@phdthesis{Araragi2013, author = {Araragi, Naozumi}, title = {Electrophysiological investigation of two animal models for emotional disorders - serotonin transporter knockout mice and tryptophan hydroxylase 2 knockout mice}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-83265}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {Serotonin (5-HT) has been implicated in the regulation of emotions as well as in its pathological states, such as anxiety disorders and depression. Mice with targeted deletion of genes encoding various mediators of central serotonergic neurotransmission therefore provides a powerful tool in understanding contributions of such mediators to homeostatic mechanisms as well as to the development of human emotional disorders. Within this thesis a battery of electrophysiological recordings were conducted in the dorsal raphe nucleus (DRN) and the hippocampus of two murine knockout lines with deficient serotonergic systems. Serotonin transporter knockout mice (5-Htt KO), which lack protein responsible for reuptake of 5-HT from the extracellular space and tryptophan hydroxylase 2 knockout (Tph2 KO) mice, which lack the gene encoding the neuronal 5-HT-synthesising enzyme. First, 5-HT1A receptor-mediated autoinhibition of serotonergic neuron firing in the DRN was assessed using the loose-seal cell-attached configuration. Stimulation of 5-HT1A receptors by a selective agonist, R-8-hydroxy-2-(di-n-propylamino)tetralin (R-8-OH-DPAT), showed a mild sensitisation and a marked desensitisation of these receptors in Tph2 KO and 5-Htt KO mice, respectively. While application of tryptophan, a precursor of 5-HT and a substrate of Tph2, did not cause autoinhibition in Tph2 KO mice due to the lack of endogenously produced 5-HT, data from 5-Htt KO mice as well as heterozygous mice of both KO mice lines demonstrated the presence of autoinhibitory mechanisms as normal as seen in wildtype (WT) controls. When the Tph2-dependent step in the 5-HT synthesis pathway was bypassed by application of 5-hydroxytryptophan (5-HTP), serotonergic neurons of both Tph2 KO and 5-Htt KO mice showed decrease in firing rates at lower concentrations of 5-HTP than in WT controls. Elevated responsiveness of serotonergic neurons from Tph2 KO mice correspond to mild sensitisation of 5-HT1A receptors, while responses from 5-Htt KO mice suggest that excess levels of extracellular 5-HT, created by the lack of 5-Htt, stimulates 5-HT1A receptors strong enough to overcome desensitisation of these receptors. Second, the whole-cell patch clamp recording data from serotonergic neurons in the DRN showed no differences in basic electrophysiological properties between Tph2 KO and WT mice, except lower membrane resistances of neurons from KO mice. Moreover, the whole-cell patch clamp recording from CA1 pyramidal neurons in the hippocampus of 5-Htt KO mice showed increased conductance both at a steady state and at action potential generation. Lastly, magnitude of long-term potentiation (LTP) induced by the Schaffer collateral/commissural pathway stimulation in the ventral hippocampus showed no differences among Tph2 KO, 5-Htt KO, and WT counterparts. Taken together, lack and excess of extracellular 5-HT caused sensitisation and desensitisation of autoinhibitory 5-HT1A receptors, respectively. However, this may not directly translate to the level of autoinhibitory regulation of serotonergic neuron firing when these receptors are stimulated by endogenously synthesised 5-HT. In general, KO mice studied here showed an astonishing level of resilience to genetic manipulations of the central serotonergic system, maintaining overall electrophysiological properties and normal LTP inducibility. This may further suggest existence of as-yet-unknown compensatory mechanisms buffering potential alterations induced by genetic manipulations.}, subject = {Serotonin}, language = {en} } @phdthesis{Asthana2013, author = {Asthana, Manish}, title = {Associative learning - Genetic modulation of extinction and reconsolidation and the effects of transcranial Direct Current Stimulation (tDCS)}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-84158}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {Scientific surveys provide sufficient evidence that anxiety disorders are one of the most common psy-chiatric disorders in the world. The lifetime prevalence rate of anxiety disorder is 28.8\% (Kessler, et al., 2005). The most widely studied anxiety disorders are as follows panic disorder (PD), post-traumatic stress disorder (PTSD), obsessive-compulsive disorder (OCD), social phobia (or social anxiety disorder), specific phobias, and generalized anxiety disorder (GAD). (NIMH Article, 2009). Classical conditioning is the stable paradigm used from the last one century to understand the neurobi-ology of fear learning. Neurobiological mechanism of fear learning is well documented with the condi-tioning studies. In the therapy of anxiety disorders, exposure based therapies are known to be the most effective approaches. Flooding is a form of exposure therapy in which a participant is exposed to the fear situation and kept in that situation until their fear dissipates. The exposure therapy is based on the phenomena of extinction; this means that a conditioned response diminishes if the conditioned stimulus (CS) is repeatedly presented without an unconditioned stimulus (UCS). One problem with extinction as well as with exposure-based therapy is the problem of fear return (for e.g. renewal, spontaneous recov-ery and reinstatement) after successful extinction. Therefore, extinction does not delete the fear memory trace. It has been well documented that memory processes can be modulated or disrupted using several sci-entific paradigms such as behavioral (for e.g. exposure therapy), pharmacological (for e.g. drug manipu-lation), non-invasive stimulation (for e.g. non-invasive stimulation such as electroconvulsive shock (ECS), transcranial magnetic stimulation (TMS), transcranial direct current stimulation (tDCS), etc. However, modulation of memory processes after reactivation or via non-invasive stimulation is still not clear, which is the focus of the current study. In addition, study of genetic variant suggests that genetic differences play a vital role in the psychiatric disorder especially in fear learning. Hence, it is also one of the concerns of the current dissertation to investigate the interaction between gene and reconsolidation of memory. With respect to fear-conditioning, there are three findings in the current dissertation, which are as fol-lows: (i) In the first study we investigated that non-invasive weak electrical stimulation interferes with the consolidation process and disrupts the fear consolidation to attain stable form. This might offer an effective treatment in the pathological memories, for e.g. PTSD, PD, etc. (ii) In the second study we demonstrated whether a brief single presentation of the CS will inhibit the fear recovery. Like earlier studies we also found that reactivation followed by reconsolidation douses fear return. Attenuation of fear recovery was observed in the reminder group compared to the no-reminder group. (iii) Finally, in our third study we found a statistically significant role of brain derived neurotrophic factor (BDNF) polymorphism in reconsolidation. Results of the third study affirm the involvement of BDNF variants (Met vs. Val) in the modulation of conditioned fear memory after its reactivation. In summary, we were able to show in the current thesis modulation of associative learning and recon-solidation via transcranial direct current stimulation and genetic polymorphism.}, subject = {Konditionierung}, language = {en} } @phdthesis{Brede2013, author = {Brede, Christian}, title = {Peripheral alloantigen expression directs the organ specific T cell infiltration after hematopoietic cell transplantation}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-85365}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {In acute graft-versus-host disease (GVHD) alloreactive donor T cells selectively damage skin, liver, and the gastrointestinal tract while other organs are rarely affected. The mechanism of this selective target tissue infiltration is not well understood. We investigated the importance of alloantigen expression for the selective organ manifestation by examining spatiotemporal changes of cellular and molecular events after allogeneic hematopoietic cell transplantation (allo-HCT). To accomplish this we established a novel multicolor light sheet fluorescence microscopy (LSFM) approach for deciphering immune processes in large tissue specimens on a single-cell level in 3 dimensions. We combined and optimized protocols for antibody penetration, tissue clearing, and triple-color illumination to create a method for analyzing intact mouse and human tissues. This approach allowed us to successfully quantify changes in expression patterns of mucosal vascular addressin cell adhesion molecule-1 (MAdCAM-1) and T cell responses in Peyer's patches following allo-HCT. In addition, we proofed that LSFM is suitable to map individual T cell subsets after HCT and detected rare cellular events. We employed this versatile technique to study the role of alloantigen expression for the selective organ manifestation after allo-HCT. Therefore, we used a T cell receptor (TCR) transgenic mouse model of GVHD that targets a single peptide antigen and thereby mimics a major histocompatibility complex (MHC)-matched single antigen mismatched (miHAg-mismatched) HCT. We transplanted TCR transgenic (OT-I) T cells into myeloablatively conditioned hosts that either express the peptide antigen ovalbumin ubiquitously (βa-Ova) or selectively in the pancreas (RIP-mOva), an organ that is normally not affected by acute GVHD. Of note, at day+6 after HCT we observed that OT-I T cell infiltration occurred in an alloantigen dependent manner. In βa-Ova recipients, where antigen was ubiquitously expressed, OT-I T cells infiltrated all organs and were not restricted to gastrointestinal tract, liver, and skin. In RIP-mOva recipients, where cognate antigen was only expressed in the pancreas, OT-I T cells selectively infiltrated this organ that is usually spared in acute GVHD. In conditioned RIP-mOva the transfer of 100 OT-I T cells sufficed to effectively infiltrate and destroy pancreatic islets resulting in 100\% mortality. By employing intact tissue LSFM in RIP-mOva recipients, we identified very low numbers of initial islet infiltrating T cells on day+4 after HCT followed by a massive T cell migration to the pancreas within the following 24 hours. This suggested an effective mechanism of effector T cell recruitment to the tissue of alloantigen expression after initial antigen specific T cell encounter. In chimeras that either expressed the model antigen ovalbumin selectively in hematopoietic or in parenchymal cells only, transplanted OT-I T cells infiltrated target tissues irrespective of which compartment expressed the alloantigen. As IFN-γ could be detected in the serum of transplanted ovalbumin expressing recipients (βa-Ova, βa-Ova-chimeras and RIP-mOva) at day+6 after HCT, we hypothesized that this cytokine may be functionally involved in antigen specific OT-I T cell mediated pathology. In vitro activated OT-I T cells responded with the production of IFN-γ upon antigen re-encounter suggesting that IFN-γ might be relevant in the alloantigen dependent organ infiltration of antigen specific CD8+ T cell infiltration after HCT. Based on these data we propose that alloantigen expression plays an important role in organ specific T cell infiltration during acute GVHD and that initial alloreactive T cells recognizing the cognate antigen propagate a vicious cycle of enhanced T cell recruitment that subsequently culminates in the exacerbation of tissue restricted GVHD.}, subject = {Alloantigen}, language = {en} } @phdthesis{Brill2013, author = {Brill, Martin Fritz}, title = {Processing and plasticity within the dual olfactory pathway in the honeybee brain}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-85600}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {In their natural environment animals face complex and highly dynamic olfactory input. This requires fast and reliable processing of olfactory information, in vertebrates as well as invertebrates. Parallel processing has been shown to improve processing speed and power in other sensory systems like auditory or visual. In the olfactory system less is known about olfactory coding in general and parallel processing in particular. With its elaborated olfactory system and due to their specialized neuroanatomy, honeybees are well-suited model organism to study parallel olfactory processing. The honeybee possesses a unique neuronal architecture - a dual olfactory pathway. Two mirror-imaged output projection neuron (PN) pathways connect the first olfactory processing stage, the antennal lobe (analog to the vertebrates olfactory bulb, OB), with the second, the mushroom body (MB) known to be involved in orientation and learning and memory, and the lateral horn (LH). The medial antennal lobe-protocerebral tract (m-APT) first innervates the MB and thereafter the LH, while the other, the lateral-APT (l-APT) projects in opposite direction. The neuroanatomy and evolution of these pathways has been analyzed, yet little is known about its physiology. To analyze the function of the dual olfactory pathway a new established recording method was designed and is described in the first chapter of this thesis (multi-unit-recordings). This is now the first time where odor response from several PNs of both tracts is recorded simultaneously and with high temporal precision. In the second chapter the PN odor responses are analyzed. The major findings are: both tracts responded to all tested odors but with differing characteristics. Since recent studies describe the input to the two tracts being rather similar, the results now indicate differential odor processing along the tracts, therefore this is a good indicator for parallel processing. PNs of the m-APT process odors in a sparse manner with delayed response latencies, but with high odor-specificity. PNs of the l-APT in contrast respond to several odor stimuli and respond in general faster. In some PN originating from both tracts, characteristics of odor-identity coding via response latencies were found. Analyzing the over-all dynamic range of the PNs both l- and m-APT PNs were tested over a large odor concentration range (10-6 to 10-2) (3. chapter). The PNs responded with linear and non-linear correlation of the response strength to the odor concentration. In most cases the l-APT is comparatively more sensitive to low odor concentrations. Response latency decreases with increasing odor concentration in both tracts. Alternative coding principles and elaboration on the hypothesis whether the dual olfactory pathway may contribute coincidental innervation to the next higher-order neurons, the Kenyon cells (KC), is subject of the 4. chapter. Cross-correlations and synchronous responses of both tracts show that in principle odors may be coded via temporal coding. Results suggest that odor processing is enhanced if both tracts contribute to olfactory coding together. In another project the distribution of the inhibitory neurotransmitter GABA (gamma-aminobutyric acid) was measured in the bee's MB during adult maturation (5. chapter). GABAergic inhibition is of high importance in odor coding. An almost threefold decrease in the total amount of GABAergic innervation was found during adult maturation in the l- and m-APT target region, in particular at the change in division of labor during the transition from a young nurse bee to an older forager bee. The results fit well into the current understanding of brain development in the honeybee and other social insects during adult maturation, which was described as presynaptic pruning and KC dendritic outgrowth. Combining anatomical and functional properties of the bee's dual olfactory pathway suggests that both rate and temporal coding are implemented along two parallel streams. Comparison with recent work on analog output pathways of the vertebrate's OB indicates that parallel processing of olfactory information may be a common principle across distant taxa.}, subject = {Tierphysiologie}, language = {en} } @phdthesis{Busch2013, author = {Busch, Martin}, title = {Aortic Dendritic Cell Subsets in Healthy and Atherosclerotic Mice and The Role of the miR-17~92 Cluster in Dendritic Cells}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-71683}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {Atherosclerosis is accepted to be a chronic inflammatory disease of the arterial vessel wall. Several cellular subsets of the immune system are involved in its initiation and progression, such as monocytes, macrophages, T and B cells. Recent research has demonstrated that dendritic cells (DCs) contribute to atherosclerosis, too. DCs are defined by their ability to sense and phagocyte antigens, to migrate and to prime other immune cells, such as T cells. Although all DCs share these functional characteristics, they are heterogeneous with respect to phenotype and origin. Several markers have been used to describe DCs in different lymphoid and non-lymphoid organs; however, none of them has proven to be unambiguous. The expression of surface molecules is highly variable depending on the state of activation and the surrounding tissue. Furthermore, DCs in the aorta or the atherosclerotic plaque can be derived from designated precursor cells or from monocytes. In addition, DCs share both their marker expression and their functional characteristics with other myeloid cells like monocytes and macrophages. The repertoire of aortic DCs in healthy and atherosclerotic mice has just recently started to be explored, but yet there is no systemic study available, which describes the aortic DC compartment. Because it is conceivable that distinct aortic DC subsets exert dedicated functions, a detailed description of vascular DCs is required. The first part of this thesis characterizes DC subsets in healthy and atherosclerotic mice. It describes a previously unrecognized DC subset and also sheds light on the origin of vascular DCs. In recent years, microRNAs (miRNAs) have been demonstrated to regulate several cellular functions, such as apoptosis, differentiation, development or proliferation. Although several cell types have been characterized extensively with regard to the miRNAs involved in their regulation, only few studies are available that focus on the role of miRNAs in DCs. Because an improved understanding of the regulation of DC functions would allow for new therapeutic options, research on miRNAs in DCs is required. The second part of this thesis focuses on the role of the miRNA cluster miR- 17~92 in DCs by exploring its functions in healthy and atherosclerotic mice. This thesis clearly demonstrates for the first time an anti-inflammatory and atheroprotective role for the miR17-92 cluster. A model for its mechanism is suggested.