tests, no relevant differences were found for anxiety-like behavior after SNI in B7-H1 ko and WT mice. Also, there was no indication of depression-like behavior after SNI or any effect of SNI on cognition in both genotypes. The injured nerves of B7-H1 ko and WT mice showed higher miR-21 expression and invasion of macrophages and T cells 7 days after SNI without intergroup differences. Perineurial miR-21 inhibitor injection reversed SNI-induced mechanical and heat hypersensitivity in old B7-H1 ko and WT mice. This study reveals that reduced mechanical thresholds and heat withdrawal latencies are associated with miR-21 induction in the tibial and common peroneal nerve after SNI, which can be reversed by perineurial injection of a miR-21 inhibitor. Contrary to expectations, miR-21 expression levels were not higher in B7-H1 ko compared to WT mice. Thus, the B7-H1 ko mouse may be of minor importance for the study of miR-21 related pain. However, these results spot the contribution of miR-21 in the pathophysiology of neuropathic pain and emphasize the crucial role of miRNA in the regulation of neuronal and immune circuits that contribute to neuropathic pain.},
  subject      = {neuropathic pain},
  language  = {en}
}
@phdthesis{Iltzsche2017,
  author    = {Iltzsche, Fabian},
  title     = {The Role of DREAM/MMB-mediated mitotic gene expression downstream of mutated K-Ras in lung cancer},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-154108},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {The evolutionary conserved Myb-MuvB (MMB) multiprotein complex has an essential role in transcriptional activation of mitotic genes. MMB target genes as well as the MMB associated transcription factor B-Myb and FoxM1 are highly expressed in a range of different cancer types. The elevated expression of these genes correlates with an advanced tumor state and a poor prognosis. This suggests that MMB could contribute to tumorigenesis by mediating overexpression of mitotic genes. Although MMB has been extensively characterized biochemically, the requirement for MMB to tumorigenesis in vivo remains largely unknown and has not been tested directly so far. In this study, conditional knockout of the MMB core member Lin9 inhibits tumor formation in vivo in a mouse model of lung cancer driven by oncogenic K-Ras and loss of p53. The incomplete recombination observed within tumors points towards an enormous selection pressure against the complete loss of Lin9. RNA interference (RNAi)-mediated depletion of Lin9 or the MMB associated subunit B-Myb provides evidence that MMB is required for the expression of mitotic genes in lung cancer cells. Moreover, it was demonstrated that proliferation of lung cancer cells strongly depends on MMB. Furthermore, in this study, the relationship of MMB to the p53 tumor suppressor was investigated in a primary lung cancer cell line with restorable p53 function. Expression analysis revealed that mitotic genes are downregulated after p53 re-expression. Moreover, activation of p53 induces formation of the repressive DREAM complex and results in enrichment of DREAM at mitotic gene promoters. Conversely, MMB is displaced at these promoters. Based on these findings the following model is proposed: In p53-negative cells, mitogenic stimuli foster the switch from DREAM to MMB. Thus, mitotic genes are overexpressed and may promote chromosomal instability and tumorigenesis. This study provides evidence that MMB contributes to the upregulation of G2/M phase-specific genes in p53-negative cells and suggests that inhibition of MMB (or its target genes) might be a strategy for treatment of lung cancer.},
  subject      = {Nicht-kleinzelliges Bronchialkarzinom (NSCLC)},
  language  = {en}
}
@phdthesis{Gupta2017,
  author    = {Gupta, Sanjay Kumar},
  title     = {The human CCHC-type Zinc Finger Nucleic Acid Binding Protein (CNBP) binds to the G-rich elements in target mRNA coding sequences and promotes translation},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-142917},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {The genetic information encoded with in the genes are transcribed and translated to give rise to the functional proteins, which are building block of a cell. At first, it was thought that the regulation of gene expression particularly occurs at the level of transcription by various transcription factors. Recent discoveries have shown the vital role of gene regulation at the level of RNA also known as post-transcriptional gene regulation (PTGR). Apart from non-coding RNAs e.g. micro RNAs, various RNA binding proteins (RBPs) play essential role in PTGR. RBPs have been implicated in different stages of mRNA life cycle ranging from splicing, processing, transport, localization and decay. In last 20 years studies have shown the presence of hundreds of RBPs across eukaryotic systems many of which are widely conserved. Given the rising number of RBPs and their link to human diseases it is quite evident that RBPs have major role in cellular processes and their regulation. The current study is aimed to describe the so far unknown molecular mechanism of CCHC-type Zinc Finger Nucleic Acid Binding Protein (CNBP/ZNF9) function in vivo. CNBP is ubiquitously expressed across various human tissues and is a highly conserved RBP in eukaryotes. It is required for embryonic development in mammals and has been implicated in transcriptional as well as post-transcriptional gene regulation; however, its molecular function and direct target genes remain elusive. Here, we use multiple systems-wide approaches to identify CNBP targets and document the consequences of CNBP binding. We established CNBP as a cytoplasmic RNA-binding-protein and used Photoactivatable Ribonucleoside Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) to identify direct interactions of CNBP with 4178 mRNAs. CNBP preferentially bound a G-rich motif in the target mRNA coding sequences. Functional analyses, including ribosome profiling, RNA sequencing, and luciferase assays revealed the CNBP mode of action on target transcripts. CNBP binding was found to increase the translational efficiency of its target genes. We hypothesize that this is consistent with an RNA chaperone function of CNBP helping to resolve secondary structures, thus promoting translation. Altogether this study provides a novel mechanism of CNBP function in vivo and acts as a step-stone to study the individual CNBP targets that will bring us closer to understand the disease onset.},
