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Role of membrane phospholipid remodeling, oxidative stress and PKC-δ upregulation in cardioprotection induced by chronic hypoxia
Ježková, Jana ; Kalous, Martin (referee)
Role of membrane phospholipid remodeling, oxidative stress and PKC- δ upregulation in cardioprotection induced by chronic hypoxia Introduction: Cardiovascular disease is the main cause of morbidity and mortality in industrially developed countries. Therefore, a number of experimental and clinical studies deal with the issue of how to increase tolerance to myocardial ischemia/reperfusion (I/R) injury. The present studies are concerned with mechanisms involved in the induction of endogenous cardioprotection after adaptation to intermittent altitude (IHA) hypoxia and influence of fat diets enriched with different fatty acids (FA) composition. We focused on changes in the composition of serum and heart lipids, the expression of PKC and on markers of oxidative stress and antioxidant enzyme activity in the myocardium. We also studied the role of reactive oxygen species (ROS) in the cardioprotective effect of IHA hypoxia using the antioxidant N-acetylcysteine (NAC). Methods: Adult male Wistar rats were exposed to IHA hypoxia in hyperbaric chamber, altitude 7000 m, 8 hours per day, 5 days a week, 25 exposures. Animals were fed by standard diet and diet enriched with 10% fat (saturated - SFA and MUFA mononemnasycené), 10% corn oil (n-6 polyunsaturated MK PUFA) and 10% fish oil (n-3 PUFA) in the diet experiment....
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Role of nuclear-encoded subunits of cytochrome c oxidase
Čunátová, Kristýna ; Pecina, Petr (advisor) ; Kalous, Martin (referee)
Cytochrome c oxidase (COX) is the terminal enzyme of the mitochondrial electron transport chain and its main function is transfer of electrons to the terminal acceptor, oxygen. Moreover, COX contributes to the generation of proton gradient, which is indispensable for the production of vast majority of ATP molecules in the mammalian cells. The present work summarizes available data concerning the structure and function of nuclear-encoded subunits, which are specific for the eukaryotic form of COX. Particular emphasis is put on the role of subunit Cox4 and its regulatory properties within the enzyme. The study also describes other proteins associated with the enzyme, which are able to regulate the activity of COX, but are mainly involved in formation and stabilization of the respiratory supercomplexes. Based on the summarized data, three levels model of quaternary COX structure is postulated. They correspond to subunits of the catalytic center, nuclear-encoded subunits and substoichiometrically asociated proteins, which may give rise to several forms of COX with varying composition and differentially regulated function.
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B-1 lymphocyte population and their role in the development of autoimmune diseases
Jabůrek, Filip ; Hájková, Michaela (advisor) ; Kalous, Martin (referee)
B-1 lymphocytes are specific type of B cells, development of witch occurs primarily in neonatal period of life. Later, the population is maintained through self-renewel. B-1 lymphocytes differ from classic folicular B lymphocytes in development from a distinct progenitor, expression of specific surface markers and production of polyreactive natural immunoglobulins. Since the discovery linking B-1 lymphocytes to the development of autoimmune diseases there was a shift in perspective on the B-1 lymphocytes and revaluation of the known facts. The aim of this thesis is to present a summary of current knowledge about B-1 lymphocytes, mechanisms of their effect on the development of autoimmune diseases and to outline the possible application of these findings in therapeutical practice. Key words: B-1 lymphocytes, autoimmune diseases, lupus, leukemia, SLE, B-CLL
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Cytosolic hydrogenase in Trichomonas vaginalis
Dohnálková, Alena ; Hrdý, Ivan (advisor) ; Kalous, Martin (referee)
Trichomonas vaginalis is a flagellated microaerophilic protozoan from the group Excavata that cause trichomoniasis, the most common nonviral sexually transmitted disease in the world. This thesis deals with the study of hydrogenases, enzymes catalyzing reversible conversion of protons and electrons to molecular hydrogen. In T. vaginalis, hydrogenases have been identified so far only in hydrogenosomes, modified anaerobic mitochondia that are involved in energy metabolism. We proved the presence of this enzyme also in the cytosol of T. vaginalis. Among several hydrogenase paralogues present in the genome, we selected an appropriate gene for the putative cytosolic hydrogenase (C-Hyd) and verified its cytosolic localization in the cells with overexpressed C-Hyd protein. Based on the determination of hydrogenase activities in different cell compartments and fractions obtained by affinity chromatography, we demonstrated the hydrogenase activity of C-Hyd protein, which means that C-Hyd is a functional hydrogenase. Identification of hydrogenase in T. vaginalis cytosol changes our understanding of trichomonad core metabolism and opens the door for the research of unexplored metabolic capabilities of this parasite.
