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Molecular consequences of electron transport chain deficiency in proliferating and quiescent cells
Magalhães Novais, Silvia Carina ; Rohlena, Jakub (advisor) ; Panicucci Zíková, Alena (referee) ; Eelen, Guy (referee)
Oxidative phosphorylation (OXPHOS) system is best known for producing ATP in mitochondria. As a major metabolic hub, OXPHOS also supports other biological functions that are unrelated to ATP synthesis. Are these additional roles of OXPHOS unique to different cell states, such as to proliferation and quiescence, two of the most contrasting states of a mammalian cell? In this thesis we used genetic models of OXPHOS deficiency to show that in proliferating cells and in tumors OXPHOS primarily supports biosynthesis of nucleotides, whereas in quiescent cells OXPHOS provides oxidative stress resistance. Mechanistically, in growing tumors OXPHOS drives dihydroorotate dehydrogenase (DHODH), an enzyme crucial for de novo synthesis of pyrimidines. Complex II of OXPHOS also acts as a sensor of mitochondrial dysfunction and regulates pyrimidine synthesis for energy conservation. In contrast, in quiescent cells OXPHOS maintains autophagy by continuous low level ROS generation and together with the antioxidant defense provides constitutive oxidative stress protection. In summary, beyond ATP production OXPHOS has additional vital roles tailored to the particular requirements of quiescence and proliferation, with relevance for pathophysiology in cancer and vascular biology.
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Supercomplexes in the respiratory chain of mitochondria
Mikulová, Tereza ; Houštěk, Josef (advisor) ; Holzerová, Kristýna (referee)
Mitochondria are very important organelles of eukaryotic cell. In mitochondria, there are located many metabolic reactions including oxidative phosphorylation (OXPHOS). In this process, respiratory chain enzyme complexes couple the oxidation of NADH and FADH2 to vectorial proton transport across the inner mitochondrial membrane. ATP synthase then uses the resulting electrochemical potential to synthesize ATP from ADP and phosphate. Recent studies of the OXPHOS demonstrate higher structures of complexes so-called supercomplexes which facilitate substrate channeling. Formation of supercomplexes plays a role in the assembly and stability of the complexes, suggesting that the supercomplexes are the functional state of the respiratory chain.
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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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The content of components of ATP synthasome in different rat tissues and in patients with defects in ATP synthase
Mikulová, Tereza ; Houštěk, Josef (advisor) ; Kalous, Martin (referee)
The complexes of oxidative phosphorylation (OXPHOS) are situated in the inner mitochondrial membrane in higher structural and functional complexes, so-called supercomplexes, which facilitates substrate channeling. ATP synthase is also able to organize in higher structures. In mammalian mitochondria, ATP synthase is usually present in a dimeric form. There is evidence of its trimerization and even tetramerization. Furthermore, it seems that ATP synthase catalyzing the phosphorylation of ADP to ATP, adenine nucleotide translocator (ANT) ensuring the exchange of ADP for newly synthesized ATP across the inner mitochondrial membrane and phosphate carrier (PiC) allowing the import of inorganic phosphate (Pi) into the matrix of mitochondria are assembled in a supercomplex called ATP synthasome. This association among the components of phosphorylative apparatus seems to increase the efficiency of processes leading to the ATP synthesis. First, we studied amounts of the components of phosphorylative apparatus in connection with various ATP synthase contents among mitochondria isolated from nine rat tissues. Mitochondrial proteins were separated by denaturing electrophoresis (SDS-PAGE) and their content was analyzed using specific antibodies. In agreement with our expectations, the highest content of...
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Study of expression and maturation of mitochondrial oxidative phosphorylation system during mammal's prenatal period
Mrhálková, Andrea ; Hůlková, Martina (advisor) ; Ješina, Pavel (referee)
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.
