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The study of the changes of hepatocyte energy metabolism: the effect of oxidative stress and triiodthyronine
Endlicher, René ; Červinková, Zuzana (advisor) ; Kalous, Martin (referee) ; Rauchová, Hana (referee)
Changes of energy metabolism of hepatocytes: The effect of oxidative stress and triiodothyronine Liver is a vital organ performing numerous essential functions. Due to its position in the blood circulation, liver is the first organ incessantly exposed to a great number of toxic substances. Respiratory chain located in mitochondria is a frequent target of toxic action of these substances. There are various mechanisms that participate in hepatocyte damage, nevertheless the most important mechanism of hepatotoxic effect is oxidative stress induced by increased production of free radicals. Impact of oxidative stress on hepatocytes is very complex and still not fully elucidated. The aim of my thesis was to investigate the effect of oxidative stress on energy metabolism of rat hepatocytes using isolated hepatocytes and isolated mitochondria. We evaluated the effect of oxidative stress on the activity of mitochondrial enzymes and function of mitochondrial permeability transition pore (MPTP), respectively. Opening of this pore induces activation of apoptotic and necrotic processes. Our results document selective action of oxidative stress on the activity of various mitochondrial enzymes. Tert-butylhydroperoxide (t-BHP) causes significant inhibition of NADH-dependent substrates, while oxidation of...
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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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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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Non-pharmacological approaches towards treatments of mitochondrial disorders
Kudrnovská, Barbora ; Mráček, Tomáš (advisor) ; Kalous, Martin (referee)
Mitochondrial diseases are characterized by a high degree of genotypic and phenotypic heterogeneity, but as a whole group, they represent one of the most common forms of inborn errors of metabolism. Treatment of these diseases is still very limited, despite massive body of ongoing research projects, which are focussed on new therapeutic approaches. Besides pharmacological methods, there are also non-pharmacological approaches such as certain diet regimens, physical activity or exposition to hypoxia. Due to their relatively generalized mechanism of action, these methods may offer benefits to a wider range of mitochondrial patients, despite different primary genetic defects. This work summarizes the existing knowledge about the use of exercise, ketogenic diet, and hypoxia application as possible therapeutic approaches towards the treatment of mitochondrial diseases. Keywords: mitochondria, mitochondrial diseases, therapy, exercise, ketogenic diet, hypoxia
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Regulation and Disorders of Mammalian Cytochrome c Oxidase
Kovářová, Nikola ; Houštěk, Josef (advisor) ; Stibůrek, Lukáš (referee) ; Kalous, Martin (referee)
Cytochrome c oxidase (COX) represents the terminal enzyme complex of respiratory chain metabolic pathway and it occurs as monomer, dimer or as a part of respiratory supercomplexes in the inner mitochondrial membrane. COX assembly process is complicated, highly regulated and depends on many ancillary proteins. Mutations in COX subunits, which are encoded by mitochondrial and nuclear DNA, or in genes encoding its assembly proteins are frequent cause of very severe mitochondrial disorders. SURF1 assembly protein participates in the first steps of COX assembly, but its exact function is not yet clarified. In humans, mutations of SURF1 gene lead to severe COX defect and fatal neurodegenerative disorder, Leigh syndrome. Knockout of SURF1 gene in mouse causes isolated COX defect as well, but less pronounced and without involvement of CNS. The aim of the thesis was detailed analysis of disturbed COX biogenesis in a condition of SURF1 gene mutations or SURF1 gene knockout, from assembly of COX monomer to interaction of COX into supercomplexes, and to the impact of isolated COX defect on other OXPHOS complexes. Mutations of SURF1 gene in patient's fibroblasts led to marked accumulation of COX assembly intermediates and to a defect in formation of functional COX monomer, which was preferentially built into an...
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The relation between n-3 polyunsaturated fatty acids and cellular sensors of energetic state AMPK and SIRT1
Zouhar, Petr ; Kalous, Martin (referee)
Relation between n-3 polyunsaturated fatty acids and cellular sensors of energetic state Petr Zouhar Abstract The regulatory proteins, which are able to react to energetic state of the cell by feed-back mechanism, are important factors in regulation of metabolic processes. Big attention is focused on the AMP activated kinase (AMPK) and the NAD+ activated deacetylase SIRT1. These enzymes interact together and their stimulation increases mitochondrial biogenesis and fatty acid oxidation. Due to this fact they function beneficially against the onset of obesity, insulin resistance and ageing. Fasting, exercise and some antidiabetogenic drugs act through these regulators. n-3 polyunsaturated fatty acids (PUFA) are also believed to be beneficial because of their stimulative effects on mitochondrial biogenesis and β-oxidation. Our previous work has showed that intake of higher doses of n-3 polyunsaturated fatty acids (PUFA) in diet leads to increase of AMPK activity in white adipose tissue. New results presented in this thesis show that SIRT1 is essential for increase of β-oxidation stimulators (PPARα etc) expression in response to n-3 PUFA in diet. n-3 PUFA also improve the metabolic profile synergistically in combination with calorie restriction. It occurs probably through the activation of SIRT1/AMPK/PGC-1α...
