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Proteomic approaches in cancer biology
Lorková, Lucie ; Petrák, Jiří (advisor) ; Šulc, Miroslav (referee) ; Kovářová, Hana (referee)
Proteomics as a modern comprehensive approach to the analysis of proteomes was applied in three projects aimed at diagnosis and therapy of cancer. The aim of the first the project was to find a new diagnostic biomarker for ovarian cancer. Two different comparative proteomic approaches were used for comparative analysis of sera from patients diagnosed with ovarian cancer and from healthy age-matched women. We identified -1-antitrypsin with increased concentration in patien sera, and apolipoprotein A4 and retinol-binding protein 4 (RBP4) with significantly decreased concentration in patients. The significantly decerased concentration of RBP4 in patients is a new observation. We propose that RBP4 is either decreased in ovarian cancer patients as a result of its reduced production by ovary or it may reflect less specific systemic changes, for instance early onset of cancer cachexia. The second project was focused on gaining insight into the molecular mechanism of cytarabine resistance in mantle cell lymphoma (MCL). Proteomic and transcriptomic analyses of cytarabine-resistant cells revealed marked downregulation of deoxycytidine kinase (DCK) - a protein essential to intracellular activation of purine and pyrimidine nucleosides and their analogues including cytarabine. The cytarabine-resistant MCL...
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Political image: marketing tool or personal conviction?
Petrák, Jiří ; Perottino, Michel (advisor) ; Brunclík, Miloš (referee)
Fashion as a phenomenon was always connected with the human society. In this work I answer the role of men's fashion in politics within the cultural context in the Czech Republic. In this part I use the theory of representation from Hanna Pitkin, concretely the part about symbolic representation. It also uses theories from political marketing. My own research was done through the interviews with politicians from investigated political parties. This paper offers an answer which role plays the fashion in politics and how it is connected with the ideological background of the individual politician. Key words Men's fashion, political representation, political marketing.
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Proteomic analysis of myocardial integral membrane proteins
Oliva, Tomáš ; Petrák, Jiří (advisor) ; Pompach, Petr (referee)
Cardiovascular diseases (CVD) are the leading cause of morbidity and mortality in Europe. Over 4 million people die from CVDs annually and another 11 million people develops CVDs every year. These numbers show that there is a need for better diagnostic, prognostic and predictive biomarkers and, more importantly, a need for new and more efficient drugs. Integral membrane proteins (IMPs) are ideal candidates for new drug targets. However, a study of IMPs represents a major challenge in current proteomics. This challenge is associated with the low abundance of IMPs, their low solubility in aqueous solvents and the absence of trypsin cleavage sites in their transmembrane segments. To overcome these issues, methods that selectively target either N-glycosylated extra-membrane segments (CSC, SPEG, N-glyco-FASP) or transmembrane segments (hpTC) were developed. In this thesis we employed a combination of two N-glyco-capture methods (SPEG and N-glyco-FASP) performed on two different samples (membrane-enriched fraction and total tissue lysate) with analysis of membrane-embedded IMP segments by hpTC and with standard non-targeted "detergent+trypsin" approach to analyze rat myocardial membrane proteome. We also performed an evaluation of employed methods for preparation of membrane fraction by western blot...
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Role of PIM oncogenes in the biology and chemoresistance of aggressive lymphomas
Kořínková, Klára ; Klener, Pavel (advisor) ; Petrák, Jiří (referee)
Proviral integration site for Moloney murine leukemia virus (PIM) kinases are serine/threonine kinases and oncoproteins involved in tumorigenesis of many solid and hematopoietic malignancies including mantle cell lymphoma (MCL) and diffuse-large B-cell lymphoma (DLBCL). They were shown to promote growth and survival of cancer cells by phosphorylation of proteins involved in cell cycle regulation, transcription, translation and apoptosis. Their potential as therapeutic target is, however, complicated by existence of overlapping signaling pathways. Here we show that inhibition of PIM kinases with AZD1208, highly selective pan-PIM inhibitor, reduces growth of cell lines derived from MCL and DLBCL patients. Inhibition of PIM kinases results in decreased phosphorylation of proteins involved in apoptosis and cap-dependent translation. We further showed that concurrent inhibition of PIM-kinases with AZD1208 and signaling from B-cell receptor with ibrutinib (a Bruton's tyrosine kinase (BTK) inhibitor) or idelalisib (a phosphatidylinositol 3-kinase (PI3K) inhibitor) synergistically reduces growth of MCL cell lines. Combination of AZD1208 and ibrutinib was accompanied by decreased phosphorylation of eukaryotic translation initiation factor 4E binding protein 1 (4E-BP1) and decreased expression of...
