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Whereas most currently used antibiotics act by interfering with essential bacterial processes, a smaller group of antibacterials disturbs the integrity of the cell membrane. Since fatty acids are a vital component of membrane phospholipids, the type-II fatty acid biosynthesis pathway (FAS-II) of bacteria constitutes a promising drug target. The front-line anti-tuberculosis prodrug isoniazid blocks the FAS-II pathway in M. tuberculosis thereby leading to morphological changes and finally to cell lysis. When it became evident that the enoyl-ACP reductase in the FAS-II pathway is the target of the activated isoniazid, several programs were initiated to develop novel inhibitors directed against this protein in different pathogens. The S. aureus enoyl-ACP reductase (saFabI) is of particular interest since three promising drug candidates inhibiting this homologue have reached clinical trials. However, despite these prospects, no crystal structures of saFabI were publicly available at the time the present work was initiated. Thus, one major goal of this thesis was the generation of high-resolution atomic models by means of X-ray crystallography. The development of a highly reproducible approach to co-crystallize saFabI in complex with NADP+ and diphenyl ether-based inhibitors led to crystal structures of 17 different ternary complexes. Additional crystallographic experiments permitted the view into two apo-structures and two atomic models of saFabI in complex with NADPH and 2-pyridone inhibitors. Based on the established saFabI structure, molecular dynamics (MD) simulations were performed to improve our understanding of the conformational mobility of this protein. Taken together, these investigations of the saFabI structure and its flexibility served as an ideal platform to address important questions surrounding substrate and inhibitor recognition by this enzyme. Intriguingly, our saFabI structures provide several vastly different snapshots along the reaction coordinate of ligand binding and hydride transfer, including the closure of the flexible substrate binding loop (SBL). The extraordinary mobility of saFabI was confirmed by MD simulations suggesting that conformational motions indeed play a pivotal role during substrate delivery and turnover. A water chain linking the active site with a water-basin inside the homo-tetrameric enzyme was found likely to be crucial for the closure and opening of the SBL and, thus, for the catalyzed reaction. Notably, the induced-fit ligand binding process involves a dimer-tetramer transition, which could be related to the observed positive cooperativity of cofactor and substrate binding. Overall, saFabI displays several unique characteristics compared to FabI proteins from other organisms that might be necessary for the synthesis of branched-chain fatty acids, which in turn are required for S. aureus fitness in vivo. This finding may explain why S. aureus is sensitive to FAS-II inhibitors even in the presence of exogenous fatty acids. Accordingly, saFabI remains a valid drug target and our structures can be used as a molecular basis for rational drug design efforts. In fact, binding affinity trends of diphenyl ether inhibitors and, more importantly, the correlated residence times could be rationalized at the molecular level. Furthermore, the structure of saFabI in complex with the 2-pyridone inhibitor CG400549 revealed unique interactions in the wider binding crevice of saFabI compared to other FabI homologues explaining the narrow activity spectrum of this clinical candidate with proven human efficacy. In summary, these studies provide an ideal platform for the development of new, effective saFabI inhibitors as exemplified by the promising 4-pyridone PT166. In the context of this dissertation, crystal structures of the condensing enzyme KasA in complex with several analogs of the naturally occurring inhibitor thiolactomycin have been solved.
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.
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.
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.
Myocardial infarction (MI) is a leading cause of death worldwide. Timely restoration of coronary blood flow to ischemic myocardium significantly reduces acute infarct mortality and attenuates ventricular remodeling. However, surviving MI patients frequently develop heart failure, which is associated with reduced quality of life, high mortality rate (10% annually), as well as high healthcare expenditures. The main processes involved in the evolution of heart failure post-MI are the great loss of contractile cardiomyocytes during ischemia-reperfusion and the subsequent complex structural and functional alterations, which are rooted in modifications at molecular and cellular levels in both the infarcted and non-infarcted myocardium. However, we still lack efficient treatments to prevent the development and progression of left ventricular remodeling. The improved survival rate of acute MI patients combined with the lack of effective therapy for post-MI remodeling contributes to the high prevalence of heart failure. Cardiac Magnetic Resonance Imaging (MRI) is an important tool for diagnosis and assessment of MI. With the advancement of this technology, the frontier of MRI has been extended to probing molecular and cellular events in vivo and non-invasively. In combination with assessment of morphology and function, the visualization of essential molecular and cellular markers in vivo could provide comprehensive, multifaceted views of the healing process in infarcted hearts, which might give new insight for the treatment of acute MI. In this thesis, molecular and cellular cardiac MRI methods were established to visualize and investigate inflammation and calcium flux in the healing process of acute MI in vivo, in a clinically relevant rat model.
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.
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 naïve 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.
Role of Hypoxia-Inducible Factor (HIF) 1α in Dendritic Cells in Immune Regulation of Atherosclerosis
(2013)
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.
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 naïve 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.
During development of the nervous system, spontaneous Ca2+ transients are observed that regulate the axon growth of motoneurons. This form of spontaneous neuronal activity is reduced in motoneurons from a mouse model of spinal muscular atrophy and this defect correlates with reduced axon elongation. Experiments from our group demonstrated that voltage-gated sodium channel pore blockers decrease spontaneous neuronal activity and
axon growth in cultured motoneurons, too. In these experiments, saxitoxin was more potent than tetrodotoxin. We identified the saxitoxin-sensitive/tetrodotoxin-insensitive voltage-gated sodium channel NaV1.9 as trigger for the opening of voltage-gated calcium channels. In motoneurons, expression of NaV1.9 was verified via quantitative RT-PCR. Immuno labelling
experiments revealed enrichment of the channel in axonal growth cones and at the nodes of Ranvier of isolated nerve fibres from wild type mice. Motoneurons from NaV1.9 knock-out mice show decreased spontaneous activity and reduced axonal elongation. This growth defect can be rescued by NaV1.9 overexpression. In motoneurons from Smn-deficient mice, NaV1.9 distribution appeared to be normal.
Recently, patients carrying a missense mutation in the NaV1.9-encoding gene SCN11A were identified. These patients are not able to feel pain and suffer from muscular weakness and a delayed motor development. Molecular biological work during this dissertation supported the analysis of this mutation in a mouse model carrying the orthologous alteration in the Scn11a
locus. The cooperation study confirmed that a gain-of-function mechanism underlies the NaV1.9-mediated channelopathy, thus suggesting a functional role of NaV1.9 in human motoneurons.
An earlier study showed in hippocampal neurons that the receptor tyrosine kinase tropomyosin receptor kinase B (TrkB) can open the NaV1.9 channel. TrkB is localized in
growth cones of motoneurons and subsequently found in close proximity to NaV1.9. In order to proof whether TrkB is involved in spontaneous excitability in motoneurons, TrkB knock-out mice were analysed. Isolated motoneurons from TrkB knock-out mice show a reduced spontaneous activity and axon elongation. It remains to be studied whether TrkB and NaV1.9 are functionally connected.
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.
Atherosclerosis is an active and progressive condition where the vascular cell adhesion molecules as VCAM-1 play a vital role controlling the recruitment of immune cells within the early and advanced plaques. Therefore targeting of VCAM-1 molecules with specific contrast agent bears the possibility to monitor the VCAM-1 expression, visualize the plaque progression starting at the early alterations, and help to establish early prevention of atherosclerosis before the origin of the thrombus formation, of which late recognition leads to myocardial infarction. Furthermore noninvasive magnetic resonance imaging (MRI) offers the benefit of combining the molecular and anatomic data and would thus enable specific detection of VCAM-1 targeted iron oxide contrast agent within inflammatory process of atherosclerosis. This thesis exactly presents the VCAM-1 concept as a suitable molecular approach and the potential of specific ultrasmall superparamagnetic iron oxide (USPIO) conjugated to the VCAM-1 binding peptide over unspecific non-targeted USPIO particles for evaluation of atherosclerosis. This work firstly demonstrated that selection of VCAM-1 molecules offers a good and potential strategy for imaging of atherosclerosis, as these vascular cell adhesion molecules are highly expressed in the early phase of inflammation and also continuously up-regulated within the advanced plaques. Secondly, this thesis showed the proof of principle and capability of the newly designed USPIO contrast agent conjugated to the specific cyclic peptide for VCAM-1 recognition. The experimental studies including ultra-high field MRI enabled further ex vivo and in vivo detection of applied USPIO-VCAM-1 particles within the aortic root region of early and advanced atherosclerotic plaques of 12 and 30 week old apolipoprotein E deficient (ApoE-/-) mice. Using a combination of histology and electron microscopy, this study for the first time pointed to distribution of targeted USPIO-VCAM-1 particles within plaque cells expressing VCAM-1 not only in luminal regions but also in deeper medial smooth muscle cell areas. Hence functionalized USPIO particles targeting VCAM-1 molecules allow specific and sensitive detection of early and advanced plaques at the molecular level, giving the new possibilities for early recognition of atherosclerotic plaques before the appearance of advanced and prone to rupture lesions. In contrast to the functionalized USPIO-VCAM-1, utilized non-targeted USPIO particles did not succeed in early plaque 6 identification limiting visualization of atherosclerosis to advanced forms in atherosclerotic ApoE-/- mice.
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.
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.
Sustained anxiety is considered as a chronic and future-oriented state of apprehension that does not belong to a specific object. It is discussed as an important characteristic of anxiety disorders including panic disorder, generalized anxiety disorder (GAD) and posttraumatic stress disorder (PTSD). Experimentally, sustained anxiety can be induced by contextual fear conditioning in which aversive events are unpredictably presented and therefore the whole context becomes associated with the threat. This thesis aimed at investigating important mechanisms in the development and maintenance of sustained anxiety: (1) facilitated acquisition and resistant extinction of contextual anxiety due to genetic risk factors (Study 1), and (2) the return of contextual anxiety after successful extinction using a new reinstatement paradigm (Study 2). To this end, two contextual fear conditioning studies were conducted in virtual reality (VR). During acquisition one virtual office was paired with unpredictable mildly painful electric stimuli (unconditioned stimulus, US), thus becoming the anxiety context (CXT+). Another virtual office was never paired with any US, thus becoming the safety context (CXT-). Extinction was conducted 24 h later, i.e. no US was presented, and extinction recall was tested another 24 h later on Day 3. In both studies context-evoked anxiety was measured on three different response levels: behavioral (anxiety-potentiated startle reflex), physiological (skin conductance level), and verbal (explicit ratings). In Study 1, participants were stratified for 5-HTTLPR (S+ risk allele vs. LL no risk allele) and NPSR1 rs324981 (T+ risk allele vs. AA no risk allele) polymorphisms, resulting in four combined genotype groups with 20 participants each: S+/T+, S+/LL, LL/T+, and LL/AA. Results showed that acquisition of anxiety-potentiated startle was influenced by a gene × gene interaction: only carriers of both risk alleles (S+ carriers of the 5-HTTLPR and T+ carriers of the NPSR1 polymorphism) exhibited significantly higher startle magnitudes in CXT+ compared to CXT-. However, extinction recall as measured with anxiety-potentiated startle was not affected by any genotype. Interestingly, the explicit anxiety level, i.e. valence and anxiety ratings, was only influenced by the NPSR1 genotype, in a way that no risk allele carriers (AA) reported higher anxiety and more negative valence in response to CXT+ compared to CXT-, whereas risk allele carriers (T+) did not. Study 2 adopted nearly the same paradigm with the modification that one group (reinstatement group) received one unsignaled US at the beginning of the experimental session on Day 3 before seeing CXT+ and CXT-. The second group served as a control group and received no US, but was immediately exposed to CXT+ and CXT-. Results showed a return of anxiety on the implicit and explicit level (higher startle responses and anxiety ratings in response to CXT+ compared to CXT-) in the reinstatement group only. Most important, the return of contextual anxiety in the reinstatement group was associated with a change of state anxiety and mood from extinction to test, that is the more anxiety and negative mood participants experienced before the reinstatement procedure, the higher their return of anxiety was. In sum, results of Study 1 showed that facilitated contextual fear conditioning on an implicit behavioral level (startle response) could be regarded as an endophenotype for anxiety disorders, which can contribute to our understanding of the etiology of anxiety disorders. Results of Study 2 imply that anxiety and negative mood after extinction could be an important facilitator for the return of anxiety. Furthermore, the present VR-based contextual fear conditioning paradigm seems to be an ideal tool to experimentally study mechanisms underlying the acquisition and the return of anxiety. Future studies could investigate clinical samples and extend the VR paradigm to evolutionary-relevant contexts (e.g., heights, darkness, open spaces).
The Staphylococcus aureus two component system (TCS) sae governs expression of numerous virulence factors, including Eap (extracellular adherence protein), which in turn among other functions also mediates invasion of host cells. The sae TCS is encoded by the saePQRS operon, with saeS coding for the sensor histidine kinase (SaeS) and saeR encoding the response regulator (SaeR). The saeRS system is preceded by two additional open reading frames (ORFs), saeP and saeQ, which are predicted to encode a lipoprotein (SaeP) and a membrane protein (SaeQ), respectively. Earlier, we have shown that SDS-containing subinhibitory concentrations of biocides (Perform®) and SDS alone activate sae transcription and increase cellular invasiveness in S. aureus strain Newman. The effect is associated with an amino acid exchange in the N-terminus of SaeS (L18P), specific to strain Newman.
In this work, the role of whether the two additional genes, saePQ coding for the accessory proteins SaeP and SaeQ, respectively, are involved in SDS-mediated saeRS was investigated. It could demonstrated that the lack of the SaeP protein resulted in an increased saeRS transcription without SDS stress in both SaeSL/P variants, while the SDS effect was less pronounced on sae and eap expression compared to the Newman wildtype, suggesting that the SaeP protein represses the sae system. Also, SDS-mediated inductions of sae and eap transcription along with enhanced invasion were found to be dependent on presence of the SaeSP variant in Newman wildtype. On the other hand, the study also shows that the saePQ region of the sae operon is required for fully functional two-component system saeRS under normal growth conditions, but it is not involved in SDS-mediated activation of the saeS signaling and sae-target class I gene, eap.
In the second approach, the study investigates whether SDS-induced sae expression and host cell invasion is common among S. aureus strains not carrying the (L18P) point mutation. To demonstrate this strain Newman, its isogenic saeS mutants, and various S. aureus isolates were analysed for sae, eap expression and cellular invasiveness. Among the strains tested, SDS exposure resulted only in an increase of sae transcription, Eap production and cellular invasiveness in strain Newman wild type and MRSA strain ST239-635/93R, the latter without an increase in Eap. Interestingly, the epidemic community-associated MRSA strain, USA300 LAC showed a biphasic response in sae transcription at different growth stages, which, however, was not accompanied by increased invasiveness. All other clinical isolates investigated displayed a decrease of the parameters tested. While in strain Newman the SDS effect was due to the saeSP allele, this was not the case in strain ST239-635/93R and the biphasic USA300 strains. Also, increased invasiveness of ST239-635/93R was found to be independent of Eap production. Furthermore, to investigate the global effect of SDS on sae target gene expression, strain Newman wild-type and Newman ∆sae were treated with SDS and analyzed for their transcription profiles of sae target genes using microarray assays. We could show that subinhibitory concentrations of SDS upregulate and downregulate gene expression of several signaling pathways involved in biosynthetic, metabolic pathways as well as virulence, host cell adherence, stress reponse and many hypothetical proteins.
In summary, the study sheds light on the role of the upstream region saePQ in SDS-mediated saeRS and eap expression during S. aureus SDS stress. Most importantly, the study also shows that subinhibitory SDS concentrations have pronounced strain-dependent effects on sae transcription and subsequent host cell invasion in S. aureus, with the latter likely to be mediated in some strains by other factors than the known invasin Eap and FnBP proteins. Moreover, there seems to exist more than the saeSP-mediated mechanism for SDS-induced sae transcription in clinical S. aureus isolates. These results help to further understand and clarify virulence and pathogenesis mechanisms and their regulation in S. aureus.
Molecular and developmental characterization of the Echinococcus multilocularis stem cell system
(2014)
The metacestode larva of Echinococcus multilocularis is the causative agent of alveolar echinococcosis (AE), one of the most dangerous zoonotic diseases in the Northern Hemisphere. Unlike “typical” metacestode larvae from other tapeworms, it grows as a mass of interconnected vesicles which infiltrates the liver of the intermediate host, continuously forming new vesicles in the periphery. From these vesicles, protoscoleces (the infective form for the definitive host) are generated by asexual budding. It is thought that in E. multilocularis, as in other flatworms, undifferentiated stem cells (so-called germinative cells in cestodes and neoblasts in free-living flatworms) are the sole source of new cells for growth and development. Therefore, this cell population should be of central importance for the progression of AE.
In this work, I characterized the germinative cells of E. multilocularis, and demonstrate that they are indeed the only proliferating cells in metacestode vesicles. The germinative cells are a population of undifferentiated cells with similar morphology, and express high levels of transcripts of a novel non-autonomous retrotransposon family (ta-TRIMs). Experiments of recovery after hydroxyurea treatment suggest that individual germinative cells have extensive self-renewal capabilities. However, germinative cells also display heterogeneity at the molecular level, since only some of them express conserved homologs of fgfr, nanos and argonaute genes, suggesting the existence of several distinct sub-populations. Unlike free-living flatworms, cestode germinative cells lack chromatoid bodies. Furthermore, piwi and vasa orthologs are absent from the genomes of cestodes, and there is widespread expression of some conserved neoblast markers in E. multilocularis metacestode vesicles. All of these results suggest important differences between the stem cell systems of free-living flatworms and cestodes.
