Refine
Has Fulltext
- yes (64)
Is part of the Bibliography
- yes (64)
Year of publication
Document Type
- Doctoral Thesis (61)
- Journal article (3)
Language
- English (64) (remove)
Keywords
- Maus (64) (remove)
Institute
- Graduate School of Life Sciences (32)
- Theodor-Boveri-Institut für Biowissenschaften (14)
- Rudolf-Virchow-Zentrum (9)
- Neurologische Klinik und Poliklinik (6)
- Institut für Medizinische Strahlenkunde und Zellforschung (4)
- Klinik und Poliklinik für Psychiatrie, Psychosomatik und Psychotherapie (4)
- Institut für Virologie und Immunbiologie (3)
- Institut für Klinische Biochemie und Pathobiochemie (2)
- Lehrstuhl für Molekulare Psychiatrie (2)
- Medizinische Fakultät (2)
- Pathologisches Institut (2)
- Fakultät für Biologie (1)
- Institut für Klinische Neurobiologie (1)
- Institut für Pharmakologie und Toxikologie (1)
- Institut für Pharmazie und Lebensmittelchemie (1)
- Julius-von-Sachs-Institut für Biowissenschaften (1)
- Klinik und Poliklinik für Dermatologie, Venerologie und Allergologie (1)
- Medizinische Klinik und Poliklinik II (1)
- Physikalisches Institut (1)
Sonstige beteiligte Institutionen
Food borne pathogens that cause systemic disease must cross the intestinal barrier. Many of these pathogens, eg Salmonella typhimurium and Shigella flexneri, use M cells, found only within the follicle associated epithelium (FAE) that overlies Peyer’s patches and other lymphoid follicles, to enter the host. This study is primarily an investigation into the interaction of S. typhimurium and Listeria monocytogenes with the intestinal epithelium, representing the early stage of an infection.
The transmission of proliferative and developmental signals from activated cell-surface receptors to initiation of cellular responses in the nucleus is synergically controlled by the coordinated action of a diverse set of intracellular signalling proteins. The Ras/Raf/MEK/MAPK signalling pathway has been shown to control the expression of genes which are crucial for the physiological regulation of cell proliferation, differentiation and apoptosis. Within this signalling cascade, the Raf protein family of serine/threonine kinases serves as a central intermediate which connects to many of other signal transduction pathways. To elucidate the signalling functions of the different Raf kinases in motoneurons during development, the expression, distribution and subcellular localization of Rafs in the spinal cord and the facial nucleus in brainstem of mice at various embryonic and postnatal stages were investigated. Moreover, we have investigated the intracellular redistribution of Raf molecules in isolated motoneurons from 13 or 14 day old mouse embryos, after addition or withdrawal of neurotrophic factors to induce Raf kinases activation in vitro. Furthermore, in order to investigate the potential anti-apoptotic function of Raf kinases on motoneurons, we isolated motoneurons from B-raf-/- and c-raf-1-/- mouse embryos and analysed the survival and differentiation effects of neurotrophic factors in motoneurons lacking B-Raf and c-Raf-1. We provide evidence here that all three Raf kinases are expressed in mouse spinal motoneurons. Their expression increases during the period of naturally occurring cell death of motoneurons. In sections of embryonic and postnatal spinal cord, motoneurons express exclusively B-Raf and c-Raf-1, but not A-Raf, and subcellularly Raf kinases are obviously colocalized with mitochondria. In isolated motoneurons, most of the B-Raf or c-Raf-1 immunoreactivity is located in the perinuclear space but also in the nucleus, especially after activation by addition of CNTF and BDNF in vitro. We found that c-Raf-1 translocation from the cytosol into the nucleus of motoneurons after its activation by neurotrophic factors is a distinct event. As a central finding of our study, we observed that the viability of isolated motoneurons from B-raf but not c-raf-1 knockout mice is lost even in the presence of CNTF and other neurotrophic factors. This indicates that B-Raf but not c-Raf-1, which is still present in B-raf deficient motoneurons, plays a crucial role in mediating the survival effect of neurotrophic factors during development. In order to prove that B-Raf is an essential player in this scenario, we have re-expressed B-Raf in mutant sensory and motor neurons by transfection. The motoneurons and the sensory neurons from B-raf knockout mouse which were transfected with exogenous B-raf gene revealed the same viability in the presence of neurotrophic factors as primary neurons from wild-type mice. Our results suggest that Raf kinases have important signalling functions in motoneurons in mouse CNS. In vitro, activation causes redistribution of Raf protein kinases, particularly for c-Raf-1, from motoneuronal cytoplasm into the nucleus. This redistribution of c-Raf-1, however, is not necessary for the survival effect of neurotrophic factors, given that B-raf-/- motor and sensory neurons can not survive despite the presence of c-Raf-1. We hypothesize that c-Raf-1 nuclear translocation may play a direct role in transcriptional regulation as a consequence of neurotrophic factor induced phosphorylation and activation of c-Raf-1 in motoneurons. Moreover, the identification of target genes for nuclear translocated c-Raf-1 and of specific cellular functions initiated by this mechanism awaits its characterization.
