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Structural and functional analysis of Francisella tularensis virulence factors
Šenitková, Iva ; Hernychová, Lenka (referee)
The presented thesis is focused on the relationship between the structure and the function of Francisella tularensis subsp. holarctica strain FSC200 FTS_1067 lipoprotein. This protein with unique structure constituted by two functional domains is involved in the virulence of bacteria, nevertheless the roles of both domains have not been previously clarified yet. Although this protein is usually denoted as a factor of virulence, the previous studies have shown that its substrates are the real virulence factors and that FTS_1067 protein is necessary for their proper function. We created and after that characterized in vivo and in vitro the new mutant strain with the deletion of DSBA-like domain. Afterwards we prepared the recombinant protein with the same deletion and made several tests focused on the understanding of the protein function. From the analyses of these results and their comparison with previous studies we elaborated the first comprehensive report about the roles of both domains in the context of the whole protein. To supplement the information about the involvement of the FTS_1067 protein in the mechanism of virulence we tried to verify its potencial substrates by using bacterial two hybrid assay.
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Characterization of protein structures using chemical cross-linking and mass spectrometry.
Kukačka, Zdeněk ; Novák, Petr (advisor) ; Rozbeský, Daniel (referee) ; Hernychová, Lenka (referee)
Some proteins require presence of their specific ligand, cofactor or prosthetic group for their activity. Binding of this specific molecule can cause conformational changes which permit to perform their function. In some occasions the identification of conformational changes could be really challenging task. In this thesis we describe the novel approach for monitoring structural changes in proteins using chemical cross-linking and high resolution mass spectrometry and its application on model calmodulin system. It is demonstrated that analysis using isotope-labelled cross-linking agents enabled us to get insight into the structural rearrangement caused by presence or absence of the protein ligand. However, it is shown that the method has potential drawback due to limited enzymatic proteolysis. The novel approach that also makes it possible to quantify the changes in protein structure was used together with other methods for characterization of the neutral trehalase Nth1 in complex with Bmh1 protein (yeast isoform of protein 14-3-3). The results revealed that Bmh1 induce structural rearrangement of Nth1 molecule with changes within the EF- hand like motif which is essential for the activation process.
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Proteome analysis of secerned proteins of Francisella tularensis
Konečná, Klára ; Hernychová, Lenka (advisor) ; Krejsek, Jan (referee) ; Bílková, Zuzana (referee)
Title of dissertation thesis: Proteome analysis of secerned proteins of Francisella tularensis The facultative intracellular bacterium Francisella tularensis is the causal agent of the infectious disease called tularaemia. Despite the available wide range of new knowledge focused on bacterium Francisella, till now, mechanisms of tularaemia disease pathogenesis were not completely clarified. The contents of our work was based upon analysis and identification of culture filtrate proteins of bacterium F. tularensis of three strains (LVS, FSC00, SchuS4). Among identified proteins, there were seeked protein candidates for secretion and proteins, which could help with explanation of molecular mechanisms of disease pathogenesis caused by F. tularensis. The best protein candidate for secretion is enzyme acid phosphatase with proven important role in bacterium F. tularensis escape from phagosome. The attention was also focused on the new described mechanism of bacterial secretion, mediated by membrane vesicles. By the help of transmission electron microscopy was demonstrated, that F. tularensis of the strains LVS and FSC200 secretes membrane vesicles into extracellular milieu. Key words: Francisella tularensis, secretion systems, cultivation filtrate proteins, secreted proteins, outer membrane vesicles
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Radiosensitization of cancer cells by modulation of autophagy: Phosphoproteomic analysis
Ondrej, Martin ; Tichý, Aleš (advisor) ; Dresler, Jiří (referee) ; Hernychová, Lenka (referee)
(ENG) Lung cancer, and in particular, non-small cell lung cancer (NSCLC), belongs to the most lethal oncological diseases worldwide. Choosing the proper therapeutic approach is, therefore, inevitable for the successful treatment of lung tumors. In this regard, radiotherapy is an essential therapeutic approach. However, like any other treatment option, radiotherapy has its limitations in the form of radioresistance. Autophagy has been generally accepted to be one of the most crucial factors in the radioresistance of tumor cells. It is, therefore, necessary to search for suitable approaches that would lead to the suppression of cytoprotective autophagy and, thus, to the radiosensitization of tumor cells. In this respect, in our study, we employed methods of quantitative phosphoproteomics along with several techniques of molecular biology to examine the underlying molecular mechanisms and signaling pathways involved in the regulation and inhibition of the autophagic process. To inhibit the cytoprotective autophagy, we used a relatively new autophagy inhibitor-Lys05, the analog of a well-described autophagy inhibitor chloroquine. As a model, we chose the radioresistant NSCLC cells (H1299; p53-negative), because they represent the most problematic group of cells within non-small cell lung tumors. The...
