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Optimization of a method for the determination of inorganic ions using capillary electrophoresis
Špačková, Simona ; Bezděková, Jaroslava (referee) ; Vaculovičová, Markéta (advisor)
Inorganic ions are one of the most important group chemicals. Their importance is crucial for all living organisms on the planet and monitoring of their content is essential for this purpose in clinical or environmental analysis but also in the food industry, agriculture or many other industries. Many analytical methods for ion detection have been designed and used, depending on the area of their use. Recently, numerous methods have been adapted to areas where not primarily intended. One such methods may be, for example, laser ablation with inductively coupled plasma mass spectrometry, which has recently begun to affect the analysis of biological samples. As a complementary method, able to detect also non-metallic ions is capillary electrophoresis. In this master thesis, the possibility of applying capillary electrophoresis with indirect photometric detection for the separation of inorganic ions in cell lysates of HeLa cells by capillary electrophoresis was verified. Main attention was paid to chloride ions. The ability to separate inorganic compounds in cellular material using salicylic acid as an absorbing anion in the base electrolyte was tested.
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Optimization of a method for the determination of inorganic ions using capillary electrophoresis
Špačková, Simona ; Bezděková, Jaroslava (referee) ; Vaculovičová, Markéta (advisor)
Inorganic ions are one of the most important group chemicals. Their importance is crucial for all living organisms on the planet and monitoring of their content is essential for this purpose in clinical or environmental analysis but also in the food industry, agriculture or many other industries. Many analytical methods for ion detection have been designed and used, depending on the area of their use. Recently, numerous methods have been adapted to areas where not primarily intended. One such methods may be, for example, laser ablation with inductively coupled plasma mass spectrometry, which has recently begun to affect the analysis of biological samples. As a complementary method, able to detect also non-metallic ions is capillary electrophoresis. In this master thesis, the possibility of applying capillary electrophoresis with indirect photometric detection for the separation of inorganic ions in cell lysates of HeLa cells by capillary electrophoresis was verified. Main attention was paid to chloride ions. The ability to separate inorganic compounds in cellular material using salicylic acid as an absorbing anion in the base electrolyte was tested.
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Recombinant expression of chloride channel from E. coliand its structure characterization
Hausner, Jiří ; Man, Petr (advisor) ; Vrbacký, Marek (referee)
Chloride channel family has been shown to play a significant role in physiological homeostasis processes. The function mechanism of these proteins has not yet been clearly understood. Their deficiency or mutation causes serious human illnesses. Our understanding of the chloride channels' transporting mechanisms can lead to better treatment of these illnesses. As mammalian chloride channels are difficult to prepare in laboratory, the experiments are usually done on homologous chloride channels from prokaryotic organisms. The structures of prokaryotic chloride channels have been solved and moreover they are produced with high yields. Most experiments currently use protein crystallography and provide a static picture of the system. This thesis is focused on the study of structural changes of an E. coli chloride channel using hydrogen/deuterium exchange. This method enables us to monitor dynamic conformation changes dependent on pH and exchanged ions. The measurements were done for the protonated (pH 4.5) and deprotonated state (pH 7.5) and/or in the presence of various anions: Cl− , SCN− , I− , F− , TAR. (tartaric anion). The obtained results justified the theories explaining the function of chloride channel as Cl− /H+ antiporter and provided new findings. Subject words biochemistry, protein...
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