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Immunohistochemical analysis of SMAD proteins expression in experimental atherogenesis.
Lasotová, Magdalena ; Nachtigal, Petr (advisor) ; Mičuda, Stanislav (referee)
Magdalena Lasotová Immunohistochemical analysis of expression of SMAD proteins in experimental atherogenesis. Diploma thesis Charles University in Prague, Faculty of Pharmacy in Hradec Králové Pharmacy Background: We observed the effect of atorvastatin on the expression of phosphorylated form of SMAD 3 in atherosclerotic plaques of ApoE / LDL recep- tor deficient mice. Methods: We used C57BL/6J female mice with double deficiency of apolipopro- tein E and LDL receptor. Animals were divided into two groups. For 8 weeks bo- th groups were fed a standard diet. Animals of the second group received addi- tion of atorvastatin 50 mg/1 kg/day. Blood samples were taken for biochemical analysis. Histological staining with oil red we have taken for the determination of lipids in atherosclerotic lesions. For immunohistochemical analysis were used samples containing semilunar valves with aorta. Detection of expression of SMAD 3 protein was performed using Avidin-Biotin method (ABC) with detecti- on using DAB. Results: Administration of atorvastatin significantly increased level of total cho- lesterol and VLDL cholesterol. Despite hypercholesterolemic effect the adminis- tration of atorvastatin resulted in significant reductions of atherosclerotic lesi- ons compared with the control group. Immunohistochemical...
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Immunohistochemical analysis of SMAD proteins expression in experimental atherogenesis.
Koubová, Michaela ; Mičuda, Stanislav (referee) ; Nachtigal, Petr (advisor)
Michaela Koubová Immunohistochemical analysis of SMAD protein expression in experimental atherogenesis Diploma thesis Charles University in Prague, Faculty of Pharmacy in Hradec Králové Pharmacy Background: The aim of this diploma thesis was to analyze SMAD 2/3 expression in mice atherosclerosis lesions and find differences in the expression between animals feeding with standard diet and diet with addition of atorvastatin. The aim was demonstrate whether atorvastatin influences SMAD 2/3 expression without hypolipidemic effects. Methods: Female C57BL/6J apolipoprotein E and LDL-receptor double deficient mice were used in the study. Biochemical analysis of blood samples, histological and immunohistochemical analysis of aorta were performed. For identification of SMAD 2/3 expression was used EnVision method with DAB visualization. Results: Biochemical analysis revealed that the eight-week administration of atorvastatin resulted in a statistically significant increase in total cholesterol. Histological analysis showed that administration of atorvastatin resulted in significant reductions in the size of atherosclerotic plaques compared with untreated group.Immunohistochemical analysis showed SMAD 2/3 expression in atherosclerotic plaques in both groups.Significant differences in the expression of SMAD...
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Spirulina platensis effects on atherogenesis in mouse model of atherosclerosis.
Hanusová, Kateřina ; Mičuda, Stanislav (referee) ; Nachtigal, Petr (advisor)
Spirulina platensis is a single cell blue-green algae of Cyanobacteria strain. Spirulina belongs among foodstuffs with the highest protein content and contains all essential amino-acids. It is also a source of some non essential amino-acids, important nutrients as for example gamma linol acid, a lot of vitamins (B1, B2, B6, biotin, etc.) and trace elements (e.g. selenium, chrome, iron, calcium). Moreover it contains natural pigments carotenoids, chlorophyll and phycocyanin. The aim of this thesis was to test potential hypolipidemic and anti- inflammatory effects of Spirulina platensis on an experimental animal model of apoE/LDLr deficient mice. Therefore the parameters of lipid spectrum in blood and VCAM-1 expressions in arterial endothelium were monitored. Mice with deficit of apolipoprotein E (apoE-/- ) and LDL receptor were weaned of and for two weeks fed with a standard diet. At the age of eight weeks they started to be fed with atherogenic diet containing 0.15% of cholesterol. This continued for eight weeks (control group). In Spirulina platensis group the mice were fed with the same atherogenic diet with daily addition of 20 mg of Spirulina platensis. Biochemical analysis of lipid spectrum as well as histochemical and imunohistochemical analyses of atherosclerotic plates and VCAM-1...