The hematopoietic-specific Rho-family GTP exchange factor (GEF) Vav-1 is a regulator of lymphocyte antigen receptor signaling and mediates normal maturation and activation of B and T cells. Recent findings suggest that Vav-1 also forms part of signaling pathways required for natural and antibody dependent cellular cytotoxicity (ADCC) of human NK cells. In this study, I show that Vav-1 is also expressed in murine NK cells. Vav-1-/- mice had normal numbers of splenic NK cells, and these displayed a similar expression profile of NK cell receptors as cells from wild type mice. Unexpectedly, IL-2-activated Vav-1-/- NK cells retained normal ADCC. Fc-receptor mediated activation of ERK, JNK, and p38 was also normal. In contrast, Vav-1-/- NK cells exhibited reduced natural cytotoxicity against EL4, C4.4.25, RMA and RMA/S. Together, these results demonstrate that Vav-1 is dispensable for mainstream NK cell development, but is required for NK cell natural cytotoxicity. Vav-2, a protein homologous to Vav-1 has also been implicated in NK cell functions. However, NK cells from Vav-2-/- mice have normal cytotoxic activities and NK cells that lack both Vav-1 and Vav-2 exhibit similar defect as Vav-1-/- cells. Thus Vav-2 has no apparent function in the development and the activation of NK cells. Although NK cell development is normal in Vav-1-/- mice, their numbers of NKT cells were dramatically diminished. Furthermore, NKT cells from Vav-1 mutant mice failed to produce IL-4 and IFNg following in vivo CD3 stimulation. A similar loss of NKT cells was observed in Vav-1-/-Vav-2-/- mice, but not in Vav-2-/- mice, suggesting that only Vav-1, and not Vav-2, is an essential regulator of NKT cell development and NK cell cytotoxicity. Similar to Vav-1, Lsc is a Rho GEF that is expressed specifically in the hematopoietic system. It contains a regulator of G-protein signaling (RGS) domain which negatively regulates the Ga12 and Ga13 subunits of G-protein coupled receptors (GPCRs). This study shows that NK and NKT cell development are normal in Lsc-/- mice. However, NK cells from mutant mice display enhanced cytotoxic responses towards a panel of tumor cells. These data implicate for the first time a RGS-containing Rho GEF in cytotoxic responses and suggest that Lsc down-modulate NK cell activation.
