@phdthesis{Mueller2017,
  author    = {M{\"u}ller, Stephanie},
  title     = {Plant thermotolerance: The role of heat stress-induced triacylglycerols in \(Arabidopsis\) \(thaliana\)},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-152829},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {Plants are exposed to high temperature, especially during hot summer days. Temperatures are typically lowest in the morning and reach a maximum in the afternoon. Plants can tolerate and survive short-term heat stress even on hot summer days. A. thaliana seedlings have been reported to tolerate higher temperatures for different time periods, a phenomenon that has been termed basal thermotolerance. In addition, plants have the inherent capacity to acclimate to otherwise lethal temperatures. Arabidopsis thaliana seedlings acclimate at moderately elevated temperatures between 32-38° C. During heat acclimation, a genetically programmed heat shock response (HSR) is triggered that is characterized by a rapid activation of heat shock transcription factors (HSFs), which trigger a massive accumulation of heat shock proteins that are chiefly involved in protein folding and protection. Although the HSF-triggered heat-shock response is well characterized, little is known about the metabolic adjustments during heat stress. The aim of this work was to get more insight into heat-responsive metabolism and its importance for thermotolerance. In order to identify the response of metabolites to elevated temperatures, global metabolite profiles of heat-acclimated and control seedlings were compared. Untargeted metabolite analyses revealed that levels of polyunsaturated triacylglycerols (TG) rapidly increase during heat acclimation. TG accumulation was found to be temperature-dependent in a temperature range from 32-50° C (optimum at 42° C). Heat-induced TG accumulation was localized in extra-chloroplastic compartments by chloroplast isolation as well as by fluorescence microscopy of A. thaliana cell cultures. Analysis of mutants deficient in all four HSFA1 master regulator genes or the HSFA2 gene revealed that TG accumulation occurred independently to HSF. Moreover, the TG response was not limited to heat stress since drought and salt stress (but not short-term osmotic, cold and high light stress) also triggered an accumulation of TGs. In order to reveal the origin of TG synthesis, lipid analysis was carried out. Heat-induced accumulation of TGs does not derive from massive de novo fatty acid (FA) synthesis. On the other hand, lipidomic analyses of A. thaliana seedlings indicated that polyunsaturated FA from thylakoid galactolipids are incorporated into cytosolic TGs during heat stress. This was verified by lipidomic analyses of A. thaliana fad7/8 transgenic seedlings, which displayed altered FA compositions of plastidic lipids. In addition, wild type A. thaliana seedlings displayed a rapid conversion of plastidic monogalactosyldiacylglycerols (MGDGs) into oligogalactolipids, acylated MGDGs and diacylglycerols (DGs). For TG synthesis, DG requires a FA from the acyl CoA pool or phosphatidylcholine (PC). Seedlings deficient in phospholipid:diacylglycerol acyltransferase1 (PDAT1) were unable to accumulate TGs following heat stress; thus PC appears to be the major FA donor for TGs during heat treatment. These results suggest that TG and oligogalactolipid accumulation during heat stress is driven by post-translationally regulated plastid lipid metabolism. TG accumulation following heat stress was found to increase basal thermotolerance. Pdat1 mutant seedlings were more sensitive to severe heat stress without prior acclimatization, as revealed by a more dramatic decline of the maximum efficiency of PSII and lower survival rate compared to wild type seedlings. In contrast, tgd1 mutants over-accumulating TGs and oligogalactolipids displayed a higher basal thermotolerance compared to wild type seedlings. These results therefore suggest that accumulation of TGs increases thermotolerance in addition to the genetically encoded heat shock response.},
  subject      = {Triglyceride},
  language  = {en}
}
@phdthesis{Schwab2017,
  author    = {Schwab, Andrea},
  title     = {Development of an osteochondral cartilage defect model},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-155617},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {The limited intrinsic self-healing capability of articular cartilage requires treatment of cartilage defects. Material assisted and cell based therapies are in clinical practice but tend to result in formation of mechanical inferior fibro-cartilage in long term follow up. If a lesion has not been properly restored degenerative diseases are diagnosed as late sequela causing pain and loss in morbidity. Complex three dimensional tissue models mimicking physiological situation allow investigation of cartilage metabolism and mechanisms involved in repair. A standardized and reproducible model cultured under controllable conditions ex vivo to maintain tissue properties is of relevance for comparable studies. Topic of this thesis was the establishment of an cartilage defect model that allows for testing novel biomaterials and investigate the effect of defined defect depths on formation of repair tissue. In part I an ex vivo osteochondral defect model was established based on isolation of porcine osteochondral explants (OCE) from medial condyles, 8 mm in diameter and 5 mm in height. Full thickness cartilage defects with 1 mm to 4 mm in diameter were created to define ex vivo cartilage critical size after 28 days culture with custom developed static culture device. In part II of this thesis hydrogel materials, namely collagen I isolated from rat tail, commercially available fibrin glue, matrix-metalloproteinase clevable poly(ethylene glycol) polymerized with heparin (starPEGh), methacrylated poly(N-(2-hydroxypropyl) methacrylamide mono-dilactate-poly(ethylene