Furthermore, I describe molecular markers for differentiated cell types, including the nervous system, which allow for the tracing of germinative cell differentiation. Using these molecular markers, a previously undescribed nerve net was discovered in metacestode vesicles. Because the metacestode vesicles are non-motile, and the nerve net of the vesicle is independent of the nervous system of the protoscolex, we propose that it could serve as a neuroendocrine system. By means of bioinformatic analyses, 22 neuropeptide genes were discovered in the E. multilocularis genome. Many of these genes are expressed in metacestode vesicles, as well as in primary cell preparations undergoing complete metacestode regeneration. This suggests a possible role for these genes in metacestode development. In line with this hypothesis, one putative neuropeptide (RGFI-amide) was able to stimulate the proliferation of primary cells at a concentration of 10-7 M, and the corresponding gene was upregulated during metacestode regeneration.
Staphylococcus aureus is a major threat to public health systems all over the globe. This second most cause of nosocomial infections is able to provoke a wide variety of different types of infection in humans and animals, ranging from superficial skin and skin structure infections to invasive disease like sepsis or pneumonia. But not enough, this pathogen is also notorious in acquiring and/or developing resistance to antimicrobial compounds, thus limiting available treatment options severely. Therefore, development of new compounds and strategies to fight S. aureus is of paramount importance. But since only 1 out of 5 compounds, which entered clinical trials, becomes a drug, the preclinical evaluation of promising compounds has to be reconsidered, too. The aim of this thesis was to address both sides of this problem: first, to improve preclinical testing by incorporating in vivo imaging technologies to the preclinical testing procedure in order to acquire additional and clearer data about efficacy of promising compounds and second, by evaluating lysostaphin, which is a promising, new option to fight S. aureus infections.
The first aim of this thesis focused on the establishment of a dual modality in vivo imaging platform, consisting of Bioluminescence Imaging (BLI) and Magnetic Resonance Imaging (MRI), to offer detailed insights into the course and gravity of S. aureus infection in the murine thigh infection model. Since luciferase-expressing S. aureus strains were generated in former studies and enabled thus bioluminescence imaging of bacterial infection, this technology should be implemented into the compound evaluation platform in order to non-invasively track the bacterial burden over time. MRI, in contrast, was only rarely used in earlier studies to visualize and measure the course of infection or efficacy of anti-bacterial therapy. Thus, the first set of experiments was performed to identify benefits and drawbacks of visualizing S. aureus infections in the mouse model by different MR methods. Native, proton-based MR imaging showed in this regard increased T2 relaxation times in the infected thigh muscles, but it was not possible to define a clear border between infected and uninfected tissue. Iron oxide nanoparticles and perfluorocarbon emulsions, two MR contrast agents or tracer, in contrast, offered this distinction. Iron oxide particles were detected in this regard by their distortion of 1H signal in proton-based MRI, while perfluorocarbon emulsion was identified by 19F MRI. Mammals do not harbor sufficient intrinsic amounts of 19F to deliver specific signal and therefore, 19F MR imaging visualizes only the signal of administered perfluorocarbon emulsion. The in vivo accumulation of perfluorocarbon emulsion can be imaged by 19F MRI and overlayed on a simultaneously acquired 1H MR image, which shows the anatomical context in clear detail. Since this is advantageous compared to contrast agent based MR methods like iron oxide particle-based MRI, further experiments were performed with perfluorocarbon emulsions and 19F MRI.
Experimental studies to elucidate the accumulation of perfluorocarbon emulsion at the site of infection showed robust 19F MR signals after administration between day 2 and at least day 8 p.i.. Perfluorocarbon emulsion accumulated in all investigated mice in the shape of a ‘hollow sphere’ at the rim of the abscess area and the signal remained stable as long as the infection prevailed. In order to identify the mechanism of accumulation, flow cytometry, cell sorting and histology studies were performed. Flow cytometry and cell sorting analysis of immune cells at the site of infection showed that neutrophils, monocytes, macrophages and dendritic cells carried contrast media at the site of infection with neutrophils accounting for the overwhelming portion of perfluorocarbon signal. In general, most of the signal was associated with immune cells, thus indicating specific immune cell dependent accumulation. Histology supported this observation since perfluorocarbon emulsion related fluorescence could only be visualized in close proximity to immune cell nuclei.
After establishing and testing of 19F MRI with perfluorocarbon emulsions as infection imaging modality, the effects of antibiotic therapy upon MR signal was investigated in order to evaluate the capability of this modality for preclinical testing procedure. Thus, the efficacy of vancomycin and linezolid, two clinically highly relevant anti - S. aureus compounds, were tested in the murine thigh infection model. Both of them showed reduction of the colony forming units and bioluminescence signal, but also of perfluorocarbon emulsion accumulation strength and volume at the site of infection, which was visualized and quantified by 19F MRI. The efficacy pattern with linezolid being more efficient in clearing bacterial infection was shown similarly by all three methods. In consequence, 19F MRI with perfluorocarbon emulsion as MR tracer proved to be capable to visualize antibacterial therapy in preclinical testing models.
The next step was consequently to evaluate a promising new compound against S. aureus infections. Thus, lysostaphin, an endo-peptidase that cleaves the cell wall of S. aureus, was tested in different concentrations alone or in combination with oxacillin for efficacy in murine thigh and catheter associated infection models. Lysostaphin only in the concentration of 5 mg/kg body weight or combined with oxacillin in the concentration of 2 mg/kg showed strong reduction of bacterial burden by colony forming unit determination and bioluminescence imaging in both models. The perfluorocarbon accumulation was investigated in the thigh infection model by 19F MRI and was strongly reduced in terms of volume and signal strength in both above-mentioned groups. In general, lysostaphin showed comparable or superior efficacy than vancomycin or oxacillin alone. Therefore, further development of lysostaphin for the treatment of S. aureus infections is recommended by these experiments. Overall, the antibiotic efficacy pattern of all applied antibiotic regimens was similar with all three applied methods, demonstrating the usefulness of MRI for antibiotic efficacy testing. Importantly, treatment with oxacillin either alone or in combination with lysostaphin resulted in stronger perfluorocarbon emulsion accumulation at the site of infection than expected compared to the results from bioluminescence imaging and colony forming unit determination. This might be an indication for immunomodulatory properties of oxacillin.
Further murine infection experiments demonstrated in this context a differential release of cytokine and chemokines in the infected thigh muscle in dependence of the applied antibacterial therapy. Especially treatment with oxacillin, but to a less degree with minocycline or linezolid, too, exhibited high levels of various cytokines and chemokines, although they reduced the bacterial burden efficiently. In consequence, possible immunomodulatory effects of antibacterial compounds have to be taken into account for future applications of imaging platforms relying on the visualization of the immune response. However, this observation opens a new field for these imaging modalities since it might be extraordinary interesting to study the immunomodulatory effects of compounds or even bacterial factors in vivo. And finally, a two modality imaging platform which combines methods to visualize on the one hand the bacterial burden and on the other hand the immune response offers an innovative, new platform to study host-pathogen interaction in vivo in a non-invasive fashion.
In summary, it could be shown that perfluorocarbon emulsions accumulate in immune cells at the site of infection in the murine S. aureus thigh infection model. The accumulation pattern shapes a ‘hollow sphere’ at the rim of the abscess area and its size and perfluorocarbon content is dependent on the severity of disease and/or efficacy of antibiotic therapy. Thus, 19F MRI with perfluorocarbon emulsions is a useful imaging modality to visualize sites and course of infection as well as to evaluate promising antibacterial drug candidates. Furthermore, since the accumulation of tracer depends on immune cells, it might be additionally interesting for studies regarding the immune response to infections, auto-immune diseases or cancer, but also to investigate the efficacy of immunomodulatory compounds and immunization.
Structural and biochemical characterization of gephyrin and various gephyrin-ligand complexes
(2014)
Efficient synaptic neurotransmission requires the exact apposition of presynaptic terminals and matching neurotransmitter receptor clusters on the postsynaptic side. The receptors are embedded in the postsynaptic density, which also contains scaffolding and regulatory proteins that ensure high local receptor concentrations. At inhibitory synapses the cytosolic scaffolding protein gephyrin assumes an essential organizing role within the postsynaptic density by the formation of self-oligomers which provide a high density of binding sites for certain -amino butyric acid type A (GABAA) and the large majority of glycine receptors (GlyR). Gephyrin contains two oligomerization domains: In isolation, the 20 kDa N-terminal G domain (GephG) and the 46 kDa E domain (GephE) trimerize and dimerize, respectively. In the full-length protein the domains are interconnected by a central ~150 amino acid linker, and only GephG trimerization is utilized, whereas GephE dimerization is prevented, thus suggesting the need for a trigger to release GephE autoinhibition, which would pave the way for the formation of higher oligomers and for efficient receptor clustering. The structural basis for this GephE autoinhibition has remained elusive so far, but the linker was reported to be sufficient for autoinhibition. This work dealt with the biochemical and structural characterization of apo-gephyrin and gephyrin in complexes with ligands which are known to promote the formation of synaptic gephyrin clusters (collybistin and neuroligin 2) and reorganize them (dynein light chain 1).
For full-length gephyrin no structural information has been available so far. Atomic force microscopy (AFM) and small-angle X-ray scattering (SAXS) analyses described in this thesis disclosed that the gephyrin trimer forms a highly flexible assembly, which, due to the long linker, can switch between compact and extended conformational states in solution, with a preference for compact states. This partial compaction and potentially GephE autoinhibition are achieved by interactions of parts of the linker with the G and E domains, as suggested by circular dichroism spectroscopy. However, the linker on its own cannot account for GephE blockage, as size exclusion chromatography experiments coupled with multi angle light scattering detection (SEC-MALS) and SAXS analyses revealed that a gephyrin variant only encompassing the linker and GephE (GephLE) forms dimers and not monomers as suggested by an earlier study. The oligomeric state of GephLE and the observation that several gephyrin variants, in which linker segments of varying length were deleted, predominantly formed trimers, suggested the presence of a linker independent mechanism of GephE dimerization blockade. Taken together, the data indicated that linker-dependent and linker-independent mechanisms mediate gephyrin autoinhibition.
In the second project gephyrin’s interaction with DYNLL1 (Dynein LC8 Light Chain 1) was characterized. DYNLL1 is a 25 kDa dimer incorporated into the dynein motor and provides two binding sites, each of which can accommodate an octapeptide derived from gephyrin’s linker region (referred to as GephDB). Originally, DYNLL1 was regarded as a cargo adaptor, linking gephyrin-GlyR complexes to the dynein motor, thus driving their retrograde transport and leading to a decrease of synaptic gephyrin-GlyR complexes.
Building on these studies, this thesis assessed the cargo hypothesis as well as the so far unclear stoichiometry of the gephyrin-DYNLL1 complex. The cargo scenario would require ternary complex formation between gephyrin, DYNLL1 and the dynein intermediate chain (DIC) of the dynein motor. However, such a complex could not be detected by analytical size exclusion chromatography (aSEC) experiments – presumably because gephyrin and DIC competed for a common binding site in DYNLL1. This finding was consistent with a single DYNLL1 dimer capturing two linker segments of a single gephyrin trimer as suggested by a 26 kDa mass increase of the gephyrin species in the presence of DYNLL1 in SEC-MALS experiments. aSEC experiments at even higher concentrations (~20 µM gephyrin and ~80 µM DYNLL1) indicated that the affinity of GephDB was significantly impaired in the context of full-length gephyrin but also in a variant that bears only GephG and the first 39 residues of the linker (GephGL220). Presumably due to avidity effects two linkers stably associated with a single DYNLL1 dimer, whereas the third DYNLL1 binding motif remained predominantly unoccupied unless high concentrations of GephGL220 (50 µM) and DYNLL1 (200 µM) were used. These findings indicate that an interplay between GephG and the N-terminal linker segment mediates the attenuation of GephDB affinity towards DYNLL1 and that preventing DYNLL1 from the induction of higher gephyrin oligomers is either advantageous for DYNLL1-mediated reorganization of gephyrin-GlyR clusters or that DYNLL1 exerts possibly two (concentration-dependent) actions on gephyrin.
The gephyrin-collybistin-neuroligin 2 complex was the subject of the third project. Previously, collybistin and gephyrin were observed to mutually trigger their translocation to the postsynaptic membrane, where the disordered cytoplasmic tail of the postsynaptic cell adhesion molecule NL2 (NL2cyt) causes the anchoring of collybistin 2 (CB2) by binding to its SH3 domain, thereby releasing SH3 domain mediated autoinhibiton of CB2 binding to the membrane phospholipid phosphatidylinositol-3-phosphate. Critical for this event is the binding of gephyrin to both CB2 and NL2, presumably via GephE.
Following up on these previous studies biochemical data presented in this thesis confirm the formation of the ternary complex. Unexpectedly, analyses by means of native polyacrylamide gel electrophoresis pointed to: (1) The existence of a complex containing NL2cyt and CB2 lacking the SH3 domain and consequently an additional NL2 binding site in CB2. (2) Attenuated gephyrin-collybistin complex formation in the presence of the SH3 domain. (3) A requirement for high NL2cyt concentrations (> 30 µM) during the formation of the ternary complex. This might allow for the regulation by other factors such as additional binding partners or posttranslational modifications. Although of preliminary character, these results provide a starting point for future studies, which will hopefully elucidate the interplay between gephyrin, collybistin, NL2 and certain GABAA receptors.
Platelet activation and aggregation are essential processes for the sealing of injured vessel walls and preventing blood loss. Under pathological conditions, however, platelet aggregation can lead to uncontrolled thrombus formation, resulting in irreversible vessel occlusion. Therefore, precise regulation of platelet activation is required to ensure efficient platelet plug formation and wound sealing but also to prevent uncontrolled thrombus formation. Rapid elevations in the intracellular levels of cations are a core signaling event during platelet activation. In this thesis, the roles of Ca2+ and Mg2+ channels in the regulation of platelet function were investigated.
Orai1, the major store-operated calcium (SOC) channel in platelets, is not only vital for diverse signaling pathways, but may also regulate receptor-operated calcium entry (ROCE). The coupling between the Orai1 signalosome and canonical transient receptor potential channel (TRPC) isoforms has been suggested as an essential step in the activation of store-operated calcium entry (SOCE) and ROCE in human platelets. However, the functional significance of the biochemical interaction between Orai and TRPC isoforms still remains to be answered. In the first part of this thesis, the functional crosstalk between Orai1 and TRPC6 was addressed. Orai1-mediated SOCE was found to enhance the activity of phospholipases (PL) C and D, to increase diacylglycerol (DAG) production and finally to regulate TRPC6-mediated ROCE via DAG, indicating that the regulation of TRPC6 channel activity seems to be independent of the physical interaction with Orai1. Furthermore, Orai1 and TRPC6 double deficiency led to a reduced Ca2+ store content and basal cytoplasmic Ca2+ concentrations, but surprisingly also enhanced ATP secretion, which may enhance Ca2+ influx via P2X1 and compensate for the severe Ca2+ deficits seen in double mutant platelets. In addition, Orai1 and TRPC6 were not essential for G protein-coupled receptor (GPCR)-mediated platelet activation, aggregation and thrombus formation.
Transient receptor potential melastatin-like 7 (TRPM7) contains a cytosolic serine/threonine protein kinase. To date, a few in vitro substrates of the TRPM7 kinase have been identified, however, the physiological role of the kinase remains unknown. In the second part of this thesis, mice with a point mutation which blocks the catalytic activity of the TRPM7 kinase (Trpm7KI) were used to study the role of the TRPM7 kinase in platelet function. In Trpm7KI platelets phosphatidylinositol-4,5-bisphosphate (PIP2) metabolism and Ca2+ mobilization were severely impaired upon glycoprotein (GP) VI activation, indicating that the TRPM7 kinase regulates PLC function. This signaling defect in Trpm7KI platelets resulted in impaired aggregate formation under flow and protected animals from arterial thrombosis and ischemic brain infarction. Altogether, these results highlight the kinase domain of TRPM7 as a pivotal signaling moiety implicated in the pathogenesis of thrombosis and cerebrovascular events.
Marine sponges (phylum Porifera) are simple, sessile, filter-feeder animals. Microbial symbionts are commonly found in the sponge internal tissue, termed the mesohyl. With respect to the microbial content, sponges are classified as either low-microbial abundance sponges (LMA), or high-microbial abundance sponges (HMA). The HMA/LMA dichotomy was explored in this Thesis using the Red Sea sponges as experimental models. A range of methods encompassing transmission electron microscopy, 16S rRNA gene deep sequencing, and metatranscriptomics was employed towards this goal. Here, particular emphasis was placed on the functional analysis of sponge microbiomes.
