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Diverse roles of B cells in the pathophysiology of rheumatoid arthritis are now well established. B cells contribute to autoimmunity by producing autoantibodies, processing autoantigen and the production of different cytokines which are involved in the inflammatory cascade. Therefore approaches to target B lymphocytes directly or indirectly are developed for clinical practice to treat autoimmune diseases including rheumatoid arthritis. Transient B cell depletion by rituximab (anti-CD20 antibody) has gained prime importance in recent years. Meanwhile anti-CD20 mediated transient B cell depletion therapy is now used with clinical efficiency in the treatment of patients with rheumatoid arthritis. Rituximab induces noteworthy changes in the homeostasis of peripheral B cell subpopulations during the repletion phase with emerging immature B cells in peripheral blood followed by normalization of the naïve B cell pool and a longterm delay in memory B cell subsets in patients with rheumatoid arthritis. Particularly IgD+CD27+ memory B cells repopulate very slowly during B cell regeneration. In a prospective clinical study, our laboratory has shown that the overall number of memory B cells correlates well to the duration of clinical response to rituximab. Little is known about the particular molecular changes in the memory B cell repertoire after rituximab therapy. To better understand peripheral memory B cell subsets, we explored in detail the somatic mutational frequency and pattern of Ig-VH3 gene rearrangements by using a single B cell sorting technique followed by nested PCR before and up to 6 years after rituximab therapy in 18 RA patients. We compared rituximab inflicted dynamics of mutational acquisition to memory B cell repopulation in 4 healthy donors and 6 non RA patients undergoing high dose chemotherapy followed by autologous or allogeneic stem cell transplantation (SCT). Firstly we analyzed the peripheral composition of memory B cell subsets. The phenotypic analysis of peripheral pre-switch (IgD+CD27+) and post-switch (IgD-CD27+) memory B cells did not reveal any quantitative differences in RA patients prior to B cell depletion therapy compared to healthy donors. However extending those studies in directly analysing the B cell immunoglobulin receptor from individual B cells of RA patients and healthy controls brought interesting results. Pre-switched and post-switched memory B cells showed a highly significant difference in the amount of mutations/sequence. The population of IgD+CD27+ memory B cells is comprised of non-mutated, low and highly mutated (median= 9 mutations/ sequence) rearranged Ig receptors whereas the IgD-CD27+ memory B cell compartment shows quite uniformly highly mutated (median 18 mutations/ sequence) sequences indicating a significant difference between these two groups (mutational frequencies 3.83±0.19% vs. 7.1±0.53%; P=0.0001). Profound changes were noted in the re-emerging pre-switch memory B cells (IgD+/ CD27+) after transient B cell depletion with rituximab. These cells showed over a time period of 6 years after treatment with rituximab significantly delayed acquisition of mutations in Ig receptors on the single B cell level. One year after a single course of rituximab 84% of single repopulating IgD+/CD27+ B cells were unmutated and no highly mutated Ig-VH gene rearrangements were found(P=0.0001). Over time increasing numbers of mutations could be detected i-e 7.8% during 2nd year of regeneration (P=0.0001), 14% after 4 years (n=2). Nevertheless even 6 years after rituximab, VH mutations in IgD+ memory B cells were still reduced with 27% highly mutated sequences compared to 52% pre therapy(P=0.0001). Post-therapy analysis of CDR3 length of regenerated IgD+ memory B cells revealed increased CDR3 length which also correlates well with elevated number of non-mutated VH gene rearrangements observed during repletion phase. In comparison patients undergoing high dose chemotherapy followed by allogeneic stem cell transplantation repopulated IgD+ memory cells earlier with higher numbers of mutations in IgD+ memory B cells. One year after transplantation Ig receptors showed already 22% highly mutated and 42 % unmutated VH rearrangements. These findings indicated that anti-CD20 mediated B cell depletion seems not only to delay the production of pre-switch memory B cells but also significantly affects the acquisition of mutations in the IgD+ memory B cell pool. In contrary to the mutational pattern of IgD+ memory B cells after rituximab class switched memory B cells repopulate in the periphery with quantitatively normal mutations in their Ig receptors. Although the numeric replenishment of these recirculating class-switched memory B cells was also reduced after rituximab, we found no delay in quantitative acquisition of mutations also an increased proportion of IgA expressing B cells in this memory B cell subset was detected. Our data showed that post-therapy mutational targeting in RGYW/WRCY motifs were significantly increased as compared with that of pre-treatment (27% before rituximab vs. 43% after therapy, P=0.0003) indicating that affinity maturation may operate differently in class-switched memory B cells before and after B cell depletion. These results indicate a normal development process with an unimpaired mechanism of mutational acquisition in class-switched memory B cells. These data argue for different requirements to undergo somatic hypermutations in IgD+ memory B cells in comparison to class switched memory B cells. To conclude, our work has demonstrated for the first time a delayed acquisition of somatic hypermutations at single Ig receptor VH gene rearrangements of IgD+ memory B cells in comparison to class-switched memory B cells. These results demonstrate that IgD+ memory B cells are particularly susceptible to anti-CD20 treatment in patients with rheumatoid arthritis. In addition antigenic pressure and/or selection are substantially reduced by rituximab therapy which is basically not seen in the class-switched memory compartment. These data are in line with the hypothesis that IgD+ memory B cells have distinct requirements for activating their mutational machinery compared to class-switched memory B cells which recover normal mutations during regeneration phase. The results have implications in understanding the pathophysiology of memory B cell in rheumatoid arthritis and may be helpful in designing new targeted therapies.