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The role of mitochondria in the pathogenesis of Huntington's disease
Řeháková, Kateřina ; Hansíková, Hana (advisor) ; Kohoutová, Michaela (referee)
Huntington's disease is a neurodegenerative disease affecting the nervous system. It is caused by the mutation of the huntingtin protein coding gene. The mutation is manifested by the multiplication of CAG triplets. Huntingtin is present more in the cytoplasm. It interacts with many proteins and has roles also in transcription and cell transport. Huntingtin also participates in correct regulation of embryonic development and development of nerve tissue. Mutant huntingtin causes oxidative stress, mitochondrial biogenesis and OXPHOS disorders. Diagnosis of Huntington's disease is based on a laboratory examination of the presence of an allele predisposing to the disease. Clinical imaging is also an important part of diagnostics. The patient exhibits uncontrollable choreatic body movements and dementia. The aim of the thesis was to describe the main characteristics of Huntington's disease with the focus of HD on mitochondrial energy metabolism. In the practical part, the aim was to analyze mitochondrial respiration by high resolution polarography in a set of 3 fibroblast lines from patients with Huntington's disease. Polarographic analyses showed that respiration of HD fibroblasts was decreased in comparison with controls. We found that complex II of oxidative phosphorylation was most affected in HD...
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Protein analysis of selected mitochondrial proteins in the muscle tissue of porcine model of Huntington's disease
Dosoudilová, Žaneta ; Klempíř, Jiří (advisor) ; Baxa, Monika (referee)
Huntington's disease (HD) is an autosomal dominant hereditary neurodegenerative disease characterized by motor, cognitive and behavioral disorders. HD is caused by expansion of CAG triplet (cytosine-adenosine-guanine) located in a gene on the short arm of the fourth chromosome. This expansion encodes an aberrant polyglutamine chain in the protein huntingtin. Physiological and mutated huntingtin (in case of HD) are expressed in almost all tissues and influences many cellular functions. The prevalence of HD in population is about 1 per 10.000. The disease is currently incurable and its mechanisms are not sufficiently understood. Besides affecting the central nervous system HD also affects peripheral tissues, including skeletal muscles. HD disrupts mitochondrial function and damages oxidative phosphorylation system, which has the task of producing energy in the form of ATP in cells. Research of transgenic minipig model for HD could help elucidate the mechanisms of disease's pathogenesis and potential therapeutic strategy. In this diploma thesis, immunodetection with help of specific antibodies to detect changes in amount of 14 selected mitochondrial proteins in skeletal muscle tissue of three age groups of transgenic HD minipigs - 24, 36 and 48 months old was used. Gradual progression in reduced...
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The content of components of ATP synthasome in different rat tissues and in patients with defects in ATP synthase
Mikulová, Tereza ; Houštěk, Josef (advisor) ; Kalous, Martin (referee)
The complexes of oxidative phosphorylation (OXPHOS) are situated in the inner mitochondrial membrane in higher structural and functional complexes, so-called supercomplexes, which facilitates substrate channeling. ATP synthase is also able to organize in higher structures. In mammalian mitochondria, ATP synthase is usually present in a dimeric form. There is evidence of its trimerization and even tetramerization. Furthermore, it seems that ATP synthase catalyzing the phosphorylation of ADP to ATP, adenine nucleotide translocator (ANT) ensuring the exchange of ADP for newly synthesized ATP across the inner mitochondrial membrane and phosphate carrier (PiC) allowing the import of inorganic phosphate (Pi) into the matrix of mitochondria are assembled in a supercomplex called ATP synthasome. This association among the components of phosphorylative apparatus seems to increase the efficiency of processes leading to the ATP synthesis. First, we studied amounts of the components of phosphorylative apparatus in connection with various ATP synthase contents among mitochondria isolated from nine rat tissues. Mitochondrial proteins were separated by denaturing electrophoresis (SDS-PAGE) and their content was analyzed using specific antibodies. In agreement with our expectations, the highest content of...
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Study of expression and maturation of mitochondrial oxidative phosphorylation system during mammal's prenatal period
Mrhálková, Andrea ; Hůlková, Martina (advisor) ; Ješina, Pavel (referee)
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.
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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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