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Regulation of epithelial plasticity by ERK1 and ERK2 isoforms
Rasl, Jan ; Vomastek, Tomáš (advisor) ; Rösel, Daniel (referee) ; Libusová, Lenka (referee)
The ERK pathway is an evolutionarily conserved three-tier signaling cascade comprised of protein kinases Raf, MEK, and ERK. These core kinases are arranged in a hierarchical order and the signal is transduced from Raf to MEK to ERK. The ERK pathway is activated by diverse extracellular signals and in response regulates many cellular processes including cell proliferation, differentiation, apoptosis, migration or epithelial plasticity. Given the role of the ERK pathway in regulating such fundamental cellular processes, the ERK pathway signaling is tightly controlled and its dysregulation has pathological consequences such as cancer development and progression. Although much is known about mechanisms underlying the signal transduction by the ERK signaling pathway, much less is known about how two highly homologous ERK1 and ERK2 isoforms contribute to the signaling by this pathway. In this thesis, I studied isoform-specific functions of ERK1 and ERK2 using epithelial Madin- Darby Canine Kidney (MDCK) cells overexpressing either ERK1 or ERK2. Obtained data show that overexpression of ERK2, but not ERK1, had significant effects on the morphology and functional phenotype of MDCK cells. Both ERK1 and ERK2 expressing cells were able to form cohesive clusters, but the only ERK2 overexpression affected...
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Synthesis and biochemical characterization of hybrid analogues of human insulin and IGF-2
Povalová, Anna ; Stiborová, Marie (advisor) ; Koberová, Monika (referee)
The ever-increasing occurrence of diabetes mellitus brings about the need for development of new therapeutic agents to provide adequate treatment for patients. An important element in this research area is elucidation how insulin works, mainly in connection with insulin-like growth factors (IGF-1 and IGF-2), which show significant structural homology to each other. In addition, their respective receptors - insulin receptor (IR) and receptor for IGF-1 and IGF-2 (IGF-1R) - exhibit very high similarity. As a result, IGF-1 and IGF-2 can bind to IR and insulin can bind to IGF-1R. Of a particular importance is the high affinity binding of IGF-2 to the isoform A of IR. Unlike insulin, which predominantly mediates glucose entry into cells, IGFs induce growth or mitogenic effects. The finding which structural determinants in insulin and IGFs are responsible for the differences in the activation of their cognate receptors could provide an explanation for different functional responses upon binding of these hormones to different target cells. Understanding of this mechanism could also help in the development of functionally selective analogues of these hormones. The aim of this study was the synthesis and characterization of analogues of human insulin extended at the C terminus of the B chain with the amino...
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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...
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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...
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Synthesis and biochemical characterization of hybrid analogues of human insulin and IGF-2
Povalová, Anna ; Stiborová, Marie (advisor) ; Koberová, Monika (referee)
The ever-increasing occurrence of diabetes mellitus brings about the need for development of new therapeutic agents to provide adequate treatment for patients. An important element in this research area is elucidation how insulin works, mainly in connection with insulin-like growth factors (IGF-1 and IGF-2), which show significant structural homology to each other. In addition, their respective receptors - insulin receptor (IR) and receptor for IGF-1 and IGF-2 (IGF-1R) - exhibit very high similarity. As a result, IGF-1 and IGF-2 can bind to IR and insulin can bind to IGF-1R. Of a particular importance is the high affinity binding of IGF-2 to the isoform A of IR. Unlike insulin, which predominantly mediates glucose entry into cells, IGFs induce growth or mitogenic effects. The finding which structural determinants in insulin and IGFs are responsible for the differences in the activation of their cognate receptors could provide an explanation for different functional responses upon binding of these hormones to different target cells. Understanding of this mechanism could also help in the development of functionally selective analogues of these hormones. The aim of this study was the synthesis and characterization of analogues of human insulin extended at the C terminus of the B chain with the amino...
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The regulation of the ERK signalling pathway by scaffold protein RACK1
Bráborec, Vojtěch ; Vomastek, Tomáš (advisor) ; Filipp, Dominik (referee)
The ERK signalling cascade comprised of protein kinases Raf, MEK and ERK is an evolutionarily conserved member of MAPK family that is activated in response to wide range of extracellular stimuli. The ERK pathway controls fundamental cellular functions including cell proliferation, differentiation, apoptosis or cell motility. To control such a diverse cellular responses by a single pathway cells have evolved regulatory mechanisms that channel the extracellular signals towards the specific biological response. Crucial to this control are non- enzymatic proteins termed scaffolds that associate with and enhance functional interaction of the components of MAPK pathways and can regulate amplitude, timing, specificity and location of signals. Scaffold protein RACK1 associates with several components of cell migration machinery including integrins, FAK, Src and the ERK pathway core protein kinases. RACK1 regulates distinct steps of cell migration such as establishment of cell polarity and focal adhesion turnover, however, the molecular mechanism by which RACK1 regulates these processes remains largely unknown. The main aim of this study was to investigate the functional role of RACK1 in cell motility, in particular to identify new effector proteins utilized by the ERK pathway and RACK1 in the regulation of...
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Molecular mechanisms of signal transduction by the ERK signaling cascade.
Bráborec, Vojtěch ; Rösel, Daniel (referee) ; Vomastek, Tomáš (advisor)
The MAPK (mitogen-activated protein kinase) cascade represents an evolutionary conserved mechanism by which cells sense extracellular signals and convert them into variety of context-dependent responses. The best studied member of the MAPK protein family is protein kinase ERK (extracellular signal-regulated kinase). Together with protein kinases Raf and MEK (MAPK/ERK kinase) comprise a prototypical signaling pathway which regulates broad-spectrum of biological processes such as cellular proliferation, differentiation, cellular migration, adhesion or apoptosis. To modulate such a multitude of distinct responses by a single pathway, cells utilize mechanisms such as signal strength and duration, distinct protein localization, communication with other signaling pathways, differential substrate selection and the selection of interactive partners. All presented means of regulation are influenced by proteins with non-enzymatic functions - scaffold proteins, protein inhibitors and anchoring proteins. These protein modulators channel the signals leading to particular cellular response, and thus represent the key element of signal transduction. Despite increasing importance of protein modulators in cellular signaling, their biological roles remain mostly unknown. The physiological importance of protein modulators is...
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