}, subject = {Aorta}, language = {en} } @article{BaeuerleinRiedelBakeretal.2013, author = {B{\"a}uerlein, Carina A. and Riedel, Simone S. and Baker, Jeanette and Brede, Christian and Jord{\´a}n Garrote, Ana-Laura and Chopra, Martin and Ritz, Miriam and Beilhack, Georg F. and Schulz, Stephan and Zeiser, Robert and Schlegel, Paul G. and Einsele, Hermann and Negrin, Robert S. and Beilhack, Andreas}, title = {A diagnostic window for the treatment of acute graft-versus-host disease prior to visible clinical symptoms in a murine model}, series = {BMC Medicine}, journal = {BMC Medicine}, doi = {10.1186/1741-7015-11-134}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-96797}, year = {2013}, abstract = {Background Acute graft-versus-host disease (aGVHD) poses a major limitation for broader therapeutic application of allogeneic hematopoietic cell transplantation (allo-HCT). Early diagnosis of aGVHD remains difficult and is based on clinical symptoms and histopathological evaluation of tissue biopsies. Thus, current aGVHD diagnosis is limited to patients with established disease manifestation. Therefore, for improved disease prevention it is important to develop predictive assays to identify patients at risk of developing aGVHD. Here we address whether insights into the timing of the aGVHD initiation and effector phases could allow for the detection of migrating alloreactive T cells before clinical aGVHD onset to permit for efficient therapeutic intervention. Methods Murine major histocompatibility complex (MHC) mismatched and minor histocompatibility antigen (miHAg) mismatched allo-HCT models were employed to assess the spatiotemporal distribution of donor T cells with flow cytometry and in vivo bioluminescence imaging (BLI). Daily flow cytometry analysis of peripheral blood mononuclear cells allowed us to identify migrating alloreactive T cells based on homing receptor expression profiles. Results We identified a time period of 2 weeks of massive alloreactive donor T cell migration in the blood after miHAg mismatch allo-HCT before clinical aGVHD symptoms appeared. Alloreactive T cells upregulated α4β7 integrin and P-selectin ligand during this migration phase. Consequently, targeted preemptive treatment with rapamycin, starting at the earliest detection time of alloreactive donor T cells in the peripheral blood, prevented lethal aGVHD. Conclusions Based on this data we propose a critical time frame prior to the onset of aGVHD symptoms to identify alloreactive T cells in the peripheral blood for timely and effective therapeutic intervention.}, language = {en} } @phdthesis{Chaudhari2013, author = {Chaudhari, Sweena M.}, title = {Role of Hypoxia-Inducible Factor (HIF) 1α in Dendritic Cells in Immune Regulation of Atherosclerosis}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-91853}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {Atherosclerosis is the underlying cause of cardiovascular diseases and a major threat to human health worldwide. It involves not only accumulation of lipids in the vessel wall but a chronic inflammatory response mediated by highly specific cellular and molecular responses. Macrophages and dendritic cells (DCs) play an essential role in taking up modified lipids and presenting them to T and B lymphocytes, which promote the immune response. Enhanced activation, migration and accumulation of inflammatory cells at the local site leads to formation of atherosclerotic plaques. Atherosclerotic plaques become hypoxic due to reduced oxygen diffusion and high metabolic demand of accumulated cells. The various immune cells experience hypoxic conditions locally and inflammatory stimuli systemically, thus up-regulating Hypoxia-inducible factor 1α. Though the role of HIF1α in macrophages and lymphocytes has been elucidated, its role in DCs still remains controversial, especially with respect to atherosclerosis. In this project work, the role of HIF1α in DCs was investigated by using a cell specific knockout mouse model where HIF1α was deleted in CD11c+ cells. Aortic root sections from atherosclerotic mice showed presence of hypoxia and up-regulation of HIF1α which co-localized with CD11c+ cells. Atherosclerotic splenic DCs also displayed enhanced expression of HIF1α, proving non-hypoxic stimulation of HIF1α due to systemic inflammation. Conditional knockout (CKO) mice lacking HIF1α in CD11c+ cells, under baseline conditions did not show changes in immune responses suggesting effects of HIF1α only under inflammatory conditions. When these mice were crossed to the Ldlr-/- line and placed on 8 weeks of high fat diet, they developed enhanced plaques with higher T-cell infiltration as compared to the wild-type (WT) controls. The plaques were of a complex phenotype, defined by increased percent of smooth muscle cells (SMCs) and necrotic core area and reduced percent of macrophages and DCs. The mice also displayed enhanced T-cell activation and a Th1 bias in the periphery. The CKO DCs themselves exhibited increased expression of IL 12 and a higher capacity to proliferate and polarize naive T cells to the Th1 phenotype in vitro. The DCs also showed decreased expression of STAT3, in line with the inhibitory effects of STAT3 on DC activation seen in previous studies. When STAT3 was overexpressed in DCs in vitro, IL 12 was down-regulated, but its expression increased significantly on STAT3 inhibition using a mutant vector. In addition, when STAT3 was overexpressed in DCs in vivo using a Cre regulated lentiviral system, the mice showed decreased plaque formation compared to controls. Interestingly, the effects of STAT3 modulation were similar in WT and CKO mice, intending that STAT3 lies downstream of HIF1α. Finally, using a chromatin immunoprecipitation assay (ChIP), it was confirmed that HIF1α binds to hypoxia responsive elements (HREs) in the Stat3 gene promoter thus regulating its expression. When DCs lack HIF1α, STAT3 expression is not stimulated and hence IL 12 production by DCs is uninhibited. This excessive IL 12 can activate naive T cells and polarize them to the Th1 phenotype, thereby enhancing atherosclerotic plaque progression. This project thus concludes that HIF1α restrains DC activation via STAT3 generation and prevents excessive production of IL 12 that helps to keep inflammation and atherosclerosis under check.}, subject = {Dendritische Zelle}, language = {en} } @phdthesis{Dunkel2013, author = {Dunkel, Nico}, title = {Regulation of virulence-associated traits of the human fungal pathogen Candida albicans by nitrogen availability}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-83076}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {Nitrogen-regulated pathogenesis describes the expression of virulence attributes as direct response to the quantity and quality of an available nitrogen source. As consequence of nitrogen availability, the opportunistic human fungal pathogen Candida albicans changes its morphology and secretes aspartic proteases [SAPs], both well characterized virulence attributes. C. albicans, contrarily to its normally non-pathogenic relative Saccharomyces cerevisiae, is able to utilize proteins, which are considered as abundant and important nitrogen source within the human host. To assimilate complex proteinaceous matter, extracellular proteolysis is followed by uptake of the degradation products through dedicated peptide transporters (di-/tripeptide transporters [PTRs] and oligopeptide transporters [OPTs]). The expression of both traits is transcriptionally controlled by Stp1 - the global regulator of protein utilization - in C. albicans. The aim of the present study was to elucidate the regulation of virulence attributes of the pathogenic fungus C. albicans by nitrogen availability in more detail. Within a genome wide binding profile of Stp1, during growth with proteins, more than 600 Stp1 target genes were identified, thereby confirming its role in the usage of proteins, but also other nitrogenous compounds as nitrogen source. Moreover, the revealed targets suggest an involvement of Stp1 in the general adaption to nutrient availability as well as in the environmental stress response. With the focus on protein utilization and nitrogen-regulated pathogenesis, the regulation of the major secreted aspartic protease Sap2 - additionally one of the prime examples of allelic heterogeneity in C. albicans - was investigated in detail. Thereby, the heterogezygous SAP2 promoter helped to identify an unintended genomic alteration as the true cause of a growth defect of a C. albicans mutant. Additionally, the promoter region, which was responsible for the differential activation of the SAP2 alleles, was delimited. Furthermore, general Sap2 induction was demonstrated to be mediated by distinct cis-acting elements that are required for a high or a low activity of SAP2 expression. For the utilization of proteins as nitrogen source it is also crucial to take up the peptides that are produced by extracellular proteolysis. Therefore, the function and importance of specific peptide transporters was investigated in C. albicans mutants, unable to use peptides as nitrogen source (opt1Δ/Δ opt2Δ/Δ opt3Δ/Δ opt4Δ/Δ opt5Δ/Δ ptr2Δ/Δ ptr22Δ/Δ septuple null mutants). The overexpression of individual transporters in these mutants revealed differential substrate specificities and expanded the specificity of the OPTs to dipeptides, a completely new facet of these transporters. The peptide-uptake deficient mutants were further used to elucidate, whether indeed proteins and peptides are an important in vivo nitrogen source for C. albicans. It was found that during competitive colonization of the mouse intestine these mutants exhibited wild-type fitness, indicating that neither proteins nor peptides are primary nitrogen sources required to efficiently support growth of C. albicans in the mouse gut. Adequate availability of the preferred nitrogen source ammonium represses the utilization of proteins and other alternative nitrogen sources, but also the expression of virulence attributes, like Sap secretion and nitrogen-starvation induced filamentation. In order to discriminate, whether ammonium availability is externally sensed or determined inside the cell by C. albicans, the response to exterior ammonium concentrations of ammonium-uptake deficient mutants (mep1Δ/Δ mep2Δ/Δ null mutants) was investigated. This study showed that presence of an otherwise suppressing ammonium concentration did not inhibit Sap2 proteases secretion and arginine-induced filamentation in these mutants. Conclusively, ammonium availability is primarily determined inside the cell in order to control the expression of virulence traits. In sum, the present work contributes to the current understanding of how C. albicans regulates expression of virulence-associated traits in response to the presence of available nitrogen sources - especially proteins and peptides - in order to adapt its lifestyle within a human host.}, subject = {Candida albicans}, language = {en} } @phdthesis{Eman2013, author = {Eman, Maher Othman Sholkamy}, title = {In Vitro and In Vivo Analysis of Insulin-Induced Oxidative Stress and DNA Damage}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-69274}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {Hyperinsulinemia, a condition with excessively high insulin blood levels, is related to an increased cancer incidence. Diabetes mellitus, metabolic syndrome, obesity and polycystic ovarian syndrome are the most common of several diseases accompanied by hyperinsulinemia. Since an elevated cancer risk especially for colon and kidney cancers, was reported for those patients, we investigated for the first time the induction of genomic damage by insulin mainly in HT29 (human colon cells), LLC-PK1 (pig kidney cells), HK2 (human kidney cells) and peripheral lymphocytes, and to confirm the genotoxicity of insulin in other cells from different tissues. To ascertain that the insulin effects were not only limited to permanent cell lines, rat primary colon, kidney, liver and fatty tissue cells were also studied. To connect the study and the findings to in vivo conditions, two in vivo models for hyperinsulinemia were used; Zucker diabetic fatty rats in a lean and diabetic state infused with different insulin concentrations and peripheral lymphocytes from type 2 diabetes mellitus patients. First, the human colon adenocarcinoma cells (HT29) showed significant elevation of DNA damage using comet assay and micronucleus frequency analysis upon treatment with 5 nM insulin in standard protocols. Extension of the treatment to 6 days lowered the concentration needed to reach significance to 0.5-1 nM. Insulin enhanced the cellular ROS production as examined by the oxidation of the dyes 2´,7´-dichlorodihydrofluorescein diacetate (H2DCF-DA) and dihydroethidium (DHE). The FPG modified comet assay and the reduction of damage by the radical scavenger tempol connected the insulin-mediatedDNA damage to ROS production. To investigate the sources of ROS upon insulin stimulation, apocynin and VAS2870 as NADPH oxidase inhibitors and rotenone as mitochondrial inhibitor were applied in combination with insulin and all of them led to a reduction of the genomic damage. Investigation of the signaling pathway started by evaluation of the binding of insulin to its receptor and to the IGF-1 receptor. The results showed the involvement of both receptors in the signaling mechanism. Following the activation of both receptors, PI3K activation occurs leading to phosphorylation of AKT which in turn activates two pathways for ROS production, the first related to mitochondria and the second through activation of Rac1 , resulting in the activation of Nox1. Both pathways could be activated through AKT or through the mitochondrial ROS which in turn could activates Nox1. Studying another human colon cancer cell line, Caco-2 and rat primary colon cells in vitro confirmed the effect of insulin on cellular chromatin. We conclude that pathophysiological levels of insulin can cause DNA damage in colon cells, which may contribute to the induction or progression of colon cancer. Second, in kidney cells, insulin at a concentration of 5 nM caused a significant increase in DNA damage in vitro. This was associated with the formation of reactive oxygen species (ROS). In the presence of antioxidants, blockers of the insulin and IGF-1 receptors, and a phosphatidylinositol 3-kinases (PI3K) inhibitor, the insulin mediated DNA damage was reduced. Phosphorylation of AKT was increased and p53 accumulated. Inhibition of the mitochondrial and NADPH oxidase related ROS production reduced the insulin mediated damage. In primary rat cells insulin also induced genomic damage. HK2 cells were used to investigate the mechanistic pathway in the kidney The signaling is identical to the one in the colon cells untill the activation of the mitochondrial ROS production, because after the activation of PI3K activation of Nox4 occurs at the same time across talk between mitochondria and Nox4 activation has been suggested and might play a role in the observed effects. In the in vivo model, kidneys from healthy, lean ZDF rats, which were infused with insulin to yield normal or high blood insulin levels, while keeping blood glucose levels constant, the amounts of ROS and p53 were elevated in the high insulin group compared to the control level group. ROS and p53 were also elevated in diabetic obese ZDF rats. The treatment of the diabetic rats with metformin reduced the DNA oxidation measured as 8-oxodG as well as the ROS production in that group. HL60 the human premyelocytic cells and cultured lymphocytes as models for the hemopoietic system cells showed a significant induction for DNA damage upon treatment with insulin. The diabetic patients also exhibited an increase in the micronucleus formation over the healthy individuals. In the present study, we showed for the first time that insulin induced oxidative stress resulting in genomic damage in different tissues, and that the source of the produced ROS differs between the tissues. If the same mechanisms are active in patients, hyperinsulinemia might cause genomic damage through the induction of ROS contributing to the increased cancer risk, against which the use of antioxidants as well as mitochondrial and NADPH oxidase inhibitors might exert protective effects with cancer preventive potential under certain conditions. Normal healthy human plasma insulin concentrations are in the order of 0.04 nM after overnight fasting and increase to less than about 0.2 nM after a meal. Pathophysiological levels can reach 1 nM and can stay above 0.2 nM for the majority of the daytime yielding condictions close to the insulin concentrations determined in the present study. Whether the observed effects also occur in vivo and whether they actually initiate or promote tumor formation remains to be determined. However, if proof of that can be obtained, our experiments with inhibitors indicate chances for pharmacological intervention applying antioxidants or enzyme inhibitors. It will not be the aim to reduce ROS in any case or as much as possible because ROS have now been recognized as important signaling molecules and participatants in immune defense, but a reduction to physiological levels instead of pathophysiological levels in the context of a disease associated with ROS overproduction might be beneficial.}, subject = {Insulin}, language = {en} } @phdthesis{Fronhofer2013, author = {Fronhofer, Emanuel Alexis}, title = {Beyond classical metapopulations: trade-offs and information use in dispersal ecology}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-85816}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {All animal and plant species must disperse in order to survive. Although this fact may seem trivial, and the importance of the dispersal process is generally accepted, the eco-evolutionary forces influencing dispersal, and the underlying movement elements, are far from being comprehensively understood. Beginning in the 1950s scientists became aware of the central role of dispersal behaviour and landscape connectivity for population viability and species diversity. Subsequently, dispersal has mainly been studied in the context of metapopulations. This has allowed researchers to take into account the landscape level, e.g. for determining conservation measures. However, a majority of theses studies classically did not include dispersal evolution. Yet, it is well known that dispersal is subject to evolution and that this process may occur (very) rapidly, i.e. over short ecological time-scales. Studies that do take dispersal evolution into account, mostly focus on eco-evolutionary forces arising at the level of populations - intra-specific competition or Allee effects, for example - and at the level of landscapes - e.g. connectivity, patch area and fragmentation. Yet, relevant ecological and evolutionary forces can emerge at all levels of biological complexity, from genes and individuals to populations, communities and landscapes. Here, I focus on eco-evolutionary forces arising at the gene- and especially at the individual level. Combining individual-based modelling and empirical field work, I explicitly analyse the influence of mobility trade-offs and information use for dispersal decisions - i.e. individual level factors - during the three phases of dispersal - emigration, transfer and immigration. I additionally take into account gene level factors such as ploidy, sexual reproduction (recombination) and dominance. Mobility-fertility trade-offs may shape evolutionarily stable dispersal strategies and lead to the coexistence of two or more dispersal strategies, i.e. polymorphisms and polyphenisms. This holds true for both dispersal distances (chapter 3) and emigration rates (chapter 4). In sessile organisms - such as trees or corals - maternal investment, i.e. transgenerational trade-offs between maternal fertility and propagule dispersiveness, can be the cause of bimodal and fat-tailed dispersal kernels. However, the coexistence of two or more dispersal strategies may be critically dependent on gene level factors, such as ploidy or dominance (chapter 4). Passively dispersing individuals may realize such multimodal dispersal kernels by mixing different dispersal vectors. Active choice of these vectors allows to optimize the kernel. As most animals have evolved some kind of memory and sensory apparatus - chemical, acoustic or optical sensors - it is obvious that these capacities should be used for dispersal decisions. Chapter 5 explores the use of chemical cues for vector choice in passively dispersed animals. I find that the neotropical phoretic flower mites Spadiseius calyptrogynae non-randomly mix different dispersal vectors, i.e. one short- and one long-distance disperser, in order to achieve fat-tailed dispersal kernels. Such kernels allow an optimal exploitation of patchily distributed habitats. In addition, this strategy increases the probability of successful immigration as the short-distance dispersal vectors show directed dispersal towards suitable habitats. Results from individual-based simulations support and explain my empirical findings. The use of memory and sensory apparatus in dispersal is also the main topic of chapter 6 which strives to bridge the gap between dispersal and movement ecology. In this part of my thesis I develop a model of non-random, memory-based animal movement strategies. Extending the movement ecology paradigm of Nathan (2008a) I postulate that four elements may be relevant for the emergence of efficient movement strategies: perception, memory, inference and anticipation. Movement strategies including these four elements optimize search efficiency at two scales: within patches and between patches. This leads to a significantly increased search efficiency over a comparable area restricted search strategy. These four chapters are completed by a general analysis of metapopulation dynamics (chapter 2). I find that although the metapopulation concept is very popular in theoretical ecology, classical metapopulations can be predicted to be rare in nature, as suggested by lacking empirical evidence. This is especially the case when gene level factors, such as ploidy and sex, are taken into account. In summary, my work analyses the effects of ecological and evolutionary forces arising at the gene- and individual level on the evolution of dispersal and movement strategies. I highlight the importance of including these limiting factors, mechanisms and processes and show how they impact the evolution of dispersal in spatially structured populations. All chapters demonstrate that these forces may have dramatic effects on resulting ecological and evolutionary dynamics. If we intend to understand animal and plant dispersal or movement, it is crucial to include eco-evolutionary forces emerging at all levels of complexity, from genes to communities and landscapes. This endeavour is certainly not purely academic. Particularly nowadays, with rapidly changing landscape structures and anticipated drastic shifts of climatic zones due to global change, dispersal is a factor that cannot be overestimated.