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The Question of Men's Fashion in Political Representation within European Context
Petrák, Jiří ; Květina, Jan (advisor) ; Hesová, Zora (referee)
Fashion as a phenomenon was always connected with the human society. In this work I answer the question of men's fashion in politics within the European cultural context. By comparing two time periods (inter-war period and current from the year of 2009) I am showing how the sight on the fashion in politics was changed and why. In this part I use the theory of representation from Hanna Pitkin, concretely the part about symbolic representation. To explain the change of social values between the two periods I use the modernization theory from Ronald Inglehart and Wayne E. Baker. This paper offers an answer which role plays the fashion in politics and how it is connected with the ideological background of the individual. Key words Men's fashion, political representation, Inglehart, Pitkin, ideology.
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Proteomic analysis of soluble and transmembrane proteins in human lymphoma cells
Vít, Ondřej ; Petrák, Jiří (advisor) ; Šulc, Miroslav (referee) ; Lenčo, Juraj (referee)
In the works presented here, we studied molecular changes associated with drug resistance in human mantle cell lymphoma (MCL) cells using proteomics. Our analyses allowed us to identify causal and/or secondary changes in protein expression associated with the development of resistance to the experimental drug TRAIL and the clinically used antimetabolites cytarabine and fludarabine. Resistance of MCL cells to the recombinant proapoptotic cytokine TRAIL was associated with downregulation of key enzymes of purine metabolism. This pathway potentially represents a molecular "weakness", which could be used as a therapeutic target for selective elimination of such resistant cells. Resistance to the pyrimidine analog drug cytarabine was associated with cross-resistance to other antinucleosides. Proteomic and transcriptomic analyses showed pronounced downregulation of deoxycytidine kinase (dCK), which activates both purine and pyrimidine antinucleosides. This change explains the cross-resistance and is the causal mechanism of resistance to cytarabine. Our observations suggest that MCL patients, who do not respond to cytarabine-based therapy, should be treated with non-nucleoside drugs. MCL cells resistant to purine-derived antinucleoside fludarabine were cross-resistant to all tested antinucleosides and...
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Proteomics as a tool for understanding molecular mechanisms of human diseases
Pospíšilová, Jana ; Petrák, Jiří (advisor) ; Šulc, Miroslav (referee) ; Kovářová, Hana (referee)
Proteomics is a set of analytical methods which enable qualitative and quantitative characterization of the proteome. Expression proteomics quantitatively compares proteomes of cells, tissues, body fluids or other biological materials to find differencies in protein expression and, based on these differencies, to describe the biological processes occuring in investigated organisms. An initial material for expression proteomic studies are complex mixtures containing thousands of proteins, which are analyzed using separation (electrophoretic and chromatographic) methods, and identified, possibly quantified using mass spectrometry. The aim of this Thesis is to demonstrate the application of the tools of expression proteomics in solving diverse challenges in biomedicine. We employed various proteomic approaches and tools for studying molecular mechanisms of human diseases using pacient biological samples, or a model organism and a cell culture. We were conducting three different research projects, namely: A quest for potencial molecular targets for selective elimination of TRAIL-resistant mantle cell lymphoma cells; Investigation of molecular mechanisms of heart failure using a rat model of the disease induced by volume overload; and Searching for diagnostically usable serum biomarkers of ovarian...
