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Molecular mechanisms of mutagenesis and resistence in CML cell lineages
Karasová, Dominika ; Čuřík, Nikola (advisor) ; Savvulidi Vargová, Karina (referee)
Chronic myeloid leukemia is a clonal haematopoietic disease, with characteristic BCR-ABL1 fusion gene. Despite the significant improvement in patient treated with tyrosine kinase inhibitors (TKI), 20-30 % of patients develop resistance. One of the main causes of treatment failure are mutations in the BCR-ABL1 kinase domain (KD). The aim of this work was to elucidate the molecular mechanisms of resistance and mutagenesis development in CML using an in vitro CML model KCL-22. The main part of this work was focused on the identification of genes involved in DNA damage response and repair, that could play a role in the process of mutagenesis of BCR-ABL1. We used the RT2 Profiler PCR Arrays method for the group of selected genes regulating DNA damage response and repair. We identified the genes XRCC6 and PARP1 whose gene expression was significantly and specifically decreased during KD BCR-ABL1 mutagenesis. Products of these genes are involved in repairing DNA double-strand breaks through non-homologous end joining (NHEJ). During study of the KD BCR-ABL1 mutagenesis we also found that clones, which developed mutations, did not show the increased BCR-ABL1 expression in the beginning of the culture compared to the clones in which mutations have not evolved. Key words: myeloid leukemia, mutation,...
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Molecular mechanisms of mutagenesis and resistence in CML cell lineages
Karasová, Dominika ; Čuřík, Nikola (advisor) ; Savvulidi Vargová, Karina (referee)
Chronic myeloid leukemia is a clonal haematopoietic disease, with characteristic BCR-ABL1 fusion gene. Despite the significant improvement in patient treated with tyrosine kinase inhibitors (TKI), 20-30 % of patients develop resistance. One of the main causes of treatment failure are mutations in the BCR-ABL1 kinase domain (KD). The aim of this work was to elucidate the molecular mechanisms of resistance and mutagenesis development in CML using an in vitro CML model KCL-22. The main part of this work was focused on the identification of genes involved in DNA damage response and repair, that could play a role in the process of mutagenesis of BCR-ABL1. We used the RT2 Profiler PCR Arrays method for the group of selected genes regulating DNA damage response and repair. We identified the genes XRCC6 and PARP1 whose gene expression was significantly and specifically decreased during KD BCR-ABL1 mutagenesis. Products of these genes are involved in repairing DNA double-strand breaks through non-homologous end joining (NHEJ). During study of the KD BCR-ABL1 mutagenesis we also found that clones, which developed mutations, did not show the increased BCR-ABL1 expression in the beginning of the culture compared to the clones in which mutations have not evolved. Key words: myeloid leukemia, mutation,...
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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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