glycol) triblock copolymer/methacrylated hyaluronic acid (MP/HA), thiol functionalized HA/allyl functionalized poly(glycidol) (P(AGE/G)-HA-SH), were tested cell free and chondrocyte loaded (20 mio/ml) as implant in 4 mm cartilage defects to investigate cartilage regeneration. Reproducible chondral defects, 8 mm in diameter and 1 mm in height, were generated with an artificial tissue cutter (ARTcut®) to investigate effect of defect depth on defect regeneration in part III. In all approaches OCE were analyzed by Safranin-O staining to visualize proteoglycans in cartilage and/or hydrogels. Immuno-histological and -fluorescent stainings (aggrecan, collagen II, VI and X, proCollagen I, SOX9, RUNX2), gene expression analysis (aggrecan, collagen II and X, SOX9, RUNX2) of chondrocyte loaded hydrogels (part II) and proteoglycan and DNA content (Part I \& II) were performed for detailed analysis of cartilage regeneration. Part I: The development of custom made static culture device, consisting of inserts in which OCE is fixed and deep well plate, allowed tissue specific media supply without supplementation of TGF � . Critical size diameter was defined to be 4 mm. Part II: Biomaterials revealed differences in cartilage regeneration. Collagen I and fibrin glue showed presence of cells migrated from OCE into cell free hydrogels with indication of fibrous tissue formation by presence of proCollagen I. In chondrocyte loaded study cartilage matrix proteins aggrecan, collagen II and VI and transcription factor SOX9 were detected after ex vivo culture throughout the two natural hydrogels collagen I and fibrin glue whereas markers were localized in pericellular matrix in starPEGh. Weak stainings resulted for MP/HA and P(AGE/G)-HA-SH in some cell clusters. Gene expression data and proteoglycan quantification supported histological findings with tendency of hypertrophy indicated by upregulation of collagen X and RunX2 in MP/HA and P(AGE/G)-HA-SH. Part III: In life-dead stainings recruitment of cells from OCE into empty or cell free collagen I treated chondral defects was seen. Separated and tissue specific media supply is critical to maintain ECM composition in cartilage. Presence of OCE stimulates cartilage matrix synthesis in chondrocyte loaded collagen I hydrogel and reduces hypertrophy compared to free swelling conditions and pellet cultures. Differences in cartilage repair tissue formation resulted in preference of natural derived polymers compared to synthetic based materials. The ex vivo cartilage defect model represents a platform for testing novel hydrogels as cartilage materials, but also to investigate the effect of cell seeding densities, cell gradients, cell co-cultures on defect regeneration dependent on defect depth. The separated media compartments allow for systematic analysis of pharmaceutics, media components or inflammatory cytokines on bone and cartilage metabolism and matrix stability.},
  subject      = {Hyaliner Knorpel},
  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{Horn2017,
  author    = {Horn, Hannes},
  title     = {Analysis and interpretation of (meta-)genomic data from host-associated microorganisms},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-152035},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {Host-microbe interactions are the key to understand why and how microbes inhabit specific environments. With the scientific fields of microbial genomics and metagenomics, evolving on an unprecedented scale, one is able to gain insights in these interactions on a molecular and ecological level. The goal of this PhD thesis was to make (meta-)genomic data accessible, integrate it in a comparative manner and to gain comprehensive taxonomic and functional insights into bacterial strains and communities derived from two different environments: the phyllosphere of Arabidopsis thaliana and the mesohyl interior of marine sponges. This thesis focused first on the de novo assembly of bacterial genomes. A 5-step protocol was developed, each step including a quality control. The examination of different assembly software in a comparative way identified SPAdes as most suitable. The protocol enables the user to chose the best tailored assembly. Contamination issues were solved by an initial filtering of the data and methods normally used for the binning of metagenomic datasets. This step is missed in many published assembly pipelines. The described protocol offers assemblies of high quality ready for downstream analysis. Subsequently, assemblies generated with the developed protocol were annotated and explored in terms of their function. In a first study, the genome of a phyllosphere bacterium, Williamsia sp. ARP1, was analyzed, offering many adaptions to the leaf habitat: it can deal with temperature shifts, react to oxygen species, produces mycosporins as protection against UV-light, and is able to uptake photosynthates. Further, its taxonomic position within the Actinomycetales was infered from 16S rRNA and comparative genomics showing the close relation between the genera Williamsia and Gordonia. In a second study, six sponge-derived actinomycete genomes were investigated for secondary metabolism. By use of state-of-the-art software, these strains exhibited numerous gene clusters, mostly linked to polykethide synthases, non-ribosomal peptide synthesis, terpenes, fatty acids and saccharides. Subsequent predictions on these clusters offered a great variety of possible produced compounds with antibiotic, antifungal or anti-cancer activity. These analysis highlight the potential for the synthesis of natural products and the use of genomic data as screening toolkit. In a last study, three sponge-derived and one seawater metagenomes were functionally compared. Different signatures regarding the microbial composition and GC-distribution were observed between the two environments. With a focus on bacerial defense systems, the data indicates a pronounced repertoire of sponge associated bacteria for bacterial defense systems, in particular, Clustered Regularly Interspaced Short Palindromic Repeats, restriction modification system, DNA phosphorothioation and phage growth limitation. In addition, characterizing genes for secondary metabolite cluster differed between sponge and seawater microbiomes. Moreover, a variety of Type I polyketide synthases were only found within the sponge microbiomes. With that, metagenomics are shown to be a useful tool for the screening of secondary metabolite genes. Furthermore, enriched defense systems are highlighted as feature of sponge-associated microbes and marks them as a selective trait.},
