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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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Protein profiling, metabolic enzymes and transmembrane signaling in the heart of spontaneously hypertensive SHR-Tg19 rat
Manakov, Dmitry ; Novotný, Jiří (advisor) ; Kuncová, Jitka (referee) ; Kalous, Martin (referee)
Cardiovascular diseases account for the majority of deaths both worldwide and in the Czech Republic. Main factors contributing heart disease development, aside age and sex, are obesity, high blood pressure and high blood cholesterol and triglyceride levels. Spontaneously hypertensive rat (SHR) was developed and used for search of genetic determinants of these traits. This commonly used rat model develops hypertension, dyslipidemia, and insulin resistance naturally which is caused by aberrant Cd36 fatty acid translocase gene. Previous studies have shown that rescue of Cd36 performed in the transgenic SHR-Tg19 strain enhances cardiac beta-adrenergic system, slightly increases heart mass and leads to higher susceptibility to arrhythmias. The present thesis had two main aims: 1) To investigate whether and how a transgenic rescue of Cd36 in SHR affects protein composition, mitochondrial function and activity of selected metabolic enzymes of the heart. 2) To study the expression and distribution of selected components of beta-adrenergic signaling system in lipid raft isolated form membranes using the TX-100 detergent. We set to compare two commonly used proteomic approaches, 2D electrophoresis with MALDI-TOF mass spectrometry and label-free LC-MS. The results did not reveal any overlap between...
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Structural composition and functional properties of mitochondrial FoF1 ATP synthase on models of specific subunits deficiencies
Efimova, Iuliia ; Mráček, Tomáš (advisor) ; Kalous, Martin (referee)
Mitochondrial ATP synthase represents the final complex of oxidative phosphorylation (OXPHOS) system located in the inner mitochondrial membrane. Its primary role is to utilize mitochondrial membrane potential (Δψm) generated by respiratory chain complexes to produce energy in the form of ATP. Mammalian ATP synthase comprises of 17 different subunits organized into membranous Fo and matrix-oriented F1 domains. Defects of complex V and their manifestation have been studied on mitochondrial, cellular, tissue and organism levels using different models, including human cell lines and cell lines derived from patient tissues. In many cases mitochondrial diseases display threshold behaviour, when genetic defect is phenotypically manifested only bellow certain threshold in particular enzyme complex activity and/or content. This work was aimed at elucidation of functional consequences of ATP synthase deficiency in HEK293 cell lines with suppressed gene expression of γ, δ or ε subunits of ATP synthase central stalk. We have analysed range of clones with respective subunits knockdown and found varying decrease in assembled ATP synthase content, which was mirrored by the decrease in individual ATP synthase subunits. The only exception was subunit Fo-c, whose levels remained unchanged or even increased. ATP...
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The function of p53 protein in mitochondria
Magdálková, Kateřina ; Kalous, Martin (advisor) ; Žurmanová, Jitka (referee)
Protein p53 is known as a tumor suppressor. In nucleus, p53 regulates the expression of its target genes, which are involved in cell cycle control, DNA repair and cell death. Protein p53 also has transcription-independent activities outside the nucleus. Under physiological conditions, certain amount of this protein can be found in mitochondria, where it is involved in mitochondrial genome integrity maintaining. Under stress conditions, p53 protein rapidly translocates to outer mitochondrial membrane or mitochondrial matrix, and takes a part in apoptotic or necrotic signaling pathway. Keywords: p53, mitochondria, mtDNA, apoptosis
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The function of p53 protein in mitochondria
Magdálková, Kateřina ; Stibůrek, Lukáš (advisor) ; Kalous, Martin (referee)
Protein p53 is known as a tumor supressor. In nucleus, p53 regulates the expression of its target genes, which are involved in cell cycle control, DNA repair and cell death. Protein p53 also has transcription-independent activities outside the nucleus. Certain amount of this protein can be found in mitochondria, where it is involved in mitochondrial genom integrity maintaining. Under stress conditions, p53 rapidly translocates to outer mitochondrial membrane, and takes a part in apoptotic signalling pathway. Keywords: p53, mitochondria, mtDNA, apoptosis
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Mitochondrial permeability transition pore and its role in cardioprotection
Ryba, Lukáš ; Kalous, Martin (advisor) ; Brabcová, Iveta (referee)
The mitochondrial permeability transition pore (MPTP) is a non-specific voltage dependent channel which is located in the inner mitochondrial membrane. High calcium concentration and oxidative stress are main inducers of MPTP opening in a tissue which is affected by ischaemia and subsequent reperfusion. Morbidity and mortality of pacients who suffer from acute myocardial infaction or cardiochirugical operation, depends on the size of ischemic- reperfusion injury (IRI). The methods of IRI attenuation are based on the inhibition of the MPTP through farmacological intervention or ischemic conditioning. This thesis summarizes the current knowledge about the MPTP structure, regulation and its role in cardioprotection. Key words: mitochondrial permeability transition pore, cardioprotection, ischemic conditioning, CsA, SAFE and RISK pathway
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