The Red Sea sponges Stylissa carteri, Xestospongia testudinaria, Amphimedon ochracea, and Crella cyathophora were classified as HMA or LMA sponges using transmission electron microscopy. The diversity, specificity, and transcriptional activity of microbes associated with the sponges S. carteri (LMA) and X. testudinaria (HMA) and seawater were investigated using 16S rRNA amplicon pyrosequencing. The microbial composition of S. carteri was more similar to that of seawater than to that of X. testudinaria, which is consistent with the observation that the sequence data set of S. carteri contained many more possibly seawater sequences (~24%) than the X. testudinaria data set (~6%). The most abundant operational taxonomic units (OTUs) were shared between all three sources (S. carteri, X. testudinaria, seawater), while rare OTUs were unique to any given source. Despite this high degree of overlap, each sponge species contained its own specific microbiota. S. carteri microbiomes were enriched of Gammaproteobacteria and members of the genus Synechococcus and Nitrospira. Enriched members of X. testudinaria microbiomes included Chloroflexi, Deferribacteres, and Actinobacteria. The transcriptional activity of sponge-associated microorganisms was assessed by comparing 16S rRNA gene with transcript amplicons, which showed a good correlation.
The microbial functional gene repertoire of sponges and seawater from the Red Sea (X. testudinaria, S. carteri) and the Mediterranean (Aplysina aerophoba, Dysidea avara) were investigated with the environmental microarray GeoChip 4. Amplicon sequencing was performed alongside in order to assess microbial diversity. The typical microbial diversity patterns characteristic of HMA (abundance of Gammaproteobacteria, Chloroflexi, Acidobacteria, Deferribacteres, and others) and LMA sponges (abundance of Alpha-, Beta-, Gammaproteobacteria, Cyanobacteria, and Bacteroidetes) were confirmed. The HMA/LMA dichotomy was stronger than any possible geographic pattern based on microbial diversity (amplicon) and functional genes (GeoChip). However upon inspection of individual genes detected by GeoChip, very few specific differences were discernible, including differences related to microbial ammonia oxidation, ammonification (higher gene abundance in sponges over seawater) as well as denitrification (lower gene abundance). Furthermore, a higher abundance of a gene, pcc, representative of archaeal autotrophic carbon fixation was noted in sponges over seawater. Thirdly, stress-related genes, in particular those related to radiation, were found in lower abundances in sponge microbiomes than in seawater. With the exception of few documented specific differences, the functional gene repertoire between the different sources appeared largely similar.
The most actively expressed genes of S. carteri microbiomes were investigated with metatranscriptomics. Prokaryotic mRNA was enriched from sponge total RNA, sequenced using Illumina HiSeq technology, and annotated with the metagenomics Rapid Annotation using Subsystem Technology (MG-RAST) pipeline. High expression of archaeal ammonia oxidation and photosynthetic carbon fixation by members of the genus Synechococcus was detected. Functions related to stress response and membrane transporters were among the most highly expressed by S. carteri symbionts. Unexpectedly, gene functions related to methylotrophy were highly expressed by gammaproteobacterial symbionts. The presence of seawater-derived microbes is indicated by the phylogenetic proximity of organic carbon transporters to orthologs of members from the SAR11 clade. In summary, the most expressed functions of the S. carteri-associated microbial community were revealed and linked to the dominant taxonomic members of the microbiome.
In conclusion, HMA and LMA Red Sea sponges were used as models to gain insights into relevant themes in sponge microbiology, i.e. diversity, specificity, and functional activities. Overall, my Thesis contributes to a better understanding of sponge-associated microbial communities, and the implications of this association to marine ecology.
The number of newly detected autoantibodies (AB) targeting synaptic proteins in neurological disorders of the central nervous system (CNS) is steadily increasing. Direct interactions of AB with their target antigens have been shown in first studies but the exact pathomecha-nisms for most of the already discovered AB are still unclear. The present study investigates pathophysiological mechanisms of AB-fractions that are associated with the enigmatic CNS disease Stiff person syndrome (SPS) and target the synaptically located proteins amphiphysin or glutamate decarboxylase 65 (GAD65).
In the first part of the project, effects of AB to the presynaptic endocytic protein amphiphysin were investigated. Ultrastructural investigations of spinal cord presynaptic boutons in an es-tablished in-vivo passive-transfer model after intrathecal application of human anti-amphiphysin AB showed a defect of endocytosis. This defect was apparent at high synaptic activity and was characterized by reduction of the synaptic vesicle pool, clathrin coated vesi-cles (CCVs), and endosome like structures (ELS) in comparison to controls. Molecular inves-tigation of presynaptic boutons in cultured murine hippocampal neurons with dSTORM microscopy after pretreatment with AB to amphiphysin revealed that marker proteins involved in vesicle exocytosis (synaptobrevin 2 and synaptobrevin 7) had an altered expression in GA-BAergic presynapses. Endophilin, a direct binding partner of amphiphysin also displayed a disturbed expression pattern. Together, these results point towards an anti-amphiphysin AB-induced defective organization in GABAergic synapses and a presumably compensatory rearrangement of proteins responsible for CME.
In the second part, functional consequences of SPS patient derived IgG fractions containing AB to GAD65, the rate limiting enzyme for GABA synthesis, were investigated by patch clamp electrophysiology and immunohistology. GABAergic neurotransmission at low and high activity as well as short term plasticity appeared normal but miniature synaptic potentials showed an enhanced frequency with constant amplitudes. SPS patient IgG after preabsorption of GAD65-AB using recombinant GAD65 still showed specific synaptic binding to neu-rons and brain slices supporting the hypothesis that additional, not yet characterized AB are present in patient IgG responsible for the exclusive effect on frequency of miniature potentials.
In conclusion, the present thesis uncovered basal pathophysiological mechanisms underlying paraneoplastic SPS induced by AB to amphiphysin leading to disturbed presynaptic architec-ture. In idiopathic SPS, the hypothesis of a direct pathophysiological role of AB to GAD65 was not supported and additional IgG AB are suspected to induce distinct synaptic malfunction.
SNAP25 (Synaptosomal-Associated Protein of 25 kDa; part of the SNARE complex) is involved in the docking and fusion of synaptic vesicles in presynaptic neurons necessary for the regulation of neurotransmitter release, as well as in axonal growth and synaptic plasticity. In humans, different single nucleotide polymorphisms of SNAP25 have repeatedly been associated with attention deficit/hyperactivity disorder (ADHD). Thus, in this study heterozygous Snap25 knockout mice were investigated as a model of ADHD.
Heterozygous (+/-) Snap25 knockout mice as well as their wild-type (+/+) littermates were reared under control conditions or underwent a Maternal Separation (MS) procedure. Starting at the age of 2 months, mice were tested for locomotor activity in a repeated long-term Open Field (OF) task, for attention deficits and impulsive behavior in the 5 Choice Serial Reaction Time Task (5CSRTT), for anxiety-like behavior in the Light-Dark Box (LDB) and for depression-like behavior in the Porsolt Forced Swim Test (FST). The brains of these mice were subsequently tested for the expression of several ADHD related genes in a quantitative Real-Time PCR (qRT-PCR) study. Another group of female mice (+/+; +/-) underwent a one hour OF test after oral administration of 45 mg/kg Methylphenidate (MPH) or placebo.
To find an optimized dosage for this MPH challenge, a pilot study was performed. Wild-type C57BL/6 mice were tested in a long-term OF with several dosages of MPH both intraperitoneally (i.p.) and orally. The brains of these animals were afterwards investigated for neurotransmitter concentrations. In this pilot study the dosages of MPH that were similarly behaviorally effective without causing symptoms of overdosing were 7.5-15 mg/kg intraperitoneally and 30-60 mg/kg orally. However, even though it was possible to find intraperitoneal and oral doses that correlate behaviorally, the neurochemistry was mostly different.
In the study on Snap25-deficient mice, unstressed controls showed a hyperactive phenotype in the second of two long-term OF sessions (60 min) spaced three weeks apart. Considering all groups, there was a significant interaction of stress and genotype in the second session, with animals subjected to MS being overall hyperactive with no genotype differences. In the training phase of the 5CSRTT only effects of stress were found, with MS animals finding and consuming fewer rewards. In the single test trial, several genotype effects became apparent, with tendencies for the number of correct nose pokes and the number of rewards eaten, and a significant effect for the number of rewards eaten directly after the correct response. In all of these variables +/- mice performed worse than their wild-type littermates. In the LDB +/- mice entered the lit compartment of the arena earlier than the controls, thus showing attenuated anxiety-like behavior. Regarding depressive-like behavior in the FST, male +/- mice spent significantly less time struggling than male +/+ mice. In the gene expression study, +/- mice had lower expression levels of Maoa and Comt, and higher expression levels of Nos1 than wild-types. Finally, the locomotor activity response to MPH was exaggerated in +/- mice as compared to controls.
Heterozygous Snap25 knockout mice show some of the behavioral characteristics of ADHD, as for example a mild hyperactivity in a familiar environment, difficulties in the correct execution of a given task and even some behavior that can be interpreted as delay aversion. Additionally, expression levels of three ADHD related genes were changed in these animals. Although the exaggerated locomotor activity response to MPH is not to be expected of an ADHD model, the difference in the response between +/+ and +/- mice nonetheless implicates a potential dysfunction of the brain dopaminergic system.
I. Nowadays, tropical landscapes experience large-scale land use intensification and land conversion driven by increasing demand for resourses. Due to the continuously high demand for tropical timber and politically intended step increase in palm oil production, multiple rounds of logging and subsequent conversion to oil palm plantations became a regionally wide-spread land conversion pattern in Southeast Asia. Although many tree species and some animals are highly threatened by logging, a great number of species groups, such as birds or mammals, have been shown to persist in logged forests. Accordingly, many ecosystem services, such as dung removal, seed dispersal or the activity of scavengers, are functionally maintained in logged forests. In contrast, oil palm plantations have been shown to not only dramatically alter the species composition and reduce biodiversity, but also curtail many crucial biotic and abiotic ecosystem functions. The focus of this dissertation was to investigate the response of anuran species richness and community composition to logging and conversion to oil palm plantation in northern Borneo (chapter II). I analysed the diet of various frog species and their change with habitat degradation. Furthermore, I assessed the shift in the trophic position of the anuran community as well as the response of anuran phylogenetic, dietary, and functional diversity to logging and conversion to oil palm plantations (chapter III). Finally, the resilience of the predator-prey interaction between an ant-specialist toad and its ant prey was analysed using shifts in species-level interactions (chapter IV).
II. This part of the study compares the species richness, relative abundance and community composition of stream anuran assemblages among primary forests, repeatedly logged forests and oil palm plantations. I used a highly standardised sampling setup applying transect-based sampling. Surprisingly, most of the anuran species native to primary forests were able to survive in logged forest streams. In contrast, on average only one third of the forest species richness was found in oil palm plantation streams. However, a high percentage of canopy cover above the plantation streams was able to mitigate this loss substantially. This study demonstrates the high conservation value of logged forests for Southeast Asian anurans. In contrast, the conversion to oil palm plantations leads to a dramatic decline of forest species. However, they have a mainly unused potential to contribute to the protection of parts of the regional anuran biodiversity if conservation-oriented management options are implemented.
III. In this part, I analysed the shifts in trophic position and multiple diversity layers of Southeast Asian stream-dependent anuran species across a gradient of disturbance from primary forest through intensively logged forest to oil palm plantation. For this purpose, I identified the diet composition of 59 anuran species by means of stomach flushing. Furthermore, I use diet composition of frog species as well as species traits to calculate dietary and functional diversity, respectively. I found that the trophic position of the entire anuran community is elevated in heavily disturbed habitats. Furthermore, species diversity, phylogenetic species variation, dietary diversity, and functional diversity were reduced. However, beyond the effect of the decreased species richness, only phylogenetic species variability and functional diversity were significantly impacted by land conversion, indicating a non-random loss of phylogenetic groups and functionally unique species. Overall, the observed changes to species interactions and functional composition suggest a greatly modified role of anurans in altered habitats and major foodweb reorganisation. Such far-reaching changes to the way species groups interact are likely to threaten local biodiversity and ecosystem functioning in natural and particularly modified habitats. However, I could also show, that small-scale habitat quality, provided by riparian reserves, is able to mitigate the negative consequences of land conversion considerably.
IV. Here I assess how logging of rain forest and conversion to oil palm plantations affect the populations of the ant-specialist giant river toad (Phrynoidis juxtaspera), and availability and composition of its ant prey. I measured canopy cover as an estimate for the degree of disturbance. I found that toad abundance decreased with increasing disturbance. At the same time, ant community composition was altered, and local ground-foraging ant species richness increased with disturbance. However, for a given amount of canopy cover, primary forest supported more ant species than altered habitats. Despite these changes, composition of ants consumed by toads was only weakly affected by habitat change, with the exception of the invasive yellow crazy ant (Anoplolepis gracilipes), which was positively selected in oil palm plantations. This suggests that predator-prey interactions can be mostly maintained with habitat disturbance despite shifts in community composition, and even that some predators are capable of exploiting new prey sources in novel ecosystems.
V. I could show that anuran diversity and their trophic interaction is negatively impacted by logging and in particular by conversion to oil palm plantations. From species richness and community composition, my study expanded to phylogenetic, dietary and functional diversity. Furthermore, I investigated the interaction of a particular toad species with its preferred prey (ants), on species level. This increasing degree of detail in my study provided comprehensive results, beyond the detail of many related studies. Overall, conservation of the remaining forest in Southeast Asia is urgently required to protect anuran biodiversity and their trophic interactions.
The infection of a eukaryotic host cell by a bacterial pathogen is one of the most intimate examples of cross-kingdom interactions in biology. Infection processes are highly relevant from both a basic research as well as a clinical point of view. Sophisticated mechanisms have evolved in the pathogen to manipulate the host response and vice versa host cells have developed a wide range of anti-microbial defense strategies to combat bacterial invasion and clear infections. However, it is this diversity and complexity that makes infection research so challenging to technically address as common approaches have either been optimized for bacterial or eukaryotic organisms. Instead, methods are required that are able to deal with the often dramatic discrepancy between host and pathogen with respect to various cellular properties and processes. One class of cellular macromolecules that exemplify this host-pathogen heterogeneity is given by their transcriptomes: Bacterial transcripts differ from their eukaryotic counterparts in many aspects that involve both quantitative and qualitative traits. The entity of RNA transcripts present in a cell is of paramount interest as it reflects the cell’s physiological state under the given condition. Genome-wide transcriptomic techniques such as RNA-seq have therefore been used for single-organism analyses for several years, but their applicability has been limited for infection studies.
The present work describes the establishment of a novel transcriptomic approach for infection biology which we have termed “Dual RNA-seq”. Using this technology, it was intended to shed light particularly on the contribution of non-protein-encoding transcripts to virulence, as these classes have mostly evaded previous infection studies due to the lack of suitable methods. The performance of Dual RNA-seq was evaluated in an in vitro infection model based on the important facultative intracellular pathogen Salmonella enterica serovar Typhimurium and different human cell lines. Dual RNA-seq was found to be capable of capturing all major bacterial and human transcript classes and proved reproducible. During the course of these experiments, a previously largely uncharacterized bacterial small non-coding RNA (sRNA), referred to as STnc440, was identified as one of the most strongly induced genes in intracellular Salmonella. Interestingly, while inhibition of STnc440 expression has been previously shown to cause a virulence defect in different animal models of Salmonellosis, the underlying molecular mechanisms have remained obscure. Here, classical genetics, transcriptomics and biochemical assays proposed a complex model of Salmonella gene expression control that is orchestrated by this sRNA. In particular, STnc440 was found to be involved in the regulation of multiple bacterial target mRNAs by direct base pair interaction with consequences for Salmonella virulence and implications for the host’s immune response. These findings exemplify the scope of Dual RNA-seq for the identification and characterization of novel bacterial virulence factors during host infection.
While TGF-β is able to regulate miRNA expression in numerous cell types, TGF-β-dependent changes in the miRNA profile of CD8+ T cells had not been studied before. Considering that TGF-β suppresses CD8+ T cell effector functions in numerous ways, we wondered whether induction of immune-regulatory miRNAs could add to the known transcriptional effects of TGF-β on immune effector molecules. In this study, we used miRNA arrays, deep sequencing and qRT-PCR to identify miRNAs that are modulated by TGF-β in human CD8+ T cells. Having found that the TGF-β-dependent downregulation of NKG2D surface expression in NK cells and CD8+ T cells does not go along with a corresponding reduction in mRNA levels, this pathway appeared to be a possible target of TGF-β-inducible miRNAs. However, this hypothesis could not be confirmed by miRNA reporter assays. Instead, we observed that DAP10 transcription is suppressed by TGF-β which in turn negatively affects NKG2D surface expression. In spite of promising preliminary experiments, technical difficulties associated with the transfection of primary NK cells and NK cell lines unfortunately precluded the final proof of this hypothesis.
