Mitochondrial ATP synthase represents the final complex of oxidative phosphorylation (OXPHOS) system located in the inner mitochondrial membrane. Its primary role is to utilize mitochondrial membrane potential (Δψm) generated by respiratory chain complexes to produce energy in the form of ATP. Mammalian ATP synthase comprises of 17 different subunits organized into membranous Fo and matrix-oriented F1 domains. Defects of complex V and their manifestation have been studied on mitochondrial, cellular, tissue and organism levels using different models, including human cell lines and cell lines derived from patient tissues. In many cases mitochondrial diseases display threshold behaviour, when genetic defect is phenotypically manifested only bellow certain threshold in particular enzyme complex activity and/or content. This work was aimed at elucidation of functional consequences of ATP synthase deficiency in HEK293 cell lines with suppressed gene expression of γ, δ or ε subunits of ATP synthase central stalk. We have analysed range of clones with respective subunits knockdown and found varying decrease in assembled ATP synthase content, which was mirrored by the decrease in individual ATP synthase subunits. The only exception was subunit Fo-c, whose levels remained unchanged or even increased. ATP...

This diploma thesis deals with identification of bacteria which are capable of producing polyhydroxyalkanoates (PHAs). Work included testing variety of genera including Pseudomonas, Lactobacillus, Bifidobacterium, thermophilic cultures and samples gathered from natural sources. Bacteria were investigated by molecular technique polymerase chain reaction – PCR. An amplification of the PHA synthase gene (phaC) was analyzed. In the first reaction phaC and 16S rRNA genes were tested at the same time. 16S rRNA gene is used as control for bacterial DNA and as an identification tool for natural source samples. This multiplex PCR used multiple primers in PCR mix. Second reaction search for amplicon specific for catalysing biosynthesis mcl-PHA (phaC1). The presence of the PHA synthase gene was verified in 11 samples which were Bifidobacterium breve CCM 7825T, Lactobacillus rhamnosus CCM 1825T, Lactobacillus zeae CCM 7069T, Lactobacillus delbrueckii subsp. bulgaricus CCM 7190T, Lactobacillus plantarum CCM 7039T, Pseudomonas gessardii, Pseudomonas fulva, Arthrobacter protophormiae, Curtobacterium flaccumfaciens, Mycobacterium neoaurum and Staphylococcus lentus.

Inhibitory factor 1 (IF1) is one of the major regulators of mitochondrial ATP synthase activity, a key enzyme of energy metabolism. Its inhibitory effects are known in conditions such as hypoxia or starvation, but the hypothesis that IF1 inhibits ATP synthase activity even under physiological conditions is still not entirely accepted. Disorders of ATP synthase regulation can be fatal to the cell and have been described, for example, in carcinogenesis and ischemia. It has also been found that silencing of the IF1 gene in pancreatic β-cells increases insulin secretion, and thus, IF1 may be important in the pathogenesis of type 2 diabetes. The goal of this work is to summarize the current knowledge about the IF1 protein and to obtain new results that will help elucidate the mechanism by which this protein regulates mitochondrial ATP synthase. Specifically, this work deals with the ratio of IF1 protein to ATP synthase in pancreatic β-cells, depending on different culture conditions. It further investigates the occurrence of post-translational modifications of the IF1 protein in pancreatic β-cells (INS- 1E model cells), which may play a role in the regulation of IF1 activity. It also deals with the cellular ATP/ADP ratio, which is one of the key factors for insulin secretion by pancreatic β-cells. An...

Postnatal adaptation of neonate to extrauterine life is among others dependent on maturation of mitochondrial oxidative phosphorylation system (OXPHOS). It depends on effective mitochondrial biogenesis during fetal developement. The inadequate capacity of mitochondrial OXPHOS system plays an important role in the neonatal mortality and morbidity. Therefore the study of mitochondrial biogenesis on molecular and biochemical level is important to improve the care of very premature neonates, especially critically ill premature neonates. This thesis has been worked out in The laboratory for study of mitochondrial disorders (Department of Pediatrics, 1st Faculty of Medicine, Charles University in Prague). The thesis is based on molecular genetic analyses, which are focused on characterisation of ATP synthase gene expression and on changes in mitochondrial DNA content during human and rat fetal development. The results provide the better insight into mitochondrial respectively ATP synthase biogenesis during human and rat fetal development.