An increase in cytosolic Ca2+ levels ([Ca2+]i) is a key event that occurs downstream of many signaling cascades in response to an external stimulus and regulates a wide range of cellular processes, including platelet activation. Eukaryotic cells increase their basal [Ca2+]i allowing extracellular Ca2+ influx into the cell, which involves different mechanisms. Store-operated Ca2+ entry (SOCE) is considered the main mechanism of extracellular Ca2+ influx in electrically non-excitable cells and platelets, and comprises an initial Ca2+ depletion from intracellular Ca2+ stores prior to activation of extracellular Ca2+ influx. Although the close relation between Ca2+ release from intracellular stores and extracellular Ca2+ influx was clear, the nature of the signal that linked both events remained elusive until 2005, when Stromal Interaction Molecule 1 (STIM1) was identified as an endoplasmic reticulum (ER) Ca2+ sensor essential for inositol (1,4,5)-trisphosphate (IP3)-mediated SOCE in vitro. However, the function of its homologue STIM2 in Ca2+ homeostasis was in general unknown. Therefore, mice lacking STIM2 (Stim2-/-) were generated in this work to study initially STIM2 function in platelets and in cells of the immune system. Stim2-/- mice developed normally in size and weight to adulthood and were fertile. However, for unknown reasons, they started to die spontaneously at the age of 8 weeks. Unexpectedly, Stim2-/- mice did not show relevant differences in platelets, revealing that STIM2 function is not essential in these cells. However, STIM2 seems to be involved in mammary gland development during pregnancy and is essential for mammary gland function during lactation. CD4+ T cells lacking STIM2 showed decreased SOCE. Our data suggest that STIM2 has a very specific function in the immune system and is involved in Experimental Autoimmune Encephalomyelitis (EAE) at early stages of the disease progression. Stim2-/- neurons were also defective in SOCE. Surprisingly, our results evidenced that STIM2 participates in mechanisms of neuronal damage after ischemic events in brain. This is the first time that the involvement of SOCE in ischemic neuronal damage has been reported. This finding may serve as a basis for the development of novel neuroprotective agents for the treatment of ischemic stroke, and possibly other neurodegenerative disorders in which disturbances in cellular Ca2+ homeostasis are considered a major pathophysiological component.