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Analysis and identification of proteins in organ dysfunction using proteomic methods
Tůma, Zdeněk ; Matějovič, Martin (advisor) ; Lopot, František (referee) ; Hernychová, Lenka (referee)
Proteomics is the large-scale study of proteins, particularly their structures and functions. Proteomics has been utilized in medicine for investigation of disease mechanisms and biomarker discovery. Instrumental methods cover sample preparation, protein and peptide separation and mass spectrometry. At present, there is no proteomic method that can be used as universal for every sample. Analytical methods need to be adapted and optimized for certain samples. The aim of this work was to create methodic procedures and to interpret results of experimental and clinical research. The first part of the thesis includes experiments utilizing proteomics to study changes in the plasma proteome clinically relevant porcine model of sepsis-induced peritonitis. Proteomic analyzes were also starting methodological strategies in experiments aimed at kidney physiology and pathophysiology of acute kidney injury during sepsis. Renal biopsies were analyzed in order to study the time course of proteome changes caused by sepsis and surgery. The second part of the thesis contains experiments studying biocompatibility. A method for elution of proteins interacting with adsorbents used in extracorporeal liver support system and with hemodialyzer capillaries was prepared. Analysis of proteins adsorbed to polysulfone...
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Use of proteomic analysis to identify potential biomarkers for cardiovascular disease
Fučíková, Alena ; Lenčo, Juraj (advisor) ; Hernychová, Lenka (referee) ; Pudil, Radek (referee)
Hypertrophic cardiomyopathy is one of the most common inherited diseases of the cardiovascular system. Although this disease is known for a long time, a suitable diagnostic procedure uncovering its early stages in patients with negative or unknown family history is still lacking. A development of the method for targeted proteomic analysis in combination with subsequent quantification of chosen hypothetical markers of hypertrophic cardiomyopathy was the main aim of this thesis. This method is able to detect very small amounts of chosen markers in the minimum amount of complex biological material. Moreover, in combination with properly used standards, targeted proteomic analysis enables quite precise quantification of many analytes in a relatively short time. Several previously described protein markers of hypertrophic cardiomyopathy were assayed and quantified using unique proteomic technique. Concurrently, a new potential protein marker - soluble fibronectin - was described. Protein concentrations were validated using enzymoimmunoanalytical method and obtained results were compared with targeted proteomic analysis findings. In the presented thesis, a new short method was developed for detection and quantification of potential markers of hypertrophic cardiomyopathy. The main objectives of this...
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The study of structure and dynamics of protein molecules and protein complexes.
Vaňková, Pavla ; Man, Petr (advisor) ; Obšilová, Veronika (referee) ; Hernychová, Lenka (referee)
5 Abstract Inside the cellular milieu, proteins are reaching their native conformation with the assistance of molecular chaperones. These high mass dynamic complexes perform other functions that are contributing to the stability of the cellular proteome. Transport of proteins into the target compartment or defence against oxidative stress rank among these as well. Dynamic structural changes of chaperones Hsp70 and NQO1 and co-chaperone Tomm34 that are participating in above mentioned processes and their interactions with ligands are characterized here by the combination of structural mass spectrometry, biophysical and molecular biology techniques. In this thesis the ATP-dependent dimerization of human inducible isoform Hsp70 (HSPA1A) and the characterization of its evolutionarily conserved dimerization interface were described. Further, highly conserved amino acid residues crucial for allosteric movements were identified. These residues create a network of contacts stabilizing both extreme conformations of HSPA1A (low and high-affinity) during the ATPase cycle. One of the co-chaperones that can affect Hsp70 ATPase cycle is TPR co-chaperone Tomm34. This co-chaperone is participating on the transport of the nuclear encoded mitochondrial precursors into the mitochondria. Here we described regulation of this...