Cellular proliferation, differentiation and survival in response to extracellular signals are controlled by the signal transduction pathway of Ras, Raf and MAP kinase. The Raf proteins are serine/threonine kinases with essential function in growth/differentiation/survival - related signal transduction events. In mammals, three functional (A-, B-, and C-Raf) genes were described. Biochemical studies suggest overlapping and differential utilization of Raf isozymes. However, the frequent co-expression of Raf isozymes and their multiple activators and effectors impedes the full understanding of their specific roles. The elucidation of these roles is important due to the involvement of the Ras/Raf/MEK/MAP kinase cascade in human disorders especially in tumor development and progression. B-Raf was shown to posses the strongest kinase activity among Raf kinases and display antiapoptotic properties. Mice deficient in B-Raf show overall growth retardation and die between E10.5 and E12.5 of vascular defects caused by excessive death of differentiated endothelial cells. To elucidate the redundancy of Raf isozymes during embryonic development and to rescue B-Raf-/- (KO) phenotype, B-Raf alleles were disrupted by introducing A-Raf cDNA under the control of endogenous B-Raf promoter. The resulting BRaf A-Raf/A-Raf (KIN) phenotype depends on genetic background. The living embryos displaying normal development but size reduction were found with low incidence at E12.5d-16.5d. All of them displayed the rescue of vascular system. One adult p20 mouse without any visible defects in development and behavior was obtained. On the other hand, the processes of neurogenesis and neural precursors migration in survived embryos were disturbed which led in some cases to underdevelopment of different brain compartments. TUNEL and cell proliferation (PCNA staining) assays revealed more apoptotic (E13.5d) and less proliferating(E12.5d cells within ventricular and sub-ventricular zones of brain ventricles and in striatum of KIN embryos. In addition, more apoptotic cells were detected in many other tissues of E13.5d and in lung of E16.5d KIN embryos but not in adult KIN mouse. p20 KIN mouse demonstrated reduced fraction of neural precursor cells in sub-granular zone of hippocampus and mature neurons in olfactory bulb. The other processes of neurogenesis were not disturbed in adult KIN animal. Fibroblasts obtained from KIN embryos demonstrated less proliferative ability and were more susceptible to apoptotic stimuli compared to WT. This was accompanied by the reduction of active ERK and Akt required for survival, and with decrease of inactive phosphorylated BAD. The kinetic of both ERK and Akt phosphorylation upon serum stimulation was delayed. All these data indicate that moderate A-Raf kinase activity can prevent the endothelial apoptosis but is not enough to completely rescue the other developmental consequences.
In vitro and in vivo studies on the activating platelet collagen receptor glycoprotein VI in mice
(2003)
The work summarized here focused on the characterization of the murine platelet collagen receptor glycoprotein (GP) VI and was performed to evaluate its potential as an antithrombotic target. The first mAb against (mouse) GPVI, JAQ1, was generated and used to demonstrate that GPVI requires the FcRgamma-chain for its expression and function and that this receptor is the central molecule in collagen-induced platelet activation. Blocking the major collagen binding site on GPVI with JAQ1 revealed the presence of a second activatory epitope within collagen. Additionally, the collagen receptor integrin alpha2beta1 was found to be required for activation via this second pathway but not to be essential for collagen-induced activation of normal platelets. In studies with mice expressing reduced levels of the GPVI-FcRgamma-complex, differential responses to GPVI ligands were observed. Most importantly, the striking difference between platelet responses to collagen and the GPVI specific synthetic collagen related peptide (CRP) confirmed the supportive role of other collagen receptor(s) on platelets. Irrespective of yet undefined additional receptors, studies with mice deficient in GPVI (FcRgamma-chain) or alpha2beta1 showed that GPVI, but not alpha2beta1 is essential for platelet-collagen interaction. Based on these results, the model of platelet attachment to collagen was revised establishing GPVI as the initial activating receptor which upregulates the activity of integrins, thus enabling firm attachment of platelets to the ECM. While the mAb JAQ1 had only limited inhibitory effects on collagen-induced activation in vitro, its in vivo application to mice resulted in completely abolished platelet responses to collagen and the GPVI specific agonists CRP and convulxin. This effect was found to be due to antibody-induced irreversible down-regulation of GPVI on circulating platelets for at least two weeks. Further studies revealed that GPVI depletion occurs independently of the targeted epitope on the receptor and does not require the divalent form of IgG as it was also induced by mAbs (JAQ2, JAQ3) or the respective Fab fragments directed against epitopes distinct from the major collagen binding site. The internalization of GPVI in vivo resulted in a long-term protection of the mice from lethal collagen-dependent thromboembolism whereas it had only moderate effects on the bleeding time, probably because the treatment did not affect other activation pathways. These results establish GPVI as a potential pharmacological target for the prevention of ischemic cardiovascular diseases and may open the way for a completely new generation of antithrombotics.
