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...

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...

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...

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...

Introduction: Diagnosing inborn metabolic diseases, as a large subgroup of rare diseases, due to their rarity and wide variety of clinical manifestations, can be demanding and often prolonged. Objective: The aim of this work is, with the regard to clinical, biochemical and genetical aspects of selected rare diseases, to contribute to their rapid detection, widen the features of the natural course of the disease and contribute to their preventability. Material: This work includes cohort studies of patiens with cardiac manifestations in mitochondrial diseases, namely a group of 48 patients with TMEM70 protein deficiency, a group of 4 patients with Barth syndrome and individual cases of rare mitochondrial cardiomyopathies, thimidine kinase 2 deficiency and alanyl tRNA synthetase 2 deficiency. By determining the frequency, severity and type of heart disease, the phenotype was expanded, and the design of a therapeutic algorithm then made a positive impact on the prognosis of these patients. The work is also focused on the role of cardiac disease in the differential diagnosis of other genetically determined rare diseases, Marfan's syndrome and especially Pompe disease, where the emphasis is on early diagnosis, mainly due to the existence of an effective therapy. Focus on a broader differential diagnosis...

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...