}, subject = {Metapopulation}, language = {en} } @phdthesis{Gajewska2013, author = {Gajewska, Agnieszka}, title = {Einfluss von ADORA2A Rezeptorgen Polymorphismus und Koffein auf emotionale und fr{\"u}he Informationsverarbeitungsprozesse - Ein mehrstufiges Modell f{\"u}r die Pathogenese der Panikst{\"o}rung}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-77449}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {Die komplexe Pathogenese von Angst und insbesondere der Panikst{\"o}rung wird sowohl von genetischen Faktoren wie dem Adenosin A2A Rezeptorgen (ADORA2A) 1976T/C Polymorphismus (rs5751876) als auch von neuropsychologischen Faktoren wie einer verzerrten Emotionsverarbeitung und Defiziten in der fr{\"u}hen Informationsverarbeitung beeinflusst. Ziel der vorliegenden doppelblinden, Placebo-kontrollierten Studie war, ein mehrstufiges pathogenetisches Angstmodell zu etablieren, in dem der Einfluss von 300 mg Koffeinzitrat - einem Antagonisten am Adenosin A2A Rezeptor - versus Placebo 1) auf den emotionspotenzierten Startlereflex (negative, neutrale und positive Bilder aus dem International Affective Picture System (IAPS) sowie zus{\"a}tzlich panikspezifisches Bildmaterial) an 115 gesunden Probanden (m = 57, w = 58) und 2) auf die fr{\"u}he Informationsverarbeitung (Prepulse-Modifikation (PPM)-Paradigma mit Interstimulus Intervallen (ISI) von 60, 120, 240, 480 und 2000 ms) an vorwiegend derselben Stichprobe von 114 gesunden Probanden (m = 57, w = 57) getestet wurde. Die Probanden wurden dabei f{\"u}r die genetische ADORA2A 1976T/C Variante stratifiziert und mittels des Angstsensitivit{\"a}ts-Index (ASI) f{\"u}r Angstsensitivit{\"a}t (AS) charakterisiert. Zus{\"a}tzlich zum erwarteten Haupteffekt der Bildkategorien (h{\"o}chste Startlemagnituden f{\"u}r negative, niedrigste f{\"u}r positive Bilder) konnte eine Genotyp X Intervention Interaktion auf die Bildkategorien beobachtet werden: Sowohl Tr{\"a}gerschaft des ADORA2A 1976TT Risikogenotyps unter Placebo als auch der Konsum von Koffein bei ADORA2A 1976CC/CT Nicht-Risikogenotyptr{\"a}gerschaft stellten ein Risiko f{\"u}r eine {\"a}hnliche, d.h. undifferenzierte physiologische Erregung in Antwort auf negative und neutrale Reize und damit wom{\"o}glich f{\"u}r eine erh{\"o}hte Angstbereitschaft dar. In {\"U}bereinstimmung mit dem hypothetisierten multifaktoriellen Risikomodell potenzierte Koffein in Synergie mit dem ADORA2A 1976TT Risikogenotyp die Startlereaktion spezifisch f{\"u}r negative emotionale Reize. Dieser Effekt wurde maßgeblich durch eine hohe Angstsensitivit{\"a}t verursacht. Die h{\"o}chsten Startlemagnituden nach Koffeineinnahme bei negativen Bildern zeigten sich insbesondere in der weiblichen Stichprobe. Bei panikspezifischen Bildern f{\"u}hrte Koffein bei ADORA2A 1976CC/CT Nicht-Risikogenotyptr{\"a}gern dazu, dass im Vergleich zu Placebo weniger zwischen negativen und Panikbildern unterschieden wurde. Bei ADORA2A 1976TT Risikogenotyptr{\"a}gern ergaben sich bzgl. der panikspezifischen Bilder weder unter Koffein- noch unter Placebobedingungen Unterschiede. Bez{\"u}glich der fr{\"u}hen Informationsverarbeitung konnte eine Vierfachinteraktion zwischen Genotyp, Intervention, Geschlecht und ISI beobachtet werden. Eine Stratifikation nach ISI ergab, dass die Prepulse Inhibition (PPI) nach Koffeineinnahme f{\"u}r das ISI von 120 ms und von 240 ms bei weiblichen ADORA2A 1976TT Risikogenotyptr{\"a}gern im Vergleich zu m{\"a}nnlichen ADORA2A 1976TT Homozygoten eingeschr{\"a}nkt war, w{\"a}hrend es keine signifikanten Effekte bei ADORA2A 1976CC/CT Nicht-Risikogenotyptr{\"a}gern oder in der Placebogruppe gab. Nur bei hoch {\"a}ngstlichen Probanden konnte ein signifikanter Interventionseffekt mit verminderter Prepulse Fazilitation (PPF; ISI von 2000 ms) unter Koffein beobachtet werden. Unsere Ergebnisse weisen auf ein komplexes, mehrstufiges und potenziell geschlechtsspezifisches pathogenetisches Angstmodell hin, bei dem genetische und biochemische Faktoren interaktiv das Risiko f{\"u}r defizit{\"a}re emotionale Verarbeitungsprozesse und somit m{\"o}glicherweise auch f{\"u}r Angstst{\"o}rungen erh{\"o}hen. Die Ergebnisse zeigen weiterhin, dass weibliche ADORA2A 1976TT Homozygote unter Koffein eine eingeschr{\"a}nkte F{\"a}higkeit haben, irrelevante sensorische Informationen zu filtern, was die Rolle des adenosinergen Systems bei der Pathogenese von Angst zus{\"a}tzlich st{\"u}tzt. Durch die Definition von multifaktoriellen Risikoprofilen f{\"u}r Angst und insbesondere die Panikst{\"o}rung, wie in der vorliegenden Arbeit exemplarisch demonstriert, k{\"o}nnen in Zukunft Fortschritte in der individuellen Prim{\"a}r- und Sekund{\"a}rpr{\"a}vention erzielt werden.}, subject = {Paniksyndrom}, language = {de} } @phdthesis{Geissler2013, author = {Geissler, Julia Maria}, title = {Neuropsychological Endophenotypes of Attention-Deficit/Hyperactivity Disorder}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-79221}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {Attention-Deficit/Hyperactivity Disorder (ADHD) endophenotypes as a link between phenotype and genotype were the focus of the present work. Candidate endophenotypes were investigated via neuropsychological tasks during the simultaneous recording of a 21-channel electroencephalogram. Since endophenotypes are assumed to more closely reflect genetic variation, the influence of ADHD-associated genes Catechol-O-methyl transferase (COMT), the dopamine transporter (DAT, SLC6A3) and Latrophilin-3 (LPHN3) was analysed. Response inhibition was assessed with a cued Continuous Performance Test, for working memory we used an n-back task, sensory gating was measured via the paired clicks paradigm and response time variability (RTV) was quantified by the standard deviation of reaction times. The sample comprised medicated (N=36) and unmedicated (N=42) ADHD patients and matched control children and adolescents (N=41). The electrophysiological correlate of response inhibition was the centroid location during response execution and inhibition, and the degree of anteriorization (NGA). Sensory gating reflects the attenuation of the P50 response to the second of two auditory stimuli presented in short succession. Working memory was examined during target and non-target trials, reflecting specific information processing stages: early sensory processing (P100 and N100), selection of material (P150), memory retrieval (N300), event categorization (P300) and updating of working memory content (P450). Performance was quantified in terms of omission errors reflecting inattention and false alarms reflecting impulsivity, as well as speed and variability of reactions. Unmedicated ADHD patients had more omission errors and more variable reaction times, pointing to difficulties with attention and state regulation. NGA did not prove an optimal endophenotype candidate, since it was not yet developed in approximately half of the examined children and adolescents. It was independent of diagnosis; however ADHD risk alleles for DAT conferred lower NGA as well as more variable reaction times across groups. DAT genotype interacted with diagnosis on the level of centroid location, however, it did not manifest in performance deficits. In the case of sensory gating, homozygosity for the DAT allele associated with ADHD (10R) conferred impairment. ADHD was only relevant in participants without genetic risk, where patients without medication struggled most with suppression. In the working memory task, DAT modulated the timing of material selection in interaction with cognitive load and diagnosis: under high load unmedicated patients showed delayed responses, while under low load risk carriers on medication had faster responses than controls. Early processing and event-categorization were stronger in unmedicated ADHD with risk genotype, but dampened without risk. An interesting trend emerged for LPHN3, where carrying all risk variants was associated with higher NGA in ADHD patients irrespective of medication. This warrants further study, as the haplotype also exerts a positive influence on sensory gating specifically in patients. At the same time within the genetic risk group, unmedicated patients had the weakest NGA. However, the LPHN3 risk haplotype effected more posterior Go centroids, putatively facilitating response execution, which is supported by a higher number of false alarms. When inhibition was required, the risk variants led to more posterior centroids in unmedicated compared to medicated patients as well as controls, speaking to differences in inhibition-related brain activation. While as expected the risk haplotype led to compromised gating in unmedicated ADHD, this was reversed in healthy controls where the haplotype was acting in a protective manner with enhanced filtering. During working memory operations, the risk haplotype showed stronger N300 responses suggesting investment of more resources. While COMT did not exert an influence on NGA directly, carriers of the risk allele (met) had more posterior centroids both during response execution and inhibition, and displayed more variable responses in addition to being more prone to false alarms. Unmedicated patients produced smaller P300 during successful execution of responses than controls in absence of the risk allele, while with risk they had shorter latencies and presumably tend towards premature reactions. Additionally, it brought out impairments in sensory gating, thus making unmedicated patients less able to filter out irrelevant information, while they were able to compensate with the protective genotype. The influence of COMT on sensory gating seems to be specific for ADHD, as this gene was of no consequence in healthy controls. In the working memory task, met was beneficial for updating as reflected by P450 amplitude. In ADHD irrespective of medication COMT did not change P450 strength, but for controls this effect was observed.}, subject = {Aufmerksamkeits-Defizit-Syndrom}, language = {en} } @phdthesis{GlotzbachSchoon2013, author = {Glotzbach-Schoon, Evelyn}, title = {Contextual fear conditioning in humans: The return of contextual anxiety and the influence of genetic polymorphisms}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-87955}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {Als Angst bezeichnet man einen nicht auf spezifische Objekte gerichteten l{\"a}nger anhaltenden zukunfts-orientierten Zustand der Besorgnis. Diese ist kennzeichnend f{\"u}r Angstst{\"o}rungen wie Panikst{\"o}rung, generalisierte Angstst{\"o}rung und Posttraumatische Belastungsst{\"o}rung (PTBS). Experimentell kann Angst durch kontextuelle Furchtkonditionierung ausgel{\"o}st werden. Bei dieser Art der Konditionierung werden aversive Ereignisse als unvorhersehbar erlebt, wodurch der gesamte Kontext mit der Gefahr assoziiert wird. Diese Arbeit hat zum Ziel, Mechanismen der Entstehung und Aufrechterhaltung von Kontextangst zu untersuchen. Dies sind zum einem erleichterte Akquisition von Kontextkonditionierungen und deren fehlerhafte Extinktion. Hier ist vor allem die Fragestellung relevant, wie dies durch genetische Varianten moduliert wird (Studie 1). Zum anderen soll die Wiederkehr der Angst nach der Extinktion mit einem neuen Reinstatement-Paradigma untersucht werden (Studie 2). Zur Untersuchung dieser Forschungsfragen wurden zwei kontextuelle Furchtkonditionierungsstudien in virtueller Realit{\"a}t (VR) durchgef{\"u}hrt. W{\"a}hrend der Akquisition wurden leicht schmerzhafte elektrische Reize (unkonditionierter Stimulus, US) unvorhersehbar pr{\"a}sentiert, w{\"a}hrend die Probanden in einem virtuellen B{\"u}roraum waren. Dadurch wurde dieser Raum zum Angstkontext (CXT+). Ein zweiter B{\"u}roraum wurde nie mit dem US gepaart, deshalb wurde dieser Raum zum Sicherheitskontext (CXT-). Die Extinktion, in der die Kontexte ohne US pr{\"a}sentiert wurden, fand 24 h sp{\"a}ter statt, und ein Test zum Abruf der Extinktion bzw. zur Wiederkehr der Angst nochmals 24 h sp{\"a}ter. In beiden Studien wurde die Angst auf drei verschiedenen Ebenen gemessen: Verhalten (angstpotenzierter Schreckreflex), Physiologie (tonische Hautleitf{\"a}higkeit), und verbale Ebene (explizite Ratings). Die Probanden f{\"u}r Studie 1 wurden anhand der 5-HTTLPR (S+ Risikoallel vs. LL nicht-Risikoallel) und NPSR1 rs324981 (T+ Risikoallel vs. AA nicht-Risikoallel) Polymorphismen stratifiziert, sodass vier kombinierte Genotyp Gruppen (S+/T+, S+/LL, LL/T+ und LL/AA) mit je 20 Probanden vorlagen. Es zeigte sich, dass der angstpotenzierte Schreckreflex durch die Interaktion zwischen beiden genetischen Polymorphismen moduliert wurde. Nur Tr{\"a}ger beider Risikoallele (S+ Tr{\"a}ger des 5-HTTLPR und T+ Tr{\"a}ger des NPSR1 Polymorphismus) zeigten einen h{\"o}heren Schreckreflex im CXT+ als im CXT- w{\"a}hrend der Akquisition. Der Abruf der Extinktion an Tag 3, gemessen anhand des Schreckreflexes, wurde allerdings nicht durch die Genotypen moduliert. Interessanterweise zeigte sich auf dem expliziten Angstlevel (Valenz- und Angstratings) nur ein Einfluss des NPSR1 Polymorphismus, und zwar bewerteten die nicht-Risikoallel Tr{\"a}ger (AA) den CXT+ mit negativerer Valenz und h{\"o}herer Angst im Vergleich zum CXT-; die Risikoallel Tr{\"a}ger (T+) taten dies nicht. In der zweiten Studie wurde fast das gleiche Paradigma benutzt wie in der ersten Studie mit der Ausnahme, dass eine Versuchsgruppe (Reinstatementgruppe) den US noch einmal am Anfang des dritten Untersuchungstages vor der Pr{\"a}sentation von CXT+ und CXT- appliziert bekam. Die zweite Versuchsgruppe (Kontrollgruppe) erhielt keinen US, sondern wurde direkt durch CXT+ und CXT- gef{\"u}hrt. Es zeigte sich, dass nur in der Reinstatementgruppe die Angst auf impliziter und expliziter Ebene wiederkehrte, d.h. die Probanden zeigten einen h{\"o}heren Schreckreflex und h{\"o}here Angstratings auf den CXT+ im Vergleich zum CXT-. Wichtig war vor allem, dass die Wiederkehr der Angst in der Reinstatementgruppe mit der Ver{\"a}nderung der Zustandsangst und der Stimmung (von der Extinktion zum Test) korrelierte. D.h. je gr{\"o}ßer die Angst und je negativer die Stimmung wurden, desto h{\"o}her war die Wiederkehr der Angst. Zusammengefasst belegt Studie 1, dass erleichterte kontextuelle Furchtkonditionierung auf impliziter Ebene (Schreckreflex) ein Endoph{\"a}notyp f{\"u}r Angstst{\"o}rungen sein k{\"o}nnte, was zu unserem Verst{\"a}ndnis der {\"A}tiologie von Angstst{\"o}rungen beitragen k{\"o}nnte. Die Ergebnisse der zweiten Studie legen nahe, dass eine {\"a}ngstliche und negative Stimmung nach der Extinktion die R{\"u}ckkehr von Angst beg{\"u}nstigen k{\"o}nnte. Dar{\"u}ber hinaus scheint das VR-basierte kontextuelle Furchtkonditionierungsparadigma ein geeignetes Mittel zu sein, um Mechanismen der Angstentstehung und Angstwiederkehr experimentell zu erforschen. Weiterf{\"u}hrende Studien k{\"o}nnten nun auch Angstpatienten untersuchen und das Paradigma auf evolution{\"a}r-relevante Kontexte (z.B. H{\"o}he, Dunkelheit, weite Pl{\"a}tze) ausweiten.}, subject = {Angst}, language = {en} } @phdthesis{Gschwendtner2013, author = {Gschwendtner, Kathrin M.}, title = {Von den Genen zum Verhalten: Der Einfluss des COMT Val158Met Polymorphismus auf visuell-r{\"a}umliche Aufmerksamkeitslenkung bei emotionalen Verarbeitungsprozessen}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-83278}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {Der Catechol-O-Methyltransferase (COMT) Val158Met Polymorphismus (rs4680) ist am Abbau von Dopamin und Noradrenalin im menschlichen Gehirn beteiligt. In bisherigen Studien konnte gezeigt werden, dass das Met-Allel mit einer erh{\"o}hten Reaktivit{\"a}t auf negative Stimuli assoziiert ist. Auf Basis der Tonischen/ Phasischen Dopaminhypothese wird postuliert, dass diese erh{\"o}hte Reaktivit{\"a}t auf negative Reize durch defizit{\"a}re Disengagementprozesse verursacht sein k{\"o}nnte. Das Ziel dieser Arbeit war es daher, diese theoretische Annahme mithilfe von Blickbewegungsmessungen zu {\"u}berpr{\"u}fen und zu untersuchen, ob die erh{\"o}hte Reaktivit{\"a}t sich auch in verl{\"a}ngerten Disengagementlatenzen von negativen Reizen widerspiegelt. Es wurden daf{\"u}r drei Studien durchgef{\"u}hrt, in denen eine adaptierte Version der emotionalen Antisakkadenaufgabe in Verbindung mit einer Blickbewegungsmessung eingesetzt wurde. In der zweiten Studie wurde zus{\"a}tzlich eine EEG-Messung durchgef{\"u}hrt. Außerdem wurde in der dritten Studie die Aufmerksamkeitslokation manipuliert. In der ersten und zweiten Studie zeigte sich nicht wie erwartet ein linearer Effekt in Relation zum COMT Val158Met Polymorphismus, sondern ein Heterosiseffekt. Dieser Effekt zeigte sich nur in der einfacheren Prosakkadenbedingung. In der ersten Studie wurde der Heterosiseffekt bei negativen Reizen gefunden, wohingegen in der zweiten Studie der Heterosiseffekt nur in einer EEG- Komponente, der Early Posterior Negativity (EPN), aber sowohl bei positiven als auch negativen Reizen gefunden wurde. In der dritten Studie zeigte sich kein Genotypeffekt. Es wird vermutet, dass der COMT Effekt in der emotionalen Verarbeitung aufgabenspezifisch sein k{\"o}nnte und daher, neben linearen Zusammenh{\"a}ngen, unter bestimmten Umst{\"a}nden auch ein Heterosiseffekt auftreten kann. Die Ergebnisse sollten nicht auf eine m{\"a}nnliche Stichprobe generalisiert werden, da in allen Studien lediglich weibliche Versuchspersonen teilnahmen.