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Molecular mechanism of reactive oxygen species production by flavin dehydrogenases of mitochondrial respiratory chain.
Holzerová, Eliška ; Mráček, Tomáš (advisor) ; Kalous, Martin (referee)
The aim of this thesis is to investigate molecular mechanism of reactive oxygen species production by flavin dehydrogenases mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH) and succinate dehydrogenase (SDH). Together, they represent important source of reactive oxygen species in mammalian mitochondria, but the mechanism of electron leak is still poorly understood. Because mechanisms of reactive oxygen species production by other complexes of respiratory chain are better characterized, they can serve as case studies to get insight into mechanisms of reactive oxygen species by flavin dehydrogenases. Relevant knowledge is therefore summarized in the first part of the thesis. To study the production of reactive oxygen species by the isolated flavin dehydrogenases, we used brown adipose tissue mitochondria solubilized by digitonin as a model. Enzyme activity measurements, hydrogen peroxide production studies by Amplex UltraRed fluorescence and luminol luminescence revealed flavin as the most likely source of electron leak in SDH under in vivo conditions, while we propose coenzyme Q binding site as the site of reactive oxygen species production in the case of mGPDH. Distinct mechanism of this production by the two dehydrogenases is also apparent from induction of reactive oxygen species...
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Application of Mass Spectrometry for Analysis of Biologically Active and Clinically Significant Compounds.
Štícha, Martin ; Jelínek, Ivan (advisor) ; Smrček, Stanislav (referee) ; Tůma, Petr (referee)
- 8 - ABSTRACT (EN) The thesis is submitted as a commented set of reviewed publications documenting and depicting the possibilities of mass spectrometry in the field of chemical, biological and pharmaceutical research; namely for the purposes of structure elucidation of selected organometallic complexes, analyses of drugs and their metabolites, monitoring of important biological markers. In course of experimental work, the following objectives were studied and solved: Proposal and realization of micro-scale preparation of selected rhenium complexes with aromatic ligands, utilizing tetrabutyammonium tetrachlorooxorhenate as a starting material; preparation and structure characterization of oxorhenium(V) complexes with 1,2-dihydroxybenzene, 1,2,3-trihydroxybenzene, and 2,3- dihydroxynaphtalene as ligands by means of ESI/MS, APPI/MS and LDI-MS; ESI/MS and UV/Vis study of kinetic behavior of complexes arising from the reaction of tetrabutylamonnium tetrachlooxorhenate with pyrogallol and catechol as ligands. Special aim was devoted to the study of subsequent chemical transformation of primarily formed Re(V) complexes; structure characterization of selected ferrocene complexes with copper, gold and silver by means of ESI/MS. Proposal of methodology of structure characterization and quantification of the...
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The role of mitochondrial complex II in cancer cell biology
Kraus, Michal ; Neužil, Jiří (advisor) ; Kašpárek, Petr (referee)
Mitochondria are essential organelles for most eukaryotic cells, containing intricate networks of numerous proteins. These include, among others, complexes I-IV of the electron transport chain. Being at the crossroads of the tricarboxylic acid cycle and the respiratory chain, mitochondrial complex II plays a key role in cellular metabolism. The protein complex, also known as succinate dehydrogenase, is capable of not only succinate oxidation and electron transfer but also contributes to the production of reactive oxygen species. Mitochondrial complex II consists of four subunits, SDHA-D, and four dedicated protein assembly factors SDHAF1-4 that participate in complex II biogenesis. Mutations and epigenetic modulations of genes coding for succinate dehydrogenase subunits or assembly factors are associated with pathological conditions such as neurodegenerative diseases, or may result in tumor formation. However, inborn complex-II-linked mitochondrial pathologies are rather understudied, compared to diseases with causative errors of other mitochondrial complexes, presumably due to the fact that none of complex II subunits is encoded in the mitochondrial genome. Recent studies have shown that impairment of mitochondrial complex II function or assembly leads to accumulation of alternative assembly forms...
