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Role of Smarca5 (Snf2h) chromation remodeling ATPase in hematopoitic development and erythropoiesis
Kokavec, Juraj ; Stopka, Tomáš (advisor) ; Divoký, Vladimír (referee) ; Kořínek, Vladimír (referee)
The Imitation Switch (ISWI) nuclear ATPase Smarca5 (Snf2h) is one of the most conserved chromatin remodeling factors. It exists in a variety of oligosubunit complexes that move DNA with respect to the histone octamer to generate regularly spaced nucleosomal arrays. Smarca5 interacts with different accessory proteins and represents a molecular motor for DNA replication, repair and transcription. We deleted Smarca5 at the onset of definitive hematopoiesis (Vav1-iCre) and observed that animals die during late fetal development due to anemia. Hematopoietic stem and progenitor cells (HSPCs) accumulated but their maturation towards erythroid and myeloid lineages was inhibited. Proerythroblasts were dysplastic while basophilic erythroblasts were blocked in G2/M and depleted. Smarca5 deficiency led to increased p53 levels, its activation at two residues, one associated with DNA damage (S-18) second with CBP/p300 (K376Ac), and finally activation of the p53 targets. We also deleted Smarca5 in committed erythroid cells (Epor-iCre) and observed that animals were anemic postnatally. Furthermore, 4- OHT-mediated deletion of Smarca5 in the ex vivo cultures confirmed its requirement for erythroid cell proliferation. Thus, Smarca5 plays indispensable roles during early hematopoiesis and erythropoiesis.
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Transcription factors CSL and their role in the yeast Schizosaccharomyces pombe
Oravcová, Martina ; Převorovský, Martin (advisor) ; Heidingsfeld, Olga (referee) ; Krásný, Libor (referee)
Proteins of the CSL family (CBF1/RBP-Jκ/Suppressor of Hairless/LAG-1) act as effectors of the Notch signalling pathway in metazoan organisms. They function as repressors or activators of gene transcription in the framework of this pathway and influence many developmental processes. Metazoan CSL proteins can regulate gene expression Notch-independently as well. Notch-independent functions of CSL proteins might be evolutionarily ancestral and in cells and organisms may be important equally as Notch-dependent functions. Presence of CSL proteins was identified in several fungal species, organisms lacking the Notch signalling pathway components and most of known metazoan interacting partners of CSL proteins. CSL paralogs of the fission yeast Schizosaccharomyces pombe, cbf11 and cbf12, are non-essential genes encoding proteins localized in the nucleus of the cell. They exert antagonistic effects on regulation of processes like coordination of nuclear and cellular division and cell cycle progression, ploidy maintenance, cell adhesion and other. In this study, we have proved that both CSL paralogs are able to sequence-specifically bind the CSL-response element DNA in vitro and Cbf11 in vivo as well. Both proteins could activate gene expression in vivo and perform the function of transcription factors....
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Cytokinines and their role in plant cell division, with accent on G2/M transition
Prášilová, Jana ; Ševčíková, Hana (advisor) ; Bíšová, Kateřina (referee)
The eukaryotic cell cycle is well understood mainly in yeasts and animals. Basic regulatory mechanisms, with cyclin-dependent kinases (CDKs) playing crucial roles, are similar in all eukaryotes including plants. CDKs operate mainly at the key cell cycle checkpoints, G1/S and G2/M. Phosphorylation and dephosphorylation of CDKs by kinases and phosphatases have both negative and positive effect. Negative regulator at the G2/M transition is WEE1 kinase which phosphorylates conserved amino acid residues T14 and Y15 of CDK. Phosphatase CDC25 removes this inhibitory phosphate in yeasts and animals and forces cells into mitosis. Plant cell cycle exhibits remarkable differences. Importantly, it is controlled by phytohormones, and some key points of regulation remain obscure - a functional plant homologue of yeast CDC25 phosphatase has not been found in plants yet though Y15 inhibitory phosphorylation by WEE1 kinase blocks mitosis entry in plants as well. Thus, the regulatory mechanism of G2/M transition in plant cells is still to be found. Phytohormones play a key role, not only in the plant cell cycle, but in whole plant development. Interplay between the two groups of phytohormones: auxins and cytokinins, is crucial. Especially cytokinins significantly influence the regulation of G2/M checkpoint. It is...
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Role of intestinal circadian clock in epithelial transport, proliferation, and tumourigenesis
Soták, Matúš ; Pácha, Jiří (advisor) ; Bendová, Zdeňka (referee) ; Herichová, Iveta (referee)
AABBSSTTRRAACCTT The molecular circadian clock enables anticipation of environmental changes. In mammals, clocks are ubiquitously present in almost all tissues and they are comprised of transcriptional-translational feedback loops of the so-called clock genes. The central clock represents the intrinsic pacemaker which is located in suprachiasmatic nuclei (SCN) of hypothalamus and synchronizes peripheral clocks. Clockwork system in alimentary tract and its regulatory link to intestinal functions are poorly understood. Therefore the objective of the thesis was to characterize molecular clock in particular parts of the rat intestine and to elucidate its link to the intestinal transport, regulation of cell cycle and neoplastic transformation in colonic tissue. We used quantitative RT-PCR (qPCR) to determine circadian profiles of mRNA expression of clock genes in the epithelium of duodenum, jejunum, ileum, and colon of rat. Furthermore, we analysed the expression of genes coding sodium chloride transporters and channels as well as cell cycle regulators in colon. To focus more precisely on different structures of intestinal epithelia we used laser capture microdissection. In addition, we performed Ussing chamber measurements to determine the colonic electrogenic transport. To study the contribution of circadian...
