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Developmental, pathobiochemical and molecular aspects of selected inborn errors of metabolism
Kolářová, Hana ; Honzík, Tomáš (advisor) ; Drahota, Zdeněk (referee) ; Morava Kozicz, Eva (referee)
Inborn errors of metabolism represent a heterogenous group of rare conditions, most having an incidence of less than 1 in 100,000 births. Because of their low prevalence, they are on the margin of attention of general research and even more so of large pharmaceutical companies. Study of rare diseases is the only way to design therapeutic options in order to improve quality of life of affected patients. Present Thesis particularly focuses on disturbances in mitochondrial energy metabolism. The main goals were the characterization of mitochondrial biogenesis within foetal development, as well as in childhood and adulthood. Another aim was to define clinical, biochemical and molecular aspects of mitochondrial optic neuropathies in childhood and adulthood. This work supported the earlier observations that gestational week 22 is the edge of viability, which has to be taken into account in upcoming discussions about guidelines on resuscitation of preterm neonates. Secondly, over last four years, we managed to examine and describe large cohort of patients with optic neuropathies based on a mitochondrial dysfunction. We have managed to characterize the biochemical and molecular-genetic background in more than 200 patients, and both selected cases (LHON/MELAS overlap syndrome) and cohort studies (MELAS,...
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Mitochondrial cytochrome c oxidase: cyanide inhibition and role of assembly factor Surf1 defect
Nůsková, Hana ; Drahota, Zdeněk (advisor) ; Kalous, Martin (referee)
The activity of mitochondrial cytochrome c oxidase (COX) can be affected by either exogenous or endogenous factors. The most efficient and in the environment abundant compound that inhibits COX is cyanide. The very frequent cause of COX deficiency in humans is represented by a defect in the SURF1 gene. The mechanism of cyanide inhibitory effect on COX as well as the conditions for its recovery are not yet fully explained. Three parameters of COX function, namely the transport of electrons (oxygen consumption), the transport of protons (mitochondrial membrane potential, m) and the enzyme affinity to oxygen (p50 value), were studied with regard to the inhibition by KCN and its reversal by pyruvate. The function of COX was analysed in intact isolated rat liver mitochondria, both within the respiratory chain and as a sole enzyme, using succinate or an artificial electron donor ascorbate + TMPD as a substrate. 250 M KCN completely inhibited both electron- and proton-transporting function of COX, and this inhibition was reversible as proved with washing of mitochondria. The addition of 60 mM pyruvate induced the maximal recovery of both parameters to 60 - 80 % of original values. Using KCN in the low concentration range up to 5 M, a profound, 30-fold decrease of COX affinity to oxygen was observed....
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Effect of ectopic synthesis of mitochondrial uncoupling protein 1 in white adipose tissue on whole-body metabolism in mice
Janovská, Petra ; Kopecký, Jan (advisor) ; Drahota, Zdeněk (referee) ; Kalous, Martin (referee)
The prevention and treatment of obesity is a major problem of health care systems in affluent societies. Metabolism of adipose tissue belongs to the therapeutical targets, since accumulation of adipose tissue is the basis of obesity development. Experiments using transgenic mice with ectopic expression of brown- fat uncoupling protein 1 (UCP1) in white adipose tissue (WAT), verified a concept that obesity could be ameliorated by increasing energy expenditure in WAT. The goal of the experiments of this PhD Thesis was to characterize in detail the phenotype of this unique animal model of obesity resistance. We have shown that mitochondrial uncoupling in WAT resulted in increased oxidation of fatty acids (FA), in face of decreased lipogenesis and induced mitochondrial biogenesis in this tissue. In further studies, we aimed to modulate propensity to obesity be increasing FA oxidation in WAT in response to physiological stimuli. This could be accomplished in response to the combination treatment using n-3 polyunsaturated fatty acids (n-3 PUFA) and mild calorie restriction in mice fed high-fat diet. Synergistic induction of mitochondrial oxidative capacity and lipid catabolism in epididymal WAT was associated with suppression of low-grade inflammation of WAT, which is typical for obesity. The improvement of lipid...
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The molecular and biochemical basis of primary coenzyme Q10 deficiency
Spáčilová, Jana ; Veselá, Kateřina (advisor) ; Drahota, Zdeněk (referee)
This diploma thesis focus on disorders of coenzyme Q10, which lead to decrease of its concentration in certain patient`s tissues (CoQ10 deficiency). This thesis introduces two molecular-genetics methods for CoQ10 deficiency analysis. The first one is a screening method - High Resolution Melting Analysis, which is a useful tool for screening of a large group of patiens with possible mutations in PDSS1, PDSS2, COQ2, COQ9, CABC1 and APTX genes, which encode some enzymes of CoQ10 biosynthetic pathway. The second one is direct sequencing of coding sequences of these genes including intron sequences neighbouring exons. This work also describes a model of CoQ10 deficiency applied in vitro using cultured human skin fibroblasts and HEK293 cells. In this case CoQ10 deficiency is evoked by CoQ10 biosynthesis inhibitor - 4 aminobenzoic acid (concentration 1 mmol.dm-3) . 4 days treatment by 4 aminobenzoic acid caused decrease in CoQ10 level in fibroblasts and HEK293 cells (60 - 70 % of control, resp. 41 % of control in case of HEK293 cells), although cell viability and morfology of mitochondria remained unchanged. Our results declare that there was slightly increased reactive oxygen species concentration after treatment, especially the amount of superoxide radicals; nevertheless the ultrastructure of mitochondria stayed...