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Succinate dehydrogenase as a hypoxia sensor in pulmonary circulation
Tichý, Václav ; Hampl, Václav (advisor) ; Žurmanová, Jitka (referee)
Hypoxic pulmonary vasoconstriction (HPV) is a local physiological mechanism in lungs that optimalises blood oxygenation during alveolar hypoxia. Arterioles in the affected region increase flow resistance which redirects blood to better ventilated parts of the lung. During global hypoxia - e.g. in high altitude or in chronic pulmonary illnessess - this mechanism doesn't work, as the blood cannot be redirected elsewhere. The pressure in pulmonary artery rises which leads to right heart hypertrophy and ultimately to cor pulmonale. This mechanism has been studied for decades, but specific signalling pathways still lack full description and therapeutical solutions are not available. This thesis offers description of selected properties of pulmonary circulation and patophysiological context of pulmonary hypertension, introduces the reader to HPV localization and signalization, and discusses its most important steps from decreased oxygen availability to vessel constriction. The practical part of this work explores Succinate dehydrogenase (SDH) - complex coupling Kreb's cycle to electron transport chain - as a primary detection site of hypoxia in pulmonary artery smooth muscle cells. We decided to test this hypothesis in isolated rat lungs by measuring if malonate (SDH inhibitor) causes vasoconstriction as...
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Application of Mass Spectrometry for Analysis of Biologically Active and Clinically Significant Compounds.
Štícha, Martin ; Jelínek, Ivan (advisor) ; Smrček, Stanislav (referee) ; Tůma, Petr (referee)
- 8 - ABSTRACT (EN) The thesis is submitted as a commented set of reviewed publications documenting and depicting the possibilities of mass spectrometry in the field of chemical, biological and pharmaceutical research; namely for the purposes of structure elucidation of selected organometallic complexes, analyses of drugs and their metabolites, monitoring of important biological markers. In course of experimental work, the following objectives were studied and solved: Proposal and realization of micro-scale preparation of selected rhenium complexes with aromatic ligands, utilizing tetrabutyammonium tetrachlorooxorhenate as a starting material; preparation and structure characterization of oxorhenium(V) complexes with 1,2-dihydroxybenzene, 1,2,3-trihydroxybenzene, and 2,3- dihydroxynaphtalene as ligands by means of ESI/MS, APPI/MS and LDI-MS; ESI/MS and UV/Vis study of kinetic behavior of complexes arising from the reaction of tetrabutylamonnium tetrachlooxorhenate with pyrogallol and catechol as ligands. Special aim was devoted to the study of subsequent chemical transformation of primarily formed Re(V) complexes; structure characterization of selected ferrocene complexes with copper, gold and silver by means of ESI/MS. Proposal of methodology of structure characterization and quantification of the...
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Molecular mechanism of reactive oxygen species production by flavin dehydrogenases of mitochondrial respiratory chain.
Holzerová, Eliška ; Mráček, Tomáš (advisor) ; Kalous, Martin (referee)
The aim of this thesis is to investigate molecular mechanism of reactive oxygen species production by flavin dehydrogenases mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH) and succinate dehydrogenase (SDH). Together, they represent important source of reactive oxygen species in mammalian mitochondria, but the mechanism of electron leak is still poorly understood. Because mechanisms of reactive oxygen species production by other complexes of respiratory chain are better characterized, they can serve as case studies to get insight into mechanisms of reactive oxygen species by flavin dehydrogenases. Relevant knowledge is therefore summarized in the first part of the thesis. To study the production of reactive oxygen species by the isolated flavin dehydrogenases, we used brown adipose tissue mitochondria solubilized by digitonin as a model. Enzyme activity measurements, hydrogen peroxide production studies by Amplex UltraRed fluorescence and luminol luminescence revealed flavin as the most likely source of electron leak in SDH under in vivo conditions, while we propose coenzyme Q binding site as the site of reactive oxygen species production in the case of mGPDH. Distinct mechanism of this production by the two dehydrogenases is also apparent from induction of reactive oxygen species...
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