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The study of mitochondrial energy-metabolism maturation
Křížová, Jana ; Hansíková, Hana (advisor) ; Pecina, Petr (referee) ; Rauchová, Hana (referee)
During intrauterine development in mammals, the fetus is exposed to a hypoxic environment. To allow proper postnatal adaptation to external conditions, a rapid transition from glycolytic to oxidative metabolism by mitochondria is required in fetal tissues after birth. Mitochondrial maturation is a complex process that is not only transcriptionally regulated. Using techniques such as microarray analysis, quantitative PCR, measurement of enzyme activities or coenzyme Q (CoQ) levels, we have described the acceleration of mitochondrial metabolism in rat liver tissue and skeletal muscle during the perinatal period and correlated the results with those in humans. Of the 1546 rat mitochondrial genes tested, we found statistically significant differences in the expression of 1119 in liver and 827 in muscle. The most significant shift in expression occurred in the rat liver between 20th and 22nd day of gestation, suggesting that the rat fetus is ready for the transition to external conditions at least 2 days before birth. Changes in CoQ levels in both rats and humans show that the amount of CoQ is low inthe prenatal period and increases after birth in both tissues. We have described the atypical kinase Coq8ap as an enzyme whose expression increases significantly after birth. It was previously predicted to...
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Study of etiopathology of mitochondrial disorders
Rákosníková, Tereza ; Tesařová, Markéta (advisor) ; Pecina, Petr (referee) ; Kalous, Martin (referee)
Mitochondrial disorders are a clinically, biochemically and genetically heterogeneous group of inherited disorders with a prevalence of about 1:5 000 live births. A common sign of those disorders is disruption of mitochondrial energetic metabolism. To this day, more than 400 genes have been associated with mitochondrial disorders, but 45% of patients are still without a genetic diagnosis. Using next-generation sequencing, new candidate genes or variants are found. To confirm the causality of those newly found genes or variants, biochemical characterisation using a plethora of various methods is necessary. The first aim of this thesis was to study the function of ACBD3 protein on mitochondrial energetic metabolism in non-steroidogenic cells HEK293 and HeLa and to confirm the causality of the ACBD3 gene in a patient with combined oxidative phosphorylation (OXPHOS) deficit. The second aim was to confirm the causality of two novel variants in MT-ND1 and MT-ND5 genes, which encode structural subunits of complex I (CI) of the respiratory chain. The third aim of the thesis was to study the formation of supercomplexes (SCs) in patients with rare metabolic diseases. Using functional studies, we showed in this thesis that ACBD3 protein has no essential function in mitochondria but plays an important role in...
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The role of c14orf2 protein in structure and function of mammalian ATP synthase
Ho, Dieu Hien ; Pecina, Petr (advisor) ; Panicucci Zíková, Alena (referee)
The F1Fo-ATP synthase (EC 3.6.3.14) is a key enzyme of the mitochondrial oxidative phosphorylation system (OXPHOS) - using the proton gradient generated by the respiratory chain it synthetizes approximately 90 % of cellular ATP. The subunit arrangement of its Fo domain has not been yet described in detail. At present, the research on ATP synthase research is focused mostly on revealing the structure of the proton channel a so that it is possible to precisely define the molecular mechanism of the ATP synthase rotation generation. The role of the supernumery subunits of Fo domain represents another unresolved issue. These proteins specific for eukaryotic ATP synthases are not essential for synthetic activity, instead they are putatively involved in assembly or stabilization of the enzyme complex. One of such subunits is the nuclear encoded MLQ protein (or also 6.8 kDa proteolipid or MP68), which is conserved only in vertebrates. The aim of this diploma thesis was to reveal the role of this subunit in the structure, assembly and function of the F1Fo-ATP synthase. For these purposes, cellular model of the HEK293 line with the deficiency of the MLQ protein was established employing the CRISPR/Cas9 method with paired nickases (the knock-out MLQ, MLQ KO) as part of the thesis. Three chosen MLQ KO lines...