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Structure and physiological role of the mitochondrial permeability transition pore
Eliáš, Jan ; Mráček, Tomáš (advisor) ; Kalous, Martin (referee)
Mitochondrial permeability transition pore (mPTP) is Ca2+ dependent channel localised in the inner mitochondrial membrane. One of its defining characteristics is inhibition by nanomolar concentrations of immunosuppressant cyclosporine A (CsA). Together with additional interacting proteins, which regulate its opening, mPTP forms a permeability transition protein complex. Persistent opening of mPTP is accompanied by mitochondrial swelling and a subsequent collapse of organelle, which precedes release of proapoptotic proteins and programmed cell death. Channel forming unit of mPTP remains unknown, despite intense and long-lasting study. Numerous proteins were proposed to play a role of channel forming subunit of mPTP, including complex of ANT and VDAC, ANT alone, PiC or even ATP synthase. Despite the fact, that molecular structure remains elusive, mPTP seems to play a role in a range of pathophysiological processes or diseases associated with them. Among others this includes ischemia/reperfusion injury, neurological and muscle dystrophies, or tumorigenesis. Keywords: mitochondria, mitochondrial permeability transition pore, cyclosporine A, programmed cell death, ATP synthase, oxidative phosphorylation apparatus.
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Antioxidant system in hypoxic heart
Sotáková, Dita ; Žurmanová, Jitka (advisor) ; Kalous, Martin (referee) ; Babula, Petr (referee)
The cardiovascular disease, particularly acute myocardial infarction, is the most common cause of death worldwide. It is well documented that adaptation to chronic hypoxia increases resistance to ischemia-reperfusion (I/R) injury in heart tissue. Reactive oxygen species (ROS) play an important signalling role by the activation of the protective pathways during I/R, although, the excess of ROS during reperfusion leads to cardiac tissue injury. As the cellular antioxidant system is responsible for the maintenance of redox homeostasis, the main aim of this thesis was to investigate the relationship between myocardial tolerance to I/R injury and regulation of main components of antioxidant systems, related transcription factors and their target genes in protective and non- protective regimens of chronic hypoxia. We found differences in cardioprotective phenotype in rats exposed to three regimens of chronic normobaric hypoxia (FiO2 0.1, 3 weeks). The adaptation to continual (CNH) and intermittent (CNH-8; 8 h/day) regimen of hypoxia increased myocardial resistance to I/R damage, whereas 1-hour daily interruption of hypoxic adaptation (INH-23) abolished cardioprotective effect and decreased the ratio of reduced and oxidized glutathione (GSH/GSSG). Both cardioprotective regimens significantly increased...
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Functional characterization of LACE1 APTase and mitochondrial AAA proteases YME1L and AFG3L2 in mitochondrial protein homeostasis.
Tesařová, Jana ; Stibůrek, Lukáš (advisor) ; Kalous, Martin (referee) ; Pecina, Petr (referee)
Mitochondrial protein homeostasis is crucial for cellular function and integrity. It is ensured by many specific mitochondrial proteases with possible chaperone functions located across the various mitochondrial subcompartments. In the first part, we have focused on characterization of functional overlap and cooperativity of proteolytic subunits AFG3L2 and YME1L of the mitochondrial inner membrane complexes m- and i-AAA in HEK293 cells. The double AFG3L2/YME1L knockdown cells showed severe alteration in OPA1 protein processing, marked elevation in OMA1 protease and severe reduction in SPG7. Our results reveal cooperative and partly redundant involvement of AFG3L2 and YME1L in the maintenance of mitochondrial protein homeostasis and further emphasize their importance for mitochondrial and cellular function and integrity. The aim of the second part was to characterize the cellular function of LACE1 (lactation elevated 1) in mitochondrial protein homeostasis. LACE1 protein is a human homologue of yeast Afg1 (ATPase family gene 1) ATPase. We show that LACE1 is a mitochondrial integral membrane protein that exists as a part of three complexes of approximately 140, 400 and 500 kDa. We demonstrate that LACE1 mediates degradation of nuclear-encoded complex IV subunits COX4, COX5A and COX6A. Using affinity...
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