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Evolution of life - the RNA world
Čvirik, Rastislav ; Markoš, Anton (advisor) ; Kalous, Martin (referee)
Origin and evolution of life represents complex and not yet resolved question in a field of natural sciences. One of the possible explanation is held by "The RNA world theory". In its general notion, RNA molecule can function both as genetic carrier and catalyst. Further it assumes, that in early development of life, genetic continuity was assured by the replication of RNA whereas proteins and DNA only appeared later. In a light of recent reasearch there is now a strong evidence supporting existence of this world. However, there are still several problems associated with prebiotic chemistry reactions and number of unresolved paradoxes. Although this teory does not directly explain life's origins, it offers us an alternative how such event may occured on primitive Earth. Powered by TCPDF (www.tcpdf.org)
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New components and functions of mitochondrial ATP synthase.
Ho, Dieu Hien ; Pecina, Petr (advisor) ; Kalous, Martin (referee)
The system of oxidative phosphorylation, or respiratory chain in mitochondria gives the eukaryotic cell total majority of the energy it receives and uses in the form of ATP. F1Fo-ATP synthase, powered by the proton-motive force is directly responsible for the ATP synthesis. Diseases connected to the ATP synthesis can have even lethal consequences. There is therefore no doubt about the need for a detailed analysis of the structure of this enzyme. What is left is to reveal the structure of the transmembrane domains, which are not involved in the synthesis itself, but they can for example work as stabilisers or assembly factors. Outside the synthesis activity the dimers of F1Fo-ATP synthase are apparently taking part in the formation of the cristae of the inner membrane of a mitochondrion. Recently, the role of the enzyme is also considered in the creation of the mitochondrial permeability transition pore.
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Alternative 2-keto acid oxidoreductases (KOR) in Trichomonas vaginalis: Artifact of histochemical staining
Zedníková, Věra ; Kalous, Martin (referee)
In 1999, Brown et al reported that Trichomonas vaginalis possesses two new enzymes of carbohydrate metabolism, alternative 2-keto acid oxidoreductases (KOR). These enzymes preferentially used indolepyruvate in a reaction that resembled that of pyruvate:ferredoxin oxidoreductase (PFO). However, the KOR did not use ferredoxin as electron acceptor and remained active in metronidazole-resistant trichomonads lacking PFO. Therefore, it was proposed that the KORs may help trichomonads to survive in the presence of metronidazole. In the study from 2012, we showed that the apparent KOR activity was caused by the non-enzymatic reduction of the indicator dye, nitroblue tetrazolium (NBT), by indolepyruvate, which is facilitated by Triton X-100 used to prepare the membrane fractions. The low level indolepyruvate-dependent activity that is present in T. vaginalis strains sensitive to metronidazole is catalyzed by PFO, which was verified using the pure enzyme. Presumed activity of alternative 2-keto acid oxidoreductases was caused by an experimental artifact and these enzymes do not exist in T. vaginalis.
