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Molecular mechanism of animal cells adaptation on hyperosmotic induced stress
Novotná, Jana ; Vávra, Jiří (advisor) ; Převorovský, Martin (referee)
Various types of cells animal tissues consist of need to adapt to intracellular as well as extracellular osmotic changes in order to maintain homeostasis. Hypertonicity (increased osmolarity) is one of the factors activating complex cellular reactions. In order to manage such stress, a cell needs to incorporate regulation pathways that can either regulate ion transporters through WNK kinases or activate gene expression of transporters of compatible osmolytes through the transcription factor TonEBP/NFAT5. Physiologically exposed to hypertonic conditions are cells in renal medulla, cartilage, inner ear tissues, in specialised neurons or buccal epithelium. This work includes basic and current knowledge about the adaption of cells to increased osmolarity of outer environment. Key words: Hyperosmolarity, osmotic stress, TonEBP/NFAT5, WNK kinases, NF-κB, HSP70
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Recombinant preparation of DNA binding domain of transcription factor TEAD1
Kúdelová, Veronika ; Novák, Petr (advisor) ; Dračínská, Helena (referee)
TEAD proteins belong to a significant family of transcription factors that contribute to the regulation of organism growth and cell differentiation during its development by activating the expression of a wide variety of genes. This family shares two highly conserved sites, the TEA DNA binding domain, after which the proteins have been named, and the domain by which transcription factors bind other coactivators. Because TEAD proteins are not able to activate transcription themselves, they interact with a number of coactivators. These coactivators allow the transcription of the gene of interest to be regulated. Failure of TEAD protein activity regulation can lead to cancer. Therefore, TEAD family proteins nowadays play an important role in the development of new anticancer drugs. One way of inhibiting these proteins is to block the active site in their DNA binding domain, thus, to block their binding to DNA. This bachelor thesis deals with recombinant expression of said DNA binding domain of transcription factor TEAD1, which is extended by amino acids in unstructured regions. After finding suitable conditions of protein production, we proceeded to large volume production which was followed by purification and protein identity verification. Finally, the ability of the produced protein to interact...
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Structural characterization of dsDNA and transcription factor interactions
Kadavá, Tereza ; Novák, Petr (advisor) ; Hubálek, Martin (referee)
Transcription factors from TEAD protein family play an important role in various eukaryotic regulatory processes. They are partly responsible for cell differentiation and apoptosis due to their involvement in the Hippo signalling pathway. Scientific interest in this protein family is mainly because they are associated with carcinogenesis. DNA-binding domain of TEAD transcription factors interacts with M-CAT element of double-stranded DNA, which is present also in enhancer and exon of C-MYC gene. Interaction between TEAD1 DNA-binding domain and C-MYC dsDNA was investigated in this bachelor's thesis mainly by ion mobility in connection with mass spectrometry using native nESI ionization. For the further analysis of TEAD1-DBD and its complexes with dsDNA, the protein was firstly recombinantly expressed by the optimized protocol in our laboratory. Investigation of TEAD1-DBD and its complexes was done using two IM-MS activations. Firstly, the collision-induced unfolding was performed and afterwards perturbation of the sample by preheating nESI emitter was done. The first activation led to the collision- induced unfolding of some charge states of TEAD1-DBD protein and both protein-DNA complexes. The second activation performed by heating of nESI emitters led to temperature melting of both dsDNA and...
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Study of the mammalian oncogenic transcription factors in the yeast model
Novák, Josef ; Zámostná, Blanka (advisor) ; Šťovíček, Vratislav (referee)
Yeast serves as a useful tool for studying cellular processes and therefore a large amount of techniques and protocols has been developed. There are special methods for studying the transcriptional factors in yeast, such as modified yeast two-hybrid screens, yeast one-hybrid screens and systems studying ability of transcription factors to transactivate a reporter gene. Oncogenes from AP-1 complex, Myc and Myb protein families are described in this work. Using a yeast model the structural-functional properties of proteins can be easily studied and in some cases even their ability of oncogenic transformation can be predicted (FASAY or ability of c-Myc to transactivate a reporter gene). However, results from yeast models must be confirmed in mammalian cells. 1
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Cis and trans elements in plant response to drought (with a particular focus on photosynthetic genes)
Panchártek, Daniel ; Holá, Dana (advisor) ; Feketová, Zuzana (referee)
Abiotic stresses, such as drought, high salinity and cold can strongly affect plant development and growth. Cis and trans elements are one of the options how plants regulate their own metabolism in those cases. That's the way how the expression of many target genes is induced. The products of these genes function not only in stress tolerance but also in general stress response. Many transcription factors and regulatory proteins (trans elements) are involved in these adaptions; those often interact with specific sequences in gene promoters (cis elements). Recenly, a progress has been made in analyzing the signal paths and complex cascades of gene expression regulation, although a little is still known about this regulation during drought conditions. Some of these target genes code products participating in photosyntesis and the regulation of their expression can significantly affect this process. This essay briefly describes main cis and trans elements of plant response to drought (with a particular focus on the regulation of photosyntetic genes). Keywords: drought, gene expression, cis-element, transcription factor, abcisic acid, photosyntesis, promoter.
