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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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Intracellular signalling of Chk2 kinase and impact of its defects in oncogenesis
Stolařová, Lenka ; Kleibl, Zdeněk (advisor) ; Brábek, Jan (referee)
Chk2 (checkpoint kinase 2), a regulatory protein of the cell cycle checkpoints, is coded by CHEK2 gene. Chk2 belongs to serine/threonine kinase family and its dominant activity is in regulation and signal distribution of intracellular response to DNA damage. The upstream regulator of Chk2 protein is the ATM kinase that activates Chk2 by its phosphorylation on Thr68 localized in FHA domain. This in turn leads to the conformation change inducing homodimerization of Chk2 protomers and their activating phosphorylation within their kinase domains. Upon phosphorylation, catalytically active Chk2 protomers dissociate and phosphorylate various intracellular proteins (incl. p53, E2F-1, BRCA1, Cdc25A a C, BRCA2 a PLK3). By regulation of these proteins, Chk2 contributes to the cell cycle arrest, regulation of DNA repair and apoptosis. Germline mutations in CHEK2 gene were identified with the increased frequency in many human cancers, including breast and colorectal cancer. Hence, the failure of Chk2 intracellular activity contributes to the process of malignant transformation.
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Vztah mezi genetickými polymorfismy DNA reparačních genů a jejich expresí u zdravé populace (s výhledem na stanovení u onkologických pacientů).
Hánová, Monika ; Vodička, Pavel (advisor) ; Bencko, Vladimír (referee) ; Černá, Marie (referee)
DNA damage response is a complex system responsible for protection of a cell against internal and external DNA damaging agents and in maintaining genome integrity. Many of genes participating in DNA damage response pathways are polymorphic. Genetic polymorphisms in coding and regulatory regions may have impact on the function of proteins encoded by the genes. Phenotypic effect of single nucleotide polymorphisms (SNPs) is subject of investigation in connection with the ability of a cell to manage genotoxic stress and subsequently, in relation to cancer susceptibility. The aim of this thesis was to evaluate the association between SNPs in DNA repair genes (hOGG1, XRCC1, XPC) and cell cycle genes (TP53, p21CDKN1A , BCL2 and BAX) and their mRNA expression in peripheral blood lymphocytes from individuals occupationally exposed to styrene and control individuals. The aim was extended to analyses of relationships between mRNA expression levels of the above-mentioned genes and markers of exposure to styrene (concentration of styrene in blood and in air), markers of DNA damage (single strand breaks - SSBs, and endonuclease III specific sites - Endo III sites) and the base excision repair (BER) capacity, by means of γ-irradiation specific DNA repair rates and oxidative repair. Study on the group of healthy...
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Kinázová signalizace v meióze I savčích oocytů
Brzáková, Adéla ; Šolc, Petr (advisor) ; Dráber, Pavel (referee)
PLK1 belongs to the extended family of serine/threonine kinases controlling the cell cycle. It is well known for its role in the control of mitosis and contributes also to the regulation of meiotic division. On a basis of Live Cell Imaging (LCI) experiments we can describe the phenotype of the oocytes with PLK1 inhibited by small molecular inhibitor BI2536. PLK1 inhibition leads to delayed nuclear envelope breakdown (NEBD) and chromatin condensation (CC) and also causes desynchronization of NEBD and CC; in contrast to control oocytes, PLK1 inhibited oocytes break down their nuclear envelope with chromatin almost fully condensed. Also duration of these two early nuclear events is prolonged in oocytes with inhibited PLK1. In contrast to somatic cells, PLK1 inhibition in mouse oocytes does not prevent assembly of spindle with two distinct poles but affects the final spindle volume. Similar to somatic cells, mouse oocytes with PLK1 inhibited from the beginning of the meiotic maturation stay arrested in metaphase I but in the case of mouse oocytes, this block is not dependent on Spindle Assembly Checkpoint (SAC) persisting activity. When mouse oocytes are synchronized on metaphase I/anaphase I transition by proteasome inhibition and then PLK1 kinase activity is inhibited, about 2/3 of the oocytes stay arrested...
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Modelling of Cell Colony Dynamics
Bělehrádek, Stanislav ; Škutková, Helena (referee) ; Sedlář, Karel (advisor)
The content of the thesis is a description of intracellular processes responsible for cell cycle regulation and reactions of cells to external and internal stimuli. Thoroughly described are important signaling pathways with appropriate methods, which can be used to simulate them in silico. From these cellular processes, a cell cycle model is created and implemented in a tool programmed in C ++ with OpenGL used for visualization. The model is then tested for various cell processes including HeLa cells growth. Finally, the results are compared with the behavior of living cells.
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Dynamics of selected DNA damage response proteins
Benada, Jan ; Hodný, Zdeněk (advisor) ; Kuthan, Martin (referee)
DNA damage response (DDR) represents a vital signaling network that protects genome integrity and prevents development of cancer. Therefore the study of DDR is of a crucial clinical importance and DDR proteins are promising therapeu- tic targets. Although the great advances have been made mapping out interac- tions between individual DDR proteins, better understanding of complex behav- ior of this network is still needed. One approach, which might help us in this task, is to describe the dynamics of key proteins under different conditions. The first objective of this study was to investigate whether the temporal dynamics of selected DDR proteins differ upon different genotoxic insults, particularly upon γ- irradiation and UV-C irradiation. We showed that under certain insult some DDR proteins exhibit a monotone continuous activation pulse, while the activation of others triggers a series of pulses. We observed a previously described pulsative dynamics of p53 after γ-irradiation in MCF7 cells. Interestingly, we detected a monotone increase of p53 in U2OS after γ-irradiation and similar dynamics upon UV-C irradiation. We suggest that p53 dynamics depends on the presence or ab- sence of effective negative feedback loops between the upstream p53-activating kinases and Wip1 phosphatase. In the second...
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