Instead, we focused on the TGF-β-induced changes in the miRNome of CD8+ T cells and confirmed the induction of the miR-23a cluster members, namely miR-23a, miR-27a and miR-24 by three different techniques. Searching for potential targets of these miRNAs which could contribute to the immunosuppressive action of TGF-β in T cells, we identified and confirmed a previously unknown regulation of IFN-γ mRNA by miR-27a and miR-24. Newly generated miRNA reporter constructs further revealed that LAMP1 mRNA is a target of miR-23a. Upon modulation of the miR-23a cluster in CD8+ T cells by the respective miRNA antagomirs and mimics, significant changes in IFN-γ expression confirmed the functional relevance of our findings. Effects on CD107a/LAMP1 expression were, in contrast, rather minimal. Still, overexpression of the miR-23a cluster attenuated the cytotoxic activity of antigen-specific CD8+ T cells. Taken together, these functional data reveal that the miR-23a cluster not only is induced by TGF-β, but also exerts a suppressive effect on CD8+ T-cell effector functions, even in the absence of TGF-β signaling.
In this work, a model-based acceleration of parameter mapping (MAP) for the determination of the tissue parameter T1 using magnetic resonance imaging (MRI) is introduced. The iterative reconstruction uses prior knowledge about the relaxation behavior of the longitudinal magnetization after a suitable magnetization preparation to generate a series of fully sampled k-spaces from a strongly undersampled acquisition. A Fourier transform results in a spatially resolved time course of the longitudinal relaxation process, or equivalently, a spatially resolved map of the longitudinal relaxation time T1.
In its fastest implementation, the MAP algorithm enables the reconstruction of a T1 map from a radial gradient echo dataset acquired within only a few seconds after magnetization preparation, while the acquisition time of conventional T1 mapping techniques typically lies in the range of a few minutes. After validation of the MAP algorithm for two different types of magnetization preparation (saturation recovery & inversion recovery), the developed algorithm was applied in different areas of preclinical and clinical MRI and possible advantages and disadvantages were evaluated.
Fear conditioning is an efficient model of associative learning, which has greatly improved our knowledge of processes underlying the development and maintenance of pathological fear and anxiety. In a differential fear conditioning paradigm, one initially neutral stimulus (NS) is paired with an aversive event (unconditioned stimulus, US), whereas another stimulus does not have any consequences. After a few pairings the NS is associated with the US and consequently becomes a conditioned stimulus (CS+), which elicits a conditioned response (CR).
The formation of explicit knowledge of the CS/US association during conditioning is referred to as contingency awareness. Findings about its role in fear conditioning are ambiguous. The development of a CR without contingency awareness has been shown in delay fear conditioning studies. One speaks of delay conditioning, when the US coterminates with or follows directly on the CS+. In trace conditioning, a temporal gap or “trace interval” lies between CS+ and US. According to existing evidence, trace conditioning is not possible on an implicit level and requires more cognitive resources than delay conditioning.
The associations formed during fear conditioning are not exclusively associations between specific cues and aversive events. Contextual cues form the background milieu of the learning process and play an important role in both acquisition and the extinction of conditioned fear and anxiety. A common limitation in human fear conditioning studies is the lack of ecological validity, especially regarding contextual information. The use of Virtual Reality (VR) is a promising approach for creating a more complex environment which is close to a real life situation.
I conducted three studies to examine cue and contextual fear conditioning with regard to the role of contingency awareness. For this purpose a VR paradigm was created, which allowed for exact manipulation of cues and contexts as well as timing of events. In all three experiments, participants were guided through one or more virtual rooms serving as contexts, in which two different lights served as CS and an electric stimulus as US. Fear potentiated startle (FPS) responses were measured as an indicator of implicit fear conditioning. To test whether participants had developed explicit awareness of the CS-US contingencies, subjective ratings were collected.
The first study was designed as a pilot study to test the VR paradigm as well as the conditioning protocol. Additionally, I was interested in the effect of contingency awareness. Results provided evidence, that eye blink conditioning is possible in the virtual environment and that it does not depend on contingency awareness. Evaluative conditioning, as measured by subjective ratings, was only present in the group of participants who explicitly learned the association between CS and US.
To examine acquisition and extinction of both fear associated cues and contexts, a novel cue-context generalization paradigm was applied in the second study. Besides the interplay of cues and contexts I was again interested in the effect of contingency awareness. Two different virtual offices served as fear and safety context, respectively. During acquisition, the CS+ was always followed by the US in the fear context. In the safety context, none of the lights had any consequences. During extinction, a additional (novel) context was introduced, no US was delivered in any of the contexts. Participants showed enhanced startle responses to the CS+ compared to the CS- in the fear context. Thus, discriminative learning took place regarding both cues and contexts during acquisition. This was confirmed by subjective ratings, although only for participants with explicit contingency awareness. Generalization of fear to the novel context after conditioning did not depend on awareness and was observable only on trend level.
In a third experiment I looked at neuronal correlates involved in extinction of fear memory by means of functional magnetic resonance imaging (fMRI). Of particular interest were differences between extinction of delay and trace fear conditioning. I applied the paradigm tested in the pilot study and additionally manipulated timing of the stimuli: In the delay conditioning group (DCG) the US was administered with offset of one light (CS+), in the trace conditioning group (TCG) the US was presented 4s after CS+ offset. Most importantly, prefrontal activation differed between the two groups. In line with existing evidence, the ventromedial prefrontal cortex (vmPFC) was activated in the DCG. In the TCG I found activation of the dorsolateral prefrontal cortex (dlPFC), which might be associated with modulation of working memory processes necessary for bridging the trace interval and holding information in short term memory.
Taken together, virtual reality proved to be an elegant tool for examining human fear conditioning in complex environments, and especially for manipulating contextual information. Results indicate that explicit knowledge of contingencies is necessary for attitude formation in fear conditioning, but not for a CR on an implicit level as measured by FPS responses. They provide evidence for a two level account of fear conditioning. Discriminative learning was successful regarding both cues and contexts. Imaging results speak for different extinction processes in delay and trace conditioning, hinting that higher working memory contribution is required for trace than for delay conditioning.
The integrity of our genome is continuously endangered by DNA damaging factors. Several cellular mechanisms have evolved to recognize and remove different types of DNA lesions. Despite the wealth of information on the three-dimensional structure and the catalytic mechanism of DNA repair enzymes, the essential process of target site search and identification remains more elusive. How can a small number of repair proteins find and detect the rare sites of damage rapidly and efficiently over an excess of millions of undamaged bases?
To address this pivotal question in DNA repair, I focused on the central players from the two DNA damage excision repair pathways in my studies: nucleotide excision repair (NER) and base excision repair (BER). As examples for completely different approaches of damage search, recognition and verification, I compared the NER protein Xeroderma pigmentosum group D (XPD) with the BER proteins human thymine DNA glycosylase (hTDG) and human 8-oxoguanine glycosylase (hOgg1).
In particular, the single molecule approach of atomic force microscopy (AFM) imaging and complementary biochemical and biophysical techniques were applied. I established a simple, optimized preparation approach, which yields homogeneous and pure samples of long (several hundreds to thousands of base pairs) DNA substrates suitable for the AFM studies with DNA repair proteins. Via this sample preparation, a single target site of interest can be introduced into DNA at a known position, which allows separate analysis of specific protein-DNA complexes bound to the lesion site and nonspecific complexes bound to non-damaged DNA.
The first part of the thesis investigates the XPD protein involved in eukaryotic NER. In general, the NER mechanism removes helix-distorting lesions – carcinogenic UV light induced photoproducts, such as cyclobutane pyrimidine dimers (CPDs) as well as bulky DNA adducts. The 5’-3’ helicase XPD has been proposed to be one of the key players in DNA damage verification in eukaryotic NER, which is still a matter of hot debate. In the studies, I focused on XPD from the archaeal species Thermoplasma acidophilum (taXPD), which shares a relatively high sequence homology with the sequence of the human protein and may serve as a good model for its eukaryotic counterpart. Based on AFM experiments and accompanying DNA binding affinity measurements with the biosensor technology Biolayer Interferometry (BLI), a clear role of XPD in damage verification was deciphered. Specifically, the data suggested that the ATP-dependent 5’-3’ helicase activity of XPD was blocked by the presence of damage leading to stalled XPD-DNA damage verification complexes at the lesion sites.
Successful damage verification led to ATP-dependent conformational changes visible by a significant transition in DNA bend angles from ~ 50° to ~ 65° at the site of the bound protein. Remarkably, this DNA bend angle shift was observed both in the presence of ATP and ATPγs (non-hydrolyzable ATP analog) indicating that ATP-binding instead of ATP hydrolysis was sufficient to induce repair competent conformational changes of XPD. Most importantly, detailed protein binding position and DNA bend angle analyses revealed for the first time that XPD preferably recognizes a bulky fluorescein lesion on the translocated strand, whereas a CPD lesion is preferentially detected on the opposite, non-translocated strand. Despite the different recognition strategies for both types of damages, they share a common verification complex conformation, which may serve as a signal for the recruitment of further NER factors.
In the second part of the thesis, AFM imaging and a 2-Aminopurine fluorescence-based base-flipping assay were combined to investigate damage search and recognition by DNA glycosylases in BER. Exemplarily, I chose to study hTDG as a representative of the vast glycosylase family. hTDG excises thymine and uracil from mutagenic G:T and G:U mispairs contributing to cancer and genetic disease. The AFM data suggested that hTDG uses the intrinsic flexibility of G:T and G:U wobble pairs for initial damage sensing, while scanning DNA as a search complex (SC, slightly bent DNA). Remarkably, hTDG has been indicated to continuously switch between the search and interrogation conformation (IC, stronger bent DNA) during damage search. In the IC, target bases are interrogated by extrahelical base flipping, which is facilitated by protein-induced DNA bending and enhanced DNA flexibility at mismatches. AFM and fluorescence analyses revealed that the flipped base is stabilized via hTDG’s arginine finger. Correct target bases are perfectly stabilized within the enzyme’s catalytic pocket resulting in prolonged residence time and enhanced excision probability. To test for the generalizability of the proposed hTDG damage search model to BER glycosylases, identical studies were performed with a second glycosylase, hOgg1. The data on hOgg1, which removes structurally more stable 8-oxoguanine lesions, supported the hypothesis developed for lesion recognition by hTDG as a common strategy employed by BER glycosylases
Stress has been shown to influence neuroplasticity and is suspected to increase the risk for psychiatric disorders such as major depression and anxiety disorders. Additionally, the short variant of the human serotonin transporter (5-HTT) length polymorphism (5-HTTLPR) is suggested to increase the risk for the development of such disorders. While stress as well as serotonergic signaling are not only discussed to be involved in the development of psychiatric disorders, they are also known to influence hippocampal adult neurogenesis (aN). Therefore, it has long been suspected that aN is involved in the etiology of these illnesses. The exact role of aN in this context however, still remains to be clarified.
In the present doctoral thesis, I am introducing two different studies, which had been carried out to assess possible changes in neuroplasticity and behavior as a result of 5-HTT genotype by stress interactions. In both studies, animals of the 5-HTT knock-out (5-HTT-/-) mouse line were used, which have been found to exhibit increased anxiety- and depression-related behavior, an altered stress response and decreased aggressive behavior. The aim of the first study, the so-called Spatial Learning study, had been to evaluate whether mice with altered levels of brain 5-HT as a consequence of lifelong 5-HTT deficiency perform differently in two spatial memory tests, the Morris Water Maze (WM) and the Barnes Maze (BM) test prospectively differing in aversiveness. Mice of the Spatial Learning study were of male sex and six months of age, and where subjected to a total of 10 (BM) or 15 (WM) trials. My particular interest was to elucidate if there are genotype by treatment interactions regarding blood plasma corticosterone levels and, if neurobiological equivalents in the brain to the found behavioral differences exist. For this purpose I carried out a quantitative immunohistochemistry study, investigating stem cell proliferation (via the marker Ki67) and aN (via the immature neuron marker NeuroD), as well as expression of the two immediate early genes (IEGs) Arc and cFos as a markers for neuronal activity in the hippocampus. The aim of the second study, the chronic mild stress (CMS) study had been to evaluate whether the innate divergent depression-like and anxiety-like behavior of mice with altered levels of brain 5-HT as a consequence of 5-HTT-deficiency is altered any further after being subjected to a CMS paradigm. Two cohorts of one-year-old female mice had been subjected to a variety of unpredictable stressors. In order to exclude possible interfering influences of behavioral testing on corticosterone levels and the outcome of the quantitative immunohistochemistry study the first cohort had been behaviorally tested after CMS while the second one had remained behaviorally untested. The objective of my part of the study was to find out about possible genotype by treatment interactions regarding blood plasma corticosterone as well as regarding aN in the hippocampus of the mice that had been subjected to CMS. For this purpose I performed a quantitative immunohistochemistry study in order to investigate the phenomenon of adult neurogenesis (via Ki67, NeuroD and the immature neuron marker DCX).
Both studies led to interesting results. In the CMS study, we could not replicate the increased innate anxiety- and depression-like behavior in 5-HTT-/- mice known from the literature. However, with regard to the also well documented reduced locomotor activity, as well as the increased body weight of 5-HTT-/- mice compared to their 5-HTT+/- and 5-HTT+/+ littermates, we could demonstrate that CMS leads to increased explorative behavior in the Open Field Test and the Light/Dark Box primarily in 5-HTT+/- und 5-HTT+/+ mice. The Spatial learning study revealed that increased stress sensitivity of 5-HTT-/- mice leads to a poorer performance in the WM test in relation to their 5-HTT+/+ and 5-HTT+/- littermates. As the performance of 5-HTT-/- mice in the less aversive BM was undistinguishable from both other genotypes, we concluded that the spatial learning ability of 5-HTT-/- mice is comparable to that of both other genotypes. As far as stress reactivity is concerned, the experience of a single trial of either the WM or the BM resulted in increased plasma corticosterone levels, irrespective of the 5-HTT genotype. After several trials 5-HTT-/- mice exhibited higher corticosterone concentrations compared with both other genotypes in both tests. Blood plasma corticosterone levels were highest in 5-HTT-/- mice tested in the WM indicating greater aversiveness of the WM and a greater stress sensitivity of 5-HTT deficient mice. In the CMS study, the corticosterone assessment of mice of cohort 1, which had undergone behavioral testing before sacrifice, resulted in significantly elevated corticosterone levels in 5-HTT-/- mice in relation to their 5-HTT+/+ controls. Contrary, corticosterone levels in mice of cohort 1, which had remained behaviorally untested, were shown to be elevated / increased after CMS experience regardless of the 5-HTT genotype. Regarding neuroplasticity, the Spatial Learning study revealed higher baseline levels of cFos- and Arc-ir cells as well as more proliferation (Ki67-ir cells) and higher numbers of neuronal progenitor cells (NeuroD-ir cells) in 5-HTT-/- compared to 5-HTT+/+ mice. Moreover, in 5-HTT-/- mice we could demonstrate that learning performance in the WM correlates with the extent of aN. The CMS study, in which aN (DCX-ir cells), has also been found to be increased in 5-HTT-/- mice compared to their 5-HTT+/+ littermates, yet only in control animals, did show hampered proliferation (Ki67-ir cells) in the hippocampus of all 5-HTT genotypes following CMS experience. Interestingly, the number of immature neurons (DCX-ir cells) was diminished exclusively in 5-HTT-/- mice in response to CMS.
From the Spatial Learning study we concluded, that increased IEG expression and aN levels observed in the hippocampus of 5-HTT deficient mice can be the neurobiological correlate of emotion circuit dysfunction and heightened anxiety of these mice and that 5-HTT-/- animals per se display a “stressed” phenotype as a consequence of long-life 5-HTT deficiency. Due to the different age and sex of the mice in the two studies, they cannot be compared easily. However, although the results of the CMS study seem to contradict the results of the Spatial Learning study at the first glance, they do support the conclusion of the Spatial Learning study by demonstrating that although CMS does have an impact on 5-HTT-/- mice on the neurobiological level (e.g. manifesting in a decrease of DXC-ir cells following CMS) CMS experience cannot add onto their heightened inborn stress-level and is almost ineffective regarding further changes of the behavior of 5-HTT-deficient mice. I thus propose, that 5-HTT-/- mice as a result of lifelong altered 5-HT signaling display a stressed phenotype which resembles a state of lethargy and is paralleled by baseline heightened IEG expression and aN. It cannot be altered or increased by CMS, but it becomes most visible in stressful situations such as repeated spatial learning tests like the WM in which locomotor activity is required.
MicroRNAs are endogenous ≈22 nt long non coding RNA molecules that modulate gene expression
at the post transcriptional level by targeting mRNAs for cleavage or translational repression.
MicroRNA-mRNA interaction involves a contiguous and perfect pairing within complementary
sites usually in the 3’ UTR of the target mRNA. Heart failure is associated with myocyte
hypertrophy and death, due to compensatory pathological remodeling and minimal functional repair
along with microRNA deregulation.
