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Oxidative phosphorylation system in rare types of mitochondrial diseases
Zdobinský, Tomáš ; Tesařová, Markéta (advisor) ; Pecinová, Alena (referee)
In their bioenergetic metabolism mammalian cells are primarily dependent on ATP production through the oxidative phosphorylation system (OXPHOS). Defects of OXPHOS function can lead to occurrence of mitochondrial disorders with different severity and diverse symptoms. Most severely affected are usually tissues with high energy demand which are also difficult to access for biochemical and other examinations. The aim of this thesis was mainly to characterize the effects of mutations in seven different genes (OPA1, DARS2, NDUFS8, NR2F1, HTRA2, MGME1, POLG) on bioenergetic metabolism and mitochondrial network structure of skin fibroblasts from eight different patients diagnosed with mitochondrial disorders. The main method used was measurement of oxygen uptake by permeabilized cells using highly sensitive polarography. Significant changes in fibroblast respiration of four patients were found. Changes in mitochondrial network morphology were found in two of those and two other patient cell lines compared to controls using fluorescent microscopy and different cultivating conditions. Skin fibroblasts are relatively easy to obtain and offer a number of benefits for both diagnostic and study purposes. The results of this work illustrate the possibilities of their use for validation of potential causal...
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Implementation of industrial X-ray computed microtomography in developmental biology
Tesařová, Markéta ; Křivánek,, Jan (referee) ; Novotná, Marie (advisor)
Rentgenová počítačová mikrotomografie (CT) je metoda pro trojrozměrné (3D) zobrazování vnitřní struktury objektů. Laboratoř rentgenové počítačové mikro a nanotomografie na CEITEC VUT (Středoevropský technologický institut, Vysoké učení technické v Brně) se zaměřuje zejména na nedestruktivní analýzu v průmyslovém odvětví. Ukázalo se ale, že tato metoda nachází široké uplatnění i v dalších vědeckých oblastech včetně vývojové biologie. Tato práce poskytuje propojení mezi materiálovými a živými vědami. Kompletní proces vedoucí k vytvoření 3D modelu různých anatomických struktur je detailně popsán. Tento postup zahrnuje kontrastování vzorků, samotné CT měření a analýzu dat. Právě zpracování dat je mnohdy považováno za nejdůležitější část v rentgenové počítačové tomografii a proto je této oblasti věnována v práci největší část. Úpravou měřicích parametrů byl dále optimalizován CT přístroj GE v|tome|x L 240 za účelem umožnění automatické segmentace. Navíc je diskutována možnost měření na synchrotronu za účelem zvýšení prostorového rozlišení a diferenciálního kontrastu. Předpokládá se, že v blízké budoucnosti získají průmyslové CT přístroje podobné zobrazovací vlastnosti, a to jak díky nedávnému vývoji v oblasti hardwaru, tak v oblasti zpracování dat.
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Deletions in human mitochondrial DNA and causes of their formation
Zdobinský, Tomáš ; Tesařová, Markéta (advisor) ; Kazantsev, Dmitry (referee)
Mitochondria are organelles of eukaryotic cells that primarily provide energy metabolism, but also participate in metabolic processes such as biosynthesis of amino acids, heme groups, Fe-S clusters etc. Mitochondrial disorders represent heterogeneous group of diseases which can occur in both child and adult life. They affect various tissues and organs in different ways, most often manifesting themselves as disorders of nervous system, skeletal muscle, liver, kidneys or endocrine system. Mitochondrial DNA deletions contribute to pathogenesis of many of those diseases and they are a symptom of several defined syndromes. They most likely arise as a result of replication stalling resulting in a double strand break of DNA. This can be caused primarily by pathogenic changes in replication apparatus and nucleotide metabolism proteins. The aim of this work is to summarize the knowledge about mitochondria and structure and replication of their genome, but also to create a summary of the most important proteins whose mutation leads to mitochondrial diseases accompanied by deletions in mtDNA and to outline the mechanism by which they arise.