}, subject = {Dopaminstoffwechsel}, language = {de} } @phdthesis{Heimberger2013, author = {Heimberger, Tanja}, title = {Der Hitzeschock-Transkriptionsfaktor 1 (HSF1) als neues potenzielles Ziel im Multiplen Myelom}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-83390}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {Die evolutionär hoch konservierte Hitzeschock-Antwort (heat shock stress response, HSR) ermöglicht Zellen sich an Stresssituationen anzupassen und so dem programmierten Zelltod zu entgehen. Die Regulation der HSR unterliegt dem Hitzeschock-Transkriptionsfaktor 1 (HSF1), der nach einem Stress-Impuls umgehend die Synthese der Hitzeschock-Proteine (HSP) initiiert. Als molekulare Chaperone assistieren die HSP bei der Faltung und den intrazellulären Transport ihrer Klientenproteine und erhalten so die lebensnotwendigen zellulären Funktionen aufrecht. Während das HSF1/HSP-System vorteilhaft f{\"u}r normale Zellen ist, kann es aber auch den Prozess der malignen Transformation unterst{\"u}tzen. In verschiedenen Tumorentitäten wurde eine Abhängigkeit der malignen Zellen von HSP, vereinzelt auch von HSF1, beschrieben. Im multiplen Myelom (MM) stabilisieren HSP u.a. Klientenproteine in einem komplexen onkogenen Signalnetzwerk und erhalten so eine aberrante Signalweiterleitung aufrecht. Wegen dieser wichtigen Funktion ist die Inhibition der HSP (insbesondere HSP90) bereits ein therapeutischer Ansatzpunkt, der jedoch im MM noch nicht zu dem erhofften Erfolg f{\"u}hrte. Dar{\"u}ber hinaus wurde beobachtet, dass es zu einer Induktion der HSP nach einer Behandlung mit neuen, antitumoralen Medikamenten (Proteasom-, HDAC- und HSP90-Inhibitoren) kommt. Diese kompensatorische Hochregulation der HSP ist assoziiert mit einem Resistenzverhalten gegen{\"u}ber der Therapie und ist somit unerw{\"u}nscht. Die bisherigen Untersuchungen legen aber auch nahe, dass HSF1 selbst eine wichtige Funktion bei der malignen Transformation einnimmt. So wurde gezeigt, dass der funktionelle Verlust von HSF1 vor der onkogenen Ras oder mutierten Trp53 getriebenen Tumorigenese sch{\"u}tzt. In der vorliegenden Arbeit wurden daher die Expression von HSF1, seine Rolle in der HSP-Regulierung und den Beitrag zum Überleben und Resistenzen in MM-Zellen analysiert. Untersuchungen der HSF1-Expression in Knochenmarkbiopsien von Myelompatienten und in Myelomzelllinien zeigten, dass in ca. 50 \% der untersuchten Biopsien und Zelllinien eine hohe HSF1-Expression vorhanden ist. Sowohl der shRNA-vermitteltem Knockdown von HSF1 als auch die pharmakologische Inhibition mit Triptolid induzierten Apoptose in MM-Zellen. Durch Microarrayanalysen nach shRNA-vermitteltem Knockdown und der anschließenden Verifikation {\"u}ber Western Blot konnte gezeigt werden, dass nach der HSF1-Depletion zahlreiche HSP (HSP90, HSP70, HSP40 and HSP27) vermindert exprimiert wurden. Einzelne Knockdown Experimente der HSP40 und HSP27 f{\"u}hrten zu moderaten Zellsterben, so dass geschlussfolgert werden kann, dass die gleichzeitige Minderung multipler HSP, durch die Depletion von HSF1, zu einem summierten starken apoptotischen Effekt f{\"u}hrt. In weiteren Studien stellte sich heraus, dass in MM-Zellen, trotz des deregulierten Systems und der aberrant hohen Expression der HSP, eine Stressantwort ausgelöst werden kann. Dies konnte durch den „klassischen" Hitzschock und durch die Behandlung mit pharmakologischen Inhibitoren von HSP90 und des Proteasoms erreicht werden. Diese unerw{\"u}nschte Reaktion auf Therapeutika wird vermutlich durch einen kompensatorischen Zellrettungsmechanismus ausgelöst, der zu Resistenzen gegen{\"u}ber der Behandlung f{\"u}hren kann. Durch die Inhibition von HSF1 mit Triptolid und durch shRNA-vermitteltem Knockdown, konnte diese zelluläre Antwort unterbunden werden. Dar{\"u}ber hinaus f{\"u}hrte die pharmakologische Inhibition von HSF1 in Kombination mit HSP90 (mit NVP-AUY922) oder Proteasom-Inhibition (mit Bortezomib) zu einem verstärkten apoptotischen Effekt in MM-Zellen. Zusammenfassend deuten diese Ergebnisse darauf hin, dass HSF1 essenziell f{\"u}r die Regulation der HSP im MM ist. Dar{\"u}ber hinaus kann die Inhibition von HSF1, besonders in Kombination mit HSP90- oder Proteasom-Inhibition, eine neue hoffnungsvolle Therapieoption f{\"u}r Myelompatienten darstellen.}, subject = {Plasmozytom}, language = {de} } @phdthesis{Heydenreich2013, author = {Heydenreich, Nadine}, title = {Studies on the contact-kinin system and macrophage activation in experimental focal cerebral ischemia}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-94534}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {Traditionally, ischemic stroke has been regarded as the mere consequence of cessation of cerebral blood flow, e.g. due to the thromboembolic occlusion of a major brain supplying vessel. However, the simple restoration of blood flow via thrombolysis and/or mechanical recanalization alone often does not guarantee a good functional outcome. It appears that secondary detrimental processes are triggered by hypoxia and reoxygenation, which are referred to as ischemia/reperfusion (I/R) injury. During recent years it became evident that, beside thrombosis inflammation and edema formation are key players in the pathophysiology of cerebral ischemia. The contact-kinin system represents an interface between thrombotic, inflammatory and edematous circuits. It connects the intrinsic coagulation pathway with the plasma kallikrein-kinin system (KKS) via coagulation factor FXII. The serine protease inhibitor C1-inhibitor (C1-INH) has a wide spectrum of inhibitory activities and counteracts activation of the contact-kinin system at multiple levels. The first part of the thesis aimed to multimodally interfere with infarct development by C1-INH and to analyze modes of actions of human plasma derived C1-INH Berinert® P in a murine model of focal cerebral ischemia. It was shown that C57BL/6 mice following early application of 15.0 units (U) C1-INH, but not 7.5 U developed reduced brain infarctions by ~60\% and less neurological deficits in the model of transient occlusion of the middle cerebral artery (tMCAO). This protective effect was preserved at more advanced stages of infarction (day 7), without increasing the risk of intracerebral bleeding or affecting normal hemostasis. Less neurological deficits could also be observed with delayed C1-INH treatment, whereas no improvement was achieved in the model of permanent MCAO (pMCAO). Blood-brain-barrier (BBB) damage, inflammation and thrombosis were significantly improved following 15.0 U C1-INH application early after onset of ischemia. Based on its strong antiedematous, antiinflammatory and antithrombotic properties C1-INH constitutes a multifaceted therapeutic compound that protects from ischemic neurodegeneration in 'clinically meaningful' settings. The second part of the thesis addresses the still elusive functional role of macrophages in the early phase of stroke, especially the role of the macrophage-specific adhesion molecule sialoadhesin (Sn). For the first time, sialoadhesin null (Sn-/-) mice, homozygous deficient for Sn on macrophages were subjected to tMCAO to assess the clinical outcome. Neurological and motor function was significantly improved in Sn-/- mice on day 1 after ischemic stroke compared with wildtype (Sn+/+) animals. These clinical improvements were clearly detectable even on day 3 following tMCAO. Infarctions on day 1 were roughly the same size as in Sn+/+ mice and did not grow until day 3. No intracerebral bleeding could be detected at any time point of data acquisition. Twenty four hours after ischemia a strong induction of Sn was detectable in Sn+/+ mice, which was previously observed only on perivascular macrophages in the normal brain. Deletion of Sn on macrophages resulted in less disturbance of the BBB and a reduced number of CD11b+ (specific marker for macrophages/microglia) cells, which, however, was not associated with altered expression levels of inflammatory cytokines. To further analyze the function of macrophages following stroke this thesis took advantage of LysM-Cre+/-/IKK2-/- mice bearing a nuclear factor (NF)-ϰB activation defect in the myeloid lineage, including macrophages. Consequently, macrophages were not able to synthesize inflammatory cytokines under the control of NF-ϰB. Surprisingly, infarct sizes and neurological deficits upon tMCAO were roughly the same in conditional knockout mice and respective wildtype littermates. These findings provide evidence that macrophages do not contribute to tissue damage and neurological deficits, at least, not by release of inflammatory cytokines in the early phase of cerebral ischemia. In contrast, Sn which is initially expressed on perivascular macrophages and upregulated on macrophages/microglia within the parenchyma following stroke, influenced functional outcome.}, subject = {Blut-Hirn-Schranke}, language = {en} } @phdthesis{Hofmann2013, author = {Hofmann, Sebastian}, title = {Studies on the function and regulation of CD84, GPVI and Orai2 in genetically modified mice}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-87949}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {Platelet activation and aggregation at sites of vascular injury are essential processes to limit blood loss but they also contribute to arterial thrombosis, which can lead to myocardial infarction and stroke. Stable thrombus formation requires a series of events involving platelet receptors which contribute to adhesion, activation and aggregation of platelets. Regulation of receptor expression by (metallo-)proteinases has been described for several platelet receptors, but the molecular mechanisms are ill-defined. The signaling lymphocyte activation molecule (SLAM) family member CD84 is expressed in immune cells and platelets, however its role in platelet physiology was unclear. In this thesis, CD84 deficient mice were generated and analyzed. In well established in vitro and in vivo assays testing platelet function and thrombus formation, CD84 deficient mice displayed phenotypes indistinguishable from wild-type controls. It was concluded that CD84 in platelets does not function as modulator of thrombus formation, but rather has other functions. In line with this, in the second part of this thesis, a novel regulation mechanism for platelet CD84 was discovered and elucidated. Upon platelet activation, the N-terminus of CD84 was found to be cleaved exclusively by the a disintegrin and metalloproteinase 10 (ADAM10), whereas the intracellular part was cleaved by calpain. In addition, regulation of the platelet activating collagen receptor glycoprotein VI (GPVI) was studied and it was shown that GPVI is in contrast to CD84 differentially regulated by ADAM10 and ADAM17. A novel role of CD84 under pathophysiological conditions was revealed as CD84 deficient mice were protected from ischemic stroke in the model of transient middle cerebral artery occlusion and this protection was based on the lack of CD84 in T cells. Ca2+ is an essential second messenger that facilitates activation of platelets and diverse functions in different eukaryotic cell types. Store-operated Ca2+ entry (SOCE) represents the major mechanism leading to rise in intracellular Ca2+ concentration in non-excitable cells. The Ca2+ sensor STIM1 (stromal interaction molecule 1) and the SOC channel subunit protein Orai1 are established mediators of SOCE in platelets. STIM2 is the major STIM isoform in neurons, but the role of the SOC channel subunit protein Orai2 in platelets and neurons has remained elusive. In the third part of this thesis, Orai2 deficient mice were generated and analyzed. Orai2 was dispensable for platelet function, however, Orai2 deficient mice were protected from ischemic neurodegeneration and this phenotype was attributed to defective SOCE in neurons.}, subject = {Thrombozyt}, language = {en} } @phdthesis{KannenCardoso2013, author = {Kannen Cardoso, Vinicius}, title = {The role of Fluoxetine against preneoplastic lesions and tumors in colon tissue}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-77589}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {Introduction: Colon cancer is one of the major human malignancies worldwide, and much effort has been applied to understand the process of colon carcinogenesis, as well as the role of potential treatments and co-therapeutical agents against it. A growing body of evidence suggests that the use of fluoxetine (FLX), an antidepressant belonging to the selective serotonin reuptake inhibitors (SSRIs), may be associated with a reduced colon cancer risk. However, controversial opinions have been published and an identification of the mechanisms of the activity of FLX on colon cells would help in the clarification of this controversy. Objectives: Using several in vitro and in vivo-based methods and analyses, we aimed to verify whether FLX has antioxidant, pro-oxidant or DNA-damaging potential in standard toxicological assays; to check whether and how FLX could prevent and reduce colon preneoplastic lesions; to ascertain whether FLX has any oncostatic potential against colon tumors; and, to investigate whether FLX activity could be comparable with a known and current applied chemotherapeutic agent against colon cancer. Results: FLX did not have any antioxidant potential in our experiments. Although it did not induce reactive oxygen species (ROS) generation or DNA-damage in fibroblast and colon tumor cell lines, FLX reduced dysplasia and proliferation in two different carcinogen models. Further, a significant decrease in colon stromal reactivity and angiogenesis was found in both carcinogen-induced preneoplasia models. In a xenograft model of colon cancer, FLX shrank tumors, reduced tumor proliferation, arrested cancer cells at the G0/G1 cell-cycle phase, and took ROS generation under control. Such effects were detected together with an intracellular acidification and loss of mitochondrial membrane potential in FLX-treated cells. Modulating mitochondrial respiratory chain, HIF-1 expression and Akt/mTOR signaling pathway, FLX was found to reduce colon tumors similar to the widely used chemotherapeutic agent 5-Fluoracil activity. Conclusion: Our collective data suggest that FLX is a remarkable chemopreventive and oncostatic agent against colon preneoplastic lesions and tumors, acting without DNA-damage or ROS generation.}, subject = {Fluoxetin}, language = {en} } @phdthesis{Kaufmann2013, author = {Kaufmann, Tobias}, title = {Brain-computer interfaces based on event-related potentials: toward fast, reliable and easy-to-use communication systems for people with neurodegenerative disease}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-83441}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {Objective: Brain Computer Interfaces (BCI) provide a muscle independent interaction channel making them particularly valuable for individuals with severe motor impairment. Thus, different BCI systems and applications have been proposed as assistive technology (AT) solutions for such patients. The most prominent system for communication utilizes event-related potentials (ERP) obtained from the electroencephalogram (EEG) to allow for communication on a character-by-character basis. Yet in their current state of technology, daily life use cases of such systems are rare. In addition to the high EEG preparation effort, one of the main reasons is the low information throughput compared to other existing AT solutions. Furthermore, when testing BCI systems in patients, a performance drop is usually observed compared to healthy users. Patients often display a low signal-to-noise ratio of the recorded EEG and detection of brain responses may be aggravated due to internally (e.g. spasm) or externally induced artifacts (e.g. from ventilation devices). Consequently, practical BCI systems need to cope with mani-fold inter-individual differences. Whilst these high demands lead to increasing complexity of the technology, daily life use of BCI systems requires straightforward setup including an easy-to-use graphical user interface that nonprofessionals can handle without expert support. Research questions of this thesis: This dissertation project aimed at bringing forward BCI technology toward a possible integration into end-users' daily life. Four basic research questions were addressed: (1) Can we identify performance predictors so that we can provide users with individual BCI solutions without the need of multiple, demanding testing sessions? (2) Can we provide complex BCI technology in an automated, user-friendly and easy-to-use manner, so that BCIs can be used without expert support at end-users' homes? (3) How can we account for and improve the low information transfer rates as compared to other existing assistive technology solutions? (4) How can we prevent the performance drop often seen when bringing BCI technology that was tested in healthy users to those with severe motor impairment? Results and discussion: (1) Heart rate variability (HRV) as an index of inhibitory control (i.e. the ability to allocate attention resources and inhibit distracting stimuli) was significantly related to ERP-BCI performance and accounted for almost 26\% of variance. HRV is easy to assess from short heartbeat recordings and may thus serve as a performance predictor for ERP-BCIs. Due to missing software solutions for appropriate processing of artifacts in heartbeat data (electrocardiogram and inter-beat interval data), our own tool was developed that is available free of charge. To date, more than 100 researchers worldwide have requested the tool. Recently, a new version was developed and released together with a website (www.artiifact.de). (2) Furthermore, a study of this thesis demonstrated that BCI technology can be incorporated into easy-to-use software, including auto-calibration and predictive text entry. Na{\"i}ve, healthy nonprofessionals were able to control the software without expert support and successfully spelled words using the auto-calibrated BCI. They reported that software handling was straightforward and that they would be able to explain the system to others. However, future research is required to study transfer of the results to patient samples. (3) The commonly used ERP-BCI paradigm was significantly improved. Instead of simply highlighting visually displayed characters as is usually done, pictures of famous faces were used as stimulus material. As a result, specific brain potentials involved in face recognition and face processing were elicited. The event-related EEG thus displayed an increased signal-to-noise ratio, which facilitated the detection of ERPs extremely well. Consequently, BCI performance was significantly increased. (4) The good results of this new face-flashing paradigm achieved with healthy participants transferred well to users with neurodegenerative disease. Using a face paradigm boosted information throughput. Importantly, two users who were highly inefficient with the commonly used paradigm displayed high accuracy when exposed to the face paradigm. The increased signal-to-noise ratio of the recorded EEG thus helped them to overcome their BCI inefficiency. Significance: The presented work at hand (1) successfully identified a physiological predictor of ERP-BCI performance, (2) proved the technology ready to be operated by na{\"i}ve nonprofessionals without expert support, (3) significantly improved the commonly used spelling paradigm and (4) thereby displayed a way to effectively prevent BCI inefficiency in patients with neurodegenerative disease. Additionally, missing software solutions for appropriate handling of artifacts in heartbeat data encouraged development of our own software tool that is available to the research community free of charge. In sum, this thesis significantly improved current BCI technology and enhanced our understanding of physiological correlates of BCI performance.