Original title: Vliv oxidačního stresu na bakteriální kultury
Translated title: Influence of oxidative pressure on bacterial cells
Authors: Dugová, Hana ; Pernicová, Iva (referee) ; Obruča, Stanislav (advisor)
Document type: Bachelor's theses
Year: 2018
Language: slo
Publisher: Vysoké učení technické v Brně. Fakulta chemická
Abstract: [slo] [eng]
Táto bakalárska práca sa zaoberá vplyvom oxidačného stresu na bakteriálny kmeň Cupriavidus necator v dvoch podobách: Cupriavidus necator H16 tvoriaci PHB granule a Cupriavidus necator PHB-4 ako mutant, ktorý nedokáže granule syntetizovať. Práca porovnáva vplyvy vyvolávajúce oxidačný stres, ktorý sa prejavuje výskytom ROS v bunke. Bunky boli ďalej analyzované na prietokovom cytometre a fluorescenčnom mikroskope. V analýze bolo potrebné pracovať s fluorescenčnými sondami rôzneho typu. Na vytvorenie oxidačného stresu boli použité viaceré koncentrácie peroxidu vodíka, ďalej bola vyskúšaná Fentonova reakcia s použitím síranu železnato–amonného a nakoniec hexahydrát chloridu kobaltnatého. Na zistenie viability bakteriálnych buniek sa používala fluorescenčná sonda propidium jodid, ako lipofilné farbivo bolo testované BODIPY. Na záver to boli fluorescenčné sondy na detekciu ROS v bunke H2DCFDA a CM–H2DCFDA, tieto sondy museli byť optimalizované pre danú techniku. Záverom práce je potvrdenie hypotézy, že schopnosť produkcie PHA granúl zvyšuje odolnosť C. necator proti oxidačnému stresu. This bachelor thesis deals with the impact of oxidative pressure on the bacterial strain Cupriavidus necator in its two forms: Cupriavidus necator H16 producing PHB granules and Cupriavidus necator PHB-4 as a mutant that is not capable of producing granules. The thesis compares different influences causing oxidative stress that is demonstrated by the occurrence of ROS in the cell. Furthermore, the cells were analysed with a flow cytometer and a fluorescence microscope. During the analysis it was necessary to use different types of fluorescence probes. The oxidative stress was created by means of hydrogen peroxide at various concentrations. Further tests focused on Fenton’s reaction including ammonium iron sulfate and cobalt chloride hexahydrate. Propidium iodide as a fluorescence probe was used to determine the viability of the bacterial cells, and BODIPY was tested as a lipophilic dye. Finally, the ROS in the cell was detected by H2DCFDA and CM–H2DCFDA, fluorescence probes that had to be optimised for the respective technique. The result of this bachelor thesis is the confirmation of the hypothesis that PHA granules production increase the resistance of Cupriavidus necator against oxidative stress.
Keywords: Cupriavidus necator; flow cytometry; oxidative stress; polyhydroxyalkanoates; reactive oxygen species

Institution: Brno University of Technology (web)
Document availability information: Fulltext is available in the Brno University of Technology Digital Library.
Original record: http://hdl.handle.net/11012/82637

Permalink: http://www.nusl.cz/ntk/nusl-378704


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Universities and colleges > Public universities > Brno University of Technology
Academic theses (ETDs) > Bachelor's theses