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Liver gangliosides in cholestasis induced by bile duct ligation.
Hynková, Barbora ; Entlicher, Gustav (advisor) ; Ledvinová, Jana (referee)
Gangliosides are sialic acid-containing glycosphingolipids located on the cell surface of all animal cell types. They play a role as receptor molecules, share in cell-to-cell interaction and protect the cell against harmful environmental factors by increasing of rigidity of cell surface. This diploma thesis studies an influence of experimental cholestasis on hepatic ganglioside composition. Cholestasis was induced by bile duct ligation in Wistar rats. A significant increase of total lipid bound sialic acid and b-series gangliosides (GD1b, GT1b, event. GD3) was found in cholestatic liver when compared with controls. These results found in obstructive cholestasis correspond with the results Majer et al. Biomed. Chromatogr., 21, 446-450 (2007), described in 17ethinylestradiol induced cholestasis, but the increase of b- series gangliosides was milder in our study. As a second point, an effect of modulated heme-oxygenase 1 (HO-1) activity was investigated in cholestatic rats (HO-1 activator- hemine, HO- 1 inhibitor- Sn- mesoporphyrin). An increase of a total lipid sialic acid was found in Sn-mesoporphyrin treated animals but without significant changes in gangliosides composition. Lipid sialic acid and gangliosides were not changed in animals with hemine activated HO-1. Expression of mRNA of key...
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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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The effect of free radicals on ageing
Lukšanová, Hana ; Wilhelm, Jiří (advisor) ; Entlicher, Gustav (referee) ; Macháčková, Ivana (referee)
Cílem této studie bylo ověřit platnost Harmanovy teorie stárnutí, založené na působen í volných ra dikálů, na nových modelech stárnutí rostlin a kvasinek, jelikož tyto organizmy byly nedávno přijaty mezinárodní komunitou jako uznávané modely pro studium mechanizmu stárnutí na molekulární úrovni. Vzhledem k tomu, že v Harmanově teorii stárnutí má centrální význam endogenní produkce volných radikálů, zaměřila jsem se ve své studii na sledování časového průbě hu vzniku jejich koncových produktů . Paralelně jsem sledovala změny antioxid a ční ch systémů, které rozhodují o rozsahu poškození vyvolaném volnými radikály. V poslední době se ukazuje, že procesů stárnutí se účastní i reaktivní slo u čen iny dusíku, a proto jsem zaměři l a pozornost i na mechanizmy zahrnující účast těchto sloučenin. Jako modelové organizmy jsem použila fazol (Phaseo/us vu/garis L.), trasgenní rostliny tabáku (Nicotiana tabacum L. ) s pozměněnou koncentrací cytokininů, husen íček rolní (Arabidopsis tha/iana) a kvasinky (Saccharomyces cerevisiae). Powered by TCPDF (www.tcpdf.org)
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Recombinant aspartic proteases of blood-feeding parasites
Váchová, Jana ; Entlicher, Gustav (referee) ; Konvalinka, Jan (advisor)
The blood fluke Schistosoma mansoni and the hard tick Ixodes ricinus produce an aspartic protease cathepsin D which initiates degradation of hemoglobin, their key nutrient. First, in the presented work, the protocol for refolding and activation of the zymogen of cathepsin D from I. ricinus (IrCatD) was developed and optimized. In acidic pH the propeptide of IrCatD zymogen was removed by an auto-activation mechanism. Further, a kinetic assay with fluorogenic substrates was employed to study functional properties of IrCatD including pH optimum, substrate and inhibition specificities. Second, two isoforms of cathepsin D from S. mansoni (SmCatD) were produced using recombinant expression in E. coli. These recombinant proteases were isolated from inclusion bodies using affinity chromatography under denaturating conditions, and protocol for their refolding was developed and optimized. The studied aspartic proteases are pharmacological targets: inhibitors of SmCatD represent potential chemotherapeutics for the treatment of schistosomiasis, and IrCatD is a candidate antigen for the development of novel anti-tick vaccines.
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Cathepsin L from the hard tick Ixodes ricinus
Talacko, Pavel ; Entlicher, Gustav (referee) ; Konvalinka, Jan (advisor)
Ticks are globally important parasites involved in transmission of a wide variety of infectious agents. The most common tick species found in Europe is the hard tick Ixodes ricinus, which transmits bacterium Borrelia burgdorferi (a causative agent of Lyme disease) or tick-borne encephalitis virus. Cathepsin proteases are important in the process of digestion of blood proteins in the tick gut. This work is focused on cathepsin L, an important digestive cysteine protease of ticks. Recombinant I. ricinus cathepsin L was expressed in Pichia pastoris and separated from the culture medium by chromatographic purification. N-terminal protein sequencing and labeling by activity-based probe Green-DCG-04 were used for characterization of purified cathepsin L. Substrate and inhibitor specificity were analyzed using peptide substrates and inhibitors. This analysis showed that Z-FR-AMC is a suitable substrate with pH optimum 3.5, and that Z-FF-DMK is an efficient inhibitor. It was demonstrated that cathepsin L cleaves protein substrates in strongly acidic environment (pH 3.5-4.5). Cathepsin L-like proteolytic activity was demonstrated in salivary gland extract and in saliva of the I. ricinus tick. The presence of a cathepsin protease in tick saliva is reported here for the first time. This finding suggests that...
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The Role of Human Sco1, Sco2, Surf1 and Oxa1l in the Biogenesis of the Mitochondrial Oxidative Phosphorylation System
Stibůrek, Lukáš ; Zeman, Jiří (advisor) ; Entlicher, Gustav (referee) ; Pelouch, Václav (referee)
Disertační práce byla vypracována v Laboratoři pro studium mitochondriálních poruch, Kliniky dětského a dorostového lékařství a 1.LF UK v Praze. Studium mitochondriální biogeneze a jejích poruch, a to především s ohledem na biogenezi systému oxidativní fosforylace (OXPHOS), zaznamenalo v uplynulém desetiletí mimořádný pokrok. Znalost kompletní sekvence lidského genomu, spolu s výsledky elegantních kvasinkových studií zaměřených na identifikaci respiračně významných genových produktů, dovolila molekulární identifikaci a následné studium biochemické podstaty celé řady lidských mitochondriálních patologií. Tyto studie odhalují molekulární a biochemické mechanismy etiopatogeneze těchto chorob a současně přinášejí nové unikátní informace o lidské mitochondriální biogenezi jako takové. Výsledky a původní publikace prezentované v této disertační práci se týkají snahy o pochopení molekulárních rolí lidských asemblačních faktorů Sco1, Sco2, Surf1 a Oxa1l v biogenezi multimerních membránových komplexů lidského systému OXPHOS. Prezentovaná data ukazují, že lidské asemblační faktory Sco1 a Sco2 fungují vysoce tkáňově specifickým způsobem na úrovni odlišných posttranslačních kroků maturace podjednotky Cox2 cytochrom c oxidázy (CcO). Prezentovaná data dále naznačují, že oba výše zmíněné lidské SCO proteiny, a asemblační...
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