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Energy Metabolism in Obesity: Metabolic Flexibility and Dietary Fat
Bardová, Kristina ; Kopecký, Jan (advisor) ; Drahota, Zdeněk (referee) ; Žurmanová, Jitka (referee)
Abstract Adipose tissue is an important homeostatic tissue within the body. It not only buffers FA availability in the organism, but also releases important autocrine, paracrine or endocrine factors influencing energy metabolism. The biology of adipose tissue is closely related and underlies whole-body metabolic consequences of obesity, such as type II. diabetes. Obesity and type II. diabetes causes and maybe are caused by metabolic inflexibility, the inability of organism to adapt fuel oxidation to fuel availability. The intersection of adipose tissue biology, obesity and its metabolic consequences and theory of metabolic flexibility is discussed in this PhD. thesis. Five articles dealing with above mentioned topics are included. The general goal of this study was to compare several approaches for metabolic flexibility assessment with respect to overall energy homeostasis. The specific goals, delineated by included articles, were (i) to evaluate the influence of n-3 long chain fatty acids (n-3 LC-PUFA), rosiglitazone, and their combination, on metabolic flexibility at a whole-body and cellular level; (ii) to evaluate impact of high-fat feeding on metabolic flexibility of male and female mice; (iii) to evaluate the impact of chenodeoxycholic acid (CDCA)...
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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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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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The role of mitochondrial creatine kinase and hexokinase in cardioprotective mechanisms induced by chronic hypoxia
Wasková, Petra ; Žurmanová, Jitka (advisor) ; Drahota, Zdeněk (referee) ; Nováková, Olga (referee)
IN ENGLISH The ischemia-reperfusion (I/R) injury, which is a consequence of myocardial infarction, represents a major cause of death worldwide. One of the most effective cardioprotective interventions increasing the resistance of hearts to the I/R injury is the adaptation to a chronic hypoxia (CH). However, the molecular mechanisms of CH are still not well understood. The most important factors responsible for the I/R injury are reactive oxygen species (ROS) produced by complexes I and III within the mitochondrial electron transport chain. Potential candidates maintaining ROS at a low level are mitochondrial creatine kinase (mtCK) and two hexokinase isoforms (HK1 and HK2). These enzymes highly support the mitochondrial oxidative phosphorylation by increasing the availability of ADP for complex V of the respiratory chain. In addition, the HK binding to mitochondria inhibits binding of the pro- apoptotic protein BAX, thereby protecting cardiac cells against apoptosis. Besides the mitochondrial CK isoform, there are two cytosolic CK (CKM and CKB) present in cardiomyocytes that help to maintain energy homeostasis. Based on the known anatomical and physiological differences between the left (LV) and the right (RV) ventricles, the first study focused on the comparing ventricles in terms of the energy...
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The study of fluorescent products in the mitochondria after an attack by free radicals
Ivica, Joško ; Wilhelm, Jiří (advisor) ; Entlicher, Gustav (referee) ; Drahota, Zdeněk (referee)
Lipofuscin-like pigments are products of reactions involving free radical attack onto molecules with nucleophilic groups. They can be for med, for example, in the reactions between lipid peroxidation decomposition products, such as aldehydes, and amino-group containing compounds, e.g. phospholipids, peptides. Owing to their intrinsic fluorescent properties LFP can be easily measured. LFP are relatively stable and therefore have been successfully used as robust markers of oxidative damage. We undertook the metabolomic studies, where fluorescent LFP were first analysed spectrofluorimetrically by using tridimensional and differential fluorescence spectral arrays. After that, certain LFP were analysed by means of high performance liquid chromatography, in order to resolve the mixture of compounds into distinct fractions. For this purpose we used LFP prepared after isolated heart itochondria had been exposed in vitro to oxidative stress initiated by various triggers. LFP were also analysed during early development in rat brain, which is accompanied by transient increase in oxygen concentration, and in erythrocytes from patients with Alzheimer's disease. We developed HPLC methods for qualitative analysis of LFP of different origin. This analysis unfolded that LFP indeed consist of many chromatographically...
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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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The study of molecular and biochemical bases of cytochrome c oxidase deficiency
Veselá, Kateřina ; Zeman, Jiří (advisor) ; Farghali, Hassan (referee) ; Drahota, Zdeněk (referee)
This thesis has been worked out in The laboratory for study of mitochondrial disorders (Department of Pediatrics, 1st faculty of Medicine, Charles Univezity in Praha), which serves as the diagnostic center for patients from Czech and Slovak Republics. During the last years, more than 40 children with isolated COX deficiency were diagnosed in our lab. But molecular background except 12 patients with mutations in SURF1 gene was remaining unknown. Due to the lack of adequate treatment for these patients, the genetic counseling and the possibility of prenatal diagnostics have high importance for the families. The possible dual origin of the defect with different hereditary aspects makes the genetic counseling in the affected families complicated and prenatal diagnostics based only on biochemical analyses very problematical if even possible. This work had been arisen basically from the necessity to find the molecular background of isolated COX deficiency in our patients. In addition of simple characterization of molecular background and optimalization of methods for routine diagnostics, we were able to study the impact of several mutations in nuclear genes for COX assembly factors on the biochemical, structural and histochemical level in affected tissues.
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