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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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