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Structural and functional analysis of Francisella tularensis virulence factors
Šenitková, Iva ; Hernychová, Lenka (referee)
The presented thesis is focused on the relationship between the structure and the function of Francisella tularensis subsp. holarctica strain FSC200 FTS_1067 lipoprotein. This protein with unique structure constituted by two functional domains is involved in the virulence of bacteria, nevertheless the roles of both domains have not been previously clarified yet. Although this protein is usually denoted as a factor of virulence, the previous studies have shown that its substrates are the real virulence factors and that FTS_1067 protein is necessary for their proper function. We created and after that characterized in vivo and in vitro the new mutant strain with the deletion of DSBA-like domain. Afterwards we prepared the recombinant protein with the same deletion and made several tests focused on the understanding of the protein function. From the analyses of these results and their comparison with previous studies we elaborated the first comprehensive report about the roles of both domains in the context of the whole protein. To supplement the information about the involvement of the FTS_1067 protein in the mechanism of virulence we tried to verify its potencial substrates by using bacterial two hybrid assay.
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Analysis and identification of proteins in organ dysfunction using proteomic methods
Tůma, Zdeněk ; Matějovič, Martin (advisor) ; Lopot, František (referee) ; Hernychová, Lenka (referee)
Proteomics is the large-scale study of proteins, particularly their structures and functions. Proteomics has been utilized in medicine for investigation of disease mechanisms and biomarker discovery. Instrumental methods cover sample preparation, protein and peptide separation and mass spectrometry. At present, there is no proteomic method that can be used as universal for every sample. Analytical methods need to be adapted and optimized for certain samples. The aim of this work was to create methodic procedures and to interpret results of experimental and clinical research. The first part of the thesis includes experiments utilizing proteomics to study changes in the plasma proteome clinically relevant porcine model of sepsis-induced peritonitis. Proteomic analyzes were also starting methodological strategies in experiments aimed at kidney physiology and pathophysiology of acute kidney injury during sepsis. Renal biopsies were analyzed in order to study the time course of proteome changes caused by sepsis and surgery. The second part of the thesis contains experiments studying biocompatibility. A method for elution of proteins interacting with adsorbents used in extracorporeal liver support system and with hemodialyzer capillaries was prepared. Analysis of proteins adsorbed to polysulfone...
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Proteome analysis of secerned proteins of Francisella tularensis
Konečná, Klára ; Hernychová, Lenka (advisor) ; Krejsek, Jan (referee) ; Bílková, Zuzana (referee)
Title of dissertation thesis: Proteome analysis of secerned proteins of Francisella tularensis The facultative intracellular bacterium Francisella tularensis is the causal agent of the infectious disease called tularaemia. Despite the available wide range of new knowledge focused on bacterium Francisella, till now, mechanisms of tularaemia disease pathogenesis were not completely clarified. The contents of our work was based upon analysis and identification of culture filtrate proteins of bacterium F. tularensis of three strains (LVS, FSC00, SchuS4). Among identified proteins, there were seeked protein candidates for secretion and proteins, which could help with explanation of molecular mechanisms of disease pathogenesis caused by F. tularensis. The best protein candidate for secretion is enzyme acid phosphatase with proven important role in bacterium F. tularensis escape from phagosome. The attention was also focused on the new described mechanism of bacterial secretion, mediated by membrane vesicles. By the help of transmission electron microscopy was demonstrated, that F. tularensis of the strains LVS and FSC200 secretes membrane vesicles into extracellular milieu. Key words: Francisella tularensis, secretion systems, cultivation filtrate proteins, secreted proteins, outer membrane vesicles
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