}, subject = {Gehirn-Computer-Schnittstelle}, language = {en} } @phdthesis{Kuehn2013, author = {K{\"u}hn, Andrea}, title = {The molecular interplay of proteins expressed in the sexual stages and the induction of gamete formation in the malaria parasite Plasmodium falciparum}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-98028}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {Transmission of the malaria parasite from man to the mosquito requires the formation of sexual parasite stages, the gametocytes. The gametocytes are the only parasite stage that is able to survive in the mosquito midgut and to undergo further development - gamete formation and fertilization. Numerous sexual stage-specific proteins have been discovered, some of which play crucial roles for parasite transmission. However, the functions of many sexual stage proteins remain elusive. Amongst the sexual stage-specific proteins are the proteins of the PfCCp proteins family, which exhibit numerous adhesion domains in their protein structures. For four members of the protein family, PfCCp1 to PfCCp4 gene-disruptant parasite lines had been already studied. Amongst these, PfCCp2 and PfCCp3 showed an important role for development of the parasites in the mosquito. In the present work the study of gene-disrupted parasites of the PfCCp Protein family was completed. PfCCp5-KO and PfFNPA-KO parasite lines were characterized to a great extent and many properties were similar to those of other PfCCp proteins. The co-dependent expression previously reported to be a phenomenon of PfCCp proteins was also observed in these two mutants, although to lesser extent. When either PfCCp5 or PfFNPA were absent, all other proteins were detected in reduced abundance only. Co-dependent expression manifests exclusively on the protein level. Transcript levels were not altered as RT-PCR showed. Amongst PfCCp proteins numerous proteinproteins interactions are taking place. The previously described multimeric protein complexes also include further sexual stage-specific proteins like Pfs230, Pfs48/45 and Pfs25. Recently, a new component of PfCCp-based multimeric protein complexes had been identified. The protein was named PfWLP1 (WD repeat protein-like protein 1) due to its possession of several WD40 repeats. In the present study expression of this uncharacterized protein was investigated via indirect IFA. It was expressed in asexual blood stages and gametocytes. Upon gamete formation and fertilization its expression ceased. Another sexual stage protein studied in this work was PfactinII. It was shown to be exclusively expressed in sexual stages. In gametocytes it co-localizes with Pfs230 and correct localization of PfactinII depends on presence of Pfs230. Transcript analysis by means of RT-PCR revealed the expression of several components of the IMC in gametocytes. Furthermore, five or six myosin genes encoded in the P. falciparum genome were detected in gametocytes. Gametocyte egress was studied on the ultrastructural level via transmission electron microscopy and an inside-out type of egress was observed. Firstly, the membrane of the parasitophorous vacuole (PVM) was lysed and only thereafter the membrane of the red blood cell (RBCM) ruptured. Furthermore, a new inductor of gametogenesis was identified: The K+/H+ ionophore nigericin induced gametocytes activation in the absence of xanthurenic acid (XA), which is responsible for gamtetocyte activation in the mosquito midgut. Selective permeabilization of RBCM and PVM by the mild detergent saponin, showed that in the absence of these membranes male gametocytes were still able to perceive both XA and the drop in temperature. Thus, the receptors for both factors signaling the parasite transmission to the mosquito, seem to be of parasitic origin. LC/MS/MS analysis confirmed the ability of RBCs to take up XA. With malaria eradication on the agenda of malaria research targeting the sexual stages becomes a crucial part of intervention strategies. The sexual stages are especially attractive target as they represent a population bottleneck. The here reported findings on P. falciparum gametocytes provide several potential candidate proteins for developing tools to interrupt transmission from man to mosquito. Such tools might include Transmission blocking vaccines and drugs.}, subject = {Malaria}, language = {en} } @phdthesis{Langenhorst2013, author = {Langenhorst, Daniela}, title = {Induktion und Aktivierung regulatorischer T-Zellen durch superagonistische Stimulation des CD28 Molek{\"u}ls}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-96700}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {Regulatorische T-Zellen (Tregs) spielen eine ntscheidende Rolle beim Erhalt der Immunhom{\"o}ostase und bei der Kontrolle {\"u}berschießender Immunantworten. Sie k{\"o}nnen anhand ihres Entstehungsortes in im Thymus generierte nat{\"u}rliche Tregs (nTregs) und in der Peripherie generierte induzierte Tregs (iTregs) unterteilt werden. Ihr Ph{\"a}notyp wie auch ihre Funktion werden zu einem großen Teil durch den transkriptionellen Masterregulator Foxp3 kontrolliert. Das kostimulatorische Molek{\"u}l CD28 wird von nTregs f{\"u}r die Differenzierung ben{\"o}tigt und von Tregs und konventionellen T-Zellen (Tkons) f{\"u}r ihre Aktivierung. Superagonistische CD28 spezifische monoklonale Antik{\"o}rper (CD28SA) aktivieren T-Zellen im Gegensatz zu konventionellen anti-CD28 Antik{\"o}rpern ohne zus{\"a}tzliche Ligation des T-Zellrezeptors. Die in vivo Applikation des CD28SA bewirkt eine starke Aktivierung der Tregs und eine pr{\"a}ferentielle Expansion der Tregs gegen{\"u}ber Tkons. Dies erkl{\"a}rt die pr{\"a}ventive und therapeutische Wirkung der CD28SA Behandlung in verschiedenen Krankheitsmodellen bei Nagern. Die erste Anwendung des humanisierten CD28SA TGN1412 f{\"u}hrte in den Testpersonen jedoch zu einem unerwarteten „Cytokine-Release Syndrom". Daher wurde hier am Mausmodell der Zusammenhang zwischen Treg Aktivierung und systemischer Zytokinaussch{\"u}ttung n{\"a}her untersucht. Es konnte gezeigt werden, dass die CD28SA vermittelte Proliferation der T-Zellen abh{\"a}ngig vom CD28 Signal und von parakrinem Interleukin (IL)-2 ist. Durch die in vivo Depletion der Tregs vor der CD28SA Injektion wurde deutlich, dass es auch in M{\"a}usen nach CD28SA Stimulation zu einer systemischen Aussch{\"u}ttung pro-inflammatorischer Zytokine kommt, die jedoch, im Gegensatz zum humanen System, von Tregs effektiv kontrolliert werden kann. Um die ussch{\"u}ttung pro-inflammatorischer Zytokine zu verhindern, w{\"a}re eine zus{\"a}tzliche prophylaktische Behandlung mit Corticosteroiden m{\"o}glich, da diese auch in hohen Dosen die CD28SA vermittelte Aktivierung und Expansion der Tregs nicht beeinflussen. Neben der Expansion wird durch die Stimulation mit CD28SA auch die Produktion des anti-inflammatorischen Zytokins IL-10 in Tregs induziert und so eine genauere Untersuchung des Ursprungs und des Schicksals IL-10 produzierender Tregs erm{\"o}glicht. Diese Tregs exprimieren im Vergleich zu IL-10 negativen Tregs ein h{\"o}heres Niveau an Molek{\"u}len, die mit einer supprimierenden Aktivit{\"a}t verbunden sind. Zudem werden IL-10 Produzenten aufgrund der Ver{\"a}nderung im Expressionsmuster der Migrationsrezeptoren nach der Stimulation von einem lymphknotensuchenden CCR7+CCR5-CCR6- zu einem entz{\"u}ndungssuchenden CCR7-CCR5+CCR6+ Ph{\"a}notyp verst{\"a}rkt in Bereiche mit stattfindender Immunantwort rekrutiert. Schließlich sind IL-10 produzierende Tregs von CD28SA stimu2 lierten M{\"a}usen in vitro st{\"a}rker apoptoseanf{\"a}llig als die IL-10 negativen Tregs. Die Aktivierung der Tregs scheint somit die terminale Differenzierung zu einem IL-10 produzierenden Effektorph{\"a}notyp mit begrenzter Lebensdauer zu induzieren. Dies f{\"u}hrt auch zur Beendigung der Immunsuppression. Die Kombination aus schwachem TZR und starkem CD28 Signal, die die CD28SA Stimulation in naiven T-Zellen ausl{\"o}st, induziert zumindest in vitro abh{\"a}ngig von IL-2 und TGFβ effizient die Expression von Foxp3. Die so generierten iTregs haben, {\"a}hnlich wie konventionell in vitro erzeugte iTregs, in Bezug auf die Expression von Oberfl{\"a}chenmolek{\"u}len und den Methylierungsstatus bestimmter Regionen des Foxp3 Gens einen Ph{\"a}notyp, der zwischen dem von Tkons und Tregs liegt. Da auch die supprimierende Aktivit{\"a}t der iTregs geringer ist als die der ex vivo Tregs bedarf es einer weiteren Optimierung des Stimulationsprotokolls, um diese Zellen f{\"u}r therapeutische Zwecke verwenden zu k{\"o}nnen. Zusammenfassend zeigt diese Arbeit, dass die superagonistische Stimulation des CD28 Molek{\"u}ls ein vielseitig einsetzbares Instrument ist. Einerseits k{\"o}nnen durch die CD28SA Stimulation Tregs polyklonal aktiviert und f{\"u}r therapeutische Zwecke mobilisiert werden und andererseits kann die besondere Art der T-Zellstimulation auch dazu genutzt werden, neue Aspekte von nTregs und iTregs zu untersuchen.}, subject = {Antigen CD28}, language = {de} } @phdthesis{Michalska2013, author = {Michalska, Marta}, title = {Molecular Imaging of atherosclerosis}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-73243}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {Atherosklerose ist eine aktive und progressive Erkrankung, bei der vaskul{\"a}re Adh{\"a}sionsmolek{\"u}le wie VCAM-1 eine entscheidende Rolle durch Steuerung der Rekrutierung von Immunzellen in den fr{\"u}hen und fortgeschrittenen Plaques spielen. Ein zielgerichteter Einsatz von VCAM-1-Molek{\"u}len mit spezifischen Kontrastmitteln ist daher eine M{\"o}glichkeit, die VCAM-1-Expression zu kontrollieren, Plaquewachstum ab einem fr{\"u}hen Zeitpunkt zu visualisieren und eine fr{\"u}he Pr{\"a}vention von Atherosklerose vor Beginn der Thrombusbildung zu etablieren. Des Weiteren bietet die nichtinvasive Magnetresonanz (MR)-Bildgebung den Vorteil der Kombination molekularer und morphologischer Daten. Sie erm{\"o}glicht, mithilfe von entwickelten VCAM-1-markierten Eisenoxidpartikeln, den spezifischen Nachweis entz{\"u}ndlicher Prozesse w{\"a}hrend der Atherosklerose. Diese Arbeit belegt, dass mit dem VCAM-1-Konzept eine vielversprechende Herangehensweise gefunden wurde und dass das, mit spezifischen superparamagnetischen Eisenoxid (USPIO) konjugierte VCAM-1-Peptid, gegen{\"u}ber unspezifischer USPIOs ein erh{\"o}htes Potenzial bei der Untersuchung der Atherosklerose in sich tr{\"a}gt. Im ersten Teil der Arbeit konnte im Mausmodell gezeigt werden, dass gerade das VCAM-1-Molek{\"u}l ein sinnvoller Ansatzpunkt zur Darstellung und Bildgebung von Atherosklerose ist, da in der fr{\"u}hen Phase der Entz{\"u}ndung die vaskul{\"a}ren Zelladh{\"a}sionsmolek{\"u}le {\"u}berexprimiert und auch kontinuierlich, w{\"a}hrend der fortschreitenden Plaquebildung, hochreguliert werden. Weiterhin beschreibt diese Arbeit die Funktionst{\"u}chtigkeit und das Verm{\"o}gen des neu gestalteten USPIO Kontrastmittels mit dem zyklischen Peptid, in seiner Spezialisierung auf die VCAM-1 Erkennung. Experimentelle Studien mit ultra-Hochfeld-MRT erm{\"o}glichten weitere ex vivo und in vivo Nachweise der eingesetzten USPIO-VCAM-1-Partikel innerhalb der Region um die Aortenwurzel in fr{\"u}hen und fortgeschrittenen atherosklerotischen Plaques von 12 und 30 Wochen alten Apolipoprotein E-defizienten (ApoE-/-) M{\"a}usen. Mit ihrer Kombination aus Histologie und Elektronenmikroskopie zeigt diese Studie zum ersten Mal die Verteilung von VCAM-1-markierten USPIO Partikeln nicht nur in luminalem Bereich der Plaques, sondern auch in tieferen Bereichen der medialen Muskelzellen. Dieser spezifische und sensitive Nachweis der fr{\"u}hen und fortgeschrittenen Stadien der Plaquebildung bringt auf molekularer Ebene neue M{\"o}glichkeiten zur Fr{\"u}herkennung von atherosklerotischen Plaques vor dem Entstehen von 8 Rupturen. Im Gegensatz zum USPIO-VCAM-1-Kontrastmittel scheiterten unspezifische USPIO Partikel an der Identifikation fr{\"u}her Plaqueformen und begrenzten die Visualisierung von Atherosklerose auf fortgeschrittene Stadien in ApoE-/- M{\"a}usen.}, subject = {VCAM}, language = {en} } @phdthesis{Mishra2013, author = {Mishra, Shambhavi}, title = {Structural and Functional Characterization of the Enzymes Involved in the Menaquinone Biosynthesis and Benzoate Degradation}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-90848}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {The present work illustrates the structural and biochemical characterization of two diverse proteins, BadI and MenD from Rhodopseudomonas palustris and Staphylococcus aureus, respectively. BadI or 2-ketocyclohexanecarboxyl-CoA is one of the key enzymes involved in the anaerobic degradation of aromatic compounds. The degradation of aromatic compounds is a vital process for the maintenance of the biogeochemical carbon cycle and bioremediation of xenobiotic compounds, which if present at higher concentrations can cause potential hazards to humans. Due to the relatively inert nature of aromatic compounds, enzymes catalyzing their degradation are of special interest for industrial applications. BadI is one of the key enzymes involved in the anaerobic degradation of aromatic compounds into an aliphatic moiety. The major focus of this study was to provide mechanistic insights into the reaction catalyzed by BadI. BadI belongs to the crotonase superfamily and shares high sequence homology with the family members of MenB or dihydroxynaphthoate synthase. BadI is known to catalyze the cleavage of the cyclic ring of 2-ketocyclohexane carboxyl-CoA by hydrolyzing the C-C bond leading to the formation of the aliphatic compound pimelyl CoA. On the other hand MenB catalyzes the condensation reaction of o-succinylbenzoyl-CoA to dihydroxylnaphthoyl-CoA. A comprehensive amino acid sequence analysis between BadI and MenB showed that the active site residues of MenB from Mycobacterium tuberculosis (mtMenB) are conserved in BadI from Rhodopseudomonas palustris. MenB is involved in the menaquinone biosynthesis pathway and is a potential drug target against Mycobacterium tuberculosis as it has no known human homologs. Due to the high homology between MenB and BadI and the inability to obtain MenB-inhibitor complex structures we extended our interest to BadI to explore a potential substitute model for mtMenB as a drug target. In addition, BadI possesses some unique mechanistic characteristics. As mentioned before, it hydrolyzes the substrate via a retro Dieckmann's reaction contrasting its closest homolog MenB that catalyzes a ring closing reaction through a Dieckmann's reaction. Nevertheless the active site residues in both enzymes seem to be highly conserved. We therefore decided to pursue the structural characterization of BadI to shed light on the similarities and differences between BadI and MenB and thereby provide some insights how they accomplish the contrasting reactions described above. We determined the first structures of BadI, in its apo and a substrate mimic bound form. The crystal structures revealed that the overall fold of BadI is similar to other crotonase superfamily members. However, there is no indication of domain swapping in BadI as observed for MenB. The absence of domain swapping is quite remarkable because the domain swapped C-terminal helical domain in MenB provides a tyrosine that is imperative for catalysis and is also conserved in the BadI sequence. Comparison of the active sites revealed that the C-terminus of BadI folds onto its core in such a way that the conserved tyrosine is located in the same position as in MenB and can form interactions with the ligand molecule. The structure of BadI also confirms the role of a serine and an aspartate in ligand interaction, thus validating that the conserved active site triad participates in the enzymatic reaction. The structures also reveal a noteworthy movement of the active site aspartate that adopts two major conformations. Structural studies further illuminated close proximity of the active site serine to a water and chlorine molecule and to the carbon atom at which the carbonyl group of the true substrate would reside. Biochemical characterization of BadI using enzyme kinetics validated that the suggested active site residues are involved in substrate interaction. However, the role of these residues is very distinct, with the serine assuming a major role. Thus, the present work ascertain the participation of putative active site residues and demonstrates that the active site residues of BadI adopt very distinctive roles compared to their closest homolog MenB. The MenD protein also referred to as SEPHCHC (2-succinyl-5-enolpyruvyl-6- hydroxy-3-cyclohexene-1-carboxylic acid) synthase is one of the enzymes involved in menaquinone biosynthesis in Staphylococcous aureus. Though S. aureus is usually considered as a commensal it can act as a remarkable pathogen when it crosses the epithelium, causing a wide spectrum of disorders ranging from skin infection to life threatening diseases. Small colony variants (SCVs), a slow growing, small sized subpopulation of the bacteria has been associated with persistent, recurrent and antibiotic resistant infections. These variants show autotrophy for thiamine, menaquinone or hemin. Menaquinone is an essential component in the electron transport pathway in gram-positive organisms. Therefore, enzymes partaking in this pathway are attractive drug targets against pathogens such as Mycobacterium tuberculosis and Bacillus subtilis. MenD, an enzyme catalyzing the first irreversible step in the menaquinone biosynthetic pathway has been implicated in the SCV phenotype of S. aureus. In the present work we explored biochemical and structural properties of this important enzyme. Our structural analysis revealed that despite its low sequence identity of 28\%, the overall fold of staphylococcal MenD (saMenD) is similar to Escherichia coli MenD (ecMenD) albeit with some significant disparities. Major structural differences can be observed near the active site region of the protein and are profound in the C-terminal helix and a loop near the active site. The loop contains critical residues for cofactor binding and is well ordered only in the ecMenD-ThDP structure, while in the apo and substrate bound structures of ecMenD the loop is primarily disordered. In our saMenD structure the loop is for the first time completely ordered in the apo form and displays a novel conformation of the cofactor-binding loop. The loop adopts an unusual open conformation and the conserved residues, which are responsible for cofactor binding are located too far away to form a productive complex with the cofactor in this conformation. Additionally, biochemical studies in conjugation with the structural data aided in the identification of the substrate-binding pocket and delineated residues contributing to its binding and catalysis. Thus the present work successfully divulged the unique biochemical and structural characteristics of saMenD.