Global studies show the effect of polymorphism of selected genes on dairy production and fatty acid spectrum. The aim of the diploma thesis was genotyping of candidate FASN locus with a focus on milk yield and fatty acid spectrum depending on genotype. The thesis describes the characteristics of cow's milk, milk fat and fatty acids in milk fat. Furthermore, the thesis deals with the genome of cattle and the potential influence of polymorphism of candidate genes affecting fatty acids of milk fat. Genotypes for FASN were determined by the PCR-RFLP method, the milk yield of selected dairy cattle was statistically evaluated according to the milk production indices for 1st lactation in individual breeds, and the determination of fatty acids was performed by spectrophotometry followed by statistical evaluation. The resulting genotypes in selected breeds were only two, the GG genotype with a higher relative frequency than the genotype AG. There is no statistically significant difference between FASN genotypes depending on the milk yield and the fatty acids spectrum.

This diploma thesis is focused on isolation and identification of thermophilic microorganisms capable of production of polyhydroxyalkanoates (PHA) in the sample of activated sludge from wastewater treatment. 6 culture samples were isolated from activated sludge by means of cultivation technics and methods of molecular biology. They were closer specified by comparing nucleotide sequences of 16S-rRNA gene and assigned to bacterial genus Anoxybacillus. The production of PHA by this genus was not reported in literature so far. Samples were confirmed to contain phaC gene that codes the enzyme PHA-synthase and they also gave a positive response to staining colonies with Nile red, what refers to presence of intracellular lipidic structures. However, the PHA production by isolates was not successful. The reason should be an inappropriate production medium or conditions. The positive phenotype result of Nile red dyeing was probably achieved by production of huge amount of lipids by bacterial cells that provides similar fluorescence than PHA granules.

This diploma thesis focuses on the molecular characterization of selected PHA producers. Within this work, the PHA producing thermophilic isolates originating from the samples of activated sludge and compost were identified and characterized using molecular biological methods. By sequencing the 16S rRNA gene, the thermophilic isolates were identified and taxonomically classified into the Firmicutes bacterial phylum. In these bacterial isolates, the ability to produce PHA at the genotype level was determined by conventional PCR detection of the phaC gene encoding PHA synthase, which is a key enzyme in PHA biosynthesis. Class I, II and IV PHA synthases were detected in most of the isolated bacteria, wherein class I and II PHA synthases are not characteristic for these bacterial genera. The largest proportion of isolates was identified for the species of thermophilic bacterium Aneurinibacillus thermoaerophilus, in which class IV PHA synthase was detected. In the second part of the diploma thesis, the RT-qPCR method was implemented to study the expression of selected genes of the bacterium Cupriavidus necator H16 involved in PHA metabolism. As part of the implementation of this method, PCR-based detection of selected genes was optimized and quantification of genes using real-time PCR was performed. The tested method included steps of RNA isolation, cDNA synthesis and quantification of gene segments for which the critical points of the method were determined based on the obtained data.

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...

1 Abstract It has been estimated that the mammalian mitochondrial proteome consists of ~1500 distinct proteins and approximately one quarter of them is still not fully characterized. One of these proteins is TMEM70, protein involved in the biogenesis of the eukaryotic F1Fo-ATP synthase. TMEM70 mutations cause isolated deficiency of ATP synthase often resulting in a fatal neonatal mitochondrial encephalocardiomyopathies in patients. To understand the molecular mechanism of TMEM70 action, we generated constitutive Tmem70 knockout mice, which led to embryonic lethal phenotype with disturbed ATP synthase biogenesis. Subsequently generated inducible Tmem70 mouse knockout was lethal by the week 8 post induction. It exhibited primarily impaired liver function, which contrasts with the predominantly cardiologic phenotype at disease onset in humans. Liver mitochondria revealed formation of labile ATP synthase subcomplexes lacking subunit c. Thus, in case of TMEM70 deficiency c-oligomer was not incorporated into ATP synthase, which led to critical impairment of mitochondrial energy provision, analogous to TMEM70 dysfunction in humans. In TMEM70 deficient models, the ATP synthase deficiency reached the 'threshold' for its pathologic presentation, which we quantified at 30 %. We observed compensatory increases in the...