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Utilization of protein radical foootprinting for stuctural biology
Polák, Marek ; Novák, Petr (advisor) ; Junková, Petra (referee)
(In English) The reaction of highly reactive oxygen radicals with protein solvent-accessible residues can be utilized to map protein landscape. Fast photochemical oxidation of proteins (FPOP) is an MS- based technique, which utilizes highly reactive radical species to oxidize proteins and map protein surface or its interactions with their interaction partners. In this work, FPOP was employed to study protein-DNA interactions. First, a full-length of FOXO4-DBD was successfully expressed and purified. The ability of the protein to bind its DNA-response element was verified by electrophoretic and MS-based techniques, respectively. Optimal experimental conditions were achieved to oxidize the protein itself and in the presence of DNA, respectively. Oxidized samples were analyzed by bottom-up and top-down approach. In the bottom-up experiment, modification of individual residues was precisely located and quantified. Different extend of modification was observed for protein alone and in complex with DNA. To avoid experimental artifacts analyzing multiply oxidized protein, standard bottom up approach was replaced by a progressive top-down technology. Only a singly oxidized protein ion was isolated, and further fragmented by collision-induced dissociation (CID) and electron-capture dissociation (ECD),...
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Structure and interactions of selected forkhead transcription factors
Kohoutová, Klára ; Obšil, Tomáš (advisor) ; Hrabal, Richard (referee)
This diploma thesis is a part of a project aiming to develop and study specific inhibitors of FOXO3 transcriptional activity. FOXO3 belongs together with FOXO1, FOXO4 and FOXO6 to FOXO subfamily of forkhead family transcription factors. FOXO transcription factors are evolutionary conserved proteins playing important roles in numerous cellular processes, such as apoptosis, cell cycle regulation and metabolism. Due to their ability to induce apoptosis and to block the cell cycle they are considered tumor suppresors. However, it has been shown that increased activity of FOXO proteins is connected with many kinds of cancer. In such cases FOXO proteins function to maintain cell homeostasis. They promote tumor resistance against chemotherapy as well as they speed up its growth. The aim of this project is to develop specific inhibitors able to bind to FOXO3 DNA-binding domain (DBD, residues 156-269) and to block its interaction with target DNA. Development of specific inhibitors of FOXO3 transcriptional programme requires knowledge of solution structure of all FOXO DBDs and detailed insight into their interaction with target DNA. So far crystal structures of complexes of FOXO1, FOXO3 and FOXO4 with target DNA and solution NMR structures of apo DBDs of FOXO3 and FOXO4 have been solved. One of the goals of...
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Regulation of transcription by proteins of the Early growth response and Myb families
Čermák, Vladimír ; Dvořák, Michal (advisor) ; Vomastek, Tomáš (referee) ; Elleder, Daniel (referee)
The regulation of transcription of tens of thousands of genes in a vertebrate organism is an enormously complex phenomenon which entails the participation of thousands of various regulatory proteins. The largest functional category of these regulators is accounted for by sequence-specific DNA-binding proteins known as transcription factors. Proteins of the EGR and Myb families of transcription factors are long-studied regulators of a variety of physiological processes including cellular proliferation and differentiation. The structural and physical aspects of their function have been well characterized. Their cell-type specific participation in complex gene-regulatory networks, on the other hand, is still incompletely understood and represents a major challenge in the respective research areas. Preliminary analysis of gene expression data from metastasizing PR9692 and non- metastasizing PR9692-E9 chicken sarcoma cell lines revealed that the transcription factor EGR1 is expressed at a higher level in metastasizing cells and can thus take part in the regulatory processes that underlie the differences between the two cell lines. Further investigation demonstrated that the introduction of exogenous EGR1 into PR9692-E9 cells restored their metastatic potential to a level indistinguishable from PR9692...
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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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Regulation of transcription in mycobacteria.
Páleníková, Petra ; Krásný, Libor (advisor) ; Mrvová, Silvia (referee)
The bacterial cell has to be able to cope with environmental changes. Adaptation to these changes is achieved by changes in gene expression. Gene expression is regulated mostly at the level or transcription initiation. Transcription initiation depends on the sequence of promoters and is regulated by alternative sigma factors and many transcription factors acting either as activators or repressors. This work describes various ways of transcription regulation in the bacterial genus Mycobacterium that includes deathly pathogens such as M. tuberculosis and M. leprae. The typical characteristics of this genus are poorly conserved promoters, a high number of sigma and transcription factors, the presence of two-component systems and a lot of small RNAs that have not been characterized in detail so far.
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