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Mitochondrial production of reactive oxygen species and its role in physiological regulations
Holzerová, Eliška ; Mráček, Tomáš (advisor) ; Kalous, Martin (referee)
The production of mitochondrial reactive oxygen species and the resulting oxidative stress is an important phenomenon driving long-lasting research and intense discussions. Knowledge of exact mechanisms of reactive oxygen species production and pathways leading to their formation could help us to directly affect their production, a task with potential terapeutic implications. The molecular nature of the production of reactive oxygen species by some enzymes has already been well documented, but others still remain controversial and current theories are obviously far from the truth. Much more interesting is the question of physiological importace of this production. The reactive oxygen species were considered harmful factors clearly distorting the integrity of the organism for a long time. However, recent research suggest that their existence can also be beneficial and effective. Evidently they can serve as a signaling molecules in several metabolic and regulatory pathways occurring in the organism. This bachelor thesis offers insight into the current state of knowledge. It focuses on the most detailed description of the reactive oxygen species production by mitochondrial respiratory chain enzymes. Furthermore, it deals with some signaling cascades, where involvement of mitochondrially generated...
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Mitochondrial dynamics in myocardium.
Weissová, Romana ; Nováková, Olga (advisor) ; Kalous, Martin (referee)
The heart is an absolutely vital body organ, which requires sufficient amount of active mitochondria for its energy demanding activity. The functionality of a mitochondrial population is maintained through mitochondrial turnover, encompassing mitophagy removing damaged mitochondria and mitochondrial biogenesis responsible for the emergence of new organelles. Dynamic processes of mitochondrial fusion and fission can also contribute to the maintenance of a healthy mitochondrial population. Mitochondrial fusion and fission have not yet been proven in cardiomyocytes, although these cells possess all the proteins required for these events. These processes, however, take on the importance during pathological conditions, when changes in the amount of protein applied in the mitochondrial dynamics occur. The modification in mitochondrial phenotype leads to the cell damage. Understanding the role of mitochondrial dynamics in myocardium may contribute to the development of new heart diseases treatments.
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Biochemical and molecular studies of cytochrome c oxidase and ATP synthase deficiencies
Fornůsková, Daniela ; Zeman, Jiří (advisor) ; Hyánek, Josef (referee) ; Stiborová, Marie (referee)
Mgr. Daniela Fornuskova PhD thesis Biochemical and molecular studies of cytochrome c oxidase and ATP synthase deficiencies ABSTRACT The mammalian organism fully depends on the oxidative phosphorylation system (OXPHOS) as the major energy (ATP) producer of the cell. Disturbances of OXPHOS may be caused by mutations in either mitochondrial DNA (mtDNA) or nuclear DNA (nDNA). One part of the thesis is focused on the role of early and late assembled nuclear-encoded structural subunits of cytochrome c oxidase (CcO) as well as Oxa1l, the human homologue of the yeast mitochondrial Oxa1 translocase, in the biogenesis and function of the human CcO complex using stable RNA interference of COX4, COX5A, COX6A1 and OXA1L, as well as expression of epitope-tagged Cox6a, Cox7a and Cox7b, in HEK (human embryonic kidney)- 293 cells. Our results indicate that, whereas nuclear- encoded CcO subunits Cox4 and Cox5a are required for the assembly of the functional CcO complex, the Cox6a subunit is required for the overall stability of the holoenzyme. In OXA1L knockdown HEK-293 cells, intriguingly, CcO activity and holoenzyme content were unaffected, although the inactivation of OXA1 in yeast was shown to cause complete absence of CcO activity. In addition, we compared OXPHOS protein deficiency patterns in mitochondria from skeletal...