Extracellular signals are translated and amplified via cascades of serially switched protein kinases, MAP kinases (MAPKs). One of the MAP pathways, the classical RAS/RAF/MEK/ERK pathway, transduces signals from receptor tyrosine kinases and plays a central role in regulation of cell proliferation. RAF kinases (A-, B- and C-RAF) function atop of this cascade and convert signals emanating from conformational change of RAS GTPases into their kinase activity, which in turn phosphorylates their immediate substrate, MEK. Disregulated kinase activity of RAF can result in tumor formation, as documented for many types of cancer, predominantly melanomas and thyroid carcinomas (B-RAF). A-RAF is the least characterized RAF, possibly due to its low intrinsic kinase activity and comparatively mild phenotype of A-RAF knockout mice. Nevertheless, the unique phenotype of araf -/- mice, showed predominantly neurological abnormalities such as cerebellum disorders, suggesting that A-RAF participates in a specific process not complemented by activities of B- and CRAF. Here we describe the role of A-RAF in membrane trafficking and identify its function in a specific step of endocytosis. This work led to the discovery of a C-terminally truncated version of A-RAF, AR149 that strongly interfered with cell growth and polarization in yeast and with endocytosis and actin polymerization in mammalian cells. As this work was in progress two splicing isoforms of ARAF, termed DA-RAF1 and DA-RAF2 were described that act as natural inhibitors of RAS-ERK signaling during myogenic differentiation (Yokoyama et al., 2007). DA-RAF2 contains the first 153 aa of A-RAF and thus is nearly identical with AR149. AR149 localized specifically to the recycling endosomal compartments as confirmed by colocalization and coimmunoprecipitation with ARF6. Expression of AR149 interferes with recycling of endocytosed transferrin (Tfn) and with actin polymerization. The endocytic compartment, where internalized Tfn is trapped, was identified as ARF6- and RAB11- positive endocytic vesicles. We conclude that the inhibition of Tfn trafficking in the absence of A-RAF or under overexpression of AR149 occurs between tubular- and TGNassociated recycling endosomal compartments. siRNA-mediated depletion of endogenous A-RAF or inhibition of MEK by U0126 mimic the AR149 overexpression phenotype, supporting a role of ARAF regulated ERK signalling at endosomes that is controlled by AR149 and targets ARF6. Our data additionally suggest EFA6 as a partner of A-RAF during activation of ARF6. The novel findings on the A-RAF localization and the interaction with ARF6 have led to a new model of ARAF function were A-RAF via activation of ARF6 controls the recycling of endocytic vesicles.Endocytosis and rapid recycling of synaptic vesicles is critically important for the physiological function of neurons. The finding, that A-RAF regulates endocytic recycling open a new perspective for investigation of the role of A-RAF in the nervous system.
The RS1 protein (gene RSC1A1) participates in regulation of Na+-D-glucose cotransporter SGLT1 and some other solute carriers. In subconfluent LLC-PK1 cells, RS1 inhibits release of SGLT1 from the trans-Golgi network and transcription of SGLT1. In subconfluent cells, RS1 is localized in the nucleus and the cytoplasm whereas confluent cells contain predominantly cytoplasmic RS1. In the present study, the mechanism and regulation of confluence-dependent nuclear location of RS1 was investigated. Confluence dependent nuclear location of RS1 was shown to be regulated by the cell cycle. A nuclear shuttling signal (NS) in pRS1 was identified that ensures confluence-dependent distribution of pRS1 and comprises nuclear localization signal (NLS) and nuclear export signal (NES). The NLS and NES of RS1 mediate translocation into and out of the nucleus via importin ß1 and CRM1, respectively, and the nuclear/cytoplasmic distribution of the RS1 protein is determined by the nuclear export activity. The adjacent protein kinase C (PKC) phosphorylation site at serine 370 of pRS1 was shown to control nuclear localization driven by NS and is necessary for the differential localization of RS1 in quiescent versus proliferating cells. Basing on the data of site-directed mutagenesis, PKC activation experiments and mass spectrometry analysis of RS1 phosphorylation, the following model of the regulation of RS1 nuclear location in LLC-PK1 cells was proposed. In subconfluent cells, RS1 is actively imported into the nucleus whereas nuclear export of RS1 is not active leading to accumulation of RS1 in the nucleus. After confluence, phosphorylation of serine 370 of pRS1 by PKC takes place leading to enhancement of RS1 nuclear export and predominantly cytoplasmic distribution of the protein in the confluent cells. The confluence-dependent regulation of RS1 localization may control SGLT1 expression during regeneration of enterocytes in small intestine and during regeneration of renal tubular cells after hypoxemic stress. Moreover, the gene expression profiling of mouse embryonic fibroblasts with RS1-/- genotype suggests that transcriptional regulation by RS1 might be important for the cell cycle and cell division. Since RS1 localization depends on the cell cycle, RS1 might play a role in the regulation of the solute carriers during specific phases of the cell cycle.
