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Aspects of Gene Regulation of CYP3A4 in Hepatic Tissue.
Krausová, Lucie ; Štaud, František (advisor) ; Mičuda, Stanislav (referee) ; Skálová, Lenka (referee)
1 Summary CYP3A4 is an important enzyme involved in elimination of majority of metabolized xenobiotics. It plays a major role in the detoxification system of the human body, therefore it is responsible for many drug-drug interactions (DDIs). DDI present a complication of current pharmacotherapy, in the extreme they can lead in failure of therapy or in life-threatening toxic effects. DDIs are caused by changes in enzymatic activity of CYP3A4, which is highly variable among individuals. An important mechanism of modulating CAP3A4 activity is the regulation of inducible transcription by nuclear receptors, especially PXR, CAR and GR. The structure of CYP3A4 promoter and mechanisms of transcriptional regulation has been studding intensively for many years, but the research of relationship of nuclear receptors and transcriptional cofactors in CYP3A4 transactivation is still incomplete. Present work contributes to elucidation of some questions concerning the effects of azole antimycotics on CYP3A4 transcription via PXR, potency of valproic acid to activate PXR and CAR or determinants of CYP3A4 expression via GR in placental cells. The experiments were performed with up-to-date molecular biology methods and using in vitromodels of the primary human hepatocytes and hepatoma cell lines. To the aims of the doctoral...
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Influence of Cholestatic Liver Injury on the Elimination and Transport of Drugs
Brčáková, Eva ; Štaud, František (advisor) ; Tilšer, Ivan (referee) ; Lotková, Halka (referee)
The liver is a unique organ with a number of vital functions. Pivotal one is its participation on bile formation and secretion, import, detoxification and excretion of endogenous substances and xenobiotics. Bile formation is essential for both absorption of lipids in intestine and excretion of various endogenous compounds and xenobiotics (e.g. bile acids, bilirubin, cholesterol, phospholipids and drugs). This function is markedly impaired during extrahepatic and intrahepatic cholestasis with partial or complete stoppage of bile flow. Consequently, hepatic and further systemic accumulation of toxic biliary constituents, such as bile acids and bilirubin, occurs. In an effort to compensate this situation, spontaneous anti-cholestatic mechanisms are activated, which provide alternative excretory routes for toxic accumulating compounds (e.g. renal elimination of bile acids and xenobiotics into urine). These mechanisms include changes in the expression, localization and function of respective transporters in liver and kidneys. Another mechanism with a significant impact on bile formation and transport of compounds between bile and blood is blood-biliary barrier formed by connection of hepatocytes by "tight-junctions" and "gap-junctions". While "gap-junctions" exchange substances among cells,...
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The effect of daunorubicine on expresion of adhesive molecules in rabit model of anthracycline toxicity
Nosková, Zuzana ; Nachtigal, Petr (advisor) ; Štaud, František (referee)
The aim of this thesis was to evaluate possible changes of cell adhesion molecules expression in rabbit aorta after the administration of Daunorubicin. We focused on the expression of VCAM-1 and ICAM-1. Chronic anthracycline cardiotoxicity was induced by repeated administration of daunorubicinu (3 mg/kg=50 mg/m2 i.v., 1x week) for the period of 10 weeks. We focused on the monitoring VCAM-1 and ICAM-1 expression in rabbit aorta. Ten daunorubicin groups were compared with control rabbits. The animals were killed 24 hours and/or 7 days after the administration of daunorubicin. Immunohistochemical analysis showed no expression of VCAM-1 in any control or experimental groups. On the contrary the expression of ICAM-1 was detected in control and experimental groups. There was no significant difference between experimental and control rabbits with respect to the intensity and staining patterns. In conclusion the administration of daunorubicin did not affect either VCAM-1 or ICAM-1 expression in aorta suggesting that endothelial dysfunction in aorta is not triggered by daunorubicin in vivo.
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Pharmacological influence of endothelial dysfunction in experimental model of diabetic rat
Přidalová, Hana ; Nachtigal, Petr (advisor) ; Štaud, František (referee)
The aim of this thesis was to evaluate the expression of cell adhesion molecules in femoral artery after induction of diabetes and after the administration of sulodexide. We focused on the expression of PECAM-1, ICAM-1 and VCAM-1 vessel endothelium. The analysis was provided by means of immnohistochemical staining. Male Wistar rats were divided into three groups. Control group received saline, the second group received sulodexide (SLX, 100 UI/kg/den). Diabetes was induced by the streptozotocine injections (30mg/kg/den i.p.) during consecutive free days. The treatment was administered for 10 weeks. Biochemical results confirmed the induction of diabetes; however the suledoxide treatment did not decrease glucose levels when compared with non-treated diabetic animals. The ICAM-1 expression was slightly increased in diabetic treated rats when compared with control rats however suledoxide treatment did not affect ICAM-1 expression. VCAM-1 expression was not detected in any rat from control, diabetes and/or suledoxide treated rats. In conclusion the results of this pilot study showed that diabetes induced by the administration of streptozotocine did not resulted in significant induction of endothelial dysfunction. Thus the possible effect of suledoxide treatment could not be evaluated in this study.
