|
|
The regulation of primary response genes by the ERK signaling pathway
Chvalová, Věra ; Vomastek, Tomáš (advisor) ; Doubravská, Lenka (referee)
The ERK signaling pathway represents an evolutionary conserved mechanism that enables cells to perceive various extracellular signals and convert them to a diverse array of biological outcomes such as proliferation, differentiation, cell cycle control, apoptosis or cell migration. Key components of this pathway are protein kinases Raf, MEK and the effector protein kinase ERK. In addition to its physiological role, continuous activation of the ERK pathway caused by somatic mutations of some of its components or upstream regulators appears to be significant cause of many human tumor diseases. That is why this pathway plays an important role also from the biomedical viewpoint. The multistep changes in gene expression are primarily responsible for these physiological and pathological events. Changes in genes expression are induced by activated kinase ERK that after translocation into the nucleus phosphorylates transcription factors (TFs) whose activation, in turn, leads to transcription of so-called immediate early genes (IEGs), many of which also code for other TFs (e.g. c-Fos, c-Jun or c-Myc). The latter TFs then regulate expression of further genes for structural and signaling proteins. This causes global changes in gene expression and leads to functional reprogramming of the cells. This thesis...
|
|
| |
|
|
Spatiotemporal regulation of Lck activity in the initiation of TCR signalling
Ballek, Ondřej ; Filipp, Dominik (advisor) ; Černý, Jan (referee) ; Vomastek, Tomáš (referee)
Ph.D. Thesis: Spatiotemporal regulation of Lck aktivity in the TCR signalling Ondřej Ballek Abstract Arguably, the most studied cell types of immune system are T-cells. They are key players of adaptive immunity responsible for targeted action against pathogens or other danger signals. Due to their central importance, any alteration in the regulation of their activity leads often to immunopathology. Thus, the knowledge how to harness their bio-destructive effector functions is of critical importance. Up today, there is only limited consensus on the nature of molecular mechanisms controlling the initiation of T-cell activation. When T-cell receptor (TCR) recognizes its cognate antigen presented on antigen presenting cell (APC), the activation signal is transmitted through the plasma membrane and subsequent phosphorylation of cytoplasmic chains of TCR complex ensues. This is commonly considered as the first biochemical sign of T-cell activation, the process called TCR triggering. How the activation signal gets into the cell and which molecular mechanisms control TCR triggering are two fundamental, yet still unanswered questions. In this study we focused mainly on the latter one. Working within this experimental framework, we investigated three particular problems. The first one concerns the spatiotemporal...
|
|
|
Signaling mechanism in nuclear reorientation and its functional singnificance in cell migration.
Maninová, Miloslava ; Vomastek, Tomáš (advisor) ; Hodný, Zdeněk (referee) ; Jaroš, Josef (referee)
The establishment of cellular polarity is first critical step of directional cell migration. The process of cellular polarization requires many signaling pathways that are differently regulated at the cell front and at the rear side and enables creation of typical asymmetrical profile of migrating cell. During the polarization cell forms the leading edge and trailing rear and relocalizes the intracellular organelles to such a position that is optimal for directional movement. In many migrating cells cell nucleus is usually located at the cell rear and microtubule organizing center localizes between the nucleus and the leading edge of the cell. This cellular arrangement is prerequisite for directional cell migration. We have shown that during cell polarization cell also reorients the nucleus to the direction of migration. The nuclear reorientation is temporally restricted rotation of the cell nucleus that aligns the longer nuclear axis with the axis of migration. Nuclear reorientation promotes the establishment of cellular polarity and facilitates the movement of the cell. The nuclear reorientation requires the physical linkage of the nucleus to cell cytoskeleton mediated by LINC (Linker of Nucleoskeleton and Cytoskeleton) complex. We have shown that LINC complex anchors the nucleus to actin stress...