Recent progresses and developments in molecular biology provide a wealth of new but insufficiently characterised data. This fund comprises amongst others biological data of genomic DNA, protein sequences, 3-dimensional protein structures as well as profiles of gene expression. In the present work, this information is used to develop new methods for the characterisation and classification of organisms and whole groups of organisms as well as to enhance the automated gain and transfer of information. The first two presented approaches (chapters 4 und 5) focus on the medically and scientifically important enterobacteria. Its impact in medicine and molecular biology is founded in versatile mechanisms of infection, their fundamental function as a commensal inhabitant of the intestinal tract and their use as model organisms as they are easy to cultivate. Despite many studies on single pathogroups with clinical distinguishable pathologies, the genotypic factors that contribute to their diversity are still partially unknown. The comprehensive genome comparison described in Chapter 4 was conducted with numerous enterobacterial strains, which cover nearly the whole range of clinically relevant diversity. The genome comparison constitutes the basis of a characterisation of the enterobacterial gene pool, of a reconstruction of evolutionary processes and of comprehensive analysis of specific protein families in enterobacterial subgroups. Correspondence analysis, which is applied for the first time in this context, yields qualitative statements to bacterial subgroups and the respective, exclusively present protein families. Specific protein families were identified for the three major subgroups of enterobacteria namely the genera Yersinia and Salmonella as well as to the group of Shigella and E. coli by applying statistical tests. In conclusion, the genome comparison-based methods provide new starting points to infer specific genotypic traits of bacterial groups from the transfer of functional annotation. Due to the high medical importance of enterobacterial isolates their classification according to pathogenicity has been in focus of many studies. The microarray technology offers a fast, reproducible and standardisable means of bacterial typing and has been proved in bacterial diagnostics, risk assessment and surveillance. The design of the diagnostic microarray of enterobacteria described in chapter 5 is based on the availability of numerous enterobacterial genome sequences. A novel probe selection strategy based on the highly efficient algorithm of string search, which considers both coding and non-coding regions of genomic DNA, enhances pathogroup detection. This principle reduces the risk of incorrect typing due to restrictions to virulence-associated capture probes. Additional capture probes extend the spectrum of applications of the microarray to simultaneous diagnostic or surveillance of antimicrobial resistance. Comprehensive test hybridisations largely confirm the reliability of the selected capture probes and its ability to robustly classify enterobacterial strains according to pathogenicity. Moreover, the tests constitute the basis of the training of a regression model for the classification of pathogroups and hybridised amounts of DNA. The regression model features a continuous learning capacity leading to an enhancement of the prediction accuracy in the process of its application. A fraction of the capture probes represents intergenic DNA and hence confirms the relevance of the underlying strategy. Interestingly, a large part of the capture probes represents poorly annotated genes suggesting the existence of yet unconsidered factors with importance to the formation of respective virulence phenotypes. Another major field of microarray applications is gene expression analysis. The size of gene expression databases rapidly increased in recent years. Although they provide a wealth of expression data, it remains challenging to integrate results from different studies. In chapter 6 the methodology of an unsupervised meta-analysis of genome-wide A. thaliana gene expression data sets is presented, which yields novel insights in function and regulation of genes. The application of kernel-based principal component analysis in combination with hierarchical clustering identified three major groups of contrasts each sharing overlapping expression profiles. Genes associated with two groups are known to play important roles in Indol-3 acetic acid (IAA) mediated plant growth and development as well as in pathogen defence. Yet uncharacterised serine-threonine kinases could be assigned to novel functions in pathogen defence by meta-analysis. In general, hidden interrelation between genes regulated under different conditions could be unravelled by the described approach. HMMs are applied to the functional characterisation of proteins or the detection of genes in genome sequences. Although HMMs are technically mature and widely applied in computational biology, I demonstrate the methodical optimisation with respect to the modelling accuracy on biological data with various distributions of sequence lengths. The subunits of these models, the states, are associated with a certain holding time being the link to length distributions of represented sequences. An adaptation of simple HMM topologies to bell-shaped length distributions described in chapter 7 was achieved by serial chain-linking of single states, while residing in the class of conventional HMMs. The impact of an optimisation of HMM topologies was underlined by performance evaluations with differently adjusted HMM topologies. In summary, a general methodology was introduced to improve the modelling behaviour of HMMs by topological optimisation with maximum likelihood and a fast and easily implementable moment estimator. Chapter 8 describes the application of HMMs to the prediction of interaction sites in protein domains. As previously demonstrated, these sites are not trivial to predict because of varying degree in conservation of their location and type within the domain family. The prediction of interaction sites in protein domains is achieved by a newly defined HMM topology, which incorporates both sequence and structure information. Posterior decoding is applied to the prediction of interaction sites providing additional information of the probability of an interaction for all sequence positions. The implementation of interaction profile HMMs (ipHMMs) is based on the well established profile HMMs and inherits its known efficiency and sensitivity. The large-scale prediction of interaction sites by ipHMMs explained protein dysfunctions caused by mutations that are associated to inheritable diseases like different types of cancer or muscular dystrophy. As already demonstrated by profile HMMs, the ipHMMs are suitable for large-scale applications. Overall, the HMM-based method enhances the prediction quality of interaction sites and improves the understanding of the molecular background of inheritable diseases. With respect to current and future requirements I provide large-scale solutions for the characterisation of biological data in this work. All described methods feature a highly portable character, which allows for the transfer to related topics or organisms, respectively. Special emphasis was put on the knowledge transfer facilitated by a steadily increasing wealth of biological information. The applied and developed statistical methods largely provide learning capacities and hence benefit from the gain of knowledge resulting in increased prediction accuracies and reliability.