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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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Supercomplexes in the respiratory chain of mitochondria
Mikulová, Tereza ; Holzerová, Kristýna (referee) ; Houštěk, Josef (advisor)
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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Mitochondrial cytochrome c oxidase: cyanide inhibition and role of assembly factor Surf1 defect
Nůsková, Hana ; Kalous, Martin (referee) ; Drahota, Zdeněk (advisor)
The activity of mitochondrial cytochrome c oxidase (COX) can be affected by either exogenous or endogenous factors. The most efficient and in the environment abundant compound that inhibits COX is cyanide. The very frequent cause of COX deficiency in humans is represented by a defect in the SURF1 gene. The mechanism of cyanide inhibitory effect on COX as well as the conditions for its recovery are not yet fully explained. Three parameters of COX function, namely the transport of electrons (oxygen consumption), the transport of protons (mitochondrial membrane potential, m) and the enzyme affinity to oxygen (p50 value), were studied with regard to the inhibition by KCN and its reversal by pyruvate. The function of COX was analysed in intact isolated rat liver mitochondria, both within the respiratory chain and as a sole enzyme, using succinate or an artificial electron donor ascorbate + TMPD as a substrate. 250 M KCN completely inhibited both electron- and proton-transporting function of COX, and this inhibition was reversible as proved with washing of mitochondria. The addition of 60 mM pyruvate induced the maximal recovery of both parameters to 60 - 80 % of original values. Using KCN in the low concentration range up to 5 M, a profound, 30-fold decrease of COX affinity to oxygen was observed....
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Distribution of mitochondrial uncoupling proteins in selected tissues from mice and rat
Alán, Lukáš ; Flachs, Pavel (referee) ; Ježek, Petr (advisor)
Mitochondrial uncoupling proteins (UCPs) belong to the superfamily of mitochondrial anion-carriers. The longest known is UCP1, predominantly expressed in brown adipose tissue, where it takes part in nonshivering thermogenesis. In the late 1990s were discovered other sequence homologs of UCP1 with tissue specific distribution. The Function of these "new" uncoupling proteins is still uncertain. It is assumed that each of the isoforms has a specific function depending on the type of tissue. This thesis showed differences in tissue transcription pattern between rat and mice using RT-PCR absolute quantification. Significant differences in pattern were found in lungs, brain and muscle. In each case UCP expression was higher in mice tissues. Mice lungs express mainly UCP2. The difference in mice brain is caused by ucp4 and ucp5 genes transcription and finally in muscle is highest content of UCP3 mRNA. We investigated whether any of ucp transcript can complement ucp2 transcripton in spleen or lungs of ucp2 -/- mice. We did not find any difference which can explain, that in isolated lung mitochondria of fasted ucp2-/- mice were uncoupled in state 4. In the last project, we found relationship between ucp2 transcription in insulinoma INS-1E cells and oxygen levels of the cultivation atmosphere.
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Steatosis of liver and mitochondrial dysfunction
Páleníčková, Eliška ; Balková, Patricie (referee) ; Novák, František (advisor)
Aim: To determine the effect of diet-induced steatosis in the development of mitochondrial dysfunction in the liver and hepatic sensitivity to the partial ischemia. Methods: Male Wistar rats (361 ± 8.8 g) were fed standard (SD) or high-fat diet (HFD). Partial ischemia was induced by short-term clamp (20 min) of vein porta two days before the end of the experiment. Results: Ten-week HFD administration lead due to increased ketogenesis the altered glucose tolerance elevated serum NEFA. We demonstrated the inhibitory effect of HFD on the respiratory capacity of mitochondria in vitro. HFD negatively affected the activity of antioxidant systems and stimulated the formation of lipoperoxides. Partial ischemia had no efect on the mitochondrial oxidative capacity but significantly elevated the oxidative stress. Conclusion: HFD administration lead to the development of fatty liver that was still not accompanied by biochemical markers of liver injury. Nevertheless, we proved the impairment of to mitochondrial respiratory capacity, signs of structural damage of mitochondria and the increased sensitivity to oxidative damage of the liver. Subject words: biochemistry, physiology Keywords: mitochondria, HFD, ischemia, respiratory chain, antioxidant systém, ROS
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Evolution of eukaryotic ABC transporters
Žárský, Vojtěch ; Doležal, Pavel (referee) ; Tachezy, Jan (advisor)
In the past twenty years there has been a lot of research done on ABC transporters. This group of membrane transporters was recognized as highly important due to its ubiquity in living organisms and due to the involvement of some of the ABC transporters in multidrug resistance of cancer cells and pathogens against chemotherapeutics. This medical aspect of ABC transporters was naturally the most important one for the majority of researchers. On the other hand, the biological aspects and evolution of many ABC transporters remained untouched. In this work I give an overview of ABC transporters of parasitic protozoa and focus on the evolutionary aspect of eukaryotic transporters and on mitochondrial ABC transporters, which are the most conserved ones among eukaryotic ABC transporters.
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