|
|
|
Radiation-induced plasticity of prostate cancer cells
Kyjacová, Lenka ; Hodný, Zdeněk (advisor) ; Bouchal, Jan (referee) ; Vomastek, Tomáš (referee)
Resistance of various cancers to conventional therapies including radio- and chemo- therapy is one of the most investigated phenomena in the molecular and clinical oncology. Recurrent disease is characterized by the presence of metastases, which are responsible for 90% of cancer-related mortality. Fractionated ionizing radiation (fIR) combined with surgery or hormone therapy represent the first-choice treatment for medium to high risk localized prostate carcinoma (PCa). In PCa, the failure of radiotherapy (RT) is often caused by radioresistance and further dissemination of escaping (surviving) cells. To investigate the radioresistance-associated phenotype, we exposed four metastasis- derived human PCa cell lines (DU145, PC-3, LNCaP, and 22RV1) to clinically relevant daily fractions of ionizing radiation (fIR; 35 doses of 2 Gy) resulting in generation of two surviving populations: adherent senescent-like cells expressing common senescence-associated markers and non-adherent anoikis-resistant stem cell-like cells with active Notch signaling and expression of stem cell markers CD133, Oct-4, Sox2, and Nanog. While the radioresistant adherent cells were capable to resume proliferation shortly after the end of irradiation, the non- adherent cells started to proliferate only after their reattachment...
|
|
|
Regulation of leukocyte signal transduction by adapter proteins with special focus on Csk anchoring proteins
Drobek, Aleš ; Brdička, Tomáš (advisor) ; Brábek, Jan (referee) ; Vomastek, Tomáš (referee)
Signaling through leukocyte receptors is an integral part of immune system homeostasis and it is crucial for immune response. Signal initiation and propagation has to be properly spatiotemporally controlled not to trigger pathological immune activation. Important part in the initiation and regulation of signaling is played by adaptor proteins. These adaptors can be involved in positive or negative control, or both, depending on what effector enzymes they bind. In presented work we studied the involvement of two adaptor proteins PSTPIP2 and LST1/A in leukocyte signaling. Both adaptors interact with several inhibitory enzymes which implicates these proteins in negative regulation of signaling. We extended current understanding of why the deficiency in the expression of one of these adaptors, PSTPIP2, results in the initiation and progression of autoinflammatory disorder chronic multifocal osteomyelitis disease in mice by describing its interactions with Csk kinase and SHIP1 phosphatase. In particular we have shown that PSTPIP2 deficiency leads to increased activity of signaling pathways and increased processing of IL-1β in neutrophils in response to a variety of stimuli and that binding to SHIP1 contributes to PSTPIP2-mediated suppression of inflammation. Our biochemical analysis of the second of...
|
|
|
Elucidation of ERK1 and ERK2 protein kinases effect on cap-independent translation initiation
Přibyl, Miroslav ; Vopálenský, Václav (advisor) ; Vomastek, Tomáš (referee)
Protein kinases ERK1 and ERK2 are one of the most studied proteins in cell signalling. Both proteins are involved in a plethora of processes, such as phosphorylation and activation of kinases as part of signalling pathways. Enzymes ERK1 and ERK2 are part of MAPK/ERK signalling cascade, connected to many cellular including cell proliferation, cell growth or differentiation. The MAPK/ERK signalling cascade is often activated in different types of tumors, making it a candidate for developing new chemical inhibitors. One of the important questions in fundamental research of ERK1 and ERK2 protein kinases is the search for difference between these proteins. Current knowledge points to redundancy of both proteins, howver several examples suggest otherwise. Recently, the work presented in Casanova et al. 2012 indirectly suggests divergent effect of ERK1 and ERK2 on cap-independent translation initiation. In the Laboratory of RNA biochemistry we focus on HCV IRES (Hepatitis C Virus Internal Ribosome Entry Site) dependent translation initiation. This diploma thesis lead to establish RNA interference method in our laboratory and to establish reporter system to study ERK1 and ERK2 effect on HCV IRES dependent translation initiation. Based on our data acquired during our research, we present in this work...