Mycobacterium tuberculosis is the causative agent of tuberculosis and responsible for more than eight million new infections and about two million deaths each year. Novel chemotherapeutics are urgently needed to treat the emerging threat of multi drug resistant and extensively drug resistant strains. Cell wall biosynthesis is a widely used target for chemotherapeutic intervention in bacterial infections. In mycobacteria, the cell wall is comprised of mycolic acids, very long chain fatty acids that provide protection and allow the bacteria to persist in the human macrophage. The type II fatty acid biosynthesis pathway in Mycobacterium tuberculosis synthesizes fatty acids with a length of up to 56 carbon atoms that are the precursors of the critical mycobacterial cell wall components mycolic acids. KasA, the mycobacterial ß-ketoacyl synthase and InhA, the mycobacterial enoyl reductase, are essential enzymes in the fatty acid biosynthesis pathway and validated drug targets. In this work, KasA was expressed in Mycobacterium smegmatis, purified and co-crystallized in complex with the natural thiolactone antibiotic thiolactomycin (TLM). High-resolution crystal structures of KasA and the C171Q KasA variant, which mimics the acyl enzyme intermediate of the enzyme, were solved in absence and presence of bound TLM. The crystal structures reveal how the inhibitor is coordinated by the enzyme and thus specifically pinpoint towards possible modifications to increase the affinity of the compound and develop potent new drugs against tuberculosis. Comparisons between the TLM bound crystal structures explain the preferential binding of TLM to the acylated form of KasA. Furthermore, long polyethylene glycol molecules are bound to KasA that mimic a fatty acid substrate of approximately 40 carbon atoms length. These structures thus provide the first insights into the molecular mechanism of substrate recognition and reveal how a wax-like substance can be accommodated in a cytosolic environment. InhA was purified and co-crystallized in complex with the slow, tight binding inhibitor 2-(o-tolyloxy)-5-hexylphenol (PT70). Two crystal structures of the ternary InhA-NAD+-PT70 were solved and reveal how the inhibitor is bound to the substrate binding pocket. Both structures display an ordered substrate binding loop and corroborate the hypothesis that slow onset inhibition is coupled to loop ordering. Upon loop ordering, the active site entrance is more restricted and the inhibitor is kept inside more tightly. These studies provide additional information on the mechanistic imperatives for slow onset inhibition of enoyl ACP reductases.
