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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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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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The role of Hippo Signalling pathway in tumor cell metabolism
Lettlová, Sandra ; Stiborová, Marie (advisor) ; Dračínská, Helena (referee)
Vitamine E analogues α-tocopheryl succinate (α-TOS) and mitochondrially targeted vitamine E succinate (MitoVES) are anti-cancer agents from the group of "mitocans", the compounds acting via mitochondria which present a promising invariant target for cancer cell therapy. α-TOS and MitoVES induce apoptosis selectively in various cancer cell types involving generation of reactive oxygen species (ROS). Generated superoxid anion radicals in response to α-TOS and MitoVES are believed to be converted into hydrogen peroxide that is known to activate Mammalian sterile 20-like kinase (Mst1), the central component of Hippo signalling pathway, that presents an universal size control mechanism in all metazoans and its deregulation is linked to tumourigenesis. MitoVES and α-TOS were both reported to activate Mst1 that phosphorylates Forkhead box O1 (FoxO1) transcription factor resulting in its transport to nucleus where induce the expression of pro-apoptotic genes, including NOXA, and thus promote apoptosis. The target of Hippo signalling pathway is transcriptional co- activator Yes-associated protein (Yap) which was found in Drosophila melanogaster to regulate the expression of transcription factor c-Myc which is known as the most prominent human oncogene. This thesis focused on involvement of Hippo signalling...
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The role of mitochondrial metabolism in initiation and adaptation to hypoxic conditions.
Rohlenová, Terezie ; Novák, Petr (advisor) ; Rohlena, Jakub (referee)
We can meet pathological hypoxia in the cases of hearth attack, ischemic stroke, but also during tumor invasion, thanks to insufficient angiogenesis. The activation of HIF- 1 factor during hypoxic conditions is crucial for the cell survival. This factor modulates energetic metabolism in favor of fast progressing glycolysis (with the contribution of glutaminolysis) which provides to cell enough ATP and "building blocks", while suppressing Krebs cycle and respiration because of shortage of oxygen. The thesis studies energetic metabolism of HepG2 cells (derived from liver carcinoma) which are cultivated in the media with various energetic substrates, i. e. glucose or galactose (always together with glutamine and pyruvate) under the hypoxic conditions (5% O2). HepG2 cells use particularly oxidative metabolism for ATP and "building blocks" production under the normoxic conditions while hypoxic environment causes metabolic shift in glycemic condition. Interestingly, cells cultured in galactose (glutamine) didn't switch the energy metabolism from oxidative to aerobic glycolysis such as cells cultivated in glucose, although HIF-1 factor was stabilized. We found that enhanced activity and integrity of mitochondria, enhanced maximal capacity and reserve capacity of respiration chain correlates with...
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Mitochondrial RNA in mammalian cell
Balvín, Sebastian ; Ježek, Petr (advisor) ; Magner, Martin (referee)
Mitochondrion is an important organelle maintaining energy metabolism of the cell and participating in signalization, cell cycle and apoptosis. It's pathology causes several diseases. Replication, transcription and translation take place in mitochondria, similarly like in nucleus, though there are only 13 protein coding genes. However, these processes, as well as mitochondrial DNA and RNA, vary significantly from those present in nucleus. Mitochondrial DNA is circular and both strands are replicated separately. Mitochondria form polycistronic transcripts, which are subsequently processed by tRNA. Mitochondrial ribosome evolved from prokaryotic one, but contains only half as much rRNA. Missing rRNAs are replaced by ribosomal proteins. These ribosomes contain even more proteins compared to much larger cytoplasmatic eukaryotic ones. This work is focused on current topic of mitochondrial molecular genetics: mitochondrial rRNA and ribosome, especially ribosomal assembly. In this process mitochondrial rRNAs interact with nuclear encoded proteins. The whole process probably takes place on the inner mitochondrial membrane close to the nucleoid. Our understanding to whole mechanism can help us to find a way how to cure mitochondrial pathologies.
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The role of mitochondria in adaptation to chronic hypoxia in the spontaneously hypertensive and conplastic rats.
Weissová, Romana ; Kalous, Martin (advisor) ; Rauchová, Hana (referee)
Adaptation to chronic hypoxia provides cardioprotective effects. Molecular mechanism of this phenomenon is not yet completely understood, but it is known that cardiac mitochondria play an essential role in induction of protective effects. The purpose of this diploma thesis is to study effects of continuous normobaric hypoxia (CNH; 10 % O2, 21 days) on spontaneously hypertensive rats (SHR) and conplastic strain that is derived from SHR. These animals have nuclear genome of SHR strain and mitochondrial genome of Brown Norway (BN) strain. Cardiac homogenate was used to measure enzymatic activity of malate dehydrogenase (MDH), citrate synthase (CS), NADH-cytochrome c oxidoreductase, succinate-cytochrome c oxidoreductase and cytochrome oxidase (COX). Using Western blot procedure the protein amount of antioxidant enzymes was measured - manganese superoxide dismutase and copper-zinc superoxide dismutase (MnSOD, Cu/ZnSOD), catalase and chosen subunits of oxidative phosphorylation complexes (Ndufa9, Sdha, Uqcrc2, COX-4, MTCO1, Atp5a1). Under normoxic conditions the conplastic strain has lower amount of complex IV subunit MTCO1 in comparison with SHR. This subunit is encoded by mitochondrial DNA and it is one of the seven protein-coding genes in conplastic strain that differ from SHR. Adaptation to hypoxia causes an...