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Activation and regulation of cell death in senescent cancer cells.
Holíček, Peter ; Anděra, Ladislav (advisor) ; Drbal, Karel (referee)
Cellular senescence is a distinct cell state, characteristic by cessation of cell proliferation and it is accompanied by specific morphological and biochemical alterations. Increasing and persisting incidence of senescence cells has been shown to have detrimental effect on an organism largely contributing to its ageing. Senescent cells also positively support tumour growth and can even stimulate carcinogenic transformation of surrounding cells. Moreover, senescence can be induced even in tumour cells spontaneously or by chemotherapy. Regardless of an initial stimuli and type of cells, there are two main senescence inducing pathways p16/pRb and p53/p21. Both senescent cells as well as senescent cancer cells seems to have modified apoptotic signalling at the level of mitochondria and Bcl-2 family proteins. In this study, we aimed to analyse effect of senescent state as well as pre-senescent (growth arrested state) induced by p16/pRb and p53/p21 signalling pathways on the response of H28 mesothelioma cancer cells-derived clonal cultures to various cell death-inducing stimuli. By inducible expression of p16 and p21 proteins in doxycycline-dependent manner, we forced cells to acquire senescent-like phenotype, which we detailly characterised. Our results showed that senescent-like phenotype, manifests...
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Transcription factors CSL and their role in the yeast Schizosaccharomyces pombe
Oravcová, Martina
Proteins of the CSL family (CBF1/RBP-Jκ/Suppressor of Hairless/LAG-1) act as effectors of the Notch signalling pathway in metazoan organisms. They function as repressors or activators of gene transcription in the framework of this pathway and influence many developmental processes. Metazoan CSL proteins can regulate gene expression Notch-independently as well. Notch-independent functions of CSL proteins might be evolutionarily ancestral and in cells and organisms may be important equally as Notch-dependent functions. Presence of CSL proteins was identified in several fungal species, organisms lacking the Notch signalling pathway components and most of known metazoan interacting partners of CSL proteins. CSL paralogs of the fission yeast Schizosaccharomyces pombe, cbf11 and cbf12, are non-essential genes encoding proteins localized in the nucleus of the cell. They exert antagonistic effects on regulation of processes like coordination of nuclear and cellular division and cell cycle progression, ploidy maintenance, cell adhesion and other. In this study, we have proved that both CSL paralogs are able to sequence-specifically bind the CSL-response element DNA in vitro and Cbf11 in vivo as well. Both proteins could activate gene expression in vivo and perform the function of transcription factors....
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Role of intestinal circadian clock in epithelial transport, proliferation, and tumourigenesis
Soták, Matúš
AABBSSTTRRAACCTT The molecular circadian clock enables anticipation of environmental changes. In mammals, clocks are ubiquitously present in almost all tissues and they are comprised of transcriptional-translational feedback loops of the so-called clock genes. The central clock represents the intrinsic pacemaker which is located in suprachiasmatic nuclei (SCN) of hypothalamus and synchronizes peripheral clocks. Clockwork system in alimentary tract and its regulatory link to intestinal functions are poorly understood. Therefore the objective of the thesis was to characterize molecular clock in particular parts of the rat intestine and to elucidate its link to the intestinal transport, regulation of cell cycle and neoplastic transformation in colonic tissue. We used quantitative RT-PCR (qPCR) to determine circadian profiles of mRNA expression of clock genes in the epithelium of duodenum, jejunum, ileum, and colon of rat. Furthermore, we analysed the expression of genes coding sodium chloride transporters and channels as well as cell cycle regulators in colon. To focus more precisely on different structures of intestinal epithelia we used laser capture microdissection. In addition, we performed Ussing chamber measurements to determine the colonic electrogenic transport. To study the contribution of circadian...
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The effect of synthetic modified mRNAs induced proliferation on pancreatic beta cells
Veľasová, Adriana ; Koblas, Tomáš (advisor) ; Bořek Dohalská, Lucie (referee)
Diabetes mellitus is a chronic disease caused by the loss of pancreatic beta cells due to autoimmune destruction or increased apoptosis. Beta-cell deficiency results in reduced insulin production, which plays an important role in glucose metabolism. The number of beta-cells in the body is one of the main factors that influence the development of this chronic disease. Therefore, it is necessary to find a way by which the number of beta-cells of the organism can be increased and thus the insulin production can be restored in a natural way without any need for the use of insulin infusions. However, the ability of beta-cells to divide decreases with age and is virtually nil in adulthood. The study of the cell cycle, especially the early and late cyclins and cyclin-dependent kinases, which act as cell cycle regulators, thus appears to be a promising way to restore natural insulin-producing tissues. In order to increase the number of beta cells entering the cell cycle, we focused on studying the effect of in vitro transcribed (IVT) mRNAs, encoding cyclins type D and cyclin dependent kinases 4 and 6 on stimulating cell division of isolated beta-cells. We found that transfection IVT mRNAs for type D cyclins in combination with cyclin-dependent kinases 4 and 6 significantly increased the proliferation of beta-cells...
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