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Ultra-weak photon emission from biological samples
Vahalová, Petra ; Cifra, Michal (advisor) ; Rác, Marek (referee) ; Šachl, Radek (referee)
Oxidative processes are an inseparable part of the life of almost all living organisms. They help to maintain homeostasis but they can also stand behind various dysfunctions or diseases. Therefore, an effective method for monitoring oxidative processes in biosamples is an essential tool for medicine, agriculture, and food industry. In this thesis, an overview of available methods for monitoring oxidation in biosamples with a special focus on biological autoluminescence (BAL) is provided. This thesis uses the term BAL to encapsulate various synonyms including the commonly used term ultra-weak photon emission. BAL correlations with various physical, chemical, and biological factors (as original research and an overview from other authors) are also presented. Specifically, the relationships between spontaneous BAL of yeast Saccharomyces cerevisiae and the selected physical and chemical parameters (pH, oxygen partial pressure, and cell concentration) during cell growth were established. Additionally, the correlation of BAL intensity from yeast cells or protein bovine serum albumin (BSA) and the number of reactive oxygen species (ROS) that originated as a result of the Fenton reaction were measured. Physical enhancement of BAL from BSA by pulsed electric field was also studied and a basic reaction...
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The effect of oxygen on the triplet state lifetime of carotenoids in photosynthetic complexes
Vahalová, Petra ; Pšenčík, Jakub (advisor) ; Bína, David (referee)
The aim of this study was to find a suitable method of preparation of anaerobic conditions in or- der to determine intrinsic lifetimes of carotenoid triplet states in photosynthetic light-harvesting complexes. We have compared the following three methods: (1) blowing an inert gas (nitrogen) above the surface of a liquid sample, (2) removing oxygen by means of chemical reactions catalyzed by glucose oxidase and catalase and (3) use of sodium dithionite. Each method was first tested on meso-tetra (4-sulfonatophenyl) porphine and then applied on the main light-harvesting complex of Amphidinium carterae. The lifetimes of the triplet states of carotenoids in the light-harvesting complex were determined from kinetics measured directly by a photomultiplier tube and from transient absorption spectra measured at different delays after the excitation pulse by an intensi- fied CCD camera. The effects of the used substances on the sample stability were evaluated by measuring steady-state absorption spectra and pH. Blowing the sample with nitrogen was time- consuming and less efficient in attaining anaerobic conditions compared to the other two methods. In contrast, anaerobic conditions were obtained in a relatively short time after the application of so- dium dithionite; however, it caused acidification of the sample ralated...
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Quenching of chlorophyll triplet states by carotenoids in thylakoid membranes
Vahalová, Petra ; Pšenčík, Jakub (advisor) ; Vácha, František (referee)
Photosynthesis is one of the most important biological processes. The photosynthetic apparatus is protected against formation of reactive singlet oxygen by carotenoids. In this work, thylakoid membranes from spinach were studied, and the main aim was to explore the effect of a partial carotenoid removal on the protective function. Carotenes, which are nonpolar carotenoids, were removed from a part of the sample by hexane. About 90 % of carotenes were removed, as revealed by liquid chromatography. However, the high efficiency of chlorophyll triplet state quenching was not affected by the carotene removal. Therefore, it was concluded that the remaining carotenes are probably localized around reaction centres, where they are not accessible to hexane and where their protective function is the most important. At the same time, changes in optical spectra were observed, which were ascribed to structural modifications caused by the carotene removal from the outer parts of the complexes. The carotenoids removal probably caused detachment of outer light-harvesting complexes. This interpretation is supported by the fact that similar changes were observed in experiments with a detergent dodecyl maltoside. 1
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Quenching of chlorophyll triplet states by carotenoids in thylakoid membranes
Vahalová, Petra ; Pšenčík, Jakub (advisor) ; Vácha, František (referee)
Photosynthesis is one of the most important biological processes. The photosynthetic apparatus is protected against formation of reactive singlet oxygen by carotenoids. In this work, thylakoid membranes from spinach were studied, and the main aim was to explore the effect of a partial carotenoid removal on the protective function. Carotenes, which are nonpolar carotenoids, were removed from a part of the sample by hexane. About 90 % of carotenes were removed, as revealed by liquid chromatography. However, the high efficiency of chlorophyll triplet state quenching was not affected by the carotene removal. Therefore, it was concluded that the remaining carotenes are probably localized around reaction centres, where they are not accessible to hexane and where their protective function is the most important. At the same time, changes in optical spectra were observed, which were ascribed to structural modifications caused by the carotene removal from the outer parts of the complexes. The carotenoids removal probably caused detachment of outer light-harvesting complexes. This interpretation is supported by the fact that similar changes were observed in experiments with a detergent dodecyl maltoside. 1
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The effect of oxygen on the triplet state lifetime of carotenoids in photosynthetic complexes
Vahalová, Petra ; Pšenčík, Jakub (advisor) ; Bína, David (referee)
The aim of this study was to find a suitable method of preparation of anaerobic conditions in or- der to determine intrinsic lifetimes of carotenoid triplet states in photosynthetic light-harvesting complexes. We have compared the following three methods: (1) blowing an inert gas (nitrogen) above the surface of a liquid sample, (2) removing oxygen by means of chemical reactions catalyzed by glucose oxidase and catalase and (3) use of sodium dithionite. Each method was first tested on meso-tetra (4-sulfonatophenyl) porphine and then applied on the main light-harvesting complex of Amphidinium carterae. The lifetimes of the triplet states of carotenoids in the light-harvesting complex were determined from kinetics measured directly by a photomultiplier tube and from transient absorption spectra measured at different delays after the excitation pulse by an intensi- fied CCD camera. The effects of the used substances on the sample stability were evaluated by measuring steady-state absorption spectra and pH. Blowing the sample with nitrogen was time- consuming and less efficient in attaining anaerobic conditions compared to the other two methods. In contrast, anaerobic conditions were obtained in a relatively short time after the application of so- dium dithionite; however, it caused acidification of the sample ralated...
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