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The role of nuclear-encoded subunits of cytochrome c oxidase in mitochondrial metabolism
Čunátová, Kristýna ; Pecina, Petr (advisor) ; Gahura, Ondřej (referee) ; Ugalde, Cristina (referee)
Mitochondria, 'the powerhouses of the cell', house the integral metabolism pathway of oxidative phosphorylation to produce the majority of cellular energy. Mammalian cytochrome c oxidase, also called complex IV (cIV), is indispensable for the overall oxidative phosphorylation function as the terminal oxidase, and for its regulation to sustain energetic needs. Since cIV is a multimeric enzyme composed of subunits encoded by nuclear and mitochondrial genomes, its biogenesis is a complicated process, which needs to be coordinated to complete a fully functional complex. Further, the setup of individual nuclear-encoded subunits isoforms of cIV may fine-tune cIV function based on the tissue or the environment context. Despite the physiological and pathological relevance of cIV composition, biogenesis, and the secondary deficiencies triggered by cIV defects, nuclear-encoded subunits' function remains poorly understood. At first, mammalian COX4 subunit isoforms with tissue- and oxygen-dependent expression were studied in the HEK293 cellular model with an exclusive expression of COX4I1 or COX4I2 isoform. Remarkably, the COX4I2 isozyme showed lower affinity to oxygen, which may imply regulation of cIV activity under hypoxia, and is of physiological relevance for the oxygen-sensing mechanism. Further,...
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Relation between n-3 polyunsaturated fatty acids and cellular sensors of energetic state
Zouhar, Petr ; Flachs, Pavel (advisor) ; Pecina, Petr (referee)
The important factor in regulation of metabolic processes is regulatory proteins, which are able to react by feed-back to energetic state of the cell. Big attention is focused on the AMP activated kinase (AMPK) and NAD+ activated deacetylase SIRT1. These enzymes interact together and their stimulation increases mitochondrial biogenesis and fatty acid oxidation. Due to this it functions beneficially against the onset of obesity, insulin resistance and ageing. Fasting, exercise and some antidiabetogenic drugs act by these regulators. n-3 polyunsaturated fatty acids (PUFA) are also known because of their stimulative effects on mitochondrial biogenesis and -oxidation. Previous work of our group have showed that intake of higher dose of n-3 polyunsaturated fatty acids (PUFA) in diet lead to increase in activity of AMPK in white adipose tissue. New results presented in this thesis show that SIRT1 is essential for increase in expression of stimulators of -oxidation (PPAR etc) in response to n-3 PUFA in diet. n-3 PUFA futher improve the metabolic profile synergistically with calorie restriction probably through SIRT1.
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The role of tissue specific isoforms of subunit 4 in assembly and function of cytochrome c oxidase
Čunátová, Kristýna ; Pecina, Petr (advisor) ; Stibůrek, Lukáš (referee)
Oxidative phosphorylation apparatus (OXPHOS) is responsible for production of majority of ATP in mammalian organisms. This process, occurring in the inner mitochondrial membrane, is partly regulated by nuclear-encoded subunits of cytochrome c oxidase (COX), the terminal enzyme of electron transport chain. Cox4 subunit, participating in OXPHOS regulation, is an early-assembly state subunit, which is necessary for incorporation of Cox2 catalytic subunit, thus for assembly of catalytically functional COX enzyme. Moreover, regulated expression of two isoforms (Cox4i1, Cox4i2) of Cox4 subunit is hypothesized to optimize respiratory chain function according to tissue oxygen supply. However, the functional impact of the isoform switch for mammalian tissues and cells is still only partly understood. In the present thesis, unique HEK293 cell line-based model with complete absence of subunit Cox4 (knock-out, KO) was prepared employing novel CRISPR CAS9-10A paired nickase technology and further characterized. Knock-out of both isoforms Cox4i1 and Cox4i2 (COX4i1/4i2 KO clones) showed general decrease of majority of Cox subunits resulting in total absence of fully assembled COX. Moreover, detected Complex I subunits as well as the content of assembled Complex I were decreased in COX4i1/4i2 KO clones. On the...