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Proteomic approaches in cancer biology
Lorková, Lucie ; Petrák, Jiří (advisor) ; Šulc, Miroslav (referee) ; Kovářová, Hana (referee)
Proteomics as a modern comprehensive approach to the analysis of proteomes was applied in three projects aimed at diagnosis and therapy of cancer. The aim of the first the project was to find a new diagnostic biomarker for ovarian cancer. Two different comparative proteomic approaches were used for comparative analysis of sera from patients diagnosed with ovarian cancer and from healthy age-matched women. We identified -1-antitrypsin with increased concentration in patien sera, and apolipoprotein A4 and retinol-binding protein 4 (RBP4) with significantly decreased concentration in patients. The significantly decerased concentration of RBP4 in patients is a new observation. We propose that RBP4 is either decreased in ovarian cancer patients as a result of its reduced production by ovary or it may reflect less specific systemic changes, for instance early onset of cancer cachexia. The second project was focused on gaining insight into the molecular mechanism of cytarabine resistance in mantle cell lymphoma (MCL). Proteomic and transcriptomic analyses of cytarabine-resistant cells revealed marked downregulation of deoxycytidine kinase (DCK) - a protein essential to intracellular activation of purine and pyrimidine nucleosides and their analogues including cytarabine. The cytarabine-resistant MCL...
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The role of caudal organ in chemical communication in field mice of the genus Apodemus
Obstová, Lucie ; Stopka, Pavel (advisor) ; Petrák, Jiří (referee)
This thesis deals with the chemical communication of wood mouse of genus Apodemus with a focus on the special sebaceous gland on the ventral side of the tail - the caudal organ. Role of the caudal organ in chemical communication is unclear. To illustrate the function of the sebaceous glands, I decided to use metods of 454 pyrosequencing, proteomic analysis and qPCR. I created a database of transcriptionally active genes of caudal organ using 454 pyrosequencing. In proteomic analysis, I focused on proteins with a molecular weight 14-18kDa to verify the presence of the lipocalins, proteins involved in chemical communication. I compared the relative mRNA abundance of selected genes between individuals of the opposite sex using qPCR. In my research, I found that the caudal organ is active organ and caudal organ could participate in chemical communication. I detected a different relative mRNA abundance of genes in males and females at the mRNA level, which could play a role in differences in development of caudal organ between individuals of the opposite sex. Finally, I suggested possible ways of using the caudal organ in the chemical communication in wood mouse genus Apodemus from the obtained results. Key words: caudal organ, Apodemus, chemical communication, FABP5, Scd1
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Cytochrome P-450: Study of structure and interactions using chemical modification, photo-initiated cross-linking and mass spectrometry
Ječmen, Tomáš ; Šulc, Miroslav (advisor) ; Petrák, Jiří (referee) ; Šebela, Marek (referee)
ABSTRACT Mixed function oxygenase system participates in biosynthesis of endogenous and metabolism of exogenous substances (e.g. drugs or chemical procarcinogens) in an organism. Substrates are biotransformed by terminal oxygenases - cytochromes P450 (P450). Catalytic properties of certain P450s (e.g. studied isoform 2B4) are altered in the presence of a redox partner - cytochrome b5 (cyb5). Both cytochromes are anchored by hydrophobic domains in a lipid membrane of endoplasmic reticulum whereas their catalytic domains are exposed to cytosol. Two zero-length cross-linking approaches were employed to extend present knowledge of P450 2B4 and cyb5 protein structure and protein-protein interactions: (1) interlinking of carboxylate and primary amine groups of amino acids by water soluble 1- ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and (2) photo-initiated cross-linking by photo-labile methionine analog (pMet), which links to any amino acid after activation by UV-irradiation, either in hydrophilic or hydrophobic environment. pMet was incorporated to methionine site(s) of cyb5 during recombinant expression in E. coli, which was carried out in limit medium supplemented with amino acid analog. Optimization of experimental conditions led to ~20-30% substitution of the natural amino acid. Covalent...
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