In this study, we identified candidate microRNAs based on their expression strength in
cardiomyocytes and by their ability to regulate hypertrophy. Expression profiling from early and
late stages of heart failure showed several deregulated microRNAs. Of these microRNAs, miR-378
emerged as a potentially interesting microRNA that was highly expressed in the mouse heart and
downregulated in the failing heart. Antihypertrophic activity of miR-378 was first observed by
screening a synthetic miR library for morphologic effects on cardiomyocytes, and validated in vitro proving the tight control of hypertrophy by this miR. We combined bioinformatic target prediction analysis and microarray analysis to identify the targets of miR-378. These analyses suggested that factors of the MAP kinase pathway were enriched among miR-378 targets, namely MAPK1 itself (also termed ERK2), the insulin-like growth factor receptor 1 (IGF1R), growth factor receptor bound protein 2 (GRB2) and kinase suppressor of ras 1 (KSR1). Regulation of these targets by miR-378 was then confirmed by mRNA and protein expression analysis. The use of luciferase reporter constructs with natural or mutated miR-378 binding sites further validated these four proteins as direct targets of miR-378. RNA interference with MAPK1 and the other three targets prevented the prohypertrophic effect of antimiR-378, suggesting that they functionally relate to miR-378. In vivo restoration of disease induced loss of miR-378 in a pressure overload mouse model of hypertrophy using adeno associated virus resulted in partial attenuation cardiac hypertrophy and significant improvement in cardiac function along with reduced expression of the four targets in heart.
We conclude from these findings that miR-378 is an antihypertrophic microRNA in cardiomyocytes, and the main mechanism underlying this effect is the suppression of the MAP kinase-signaling pathway on four distinct levels. Restoration of disease-associated loss of miR-378 through cardiomyocyte-targeted AAV-miR-378 may prove as an effective therapeutic strategy in myocardial disease.
Theories of attention deficit hyperactivity disorder (ADHD) aetiology have placed a focus on impaired behavioural inhibition presumably leading to executive function (EF) deficits. Neuroimaging studies report neurophysiological findings consistent with these hypothesised impairments, and investigations of functional brain activation from a network perspective report hypoactivation in the frontoparietal network as well as hyperactivation in the dorsal attention network. Studies investigating the acute effects of stimulant medication on EF show an improvement on behavioural EF measures including working memory. In addition, methylphenidate (MPH) was shown to up-regulate the task-positive/ frontoparietal network in children and adolescents with ADHD. So far, there are only few studies investigating the impact of ADHD on behavioural and neurophysiological EF measures as well as the effect of several weeks of stimulant medication in adult patients.
The importance of the catechol-O-methyltransferase (COMT) enzyme for subcortical and cortical dopaminergic and noradrenergic functioning furthermore led to studies investigating a potential interactive impact of COMT genotype and ADHD on neuropsychological functioning, with a particular focus on working memory. The results of these studies were very heterogeneous. In addition, as none of the studies compared the results of ADHD patients to those of a healthy control group, possible differential effects of COMT in patients and healthy controls could not be examined.
The aim of this dissertation was to investigate selective attention properties of the central executive component during a working memory task and to transfer this task to fMRI. A third study then aimed to investigate the effects of adult ADHD (aADHD), MPH, and COMT genotype on working memory with a particular focus on activation of the task-positive network during the analysis of the fMRI data.
The first study (EEG) could replicate and extend the results from previous research. This study could furthermore connect the overall activation in frontal areas to suppression efficiency in posterior visual areas as well as establish the impact of hyperactive/ impulsive ADHD symptoms on task performance. The second study (fMRI) allowed the successful transfer of the paradigm to fMRI, and the further replication and extension of previous findings. In addition, this study showed the sensitivity of the task to the effects of the COMT genotype. The third study (fMRI) was one of the first studies that exploratorily investigated the effects COMT in a sample of aADHD patients and a comparable healthy control group. This study showed an interactive effect of these two factors on neuropsychological measures as well as on fMRI activation during a classic n-back working memory task. In addition, this task led to more activation in the task-positive network of the aADHD group compared to a healthy control group in the absence of performance differences, pointing towards compensatory activation in the aADHD group. Furthermore, activation in the frontal cortex was increased in patients taking MPH compared to a placebo. The fMRI data from the selective attention task moreover showed decreased activation in the right DLPFC of the patient group, which was associated with reduced suppression efficiency across all participants. The clinical effect of MPH in the third study was visible but did not reach significance, which is probably attributable to a lack of experimental power.
The studies in this dissertation could successfully replicate and extend previous findings. A goal for future studies should be the further investigation of the interactive effects of COMT genotype and aADHD on neuropsychological test results and fMRI activation, but also on medication response and adverse effects. In this context, the adaptation of a network perspective during the analysis of fMRI data seems to be the best way to detect existing between-group differences.
Mammalian haloacid dehalogenase (HAD)-type phosphatases are an emerging family of enzymes with important functions in physiology and disease. HAD phosphatases can target diverse metabolites, lipids, DNA, and serine/threonine or tyrosine phosphorylated proteins with often high specificity (Seifried et al., 2013). These enzymes thus markedly enlarge the repertoire and substrate spectrum of mammalian phosphatases. However, the basis of HAD phosphatase substrate specificity is still elusive and a number of mammalian HAD phosphatases remain uncharacterized to date. This study characterizes the biochemical and structural properties of AUM (aspartate-based, ubiquitous, Mg2+-dependent phosphatase), a previously unexplored mammalian HAD phosphatase.
In vitro phosphatase assays of purified, recombinant AUM showed phosphatase activity towards para-nitrophenyl phosphate and adenine and guanine nucleotide di- and triphosphates. Inhibitor studies indicated that similar to other HAD superfamily members, the AUM-catalyzed dephosphorylation reaction proceeds via a pentacovalent phosphoaspartate intermediate. In line with an aspartate-based catalytic mechanism, AUM was insensitive to inhibitors of serine/threonine phosphatases. The characterization of the purified recombinant murine enzyme also revealed that AUM exists in equilibrium between dimers and tetramers.
AUM was identified as the closest, yet functionally distinct relative of chronophin, a pyridoxal 5’-phosphate and serine/threonine-directed phosphatase. Phylogenetic analyses showed that AUM and chronophin evolved via duplication of an ancestral gene at the origin of the vertebrates. In contrast to chronophin, AUM acts as a tyrosine-specific HAD-type phosphatase in vitro and in cells. To elucidate how AUM and chronophin achieve these distinct substrate preferences, comparative evolutionary analyses, biochemical approaches and structural analyses were combined. Swapping experiments of less homologous regions between AUM and chronophin were performed. The mutational analysis revealed residues important for AUM catalysis and specificity. A single differently conserved residue in the cap domain of AUM or chronophin is crucial for phosphatase specificity (AUML204, chronophinH182). The X-ray crystal structure of the AUM cap fused to the catalytic core of chronophin (CAC, PDB: 4BKM) was solved to 2.65 Å resolution. It presents the first crystal structure of the murine AUM capping domain. The detailed view of the catalytic clefts of AUM and chronophin reveals the structural basis of the divergent substrate specificities. These presented findings provide insights into the design principles of capped HAD phosphatases and show that their substrate specificity can be encoded by a small number of predictable residues. In addition, the catalytic properties of AUM were investigated, identifying a mechanism of reversible oxidation regulating the activity of AUM in vitro. AUM phosphatase activity is inhibited by oxidation and can be recovered by reduction. The underlying molecular mechanism was revealed by mutational analyses. The cysteines C35, C104 and C243, located in the AUM core domain, are responsible for the inhibition of AUM by oxidation. C293 mediates the redox-dependent tetramerization of AUM in vitro. Based on the chronophin and CAC structure, a direct impact of the oxidation of C35 on the nucleophile D34 is proposed. In addition, a redox-dependent disulfide bridge (C104, C243), connecting the core and cap domain of AUM may be important for an open/close-mechanism. This hypothesis is supported by CD spectroscopy experiments that demonstrate a structural change in AUM upon reduction. These data present the first evidence for the regulation of AUM catalysis by reversible oxidation. This finding is so far unique in the field of HAD phosphatases.
In this context, the first cell-based AUM activity assay was developed. For this, the artificial substrate pNPP was combined with the reducing agent DTT to create a specific AUM activity readout. This fractionation-based assay is the first tool to differentiate between cell lines or tissues with different AUM concentrations or activities.
Taken together, the presented biochemical characterization reveals the specificity determinants and catalytic properties of AUM. General insights into structural determinants of mammalian HAD phosphatase substrate recognition are provided and reversible oxidation as possible regulatory mechanism for AUM is proposed. These findings constitute a framework for further functional analyses to elucidate the biomedical importance of AUM.
The Fanconi anemia (FA) pathway is a replication-dependent DNA repair mechanism which is essential for the removal of interstrand crosslink (ICL) DNA damages in higher eukaryotes (Moldovan and D’Andrea, 2009). Malfunctions in this highly regulated repair network lead to genome instability (Deans and West, 2011). Pathological phenotypes of the disease FA which is caused by mutations in the eponymous pathway are very heterogeneous, involving congenital abnormalities, bone-marrow failure, cancer predisposition and infertility (Auerbach, 2009). The FA pathway comprises a complex interaction network and to date 16 FA complementation groups and associated factors have been identified (Kottemann and Smogorzewska, 2013). Additionally, components of nucleotide excision repair (NER), homologous recombination repair (HRR), and translesion synthesis (TLS) are involved and coordinated by the FA proteins (Niedzwiedz et al., 2004; Knipscheer et al., 2009). One of the FA proteins is the DEAH helicase FANCM. In complex with its binding partners FAAP24 and MHF1/2 it binds the stalled replication fork and activates the FA damage response (Wang et al., 2013). However, the exact steps towards removal of the ICL damage still remain elusive.
To decipher the underlying process of FA initiation by FANCM, this thesis mainly focuses on the archaeal FANCM homolog helicase-associated endonuclease for fork-structured DNA (Hef). Hef from the archaeal organism Thermoplasma acidophilum (taHef) differs from other archaeal Hef proteins and exclusively comprises an N-terminal helicase entity with two RecA and a thumb-like domain while others additionally contain a nuclease portion at the C-terminus. I solved the crystal structure of full-length taHef at a resolution of 2.43 Å. In contrast to the crystal structure of the helicase domain of Hef from Pyrococcus furiosus (pfHef), taHef exhibits an extremely open conformation (Nishino et al., 2005b) which implies that a domain movement of the RecA-like helicase motor domains of 61° is possible thus highlighting the flexibility of helicases which is required to translocate along the DNA. However, small-angle x-ray scattering (SAXS) measurements confirm an intermediate conformation of taHef in solution indicating that both crystal structures represent rather edge states. Most
importantly, proliferating cell nuclear antigen (PCNA) was identified as an interaction partner of Hef. This interaction is mediated by a highly conserved canonical PCNA interacting peptide (PIP) motif. Intriguingly, the presence of PCNA does not alter the ATPase nor the helicase activity of taHef, thus suggesting that the interaction is entirely dedicated to recruit taHef to the replication fork to fulfill its function. Due to a high level of flexibility the taHef-taPCNA complex could not be crystallized and therefore SAXS was utilized to determine a low-resolution model of this quaternary structure.
This newly discovered PCNA interaction could also be validated for the eukaryotic FANCM homolog Mph1 from the thermophilic fungus Chaetomium thermophilum (ctMph1). As the first step towards the characterization of this interaction I solved the crystal structure of PCNA from Chaetomium thermophilum (ctPCNA).
Furthermore, it was possible to achieve preliminary results on the putative interaction between the human proteins FANCM and PCNA (hsFANCM, hsPCNA). In collaboration with Detlev Schindler (Human Genetics, Würzburg) and Weidong Wang (National Institute on Aging, Baltimore, USA) co-immunoprecipitation (CoIP) experiments were performed using hsFANCM and hsPCNA expressed in HEK293 cells. Although an interaction was reproducibly observed in hydroxyurea stimulated cells
further experiments and optimization procedures are required and ongoing.
Leishmaniasis is a neglected tropical disease that can be manifested through different clinical forms, ranging from cutaneous to visceral. The host response against Leishmania spp. is greatly dependent on T cell-mediated immunity, in which T helper 1 responses are associated with macrophage activation and elimination of the parasite, while regulatory T cells and T helper 2 responses are correlated with parasite survival and persistence of infection. Leishmania uses different virulence factors as strategies for evading the immune response of the host. One of them are cathepsin-like cysteine proteases, which are currently under extensive investigation as targets for drug development. Previous studies with inhibitors of cathepsins B and L in vivo revealed an outstanding modulation of the host T helper cell response. However, the mechanisms behind these observations were not further investigated. Given the urgent need for better treatments against leishmaniasis, the aim of this study was to investigate the effects that the lack of cathepsin B and L activity have on the signals that dendritic cells use to instruct T helper cell polarization in response to infection with Leishmania major.
The cathepsin inhibitors tested showed low or no cytotoxicity in bone marrow-derived dendritic cells, and dendritic cells and macrophages could be generated from cathepsin B and cathepsin L-deficient mice without apparent alterations in their phenotype in comparison to wild-type controls. Furthermore, lack of cathepsin B and L activity showed no impact in the rate of promastigote processing by dendritic cells. Cathepsin B and cathepsin L-deficient macrophages showed no differences in parasite proliferation and capacity to produce nitric oxide in comparison to wild-type macrophages. In response to the parasite, dendritic cells treated with a cathepsin B inhibitor and dendritic cells from cathepsin B-deficient mice showed higher levels of expression of major histocompatibility complex (MHC) class II molecules than dimethyl sulfoxide (DMSO) or wild-type controls, but it was not accompanied by changes in the expression of costimulatory molecules. Wild-type dendritic cells and macrophages are not able to express the pro-inflammatory cytokine interleukin (IL)-12 in response to promastigotes. However, cells treated with a cathepsin B inhibitor or cells deficient for cathepsin B were able to express IL-12, whilethe expression of other cytokines -including IL-6 and tumor necrosis factor (TNF)-alpha-remained unchanged. These characteristics point towards a more “pro-Th1” profile of dendritic cells in the absence of cathepsin B.
This data is the first report on IL-12 regulation depending on cathepsin B. The IL-12 up-regulation observed was already present at the transcriptional level. Furthermore, it was also present in macrophages and dendritic cells in response to LPS, and the latter had a higher capacity to induce T cell helper 1 polarization in vitro than wild-type dendritic cells. The activation of different signaling pathways was analyzed, but the up-regulation of IL-12 could not be attributed to modulation of nuclear factor-kappaB (NFkappaB), p38 mitogen activated protein kinase (MAPK) and extra-cellular signal-regulated kinase (ERK)1/2 pathways. Thus, the mechanism behind IL-12 regulation by cathepsin B remains to be elucidated, and the impact of these effects is yet to be confirmed in vivo. Altogether it is tempting to speculate that cathepsin B, in addition to its role in processing endocytosed material, is involved in the modulation of the pro-inflammatory cytokine IL-12.
Besides HIV and tuberculosis, malaria still is one of the most devastating infectious diseases especially in developing countries, with Plasmodium falciparum being responsible for the frequently lethal form of malaria tropica. It is a major cause of mortality as well as morbidity, whereby pregnant women and children under the age of five years are most severely affected. Rapidly emerging drug resistances and the lack of an effective and safe vaccine hamper the combat against malaria by chemical and pharmacological regimens, and moreover the poor socio-economic and healthcare conditions in malaria-endemic countries are compromising the extermination of this deadly tropical disease to a large extent. Malaria research is still questing for druggable targets in the parasitic protozoan which pledge to be refractory against evolving resistance-mediating mutations and yet constitute affordable and compliant antimalarial chemotherapeutics.
The parasite kinome consists of members that represent most eukaryotic protein kinase groups, but also contains several groups that can not be assigned to conservative ePK groups. Moreover, given the remarkable divergence of plasmodial kinases in respect to the human host kinome and the fact that several plasmodial kinases have been identified that are essential for the intraerythrocytic developmental cycle, these parasite enzymes represent auspicious targets for antimalarial regimens. Despite elaborate investigations on several other ePK groups, merely scant research has been conducted regarding the four identified members of the cyclin-dependent kinase-like kinase (CLK) family, PfCLK-1-4. In other eukaryotes, CLKs are involved in mRNA processing and splicing by means of phosphorylation of serine/arginine-rich (SR) proteins, which are crucial components of the splicing machinery in the alternative splicing pathway. All four PfCLKs are abundantly expressed in asexual parasites and gametocytes, and stage-specific expression profiles of PfCLK-1 and PfCLK-2 exhibited nucleus-associated localization and an association with phosphorylation activity. In the course of this study, PfCLK-3 and PfCLK-4 were functionally characterized by indirect immunofluorescence, Western blot analysis and kinase activity assays. These data confirm that the two kinases are primarily expressed in the nucleus of trophozoites and both kinases possess in vitro phosphorylation activity on physiological substrates. Likewise PfCLK-1 and PfCLK-2, reverse genetic studies exhibited the indispensability of both PfCLKs on the asexual life cycle of P. falciparum, rendering them as potential candidates for antiplasmodial strategies. Moreover, this study was conducted to identify putative SR proteins as substrates of all four PfCLKs. Previous alignments revealed a significant homology of the parasite CLKs to yeast SR protein kinase Sky1p. Kinase activity assays showed in vitro phosphorylation of the yeast Sky1p substrate and SR protein Npl3p by precipitated PfCLKs. In addition, four homologous plasmodial SR proteins were identified that are phosphorylated by PfCLKs in vitro: PfASF-1, PFSRSF12, PfSFRS4 and PfSR-1. All four parasite SR splicing factors are predominantly expressed in the nuclei of trophozoites. For PfCLK-1, a co-localization with the SR proteins was verified.
