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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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Proteomic analysis of soluble and transmembrane proteins in human lymphoma cells
Vít, Ondřej
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 also...
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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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Proteomic analysis of soluble and transmembrane proteins in human lymphoma cells
Vít, Ondřej
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 also...
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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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