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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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Molecular mechanisms of G2/M checkpoint regulation
Kořínková, Klára ; Macůrek, Libor (advisor) ; Forman, Martin (referee)
Cell division is necessary for maintaining tissue homoeostasis, but at the same time its defects are closely related to the development of many diseases including cancer and premature ageing. Activation of oncogenes leads to replication stress and directly threatens genome stability. The right control of transition between interphase and mitosis is an important mechanism for the protection of genome integrity. Nuclear division is only possible with those cells in which flawless duplication of genetic information occurred. By contrast, cells with damaged DNA structure remain temporarily or permanently stopped at G2 phase of the cell cycle. The topic of this thesis is a detailed literature overview with the subject of molecular mechanisms of the G2/M transition regulation under unperturbed conditions and in the presence of damaged DNA.
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The relationship between circadian system and cell cycle
Vrtílková, Andrea ; Bendová, Zdeňka (advisor) ; Fárková, Eva (referee)
The circadian system is able to oscillate by itself owing to the transcriptional-translation feedback loop. Components of this loop do not affect just their own run, but they also have an impact on some other functions of the cell, for example cell cycle. This interaction is made by clock proteins (PER, CRY etc.) and by clock-controlled proteins (WEE1, TIM, XPA etc.). These proteins participate in the cell cycle run and have an impact on check-points. Disruption of the circadian clock can cause faults in cell cycle check-points, storing of DNA damages and increased cell apoptosis or tumor progression. Key words: circadian systém, cell cycle, WEE1, XPA, P21, C-Myc, TIM, PER
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Role of Polo-like kinases in the cell cycle and DNA damage response
Kudláčková, Radmila ; Macůrek, Libor (advisor) ; Šolc, Petr (referee)
Within the process of cell division, genetic material must be equally distributed between the two daughter cells. In the next phase, the missing portion of the genome must be synthesized. The entire cycle is regulated by cyclin-dependent kinases (Cdks) in cooperation with cyclins. If the DNA is damaged during the cell cycle, signaling pathways of checkpoints supress cycle progression and enforce the cell cycle arrest until the damage is repaired. Malfunction of the checkpoints results in tumorigenesis. Polo-like kinases (Plks) are, much like Cdks, important regulators of the cell cycle. Plks play significant role mainly in the mitosis and also in a response to the DNA damage. This thesis is focused on human homologues, nevertheless conservation of homologues among organisms is considerable, thus presented findings are of general relevance. Human cells express five proteins from the family of Polo-like kinases, from which Plk1 corresponds to Polo-like kinases of lower eukaryotes. Knowledge on the remaining four kinases is still on the rise.
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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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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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Transcription factors driving periodic gene expression during the fission yeast cell cycle
Jordáková, Anna ; Převorovský, Martin (advisor) ; Paleček, Jan (referee)
The fission yeast Schizosaccharomyces pombe plays an important role in elucidation of the mechanisms of cell cycle regulation and characterization of the relevant effector molecules involved. The cell cycle of S. pombe consists of a prolonged period of growth (G2 phase), which is followed by a nuclear division (M phase), a very short G1 phase and DNA replication (S phase). Already during S phase formation of division septum occurs. Cell cycle progression is regulated at multiple levels. Although the yeast S. pombe is an extensively studied model organism, knowledge of the transcriptional network regulating progression through the cell cycle is still incomplete. Transcription factors are very important regulators of gene expression and therefore their characterization is the subject of research. At the transcriptional level, several key transcription factors have been identified that regulate periodically oscillating and interdependent waves of gene expression during the cell cycle. This study summarizes the current state of knowledge in the field of the transcriptional regulation of periodic gene expression in the fission yeast cell cycle.
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Optimization of HEK293 cell line expression system by regulation of cell cycle and apoptosis
Poláchová, Edita ; Vaněk, Ondřej (advisor) ; Pavlíček, Jiří (referee)
Transient transfection of mammalian cell lines is an effective approach for recombinant protein production, which can provide milligrams to grams of proteins in two weeks from cloning of the corresponding cDNA. Native glycosylated proteins prepared via this approach can be used for various purposes in molecular biology, immunology or pharmaceutical industry, i.e. initial phase of pre-clinical therapeutic protein research. One of the most used mammalian host cell lines is the human embryonic kidney cell line, that can be easily cultivated and chemically transfected. The amount of proteins produced by transiently transfected human embryonic kidney cells can be enhanced by a whole range of factors, i.e. co-expression or direct addition of acidic fibroblast growth factor to the culture medium, co-expression of cell cycle regulating proteins or anti-apoptotic proteins. Expression plasmid pTW5 was prepared and further modified by gene insertion of aFGF, cell cycle regulator p18, p21 or p27 (cyclin-dependent kinase inhibitors) or apoptosis inhibitor bcl-2 or bcl-x. These plasmids were then used for optimization of HEK293T cell line expression system. The impact of every single regulator and their combinations, including hitherto undescribed effect of combination of cell cycle regulator and anti-apoptotic...
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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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The role of Ser/Thr phosphorylation of p130CAS in signaling
Dibus, Michal ; Rösel, Daniel (advisor) ; Macůrková, Marie (referee)
p130Cas is an important adaptor protein that plays an essential role in many intracellular signaling processes. Given the fact that p130Cas is a well-known substrate for wide spectrum of kinases, its function is regulated mostly by phosphorylation on tyrosine, threonine and serine residues. This work is focused on Serine/Threonine phosphorylation and its role in regulation of p130Cas signaling. Although it is known that Serine/Threonine phosphorylation of p130Cas is regulated by cell cycle, integrin-mediated cell adhesion and association with BCAR3, the mechanisms leading to the phosphorylation are still not well understood and the kinases involved in these processes are unknown. Being p130Cas an important regulator of cell migration and tumor invasiveness, understanding of these mechanisms should provide a useful tool in developing new strategies in aiming of anti-cancer drugs.
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