Finally, a library of human and microbial CLK inhibitors and the antiseptic chlorhexidine (CHX) was screened to determine their inhibitory effect on different parasite life cycle stages and on the PfCLKs specifically. Five inhibitors out of 63 compounds from the investigated library were selected that show a moderate inhibition on asexual life cycle stages with IC50 values ranging between approximately 4 and 8 µM. Noteworthy, these inhibitors belong to the substance classes of aminopyrimidines or oxo-β-carbolines. Actually, the antibiotic compound CHX demonstrated an IC50 in the low nanomolar range. Stage-of-inhibition assays revealed that CHX severely affects the formation of schizonts. All of the selected CLKs inhibitors also affect gametocytogenesis as well as gametogenesis, as scrutinized in gametocyte toxicity assays and exflagellation assays, respectively. Kinase activity assays confirm a specific inhibition of CLK-mediated phosphorylation of all four kinases, when the CLK inhibitors are applied on immunoprecipitated PfCLKs. These findings on PfCLK-inhibiting compounds are initial attempts to determine putative antimalarial compounds targeting the PfCLKs. Moreover, these results provide an effective means to generate chemical kinase KOs in order to phenotypically study the role of the PfCLKs especially in splicing events and mRNA metabolism. This approach of functionally characterizing the CLKs in P. falciparum is of particular interest since the malarial spliceosome is still poorly understood and will gain further insight into the parasite splicing machinery.
Studies on receptor signaling and regulation in platelets and T cells from genetically modified mice
(2014)
Receptors with tyrosine-based signaling motifs control essential functions of hematopoietic cells, including lymphocytes and platelets. Downstream of the platelet receptor glycoprotein (GP) VI and the T cell receptor (TCR) the immunoreceptor tyrosine-based activation motif (ITAM) initiates a signaling cascade that involves kinases, adapter and effector proteins and finally leads to cellular activation. This thesis summarizes the results of three studies investigating different aspects of receptor signaling and regulation in platelets and T cells.
In the first part, the impact of constitutive Ca2+ influx on TCR signaling and T cell physiology was investigated using a transgenic mouse line with a mutation in the Ca2+ sensor stromal interaction molecule 1 (STIM1). The elevated cytoplasmic Ca2+ level resulted in an altered phosphorylation pattern of the key enzyme phospholipase (PL) Cγ1 in response to TCR stimulation, but without affecting its enzymatic activity. Withdrawal of extracellular Ca2+ or inhibition of the phosphatase calcineurin restored the normal phosphorylation pattern. In addition, there was a decrease in the release of Th2-type cytokines interleukin 4, 5 and 13 upon stimulation in vitro.
The second part of the thesis deals with the role of the adapter protein growth factor receptor-bound protein 2 (Grb2) in platelets using a megakaryocyte/platelet-specific knockout mouse line. Loss of Grb2 severely impaired signaling of GPVI and C-type lectin-like receptor 2 (CLEC-2), a related hemITAM receptor. This was attributed to defective stabilization of the linker for activation of T cells (LAT) signalosome and resulted in reduced adhesion, aggregation, Ca2+ mobilization and procoagulant activity downstream of (hem)ITAM-coupled receptors in vitro. In contrast, the signaling pathways of G protein-coupled receptors (GPCRs) and the integrin αIIbβ3, which do not utilize the LAT signalosome, were unaffected. In vivo, the defective (hem)ITAM signaling caused prolonged bleeding times, however, thrombus formation was only affected under conditions where GPCR signaling was impaired (upon acetylsalicylic acid treatment). These results establish Grb2 as an important adapter protein in the propagation of GPVI- and CLEC-2-induced signals.
Finally, the proteolytic regulation of the immunoreceptor tyrosine-based switch motif (ITSM)-bearing receptor CD84 in platelets was investigated. This study demonstrated that in mice CD84 is cleaved by two distinct and independent proteolytic mechanisms upon platelet activation: shedding of the extracellular part, which is exclusively mediated by a disintegrin and metalloproteinase (ADAM) 10 and cleavage of the intracellular C-terminus by the protease calpain. Finally, the analysis of soluble CD84 levels in the plasma of transgenic mice revealed that shedding of CD84 by ADAM10 occurs constitutively in vivo.
Evolution of Vγ9Vδ2 T-cells
(2014)
Human Vγ9Vδ2 T cells are the major subset of blood γδ T cells and account for 1-5% of blood T cells. Pyrophosphorylated metabolites of isoprenoid biosynthesis are recognized by human Vγ9Vδ2 T cells and are called as phosphoantigens (PAg). Isopentenyl pyrophosphate (IPP) and (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) are among the few well studied PAg. IPP is found in all organisms while HMBPP is a precursor of IPP found only in eubacteria, plants and apicomplexaen parasite. Interestingly, the PAg reactive Vγ9Vδ2 T cells are so far identified only in human and higher primates but not in rodents. Hence, Vγ9Vδ2 T cells are believed to be restricted to primates. With regard to PAg recognition, a Vγ9JP recombined TCRγ chain and certain CDR3 motifs of the TCR chain are mandatory. The BTN3A1 molecule is essential for a response to PAg. BTN3 is a trans-membrane protein belonging to butyrophilin family of proteins. Though BTN3A1 was found to be essential for PAg presentation, the exact molecular basis of PAg presentation still remains unclear.
This thesis presents new data on the evolution of Vγ9Vδ2 TCR and its ligands (BTN3) as well as the genetic basis of PAg presentation to Vγ9Vδ2 TCR.
The comprehensive analysis of genomic database sequences at NCBI and other public domain databases revealed for the first time that Vγ9, Vδ2 and BTN3 genes emerged and co-evolved along with the placental mammals. Vγ9, Vδ2 and BTN3 genes are scattered across mammalian species and not restricted to primates. But interestingly, all three genes are highly conserved between phylogenetically distinct species. Moreover, the distribution pattern of Vγ9, Vδ2 TCR genes and BTN3 genes suggests a functional association between these genes representing the TCR - ligand relationship. Alpaca (Vicugna pacos), a member of the camelid family, is one among the 6 candidate non-primate species which were found to possess functional Vγ9, Vδ2 and BTN3 genes.
From peripheral lymphocytes of alpaca, Vγ9 chain transcripts with a characteristic JP rearrangement and transcripts of Vδ2 chains with a CDR3 typical for PAg-reactive TCR were identified. The transduction of αβ TCR negative mouse thymoma BW cells with alpaca Vγ9 and Vδ2 TCR chains resulted in surface expression of the TCR complex as it was deduced from detection of cell surface expression of mouse CD3. Cross-linking of alpaca Vγ9Vδ2 TCR transductants with anti-CD3ε led to IL-2 production which confirmed that alpaca Vγ9 and Vδ2 TCR chains pair to form a functional TCR. Besides the conservation of human like Vγ9 and Vδ2 TCR chains, alpaca has conserved an orthologue for human BTN33A1 as well. Interestingly, the predicted PAg binding sites of human BTN3A1 was 100% conserved in deduced amino acid sequence of alpaca BTN3A1. All together alpaca is a promising candidate for further studies as it might have preserved Vγ9Vδ2 T cells to function in surveillance of stress and infections.
This thesis also provides the sequence of Vγ9Vδ2 TCR of African green monkey (Chlorocebus aethiops), which was previously unknown. Moreover, our data indicates the lack of any species specific barrier which could hinder the PAg presentation by African monkey derived COS cells to human Vγ9Vδ2 TCR and vice versa of human cells to African green monkey Vγ9Vδ2 TCR which was in contradiction to previously reported findings.
Apart from the above, the thesis also presents new data on the genetic basis of PAg presentation to Vγ9Vδ2 T cells, which revealed that human chromosome 6 is sufficient for the presentation of exogenous and endogenous PAg. By employing human/mouse somatic hybrids, we identified the role of human chromosome 6 in PAg presentation and in addition, we observed the lack of capacity of human chromosome 6 positive hybrids to activate Vγ9Vδ2 TCR transductants in the presence of the alkylamine sec-butylamine (SBA). Investigation of Chinese hamster ovary (CHO) cells containing the human chromosome 6 also yielded similar results. This suggests that aminobisphosphonates (zoledronate) and alkylamines employ different mechanisms for activation of Vγ9Vδ2 T cells although both have been described to act by inhibition of farnesyl pyrophosphate synthase activity which is known to increase intracellular levels of the IPP.
In conclusion, this thesis suggests that Vγ9, Vδ2 and BTN3 genes controlling Vγ9Vδ2 TCR- ligand relationship emerged and co-evolved along with placental mammals; and also identified candidate non-primate species which could possess Vγ9Vδ2 T cells. Furthermore, it suggests alpaca as a promising non-primate species to investigate the physiological function of Vγ9Vδ2 T cells. With respect to PAg antigen presentation it was shown that chromosome 6 is essential and sufficient for exogenous and endogenous PAg presentation. Moreover, the alkylamine SBA and aminobisphosphonate zoledronate may engage different cellular mechanism to exert inhibition over IPP consumption. The thesis raises interesting questions which need to be addressed in future: 1) What are the environmental and evolutionary factors involved in preservation of Vγ9Vδ2 T cells only by few species? 2) What could be the functional nature and antigen recognition properties of such a conserved T cell subset? 3) What is the genetic and molecular basis of the differential capacity of human chromosome 6 bearing rodent-human hybridoma cells in activating Vγ9Vδ2 T cells in presence of SBA and aminobisphosphonates?
The haloacid dehalogenase (HAD) family of phosphatases is an ancient, ubiquitous group of enzymes, and their emerging role in human health and disease make them attractive targets for detailed analyses.
This thesis comprises the biochemical and structural characterization of chronophin, an HAD-type
phosphatase, which has been shown to act on Ser3-phosphorylated cofiln-1, a key regulator of actin dynamics, and on the Ser/Thr-phosphorylated steroid receptor co-activator 3 (SRC-3). Besides being a specific phosphoprotein phosphatase, chronophin also acts on the small molecule pyridoxal 5'-phosphate (PLP, vitamin B6), implying that chronophin serves as a regulator of a variety important physiological pathways. The analysis of chronophin was performed on different levels, ranging from intrinsic regulatory mechanisms, such as the allosteric regulation via dimerization or the characterization of specificity determinants, to modes of extrinsic modulation, including the association with putative interacting proteins or the generation of chronophin-specific inhibitors.
The association of the previously identified putative chronophin interactors calcium- and integrinbinding protein 1 (CIB1) and calmodulin was investigated using recombinantly expressed and purified proteins. These studies revealed that the interaction of chronophin with CIB1 or calmodulin is mutually exclusive and regulated by calcium. Neither CIB1 nor calmodulin had an effect on the in vitro chronophin phosphatase activity towards PLP or phospho-cofilin-1, but might regulate other functions of this important phosphatase.
The role of chronophin dimerization was studied by generating a constitutively monomeric variant,
which showed reduced PLP hydrolyzing activity. X-ray crystallographic studies revealed that dimerization is essential for the positioning of the substrate specificity loop in chronophin, unraveling a previously unknown mechanism of allosteric regulation through a homophilic interaction. This mechanism potentially applies to other enzymes of the C2a subfamily of HAD-type phosphatases, as all structurally characterized members show a conserved mode of dimerization.
The general determinants of substrate specificity in the C2a subfamily of HAD phosphatases were
investigated by performing domain swapping experiments with chronophin and its paralog AUM and
subsequent biochemical analyses of the hybrid proteins. The X-ray crystallographic structure
determination of the chronophin catalytic domain equipped with the AUM capping domain revealed the first partial structure of AUM. This structural information was then used in subsequent studies that analyzed the divergent substrate specificities of AUM and chronophin in an evolutionary context.
Finally, a set of four chronophin inhibitors were generated based on the structure of PLP and
characterized biochemically, showing moderate inhibitory effects with IC50-values in the micromolar range. These compounds nevertheless constitute valuable tools for future in vitro experiments, such as studies concerning the structure-function relationship of chronophin as a PLP phosphatase. In addition, the crystal structure of one inhibitor bound to chronophin could be solved. These results provide the basis for the further development of competitive chronophin inhibitors with increased specificity and potency.
Calcium ions can activate intracellular signalling cascades that control key functions in all types of neurons. These functions include neuronal excitability and excitation, synaptic plasticity, cell migration, transmitter release, gene transcription, and apoptosis. The major intracellular neuronal store for calcium is the endoplasmic reticulum (ER), a continuous and dynamic, membranous organelle that extends through all parts of neurons, from axons to dendrites. The calcium concentration in the ER is appr. one thousand fold higher than in the cytosol and this calcium gradient is built up by the sarco-/endoplasmic reticulum calcium ATPase (SERCA) pump that pumps calcium from the cytosol into the ER.
Despite detailed knowledge about various induced calcium signals within neurons, it was still elusive, how resting neurons maintain their ER calcium content at rest. In order to shed light on the calcium homeostasis at rest, the targeted-esterase induced dye loading (TED) technique was improved. TED allows the direct and non-disruptive visualization of ER calcium in presence of extracellular calcium, thus enabling to visualize the dynamic flow of ER calcium. TED is based on the overexpression of an ER-targeted mouse carboxylesterase. Inside the ER the carboxylesterase cleaves the acetoxymethyl ester calcium dye Fluo5N, AM, thereby converting this dye into a calcium sensitive, low-affinity, cell membrane impermeable calcium indicator that is trapped in the ER. When bound to calcium ions and excited by fluorescent light, its fluorescence intensity increases one hundredfold compared to the calcium-free state.
It was observed that calcium withdrawal from resting neurons led to a rapid loss of calcium from both the ER and the cytosol, which recovered upon calcium re-addition. It was concluded that a strong calcium influx and efflux must exist under resting conditions that maintain a constant calcium concentration in neurons at rest. TED calcium imaging could visualize this resting calcium influx event. When the inhibitor of store-operated calcium entry (SOCE), SKF-96365, was acutely added to neurons an immediate decline in ER calcium levels was observed, whereas cytosolic calcium levels remained constant. Based on these findings, a novel calcium homeostasis model is proposed in which a strong SOCE-like calcium influx and a corresponding calcium efflux maintain the ER calcium levels at rest. These fluxes are adapted to disturbances in order to maintain a constant calcium level in resting neurons.
This study visualizes for the first time the resting calcium flow into the ER. The calcium enters the neurons via a store-operated calcium entry-like mechanism, a form of calcium influx that was thought to be induced by signalling events.
Cardiac healing after myocardial infarction (MI) represents the cardinal prerequisite for proper replacement of the irreversibly injured myocardium. In contrast to innate immunity, the functional role of adaptive immunity in postinfarction healing has not been systematically addressed. The present study focused on the influence of CD4+ T lymphocytes on wound healing and cardiac remodeling after experimental myocardial infarction in mice. Both conventional and Foxp3+ regulatory CD4+ T cells (Treg cells) became activated in heart draining lymph nodes after MI and accumulated in the infarcted myocardium. T cell activation was strictly antigen-dependant as T cell receptor-transgenic OT-II mice in which CD4+ T cells exhibit a highly limited T cell
receptor repertoire did not expand in heart-draining lymph nodes post-MI. Both OT-II and major histocompatibility complex class II-deficient mice lacking a CD4+ T cell compartment showed a fatal clinical postinfarction outcome characterized by disturbed scar tissue construction that resulted in impaired survival due to a prevalence of left-ventricular ruptures. To assess the contribution of anti-inflammatory Treg cells on wound healing after MI, the Treg cell compartment was depleted using DEREG mice that specifically express the human diphtheria toxin receptor in Foxp3-positive cells, resulting in Treg cell ablation after diphtheria toxin administration. In a parallel line of experiments, a second model of anti-CD25 antibody-mediated Treg cell immuno-depletion was used. Treg cell ablation prior to MI resulted in adverse postinfarction left-ventricular dilatation associated with cardiac deterioration. Mechanistically, Treg cell depletion resulted in an increased recruitment of pro-inflammatory neutrophils and Ly-6Chigh monocytes into the healing myocardium. Furthermore, Treg cell-ablated mice exhibited an adverse activation of conventional non-regulatory CD4+ and CD8+ T cells that
showed a reinforced infiltration into the infarct zone. Increased synthesis of TNFα and IFNγ by conventional CD4+ and CD8+ T cells in hearts of Treg cell-depleted mice provoked an M1-like macrophage polarization characterized by heightened expression of healing-compromising induced NO synthase, in line with a reduced synthesis of healing-promoting transglutaminase factor XIII (FXIII), osteopontin (OPN) and transforming growth factor beta 1 (TGFβ1).