  subject      = {CNBP},
  language  = {en}
}
@phdthesis{Cherpokova2017,
  author    = {Cherpokova, Deya},
  title     = {Studies on modulators of platelet (hem)ITAM signaling and platelet production in genetically modified mice},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-120068},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {Summary Platelet activation and aggregation at sites of vascular injury is critical to prevent excessive blood loss, but may also lead to life-threatening ischemic disease states, such as myocardial infarction and stroke. Glycoprotein (GP) VI and C type lectin-like receptor 2 (CLEC-2) are essential platelet activating receptors in hemostasis and thrombo-inflammatory disease which signal through a (hem)immunoreceptor tyrosine-based activation motif (ITAM)-dependent pathway. The adapter molecules Src-like adapter protein (SLAP) and SLAP2 are involved in the regulation of immune cell receptor surface expression and signaling, but their function in platelets is unknown. As revealed in this thesis, single deficiency of SLAP or SLAP2 in mice had only moderate effects on platelet function, while SLAP/SLAP2 double deficiency resulted in markedly increased signal transduction, integrin activation, granule release, aggregation, procoagulant activity and thrombin generation following (hem)ITAM-coupled, but not G protein-coupled receptor activation. Slap-/-/Slap2-/- mice displayed accelerated occlusive arterial thrombus formation and a dramatically worsened outcome after focal cerebral ischemia. These results establish SLAP and SLAP2 as critical inhibitors of platelet (hem)ITAM signaling in the setting of arterial thrombosis and ischemic stroke. GPVI has emerged as a promising novel pharmacological target for treatment of thrombotic and inflammatory disease states, but the exact mechanisms of its immunodepletion in vivo are incompletely understood. It was hypothesized that SLAP and SLAP2 may be involved in the control of GPVI down-regulation because of their role in the internalization of immune cell receptors. As demonstrated in the second part of the thesis, SLAP and SLAP2 were dispensable for antibody-induced GPVI down-regulation, but anti-GPVI treatment resulted in prolonged strong thrombocytopenia in Slap-/-/Slap2-/- mice. The profound thrombocytopenia likely resulted from the powerful platelet activation which the anti-GPVI antibody induced in Slap-/-/Slap2-/- platelets, but importantly, not in wild-type platelets. These data indicate that the expression and activation state of key modulators of the GPVI signaling cascade may have important implications for the safety profile and efficacy of anti-GPVI agents. Small GTPases of the Rho family, such as RhoA and Cdc42, are critically involved in the regulation of cytoskeletal rearrangements during platelet activation, but little is known about the specific roles and functional redundancy of both proteins in platelet biogenesis. As shown in the final part of the thesis, combined deficiency of RhoA and Cdc42 led to marked alterations in megakaryocyte morphology and the generation of platelets of heterogeneous size and granule content. Despite severe hemostatic defects and profound thrombo¬cytopenia, circulating RhoA-/-/Cdc42-/- platelets were still capable of granule secretion and the formation of occlusive thrombi. These results implicate the existence of both distinct and overlapping roles of RhoA and Cdc42 in platelet production and function.},
  subject      = {Thrombozyt},
  language  = {en}
}
@phdthesis{Schmitt2017,
  author    = {Schmitt, Dominik},
  title     = {Structural Characterization of the TFIIH Subunits p34 and p44 from C. thermophilum},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-104851},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {Several important cellular processes, including transcription, nucleotide excision repair and cell cycle control are mediated by the multifaceted interplay of subunits within the general transcription factor II H (TFIIH). A better understanding of the molecular structure of TFIIH is the key to unravel the mechanism of action of this versatile protein complex within these pathways. This becomes especially important in the context of severe diseases like xeroderma pigmentosum, Cockayne syndrome and trichothiodystrophy, that arise from single point mutations in some of the TFIIH subunits. In an attempt to structurally characterize the TFIIH complex, we harnessed the qualities of the eukaryotic thermophile Chaetomium thermophilum, a remarkable fungus, which has only recently been recognized as a novel model organism. Homologues of TFIIH from C. thermophilum were expressed in E. coli, purified to homogeneity and subsequently utilized for crystallization trials and biochemical studies. The results of the present work include the first crystal structure of the p34 subunit of TFIIH, comprising the N-terminal domain of the protein. The structure revealed