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Analysis of mitochondrial energetic metabolism in early stages of Huntington's disease
Ďurčeková, Katarína ; Hansíková, Hana (advisor) ; Kohoutová, Michaela (referee)
Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder characterized by motor, psychiatric and cognitive symptoms. The average age of onset of HD is 40 years and affected individuals have an unfavourable prognosis. Since there is still no curative treatment available, the current treatment of HD focuses only on symptom suppression. The clinical manifestation of HD is preceded by an asymptomatic period in which continuous biochemical, structural and functional changes at the cellular level are already occurring. Our previous studies have demonstrated impaired mitochondrial parameters in lymphocytes and cultured fibroblasts of patients with advanced HD. The aim of this study was to characterize the essential components of mitochondrial energy metabolism by functional methods and to find suitable mitochondrial biomarkers to monitor progression of HD. The analysed set consisted of 12 cell lines of cultured skin fibroblasts from patients with genetically proven HD, four patients in asymptomatic, four in early and four in symptomatic stages of HD. Four lines from healthy individuals served as controls. Mitochondrial respiration and glycolytic activity were analysed by real-time monitoring of oxygen consumption and pH changes, ATP production by luminescence, and metabolic turnover...
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The study of energetic metabolism in patients with mitochondrial translation defects
Hýbl, Martin ; Hansíková, Hana (advisor) ; Čermáková, Michaela (referee)
Mitochondria are semi-autonomous organelles that contain their own DNA. Human mitochondrial DNA (mtDNA) encodes a total of 37 genes: 13 subunits of oxidative phosphorylation complexes (OXPHOS), 22 transfer RNA (tRNA) molecules and 2 ribosomal RNA (rRNA) molecules. Pathogenic mutations in genes associated with mitochondrial translation are a common cause of mitochondrial disease. These mutations can be found in mtDNA or in nuclear genes encoding ribosomal proteins, initiation, elongation and termination factors, mitochondrial tRNA-modifying enzymes and aminoacyl-tRNA synthetases. Mitochondrial aminoacyl-tRNA synthetases (mt-aaRS) are enzymes that catalyse the addition of single amino acids to specific tRNAs. The aim of the bachelor thesis was an introduction to the work in the tissue culture laboratory. To prepare samples for the following experiments, skin fibroblasts from five patients with mt-aaRS disorders (AARS2, DARS2, NARS2, SARS2) and control lines were cultured in glucose and galactose media. Subsequently, the procedure for determining the equilibrium amount of selected subunits of the OXPHOS complexes was optimized and applied to the analysis of fibroblasts from five patients with mt-aaRS disorder. When the cells were cultured in glucose medium, decreased levels of some subunits of complex...
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Mitochondrial energy generating capacity in cultured skin fibroblasts
Daňhelovská, Tereza ; Tesařová, Markéta (advisor) ; Ješina, Pavel (referee)
Mitochondrial disorders, with incidence 1:5000 live births children, are one of the most common metabolic diseases. Clinically, it is heterogeneous group of disorders caused by mutations in more than 250 genes. Diagnostic of patients with suspected mitochondrial disorder relies on broad spectrum of biochemical analysis. One of them is a measurement of Mitochondrial Energy Generating Capacity (MEGC). The principle of MEGC analysis is measuring oxidations rate of 14 C - labeled substrates in 10 different incubations. These incubations contain [1-14 C]pyruvate, [U-14 C]malate or [1,4-14 C]succinate, donors and acceptors of Acetyl-CoA and inhibitors of TCA cycle. The results of MEGC analysis provide a variety of information about mitochondrial energy metabolism (MEM) of individual in particular tissue. In diagnostic of patients with suspected mitochondrial disorder is MEGC routinely determined in skeletal muscle. The aim of this study is to optimize MEGC analysis for its use in cultures skin fibroblasts. In sum, MEGC analysis was performed in 23 patients with primary deficiency of oxidative phosphorylation (OXPHOS), in 7 patients with secondary deficiency of OXPHOS and in 15 controls cell lines. The results of MEGC in cultured skin fibroblasts were then compared with results of spectrophotometric...
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