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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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System of oxidative phosphorylation and methods for its functional analysis
Daňhelovská, Tereza ; Tesařová, Markéta (advisor) ; Míčová, Petra (referee)
The system of oxidative phosphorylation (OXPHOS) is main and essential source of energy (ATP) in eukaryotic cells. It is complex process situated in the inner mitochondrial membrane in which 4 enzymes of the respiratory chain, 2 mobile carriers and the ATP synthase participate. Defect in any part of OXPHOS may lead to the mitochondrial disorders. An incidency of the mitochondrial disorders is estimated to be 1:5000 and mitochondrial disorders are the most common inherent metabolic diseases. Clinically, it is extremely heterogeneous group of disorders affecting primarily tissue with high energy demand, for example brain, hearth and muscle. With regard to broad spectrum of clinical symptoms and relatively poor genotype-phenotype correlation of mitochondrial genetic defects, it is preferred to perform complete laboratory diagnostic tests including several biochemical and molecular genetic approaches. This bachelor thesis summarizes actual information about mechanism of OXPHOS and describes three major approaches to detect its functionality. Three major approaches are: spectrophotometric measurement of individual OXPHOS enzyme activities, measuring capacity of the mitochondrial energy generating system using various radioactive labeled substrates and measuring of oxygen consumption by polarography. To...
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Genetické příčiny deficitu cytochrom c oxidázy u dětí
Vondráčková, Alžběta ; Tesařová, Markéta (advisor) ; Brdička, Radim (referee) ; Procházková, Dagmar (referee)
Mitochondria are the key source of vital ATP molecules, which are largely produced within cells by a system of oxidative phosphorylation (OXPHOS). Genetic defects affecting any of the components of the oxidative phosphorylation system or the structure and function of mitochondria lead to mitochondrial disorders, which occur at an incidence rate of 1 in 5000 live births. Cytochrome c oxidase (COX) is the terminal enzyme and electron acceptor of a respiratory chain that catalyses oxygen to produce a water molecule. In addition to complex I deficiency, isolated or combined COX deficiency is the most common respiratory chain defect in paediatric patients, and it can arise from mutations located either in mitochondrial DNA or in nuclear genes encoding the structural subunits or corresponding assembly factors of the enzyme complex. However, the molecular basis of COX deficiency remains elusive in many patients despite advances in the identification of an increasing number of mutations and genes involved in the disease. This thesis focuses on the identification of the genetic causes of mitochondrial diseases in a cohort of 60 unrelated Czech children with clinically and laboratory confirmed COX-deficiency. With the use of a high-resolution melting analysis mutation screen, four heterozygous sequence...
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Human F1Fo-ATPsynthase deficiency
Suldovská, Sabina ; Tesařová, Markéta (advisor) ; Černá, Leona (referee)
F1FO-ATPsynthase is a key enzyme in energy metabolism of the cell. Its deficit is caused usually by mutations in two structural genes MT-ATP6 and MT-ATP8 encoded by the mitochondrial DNA or in nuclear genes ATPAF2 and TMEM70 encoding the biogenesis factors and structural gene ATP5E. Deficiency of the F1FO-ATPsynthase leads to progressive and serious phenotype affecting organs with high energy demands. The first symptoms usually occurs in neonatal age and prognosis of the disease is fatal. Mutations in these genes result in both qualitative and quantitative defects of the F1FO-ATPsynthase. The study of molecular bases of mitochondrial disorders including F1FO-ATPsynthase deficiency uses large number of biochemical and molecular-genetic methods to determine a proper diagnosis which is essential for the symptomatic therapy and genetic counselling in affected families. The aim of the diploma thesis was to characterise the F1FO-ATPsynthase deficiency in isolated mitochondria from the lines of cultured cells by the determination oligomycin- sensitive ATP-hydrolytic activity of the F1FO-ATPsynthase, enzymatic activities of the respiratory chain complexes and to analyse changes in the steady-state levels of the representative subunits and whole complex of the F1FO-ATPsynthase in comparison with controls. 3...
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