  subject      = {Bakterien},
  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}
}
@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{Schmitt2017,
  author    = {Schmitt, Dominique},
  title     = {Initial characterization of mouse Syap1 in the nervous system: Search for interaction partners, effects of gene knockdown and knockout, and tissue distribution with focus on the adult brain},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-147319},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {The synapse-associated protein of 47 kDa (Sap47) in Drosophila melanogaster is the founding member of a phylogenetically conserved protein family of hitherto unknown molecular function. Sap47 is localized throughout the entire neuropil of adult and larval brains and closely associated with glutamatergic presynaptic vesicles of larval motoneurons. Flies lacking the protein are viable and fertile and do not exhibit gross structural or marked behavioral deficiencies indicating that Sap47 is dispensable for basic synaptic function, or that its function is compensated by other related proteins. Syap1 - the mammalian homologue of Sap47 - was reported to play an essential role in Akt1 phosphorylation in various non-neuronal cells by promoting the association of mTORC2 with Akt1 which is critical for the downstream signaling cascade for adipogenesis. The function of Syap1 in the vertebrate nervous system, however, is unknown so far. The present study provides a first description of the subcellular localization of mouse Syap1 in cultured motoneurons as well as in selected structures of the adult mouse nervous system and reports initial functional experiments. Preceding all descriptive experiments, commercially available Syap1 antibodies were tested for their specificity and suitability for this study. One antibody raised against the human protein was found to recognize specifically both the human and murine Syap1 protein, providing an indispensable tool for biochemical, immunocytochemical and immunohistochemical studies. In the course of this work, a Syap1 knockout mouse was established and investigated. These mice are viable and fertile and do not show obvious changes in morphology or phenotype. As observed for Sap47 in flies, Syap1 is widely distributed in the synaptic neuropil, particularly in regions rich in glutamatergic synapses but it was also detected at perinuclear Golgi-associated sites in certain groups of neuronal somata. In motoneurons the protein is especially observed in similar perinuclear structures, partially overlapping with Golgi markers and in axons, dendrites and axonal growth cones. Biochemical and immunohistochemical analyses showed widespread Syap1 expression in the central nervous system with regionally distinct distribution patterns in cerebellum, hippocampus or olfactory bulb. Besides its expression in neurons, Syap1 is also detected in non-neuronal tissue e.g. liver, kidney and muscle tissue. In contrast, non-neuronal cells in the brain lack the typical perinuclear accumulation. First functional studies with cultured primary motoneurons on developmental, structural and functional aspects reveal no influence of Syap1 depletion on survival and morphological features such as axon length or dendritic length. Contrary to expectations, in neuronal tissues or cultured motoneurons a reduction of Akt phosphorylation at Ser473 or Thr308 was not detected after Syap1 knockdown or knockout.},
  subject      = {Synapse},
  language  = {en}
}
@phdthesis{LeBlancSoto2017,
  author    = {Le Blanc Soto, Solange},
  title     = {Role of FGF signaling in the adipogenic and osteogenic differentiation of human bone marrow stromal cells in a three-dimensional \(in\) \(vitro\) model},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-147659},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {Adult human skeletal stem cells are considered to give rise to the bone marrow stromal compartment, including bone-forming osteoblasts and marrow adipocytes. Reduced osteogenesis and enhanced adipogenesis of these skeletal progenitors may contribute to the bone loss and marrow fat accumulation observed during aging and osteoporosis, the main disorder of bone remodeling. Concordantly, in vitro evidence indicates that adipogenic and osteogenic differentiation of human bone marrow stromal cells (hBMSCs) display an inverse relationship under numerous conditions. Hence, the identification of factors modulating inversely both differentiation pathways is of great therapeutic interest. Based on mRNA expression analysis of inversely regulated genes after switching differentiation conditions, our group had previously proposed that fibroblast growth factor 1 (FGF1) might play such a modulator role in hBMSC differentiation. The main aim of this work was, therefore, to investigate the role of FGF1 signaling in the adipogenic and osteogenic differentiation of hBMSCs using a three-dimensional (3D) culture system based on collagen type I hydrogels in order to better mimic the natural microenvironment. Adipogenic and osteogenic differentiation of hBMSCs embedded in collagen gels was successfully established. Treatment with recombinant human