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Gene expression of enzymes involved in the regulation of apoptosis in rat moycardium - effect of chronic and acute hypoxia
Blahová, Tereza ; Žurmanová, Jitka (advisor) ; Kalous, Martin (referee)
Adaptation to chronic hypoxia provides myocardial protection against ischemia - reperfusion injury (IR). Cardioprotective effect of adaptation depends on the degree and duration of hypoxic exposure and daily regime of adaptation. Certain protective regimes of adaptations to hypoxia have been reported to activate proapoptotic signaling pathways and bioactive sphingolipids were recently shown to play important role in the regulation of apoptosis in the heart. We aimed to determine the mRNA level of selected genes related to apoptotic pathways and to sphingolipid metabolism in two models of hypoxic adaptation, continous normobaric hypoxia (CNH 10% O2) with different exposures (4h, 48h, 120h, 21days) and intermitent hypobaric hypoxia (IHH 7000 m, 8h/day). Both ventricles, LV and RV, were analysed after adaptation to CNH and only LV was analysed after IHH adaptation. Our results show that both types of adaptation increased mRNA of proapoptotic genes, CNH mainly in RV and IHH in LV. Furthermore, increased expressions of proapoptotic genes were accompanied by the increase of expression of enzymes producing predominantly protective kinds of sphingolipids. The exact role of apoptosis and sphingolipid signaling molecules in endogenous myocardial protection requires further research. Key words: Apoptosis,...
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The study of the changes of hepatocyte energy metabolism: the effect of oxidative stress and triiodthyronine
Endlicher, René ; Červinková, Zuzana (advisor) ; Kalous, Martin (referee) ; Rauchová, Hana (referee)
Changes of energy metabolism of hepatocytes: The effect of oxidative stress and triiodothyronine Liver is a vital organ performing numerous essential functions. Due to its position in the blood circulation, liver is the first organ incessantly exposed to a great number of toxic substances. Respiratory chain located in mitochondria is a frequent target of toxic action of these substances. There are various mechanisms that participate in hepatocyte damage, nevertheless the most important mechanism of hepatotoxic effect is oxidative stress induced by increased production of free radicals. Impact of oxidative stress on hepatocytes is very complex and still not fully elucidated. The aim of my thesis was to investigate the effect of oxidative stress on energy metabolism of rat hepatocytes using isolated hepatocytes and isolated mitochondria. We evaluated the effect of oxidative stress on the activity of mitochondrial enzymes and function of mitochondrial permeability transition pore (MPTP), respectively. Opening of this pore induces activation of apoptotic and necrotic processes. Our results document selective action of oxidative stress on the activity of various mitochondrial enzymes. Tert-butylhydroperoxide (t-BHP) causes significant inhibition of NADH-dependent substrates, while oxidation of...
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Expression of selected defects of oxidative phosphorylation system in cultivated fibroblasts
Marková, Michaela ; Hansíková, Hana (advisor) ; Kalous, Martin (referee)
AAbbssttrraacctt:: The mammalian organism is entirely dependent on ATP production by oxidative phosphorylation system (OXPHOS) on the inner mitochondrial membrane. OXPHOS is composed of respiratory chain complexes I-IV, ATP synthase and also include two electron transporters cytochrome c and coenzyme Q. Disorders of mitochondrial energy metabolism caused by OXPHOS defects are characterized by extreme heterogeneity of clinical symptoms, variability of tissues affected and the severity of the defect at the level of individual tissues. The mitochondrial disorders are not always clearly expressed at the level of available tissue or most easily available cultured fibroblasts and/or currently available methods are not capable to detect the defects on the fibroblasts level. The aim of this master thesis was to identify by biochemical methods, especially by high sensitive polarography, OXPHOS disorders in cultured fibroblasts. Cell lines from 10 patients with isolated (SURF21, SCO1 ND1, ND5) or combined defects of OXPHOS complexes whose biochemical defect was confirmed in muscle tissue as well as 14 patients with non- mitochondrial diseases (8 patients with Huntington disease, 6 patients with disorder of sulphur amino acids metabolism) were analysed. Furthermore impact of various cultivation conditions on OXPHOS...
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