Studies on platelet cytoskeletal dynamics and receptor regulation in genetically modified mice
(2009)
Platelets are produced by bone marrow megakaryocytes in a process involving actin dynamics. Actin-depolymerizing factor (ADF) and cofilin are actin-binding proteins that act as key regulators in actin turnover by promoting filament severing and depolymerization. The overall significance of ADF/cofilin function and actin turnover in platelet formation is presently unclear. In the first part of this thesis, platelet formation and function were studied in mice constitutively lacking ADF and/or mice with a conditional deficiency (Cre/loxP) in n-cofilin. To delete cofilin exclusively in megakaryocytes and platelets, cofilinfl/fl mice were crossed with PF4 (platelet factor 4)-Cre mice. While a single-deficiency in ADF or n-cofilin resulted in no or only a minor platelet formation defect, respectively, a double-deficiency in ADF and n-cofilin led to an almost complete loss of platelets. Bone marrow megakaryocytes of ADF/n-cofilin-deficient mice showed defective platelet zone formation. Interestingly, in vitro and ex vivo megakaryocyte differentiation revealed reduced proplatelet formation and absence of platelet-forming swellings. These data establish that ADF and n-cofilin have redundant but essential roles in the terminal step of platelet formation in vitro and in vivo. In the second part of the thesis, mechanisms underlying cellular regulation of the major platelet collagen receptor, glycoprotein VI (GPVI), were studied. GPVI mediates platelet activation on exposed subendothelial collagens at sites of vascular injury, and thereby contributes to normal hemostasis but also to occlusion of diseased vessels in the setting of myocardial infarction or stroke. Thus, GPVI is an attractive target for anti-thrombotic therapy, particularly because previous studies have shown that anti-GPVI antibodies induce irreversible down-regulation of the receptor in circulating platelets by internalization and ectodomain shedding. Metalloproteinases of the ADAM (a disintegrin and metalloproteinase domain) family are suspected to mediate this ectodomain shedding, but in vivo evidence for this is lacking. To study the mechanism of GPVI regulation in vivo, two mouse lines, Gp6 knock-out and Adam10fl/fl, PF4-Cre mice, were generated and in addition low TACE (TNFalpha converting enzyme) mice were analyzed. It was shown that GPVI can be cleaved in vitro by ADAM10 or TACE depending on the shedding-inducing signaling pathway. Moreover, GPVI was down-regulated in vivo upon antibody injection in ADAM10-deficient and low TACE mice suggesting that either both or an additional metalloproteinase is involved in GPVI regulation in vivo.
Integrating neurobiological markers of depression: an fMRI-based pattern classification approach
(2010)
While depressive disorders are, to date, diagnosed based on behavioral symptoms and course of illness, the interest in neurobiological markers of psychiatric disorders has grown substantially in recent years. However, current classification approaches are mainly based on data from a single biomarker, making it difficult to predict diseases such as depression which are characterized by a complex pattern of symptoms. Accordingly, none of the previously investigated single biomarkers has shown sufficient predictive power for practical application. In this work, we therefore propose an algorithm which integrates neuroimaging data associated with multiple, symptom-related neural processes relevant in depression to improve classification accuracy. First, we identified the core-symptoms of depression from standard classification systems. Then, we designed and conducted three experimental paradigms probing psychological processes known to be related to these symptoms using functional Magnetic Resonance Imaging. In order to integrate the resulting 12 high-dimensional biomarkers, we developed a multi-source pattern recognition algorithm based on a combination of Gaussian Process Classifiers and decision trees. Applying this approach to a group of 30 healthy controls and 30 depressive in-patients who were on a variety of medications and displayed varying degrees of symptom-severity allowed for high-accuracy single-subject classification. Specifically, integrating biomarkers yielded an accuracy of 83% while the best of the 12 single biomarkers alone classified a significantly lower number of subjects (72%) correctly. Thus, integrated biomarker-based classification of a heterogeneous, real-life sample resulted in accuracy comparable to the highest ever achieved in previous single biomarker research. Furthermore, investigation of the final prediction model revealed that neural activation during the processing of neutral facial expressions, large rewards, and safety cues is most relevant for over-all classification. We conclude that combining brain activation related to the core-symptoms of depression using the multi-source pattern classification approach developed in this work substantially increases classification accuracy while providing a sparse relational biomarker-model for future prediction.
