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...

1 Univerzita Karlova v Praze 1. lékařská fakulta Některé aspekty patofyziologie plicní arteriální hypertenze a její výskyt v České republice Some aspects of pathophysiology of pulmonary arterial hypertension and its epidemiology in the Czech Republic MUDr. Pavel Jansa Praha 2011 2 Abstract Pulmonary arterial hypertension (PAH) is a group of diseases characterized by a progressive increase of resistance and pressure in pulmonary vascular bed. In all types of PAH the same four pathological processes are reported: vasoconstriction, inflammation, thrombosis and remodelling. The genetic background is essential for the development of PAH. We aimed to investigate the role of polymorphisms of endothelial nitric oxide synthase (eNOS) genes in PAH. We studied 142 PAH patients and 189 healthy subjects. We examined 3 polymorphisms of the eNOS gene, including the Glu298Asp polymorphism, 27-base pair (bp) variable numbers of tandem repeats (VNTR) and -786 T/C promoter gene polymorphism. Prevalence of 27-bp VNTR allele A was higher in patients with PAH compared with healthy controls. Patients with PAH associated with connective tissue diseases had higher prevalence of AA genotype compared with other PAH subgroups. The Glu298Asp polymorphism and -786 T/C polymorphism are not associated with PAH. Thrombotic arteriopathy is...

Endothelial dysfunction is a pathologic state characterized by an altered equilibrium among vasodilatory and antithrombotic mediators and vasoconstrictive and prothrombotic mediators produced by the vascular endothelium. Multiple factors induce impaired production or increased consumption nitric oxide (NO), the key mediator of vascular homeostasis, produced by the nitric oxide synthase enzymes (NOS). Endothelial dysfunction represents one of the initial steps in the development of atherosclerosis, a chronic inflammatory disease of the vascular wall. The inducible enzyme heme oxygenase 1 (HO-1) represents one of the main cellular defense mechanisms against increased oxidative stress and decreased NO bioavailability accompanying endothelial dysfunction and atherosclerosis. We studied the genetic determinants of endothelial dysfunction and atherosclerosis by evaluating the association of the G894T endothelial NOS (eNOS) polymorphism and the HO-1 (GT)n promoter polymorphism with coronary artery atherosclerosis severity and risk profile and their evolution during hypolipidaemic treatment. In addition, we searched for genetic variations in exons 25 and 26 of eNOS gene, encoding the C-terminal part of the protein, deemed crucial for proper enzyme function and the 3'- untranslated region crucial for eNOS...

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...

Background and aims: Treatment of acute fulminant liver damage arising as a result of various origins (ischemia-reperfusion injury, toxic shock, an infectious cause or cholestasis) still remains a major clinical problem. We currently do not have available clinically proven, pharmacologically effective and universal compound for the treatment of acute liver injury. The main aim of my research work was, therefore, to test the potential hepatoprotective effect of selected cytoprotective drugs and try to find out or suggest their mechanism of action, which we have examined in the systems for the intracellular gaseous signaling molecules NO and CO, where the key enzymes for their formation are NOS / HO respectively. My PhD study had two main directions: 1) Experimental study of the relationship between HO / CO and NOS / NO systems in the environment of hepatotoxic substances on isolated primary rat hepatocytes and in rat model, 2) Evaluation of ameliorative effect of selected substances in the hepatotoxicity models and to test the relationship of this effect on changes in some parameters of cytotoxicity / cytoprotection, antioxidant parameters, gene expression of mRNA for selected genes and histological changes in the state of cells / tissues / organs. Methods: We measured urea, bilirubin and liver...

Chronic kidney disease (CKD) is a life-threating disease which arises as a frequent consequence of diabetes and hypertension. Since it is going on silently, CKD often progresses to the end-stage renal disease. It is therefore necessary to combat this disease especially due to the fact that the world population is growing old. The aim of this work was to determine the contribution of selected vasoactive systems contributing to the maintenance of high blood pressure in the developmental and established phase of CKD. Two models of CKD were used: 5/6 nephrectomy in Ren-2 transgenic rats (TGR) and stenosis of renal artery (2K1C) in Wistar rats. We demonstrated that renin-angiotensin system does not play so important role in blood pressure maintenance in both CKD models. By contrast, a more important role has sympathetic nervous system. During both the developmental and established phase of CKD, vasoconstrictor systems prevail above vasodilator NO-synthase effects. In fact, the role of NO-dependent vasodilation gradually decreased in nephrectomized TGR rats, while it was unchanged in Wistar rats with 2K1C hypertension.

Background & Aims: Oxidative stress and apoptosis are proposed mechanisms of cellular injury in studies of xenobiotic hepatotoxicity. The aim of this work is to find early signal markers of drug-induced injury of the liver by focusing on select antioxidant/oxidant and apoptotic genes. As well, to address the relationship between conventional liver dysfunction markers and the measured mRNA and protein expressions in the D-galactosamine/lipopolysaccharide and tert-butylhydroperoxide hepatotoxicity models. Furthermore, potential hepatoprotective capabilities of antioxidant polyphenols quercetin and curcumin were evaluated in relation to its modulation of the oxidative stress and apoptotic parameters in the given xenobiotic hepatotoxicity models. Methods: Biochemical markers testing the hepatic function included aminotransferases (ALT, AST) and bilirubin. Measurements of TBARS and conjugated dienes were used to assess lipoperoxidation. Plasma levels of catalase and reduced glutathione were used as indicators of the oxidative status of the cell. Real time PCR was used to analyse the mRNA expressions of the inducible nitric oxide synthase (NOS-2), heme oxygenase-1 (HO-1), superoxide dismutase (SOD-1), glutathione peroxidase (Gpx-1), caspase 3 (Casp3), BH3 interacting domain death agonist (Bid) and Bcl-2...

Part I. The aldosterone synthase gene (CYP11B2) polymorphism T-344C in blood pressure and left ventricular hypertrophy. BACKGROUND: Aldosterone is a key cardovascular hormone, it significantly influences volume, pressure and electrolyte balance. Aldosterone plays an important role in development of left ventricular (LV) hypertrophy and myocardial fibrosis. The aldosterone synthase gene (CYP11B2) is an important candidate gene region in essential hypertension. DESIGN AND METHODS: We assessed the influence of the T-344C polymorphism of aldosterone synthase - the rate-limiting enzyme in aldosterone biosynthesis - on the structure of the left ventricle in young normotensive men. The population included 113 normotensive mid-European Caucasian men aged 18-40 years (mean 27 +/- 5 years). We also studied the association of -344T/C polymorphism of the CYP11B2 gene with the presence and severity of hypertension in 369 individuals, of whom 213 were hypertensive patients (139 controlled hypertensive, 74 resistant hypertensive) and 156 were healthy normotensive subjects. The genotype was assessed using polymerase chain reaction with subsequent cleavage with restriction enzyme HAEIII (restriction fragment length polymorphism method) and visualization with ethidium bromide. Plasma renin activity (PRA) and plasma...