FGF1 (rhFGF1), as well as rhFGF2, throughout differentiation induction was found to exert a dose-dependent inhibitory effect on adipogenesis in hBMSCs. This inhibitory effect was found to be reversible and dependent on FGF receptors (FGFR) signaling, given that simultaneous pharmacological blockage of FGFRs rescued adipogenic differentiation. Additionally, matrix mineralization under osteogenic induction was also inhibited by rhFGF1 and rhFGF2 in a dose-dependent manner. A transient treatment with rhFGF1 and rhFGF2 during an expansion phase, however, enhanced proliferation of hBMSCs without affecting the differentiation capacity, although matrix mineralization under osteogenic conditions was hindered. Additionally, rhFGF1 and rhFGF2 treatments affected the matrix remodeling ability of hBMSCs, which displayed alterations in the cytoskeletal phenotype and the expression patterns of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). On the other hand, inhibition of FGFR signaling throughout differentiation induction elicited a strong enhancement of matrix mineralization under osteogenic conditions but had no significant effect on adipocyte formation under adipogenic induction. IX In conclusion, FGF1 and FGF2 signaling was found to support the expansion of bone marrow stromal precursors with adipogenic and osteogenic capacities, to hinder adipogenic and osteogenic differentiation if continuously present during differentiation induction and to alter the matrix remodeling ability of hBMSCs within a 3D collagenous microenvironment.},
  subject      = {Fettzelle},
  language  = {en}
}
@phdthesis{Moradi2017,
  author    = {Moradi, Mehri},
  title     = {Differential roles of α-, β- and γ-actin isoforms in regulation of cytoskeletal dynamics and stability during axon elongation and collateral branch formation in motoneurons},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-147453},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {In highly polarized cells like neurons, cytoskeleton dynamics play a crucial role in establishing neuronal connections during development and are required for adult plasticity. Actin turnover is particularly important for neurite growth, axon path finding, branching and synaptogenesis. Motoneurons establish several thousand branches that innervate neuromuscular synapses (NMJs). Axonal branching and terminal arborization are fundamental events during the establishment of synapses in motor endplates. Branching process is triggered by the assembly of actin filaments along the axon shaft giving rise to filopodia formation. The unique contribution of the three actin isoforms, α-, β- and γ-actin, in filopodia stability and dynamics during this process is not well characterized. Here, we performed high resolution in situ hybridization and qRT-PCR and showed that in primary mouse motoneurons α-, β- and γ-actin isoforms are expressed and their transcripts are translocated into axons. Using FRAP experiments, we showed that transcripts for α-, β- and γ-actin become locally translated in axonal growth cones and translation hot spots of the axonal branch points. Using live cell imaging, we showed that shRNA depletion of α-actin reduces dynamics of axonal filopodia which correlates with reduced number of collateral branches and impairs axon elongation. Depletion of β-actin correlates with reduced dynamics of growth cone filopoida, disturbs axon elongation and impairs presynaptic differentiation. Also, depletion of γ-actin impairs axonal growth and decreases axonal filopodia dynamics. These findings implicate that actin isoforms accomplish unique functions during development of motor axons. Depletions of β- and γ-actin lead to compensatory upregulation of other two isoforms. Consistent with this, total actin levels remain unaltered and F-actin polymerization capacity is preserved. After the knockdown of either α- or γ-actin, the levels of β-actin increase in the G-actin pool indicating that polymerization and stability of β-actin filaments depend on α- or γ-actin. This study provides evidence both for unique and overlapping function of actin isoforms in motoneuron growth and differentiation. In the soma of developing motoneurons, actin isoforms act redundantly and thus could compensate for each other's loss. In the axon, α-, β- and γ-actin accomplish specific functions, i.e. β-actin regulates axon elongation and plasticity and α- and γ-actin regulate axonal branching. Furthermore, we show that both axonal transport and local translation of α-, β- and γ-actin isoforms are impaired in Smn knockout motoneurons, indicating a role for Smn protein in RNA granule assembly and local translation of these actin isoforms in primary mouse motoneurons.},
  subject      = {Motoneuron},
  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{Hoffmann2017,
  author    = {Hoffmann, Helene},
  title     = {Identifying regulators of tumor vascular morphology},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-142348},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {In contrast to normal vessels, tumor vasculature is structurally and functionally abnormal. Tumor vessels are highly disorganized, tortuous and dilated, with uneven diameter and excessive branching. Consequently, tumor blood flow is chaotic, which leads to hypoxic and acidic regions in tumors. These conditions lower the therapeutic effectiveness and select for cancer cells that are more malignant and metastatic. The therapeutic outcome could be improved by increasing the functionality and density of the tumor vasculature. Tumor angiogenesis also shows parallels to epithelial to mesenchymal transition (EMT), a process enabling metastasis. Metastasis is a multi-step process, during which tumor cells have to invade the surrounding host tissue to reach the circulation and to be transported to distant sites. We hypothesize that the variability in the phenotype of the tumor