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Functional characterisation of new components of mitochondrial proteome.
Kovalčíková, Jana ; Vrbacký, Marek (advisor) ; Červinková, Zuzana (referee) ; Ješina, Pavel (referee)
1 Abstract It has been estimated that the mammalian mitochondrial proteome consists of ~1500 distinct proteins and approximately one quarter of them is still not fully characterized. One of these proteins is TMEM70, protein involved in the biogenesis of the eukaryotic F1Fo-ATP synthase. TMEM70 mutations cause isolated deficiency of ATP synthase often resulting in a fatal neonatal mitochondrial encephalocardiomyopathies in patients. To understand the molecular mechanism of TMEM70 action, we generated constitutive Tmem70 knockout mice, which led to embryonic lethal phenotype with disturbed ATP synthase biogenesis. Subsequently generated inducible Tmem70 mouse knockout was lethal by the week 8 post induction. It exhibited primarily impaired liver function, which contrasts with the predominantly cardiologic phenotype at disease onset in humans. Liver mitochondria revealed formation of labile ATP synthase subcomplexes lacking subunit c. Thus, in case of TMEM70 deficiency c-oligomer was not incorporated into ATP synthase, which led to critical impairment of mitochondrial energy provision, analogous to TMEM70 dysfunction in humans. In TMEM70 deficient models, the ATP synthase deficiency reached the 'threshold' for its pathologic presentation, which we quantified at 30 %. We observed compensatory increases in the...
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Pharmacological and metabolic influence on liver mitochondrial functions
Sobotka, Ondřej ; Červinková, Zuzana (advisor) ; Kuncová, Jitka (referee) ; Žurmanová, Jitka (referee)
Liver mitochondria play a crucial role in intermediary metabolism and main metabolic pathways. We evaluated the pharmacological effect on liver mitochondria in vitro using two novel anticancer drugs: 3-bromopyruvate and α-tocopheryl succinate. Metabolic influence on liver mitochondria was performed in vivo by high fat and high cholesterol diet. Toxicity of both drugs was evaluated in cell cultures of hepatocytes isolated from rat and mouse liver. The effect of anticancer drugs on liver mitochondrial functions in vitro was studied on suspensions of isolated liver mitochondria, tissue homogenate and permeabilized hepatocytes. Mitochondrial respiration was measured using high-resolution respirometry. 3-bromopyruvate caused morphological and functional damage of primary rat and mouse hepatocytes in cell cultures; this toxic effect was accompanied by an increase of reactive oxygen species production and mitochondrial dysfunction. 3-bromopyruvate decreased the oxygen consumption of mitochondria energized by substrates for complex I and complex II. α-Tocopheryl succinate caused a decrease of succinate-dependent respiration in all experimental models both in coupled and in uncoupled states. The most pronounced effect of α-tocopheryl succinate was apparent in isolated mitochondria and the least pronounced...
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Liver regeneration after partial hepatectomy in rats suffering from non-alcoholic fatty liver disease
Garnol, Tomáš ; Červinková, Zuzana (advisor) ; Pantoflíček, Tomáš (referee) ; Šiller, Jiří (referee)
1. SUMMARY Liver regeneration after partial hepatectomy in rats suffering from non-alcoholic fatty liver disease Non-alcoholic fatty liver disease (NAFLD) is currently the most frequent chronic liver disease in economically developed countries with prevalence of about 30 %. Liver resection from different reasons is nowadays common surgical procedure. Successful recovery and renewal of liver functions depend on regenerative capacity of liver remnant. The course of liver regeneration could be profoundly influenced by concomitant liver pathological processes including NAFLD. The aim of this work has been study of early phase of liver regeneration after partial hepatectomy (PHx) in rats with nutritionally induced simple steatosis. At the beginning we introduced a model of NAFLD by a high-fat diet giving to Wistar or Sprague-Dawley rats for 3 and 6 weeks, resp. This regimen induced simple steatosis without signs of inflammation, necrosis or fibrotic changes in both strains. In the second part of our study we followed if liver regeneration after PHx in rats with NAFLD is altered. Liver regeneration was assessed by bromodeoxyuridin incorporation into DNA of hepatocytes. Regeneration of liver with simple steatosis induced by 2/3 PHx was not significantly influenced in comparison to non-steatotic liver. The only...
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The US Supreme Court, its, formation and first key decisions
Červinková, Zuzana ; Seltenreich, Radim (advisor) ; Horák, Záboj (referee)
These days, the Supreme Court of the United States represents a very important and irreplaceable role in its home land: protection of constitutionality, explanation of federal laws, as well as appellate court of last resort within the framework of American courts. Today there is no doubt about its jurisdiction, but there were doubts. In the beginning of its existence no one was sure if the Court could overcome the initial obstacles which were in the way. This diploma thesis deals with the initial existence of the Supreme Court of the United States, its role within the framework of the US federal power system, and its progressive integration as equal with the legislative branch and executive power - The United States Congress and the President, respectively. The main objective of this thesis is to introduce the federal judiciary of the United States, show its differences from the continental system, and accentuate the importance of precedents. First, I focus on the history of the United States; colonization by British citizens, the American War for Independence, The Declaration of Independence, and the formation of American statehood in the 1780s, which resulted in the acceptance of one of the longest- lasting written constitutions in the world. After the historical excurse, the thesis focuses on...