The mammalian Vasodilator Stimulated Phosphoprotein (VASP) is a founding member of the Ena/VASP family of proteins that includes Drosophila Enabled (ena), the mammalian Ena homologue (Mena) and the Ena-VASP-like protein (Evl). VASP was initially discovered and characterized as a substrate for cGMP- and cAMP-dependent protein kinases (cGKs and cAKs). Ena/VASP proteins are involved in Actin-filament formation, plasma membrane protrusion, acceleration of Actin-based motility of Listeria and the establishment of cell-cell adhesion. Moreover, Ena/VASP proteins have been implicated as inhibitory factors in repulsive axon guidance and inhibition of plasma membrane activity and random motility in fibroblast. In order to study the physiological function of VASP, VASP-deficient mice had been generated in the laboratory by homologous recombination. VASP-/- mice showed hyperplasia of megakaryocytes in the bone marrow and spleen and a two-fold increase in thrombin- and collagen-induced platelet activation. To further investigate the cellular function of VASP, I established cardiac fibroblast cell lines derived from both wild type and VASP-/- mice. Both cell lines presented similar growth rates and normal contact dependent-growth inhibition but showed differences in morphology, migration and adhesion. Adherent VASP-/- cells, despite normal Mena and Evl expression levels, were highly spread. VASP-/- cells covered about twice the substrate surface area as wild type cells, while the cell volumes were unchanged. This shape difference suggests that VASP is involved in the regulation of spreading. Since the small GTPases Rac and Cdc 42 and their effector p21-activated kinase (Pak) are key regulators of lamellipodia formation and cell spreading, I analyzed this signalling pathway in VASP-/- cells stimulated with Platelet Derived Growth Factor-BB (PDGF-BB) or fetal calf serum. In wild type cells Rac and Pak were rapidly and transiently activated by PDGF or serum; however, in the absence of VASP both Rac and Pak activation was dramatically prolonged. The Rac/Pak pathway is known to play an essential role in cell motility. VASP deficient cells showed compromised migration and reorientation in a wound healing assay, probably due to enhanced Rac activity. The spreading phenotype, compromised migration and the effect observed on the Rac and Pak activities were reverted in VASP-/- cells stably transfected with full lenght human VASP, indicating a VASP dependent modulation of the Rac/Pak pathway and Rac/Pak regulated processes. Moreover, adhesion and detachment of VASP-deficient cells were significantly slower when compared to wild type cells. Preincubation of VASP+/+ cells with a cGMP analog accelerated adhesion. This acceleration did not take place in the VASP-/- cells, suggesting a VASP dependent effect. The second part of this work focused on VASP function in platelets. On the one hand I investigated the possibility of VASP-dependent Rac regulation in mouse platelets. Murine platelets are a good model for studying Rac regulation since they express high levels of VASP but not Mena/Evl and since VASP-deficient platelets show an increased platelet activation. Rac was activated by platelet agonists which was inhibited by preincubation with cGMP and cAMP analogs. Initial results which need to be extended showed that the cGMPcaused inhibition of Rac activation was VASP-dependent. Finally, in vivo platelet adhesion (platelet-vessel wall interactions) was studied using VASP-deficient mice. These studies demonstrated in-vivo that VASP down regulates platelet adhesion to the vascular wall under both physiological and pathophysiological conditions.