vasculature is controlled by the differential expression of key transcription factors. Inhibiting these transcription factors might be a promising way for angiogenic intervention and vascular re-engineering. Therefore, we investigated the interdependence of tumor-, stroma- and immune cell-derived angiogenic factors, transcription factors and resulting vessel phenotypes. Additionally, we evaluated whether transcription factors that regulate EMT are promising targets for vascular remodeling. We used formalin fixed paraffin embedded samples from breast cancer patients, classified according to estrogen-, progesterone- and human epidermal growth factor receptor (HER) 2 status. Establishing various techniques (CD34 staining, laser microdissection, RNA isolation and expression profiling) we systematically analyzed tumor and stroma-derived growths factors. In addition, vascular parameters such as microvessel size, area, circularity and density were assessed. Finally the established expression profiles were correlated with the observed vessel phenotype. As the SNAI1 transcriptional repressor is a key regulator of EMT, we examined the effect of vascular knockdown of Snai1 in murine cancer models (E0771, B16-F10 and lewis lung carcinoma). Among individual mammary carcinomas, but not among subtypes, strong differences of vascular parameters were observed. Also, little difference between lobular carcinomas and ductal carcinomas was found. Vessel phenotype of Her2 enriched carcinomas was similar to that of lobular carcinomas. Vessel morphology of luminal A and B and basal-like tumors resembled each other. Expression of angiogenic factors was variable across subtypes. We discovered an inverse correlation of PDGF-B and VEGF-A with vessel area in luminal A tumors. In these tumors expression of IL12A, an inhibitor of angiogenesis, was also correlated with vessel size. Treatment of endothelial cells with growth factors revealed an increased expression of transcription factors involved in the regulation of EMT. Knockdown of Snai1 in endothelial cells of mice increased tumor growth and decreased hypoxia in the E0771 and the B16-F10 models. In the lewis lung carcinomas, tumor vascularity and biodistribution of doxorubicin were improved. Here, doxorubicin treatment in combination with the endothelial cell-specific knockdown did slow tumor growth. This shows that SNAI1 is important for a tumor's vascularization, with the significance of its role depending on the tumor model. The methods established in this work open the way for the analysis of the expression of key transcription factors in vessels of formalin fixed paraffin embedded tumors. This research enables us to find novel targets for vascular intervention and to eventually design novel targeted drugs to inhibit these targets.},
  subject      = {Antiangiogenese},
  language  = {en}
}
@phdthesis{Meduri2017,
  author    = {Meduri, Rajyalakshmi},
  title     = {Elucidation of an intricate surveillance network for cellular U snRNP homeostasis},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-143173},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {Spliceosomal U-rich small ribonucleoprotein particles (U snRNPs) are the major building blocks of the nuclear pre-mRNA splicing machinery. The core composition of U snRNPs includes the name giving U snRNA and a set of seven common (Sm) proteins termed Sm B/B', D1, D2, D3, E, F and G. These Sm proteins are arranged in the form of a toroidal ring on the single stranded conserved sequence element in the snRNA to form the Sm core domain. Even though U snRNPs assemble spontaneously in vitro, their assembly in vivo requires an amazingly large number of trans-acting assembly factors united in the Protein Arginine Methyltransferase 5 (PRMT5) and the Survival Motor Neuron (SMN) complexes. The cytoplasmic assembly pathway of U snRNPs can be divided into the early and the late phase. The early phase is dominated by the assembly chaperone, pICln, a subunit of the PRMT5 complex. This factor binds to Sm proteins and delivers them in a pICln-bound form to the PRMT5 complex. The early assembly phase then segregates into two lines. In one assembly line, a stable hexameric ring intermediate (6S complex) composed of pICln and the five Sm proteins D1, D2, F, E and G, is formed. This intermediate forms at the PRMT5 complex but dissociates from the latter upon completion of its assembly. Within the 6S complex, these Sm proteins are pre-organized into respective spatial positions adopted in the assembled U snRNP. The other assembly line forms a protein trimer composed of pICln, Sm B/B' and D3, which unlike the 6S complex is not released from the PRMT5 complex. As a consequence of their association with pICln, Sm proteins are kinetically trapped and fail to proceed in the assembly pathway. The late phase of the U snRNP formation is dominated by the SMN complex, which resolves this kinetic trap by dissociating pICln from the pre-organized Sm proteins and, subsequently catalyzes the loading of the Sm proteins on the U snRNA. Even though basic principles of U snRNP assembly have been understood in some detail, the question arises as to why cells employ sophisticated assembly machinery for the assembly despite the reaction occurring spontaneously in vitro. A few studies have shown that the system works towards rendering specificity to the assembly reaction. However, Sm proteins in their free form expose hydrophobic surfaces to the cytosolic solvent. Hence, I reasoned that the assembly machinery of snRNPs might also prevent Sm protein aggregation. In this thesis, I describe the work that leads to the discovery of a multi-layered regulatory network for Sm proteins involving post-transcriptional and post-translational surveillance mechanisms. Here, I show that the reduced level of SMN (a key assembly factor of the late phase) leads to the initial tailback of Sm proteins over pICln followed by the transcriptional down regulation of Sm protein encoding mRNAs. In contrast, depletion of pICln, a key factor of the early phase, results in the retention of Sm proteins on the ribosomes followed by their degradation via autophagy. Furthermore, I show that exceeding levels of Sm proteins over pICln caused by overexpression results in aggregation and mis-localization of Sm proteins. Thus, my findings uncover a complex regulatory network that helps to maintain the cellular U snRNP homeostasis by either preventing or clearing the unassembled Sm protein aggregates when they are not faithfully incorporated into the U snRNPs.},
