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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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Genetic and functional characterisation of mitochondrial diseases caused by ATP synthase defects
Tauchmannová, Kateřina ; Houštěk, Josef (advisor) ; Flachs, Pavel (referee) ; Kutejová, Eva (referee)
Disorders of ATP synthase, the key enzyme of mitochondrial energy provision belong to the most severe metabolic diseases presenting mostly as early-onset mitochondrial encephalo-cardio-myopathies. Mutations in four nuclear genes can result in isolated deficiency of ATP synthase, all sharing a similar biochemical phenotype - pronounced decrease in the content of fully assembled and functional ATP synthase complex. The thesis summarises studies on two distinct causes of ATP synthase deficiency. First is TMEM70 protein, a novel ancillary factor of ATP synthase, which represents most frequent determinant of severe inborn deficiency of ATP synthase. TMEM70 is a 21 kDa protein of the inner mitochondrial membrane, facilitating the biogenesis of mitochondrial ATP synthase, possibly through TMEM70 protein region exposed to the mitochondrial matrix, but the proper regulatory mechanism remains to be elucidated. In TMEM70-lacking patient fibroblasts the low content of ATP synthase induces compensatory adaptive upregulation of mitochondrial respiratory chain complexes III and IV, interestingly by a posttranscriptional mechanisms. The second type of ATP synthase deficiency studied was mtDNA m.9205delTA mutation affecting maturation of MT-ATP8/MT-ATP6/MT-CO3 mRNA and thus biosynthesis of Atp6 (subunit a) and Cox3...
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Structural and Functional Interactions of Mitochondrial ADP-Phosphorylating Apparatus
Nůsková, Hana ; Houštěk, Josef (advisor) ; Kolarov, Jordan (referee) ; Kuda, Ondřej (referee) ; Panicucci Zíková, Alena (referee)
The complexes of the oxidative phosphorylation (OXPHOS) system in the inner mitochondrial membrane are organised into structural and functional super-assemblies, so-called supercomplexes. This type of organisation enables substrate channelling and hence improves the overall OXPHOS efficiency. ATP synthase associates into dimers and higher oligomers. Within the supercomplex of ATP synthasome, it interacts with ADP/ATP translocase (ANT), which exchanges synthesised ATP for cytosolic ADP, and inorganic phosphate carrier (PiC), which imports phosphate into the mitochondrial matrix. The existence of this supercomplex is generally accepted. Experimental evidence is however still lacking. In this thesis, structural interactions between ATP synthase, ANT and PiC were studied in detail. In addition, the interdependence of their expression was examined either under physiological conditions in rat tissues or using model cell lines with ATP synthase deficiencies of different origin. Specifically, they included mutations in the nuclear genes ATP5E and TMEM70 that code for subunit ε and the ancillary factor of ATP synthase biogenesis TMEM70, respectively, and a microdeletion at the interface of genes MT-ATP6 and MT-COX3 that impairs the mitochondrial translation of both subunit a of ATP synthase and subunit Cox3...
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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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Mitochondrial ATP synthase deficiencies of a nuclear genetic origin
Karbanová, Vendula ; Houštěk, Josef (advisor) ; Kalous, Martin (referee) ; Rossmeisl, Martin (referee)
ATP synthase represents the key enzyme of cellular energy provision and ATP synthase disorders belong to the most deleterious mitochondrial diseases affecting pediatric population. The aim of this thesis was to identify nuclear genetic defects and describe the pathogenic mechanism of altered biosynthesis of ATP synthase that leads to isolated deficiency of this enzyme manifesting as an early onset mitochondrial encephalo-cardiomyopathy. Studies in the group of 25 patients enabled identification of two new disease-causing nuclear genes responsible for ATP synthase deficiency. The first affected gene was TMEM70 that encodes an unknown mitochondrial protein. This protein was identified as a novel assembly factor of ATP synthase, first one specific for higher eukaryotes. TMEM70 protein of 21 kDa is located in mitochondrial inner membrane and it is absent in patient tissues. TMEM70 mutation was found in 23 patients and turned to be the most frequent cause of ATP synthase deficiency. Cell culture studies also revealed that enzyme defect leads to compensatory-adaptive upregulation of respiratory chain complexes III and IV due to posttranscriptional events. The second affected gene was ATP5E that encodes small structural epsilon subunit of ATP synthase. Replacement of conserved Tyr12 with Cys caused...
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Hereditary Mitochondrial Disorders Caused by Oxidative Phosphorylation Disfunction
Hanušová, Eva ; Ehler, Edvard (advisor) ; Pavlasová, Lenka (referee)
Hereditary Mitochondrial Disorders Caused by Oxidative Phosphorylation Dysfunction This bachelor thesis deals with problems of hereditary mitochondrial disorders that are caused by various dysfunctions of proteins in oxidative fosforylation known as OXPHOS complex. Following recherche provides the reader with information about the genom and the structure of mitochondria where the OXPHOS is realized and about its structure and progress. I describe basic facts about various mutations in nuclear and mitochondrial DNA that negatively affect function of OXPHOS complex and the biogenesis of mitochondria. The focus of this work is to summarize the newest data of individual diseases - their clinical manifestation, etiopatogenesis, prevalance in population and its possible treatment and prevention. Key words: hereditary, mtDNA, mitochondrial diseases, OXPHOS complex
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Význam laktátu v diagnostice mitochondriálních onemocnění u dětí
Magner, Martin ; Zeman, Jiří (advisor) ; Baxová, Alice (referee) ; Procházková, Dagmar (referee)
The lactate level assesment in various body fluids plays an important role in the diagnostics of mitochondrial disorders in children. However, the interpretation of lactate level is often difficult due to its unspecificity and variability even in particular mitochondrial disorders. Three specific aims have been stated in this PhD Thesis: 1. To analyse the role of lactate examination in the differential diagnosis between children with mitochondrial disorders and children with other diseases. 2. To study the lactate level differences in various mitochondrial syndromes. 3. To characterise the clinical and laboratory data of neonates with mitochondrial disorders and to suggest new diagnostic algorhytms. Clinical and laboratory data from patients hospitalized in the Department of Pediatrics were collected. Laboratory methods were provided in the cooperation with the Mitochondrial laboratory of the Department of Pediatrics and Institute of Inherited Metabolic Disorders. The study with lactate levels in 107 patients documented that brief seizures lasting less than 2 minutes did not increase lactate concentration in the CSF. CSF-lactate was a relialable marker in differential diagnosis in the children with mitochondrial disorders against children with epilepsy. 2. The severity of particular phenotype is more...
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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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