a von Willebrand Factor A (vWA) like fold, which is generally known to be involved in a multitude of protein-protein interactions. Structural comparison allowed to delineate similarities as well as differences to already known vWA domains, providing insight into the role of p34 within TFIIH. These results indicate that p34 assumes the role of a structural scaffold for other TFIIH subunits via its vWA domain, while likely serving additional functions, which are mediated through its C-terminal zinc binding domain and are so far unknown. Within TFIIH p34 interacts strongly with the p44 subunit, a positive regulator of the XPD helicase, which is required for regulation of RNA Polymerase II mediated transcription and essential for eukaryotic nucleotide excision repair. Based on the p34 vWA structure putative protein-protein interfaces were analyzed and binding sites for the p34 p44 interaction suggested. Continuous crystallization efforts then led to the first structure of a p34 p44 minimal complex, comprising the N-terminal vWA domain of p34 and the C-terminal C4C4 RING domain of p44. The structure of the p34 p44 minimal complex verified the previous hypothesis regarding the involved binding sites. In addition, careful analysis of the complex interface allowed to identify critical residues, which were subsequently mutated and analyzed with respect to their significance in mediating the p34 p44 interaction, by analytical size exclusion chromatography, electrophoretic mobility shift assays and isothermal titration calorimetry. The structure of the p34 p44 complex also revealed a binding mode of the p44 C4C4 RING domain, which differed from that of other known RING domains in several aspects, supporting the hypothesis that p44 contains a novel variation of this domain.},
  subject      = {DNA-Reparatur},
  language  = {en}
}
@phdthesis{Hagen2017,
  author    = {Hagen, Franziska},
  title     = {Sphingolipids in gonococcal infection},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-153852},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {Neisseria gonorrhoeae, the causative agent of the sexually transmitted disease gonorrhea, has the potential to spread in the human host and cause a severe complication called disseminated gonococcal infection (DGI). The expression of the major outer membrane porin PorBIA is a characteristic of most gonococci associated with DGI. PorBIA binds to the scavenger receptor expressed on endothelial cells (SREC-I), which mediates the so-called low phosphate-dependent invasion (LPDI). This uptake mechanism enables N. gonorrhoeae to rapidly invade epithelial and endothelial cells in a phosphate-sensitive manner. We recently demonstrated that the neutral sphingomyelinase, which catalyses the hydrolysis of sphingomyelin to ceramide and phosphorylcholine, is required for the LPDI of gonococci in non-phagocytic cells. Neutral sphingomyelinase 2 (NSM2) plays a key role in the early PorBIA signaling by recruiting the PI3 kinase to caveolin. The following activation of the PI3 kinase-dependent downstream signaling leads to the engulfment of the bacteria. As a part of this work, I could confirm the involvement of the NSM2. The role of the enzyme was further elucidated by the generation of antibodies directed against NSM2 and the construction of an epithelium-based NSM2 knockout cell line using CRISPR/Cas9. The knockout of the NSM2 strongly inhibits the LPDI. The invasion could be, however, restored by the complementation of the knockout using an NSM2-GFP construct. However, the results could not be reproduced. In this work, I could show the involvement of further members of the sphingolipid pathway in the PorBIA-mediated invasion. Lipidome analysis revealed an increase of the bioactive molecules ceramide and sphingosine due to gonococcal infection. Both molecules do not only affect the host cell, but seem to influence the bacteria as well: while ceramide seems to be incorporated by the gonococci, sphingosine is toxic for the bacteria. Furthermore, the sphingosine kinase 2 (SPHK2) plays an important role in invasion, since the inhibition and knockdown of the enzyme revealed a negative effect on gonococcal invasion. To elucidate the role of the sphingosine kinases in invasion in more detail, an activity assay was established in this study. Additionally, the impact of the sphingosine-1-phosphate lyase (S1PL) on invasion was investigated. Inhibitor studies and infection experiments conducted with a CRISPR/Cas9 HeLa S1PL knockout cell line revealed a role of the enzyme not only in the PorBIA-mediated invasion, but also in the Opa50/HSPG-mediated gonococcal invasion. The signaling experiments allowed the categorization of the SPHK and S1PL activation in the context of infection. Like the NSM2, both enzymes play a role in the early PorBIA signaling events leading to the uptake of the bacteria. All those findings indicate an important role of sphingolipids in the invasion and survival of N. gonorrhoeae. In the last part of this work, the role of the NSM2 in the inhibition of apoptosis in neutrophils due to gonococcal infection was investigated. It could be demonstrated that the delayed onset of apoptosis is independent of neisserial porin and Opa proteins. Furthermore, the influence of neisserial peptidoglycan on PMN apoptosis was analysed using mutant strains, but no connection could be determined. Since the NSM2 is the most prominent sphingomyelinase in PMNs, fulfils manifold cell physiological functions and has already been connected to apoptosis, the impact of the enzyme on apoptosis inhibition due to gonococcal infection was investigated using inhibitors, with no positive results.},
  subject      = {gonococcal},
  language  = {en}
}