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The role of human mitochondrial YME1L protease in the biogenesis of the oxidative phosphorylation system
Tesařová, Jana ; Stiborová, Marie (advisor) ; Pecina, Petr (referee)
Mitochondria are found in virtually all eukaryotic cells where their main function is the production of ATP in the oxidative phosphorylation system (OxPhos). OxPhos is build-up of both nuclear and mitochondrial encoded protein subunits. Due to the potential function threatening defects, the quality of these protein subunits is constantly under tight control by specialized proteins. The recognition and selective removal of defective mitochondrial proteins is carried out by specific mitochondrial ATP-dependent proteases. So far, four such proteolytic complexes active within distinct mitochondrial subcompartments were identified. Both i- and m-AAA protease complexes are found in the inner mitochondrial membrane. Whereas the i-AAA protease is active in the intermembrane space, the homologous m-AAA protease functions on the matrix side of the inner membrane. The aim of the present work was to characterize cellular function of the human orthologue YME1L of the yeast i-AAA protease subunit YME1 using human HEK293 cell model. We found that human YME1L is an integral membrane protein with molecular weight of approx. 600-1100 kDa, exposing the carboxy-terminal domain to intermembrane space. The HEK293 cell line with shRNA silenced expression of YME1L showed accumulation of Ndufb6 and ND1 subunits of complex...
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New components and functions of mitochondrial ATP synthase.
Ho, Dieu Hien ; Pecina, Petr (advisor) ; Kalous, Martin (referee)
The system of oxidative phosphorylation, or respiratory chain in mitochondria gives the eukaryotic cell total majority of the energy it receives and uses in the form of ATP. F1Fo-ATP synthase, powered by the proton-motive force is directly responsible for the ATP synthesis. Diseases connected to the ATP synthesis can have even lethal consequences. There is therefore no doubt about the need for a detailed analysis of the structure of this enzyme. What is left is to reveal the structure of the transmembrane domains, which are not involved in the synthesis itself, but they can for example work as stabilisers or assembly factors. Outside the synthesis activity the dimers of F1Fo-ATP synthase are apparently taking part in the formation of the cristae of the inner membrane of a mitochondrion. Recently, the role of the enzyme is also considered in the creation of the mitochondrial permeability transition pore.
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The role of adipose tissue in the whole-body energy metabolism in mice with different genetic background
Funda, Jiří ; Janovská, Petra (advisor) ; Pecina, Petr (referee) ; Žurmanová, Jitka (referee)
Adipose tissue greatly contributes to the maintenance of the whole-body energy homeostasis. White adipose tissue (WAT) is the most important storage of metabolic energy in the body, while brown adipose tissue (BAT) enables the body to survive in cold environment by transforming metabolic energy to heat. Both WAT and BAT have a critical role in the control of systemic levels of fatty acids, which is necessary for the maintenance of the energy homeostasis and for the prevention of lipotoxic damage of non-adipose tissues. Abundant lipid accumulation can lead to the development of obesity, which is often accompanied by metabolic disorders such as type 2 diabetes and by the impairment of adipose tissue metabolic functions. Healthy adipose tissue prevents from the development of metabolic disorders associated with obesity by buffering the excess of nutrients. The key processes for efficient buffering of fatty acids are futile triacylglycerols/fatty acid cycling (TAG/FA cycling) and fatty acid oxidation. These processes occur in both WAT and BAT and their rates are largely affected by a set of transcriptional regulators, especially peroxisome proliferator-activated receptors (PPARs) and their coactivators. Bioactive molecules such as hormones, polyunsaturated fatty acids (PUFA) or pharmaceutics such as...
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