|
|
|
Identification of a new mechanism of Lck regulation via its C-terminal sequence
Valečka, Jan ; Filipp, Dominik (advisor) ; Vomastek, Tomáš (referee)
T-cell activation is a complex process crucial for a proper function of immune system. It has been extensively studied and its main features are well understood. However, some of the events involved in T-cell signalling are still unclear. After T-cell receptor stimulation, Src-family kinase Lck drives the initiation of signalling by tyrosine phosphorylation. Phosphorylation of several downstream targets is dependent on the redistribution of Lck to the different compartment of the plasma membrane, called lipid rafts. In lipid rafts, active Lck is juxtaposed and activates raft-resident substrates which then trigger downstream signalling. The critical in this process is the mechanism of Lck translocation to lipid rafts which has not been studied so far and represents the topic of great academic and clinical interests. Previously, we identified the adaptor protein RACK1 as a candidate protein mediating the redistribution of Lck to lipid rafts by linking it to the microtubular network. In this thesis, we analysed the structural features and functional role of RACK1 in its interaction with Lck. We show here, using the SYF cell lines expressing the wild type and various mutated forms of Lck, that intact SH3 or SH2 domains of Lck are required for an effective RACK1-Lck complex formation. We also documented...
|
|
|
Regulation of actin cytoskeleton and epithelial cells morphology by the ERK signaling pathway
Rasl, Jan ; Vomastek, Tomáš (advisor) ; Tolde, Ondřej (referee)
The ERK signalling cascade belongs to a familly of the signalling pathways conserved in eukaryotic cells, which responds to the wide spectrum of extracellular stimuli and convert these stimuli to appropriate response. In epithelial cells the ERK signalling cascade induces disintegration of epithelial architecture and induces morphological changes leading to the gain of the autonomy of the epithelial cells. During morphological changes of the epithelial structure, the ERK signalling cascade participates in the remodelling of the actin cytoskeleton, which leads to the disassembly of cell-cell adhesions and the loss of the epithelial polarity. Subsequently ERK activates the migration programme, which enables epithelial cells to use individual mesenchymal-like mode of migration. The so called peripheral actin is one of the least explored actin structures that forms at the periphery of the epithelial cells and surrounds the colony of epithelial cells. Peripheral actin is located at the basal side of the cell and it probably takes part in the integrity of epithelial tissue. Nevertheless, up to date it is not know if and how ERK signalling cascade regulatesthe peripheral actin and if remodeling of peripheral actin takes part in the cell migration. In this thesis we show, that ERK signalling cascade is...
|
|
|
The effect of Crk SH3domain inhibition in invasiveness of cells
Tomášová, Lea ; Rösel, Daniel (advisor) ; Vomastek, Tomáš (referee)
Protooncogene Crk was found to be upregulated in tumours with aggressive and invasive potential. The adaptor protein Crk has an important role in cell signaling: it integrates signals from activated integrins and growth factors receptors via its SH2 domain and transmits the signal to its SH3 domain binding partners that activate the small GTPases Rac1, Rap1 and Ras. This leads to regulation of cell migration, proliferation and survival. The aim of this thesis project was to inhibit the Crk dependent signaling by a competitive inhibition of the Crk SH3 domain, using a high affinity CrkSH3 binding peptoid. Binding of the inhibitor to the Crk SH3 domain prevents binding of cellular Crk SH3 interaction partners and the corresponding signal transmission is impaired. In this thesis project the effect of the Crk SH3 inhibition on the invasiveness of cancer cells was analyzed. The observed inhibitory effect on cell invasion as well as on anchorage independent growth provides a proof of therapeutical relevance of targeting CrkSH3N domain by peptoide-based inhibitors. Powered by TCPDF (www.tcpdf.org)
|
guest ::