}, subject = {Benzoate}, language = {en} } @phdthesis{Niemann2013, author = {Niemann, Sylvia}, title = {Seed Coat Permeability of Active Ingredients}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-79585}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {The seed coat is the barrier controlling exchange of solutes between the plant embryo and its environment. This exchange is of importance for example in the uptake of germination inhibitors or in the uptake of agrochemicals applied as seed treatment. A thorough understanding of the basic mechanisms underlying solute permeation across the seed coat would help to improve the effectiveness of seed treatment formulations. In seed treatment formulations, additives can be used to enhance or decrease mobility or uptake of the active ingredient (AI). In the present study the seed coat barrier properties and the seed coat permeation process was examined with the model species Pisum sativum and with a set of model solutes. The lipophilic fraction of the seed coat was analysed by gas chromatography and mass spectrometry and it was found that the total lipophilic compartment of the seed coat represents 0.61 \% of the weight of a swollen seed coat. The seed is covered by a lipophilic cuticle. The seed coat coverage with cuticular waxes is ten to 18-fold lower than wax coverage of pea leaves, though. In order to examine sorption of solutes in the small lipophilic compartment of the seed coat, seed coat/water partition coefficients were determined. These cover a much smaller range than the corresponding n-octanol/water partition coefficients. The lipophilic sorption compartment as calculated from the seed coat/water partition coefficient data is smaller than the analysed total lipophilic compartment of the seed coat since not all of the lipid components can act as sorption compartment. During seed swelling, the pea seed nearly doubles its weight. The uptake of water is driven by the very low water potential of the dry seed and controlled by the seed coat hydraulic conductivity both of which increase during seed swelling. Depending on the available form of water, water uptake can take place by diffusion from air humidity or by mass flow from liquid water. Water uptake by a seed in moist sand takes place by a combination of both uptake mechanisms. The basic transport mechanism underlying solute permeation of seed coats was analysed by steady-state experiments with a newly devised experimental setup. The permeance P for permeation of the set of model compounds across isolated seed coat halves ranged from 3.34 x 10-8 m s-1 for abamectin to 18.9 x 10-8 m s-1 for caffeine. It was found that solute permeation across the seed coat takes aqueous pathways. This was concluded from the facts that molar volume instead of lipophilicity of the solutes determine permeation and that the temperature effect on permeation is very small. This is in contrast to typical leaf and fruit cuticular uptake where lipophilic pathways dominate. Solute uptake across the seed coat can take place by two different mechanisms both of which take aqueous pathways. Uptake can be by diffusion and in the presence of a bulk flow of water driven by a water potential difference also by solvent drag. The presence of the solvent drag uptake mechanism shows that the aqueous pathways form an aqueous continuum across the seed coat. These findings indicate that the seed coat covering cuticle does not form a continuous barrier enclosing the seed. In order to examine solute uptake across the seed coat under conditions close to a situation taking place in the field, the process of uptake of a seed treatment AI in the field was simulated. In the situation of a treated seed in the field, the seed treatment residue dissolves and then the AI can move either into the surrounding soil or across the seed coat into the seed. Uptake across the seed coat can take place either by diffusion or during seed swelling by the solvent drag mechanism. Since the seed treatment residue depletes over time, non-steady-state uptake takes place. To simulate these processes, laboratory scale seed treatment methods were established to produce treated seeds and isolated treated seed coat halves. Experimental setups for non-steady-state uptake experiments were established with whole treated seeds and with isolated treated seed coat halves as simplified screening tool. By modelling of the AI uptake as a first-order process the rate constant k and the final relative uptake amount Mt→∞ M0-1 were obtained. With k and Mt→∞ M0-1 a quantification and comparison of the uptake curves was possible. Both in the experiments with whole treated seeds and with isolated treated seed coats, uptake of metalaxyl-M was much faster than uptake of sedaxane. In the uptake of a seed treatment AI, not only the solute's molar volume but also its water solubility determine uptake. The solute's water solubility is important for dissolution of the AI from the seed treatment residue and thus determines availability of the AI for uptake. Water solubility also controls the possible concentration in solution and thus the driving force for diffusive uptake. Furthermore, the AI amount taken up by solvent drag is determined by concentration in the inflowing water and thus by water solubility. In the experiments with whole treated seeds the additive effects on uptake were smaller than in the experiments with isolated treated seed coats or not significant. Adigor functions as an emulsifier and can lead to a slight increase of AI mobilisation from the seed treatment residue. NeoCryl A-2099 can cause a slowed down release of the AI from the seed treatment residue. The effects of both additives were smaller than the effect caused by different AI physico-chemical properties. Therefore, the most important factor determining uptake of a seed treatment AI are the AI's physico-chemical properties, especially its water solubility.}, subject = {Samenschale}, language = {en} } @phdthesis{Paknia2013, author = {Paknia, Elham}, title = {Identification of a quality control check-point for the assembly of mRNA-processing snRNPs}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-98744}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {An essential step in eukaryotic gene expression is splicing, i.e. the excision of non-coding sequences from pre-mRNA and the ligation of coding-sequences. This reaction is carried out by the spliceosome, which is a macromolecular machine composed of small nuclear ribonucleoproteins (snRNPs) and a large number of proteins. Spliceosomal snRNPs are composed of one snRNA (or two in case of U4/6 snRNPs), seven common Sm proteins (SmD1, D2, D3, B, E, F, G) and several particle-specific proteins. The seven Sm proteins form a ring shaped structure on the snRNA, termed Sm core domain that forms a structural framework of all spliceosomal snRNPs. In the toroidal Sm core domain, the individual Sm proteins are arranged in the sequence SmE-SmG-SmD3-SmB- SmD1-SmD2-SmF from the first to the seventh nucleotide of the Sm site, respectively. The individual positions of Sm proteins in the Sm core domain are not interchangeable. snRNPs are formed in vivo in a step-wise process, which starts with the export of newly transcribed snRNA to the cytoplasm. Within this compartment, Sm proteins are synthesized and subsequently transferred onto the snRNA. Upon formation of the Sm core and further modifications of snRNA, the snRNP is imported into the nucleus to join the spliceosome. Prior to assembly into snRNPs, Sm proteins exist as specific hetero-oligomers in the cytoplasm. The association of these proteins with snRNA occurs spontaneously in vitro but requires the assistance of two major units, PRMT5- and SMN- complexes, in vivo. The early phase of assembly is critically influenced by the assembly chaperone pICln. This protein pre-organizes Sm proteins to functional building blocks and enables their recruitment onto the PRMT5 complex for methylation. Sm proteins are subsequently released from the PRMT5 complex as pICln bound entities and transferred onto the SMN-complex. The SMN complex then liberates the Sm proteins from the pICln-induced kinetic trap and allows their transfer onto the snRNA. Although the principal roles of SMN- and PRMT5 complexes in the assembly of snRNPs have been established, it is still not clear how newly translated Sm proteins are guided into the assembly line. In this thesis, I have uncovered a new facet of pICln function in the assembly of snRNPs. I have shown that newly synthesized Sm proteins are retained at the ribosome upon termination of translation. Their release is facilitated by pICln, which interacts with the cognate Sm protein hetero-oligomers at their site of synthesis on the ribosome and recruits them into the assembly pathway. Additionally, I have been able to show that the early engagement of pICln with the Sm proteins ensures the flawless oligomerization of Sm proteins and prevents any non-chaperoned release and diffusion of Sm proteins in the cytoplasm. In a second project, I have studied the mechanism of U7 snRNP assembly. This particle is a major component of the 3' end processing machinery of replication dependent histone mRNAs. A biochemical hallmark of U7 is its unique Sm core in which the two canonical Sm proteins D1 and D2 are replaced by so-called "like Sm proteins". The key question I addressed in my thesis was, how this "alternative" Sm core is assembled onto U7 snRNA. I have provided experimental evidence that the assembly route of U7 snRNPs and spliceosomal snRNPs are remarkably similar: The assembly of both particles depends on the same assembly factors and the mechanistic details are similar. It appears that formation of the U7- or spliceosomal- core specific 6S complex is the decisive step in assembly.}, subject = {Small nuclear RNP}, language = {en} } @phdthesis{Reddy2013, author = {Reddy, Edamakanti Chandrakanth}, title = {Role of differential phosphorylation of c-Jun N-terminal domain in degenerative and inflammatory pathways of CNS}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-90748}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {In this study we have investigated the possible role of c-Jun and it's activation by the JNK pathway in neuronal cell death and in the inflammatory response of activated astrocytes. The first part of this thesis focuses on the role of site specific phosphorylation of c-Jun in neuronal cell death. The second part focuses on the function of c-Jun in LPS-mediated activation of Bergmann glia cells. In the nervous system, activation of c-Jun transcription factor by different isoforms of c-Jun N-terminal kinase (JNK) functions in various cellular programs, including neurite outgrowth, repair and apoptosis. Yet, the regulatory mechanism underlying the functional dichotomy of c-Jun remains to be elucidated. Serine (S) 63/73 and threonine (T) 91/93 of c-Jun are the target phosphorylation sites for JNKs in response to various stimuli. Yet, these two groups of phosphorylation sites are differentially regulated in vivo, as the S63/73 sites are promptly phosphorylated upon JNK activation, whereas T91/93 phosphorylation requires a priming event at the adjacent T95 site. In our study, we used cerebellar granule cell (CGC) apoptosis by trophic/potassium (TK) deprivation as a model system to investigate the regulation and function of site-specific c-Jun phosphorylation at the S63 and T91/T93 JNK-sites in neuronal cell death. In this model system, JNK induces pro-apoptotic genes through the c-Jun/Ap-1 transcription factor. On the other side, a survival pathway initiated by lithium leads to repression of pro-apoptotic c-Jun/Ap-1 target genes without interfering with JNK activity. Yet, the mechanism by which lithium inhibits c-Jun activity remains to be elucidated. We found that TK-deprivation led to c-Jun phosphorylation at all three JNK sites. However, immunofluorescence analysis of c-Jun phosphorylation at single cell level revealed that the S63 site was phosphorylated in all c-Jun-expressing cells, whereas the response of T91/T93 phosphorylation was more sensitive, mirroring the switch-like apoptotic response of cerebellar granular cells (CGCs). Furthermore, we observed that lithium impaired c-Jun phosphorylation at T91/93, without interfering with S63/73 phosphorylation or JNK activation, suggesting that T91/T93 phosphorylation triggers c-Jun pro-apoptotic activity. Notably, expression of a c-Jun mutant lacking the T95-priming site for T91/93 phosphorylation (c-Jun A95) mimicked the effect of lithium on both cell death and c-Jun site-specific phosphorylation, whereas it was fully able to induce neurite outgrowth in na{\"i}ve PC12 cells. Vice-versa, a c-Jun mutant bearing aspartate-substitution of T95 overwhelmed lithium-mediate protection of CGCs from TK-deprivation, validating that inhibition of T91/T93/T95 phosphorylation underlies the effect of lithium on cell death. Mass-spectrometry analysis confirmed that c-Jun is phosphorylation at T91/T93/T95 in cells. Moreover, recombinant-JNK phosphorylated c-Jun at T91/T93 in a T95-dependent manner. Based on our results, we propose that T91/T93/T95 phosphorylation of c-Jun functions as a sensitivity amplifier of the JNK cascade, setting the threshold for c-Jun pro-apoptotic activity in neuronal cells. In the central nervous system (CNS), the c-Jun transcription factor has been mainly studied in neuronal cells and coupled to apoptotic and regenerative pathways following brain injury. Besides, several studies have shown a transcriptional role of c-Jun in activated cortical and spinal astrocytes. In contrast, little is known about c-Jun expression and activation in Bergmann glial (BG) cells, the radial cerebellar astrocytes playing crucial roles in cerebellar development and physiology. In this study, we used neuronal/glial cerebellar cultures from neonatal mice to assess putative functions of c-Jun in BG cells. By performing double immunocytochemical staining of c-Jun and two BG specific markers, S100 and GLAST, we observed that c-Jun was highly expressed in radial glial cells derived from Bergmann glia. Bergmann glia-derived cells expressed toll-like receptor (TLR 4) and treatment with bacterial lipopolysaccharide (Le et al.) induced c-Jun phosphorylation at S63, exclusively in BG cells. Moreover, LPS induced IL-1β expression and inhibition of JNK activity abolished both c-Jun phosphorylation and the increase of IL-1β mRNA. Notably, we also observed that LPS failed to induce IL-1β mRNA in neuronal/glial cerebellar cultures generated from conditional knockout mice lacking c-Jun expression in the CNS. These results indicate that c-Jun plays a central role in c-Jun in astroglial-specific induction of IL-1β. Furthermore, we confirmed in vivo that c-Jun is expressed in BG cells, during the formation of the BG monolayer. Altogether, our finding underlines a putative role of c-Jun in astroglia-mediated neuroinflammatory dysfunctions of the cerebellum.}, subject = {Jun}, language = {en} } @phdthesis{Riedel2013, author = {Riedel, Simone Stefanie}, title = {Characterization of the fluorescence protein FP635 for in vivo imaging and establishment of a murine multiple myeloma model for non-invasive imaging of disease progression and response to therapy}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-77894}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {Optical in vivo imaging methods have advanced the fields of stem cell transplantation, graft-versus-host disease and graft-versus-tumor responses. Two well known optical methods, based on the transmission of light through the test animal are bioluminescence imaging (BLI) and fluorescence imaging (FLI). Both methods allow whole body in vivo imaging of the same animal over an extended time span where the cell distribution and proliferation can be visualized. BLI has the advantages of producing almost no unspecific background signals and no necessity for external excitation light. Hence, BLI is a highly sensitive and reliable detection method. Yet, the BLI reporter luciferase is not applicable with common microscopy techniques, therefore abolishing this method for cellular resolution imaging. FLI in turn, presents the appealing possibility to use one fluorescent reporter for whole body imaging as well as cellular resolution applying microscopy techniques. The absorption of light occurs mainly due to melanin and hemoglobin in wavelengths up to 650 nm. Therefore, the wavelength range beyond 650 nm may allow sensitive optical imaging even in deep tissues. For this reason, significant efforts are undertaken to isolate or develop genetically enhanced fluorescent proteins (FP) in this spectral range. "Katushka" also called FP635 has an emission close to this favorable spectrum and is reported as one of the brightest far-red FPs. Our experiments also clearly showed the superiority of BLI for whole body imaging over FLI. Based on these results we applied the superior BLI technique for the establishment of a pre-clinical multiple myeloma (MM) mouse model. MM is a B-cell disease, where malignant plasma cells clonally expand in the bone marrow (BM) of older people, causing significant morbidity and mortality. Chromosomal abnormalities, considered a hallmark of MM, are present in nearly all patients and may accumulate or change during disease progression. The diagnosis of MM is based on clinical symptoms, including the CRAB criteria: increased serum calcium levels, renal insufficiency, anemia, and bone lesions (osteolytic lesions or osteoporosis with compression fractures). Other clinical symptoms include hyperviscosity, amyloidosis, and recurrent bacterial infections. Additionally, patients commonly exhibit more than 30\% clonal BM plasma cells and the presence of monoclonal protein is detected in serum and/or urine. With current standard therapies, MM remains incurable and patients diagnosed with MM between 2001 and 2007 had a 5-year relative survival rate of only 41\%. Therefore, the development of new drugs or immune cell-based therapies is desirable and necessary. To this end we developed the MOPC-315 cell line based syngeneic MM mouse model. MOPC-315 cells were labeled with luciferase for in vivo detection by BLI. We validated the non-invasively obtained BLI data with histopathology, measurement of idiotype IgA serum levels and flow cytometry. All methods affirmed the reliability of the in vivo BLI data for this model. We found that this orthotopic MM model reflects several key features of the human disease. MOPC-315 cells homed efficiently to the BM compartment including subsequent proliferation. Additionally, cells disseminated to distant skeletal parts, leading to the typical multifocal MM growth. Osteolytic lesions and bone remodeling was also detected. We found evidence that the cell line had retained plasticity seen by dynamic receptor expression regulation in different compartments such as the BM and the spleen.