  language  = {en}
}
@phdthesis{Fuchs2017,
  author    = {Fuchs, Benjamin Felix},
  title     = {Effects of timing and herbivory on a grass-endophyte association and its trophic interactions},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-141465},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {I.) Plant associated microorganisms can affect the plant`s interaction with herbivores and higher trophic levels. For instance, endophytic fungi infecting aerial plant parts of grass species produce bioactive alkaloids that can negatively affect species from higher trophic levels, indicating a defensive mutualism between the grass and the endophyte. However, beneficial insects can also be negatively affected by the endophyte, which might question the mutualistic effect of endophytic fungi. On the other hand, grass-endophytes are affected by environmental conditions and species interactions. Grazing can increase endophyte frequencies in natural habitats. Furthermore, endophyte mediated effects on herbivores are most pronounced during warm summers following rainy springs. In this study, we investigated whether endophyte derived alkaloids cascade up a food chain (chapter II) and whether their concentrations depend on plant age and season (chapter III). Further we analysed, whether altered herbivore phenology affects the endophytic fungus (chapter IV) and whether endophyte derived alkaloid production is induced by different herbivore species (chapter V). II.) In our first experimental study we analysed whether grass-endophyte derived alkaloids decreased the performance of two ladybird species feeding on aphids exclusively reared on endophyte infected grass (6 weeks young grass). Further, we screened species from three trophic levels (grass, herbivores and aphid predators) for their alkaloid content using two year old infected grass as diet for herbivores. We established an UPLC-MS method to detect and quantify the amount of the endophyte derived alkaloids peramine and lolitrem B extracted from the organic plant and insect material. Performance parameters of ladybirds revealed little differences between ladybirds fed on aphids reared on endophyte infected and non-infected grass, which probably resulted from low alkaloid concentrations in the young (6-weeks old) endophyte infected grass used in this part of the study. Alkaloid quantification of the two year old endophyte infected grass, herbivores and aphid predators revealed similar concentrations between grass and aphids, while aphid predators contained approximately half of that amount which still exceeded the bioactive threshold. We conclude that alkaloids produced by grass-endophytes cascade up the food chain and are responsible for fitness disadvantages of higher trophic levels. III.) In the second study we investigated the impact of plant age and seasonal timing on grass-endophyte growth and alkaloid production. Plants were sown in April of 2013 and sampled monthly over 30 consecutive months. Endophyte growth was quantified with real-time PCR (qPCR) and alkaloid concentrations with UPLC-MS. We showed that alkaloid concentrations and fungal growth followed a seasonal rhythmicity and that alkaloid concentrations increased with plant age. Alkaloid concentrations peak during summer, when also herbivore abundances are high. Consequently, we conclude that plant age and season contribute to the toxicity of endophytes on grass herbivores IV.) In the third study we simulated earlier spring arrival of aphids by enhancing aphid abundance on endophyte infected and endophyte-free grass in spring and analysed responses across three trophic levels. Enhanced aphid abundance in spring caused higher aphid abundances during the study period. Predators stayed unaffected by increased herbivore abundances; however they did level aphid numbers within two weeks after arrival on the plants, independent of aphid abundance. Grass-endophyte showed a time delayed growth, two weeks after aphid abundance peak and after predators already controlled aphid infestations on the plants. We conclude that phenology shifts of herbivorous insects can affect multi-trophic interactions leading to desynchronizations between phenologies of interacting species and mismatches in food-webs. V.) In the fourth study we analysed whether herbivores induce endophyte growth and alkaloid production and whether different types of herbivores induce specific alkaloid production. We applied three different herbivore treatments on endophyte infected grass over 18 weeks. Locust herbivory increased the insect deterring alkaloid peramine and clipping of plants (simulation of grazing livestock) increased the vertebrate toxic alkaloid lolitrem B. Aphid herbivory did not affect endophyte derived alkaloid concentrations. Endophyte responses to herbivory were species specific which indicates a primarily plant protecting role of alkaloid synthesis in endophyte infected plants and a close chemical crosstalk between interacting species. VI.) In summary, we showed that endophyte derived alkaloids affect higher trophic levels and that alkaloid concentrations in the plant depend on prevalent herbivore species, plant age and seasonal timing. Our results indicate a close chemical crosstalk between the host plant and the endophytic fungus which is susceptible to environmental changes altering the endophyte`s alkaloid production in plants. We gained insights into the grass-endophyte symbiosis in ecological contexts and conclude that several factors determine the herbivore toxic potential of endophytic fungi and thereby their plant mutualistic or parasitic character. Future studies should investigate the mechanisms behind the herbivore induced alkaloid concentration increase, shown in this thesis, especially whether plant signals mediate the endophyte response. Furthermore it would be interesting to study the induction of indirect endophyte mediated defence and how it affects multi-trophic level interactions.},