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Energy metabolism of skeletal muscle
Elkalaf, Moustafa GamalEldin Mahmoud Mohamed ; Anděl, Michal (advisor) ; Drahota, Zdeněk (referee) ; Žurmanová, Jitka (referee)
Skeletal muscle is the largest tissue in the body and plays a marked role in the homeostasis of the body metabolic state. Mitochondria have been proven to contribute to the pathophysiology of various metabolic diseases, either due to defects in their bioenergetic properties or the production of reactive oxygen species. In this work murine myoblasts C2C12 were used as a model of skeletal muscle in vitro, and rat muscle was used to prepare homogenate enriched in the mitochondrial fraction. This work investigates the changes in respiratory parameters in models where mitochondrial oxidative phosphorylation is induced by changing the available consumable substrates in the culture media, such as replacing glucose by galactose, and the effect of treating the cells with high glucose concentration during the process of differentiation on mitochondrial performance. It also investigates the changes in bioenergetic profiles in samples treated with inactive derivatives of the widely used triphenylphosphonium (TPP+) salts to target mitochondria by various probes and antioxidants. The methods used in this study included evaluating mitochondrial parameters in intact and permeabilized cells by real time measurement of the oxygen consumption rate using the extracellular flux analyzer, measuring the enzymatic...
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Effect of selected nutrients on skeletal muscle mitochondrial metabolism
Tůmová, Jana ; Anděl, Michal (advisor) ; Poledne, Rudolf (referee) ; Drahota, Zdeněk (referee)
Skeletal muscle plays an important role in the maintenance of whole-body metabolic homeostasis. Metabolic alterations of skeletal muscle contribute to the pathogenesis of a wide range of human diseases, such as obesity, type 2 diabetes and hypertension. Relative excess and suboptimal composition of nutrients negatively affect skeletal muscle metabolism and a better understanding of mechanisms involved in these changes is of central importance. The aim of the work presented in this thesis was to explore cell viability and mitochondrial respiratory parameters following experimentally induced changes in the availability or composition of selected nutrients (fatty acids and glutamine). We attempted to elucidate the mechanisms responsible for the observed changes, such as mitochondrial DNA (mtDNA) damage, or nuclear receptors activation. The studies were performed in vitro on skeletal muscle cell culture models. In addition, we examined mitochondrial function and fat accumulation in skeletal muscle of vegans, i.e. subjects consuming a strict plant-based diet. Using C2C12 skeletal muscle cells we studied the effects of free fatty acids (FFA). We found that relatively low doses of saturated palmitic acid increased hydrogen peroxide production and induced mtDNA damage, mitochondrial respiratory dysfunction...
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Developmental, pathobiochemical and molecular aspects of selected inborn errors of metabolism
Kolářová, Hana ; Honzík, Tomáš (advisor) ; Drahota, Zdeněk (referee) ; Morava Kozicz, Eva (referee)
Inborn errors of metabolism represent a heterogenous group of rare conditions, most having an incidence of less than 1 in 100,000 births. Because of their low prevalence, they are on the margin of attention of general research and even more so of large pharmaceutical companies. Study of rare diseases is the only way to design therapeutic options in order to improve quality of life of affected patients. Present Thesis particularly focuses on disturbances in mitochondrial energy metabolism. The main goals were the characterization of mitochondrial biogenesis within foetal development, as well as in childhood and adulthood. Another aim was to define clinical, biochemical and molecular aspects of mitochondrial optic neuropathies in childhood and adulthood. This work supported the earlier observations that gestational week 22 is the edge of viability, which has to be taken into account in upcoming discussions about guidelines on resuscitation of preterm neonates. Secondly, over last four years, we managed to examine and describe large cohort of patients with optic neuropathies based on a mitochondrial dysfunction. We have managed to characterize the biochemical and molecular-genetic background in more than 200 patients, and both selected cases (LHON/MELAS overlap syndrome) and cohort studies (MELAS,...
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Modulation of body fat content by targeting the lipid metabolism of adipose tissue
Šponarová, Jana ; Kopecký, Jan (advisor) ; Konvalinka, Jan (referee) ; Drahota, Zdeněk (referee)
CONCLUSIONS 1. Induction of obesiý resistance in mice by transgenic expression of UCP1 in white fat can be explained by a metabolic switch ínadipocýes, due to depressíon oť celluiar energy charge, in situ activaÍion of AMPK, down- regulation ofadipogenic genes and increase in lipid oxidation. 2. AMPK cascade is involved in the fat depot-specific metabolic responses in various fat depots to starvation. Activation of the cascade occurred in epididymal but not in subcutaneous fat in mice. The activation of AMPK in adipocytes might represent an important mechanism by which body fat stores are regulated and may contributes to regional differences in the metabolic properties ofadipose tissue depots. Dietary EPA and DHA reduce development of obesity induced in mice by high fat diets, in part due to counteracting increase in tissue cellularity, particularly in epididymal íat.Low EPA/DHA ratio potentiates the anti- adipogenic efíect. Dietary EPA and DHA induce a metabolic shift in white adipose tissue by up-regulating genes for mitochondrial proteins, including their regulatory genes PGCIa and NRF-I, and increase B-oxidation while depressing lipogenesis, preferentially in the epididymal fat in the abdomen. 3. 4. J. 21
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