The experimental work of this thesis addresses the questions of whether established cell lines injected into murine blastocysts find their way back home and seed preferentially at the site of their origin. Furthermore, can they change their fate and differentiate to unrelated cell types when exposed to the embryonic environment. This survey was based on the fact that different cell lines have different potentials in developing embryos, dependent on their cellular identity. The cell lines used in this survey were AGM region-deriving DAS 104-4, DAS 104-8 cells, yolk sac-deriving YSE cells and bone marrow-deriving FDCP mix cells. These cells were injected into mouse blastocysts. Donor cells were traced in developing embryos via specific markers. Analysis of the embryos revealed that DAS cells are promiscuous in their seeding pattern, since they were found in all analysed tissues with similar frequencies. YSE cells showed preferences in seeding yolk sac and liver. YSE donor cells in chimaeric tissues were not able to change their immuno-phenotype, indicating that they did not change their destiny. Analysis of adult mice did not reveal any of YSE-derived cells donor contribution. In contrast, FDCP mix cells mostly engrafted haematopoietic tissues, although the embryos analysed by in situ hybridization had donor signals frequently in cartilage primordia, heads, and livers. Analysis of whether FDCPmix-derived cells found in foetal livers were of haematopoietic or hepatocytes nature showed that progeny of injected FDCP mix cells do not differentiate into cells that express a hepatocyte-specific marker. Further analysis showed that FDCPmix-derived donor cells found in brain express neural or haematopoietic markers. In order to reveal if they transdifferentiate to neurons or fuse with neurons/glial cells, nuclear diameters of donor and recipient cells were determined. Comparison of the nuclear diameters of recipient and donor cells revealed no differences. Therefore this suggests that progeny of FDCP mix in brain are not fusion products. Analysis of adult mice tissues revealed that presence of FDCP mix-derived cells was the highest in brains. These results confirmed the assumption that the developmental potential of the analysed cells cannot be easily modified, even when exposed to early embryonic environment. Therefore one can conclude that the analysed cell types had different homing patterns depending on their origins.
Platelet interaction with the subendothelium is essential to limit blood loss after tissue injury. However, upon rupture of atherosclerotic plaques, this interaction may result in blood vessel occlusion leading to life threatening diseases such as myocardial infarction or stroke. Among the subendothelial matrix proteins, collagen is considered to be the most thrombogenic component as it directly activates platelets. Platelets interact with collagen, either indirectly through glycoprotein (GP) Ib-V-IX receptor complex, or directly through the major collagen receptor on the platelet surface, GPVI. The work presented here focused on studying the cellular regulation of GPVI. In addition, a possible role for GPVI in thrombus formation induced by atherosclerotic plaque material was investigated and it was found that GPVI plays an important role in this process. Using a recently published mitochondrial injury model, it was found that GPVI contains a cleavage site for a platelet-expressed metalloproteinase. Further studies showed that platelet activation by CRP, or thrombin induced down-regulation of GPIb, but not GPVI. In parallel, cellular regulation of GPV was studied and it was found that GPV is cleaved in vitro by the metalloproteinase ADAM17. In previous studies it was shown that injection of mice with the anti-GPVI mAb, JAQ1, induces GPVI down-regulation, which is associated with a strong, but transient, thrombocytopenia. Using new anti-GPVI mAbs, which bind different epitopes on the receptor, it is shown in this study that GPVI down-regulation occurs in an epitope-independent manner. Further experiments showed that antibody treatment induces a transient, but significant increase in bleeding time. Using different genetically modified mice, it is shown that, upon antibody injection, GPVI is both, shed from the platelet surface and internalized into the platelet. Signaling through the immunoreceptor tyrosine-based activation motif (ITAM) of the FcR chain is essential for both processes, while LAT and PLC2 are essential for the shedding process only. Antibody-induced increase in bleeding time and thrombocytopenia were absent in LAT deficient mice, showing that it is possible to uncouple the associated side effects from the down-regulation process. As antibody-induced GPVI internalization still occurs in LAT and PLC2 deficient mice, this suggests a novel signaling pathway downstream of GPVI that has not been described so far.