}, subject = {Fluoreszenzproteine}, language = {en} } @phdthesis{Schiebel2013, author = {Schiebel, Johannes}, title = {Structure-Based Drug Design on Enzymes of the Fatty Acid Biosynthesis Pathway}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-69239}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {W{\"a}hrend die Wirkung der meisten gebr{\"a}uchlichen Antibiotika auf einer Beeintr{\"a}chtigung wichtiger bakterieller Prozesse beruht, wirken manche Substanzen durch die St{\"o}rung der Zellmembran-Struktur. Da Fetts{\"a}uren ein essentieller Bestandteil von Membran-Phospholipiden sind, stellt die bakterielle Fetts{\"a}urebiosynthese II (FAS-II) einen relativ wenig erforschten, aber dennoch vielversprechenden Angriffspunkt f{\"u}r die Entwicklung neuer Antibiotika dar. Das wichtige Antituberkulotikum Isoniazid blockiert die mykobakterielle Fetts{\"a}urebiosynthese und ruft dadurch morphologische {\"A}nderungen sowie letztlich die Lyse des Bakteriums hervor. Eine wichtige Erkenntnis war, dass Isoniazid den letzten Schritt des FAS-II Elongationszyklus inhibiert, der durch die Enoyl-ACP Reduktase katalysiert wird. Darauf aufbauend wurden mehrere Programme ins Leben gerufen, die sich zum Ziel gesetzt hatten, neue Molek{\"u}le zu entwickeln, welche dieses Protein verschiedener Pathogene hemmen. Die S. aureus Enoyl-ACP Reduktase (saFabI) ist von besonders großem Interesse, da drei vielversprechende Inhibitoren dieses Proteins entwickelt werden konnten, die momentan in klinischen Studien eingehend untersucht werden. Trotz dieser Erfolgsaussichten waren zum Zeitpunkt, als die vorliegenden Arbeiten aufgenommen wurden, keine Kristallstrukturen von saFabI {\"o}ffentlich verf{\"u}gbar. Daher war es eines der Hauptziele dieser Doktorarbeit, auf der Basis von kristallographischen Experimenten atomar aufgel{\"o}ste Modelle f{\"u}r dieses wichtige Protein zu erzeugen. Durch die Entwicklung einer verl{\"a}sslichen Methode zur Kristallisation von saFabI im Komplex mit NADP+ und Diphenylether-Inhibitoren konnten Kristallstrukturen von 17 verschiedenen tern{\"a}ren Komplexen gel{\"o}st werden. Weitere kristallographische Experimente ergaben zwei apo-Strukturen sowie zwei Strukturen von saFabI im Komplex mit NADPH und 2-Pyridon-Inhibitoren. Basierend auf der nun bekannten saFabI-Struktur konnten Molekulardynamik-Simulationen durchgef{\"u}hrt werden, um zus{\"a}tzliche Erkenntnisse {\"u}ber die Flexibilit{\"a}t dieses Proteins zu erhalten. Die so gewonnenen Informationen {\"u}ber die Struktur und Beweglichkeit des Enzyms dienten in Folge als ideale Grundlage daf{\"u}r, den Erkennungsprozess von Substrat und Inhibitor zu verstehen. Besonders bemerkenswert dabei ist, dass die verschiedenen saFabI Kristallstrukturen Momentaufnahmen entlang der Reaktionskoordinate der Ligandenbindung und des Hydrid-Transfers repr{\"a}sentieren. Dabei verschließt der so genannte Substratbindungsloop das aktive Zentrum des Enzyms allm{\"a}hlich. Die außergew{\"o}hnlich hohe Mobilit{\"a}t von saFabI konnte durch molekulardynamische Simulationen best{\"a}tigt werden. Dies legt nahe, dass die beobachteten {\"A}nderungen der Konformation tats{\"a}chlich an der Aufnahme und Umsetzung des Substrates beteiligt sind. Eine Kette von Wassermolek{\"u}len zwischen dem aktiven Zentrum und einer wassergef{\"u}llten Kavit{\"a}t im Inneren des Tetramers scheint f{\"u}r die Beweglichkeit des Substratbindungsloops und somit f{\"u}r die katalysierte Reaktion von entscheidender Bedeutung zu sein. Außerdem wurde die erstaunliche Beobachtung gemacht, dass der adaptive Substratbindungsprozess mit einem Dimer-Tetramer {\"U}bergang gekoppelt ist, welcher die beobachtete positive Kooperativit{\"a}t der Ligandenbindung erkl{\"a}ren kann. Alles in allem weist saFabI im Vergleich zu FabI Proteinen aus anderen Organismen mehrere außergew{\"o}hnliche Eigenschaften auf, die f{\"u}r die Synthese von verzweigten Fetts{\"a}uren n{\"o}tig sein k{\"o}nnten, welche wiederum f{\"u}r die {\"U}berlebensf{\"a}higkeit von S. aureus im Wirt von Bedeutung sind. Diese Erkenntnis k{\"o}nnte erkl{\"a}ren, warum S. aureus selbst bei Anwesenheit von exogenen Fetts{\"a}uren von FAS-II Inhibitoren abget{\"o}tet werden kann. Somit k{\"o}nnen die gewonnenen atomaren saFabI Modelle einen entscheidenden Beitrag zur Entwicklung neuer Hemmstoffe dieses validierten Angriffszieles leisten. Tats{\"a}chlich konnten die neuen Strukturen genutzt werden, um die Bindungsst{\"a}rken sowie die Verweilzeiten verschiedener saFabI Inhibitoren molekular zu erkl{\"a}ren. Die Struktur von saFabI im Komplex mit dem 2-Pyridon Inhibitor CG400549 hingegen enth{\"u}llte spezifische Wechselwirkungen in der geweiteten Bindetasche des S. aureus Enzyms, welche das geringe Aktivit{\"a}tsspektrum dieses derzeit klinisch erprobten Inhibitors erkl{\"a}ren. Diese Studien schaffen somit eine ideale Voraussetzung f{\"u}r die Entwicklung neuer wirksamer saFabI Inhibitoren, was am Beispiel des 4-Pyridons PT166 belegt werden kann. Im Rahmen der vorliegenden Dissertation konnten außerdem die Strukturen des Enzyms KasA im Komplex mit mehreren Derivaten des Naturstoffs Thiolactomycin gel{\"o}st werden.}, subject = {Staphylococcus aureus}, language = {en} } @phdthesis{Schillig2013, author = {Schillig, Rebecca}, title = {Funktionelle Analyse der Zink-Cluster-Transkriptionsfaktorfamilie von Candida albicans durch artifizielle Aktivierung}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-79608}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {Der Hefepilz Candida albicans geh{\"o}rt zu den opportunistischen Infektionserregern. Er ist Teil der nat{\"u}rlichen Mikroflora der Schleimh{\"a}ute des Gastrointestinal- und Urogenitaltraktes des Menschen. Bei St{\"o}rungen des nat{\"u}rlichen Gleichgewichts dieser Flora kann es zu oberfl{\"a}chlichen Mykosen, z. B. der oropharyngealen Candidiasis (Mundsoor), kommen. Besonders immunsupprimierte Patienten, wie AIDS-Patienten, leiden h{\"a}ufig unter immer wiederkehrenden Infektionen, die mitunter auch zu schwerwiegenden Infektionsverl{\"a}ufen, bis hin zu lebensbedrohlichen systemischen Mykosen f{\"u}hren k{\"o}nnen. Zur Therapie solcher Erkrankungen werden oft Ergosterolbiosyntheseinhibitoren, wie Fluconazol, eingesetzt. Besonders bei wiederkehrenden Infektionen und wiederholender Therapie ist C. albicans in der Lage, gegen diese h{\"a}ufig verabreichten Antimykotika Resistenzen zu entwickeln. Hierbei spielen Zink-Cluster-Transkriptionsfaktoren eine zentrale Rolle. Zink-Cluster-Proteine geh{\"o}ren zu einer pilzspezifischen Familie von Transkriptionsfaktoren, die ein großes Spektrum an zellul{\"a}ren Prozessen regulieren. Die gut charakterisierten Regulatoren Upc2, Tac1 und Mrr1 geh{\"o}ren zu den Zink-Cluster-Transkriptionsfaktoren, die maßgeblich zur Resistenzentwicklung von C. albicans beitragen. Upc2 kontrolliert die Expression vieler Ergosterolbiosynthesegene, besonders die von ERG11, welches f{\"u}r die Zielstruktur des g{\"a}ngigen Antimykotikums Fluconazol kodiert. Tac1 und Mrr1 hingegen regulieren die Expression von Multidrug-Effluxpumpen, den ABC-Transportern CDR1 und CDR2 bzw. dem Major Facilitator MDR1. Gain-of-function-Mutationen in diesen Transkriptionsfaktoren resultieren in einer konstitutiven {\"U}berexpression ihrer Zielgene und sind verantwortlich f{\"u}r die Resistenz vieler klinischer Isolate. In dieser Arbeit wurde gezeigt, dass die Fusion von Mrr1 mit der Gal4-Aktivierungsdom{\"a}ne von Saccharomyces cerevisiae zu einem konstitutiv aktiven Hybridtranskriptionsfaktor f{\"u}hrte, der eine MDR1-{\"U}berexpression bewirkte und Fluconazolresistenz vermittelte. Dieses Hybridprotein vermittelte sogar eine h{\"o}here Resistenz als ein Mrr1 mit nat{\"u}rlich vorkommenden gain-of-function-Mutationen. Analoge Fusionen mit Tac1 und Upc2 resultierten ebenfalls in einer konstitutiven Aktivierung dieser Transkriptionsfaktoren, die einen starken Anstieg der Fluconazolresistenz zur Folge hatte. Daraus ergab sich die Schlussfolgerung, dass dies eine generelle Methode sein k{\"o}nnte, die Zink-Cluster-Transkriptionsfaktoren k{\"u}nstlich zu aktivieren und so ihre biologischen Funktionen zu offenbaren, ohne die genauen Bedingungen f{\"u}r ihre Aktivit{\"a}t zu kennen. Deshalb wurde auf der Basis dieser Strategie eine Bibliothek von C.-albicans-St{\"a}mmen konstruiert, in der alle 82 putativen Zink-Cluster-Transkriptionsfaktoren in dieser m{\"o}glicherweise hyperaktiven Form exprimiert werden. Untersuchungen dieser Bibliothek offenbarten neue Transkriptionsfaktoren, die Fluconazolresistenz vermittelten, aber auch noch unbekannte Regulatoren der Morphogenese und andere Ph{\"a}notypen konnten beobachtet werden. Um einen tieferen Einblick in die Funktionsweise zu bekommen, wurden die Transkriptionsprofile der vier Transkriptionsfaktoren ermittelt, die in ihrer hyperaktiven Form die h{\"o}chste Fluconazolresistenz bewirkten. Dabei stellte sich heraus, dass die zwei k{\"u}nstlich aktivierten (*) Regulatoren ZCF34* und ZNC1* die Expression der wichtigsten Multidrug-Effluxpumpe CDR1 stark hochregulierten. Der Transkriptionsfaktor mit dem vorl{\"a}ufigen Namen ZCF34 konnte im Verlauf dieser Arbeit als ein wichtiger Regulator f{\"u}r die CDR1-Expression identifiziert werden. Er ist sowohl an der Aktivierung der Expression von CDR1 beteiligt als auch f{\"u}r die basale CDR1-Promotoraktivit{\"a}t notwendig. Aus diesem Grund wurde er in MRR2 (multidrug resistance regulator 2) umbenannt. Mit der Entdeckung eines neuen Regulators der wichtigsten Multidrug-Effluxpumpe von C. albicans wurde ein wichtiger Beitrag zum Verst{\"a}ndnis der Regulation solcher Transporter geleistet. Die {\"U}berexpression dieser Pumpen ist einer der h{\"a}ufigsten Resistenzmechanismen in C. albicans. Auf diesem Wege kann Resistenz gegen strukturell v{\"o}llig unterschiedliche Antimykotika bewirkt werden. Somit stellen sowohl diese Effluxpumpen, als auch deren Regulatoren m{\"o}gliche Angriffsziele f{\"u}r die Entwicklung neuer oder Weiterentwicklung bereits vorhandener Antimykotika dar.}, subject = {Candida albicans}, language = {de} } @phdthesis{Schul2013, author = {Schul, Daniela}, title = {Spatio-temporal investigation and quantitative analysis of the BMP signaling pathway}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-84224}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {Bone Morphogenetic Proteins (BMPs) are key regulators for a lot of diverse cellular processes. During embryonic development these proteins act as morphogens and play a crucial role particularly in organogenesis. BMPs have a direct impact on distinct cellular fates by means of concentration-gradients in the developing embryos. Using the diverse signaling input information within the embryo due to the gradient, the cells transduce the varying extracellular information into distinct gene expression profiles and cell fate decisions. Furthermore, BMP proteins bear important functions in adult organisms like tissue homeostasis or regeneration. In contrast to TGF-ß signaling, currently only little is known about how cells decode and quantify incoming BMP signals. There is poor knowledge about the quantitative relationships between signal input, transducing molecules, their states and location, and finally their ability to incorporate graded systemic inputs and produce qualitative responses. A key requirement for efficient pathway modulation is the complete comprehension of this signaling network on a quantitative level as the BMP signaling pathway, just like many other signaling pathways, is a major target for medicative interference. I therefore at first studied the subcellular distribution of Smad1, which is the main signal transducing protein of the BMP signaling pathway, in a quantitative manner and in response to various types and levels of stimuli in murine c2c12 cells. Results indicate that the subcellular localization of Smad1 is not dependent on the initial BMP input. Surprisingly, only the phospho-Smad1 level is proportionally associated to ligand concentration. Furthermore, the activated transducer proteins were entirely located in the nucleus. Besides the subcellular localization of Smad1, I have analyzed the gene expression profile induced by BMP signaling. Therefore, I examined two endogenous immediate early BMP targets as well as the expression of the stably transgenic Gaussia Luciferase. Interestingly, the results of these independent experimental setups and read-outs suggest oscillating target gene expression. The amplitudes of the oscillations showed a precise concentration-dependence for continuous and transient stimulation. Additionally, even short-time stimulation of 15' activates oscillating gene-expression pulses that are detectable for at least 30h post-stimulation. Only treatment with a BMP type I receptor kinase inhibitor leads to the complete abolishment of the target gene expression. This indicated that target gene expression oscillations depend directly on BMP type I receptor kinase activity.}, subject = {Knochen-Morphogenese-Proteine}, language = {en} } @phdthesis{Srinivasan2013, author = {Srinivasan, Aruna}, title = {RS1 protein dependent and independent short and long term regulation of sodium dependent glucose transporter -1}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-85665}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {The Na+-D-glucose cotransporter in small intestine is regulated in response to food composition. Short term regulation of SGLT1 occurs post-transcriptionally in response to changes in luminal glucose. Adaptation to dietary carbohydrate involves long term regulation at the transcriptional level. The intracellular protein RS1 (gene RSC1A1) is involved in transcriptional and post-transcriptional regulation of SGLT1. RS1 contains an N-terminal domain with many putative phosphorylation sites. By Expressing SGLT1 in oocytes of Xenopus laevis it was previously demonstrated that the post-transcriptional down-regulation of SGLT1 by RS1 was dependent on the intracellular glucose concentration and activated by protein kinase C (PKC). The role of RS1 for short term regulation of SGLT1 in mouse small intestine in response to glucose and PKC was investigated comparing effects in RS1-/- mice and wildtype mice. Effects on SGLT1 activity were determined by measuring phlorizin inhibited uptake of α-methylglucoside (AMG). The involvement of RS1 in glucose dependent short term regulation could not be elucidated for technical reasons. However, evidence for RS1 independent short-term downregulation of SGLT1 after stimulation of PKC could be provided. It was shown that this downregulation includes decrease in the amount and/or in turnover of SGLT1 in the brush-border membrane as well as an increase of substrate affinity for AMG transport. Trying to elucidate the role of RS1 in long term regulation of SGLT1 in small intestine in response to glucose and fat content of the diet, wildtype and RS1-/- mice were kept for 2 months on a normo-caloric standard diet with high glucose and low fat content (ND), on a hyper-caloric glucose-galactose reduced diet with high fat content (GGRD) or on a hyper-caloric diet with a high fat and high glucose content (HFHGD). Thereafter the animals were starved overnight and SGLT1 mediated AMG uptake was measured. Independent of diet AMG uptake in ileum was smaller compared to duodenum and jejunum. In jejunum of wildtype and RS1-/- mice kept on the fat rich diets (GGRD and HFHGH) transport activity of SGLT1 was lower compared to mice kept on ND with low fat content. This result suggests an RS1 independent downregulation due to fat content of diet. Different to RS1-/- mice, the duodenum of wildtype mice showed transport activity of SGLT1 smaller in mice kept on glucose galactose reduced diet (GGRD) compared to the glucose galactose rich diets (ND and HFHGG). These data indicate that RS1 is involved in glucose dependent long term regulation in duodenum.}, subject = {Glucosetransportproteine}, language = {en} } @phdthesis{Tupak2013, author = {Tupak, Sara}, title = {Modulators of Prefrontal Fear Network Function: An Integrative View}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-85673}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {Regulating our immediate feelings, needs, and urges is a task that we are faced with every day in our lives. The effective regulation of our emotions enables us to adapt to society, to deal with our environment, and to achieve long-term goals. Deficient emotion regulation, in contrast, is a common characteristic of many psychiatric and neurological conditions. Particularly anxiety disorders and subclinical states of increased anxiety are characterized by a range of behavioral, autonomic, and neural alterations impeding the efficient down-regulation of acute fear. Established fear network models propose a downstream prefrontal-amygdala circuit for the control of fear reactions but recent research has shown that there are a range of factors acting on this network. The specific prefrontal cortical networks involved in effective regulation and potential mediators and modulators are still a subject of ongoing research in both the animal and human model. The present research focused on the particular role of different prefrontal cortical regions during the processing of fear-relevant stimuli in healthy subjects. It is based on four studies, three of them investigating a different potential modulator of prefrontal top-down function and one directly challenging prefrontal regulatory processes. Summarizing the results of all four studies, it was shown that prefrontal functioning is linked to individual differences in state anxiety, autonomic flexibility, and genetic predisposition. The T risk allele of the neuropeptide S receptor gene, a recently suggested candidate gene for pathologically elevated anxiety, for instance, was associated with decreased prefrontal cortex activation to particularly fear-relevant stimuli. Furthermore, the way of processing has been found to crucially determine if regulatory processes are engaged at all and it was shown that anxious individuals display generally reduced prefrontal activation but may engage in regulatory processes earlier than non-anxious subjects. However, active manipulation of prefrontal functioning in healthy subjects did not lead to the typical behavioral and neural patterns observed in anxiety disorder patients suggesting that other subcortical or prefrontal structures can compensate for an activation loss in one specific region. Taken together, the current studies support prevailing theories of the central role of the prefrontal cortex for regulatory processes in response to fear-eliciting stimuli but point out that there are a range of both individual differences and peculiarities in experimental design that impact on or may even mask potential effects in neuroimaging research on fear regulation.}, subject = {Neurogenetik}, language = {en} } @phdthesis{Vidal2013, author = {Vidal, Marie}, title = {b-adrenergic receptors and Erk1/2-mediated cardiac hypertrophy}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-83671}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {Chronische Aktivierung von b-Adrenorezeptoren (b-ARs) durch Katecholamine ist ein Stimulus f{\"u}r kardiale Hypertrophie und Herzinsuffizienz. Ebenso f{\"u}hrt die Expression von b1-ARs oder Gas-Proteinen in genetisch modifizierten M{\"a}usen zu Hypertrophie und Herzinsuffizienz. Allerdings f{\"u}hrt die direkte Aktivierung dem Gas nachgeschalteten Komponenten des b-adrenergen Signalwegs wie z.B. die Aktivierung der Adenylylcyclase (AC) oder der Proteinkinase A (PKA) nicht im signifikanten Ausmaß zur Herzhypertrophie. Diese Ergebnisse deuten darauf hin, dass zus{\"a}tzlich zu dem klassischen Signalweg, auch weitere durch Gas-Proteine aktivierte Komponenten in die b-adrenerg vermittelte Hypertrophieentwicklung involviert sind. Interessanterweise wurde vor kurzem ein hypertropher Signalweg beschrieben, der eine direkte Involvierung von Gbg-Untereinheiten bei der Induktion von Herzhypertrophie durch die extrazellul{\"a}r-regulierten Kinasen 1 und 2 (ERK1/2) zeigt: Nach Aktivierung Gaq-gekoppelter Rezeptoren binden Gbg-Untereinheiten an die aktivierte Raf/Mek/Erk Kaskade. Die Bindung der freigesetzten Gbg-Untereinheiten an Erk1/2 f{\"u}hrt zu einer Autophosphorylierung von Erk1/2 an Threonin 188 (bzw. Thr208 in Erk1; im folgenden ErkThr188-Phosphorylierung genannt), welche f{\"u}r die Vermittlung kardialer Hypertrophie verantwortlich ist. In dieser Arbeit konnte nun gezeigt werden, dass auch die Aktivierung von b-ARs in M{\"a}usen sowie von isolierten Kardiomyozyten zur Induktion von ErkThr188-Phosphorylierung f{\"u}hrt. Dar{\"u}berhinaus f{\"u}hrte die {\"U}berexpression von Erk2 Mutanten (Erk2T188S und Erk2T188A), die nicht an Threonin 188 phosphoryliert werden k{\"o}nnen, zu einer deutlich reduzierten Hypertrophieantwort von Kardiomyozyten auf Isoproterenol. Auch die kardiale Expression der Erk2T188S Mutante im M{\"a}usen verminderte die Hypertrophieantwort auf eine 2-w{\"o}chige Isoproterenol-Behandlung deutlich: Die linksventrikul{\"a}re Wanddicke, aber auch interstitielle Fibrose und Herzinsuffizienzmarker wie z.B. BNP waren signifikant reduziert. Weiterhin konnte in dieser Arbeit gezeigt werden, dass tats{\"a}chlich ein Zusammenspiel von Ga und Gbg-vermittelten Signalen zur Induktion von ErkThr188-Phosphorylierung und damit zur Induktion von b-adrenerg vermittelter Hypertrophie notwendig ist. W{\"a}hrend die Hemmung von Gbg-Signalen mit dem C-Terminus der GRK2 oder die Hemmung von Adenylylzyklase eine ErkThr188-Phosphorylierung und eine Hypertrophieantwort nach Isoprenalingabe effektiv reduzierten, f{\"u}hrt die alleinige Aktivierung von Adenylylzyklase nicht zu einer Hypertrophieantwort. Diese Ergebnisse k{\"o}nnten bei der Entwicklung neuer m{\"o}glicher therapeutischen Strategien zur Therapie b-adrenerg induzierter Herzhypertrophie und Herzinsuffizienz helfen.