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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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Molecular mechanism of hydrogen sulfide action during meiotic maturation of porcine oocytes
Veselá, Andrea ; Hošková, Kristýna (advisor) ; Tomáš, Tomáš (referee)
At present reproductive biotechnology methods are on the rise, but their development and application in the broader management of reproduction is dependent on obtaining a sufficient number of quality oocytes cultured in vitro. The prerequisite for this requirement is the creation of the optimal conditions in the course of culturing oocytes.
Understanding and knowledge of the processes that occur in oocyte during maturation is an important and necessary condition for optimizing the process of culturing oocytes in vitro and gaining a sufficient number of good quality oocytes in metaphase II of meiotic division. A large number of mechanisms that affect and control oocyte maturation are known, however it cannot be claimed that this process has been fully explained and studied. One factor which has a potential role in the regulation of meiotic maturation of oocytes is gasotransmitter hydrogen sulfide (H2S), a critical signaling molecule of endogenous origin.
The study of H2S led to the hypothesis that H2S actively influences the course of meiotic maturation of pig oocytes by regulating key signaling cascades. The aim of this work was to determine the involvement of H2S in the regulation of the MEK1-MAPK signaling cascade, responsible for the initiation and progress of the meiotic maturation of oocytes and the MEK1-PARP-1 cascade as signaling that supports cell viability. For this purpose, pig oocytes cultured in modified media were used, supplemented with a specific combination of enzyme inhibitors (3Ki) or in a culture medium with donor H2S. The ocytes were then subjected to immunocytochemistry staining, fluorescence microscopy and image analysis.
The results show that H2S is involved in the regulation of meiotic maturation. It confirmed the hypothesis of the endogenous production of H2S in the course of the meiotic maturation of pig oocytes and the influence of the MAPK signaling cascade. Based on the results, it is however likely that the MEK1-PARP-1 signaling cascade is not affected by H2S, unlike MAPK signaling, comprising the mentioned MEK1 as superior kinase. MAPK kinase activity is significantly lower in oocytes after treatment 3Ki. Further experiments are for a detailed understanding of these regulatory pathways and for the proper verification of the mechanism of the effects of H2S necessary, in particular for a full understanding of the target control factors by the post-translational modification of S-sulfhydration.
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