Therapeutic Treg cell activation by a superagonistic anti-CD28 monoclonal antibody stimulated Treg cell accumulation in the infarct zone and led to an increased expression of mediators inducing an M2-like macrophage polarization state, i.e. interleukin-10, interleukin-13 and TGFβ1. M2-like macrophage differentiation in the healing infarct was associated with heightened expression of scar-forming procollagens as well as scar-stabilizing FXIII and OPN, resulting in improved survival due to a reduced incidence of left-ventricular ruptures. Therapeutic Treg cell activation and the induction of a beneficial M2-like macrophage polarization was further achieved by employing a treatment modality of high clinical potential, i.e. by therapeutic administration of IL-2/ anti-IL-2 monoclonal antibody complexes. The findings of the present study suggest that therapeutic Treg cell activation and the resulting improvement of healing may represent a suitable strategy to attenuate adverse infarct expansion, left-ventricular remodeling, or infarct ruptures in patients with MI.
Background: Food craving refers to an intense desire to consume a specific kind of food of which chocolate is the most often craved one. It is this intensity and specificity that differentiates food craving from feelings of hunger. Although food craving and hunger often co-occur, an energy deficit is not a prerequisite for experiencing food craving, that is, it can also occur without being hungry. Food craving often precedes and predicts over- or binge eating which makes it a reasonable target in the treatment of eating disorders or obesity. One of the arguably most extensively validated measures for the assessment of food craving are the Food Cravings Questionnaires (FCQs), which measure food craving on a state (FCQ-S) and trait (FCQ-T) level. Specifically, the FCQ-S measures the intensity of current food craving whereas the FCQ-T measures the frequency of food craving experiences in general. The aims of the present thesis were to provide a German measure for the assessment of food craving and to investigate cognitive, behavioral, and physiological correlates of food craving. For this purpose, a German version of the FCQs was presented and its reliability and validity was evaluated. Using self-reports, relationships between trait food craving and dieting were examined. Cognitive-behavioral correlates of food craving were investigated using food-related tasks assessing executive functions. Psychophysiological correlates of food craving were investigated using event-related potentials (ERPs) in the electroencephalogram and heart rate variability (HRV). Possible intervention approaches to reduce food craving were derived from results of those studies.
Methods: The FCQs were translated into German and their psychometric properties and correlates were investigated in a questionnaire-based study (articles #1 & #2). The relationship between state and trait food craving with executive functioning was examined with behavioral tasks measuring working memory performance and behavioral inhibition which involved highly palatable food-cues (articles #3 & #4). Electrophysiological correlates of food craving were tested with ERPs during a craving regulation task (article #5). Finally, a pilot study on the effects of HRV-biofeedback for reducing food craving was conducted (article #6).
Results: The FCQs demonstrated high internal consistency while their factorial structure could only partially be replicated. The FCQ-T also had high retest-reliability which, expectedly, was lower for the FCQ-S. Validity of the FCQ-S was shown by positive relationships with current food deprivation and negative affect. Validity of the FCQ-T was shown by positive correlations with related constructs. Importantly, scores on the subscales of the FCQ-T were able to discriminate between non-dieters and successful and unsuccessful dieters (article #1). Furthermore, scores on the FCQ-T mediated the relationship between rigid dietary control strategies and low dieting success (article #2). With regard to executive functioning, high-calorie food-cues impaired working memory performance, yet this was independent of trait food craving and rarely related to state food craving (article #3). Behavioral disinhibition in response to high-calorie food-cues was predicted by trait food craving, particularly when participants were also impulsive (article #4). Downregulation of food craving by cognitive strategies in response to high-calorie food-cues increased early, but not later, segments of the Late Positive Potential (LPP) (article #5). Few sessions of HRV-biofeedback reduced self-reported food cravings and eating and weight concerns in high trait food cravers (article #6).
Conclusions: The German FCQs represent sound measures with good psychometric properties for the assessment of state and trait food craving. Although state food craving increases during cognitive tasks involving highly palatable food-cues, impairment of task performance does not appear to be mediated by current food craving experiences. Instead, trait food craving is associated with low behavioral inhibition in response to high-calorie food-cues, but not with impaired working memory performance. Future studies need to examine if trait food craving and, subsequently, food-cue affected behavioral inhibition can be reduced by using food-related inhibition tasks as a training. Current food craving and ERPs in response to food-cues can easily be modulated by cognitive strategies, yet the LPP probably does not represent a direct index of food craving. Finally, HRV-biofeedback may be a useful add-on element in the treatment of disorders in which food cravings are elevated. To conclude, the current thesis provided measures for the assessment of food craving in German and showed differential relationships between state and trait food craving with self-reported dieting behavior, food-cue affected executive functioning, ERPs and HRV-biofeedback. These results provide promising starting points for interventions to reduce food craving based on (1) food-cue-related behavioral trainings of executive functions, (2) cognitive craving regulation strategies, and (3) physiological parameters such as HRV-biofeedback.
Motivation and Aim:
Cardiovascular disease has been the leading cause of mortality and morbidity throughout the world. In developed countries, cardiovascular diseases are already responsible for a majority of deaths and will become the pre-eminent health problem worldwide (1,2). Rupture of atherosclerotic plaque accounts for approximately 70% of fatal acute myocardial infarction and sudden heart deaths. Conventional criterias for the diagnosis of “vulnerable plaques” are calcified nodules, yellow appearance of plaque, a thin cap, a large lipid core, severe luminal stenosis, intraplaque hemorrhage, inflammation, thrombogenicity, and plaque injury (3-5).
Noninvasive diagnosis of vulnerable plaque still remains a great challenge and a huge research prospect, which triggered us to investigate the feasibility of PET imaging on the evaluation of atherosclerosis. Nuclear imaging of atherosclerosis, especially co-registered imaging modalities, could provide a promising diagnostic tool including both anatomy and activities to identify vulnerable atherosclerotic plaque or early detection of inflammatory endothelium at risk. Furthermore, the development of specific imaging tracers for clinical applications is also a challenging task. The aim of this work was to assess the potential of novel PET imaging probes associated with intra-plaque inflammation on animal models and in human respectively.
Methods
In this work, several molecular imaging modalities were employed for evaluation of atherosclerosis. They included Positron emission tomography / Computed tomography (PET/CT) for human studies, and micro-PET, autoradiography and high-resolution magnetic resonance imaging (MRI) for animal studies. Radiotracers for PET imaging included the glucose analogue 18F-Fluorodeoxyglucose (18F-FDG), the somatostatin receptor avide tracer 68Ga-DOTATATE, and the Gallium-68 labeled fucoidan (68Ga-Fucoidan), which was developed as a PET tracer to detect endothelial P-selectin, which overexpressed at early stage of atherosclerosis and endothelial overlying activated plaque. Tracer’s capabilities were firstly assessed on cellular level in vitro. Subsequently, Animal studies were conducted in two animal models: 1, Apolipoprotein E (ApoE-/-) mice having severe atherosclerotic plaque; 2, Lipopolysaccharide (LPS) -induced mice for receiving acute vascular inflammation. Corresponding analyses on protein and histological level were conducted as well to confirm our results.
In human study, 16 patients with neuroendocrine tumors (NETs) were investigated on imaging vascular inflammation. These patients had undergone both 68Ga-DOTATATE PET/CT and 18F-FDG PET/CT for staging or restaging within 6 weeks. 16 patients were randomized into two groups: high-risk group and low-risk group. Uptake ratio of both tracers from two groups were compared and correlated with common cardiovascular risk factors.
Results and Conclusion
In murine study, the expression of somatostatin receptor 2, which is the main bio-target of 68Ga-DOTATATE on macrophage/monocyte was confirmed by flow cytometry and immunohistochemistry. Prospectively, high specific accumulation of 68Ga-DOTATATE to the macrophage within the plaques was observed in aorta lesions by autoradiography and by micro-PET. In study with 68Ga-fucoidan, a strong expression of P-selectin on active endothelium overlying on inflamed plaque but weaker on inactive plaques was confirmed. Specific focal uptake of 68Ga-fucoidan were detected at aorta segments by micro-PET, and correlated with high-resolution magnetic resonance imaging (MRI), which was used to characterize the morphology of plaques. 68Ga-fucoidan also showed a greater affinity to active inflamed plaque in comparison of inactive fibrous plaque, which was assessed by autoradiography. Specificity of 68Ga-DOTATATE and 68Ga-fucoidan were confirmed by ex-vivo blocking autoradiography and in vivo blocking PET imaging respectively.
In human study, focal uptake of both 18F-FDG and 68Ga-DOTATATE was detected. Analyzing concordance of two tracers’ uptake ratio, Out of the 37 sites with highest focal 68Ga-DOTATATE uptake, 16 (43.2%) also had focal 18F-FDG uptake. Of 39 sites with highest 18F-FDG uptake, only 11 (28.2%) had a colocalized 68Ga-DOTATATE accumulation. Correlated tracers’ uptake and calcium burden and risk factors, Mean target-to-background ratio (TBR) of 68Ga-DOTATATE correlated significantly with the presence of calcified plaques (r=0.52), hypertension (r=0.60), age (r=0.56) and uptake of 18F-FDG (r=0.64). TBRmean of 18F-FDG correlated significantly only with hypertension (r=0.58; p<0.05). Additionally, TBRmean of 68Ga-DOTATATE is significant higher in the high risk group while TBRmean of 18F-FDG is not.
In conclusion, we evaluated vascular inflammation of atherosclerosis non-invasively using the two PET tracers: 68Ga-DOTATATE and 68Ga-Fucoidan. 68Ga-DOTATATE show specific affinity to infiltrated macrophage within the plaques. 68Ga-Fucoidan may hold the potential to discriminate between active and inactive atherosclerotic plaques in terms of variant accumulation on different-types of plaques. PET as leading molecular imaging technique provides superiority in assessing cellular activity, which is pivotal for understanding internal activity of atherosclerotic plaques. Since diagnosis of atherosclerosis is a complex and multi-dimensional task. More integrated imaging technology such as PET/MRI, faster imaging algorithm, more efficient radiotracer are required for further development of atherosclerosis imaging,
The number of fungal infections is rising in Germany and worldwide. These infections are mainly caused by the opportunistic fungal pathogen C. albicans, which especially harms immunocompromised people. With increasing numbers of fungal infections, more frequent and longer lasting treatments are necessary and lead to an increase of drug resistances, for example against the clinically applied therapeutic fluconazole. Drug resistance in C. albicans can be mediated by the Multidrug resistance pump 1 (Mdr1), a membrane transporter belonging to the major facilitator family. However, Mdr1-mediated fluconazole drug resistance is caused by the pump’s regulator, the transcription factor Mrr1 (Multidrug resistance regulator 1). It was shown that Mrr1 is hyperactive without stimulation or further activation in resistant strains which is due to so called gain of function mutations in the MRR1 gene.
To understand the mechanism that lays behind this constitutive activity of Mrr1, the transcription factor should be structurally and functionally (in vitro) characterized which could provide a basis for successful drug development to target Mdr1-mediated drug resistance caused by Mrr1. Therefore, the entire 1108 amino acid protein was successfully expressed in Escherichia coli. However, further purification was compromised as the protein tended to form aggregates, unsuitable for crystallization trials or further characterization experiments. Expression trials in the eukaryote Pichia pastoris neither yielded full length nor truncated Mrr1 protein. In order to overcome the aggregation problem, a shortened variant, missing the N-terminal 249 amino acids named Mrr1 ‘250’, was successfully expressed in E. coli and could be purified without aggregation. Similar to the wild type Mrr1 ‘250’, selected gain of function variants were successfully cloned, expressed and purified with varying yields and with varying purity. The Mrr1 `250’ construct contains most of the described regulatory domains of Mrr1. It was used for crystallization and an initial comparative analysis between the wild type protein and the variants. The proposed dimeric form of the transcription factor, necessary for DNA binding, could be verified for both, the wild type and the mutant proteins. Secondary structure analysis by circular dichroism measurements revealed no significant differences in the overall fold of the wild type and variant proteins. In vitro, the gain of function variants seem to be less stable compared to the wild type protein, as they were more prone to degradation. Whether this observation holds true for the full length protein’s stability in vitro and in vivo remains to be determined. The crystallization experiments, performed with the Mrr1 ‘250’ constructs, led to few small needle shaped or cubic crystals, which did not diffract very well and were hardly reproducible. Therefore no structural information of the transcription factor could be gained so far.
Infections with M. tuberculosis, the causative agent of tuberculosis, are the leading cause of mortality among bacterial diseases. Especially long treatment times, an increasing number of resistant strains and the prevalence of for decades persisting bacteria create the necessity for new drugs against this disease. The cholesterol import and metabolism pathways were discovered as promising new targets and interestingly they seem to play an important role for the chronic stage of the tuberculosis infection and for persisting bacteria.
In this thesis, the 3-ketoacyl-CoA thiolase FadA5 from M. tuberculosis was characterized and the potential for specifically targeting this enzyme was investigated. FadA5 catalyzes the last step of the β-oxidation reaction in the side-chain degradation pathway of cholesterol. We solved the three dimensional structure of this enzyme by X-ray crystallography and obtained two different apo structures and three structures in complex with acetyl-CoA, CoA and a hydrolyzed steroid-CoA, which is the natural product of FadA5. Analysis of the FadA5 apo structures revealed a typical thiolase fold as it is common for biosynthetic and degradative enzymes of this class for one of the structures. The second apo structure showed deviations from the typical thiolase fold. All obtained structures show the enzyme as a dimer, which is consistent with the observed dimer formation in solution. Thus the dimer is likely to be the catalytically active form of the enzyme. Besides the characteristic structural fold, the catalytic triad, comprising two cysteines and one histidine, as well as the typical coenzyme A binding site of enzymes belonging to the thiolase class could be identified. The two obtained apo structures differed significantly from each other. One apo structure is in agreement with the characteristic thiolase fold and the well-known dimer interface could be identified in our structure. The same characteristics were observed in all complex structures. In contrast, the second apo structure followed the thiolase fold only partially. One subdomain, spanning 30 amino acids, was in a different orientation. This reorientation was caused by the formation of two disulfide bonds, including the active site cysteines, which rendered the enzyme inactive. The disulfide bonds together with the resulting domain swap still permitted dimer formation, yet with a significantly shifted dimer interface. The comparison of the apo structures together with the preliminary activity analysis performed by our collaborator suggest, that FadA5 can be inactivated by oxidation and reactivated by reduction. If this redox switch is of biological importance requires further evaluation, however, this would be the first reported example of a bacterial thiolase employing redox regulation.
Our obtained complex structures represent different stages of the thiolase reaction cycle. In some complex structures, FadA5 was found to be acetylated at the catalytic cysteine and it was in complex with acetyl-CoA or CoA. These structures, together with the FadA5 structure in complex with a hydrolyzed steroid-CoA, revealed important insights into enzyme dynamics upon ligand binding and release. The steroid-bound structure is as yet a unique example of a thiolase enzyme interacting with a complex ligand. The characterized enzyme was used as platform for modeling studies and for comparison with human thiolases. These studies permitted initial conclusions regarding the specific targetability of FadA5 as a drug target against M. tuberculosis infection, taking the closely related human enzymes into account. Additional analyses led to the proposal of a specific lead compound based on the steroid and ligand interactions within the active site of FadA5.
Burkitt lymphoma (BL) is a highly aggressive B cell malignancy. Rituximab, a humanized antibody against CD20, in a combination with chemotherapy is a current treatment of choice for B-cell lymphomas including BL. However, certain group of BL patients are resistant to Rituximab therapy. Therefore, alternative treatments targeting survival pathways of BL are needed.
In BL deregulation of MYC expression, together with additional mutations, inhibits differentiation of germinal centre (GC) B cells and drives proliferation of tumor cells. Pro-apoptotic properties of MYC are counteracted through the B-cell receptor (BCR) and phosphoinositide-3-kinase (PI3K) pathway to ensure survival of BL cells. In normal B-cells BCR triggering activates both NF-κB and NFAT-dependent survival signals. Since BL cells do not exhibit constitutive NF-κB activity, we hypothesized that anti-apoptotic NFATc1A isoform might provide a major survival signal for BL cells.
We show that NFATc1 is constitutively expressed in nuclei of BL, in BL cell lines and in Eµ-Myc–induced B cell lymphoma (BCL) cells. Nuclear residence of NFATc1 in these entities depends on intracellular Ca2+ levels but is largely insensitive to cyclosporine A (CsA) treatment and therefore independent from calcineurine (CN) activity. The protein/protein interaction between the regulatory domain of NFATc1 and DNA binding domain of BCL6 likely contributes to sustained nuclear residence of NFATc1 and to the regulation of proposed NFATc1-MYC-BCL6-PRDM1 network in B-cell lymphomas.
Our data revealed lack of strict correlation between the expression of six NFATc1 isoforms in different BL-related entities suggesting that both NFATc1/alphaA and -betaA isoforms provide survival functions and that NFATc1alpha/betaB and -alpha/betaC isoforms either do not possess pro-apoptotic properties in BL cells or these properties are counterbalanced. In addition, we show that in BL entities expression of NFATc1 protein is largely regulated at post-transcriptional level, including MYC dependent increase of protein stability.
Functionally we show that conditional inactivation of Nfatc1 gene in Eµ-Myc mice prevents development of BCL tumors with mature B cell immunophenotype (IgD+). Loss of NFATc1 expression in BCL cells ex vivo results in apoptosis of tumor cells.
