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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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The role of toll-like receptors in the pathogenesis of liver diseases
Petrášek, Jan ; Jirsa, Milan (advisor) ; Tlaskalová - Hogenová, Helena (referee) ; Červinková, Zuzana (referee)
Identifikační záznam: PETRÁŠEK, Jan. ÚLOHA TOLL-LIKE RECEPTORŮ V PATOGENEZI JATERNÍCH ONEMOCNĚNÍ. [The role of toll-like receptors in the pathogenesis of liver diseases]. Praha, 2010. 198s., Disertační práce. Univerzita Karlova v Praze, 1. lékařská fakulta, Laboratoř Experimentální Hepatologie IKEM. Vedoucí práce: Milan Jirsa. Abstrakt Společným jmenovatelem nejčastějších onemocnění jater je aktivace mechanismů vrozené imunity, které přispívají k rozvoji zánětu a poškození jaterního parenchymu. Klíčovou úlohu v rozvoji jaterního poškození hrají Toll-like receptory, jejichž charakterizace v posledním desetiletí vedla přehodnocení patofyziologie některých jaterních onemocnění. Předkládaná práce studuje význam alelických variant v genech kódujících proteiny Toll-like receptorové signální kaskády a mezibuněčné signalizace v patogenezi alkoholické nemoci jater, přináší nový pohled na probiotika v léčbě nealkoholické steatohepatitidy a nové poznatky o protizánětlivém působení interferonů I. typu u některých jaterních chorob. Abstract Recent reports suggest that majority of chronic and acute liver diseases share a significant degree of liver inflammation and injury attributable to innate immunity, activated through Toll-like receptors. Detailed characterization of Toll-like receptor sigaling cascades in the last...
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Enterohepatic circulation of bilirubin
Zelenka, Jaroslav ; Vítek, Libor (advisor) ; Entlicher, Gustav (referee) ; Červinková, Zuzana (referee)
Bilirubin is a main physiological product of heme degradation possessing important antioxidant and antiinflammatory properties. On the other hand, it could be neurotoxic during severe unconjugated hyperbilirubinemia combined with insufficiency of blood-brain barrier (neonatal jaundice). It is secreted from the body via bile and is further metabolized in the intestine. Part of the substance is reduced to urobilinoids, part is adsorbed to the intestinal content and some part could be reabsorbed back to the systemic circulation. This enterohepatically and enterosystemically circulating fraction varies in size depending on the rate of bilirubin secretion, solubility in the intestine and intensity of its intestinal metabolism. Under specific circumstances, EHC and ESC may significantly increase serum and bile bilirubin levels and influence physiological as well as pathological processes occuring in the body. Among the most important is the protective elevation of UCB levels in Gilbert syndrome subjects and dangerous increase in severity of neonatal jaundice. In the presented thesis, the mechanisms affecting EHC and ESC of bilirubin and tools for further research in BP metabolism were investigated. The solubility of intestinal UCB is strongly decreased by addition of divalent cations. However, such approach to...
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Study of signal systems with special focus on the role of nitric oxide: gene expression, regulation and pharmacological modulation in hepatocytes and adipocytes
Kutinová Canová, Nikolina ; Farghali, Hassan (advisor) ; Červinková, Zuzana (referee) ; Martásek, Pavel (referee)
Nitric oxide (NO) is involved in surprising array of vital physiological and pathophysiological phenomena. The expression of nitric oxide synthases, endothelial (eNOS) and inducible (iNOS), was demonstrated in rat liver and white adipose tissue among others. Therefore, the goals of the present thesis were to provide in vitro data about NO in the liver and white adipose tissue and to assess: 1) the effects of selective immunosuppressive drugs, cyclosporin A (CsA) and tacrolimus (FK 506), and nonspecific and specific iNOS inhibitors on NO production and iNOS expression during endotoxemic insult using primary rat hepatocyte culture; 2) spontaneous NO production under various culture conditions with comparison of its influence on functional status of hepatocytes in conventional cell culture and in hepatocyte bioreactors; 3) effects of S-nitroso-N-acetyl penicillamine (SNAP), D- galactosamine (D-GalN), lipopolysaccharide (LPS), LPS+D-GalN and thapsigargin (TG), a selective inhibitor of a sarco-endoplasmic reticulum-Ca2+ -ATPase, on apoptotic/necrotic markers in relation to NO production; 4) the effect of LPS on lipolysis in relation to iNOS stimulation; and 5) the interplay between NO production and β3-adrenoreceptor (β3- AR)/cAMP pathway on lipolysis in rat epididymal adipocyte culture. We found that CsA, FK...
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