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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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From iron metabolism in parasitic protists to drug discovery
Arbon, Dominik ; Šuťák, Róbert (advisor) ; De Koning, Harry P. (referee) ; Petrák, Jiří (referee)
Iron is essential for the correct function of various biochemical processes. Most living organisms developed elaborate strategies for homeostasis of this metal, including specific approaches for its acquisition and trafficking, to incorporate it into different pathways in various cellular compartments. Across the domain of parasitic protists, iron plays a crucial role in the interaction between pathogen and its host, where the struggle for scavenging available iron is a basis for nutritional immunity. This work summarizes the current knowledge about iron acquisition and trafficking in a spectrum of facultative and obligative unicellular parasites, with experimental discoveries of the iron acquisition strategies and response to iron-deficient conditions in parasitic protists Naegleria fowleri and Acanthamoeba castellanii. Based on this, the validity of exploiting iron chelation therapy is theoretically and experimentally assessed. Further work focuses on the utilization of mitochondrial targeting as a method of improving the potential of therapeutic compounds, including iron chelators, and describing their action in vitro and in vivo against a range of parasites.
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Oxidative Post-translational Modifications and Their Importance
Šimková, Anna ; Míšek, Jiří (advisor) ; Petrák, Jiří (referee)
Aerobic life brings the inevitable exposure of living systems to reactive oxygen, nitrogen and chlorine species. A wide variety of oxidants can alter the structure of biomolecules such as proteins, lipids and DNA. Oxidative post-translational midifications play an important role in a number of cellular processes, for example, they are involved in redox signaling, gene transcription and metabolism. The increase of oxidants in cells leads to oxidative stress, which is associated with the development of neurodegenerative, cardiovascular and autoimmune diseases in humans. Considering reactive species as specific molecular agents can shed light on the complexity of the connection between redox processes and develop the emerging field of redox medicine.
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Proteomic analysis of selected oncohematological diseases
Pimková, Kristýna ; Petrák, Jiří (referee)
Oxidative stress is an important factor in carcinogenesis of oncohematological diseases. However its role in the pathogenesis of myelodysplastic syndromes (MDS) remains unclear. In this study, we have determined the oxidative status and evaluated proteomic changes in plasma of MDS patients as a consequence of oxidative dysbalance (oxidative modifications, protein-protein interaction and complex forming). We measured the levels of total cysteine, homocysteine, cysteinyglycine, glutathione, nitrites and nitrates in the plasma from 61 MDS patients and 23 healthy donors using high performance liquid chromatography. Glutathione and nitrites levels reduced significantly while other aminothiols levels increased significantly in plasma of MDS patients. This association with oxidative stress did not correlate with iron overload. We also found enhanced levels of asymmetric dimethylarginine in serums of middle aged patients with MDS that correlate to posttranslational modifications of proteins arginyl residues. Furthermore, carbonylated proteins level was significantly elevated in MDS patients compared to healthy donors. Using mass spectrometry, 5 S-nitrosylated blood platelets proteins were identified in plasma and blood platelets of MDS patients and set of 16 plasma proteins with high probability of...
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Analysis of bone marrow plasma in order to identify new biomarkers in childhood acute lymphoblastic leukemia
Kováč, Martin ; Hrušák, Ondřej (advisor) ; Schwarz, Jiří (referee) ; Petrák, Jiří (referee)
Leukemia, a malignant disorder of lymphoid or myeloid progenitor cells, is the most frequent malignant disease of childhood. The most common subtype is acute lymphoblastic leukemia (ALL). In acute leukemia, several clinical symptoms may be caused by soluble proteins secreted by AL cells into the bone marrow (BM) microenvironment. We aimed at identifying proteins in BM plasma of children with ALL, which may be responsible for ALL aggressiveness or for microenvironment-mediated survival of ALL cells. LBMp (leukemic bone marrow plasma) at the diagnosis of ALL was compared to CBMp (control bone marrow plasma) and CPBp (control peripheral blood plasma) using cytokine antibody array and two-dimensional electrophoresis (2-D PAGE). Cytokine antibody array for 79 proteins identified 23 proteins expressed differentially with a statistical significance of p<0.05; of those, 2 proteins (TIMP-1 and LIF) withstood Bonferroni correction for multiple comparisons (p<0.00064). On the other hand little difference was observed between CBMp and CPBp. Using 2-D PAGE, we were able to detect 397 protein spots per gel, among which 16 showed statistically significant differences in protein levels, when comparing LBMp and CBMp by MALDI-TOF analysis. The highest difference has been found for haptoglobin in LBMp and retinol binding...