  language  = {en}
}
@phdthesis{Schmitt2017,
  author    = {Schmitt, Franziska},
  title     = {Neuronal basis of temporal polyethism and sky-compass based navigation in \(Cataglyphis\) desert ants},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-142049},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {Desert ants of the genus Cataglyphis (Formicinae) are widely distributed in arid areas of the palearctic ecozone. Their habitats range from relatively cluttered environments in the Mediterranean area to almost landmark free deserts. Due to their sophisticated navigational toolkit, mainly based on the sky-compass, they were studied extensively for the last 4 decades and are an exceptional model organism for navigation. Cataglyphis ants exhibit a temporal polyethism: interior workers stay inside the dark nest and serve as repletes for the first ∼2 weeks of their adult life (interior I). They then switch to nursing and nest maintenance (interior II) until they transition to become day-active outdoor foragers after ∼4 weeks. The latter switch in tasks involves a transition phase of ∼2-3 days during which the ants perform learning and orientation walks. Only after this last phase do the ants start to scavenge for food as foragers. In this present thesis I address two main questions using Cataglyphis desert ants as a model organism: 1. What are the underlying mechanisms of temporal polyethism? 2. What is the neuronal basis of sky-compass based navigation in Cataglyphis ants? Neuropeptides are important regulators of insect physiology and behavior and as such are promising candidates regarding the regulation of temporal polyethism in Cataglyphis ants. Neuropeptides are processed from large precursor proteins and undergo substantial post-translational modifications. Therefore, it is crucial to biochemically identify annotated peptides. As hardly any peptide data are available for ants and no relevant genomic data has been recorded for Cataglyphis, I started out to identify the neuropeptidome of adult Camponotus floridanus (Formicinae) workers (manuscript 1). This resulted in the first neuropeptidome described in an ant species - 39 neuropeptides out of 18 peptide families. Employing a targeted approach, I identified allatostatin A (AstA), allatotropin (AT), short neuropeptide F (sNPF) and tachykinin (TK) using mass spectrometry and immunohistology to investigate the distribution of AstA, AT and TK in the brain (manuscript 2). All three peptides are localized in the central complex, a brain center for sensory integration and high-order control of locomotion behavior. In addition, AstA and TK were also found in visual and olfactory input regions and in the mushroom bodies, the centers for learning and memory formation. Comparing the TK immunostaining in the brain of 1, 7 and 14 days old dark kept animals revealed that the distribution in the central complex changes, most prominently in the 14 day old group. In the Drosophila central complex TK modulates locomotor activity levels. I therefore hypothesize that TK is involved in the internal regulation of the interior I-interior II transition which occurs after ∼2 weeks of age. I designed a behavioral setup to test the effect of neuropeptides on the two traits: 'locomotor activity level' and 'phototaxis' (manuscript 3). The test showed that interior I ants are less active than interior II ants, which again are less active than foragers. Furthermore, interior ants are negatively phototactic compared to a higher frequency of positive phototaxis in foragers. Testing the influence of AstA and AT on the ants' behavior revealed a stage-specific effect: while interior I behavior is not obviously influenced, foragers become positively phototactic and more active after AT injection and less active after AstA injection. I further tested the effect of light exposure on the two behavioral traits of interior workers and show that it rises locomotor activity and results in decreased negative phototaxis in interior ants. However, both interior stages are still more negatively phototactic than foragers and only the activity level of interior II ants is raised to the forager level. These results support the hypothesis that neuropeptides and light influence behavior in a stage-specific manner. The second objective of this thesis was to investigate the neuronal basis of skycompass navigation in Cataglyphis (manuscript 4). Anatomical localization of the sky-compass pathway revealed that its general organization is highly similar to other insect species. I further focused on giant synapses in the lateral complex, the last relay station before sky-compass information enters the central complex. A comparison of their numbers between newly eclosed ants and foragers discloses a rise in synapse numbers from indoor worker to forager, suggesting task-related synaptic plasticity in the sky-compass pathway. Subsequently I compared synapse numbers in light preexposed ants and in dark-kept, aged ants. This experiment showed that light as opposed to age is necessary and sufficient to trigger this rise in synapse number. The number of newly formed synapses further depends on the spectral properties of the light to which the ants were exposed to. Taken together, I described neuropeptides in C. floridanus and C. fortis, and provided first evidence that they influence temporal polyethism in Cataglyphis ants. I further showed that the extent to which neuropeptides and light can influence behavior depends on the animals' state, suggesting that the system is only responsive under certain circumstances. These results provided first insight into the neuronal regulation of temporal polyethism in Cataglyphis. Furthermore, I characterized the neuronal substrate for sky-compass navigation for the first time in Cataglyphis. The high level of structural synaptic plasticity in this pathway linked to the interior-forager transition might be particularly relevant for the initial calibration of the ants' compass system.},