}, subject = {Adrenerger Rezeptor}, language = {en} } @phdthesis{Wangorsch2013, author = {Wangorsch, Gaby}, title = {Mathematical modeling of cellular signal transduction}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-87746}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {A subtly regulated and controlled course of cellular processes is essential for the healthy functioning not only of single cells, but also of organs being constituted thereof. In return, this entails the proper functioning of the whole organism. This implies a complex intra- and inter-cellular communication and signal processing that require equally multi-faceted methods to describe and investigate the underlying processes. Within the scope of this thesis, mathematical modeling of cellular signaling finds its application in the analysis of cellular processes and signaling cascades in different organisms. ...}, subject = {Mathematische Modellierung}, language = {en} } @phdthesis{Wetzel2013, author = {Wetzel, Andrea}, title = {The role of TrkB and NaV1.9 in activity-dependent axon growth in motoneurons}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-92877}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {W{\"a}hrend der Entwicklung des Nervensystems lassen sich bei Motoneuronen aktivit{\"a}tsabh{\"a}ngige Kalziumstr{\"o}me eobachten, die das Axonwachstum regulieren. Diese Form der neuronalen Spontanaktivit{\"a}t sowie das Auswachsen von Axonen sind bei Motoneuronen, die aus Tiermodellen der Spinalen Muskelatrophie isoliert werden, gest{\"o}rt. Experimente aus unserer Arbeitsgruppe haben gezeigt, dass spontane Erregbarkeit und aktivit{\"a}tsabh{\"a}ngiges Axonwachstum von kultivierten Motoneuronen auch unter Verwendung von Toxinen beeintr{\"a}chtigt sind, welche die Aktivit{\"a}t von spannungsabh{\"a}ngigen Natriumkan{\"a}len blockieren. In diesen Versuchen war die Wirkung von Saxitoxin effizienter als die Wirkung von Tetrodotoxin. Wir identifizierten den Saxitoxin-sensitiven/Tetrodotoxin-insensitiven spannungsabh{\"a}ngigen Natriumkanal NaV1.9 als Trigger f{\"u}r das {\"O}ffnen spannungsabh{\"a}ngiger Kalziumkan{\"a}le. Die Expression von NaV1.9 in Motoneuronen konnte {\"u}ber quantitative RT-PCR nachgewiesen werden und antik{\"o}rperf{\"a}rbungen offenbarten eine Anreicherung des Kanals im axonalen Wachstumskegel sowie an Ranvier'schen Schn{\"u}rringen von isolierten Nervenfasern wildtypischer M{\"a}use. Motoneurone von NaV1.9 knock-out M{\"a}usen zeigen reduzierte Spontanaktivit{\"a}t und eine Reduktion des Axonwachstums, welche durch NaV1.9 {\"U}berexpression normalisiert werden kann. In Motoneuronen von Smn-defizienten M{\"a}usen konnte keine Abweichung der NaV1.9 Proteinverteilung nachgewiesen werden. K{\"u}rzlich wurden Patienten identifiziert, die eine missense-Mutation im NaV1.9 kodierenden SCN11A Gen tragen. Diese Patienten k{\"o}nnen keinerlei Schmerz empfinden und leiden zudem an Muskelschw{\"a}che in Kombination mit einer verz{\"o}gerten motorischen Entwicklung. Im Rahmen dieser Doktorarbeit konnten molekularbiologische Untersuchungen an M{\"a}usen, welche die Mutation im orthologen Scn11a Gen tragen, zur Aufkl{\"a}rung des Krankheitsmechanismus beitragen. Die Kooperationsstudie zeigte, dass eine gesteigerte Funktion von NaV1.9 diese spezifische Kanalerkrankung ausl{\"o}st, was die Wichtigkeit von NaV1.9 in menschlichen Motoneuronen unterstreicht. Eine fr{\"u}here Studie beschrieb an hippocampalen Neuronen, dass die Rezeptortyrosinkinase tropomyosin receptor kinase B (TrkB) den NaV1.9 Kanal {\"o}ffnen kann. Im Wachstumskegel von Motoneuronen ist TrkB nachweisbar und folglich in r{\"a}umlicher N{\"a}he zu NaV1.9 zu finden. Um zu pr{\"u}fen, ob TrkB in die spontane Erregbarkeit von Motoneuronen involviert ist, wurden TrkB knock-out M{\"a}use untersucht. Isolierte Motoneurone von TrkB knock-out M{\"a}usen weisen eine Reduktion der Spontanaktivit{\"a}t und eine Verringerung des Axonwachstums auf. Ob TrkB und NaV1.9 hierbei funktionell gekoppelt sind, ist Gegenstand k{\"u}nftiger Forschung.}, subject = {Motoneuron}, language = {en} } @phdthesis{Wu2013, author = {Wu, Lingdan}, title = {Emotion Regulation in Addicted Smokers}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-85471}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {Background: Nicotine addiction is the most prevalent type of drug addiction that has been described as a cycle of spiraling dysregulation of the brain reward systems. Imaging studies have shown that nicotine addiction is associated with abnormal function in prefrontal brain regions that are important for cognitive emotion regulation. It was assumed that addicts may perform less well than healthy nonsmokers in cognitive emotion regulation tasks. The primary aims of this thesis were to investigate emotional responses to natural rewards among smokers and nonsmokers and to determine whether smokers differ from nonsmokers in cognitive regulation of positive and negative emotions. To address these aims, two forms of appraisal paradigms (i.e., appraisal frame and reappraisal) were applied to compare changes in emotional responses of smokers with that of nonsmokers as a function of appraisal strategies. Experiment 1: The aim of the first experiment was to evaluate whether and how appraisal frames preceding positive and negative picture stimuli affect emotional experience and facial expression of individuals. Twenty participants were exposed to 125 pairs of auditory appraisal frames (either neutral or emotional) followed by picture stimuli reflecting five conditions: unpleasant-negative, unpleasant-neutral, pleasant-positive, pleasant-neutral and neutral-neutral. Ratings of valence and arousal as well as facial EMG activity over the corrugator supercilii and the zygomaticus major were measured simultaneously. The results indicated that appraisal frames could alter both subjective emotional experience and facial expressions, irrespective of the valence of the pictorial stimuli. These results suggest and support that appraisal frame is an efficient paradigm in regulation of multi-level emotional responses. 8 Experiment 2: The second experiment applied the appraisal frame paradigm to investigate how smokers differ from nonsmokers on cognitive emotion regulation. Sixty participants (22 nonsmokers, 19 nondeprived smokers and 19 12-h deprived smokers) completed emotion regulation tasks as described in Experiment 1 while emotional responses were concurrently recorded as reflected by self-ratings and psychophysiological measures (i.e., facial EMG and EEG). The results indicated that there was no group difference on emotional responses to natural rewards. Moreover, nondeprived smokers and deprived smokers performed as well as nonsmokers on the emotion regulation task. The lack of group differences in multiple emotional responses (i.e., self-reports, facial EMG activity and brain EEG activity) suggests that nicotine addicts have no deficit in cognitive emotion regulation of natural rewards via appraisal frames. Experiment 3: The third experiment aimed to further evaluate smokers' emotion regulation ability by comparing performances of smokers and nonsmokers in a more challenging cognitive task (i.e., reappraisal task). Sixty-five participants (23 nonsmokers, 22 nondeprived smokers and 20 12-h deprived smokers) were instructed to regulate emotions by imagining that the depicted negative or positive scenario would become less negative or less positive over time, respectively. The results showed that nondeprived smokers and deprived smokers responded similarly to emotional pictures and performed as well as nonsmokers in down-regulating positive and negative emotions via the reappraisal strategy. These results indicated that nicotine addicts do not have deficit in emotion regulation using cognitive appraisal strategies. In sum, the three studies consistently revealed that addicted smokers were capable to regulate emotions via appraisal strategies. This thesis establishes the groundwork for therapeutic use of appraisal instructions to cope with potential self-regulation failures in nicotine addicts.}, subject = {Gef{\"u}hl}, language = {en} } @phdthesis{Ye2013, author = {Ye, Yuxiang}, title = {Molecular and Cellular Magnetic Resonance Imaging of Myocardial Infarct Healing}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-72514}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {Myokardinfarkte (MI) sind eine der h{\"a}ufigsten Todesursachen weltweit. Eine rechtzeitige Wiederherstellung des koronaren Blutflusses im isch{\"a}mischen Myokard reduziert signifikant die Sterblichkeitsrate akuter Infarkte und vermindert das ventrikul{\"a}re Remodeling. {\"U}berlebende MI-Patienten entwickeln jedoch h{\"a}ufig eine Herzinsuffizienz, die mit einer reduzierten Lebensqualit{\"a}t, hohen Sterblichkeitsrate (10\% j{\"a}hrlich), sowie hohen Kosten f{\"u}r das Gesundheitssystem einhergeht. Die Entwicklung der Herzinsuffizienz nach einem MI ist auf den hohen Verlust kontraktiler Kardiomyozyten, w{\"a}hrend der Isch{\"a}mie-Reperfusion zur{\"u}ckzuf{\"u}hren. Anschließende komplexe strukturelle und funktionelle Ver{\"a}nderungen resultieren aus Modifikationen des infarzierten und nicht infarzierten Myokards auf molekularer und zellul{\"a}rer Ebene. Die verbesserte {\"U}berlebensrate von Patienten mit akutem MI und das Fehlen effizienter Therapien, die die Entwicklung und das Fortschreiten des ventrikul{\"a}ren Remodelings verhindern, f{\"u}hren zu einer hohen Pr{\"a}valenz der Herzinsuffizienz. Die kardiale Magnetresonanztomographie (MRT) ist eine wichtige Methode zur Diagnose und Beurteilung des Myokardinfarktes. Mit dem technologischen Fortschritt wurden die Grenzen der MRT erweitert, so dass es heute m{\"o}glich ist, auch molekulare und zellul{\"a}re Ereignisse in vivo und nicht-invasiv zu untersuchen. In Kombination mit kardialer Morphologie und Funktion k{\"o}nnte die Visualisierung essentieller molekularer und zellul{\"a}rer Marker in vivo weitreichende Einblicke in den Heilungsprozess infarzierter Herzen liefern, was zu neuen Erkenntnissen f{\"u}r ein besseres Verst{\"a}ndnis und bessere Therapien des akuten MI f{\"u}hren k{\"o}nnte. In dieser Arbeit wurden Methoden f{\"u}r die molekulare und zellul{\"a}re kardiale MRT-Bildgebung der Inflammation und des Kalziumstroms im Heilungsprozess des akuten Myokardinfarktes in vivo in einem Rattenmodel mit klinischer Relevanz etabliert.}, subject = {Kernspintomografie}, language = {en} } @phdthesis{Zancolli2013, author = {Zancolli, Giulia}, title = {Amphibian diversity along the slope of Mount Kilimanjaro: from species to genes}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-91792}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {1. Since the early nineteenth century describing (and understanding) patterns of distribution of biodiversity across the Earth has represented one of the most significant intellectual challenges to ecologists and biogeographers. Among the most striking patterns of species richness are: the latitudinal and elevational gradients, with peaks in number of species at low latitudes and somewhere at mid altitudes, although other patterns, e.g. declines with increasing elevation, are often observed. Even in highly diverse tropical regions, species richness is not evenly distributed but there are "hotspots" of biodiversity where an exceptional number of species, especially endemics, are concentrated. Unfortunately, such areas are also experiencing dramatic loss of habitat. Among vertebrate taxa, amphibians are facing the most alarming number of extinctions. Habitat destruction, pollution and emergence of infectious diseases such as chytridiomycosis, are causing worldwide population declines. Responses to these drivers can be multidirectional and subtle, i.e. they may not be captured at the species but at the genetic level. Moreover, present patterns of diversity can result from the influence of past geological, climatic and environmental changes. In this study, I used a multidisciplinary and multilevel approach to understand how and to which extent the landscape influences amphibian diversity. Mount Kilimanjaro is an exceptional tropical region where the landscape is rapidly evolving due to land use changes; additionally, there is a broad lack of knowledge of its amphibian fauna. During two rainy seasons in 2011, I recorded anurans from the foothills to 3500 m altitude; in addition, I focused on two river frog species and collected tissue samples for genetic analysis and swabs for detection of chytridiomycosis, the deadly disease caused by Batrachochytrium dendrobatidis (Bd). 2. I analyzed how species richness and composition change with increasing elevation and anthropogenic disturbance. In order to disentangle the observed patterns of species diversity and distribution, I incorporated inferences from historical biogeography and compared the assemblage of Mt. Kilimanjaro and Mt. Meru (both recent volcanoes) with those of the older Eastern Arc Mountains. Species richness decreased with elevation and locally increased in presence of water bodies, but I did not detect effects of either anthropogenic disturbance or vegetation structure on species richness and composition. Moreover, I found a surprisingly low number of forest species. Historical events seem to underlie the current pattern of species distribution; the young age of Mt. Kilimanjaro and the complex biogeographic processes which occurred in East Africa during the last 20 million years prevented montane forest frogs from colonizing the volcano. 3. I focused on the genetic level of biodiversity and investigated how the landscape, i.e. elevation, topographic relief and land cover, influence genetic variation, population structure and gene flow of two ecologically similar and closely related river frog species, namely Amietia angolensis and Amietia wittei. I detected greater genetic differentiation among populations in the highland species (A. wittei) and higher genetic variation in the lowland species (A. angolensis), although genetic diversity was not significantly correlated with elevation. Importantly, human settlements seemed to restrict gene flow in A. angolensis, whereas steep slopes were positively correlated with gene flow in A. wittei. This results show that even ecologically similar species can respond differently to landscape processes and that the spatial configuration of topographic features combined with species-specific biological attributes can affect dispersal and gene flow in disparate ways. 4. River frogs of the genus Amietia seem to be particularly susceptible to chytridiomycosis, showing the highest pathogen load in Kenya and other African countries. In the last study, I collected swab samples from larvae of A. angolensis and A. wittei for Bd detection. Both species resulted Bd-positive. The presence of Bd on Mt. Kilimanjaro has serious implication. For instance, Bd can be transported by footwear of hikers from contaminated water and soil. Tourists visiting Mt. Kilimanjaro may translocate Bd zoospores to other areas such as the nearby Eastern Arc Mts. where endemic and vulnerable species may still be na{\"i}ve to the fungus and thus suffer of population declines. 5. My study significantly contributed to the knowledge of the amphibian fauna of Mt. Kilimanjaro and of East Africa in general, and it represents a valuable tool for future conservation actions and measures. Finally, it highlights the importance of using a multidisciplinary (i.e. community ecology, historical biogeography, landscape genetics, disease ecology) and multilevel (i.e. community, species, population, gene) approach to disentangle patterns of biodiversity.}, subject = {Kilimandscharo}, language = {en} } @phdthesis{Zeller2013, author = {Zeller, Mario}, title = {Dichtegewichtete Magnetresonanz-Bildgebung mit Multi-Echo-Sequenzen}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-84142}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {Das Signal-zu-Rausch-Verh{\"a}ltnis (SNR) stellt bei modernen Bildgebungstechniken in der Magnetresonanz-Tomographie heutzutage oftmals die entscheidende Limitation dar. Eine Verbesserung durch Modifikation der Hardware ist kostspielig und f{\"u}hrt meistens zu einer Verst{\"a}rkung anderer Probleme, wie zum Beispiel erh{\"o}hte Energiedeposition ins Gewebe. Im Gegensatz dazu ist Dichtegewichtung eine Methode, die eine SNR-Erh{\"o}hung durch Modifikation der Aufnahmetechnik erm{\"o}glicht. In der MR-Bildgebung erfolgt oftmals eine retrospektive Filterung des aufgenommenen Signalverlaufs, beispielsweise zur Artefaktreduktion. Damit einhergehend findet eine Ver{\"a}nderung der Modulationstransferfunktion (MTF) bzw. ihrer Fouriertransformierten, der r{\"a}umlichen Antwortfunktion (SRF), statt. Optimales SNR wird nach dem Matched Filter-Theorem erzielt, wenn die nachtr{\"a}gliche Filterung dem aufgenommenen Signalverlauf proportional ist. Dies steht dem Ziel der Artefaktreduktion entgegen. Bei Dichtegewichtung steht durch nicht-kartesische Abtastung des k-Raums mit der k-Raum-Dichte ein zus{\"a}tzlicher Freiheitsgrad zur Verf{\"u}gung. Dieser erm{\"o}glicht es, im Falle eines konstanten Signalverlaufs eine gew{\"u}nschte MTF ohne Filterung zu erreichen. Bei ver{\"a}nderlichem Signalverlauf kann ein SNR Matched Filter angewendet werden, dessen negative Einfl{\"u}sse auf die MTF durch Dichtegewichtung kompensiert werden. Somit erm{\"o}glicht Dichtegewichtung eine vorgegebene MTF und gleichzeitig ein optimales SNR. In der vorliegenden Arbeit wurde Dichtegewichtung erstmals bei den schnellen Multi-Echo-Sequenzen Turbo-Spin-Echo und Echoplanar-Bildgebung (EPI) angewendet. Im Gegensatz zu bisherigen Implementierungen muss hier der Signalabfall durch T2- bzw. T2*-Relaxation ber{\"u}cksichtigt werden. Dies f{\"u}hrt dazu, dass eine prospektiv berechnete dichtegewichtete Verteilung nur bei einer Relaxationszeit optimal ist. Bei Geweben mit abweichenden Relaxationszeiten k{\"o}nnen sich wie auch bei den kartesischen Varianten dieser Sequenzen {\"A}nderungen an SRF und SNR ergeben. Bei dichtegewichteter Turbo-Spin-Echo-Bildgebung des Gehirns konnte mit den gew{\"a}hlten Sequenzparametern ein SNR-Vorteil von 43 \% gegen{\"u}ber der kartesischen Variante erzielt werden. Die Akquisition wurde dabei auf die T2-Relaxationszeit von weißer Substanz optimiert. Da die meisten Gewebe im Gehirn eine {\"a}hnliche Relaxationszeit aufweisen, blieb der visuelle Gesamteindruck identisch zur kartesischen Bildgebung. Der SNR-Gewinn konnte in der dichtegewichteten Implementierung zur Messzeithalbierung genutzt werden. Dichtegewichtete EPI weist eine hohe Anf{\"a}lligkeit f{\"u}r geometrische Verzerrungen, welche durch Inhomogenit{\"a}ten des Hauptmagnetfeldes verursacht werden, auf. Die Verzerrungen konnten erfolgreich mit einer Conjugate Phase-Methode korrigiert werden. Dazu muss die r{\"a}umliche Verteilung der Feldinhomogenit{\"a}ten bekannt sein. Dazu ist zus{\"a}tzlich zur eigentlichen EPI-Aufnahme die zeitaufwendige Aufnahme einer sogenannten Fieldmap erforderlich. Im Rahmen dieser Arbeit konnte eine Methode entwickelt werden, welche die zur Erlangung einer Fieldmap notwendige Aufnahmedauer auf wenige Sekunden reduziert. Bei dieser Art der Fieldmap-Aufnahme m{\"u}ssen jedoch durch Atmung hervorgerufene Effekte auf die Bildphase ber{\"u}cksichtigt werden. Die Fieldmap-Genauigkeit kann durch Aufnahme unter Atempause, Mittelung oder retrospektiver Phasenkorrektur erh{\"o}ht werden. F{\"u}r die gew{\"a}hlten EPI-Sequenzparameter wurde mit Dichtegewichtung gegen{\"u}ber der kartesischen Variante ein SNR-Gewinn von 14 \% erzielt. Anhand einer funktionellen MRT (fMRI)-Fingertapping-Studie konnte demonstriert werden, dass die SNR-Steigerung auch zu einer signifikant erh{\"o}hten Aktivierungsdetektion in Teilen der Hirnareale f{\"u}hrt, die bei der Fingerbewegung involviert sind. Die Verwendung von zus{\"a}tzlicher EPI-Phasenkorrektur und iterativer Optimierung der dichtegewichteten k-Raum-Abtastung f{\"u}hrt zu weiteren Verbesserungen der dichtegewichteten Bildgebung mit Multi-Echo-Sequenzen.}, subject = {Kernspintomografie}, language = {de} }