Spreds are a new Sprouty-related family of membrane-associated proteins inhibiting the MAPK signaling pathway by interacting with Ras and Raf-1. Different studies have already demonstrated the inhibitory function of Spreds in cell culture systems, but the in vivo function of Spreds in the whole organism was still unclear. Therefore, Spred-2 knockout mice were generated using a gene trap approach. The Spred-2 deficiency was verified on RNA and protein levels and the lack of functional Spred-2 protein in mice caused a dwarf phenotype similar to achondroplasia, the most common form of human dwarfism. Spred-2-/- mice showed reduced growth and body weight, they had a shorter tibia length and showed narrower growth plates as compared to wildtype mice. Spred-2 promoter activity and protein expression were detected in chondrocytes, suggesting an important function of Spred-2 in chondrocytes and bone development. Furthermore, stimulation of chondrocytes with different FGF concentrations showed earlier and augmented ERK phosphorylation in Spred-2-/- chondrocytes as compared to Spred-2+/+ chondrocytes. These observations suggest a model, in which loss of Spred-2 inhibits bone growth by inhibiting chondrocyte differentiation through upregulation of the MAPK signaling pathway. An additional observation of Spred-2-/- mice was an increased bleeding phenotype after injuries, whereas the bleeding volume was extremely enlarged and the bleeding time was significantly prolonged. So far, hypertension as cause could be excluded, but to discover the physiological reasons for this phenotype, the different steps of the clotting cascade have to be investigated further. As the Spred-2 promoter activity studies demonstrated a high and specific Spred-2 expression in vascular smooth muscle cells and previous studies showed an interaction of Spreds with RhoA, a key regulator of vascular smooth muscle contraction, the regulation of smooth muscle contractility seems to be a good candidate of this phenomenon. Moreover, Spred-1 and Spred-2 specific antibodies were generated as important tools to study the protein expression patterns in mice. Furthermore, nothing was known about the Spred-2 promoter region and its regulation. Here, a detailed in situ analysis of the physiological promoter activity profile in the gene trapped Spred-2-deficient mouse strain was shown. In these mice, the beta-galactosidase and neomycin fusion gene (β-geo) of the gene trap vector was brought under control of the endogenous Spred-2 promoter, giving the opportunity to monitor Spred-2 promoter activity in practically every organ and their corresponding sub-compartments. X-Gal staining of sections of newborn and adult mice revealed 1) a very high Spred-2 promoter activity in neural tissues and different glands; 2) a high activity in intestinal and uterine smooth muscle cells, and kidney; 3) a low activity in heart, testis, lung, and liver; 4) an almost lacking activity in skeletal muscle and spleen, and 5) very interestingly, a very distinct and strong activity in vascular smooth muscle cells. Moreover, comparison of newborn and adult mouse organs revealed a nearly congruent Spred-2 promoter activity. These detailed data provide valuable information for further studies of the physiological functions of Spred-2 in organs showing strong Spred-2 promoter activity, which are in most of these organs still unclear. Finally, gene targeting vectors for Spred-1 and Spred-2 were cloned, to generate ES cells with a floxed exon 2 of the Spred-1 and Spred-2 gene, respectively. Now, these ES cells are valuable tools to establish conditional knockout mice. This is of major interest to investigate the physiological tissue specific functions of Spred-1 and Spred-2, especially if the double knockout mice are not viable.
In this project two novel murine autoimmune models were to be established in an attempt to further investigate the nervous system disorders of Multiple Sclerosis and Guillain Barré Syndrome. Previous experimental autoimmune encephalomyelitis (EAE) and experimental autoimmune neuritis (EAN) models have demonstrated that T cells play a major role in these diseases. Which roles CD4 and CD8 T cells specifically have in the initiation, propagation and termination of an autoimmune nervous system disorder remains controversial. To this end two transgenic mice specifically expressing the neo-antigen (Ag) ovalbumin (OVA) in either the central nervous system (CNS) or peripheral nervous system (PNS) were to be generated. The myelin basic protein (MBP) is a major component of the myelin sheath both within the CNS and the PNS. Therefore the MBP promoter was employed for its distinct regulatory elements to facilitate exclusive CNS or PNS OVA expression. The adoptive transfer of OVA specific MHCI restricted (OT-I) and MHCII restricted (OT-II) TCR Tg T cells extended the OVA Tg mouse model by allowing potentially encephalitogenic T cells to be tracked in vivo. Specificity for the target Ag should enable the dynamic role of antigen specific T cells in neuroinflammatory diseases to be revealed in more detail.