  subject      = {Cataglyphis},
  language  = {en}
}
@phdthesis{Schampel2017,
  author    = {Schampel, Andrea},
  title     = {Beneficial therapeutic effects of the L-type calcium channel antagonist nimodipine in experimental autoimmune encephalomyelitis - an animal model for multiple sclerosis},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-148952},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {Multiple sclerosis (MS) is the most prevalent neurological disease of the central nervous system (CNS) in young adults and is characterized by inflammation, demyelination and axonal pathology that result in multiple neurological and cognitive deficits. The focus of MS research remains on modulating the immune response, but common therapeutic strategies are only effective in slowing down disease progression and attenuating the symptoms; they cannot cure the disease. Developing an option to prevent neurodegeneration early on would be a valuable addition to the current standard of care for MS. Based on our results we suggest that application of nimodipine could be an effective way to target both neuroinflammation and neurodegeneration. We performed detailed analyses of neurodegeneration in experimental autoimmune encephalomyelitis (EAE), an animal model of MS, and in in vitro experiments regarding the effect of the clinically well-established L-type calcium channel antagonist nimodipine. Nimodipine treatment attenuated the course of EAE and spinal cord histopathology. Furthermore, it promoted remyelination. The latter could be due to the protective effect on oligodendrocytes and oligodendrocyte precursor cells (OPCs) we observed in response to nimodipine treatment. To our surprise, we detected calcium channel-independent effects on microglia, resulting in apoptosis. These effects were cell type-specific and independent of microglia polarization. Apoptosis was accompanied by decreased levels of nitric oxide (NO) and inducible NO synthase (iNOS) in cell culture as well as decreased iNOS expression and reactive oxygen species (ROS) activity in EAE. Overall, application of nimodipine seems to generate a favorable environment for regenerative processes and could therefore be a novel treatment option for MS, combining immunomodulatory effects while promoting neuroregeneration.},
  subject      = {Nimodipin},
  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{Oesterreich2017,
  author    = {Oesterreich, Babett},
  title     = {Preclinical development of an immunotherapy against antibiotic-resistant Staphylococcus aureus},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-123237},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2017},
  abstract  = {The Gram-positive bacterium Staphylococcus aureus is the leading cause of nosocomial infections. In particular, diseases caused by methicillin-resistant S. aureus (MRSA) are associated with higher morbidity, mortality and medical costs due to showing resistance to several classes of established antibiotics and their ability to develop resistance mechanisms against new antibiotics rapidly. Therefore, strategies based on immunotherapy approaches have the potential to close the gap for an efficient treatment of MRSA. In this thesis, a humanized antibody specific for the immunodominant staphylococcal antigen A (IsaA) was generated and thoroughly characterized as potential candidate for an antibody based therapy. A murine monoclonal antibody was selected for humanization based on its binding characteristics and the ability of efficient staphylococcal killing in mouse infection models. The murine antibody was humanized by CDR grafting and mouse and humanized scFv as well as scFv-Fc fragments were constructed for comparative binding studies to analyse the successful humanization. After these studies, the full antibody with the complete Fc region was constructed as isotype IgG1, IgG2 and IgG4, respectively to assess effector functions, including antibody-dependent killing of S. aureus. The biological activity of the humanized antibody designated hUK-66 was analysed in vitro with purified human PMNs and whole blood samples taken from healthy donors and patients at high risk of S. aureus infections, such as those with diabetes, end-stage renal disease, or artery occlusive disease (AOD). Results of the in vitro studies show, that hUK-66 was effective in antibody-dependent killing of S. aureus in blood from both healthy controls and patients vulnerable to S. aureus infections. Moreover, the biological activity of hUK-66 and hUK-66 combined with a humanized anti-alpha-toxin antibody (hUK-tox) was investigated in vivo using a mouse pneumonia model. The in vivo results revealed the therapeutic efficacy of hUK-66 and the antibody combination of hUK-66 and hUK-tox to prevent staphylococcal induced pneumonia in a prophylactic set up. Based on the experimental data, hUK-66 represents a promising candidate for an antibody-based therapy against antibiotic resistant MRSA.},
  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{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{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}
}