Stem cells with the particular potential to self renew and to differentiate into multiple cell lineages are fascinating cell types for basic and applied research. Pluripotent embryonic stem (ES) cells are derived from the inner cell mass (ICM) of preimplantation embryos. Upon differentiation ES cells can give rise to cells of ecto-, meso- and endoderm including germ cells. In contrast, multipotent adult stem cells are more restricted in their differentiation outcomes,they differentiate into cells of their tissue of origin. For example, hematopoietic stem cells (HSCs) that reside in hemogenic tissues such as the bone marrow (BM) differentiate into hemato-/lymphoid cell lineages. Upon differentiation of stem cells not the genome, but the epigenetic regulation changes. Differentiation-associated epigenetic changes generate cell types with distinct phenotypes and functions. For stem cell-based therapies it is important to deeper understand the relation between epigenome and cellular function. In the scope of this thesis I aimed to analyze cultures of differentiating stem cells with respect to gene expression, chromatin regulation and differentiation potential. For the analysis of global histone modification levels, which represent one mechanism for epigenetic regulation, fow cytometric protocols were established that allow single cell measurements. By applying this methodology decreased histone acetylation levels were shown in differentiated ES cell populations. In contrast, comparable histone acetylation levels were observed in differentiated and undifferentiated BM cells. In addition, I investigated effects of the histone deacetylase (HDAC) inhibitor trichostatin A (TSA) on murine BM cells, comprising also HSCs. Upon TSA treatment the frequency of cells with in vitro and in vivo hematopoietic activity was increased, while lineage committed cells underwent apoptosis. Next, the loss of pluripotency was assessed in differentiating ES cell cultures. Using short-term in vitro differentiation protocols marker-based analyses and functional assays were performed.Functionally pluripotency was diminished after 2 days of differentiation as assessed by colony formation, embryoid body (EB) formation and cardiomyogenic differentiation approaches. In contrast, pluripotency marker expression was reduced at later time points. Further, the application of distinct differentiation systems (aggregation EB, clonal EB or monolayer (ML) culture) had an impact on the progression and homogeneity of differentiation cultures. To further study the end of pluripotency, differentiated ES cells were placed under ES cell culture conditions. The data suggest that 3 days differentiated ES cells had passed a point of no return and failed to regain Oct4-eGFP expression and that HDAC inhibitor treatment selectively killed differentiated ES cells. Finally, I aimed to study the effect of EED - a core subunit of the histone methylating Polycomb repressive complex 2 (PRC2) - on ES cell chromatin and function. ES cells lacking EED showed loss of histone H3 lysine 27 trimethylation (H3K27me3) accompanied by increased histone acetylation and reduced H3K9me3 levels. Despite typical ES cell morphology and pluripotency marker expression, EED knockout (KO) ES cells exhibited altered nuclear heterochromatin organization, delayed chromatin mobility and a failure in proper differentiation. Conclusively, my data provide insights into the epigenetic regulation of stem cells. Particularly, the results suggest that HDAC inhibitor treatment was detrimental for differentiated BM as well as for differentiated ES cells and that ES cells after 3 days of differentiation had lost pluripotency. Further, the data demonstrate that EED KO ES cells self renewed, exhibited morphology and pluripotency marker expression similar to wild type ES cells, but failed to differentiate. This indicates an important role of EED not only for undifferentiated but also for differentiating ES cells.
Semaphorin receptors in the immunological synapse: regulation and measles virus-driven modulation
(2010)
Measles virus (MV) infection causes approximately 164,000 deaths per year worldwide (WHO, 2008). The main cause of death is MV-induced immunosuppression but the underlying mechanisms are not fully understood. It has been suggested that MV renders T cells dysfunctional by disrupting the integrity of actin dynamics while MV infection of dendritic cells results in their inability to sustain T cell activation. During neuronal development, semaphorins (SEMAs), especially SEMA3A, induce a collapse of growing dendrites via the binding to plexin-A1 (plexA1) and its coreceptor neuropilin-1 (NP-1). The collapse results from a disruption of actin dynamics. In this study, the roles of these three molecules were investigated in human immune cells and their possible role in MV induced immunosuppression. The present data have shown that plexA1 is an important component of human immunological synapse (IS). It translocated transiently to the surface of T cells after CD3/28 ligation and accumulated at the stimulatory interface between T cells and DCs (or CD3/28 coated beads). When plexA1 expression was inhibited (RNAi) or its function was disrupted (exogenous blocking or dominant negative expression), T cell expansion was reduced. Upon MV exposure, translocation of plexA1 and NP-1, another important component of IS, towards the stimulatory interface in T cells was abrogated. Moreover, MV infection interfered with plexA1/NP-1 turnover in maturing DCs and promoted early and substantial release of SEMA3A from these cells, particularly in the presence of allogenic T cells. As revealed by scanning electron microscopy, the release of SEMA3A caused a transient loss of actin-based protrusions on T cells. SEMA3A affected chemotactic migration of T cells and DCs, and reduced formation of allogenic DC/T cell conjugates. In conclusion, MV targeted SEMA receptor function both by disrupting their recruitment to the IS and by promoting a premature release of their repulsive ligand, SEMA3A. Both of which could contribute to MV-induced immunosuppression.