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Gene silencing of ICAM-1 by lysine modified oligonucleotides
Kocourková, Aneta ; Štaud, František (advisor) ; Nachtigal, Petr (referee)
Conjugation of ligands to antisense oligonucleotides is a promising approach for enhancing their effects on gene expression. In this study 2'-O-lysylaminohexyl group was linked to the uridine base, which replaces one, two or three thymine bases thus modifies the oligonucleotides. This exchange of bases was tested for improvement of silencing target protein expression. Effectivity of modifications in silencing target protein expression was examined with the alicaforsen sequence (DNA) and siRNA. Alicaforsen, currently in clinical trial 3, is a phosphorothioate targeting ICAM-1, which was the model used to evaluate the influence of modifications. The same target was chosen for siRNA to compare the efficiency of DNA and siRNA substances. For the first time, down-regulation of ICAM-1 was shown on the blood brain barrier cell line ECV304. Unmodified/modified antisense oligonucleotides and siRNA sequences were transfected into ECV304 cells with the help of a transfection agent lipofectamine 2000. After 24 hours of transfection cells were disrupted by a chemical lysis. Protein concentrations were determined by Bradford protein assay. ICAM-1 inhibition was assessed with western blot. The inhibitory effect of ICAM-1 was normalized to the corresponding actin and untreated cells. ICAM-1 protein levels were...
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Side-effects of siRNA - induced gene silencing
Feřtek, Marina ; Štaud, František (advisor) ; Nachtigal, Petr (referee)
Vedlejší účinky siRNA byly testovány na buněčných liniích po "umlčení" genu kódujícího cyklooxygenázu. Teoretickým základem tohoto experimentu byl poznatek, že siRNA mohou vyvolat imunitní odpověď. Z tohoto důvodu byla testována exprese IFN-indukovaných genů v intaktní Hep2 buněčné linii a ve třech liniích odvozených od Hep2 buněk, kde byla různými metodami vnesena siRNA. Buněčné linie byly odvozeny z Hep2 pomocí různých metod transfekce siRNA (tj. nuzkleofekce, effectene technologie a klonování). RNA byla izolována z těchto buněčných linií a cDNA byla získaná reverzní transkripcí. Genová exprese byla měřena pomocí QRT PCR a kvantifikována jako počet kopií/mikrogram RNA. Stupeň exprese byl porovnáván s intaktní linií Hep2. Jako houskeeping gen byl použit NUP. Výsledky byly vyhodnoceny pomocí softwarů REST verze 2 a rotoru Gene verze 6. Mělo být testováno devět IFN-indukovaných genů, RNL, PKR, IRF1, IRF3, IFITM, IFIT1, OAS1, OAS2 a OAS3. Z nich OAS1 a OAS2 nebylo možné izolovat a tedy měřit. Exprese PKR byla up-regulovaná; exprese všech ostatních testovaných genů byly down-regulovány. Další rozsáhlé studie jsou nezbytné pro vysvětlení těchto výsledků.
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Dysfunction of Endothelium in Relationship to Experimentally Induced
Černý, Dalibor ; Nachtigal, Petr (advisor) ; Štaud, František (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Rigorous Thesis - Abstract Name: Mgr. Dalibor Černý Year: 2007 Dysfunction of Endothelium in Relationship to Experimentally Induced Anthracycline Drugs' Toxicity Endothelial dysfunction is defined as functional lesion of endothelium, characterized by increased permeability of vessel wall, by imbalance among vasoactive, coagulative and proliferation influencing active substances, in result leading to increased expression of cell adhesion molecules VCAM-1 and ICAM-1, which are considered as standard morphological markers of its progress. Adhesion molecules VCAM-1 and ICAM-1 play an important role in building-up of inflammatory reaction and take part in various pathological stages. They are expressed by endothelial cells, by scavenger cells, macrophages and smooth muscle cells. The aim of this rigorous thesis was to examine whether long-lasting daunorubicin administration leads except of significant changes in heart also to the progress of endothelial dysfunction. Imunohistochemical methods were used to detect the expression of these adhesion molecules. Then, the selected heart parameters like LVEF (left ventriculum eject fraction), FS (fractional shortening) and dP/dtmax index, that means peak...
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Effect of combined statin therapy MDOCTM and the atherogenic / / process in an experimental model of atherosclerosis
Metelková, Marie ; Nachtigal, Petr (advisor) ; Štaud, František (referee)
The effects of combination treatment of MDOCTM and statin on atherogenic process in experimental model of atherosclerosis. Mgr. Marie Metelková MDOC™ is micro dispersed derivatives of oxidized cellulose (polyanhydroglucuronic acid - PAGA). This semi-natural, biocompatible, bioabsorbable, non-acidic, sterile powder has been in use as a topical haemostatic agent since 1998. In this rigorous work we wanted to evaluate whether MDOC™ affects cholesterol levels, inflammatory markers and cell adhesion molecule expression in both blood and vessel wall in cholesterol fed apoE-deficient mice. Male apoE-/- mice were divided into 4 groups. Control group (n=8) of mice consumed an atherogenic diet for 4 weeks. The same diet was used in other animals except MDOC™, atorvastatin and MDOC™ together with atorvastatin were added to the diet. Analysis of blood cholesterol, IL-6 in blood and ICAM-1 endothelial expression in aortic sinus were performed by means of biochemical, ELISA and immunohistochemical analysis. LDL cholesterol levels were significantly decreased in all drug treated groups when compared with control animals. Moreover MDOC™ treatment significantly increased HDL cholesterol in comparision to control group. ELISA analysis revealed significant decrease of IL-6 levels MDOC™ group and atorvastatin group....
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