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Proteomic analysis of selected oncohematological diseases
Pimková, Kristýna ; Dyr, Jan (advisor) ; Kodíček, Milan (referee) ; Petrák, Jiří (referee)
Oxidative stress is an important factor in carcinogenesis of oncohematological diseases. However its role in the pathogenesis of myelodysplastic syndromes (MDS) remains unclear. In this study, we have determined the oxidative status and evaluated proteomic changes in plasma of MDS patients as a consequence of oxidative dysbalance (oxidative modifications, protein-protein interaction and complex forming). We measured the levels of total cysteine, homocysteine, cysteinyglycine, glutathione, nitrites and nitrates in the plasma from 61 MDS patients and 23 healthy donors using high performance liquid chromatography. Glutathione and nitrites levels reduced significantly while other aminothiols levels increased significantly in plasma of MDS patients. This association with oxidative stress did not correlate with iron overload. We also found enhanced levels of asymmetric dimethylarginine in serums of middle aged patients with MDS that correlate to posttranslational modifications of proteins arginyl residues. Furthermore, carbonylated proteins level was significantly elevated in MDS patients compared to healthy donors. Using mass spectrometry, 5 S-nitrosylated blood platelets proteins were identified in plasma and blood platelets of MDS patients and set of 16 plasma proteins with high probability of...
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Identification of novel substrates of PKN3 kinase and characterization of the role of phosphorylation in the regulation of Rho GAP activity
Dibus, Michal ; Rösel, Daniel (advisor) ; Vomastek, Tomáš (referee) ; Petrák, Jiří (referee)
Protein phosphorylation represents one of the most important posttranslational modifications in signal transduction and plays a crucial role in regulation of most of the cellular processes including cell cycle, communication with extracellular environment, cell migration or apoptosis. Phosphorylation is mediated by protein kinases, deregulation of which often negatively affects development and overall homeostasis and leads to development of several diseases, including cancer. In the first part of this work we focused on identification of new substrates of PKN3 kinase, which is a known player in regulation of cytoskeletal organization and pro-malignant tumor growth. Using an analog-sensitive mutant of PKN3 we performed a phosphoproteomic screen and identified 281 proteins that could potentially be phosphorylated by PKN3. Among these, we selected ARHGAP18, a protein from Rho GAP family, for further study. We confirmed PKN3 is able to phosphorylate ARHGAP18 on Thr154, Ser156 and Thr158 and that the two proteins are able to interact with one another in an ARHGAP18 isoform-specific manner. We further showed that substitution of the three candidate sites for phosphomimicking aspartate led to the activation of ARHGAP18 GAP domain which resulted in decreased levels of active RhoA, suggesting the existence...
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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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Molecular mechanisms of Diamond-Blackfan anemia
Handrková, Helena ; Petrák, Jiří (advisor) ; Šebela, Marek (referee) ; Trka, Jan (referee)
Diamond-Blackfan anemia (DBA) is a rare congenital syndrome that presents with ane- mia and selective deficiency of erythroid precursors, while other blood lineages are usu- ally unaffected. Approximately half of the patients display additional somatic anoma- lies and growth retardation. The therapy is mostly symptomatic and is dominated by corticosteroids, other modalities include regular blood transfusions or hematopoietic stem cell transplantation. At the beginning of this work, only two DBA causal genes were known, RPS19 and RPS24, being mutated in approximately 1/4 of all DBA patients. The goals of this work were to study the consequences of the known DBA causal mutations on cellular level and to find novel DBA causal genes. To date, over a half of DBA patients have been reported to carry a mutation in one of nine known DBA causal genes, including RPS17, RPL11 and RPL5, that are reported in this dissertation. All confirmed DBA causal genes encode for ribosomal proteins (RPs) that were essential for ribosome assembly. We further hypothesized a non- ribosomal protein participating in this process might be involved in DBA pathogenesis, too. In one DBA patient, we identified a rare sequence variant in one such candidate, a protein arginine methyltransferase 3 (PRMT3). We reported that the patient PRMT3...
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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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