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Regulation of liver regeneration as assessed by means of molecular genetics
Rychtrmoc, David ; Červinková, Zuzana (advisor) ; Leníček, Martin (referee) ; Pospíšilová, Šárka (referee)
Regulation of liver regeneration as assessed by means of molecular genetics Liver regeneration is a noteworthy phenomenon of a vigorous tissue recovery following a wide variety of damaging events. Regenerative ability of the liver is unique among the mammalian visceral organs and plays its role in pathophysiology of heterogeneous hepatic diseases. The recovery process occurs in three phases of which the termination remains the least elucidated. Even the questions of regeneration termination timing and of completeness of organ build-up are still controversial despite decades of studies. The aim of the present thesis was to analyse gene expression data pertinent to late phase of liver regeneration.We evaluated the course of spontaneous recovery in rats subjected to 2/3 partial hepatectomy. Because of lack of generally agreed parameter of hepatic regeneration completeness we proposed a proprietary indicator denoted as %LReg, based on organ and whole body weight assessments performed throughout the study. Gene expression analysis was first performed using DNA microarrays and consequently by real-time RT-PCR. Whole genome scale transcriptional analysis yielded vast amount of data covering gene expression dynamics of fourteen post-surgery days after 2/3 partial hepatectomy in adult male rats. This intervention in...
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The role of Nrf2 in selected models of liver injury
Mezera, Vojtěch ; Červinková, Zuzana (advisor) ; Brůha, Radan (referee) ; Ehrmann, Jiří (referee)
The role of Nrf2 in selected models of liver injury Oxidative stress accompanies various forms of liver injury, including liver resection and toxic liver injury. Two-thirds partial hepatectomy (PHx) is an established model for the study of liver regeneration after resection. We tested the effect of epigallocatechin gallate (EGCG), a green tea antioxidant, on the early phase of liver regeneration after PHx. Male Wistar rats received either water for injections or EGCG for 3 consecutive days and then were subjected either to laparotomy or hepatectomy. We observed a lower accumulation of bromodeoxyuridine pointing to lower DNA synthesis in hepatectomized rats receiving EGCG in a dose of 50 mg/kg than in hepatectomized rats treated with water. The activity of caspases 3/7, expression of p-p53 and tissue levels of IL-6 displayed a similar trend. EGCG in a dose of 20 mg/kg had no such effects. Next, male Wistar rats were subjected either to PHx or laparotomy. Twenty-four hours after the surgery, hepatocytes were isolated and treated with various concentrations of EGCG for 24 h. Morphological criteria, cell viability tests, and albumin synthesis revealed toxicity starting at 10 µmol/L. DNA synthesis was higher in hepatocytes isolated from rats after PHx and inhibited by EGCG. Furthermore, EGCG increased...
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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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The influence of the treatment of experimental acute liver failure by FPSA/Prometheus on intracranial pressure
Pražák, Josef ; Cvachovec, Karel (advisor) ; Červinková, Zuzana (referee) ; Kieslichová, Eva (referee)
Background: Cerebral edema is a well-known and fatal complication of acute liver failure (ALF). The influence of the treatment by artificial liver devices on the cerebral damage caused by ALF is usually monitored by only measuring the intracranial pressure (ICP). The aim of this work was to determine the influence of Fractionated Plasma Separation and Adsorption (FPSA), nonbiological artificial liver device, on the intracranial pressure. The second goal of this work was to determine the potential role of cerebral microdialysis in monitoring of the efficiency of fractionated plasma separation and adsorption (FPSA) treatment of ALF. Methods: Two types of surgical resection model of ALF were used in two separate experiments in pigs. In the first study data from monitoring of the intracranial pressure of ALF group (animals with ALF only) and FPSA group (animals with ALF treated by FPSA) were compared. In the second study data from monitoring by cerebral microdialysis and ICP of the ALF group (animals with ALF only), FPSA group (animals with ALF treated by FPSA) and SHAM group (animals with only laparotomy) were compared using statistical analyses. Results: In both experiments, the ICP was significantly higher in the ALF group than in the FPSA group from the 9th hour of the experiment. In the second...
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Possibilities of UCP2 (uncoupling protein 2) induction in rat hepatocytes in in vivo conditions
Bolehovská, Radka ; Červinková, Zuzana (advisor) ; Kalous, Martin (referee) ; Novotný, Dalibor (referee)
Possibilities of UCP2 (uncoupling protein 2) induction in rat hepatocytes in in vivo conditions Introduction Uncoupling protein 2, discovered in 1997, is the first described homologue of uncoupling protein 1. Uncoupling proteins increase the permeability of inner mitochondrial membrane for protons, decrease the efficiency of energy conversion, inhibit the ATP synthesis and stimulate energy release in form of heat. Uncoupling proteins also increase the substrate oxidation and reduce production of reactive oxygen species in mitochondria. Objective, material and method The aim of this study was to establish and optimised the quntitative real-time PCR for detection of UCP2 mRNA expression kinetics in rat liver tissue. The present study was conducted to assess the effects of acute and chronic treatment with triiodothyronine and the effect of partial hepatectomy on liver uncoupling protein 2 mRNA levels in male Wistar rats. Results Intraperitoneal injection of one dose of triiodothyronine (200 μg/kg rat body weight) increased mRNA expression of uncoupling protein 2 in liver tissue almost 2-fold (P < 0.01 vs. control group) in rats 12 hours after T3 administration. Concentrations of total triiodothyronine and free triiodothyronine in serum were increased 122-fold and 77-fold (p < 0.001), respectively....
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