Together our results identify NFATc1 as an important survival factor in BL cells and, hence, as a promising target for alternative therapeutic strategies for BL.
Due to the rotation of the earth in the solar system all inhabitants of our planet are exposed to regular environmental changes since more than 3.5 billion years. In order to anticipate these predictable changes in the environment, evolutionarily conserved biological rhythms have evolved in most organisms – ranging from ancient cyanobacteria up to human beings – and also at different levels of organization – from single cells up to behavior. These rhythms are endogenously generated by so called circadian clocks in our body and entrained to the 24 h cycle by external timing cues. In multi-cellular organisms the majority of the cells in the body is equipped with such an oscillator. In mammals, the circadian system is structured in a hierarchical fashion: A central pacemaker resides in the bilateral suprachiasmatic nucleus (SCN) of the hypothalamus, while subsidiary peripheral clocks exist in nearly every tissue and organ.
In contrast to the aforementioned recurrent environmental changes most organisms are also exposed to unpredictable changes in the environment. In order to adapt to these sudden alterations the acute activation of the stress response system, involving the hypothalamic-pituitary-adrenal (HPA) axis and the sympathetic nervous system, displays a fundamental survival mechanism. However, if activation of the stress system becomes chronic, devastating somatic and affective disorders might be the consequence.
At first glance, the circadian and the stress system seem to represent two separate bodily control systems that are involved in adaptation to predictable and unpredictable stimuli, respectively. However, both systems are fundamental for survival, and thus, communicate with each other at various levels. Early studies already demonstrated that stressor exposure at different times of the diurnal cycle generates different stress effects, whereupon the type of stressor plays a pivotal role. Moreover, alterations in the SCN and peripheral circadian clocks could be shown following stressor exposure.
In cooperation with various co-workers, I investigated whether the stress responsiveness is modulated by the endogenous clock in a diurnal fashion and whether repeated psychosocial stress impacts the circadian clock depending on the time of day of stressor exposure. Therefore, male C57BL/6 mice were repeatedly exposed to a psychosocial stressor, either at the beginning of the inactive/light phase (SDL mice) or active/dark phase (SDD mice).
Subsequently, different behavioral, physiological/endocrine and immunological/ inflammatory consequences were assessed. It could be shown that the effects of repeated psychosocial stressor exposure strongly depend on the time of day of stressor exposure. The present results demonstrate that repeated daily stressor exposure has a more negative outcome when applied during the active/dark phase compared to the inactive/light phase. Stressor exposure during the active phase resulted in a loss of general activity, decreased interest in an unfamiliar conspecific, a shift towards a more pro-inflammatory body milieu, and rhythm disturbances in plasma hormones, all representing well-accepted hallmarks of depression. In contrast, C57BL/6 mice exposed to the stressor in their inactive phase exhibited minor physiological alterations that might prevent the formation of the maladaptive consequences mentioned above, thus representing beneficial adaptations.
The second focus of this thesis was put on the investigation of the effects of repeated psychosocial stressor exposure at different times of the light-dark cycle on various levels of the circadian system. An increased expression of the PERIOD2 (PER2) protein, which represents an essential core clock component, could be found in the SCN of mice repeatedly exposed to the stressor during their active phase. In consistence with the alterations in the central circadian pacemaker, the daily rhythm of different hormones and the activity rhythm were considerably affected by SDD. Mice exposed to the psychosocial stressor in their active phase showed a shifted, or absent, rhythm of the hormones corticosterone and leptin. Moreover, their activity was found to be phase-delayed, which seems to be attributable to the Period (Per) gene since Per1/Per2 double-mutants still exhibited their normal activity rhythm following 19 days of stressor exposure during the active phase. In contrast, a phase-advance in the peripheral adrenal gland clock could be seen in C57BL/6 mice subjected to the stressor during their inactive phase. This phase-shift might be required for maintaining the normal rhythmicity in hormonal release and activity.
It has previously been suggested that activation of the HPA axis upon stressor exposure at different times of the light-dark cycle is depending on whether the stressor is of physical or psychological nature. Data from the HPA axis analysis now refine previous findings, indicating that psychosocial stressors also modulate HPA axis responses based on the time of day of stressor presentation. The present results demonstrate that HPA axis activity was reduced following repeated stressor exposure during the active phase. It is reasonable to speculate that this reduced basal activity of the stress system represents a failure in HPA axis adjustment, which could contribute to the negative consequences of repeated psychosocial stressor exposure during the dark phase.
Taken together, it can be concluded that the endogenous clock in mice modulates the stress responsiveness in a circadian fashion and that repeated psychosocial stressor exposure affects the biological clock depending on the time of day of stressor presentation. Thereby, stressor exposure during the active phase results in a more negative outcome as compared to stressor experience during the inactive phase. It is assumed that the interaction between the circadian clock and the stress system is a complex issue that might ensure that the endogenous clock does not get out of synchrony in any order.
Abstract
Glioblastomas, primary brain tumors, represent a tumor entity with a dismal prognosis and a median survival of only about one year. Invasion into the healthy brain parenchyma contributes substantially to the malignancy of this type of brain tumor. Therefore, a better understanding of the mechanisms promoting the invasive behavior of these brain tumors is needed to identify new therapeutic targets.
Cofilin, an actin regulatory protein, has been shown to be an important regulator of the invasive behavior of tumor cells in other types of cancer and the actin cytoskeleton is involved in the formation of a variety of cellular structures important for cell migration and invasion. Cofilin is regulated by phosphorylation on a single residue, serine 3. The aim of this thesis was to examine the role of the cofilin regulatory phosphatase chronophin for glioma cell migration and invasion.
First, it was established that chronophin depletion in the cell line GBM6840 leads to an increase in the ratio of phosphorylated cofilin to total cofilin. Higher chronophin levels were correlated with a decrease in F-actin in the cell lines GBM6840 and U87 as measured in an actin spin down assay and in a flow cytometry based assay.
Furthermore, it was shown that knockdown of chronophin in two different cell lines, GBM6840 and DBTRG-05-MG, strongly increased their invasiveness in vitro. Expression of human chronophin in the cell line U87 decreased its invasiveness substantially. There was no difference in cell proliferation between GBM6840 and DBTRG-05-MG cells expressing a chronophin targeting shRNA or a control shRNA and U87 cells transfected with an empty vector or a human chronophin encoding plasmid. The increase in invasiveness after chronophin depletion could be correlated with an increase in directionality in cell migration under 2D culture conditions in the cell lines U87 and GBM6840. Moreover, treatment with the ROCK inhibitor Y-27632 decreased directionality in GBM6840 cells under 2D culture conditions and reduced the invasiveness of GBM6840 chronophin shRNA cells back to control levels.
Expression of a non-phosphorylatable cofilin mutant, the S3A mutant, was able to reduce invasiveness and to reduce directionality under 2D culture conditions back to control levels in GBM6840 chronophin shRNA cells.
This provides important evidence for the involvement of cofilin phosphoregulation in the phenotypes described above.
In vivo, when injected into NOD-SCID mice, chronophin depleted cells showed a dramatic growth reduction as compared to control and rescue cells.
Transciptomic characterization of GBM6840 cells by microarray analysis and subsequent comparison of the data with microarray profiles of normal brain tissues and different glioma entities identified two specifically chronophin regulated transcripts potentially involved in tumor progression and invasion, MXI1 and EDIL3. Moreover, c-myc was identified as a significantly altered transcription factor after chronophin deregulation based on the number of c-myc target molecules in the microarray dataset.
MXI1 is a potential negative regulator of c-myc dependent transcription, and was strongly downregulated after chronophin knockdown in GBM6840. In line with this, the activity of a c-myc reporter plasmid was increased after chronophin depletion in GBM6840 and reduced after chronophin expression in U87 cells.
However, the protein level of the c-myc protein was reduced after chronophin depletion in GBM6840.
Finally, anaylsis of the expression of proteases known to be important for glioblastoma pathogenesis revealed no major changes in protease expression between chronophin depleted and control cells.
Therefore, a comprehensive analysis of chronophin in the context of glioma pathogenesis has been performed in this thesis. It has been shown that chronophin depletion strongly enhanced invasiveness of glioma cells and that it induced transcriptomic changes potentially involved in tumor progression. The proteins regulating cofilin phosphorylation are therefore valuable therapeutic targets for anti-invasive therapy in glioblastomas. Inhibitors for kinases upstream of cofilin, e.g. LIMKs and ROCKs, are available, and might be promising agents for anti-invasive therapy.
Function and regulation of phospholipase D in blood platelets: in vitro and in vivo studies in mice
(2014)
Summary
Platelet activation and aggregation are crucial for primary hemostasis but can also result in occlusive thrombus formation. Agonist induced platelet activation involves different signaling pathways leading to the activation of phospholipases (PL) which produce second messengers. While the role of PLCs in platelet activation is well established, less is known about the relevance of PLDs. In the current study, the function and regulation of PLD in platelets was investigated using genetic and pharmacological approaches.
In the first part of this thesis, adhesion, activation and aggregation of platelets from mice lacking PLD2 or both PLD1 and PLD2 were analyzed in vitro and in vivo. While the absence of PLD2 resulted in slightly reduced PLD activity in platelets, it had no detectable effect on the platelet function in vitro and in vivo. However, the combined deficiency of both PLD isoforms resulted in defective alpha-granule release and protection in a model of ferric chloride induced arteriolar thrombosis, effects that were not observed in mice lacking only one PLD isoform. These results revealed, for the first time, redundant roles of PLD1 and PLD2 in platelet alpha-granule secretion and indicate that this may be relevant for pathological thrombus formation. Thus, PLD might represent a promising target for antithrombotic therapy.
Thus, this hypothesis was tested more directly in the second part of this thesis. The effects of pharmacological inhibition of PLD activity on hemostasis, thrombosis and thrombo-inflammatory brain infarction in mice were assessed. Treatment of platelets with the reversible, small molecule PLD inhibitor 5-Fluoro-2-indolyl des-chlorohalopemide (FIPI) led to a specific blockade of PLD activity that was associated with reduced -granule release and integrin activation. Mice that received FIPI at a dose of 3 mg/kg displayed reduced occlusive thrombus formation upon chemical injury of carotid arteries or mesenterial arterioles. Similarly, FIPI-treated mice had smaller infarct sizes and significantly better motor and neurological function 24 hours after transient middle cerebral artery occlusion. This protective effect was not associated with major intracerebral hemorrhage or prolonged tail bleeding times. Thus, pharmacological PLD inhibition might represent a safe therapeutic strategy to prevent arterial thrombosis or ischemic stroke.
After revealing a central role for PLD in thrombo-inflammation, the regulation of PLD activity in platelets was analyzed in the last part of the thesis. Up to date, most studies made use of inhibitors potentially exerting off-target effects and consequently PLD regulation is discussed controversially. Therefore, PLD activity in mice genetically lacking potential modulators of PLD activity was determined to address these controversies. These studies revealed that PLD is tightly regulated during initial platelet activation. While integrin outside-in signaling and Gi signaling was dispensable for PLD activation, it was found that PLC dependent pathways were relevant for the regulation of PLD enzyme activity.
Platelets are important players in haemostasis and their activation is essential to limit post-traumatic blood loss upon vessel injury. On the other hand, pathological platelet activation may lead to thrombosis resulting in myocardial infarction and stroke. Platelet activation and subsequent thrombus formation are, therefore, tightly regulated and require a well-defined interplay of platelet surface receptors, intracellular signalling molecules, cytoskeletal rearrangements and the activation of the coagulation cascade.
In vivo thrombosis and haemostasis models mimic thrombus formation at sites of vascular lesions and are frequently used to assess thrombotic and haemostatic functions of platelets. In this dissertation, different in vivo models were used in mice to address the question at what level a reduced platelet count (PC) compromises stable thrombus formation. To study this, mice were rendered thrombocytopenic by low-dose anti-GPIbα antibody treatment and subjected to a tail bleeding time assay as well as to four different in vivo thrombosis models. Haemostasis and occlusive thrombus formation in small vessels were only mildly affected even at severe reductions of the PC. In contrast, occlusive thrombus formation in larger arteries required higher PCs demonstrating that considerable differences in the sensitivity for PC reductions exist between these models.
In a second part of this study, mice were rendered thrombocytopenic by injection of high-dose anti-GPIbα antibody which led to the complete loss of all platelets from the circulation for several days. During recovery from thrombocytopenia, the newly generated platelet population was characterised and revealed a defect in immunoreceptor tyrosine-based activation motif (ITAM)-signalling. This defect translated into impaired arterial thrombus formation.
To further investigate ITAM-signalling in vivo, genetically modified mice were analysed which display a positive or negative regulation of platelet ITAM-signalling in vitro. Whereas mice lacking the adapter Grb2 in platelets showed a delayed thrombus formation in vivo after acetylsalicylic acid treatment, Clp36ΔLIM bone marrow chimeric mice and SLAP/SLAP2-deficient mice displayed pro-thrombotic properties in vivo. Finally, mice lacking the adapter protein EFhd2 were analysed in vitro and in vivo. However, EFhd2-deficient platelets showed only a minor increase in the procoagulant activity compared to control.
A precious treasure in traditional Chinese medicine (TCM), acupuncture played a vital and irreplaceable role in contributing to people’s health in the thousands of years of Chinese history, and in 2010 was officially added to the “Representative List of the Intangible Cultural Heritage of Humanity” by the United Nations. Because of the side-effects of long-term drug therapy for pain, and the risks of dependency, acupuncture has been widely accepted as one of the most important alternative choice therapies for treating varieties of acute and chronic pain-related disorders. The clinical application and scientific mechanism research of acupuncture have therefore increased intensively in the last few decades. Besides hand acupuncture, other treatment approaches e.g. electroacupuncture (EA) have been widely accepted and applied as an important acupuncture-related technique for acupuncture analgesia (AA) research. The involvement of opioid peptides and receptors in acute AA has been shown via pre-EA application of opioid receptor/peptide antagonists. However, existing publications still cannot illuminate the answer to the following question: how does sustained antinociception happen by EA treatment? The hypothesis of opioid peptide-mediated tonic AA might be able to answer the question.
In the first part of this thesis, the institution of a reproducible acupuncture treatment model as well as the endogenous opioid-related mechanisms was demonstrated. An anatomically-based three-dimensional (3D) rat model was established to exhibit a digital true-to-life organism, accurate acupoint position and EA treatment protocol on bilateral acupoint GB-30 Huantiao. The optimal EA treatment protocol (100 Hz, 2-3 mA, 0.1 ms, 20 min) at 0 and 24 h after induction of inflammatory pain by complete Freund’s adjuvant (CFA) on conscious free-moving rats was then established. EA elicited significant sustained mechanical and thermal antinociception up to 144 h. Post-EA application of opioid receptors (mu opioid receptor, MOR; delta opioid receptor, DOR) antagonists naloxone (NLX) and naltrindole (NTI), or opioid peptide antibodies anti-beta-endorphin (anti-END), met-enkephalin (anti-ENK) or -dynorphin A (anti-DYN) could also block this effect at a late phase (96 h) of CFA post-EA, which suggested opioid-dependent tonic analgesia was produced by EA. Meanwhile, EA also reduced paw temperature and volume at 72-144 h post CFA indicating anti-inflammatory effects. Nociceptive thresholds were assessed by paw pressure threshold (Randall-Sellito) or paw withdrawal latency (Hargreaves) and an anti-inflammatory effect was evaluated by measurement of plantar temperature and volume of inflamed paw.
The second part of the thesis further suggests the correlation between the chemokine CXCL10 (= interferon-gamma inducible protein 10, IP-10) and opioid peptides in EA-induced antinociception. Based on a comprehensive Cytokine Array of 29 cytokines, targeted cytokines interleukin (IL)-1alpha, interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, interleukin (IL)-4, interleukin (IL)-13, interferon (IFN)-gamma as well as CXCL10 were selected and quantified by enzyme-linked immunosorbent assay (ELISA), and real time reverse transcription-polymerase chain reaction (RT-PCR) quantification confirmed upregulation of CXCL10 mRNA at both 72 and 96 h. The following hyperalgesic assessment suggested the antinociceptive effect of CXCL10. The double immunostaining localizing opioid peptides with macrophages expressed the evident upregulation of CXCR3-receptor of CXCL10 in EA treated samples as well as the significant upregulation or downregulation of opioid peptides by repeated treatment of CXCL10 or antibody of CXCL10 via behavioral tests and immune staining. Subsequent immunoblotting measurements showed non-alteration of opioid receptor level by EA, indicating that the opioid receptors did not apparently contribute to AA in the present studies. In vitro, CXCL10 did not directly trigger opioid peptide END release from freshly isolated rat macrophages. This might implicate an indirect property of CXCL10 in vitro stimulating the opioid peptide-containing macrophages by requiring additional mediators in inflammatory tissue.
In summary, this project intended to explore the peripheral opioid-dependent analgesic mechanisms of acupuncture with a novel 3D treatment rat model and put forward new information to support the pivot role of chemokine CXCL10 in mediating EA-induced tonic antinociception via peripheral opioid peptides.