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Fotoprotektivní vlastností PHB granulí
Hesko, Ondrej ; Obruča, Stanislav (oponent) ; Mravec, Filip (vedoucí práce)
Cílem práce je studium a návrh fyzikálně-chemické metody na ověření fotoprotektivních vlastností PHB granulí pomocí bakterie Cupriavidus necator H16 a její mutantní kmen Cupriavidus necator PHB-4 lišíci se schopností produkce polyhydroxyalkanoátů. V teoretické části jsou zpracované základní informace ku polymerním agregátů a metodám využitých při zkoumaní fotoprotektivity. Byli optimalizovány metody jako nefelometrie a kontinuální fluorescenční mikrofotolýza (CFM) sloužíci k charakterizaci mechanizmu způsobu interakce světla s granulemi. V experimentální časti byly stanovovány rozptylové vlastnosti bakterií a získány charakteristické křivky. Dále byla porovnávána fotostabilita fluorescenční sondy specifická pro nukleové kyseliny v bakteriích. Tato metoda měla dokázat větší odolnost vůči UV světlu kmenu H16. Zadefinovala také možnou interakci nukleových kyselin a zkoumaných granulí.
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Electrochemical analysis of RNA: development of a method suitable for the characterization of products of non-enzymatic polymerization of cyclic nucleoside monophosphates under conditions modeling pr
Hesko, Ondrej ; Vacek,, Jan (oponent) ; Špaček,, Jan (vedoucí práce)
This thesis focuses on the optimazation of the electrochemical method, which characterizes products of untemplated nonenzymatic polymerization of 3',5' -cyclic guanosine monophosphate (cGMP) under conditions modeling prebiotic environment. An adsorptive transfer stripping techniques on carbon electrode and gel electrophoresis were used. The method was optimized on the model system of oligonucleotides located in solution of cGMP on carbon electrode, where DNA and RNA adsorb. This technique allows simple removing of interfering substances such as cGMP, which are not present in the original sample, but they do not adsorb on the surface of electrode or they adsorb weaker than oligonucleotides or polynucleotides. Analyses are based on the selective desorption of cGMP from the surface of the carbon electrode by the chemical and physical methods before the measurement of linear voltammetry itself. Detergents, such as SDS, Tween 20 and Triton x-100 with different concentrations and electrostatic repulsions of cGMP with different negative potentials on the carbon electrode were used for the selective desorption of cGMP. The selective desorption of cGMP was observed for all detergents and inserted negative potentials. Used methods were compared and the most effective detergent for selective desorption of cGMP was SDS. Desorption of oligonucleotides was minimalized by inserted positive potential on washed carbon electrode in 0,01% SDS in basic medium. This optimized method was used on electrochemical analysis of preliminary samples of untemplated nonenzymatic polymerization of 3',5' -cGMP and compared to the analysis of gel electrophoresis.
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Electrochemical analysis of RNA: development of a method suitable for the characterization of products of non-enzymatic polymerization of cyclic nucleoside monophosphates under conditions modeling pr
Hesko, Ondrej ; Vacek,, Jan (oponent) ; Špaček,, Jan (vedoucí práce)
This thesis focuses on the optimazation of the electrochemical method, which characterizes products of untemplated nonenzymatic polymerization of 3',5' -cyclic guanosine monophosphate (cGMP) under conditions modeling prebiotic environment. An adsorptive transfer stripping techniques on carbon electrode and gel electrophoresis were used. The method was optimized on the model system of oligonucleotides located in solution of cGMP on carbon electrode, where DNA and RNA adsorb. This technique allows simple removing of interfering substances such as cGMP, which are not present in the original sample, but they do not adsorb on the surface of electrode or they adsorb weaker than oligonucleotides or polynucleotides. Analyses are based on the selective desorption of cGMP from the surface of the carbon electrode by the chemical and physical methods before the measurement of linear voltammetry itself. Detergents, such as SDS, Tween 20 and Triton x-100 with different concentrations and electrostatic repulsions of cGMP with different negative potentials on the carbon electrode were used for the selective desorption of cGMP. The selective desorption of cGMP was observed for all detergents and inserted negative potentials. Used methods were compared and the most effective detergent for selective desorption of cGMP was SDS. Desorption of oligonucleotides was minimalized by inserted positive potential on washed carbon electrode in 0,01% SDS in basic medium. This optimized method was used on electrochemical analysis of preliminary samples of untemplated nonenzymatic polymerization of 3',5' -cGMP and compared to the analysis of gel electrophoresis.
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Fotoprotektivní vlastností PHB granulí
Hesko, Ondrej ; Obruča, Stanislav (oponent) ; Mravec, Filip (vedoucí práce)
Cílem práce je studium a návrh fyzikálně-chemické metody na ověření fotoprotektivních vlastností PHB granulí pomocí bakterie Cupriavidus necator H16 a její mutantní kmen Cupriavidus necator PHB-4 lišíci se schopností produkce polyhydroxyalkanoátů. V teoretické části jsou zpracované základní informace ku polymerním agregátů a metodám využitých při zkoumaní fotoprotektivity. Byli optimalizovány metody jako nefelometrie a kontinuální fluorescenční mikrofotolýza (CFM) sloužíci k charakterizaci mechanizmu způsobu interakce světla s granulemi. V experimentální časti byly stanovovány rozptylové vlastnosti bakterií a získány charakteristické křivky. Dále byla porovnávána fotostabilita fluorescenční sondy specifická pro nukleové kyseliny v bakteriích. Tato metoda měla dokázat větší odolnost vůči UV světlu kmenu H16. Zadefinovala také možnou interakci nukleových kyselin a zkoumaných granulí.
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