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Role of mitochonodrial genome in myocardial ischemia-reperfusion injury of spontaneously hypertensive rats (SHR) adapted to hypoxia.
Brabcová, Iveta ; Žurmanová, Jitka (advisor) ; Mlejnek, Petr (referee)
Diplomová práce Abstract - Iveta Brabcová Abstract Ischemia-reperfusion heart injury is one of the most significant diseases affecting mankind and therefore current research pays more attention to its prevention and knowledge of the possible mechanisms which protect the heart. Adaptation to hypoxia has been known for several decades as a cardioprotective intervention but the main issues of protective mechanisms which are induced by the adaptation are still not completely understood. An important role of mitochondria as the main producers of energy and reactive oxygen species which can play a signalizing role in these mechanisms is confirmed in many studies. For this reason a special conplastic strain SHR/OlaIpcv-mtBN/Crl was created. This strain carries the nuclear genome of spontaneously hypertensive rat (SHR) and the mitochondrial genome of normotensive, highly resistant strain Brown Norway (BN). The aim of this study was to compare the expression of selected gene transcripts in the area of energy metabolism, of genes which are related to mitochondrial biogenesis and signaling and antioxidant systems. Comparing the expression was analyzed between strains and after chronic hypoxia adaptation, which cause cardioprotective phenotype in both of these strains. Our results showed a different expression HIF-1α...
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Pozitive and negative selection of mitochondrial genome
Svoboda, Matěj ; Kupková, Kristýna (referee) ; Maděránková, Denisa (advisor)
The bachelor thesis engages with problematics of positive and negative selection of mitochondrial genome. Thesis is divided into two parts. First part grapples with theory and explanation of fundamental definitions, in particular understates mitochondrial genome, nucleotide mutation, positive and negative selection and furthermore evolution models. Second part focuses on processing mitochondrial DNA practically and on establishing functions in programming environment R. The emphasis is foremost on gene sequencing alignment and exploring substitutions. Consequently, obtained outcomes are contrasted with PAML and KaKs Calculator programme outcomes.
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Role of a SNARE protein in the biogenesis of Giardia intestinalis mitosomes.
Voleman, Luboš ; Doležal, Pavel (advisor) ; Nohýnková, Eva (referee)
SNARE proteins play essential role in most membrane fusions taking place in eukaryotic cell. They are responsible for all fusions that occur across endocytic and secretory pathways. Apart from these processes stand mitochondria and plastids. Fusion of these organelles is directed by specific protein machineries. In this work we review up-to-date information on SNARE mediated membrane fusion and fusion of outer and inner mitochondrial membranes with an emphasis on situation in flagellated protozoan parasite Giradia intestinalis. It was suggested that one of typical SNARE protein in Giardia (GiSec20) is localised to its highly reduced mitochondria called mitosomes. This protein is also essential for surviving of Giardia trophozoites. In this work we show that mitosomal localization of Gisec20 is caused by episomal expression however the protein is localised to endoplasmic reticulum under physiological conditions. Using GFP tag we were able to characterize its targeting signal which showed to be localised in transmembrane domain of GiSec20. This signal targets the protein to mitosomes of G. intestinalis and S. cerevisiae, respectively. Mitosomal localization was prevented by adding 3'UTR to gene sequence and its episomal expression. This suggests existence of targeting mechanism based on information...
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Reductive Evolution of Mitochondria - Related Organelles in Anaerobic Protist
Rada, Petr ; Tachezy, Jan (advisor) ; Embley, Martin (referee) ; Eliáš, Marek (referee)
Charles University in Prague, Faculty of Science Department of Parasitology Ph.D. study program: Parasitology Abstract of the Ph.D. Thesis Reductive Evolution of Mitochondria - Related Organelles in Anaerobic Protist Petr Rada Supervisor: Prof. RNDr. Jan Tachezy,Ph.D. Advisor: Doc. RNDr. Ivan Hrdý, Ph.D. Praha, 2011 1 ABSTRACT Trichomonas vaginalis and Giardia intestinalis are parasitic protists of the Excavata group. Both contain anaerobic forms of mitochondria called hydrogenosomes (Trichomonas) and mitosomes (Giardia). Hydrogenosomes produce hydrogen and ATP by substarte level phosphorylation and mitosomes represent the highly-reduced form of mitochondria that do not participate in cellular energy metabolism and ATP generation. Both types of organelles lost the majority of mitochondrial pathways and their genomes during the mitochondrion to hydrogenosome transition. Consequently, hydrogenosomes and mitosomes facilitate translocation of nuclearly encoded proteins into the matrix of the organelle as well as exchange of metabolites and ions across their membranes. Little is known about the membrane machineries required for the biogenesis of the organelle and metabolite exchange and the limited knowledge of mitosomal proteomes has been mostly gained from genomic analysis and localization studies of a few...
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