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Importance of Transport Proteins and Biotransformation Enzymes for Defending Role of the Placenta
Vacková, Zuzana ; Štaud, František (advisor) ; Trejtnar, František (referee) ; Mičuda, Stanislav (referee)
Placenta is a unique organ which ensures a number of vital functions necessary for normal course of pregnancy and development of a new individual. In addition to its main function of oxygen supply and nutrient and waste product exchange, placenta also serves as an endocrine, metabolic and protective organ. Placenta is considered to be one of the physiological barriers of the organism which regulates transport of both endogenous and exogenous compounds between two compartments - maternal and fetal blood circulations. Up to recently, the placental barrier was supposed to be formed only by cellular layers which separate maternal and fetal blood - syncytiotrophoblast and fetal capillary endothelium. However, it has been demonstrated that the activity of placental efflux transport proteins and metabolic enzymes contributes considerably to the protective function of placental barrier. Efflux transporters are membrane proteins which actively (along with consumption of ATP) "pump" a diversity of substrates out of the cell. It has been shown that the kinetics of transport of various substances across the placenta is affected predominantly by two transporters: P-glycoprotein (P- gp) and breast cancer resistance protein (BCRP). Compared to these transporters, placental biotransformation enzymes are considered...
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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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Novel approaches for development of in vitro liver cell models
Smutný, Tomáš ; Pávek, Petr (advisor) ; Mičuda, Stanislav (referee) ; Kollár, Peter (referee)
1 Abstract Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate: Tomáš Smutný, MSc. Supervisor: Prof. PharmDr. Petr Pávek, Ph.D. Title of doctoral thesis: Novel approaches for development of in vitro liver cell models. The liver is a main metabolizing organ in the body. Therefore, the evaluation of hepatic metabolism is a crucial step during drug development. Moreover, a liver damage induced by drugs is another task to be assessed in drug development. In vitro liver cell models allow addressing some of these concerns. Up to date, primary human hepatocytes are considered as a "gold" standard of in vitro liver cell models. Additionally, other liver model systems are used such as liver tissue slices, subcellular fractions, liver cancer cell lines and hepatocytes derived from stem cells. Despite the significant progress towards right estimation of pharmacokinetic and toxicological parameters of drug candidates during drug development, current in vitro systems still suffer from various drawbacks. One of these limitations is their insufficient similarity with in vivo-like phenotype associated with low metabolic capacity of the models. In last several years, we were victims of tremendous effort to improve existing models such as 3D models, co-culture and...
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Changes in the composition and localization of gangliosides in cholestasis associated with other markers of pathological processes in hepatocytes.
Petr, Tomáš ; Muchová, Lucie (advisor) ; Mičuda, Stanislav (referee) ; Befekadu, Asfaw (referee)
This thesis is focused on the study of glycosphingolipids in the rat liver in different types of cholestasis and the effect of oxidative stress on changes in the composition and localization of gangliosides. First, it was necessary to optimize the immunochemical detection of glycosphingolipids. GM1 ganglioside was selected as a representative of a large glycolipid family. We found that minimum water content in the fixing solution was a key condition for fixation of histological sections. Optimized method of GM1 detection was subsequently used in in vivo experiments. We have demonstrated that estrogen-induced cholestasis characterized by high concentrations of bile acids and increased oxidative stress caused changes in the synthesis and distribution of liver gangliosides. HMOX induction is associated with a reduction in oxidative stress level and accompanied by normalization in GSL content. In experiments with obstructive cholestasis, we found that changes in the distribution and synthesis of gangliosides were not strictly specific to a particular type of cholestasis. We assume that it represents a general mechanism of hepatoprotection. We also confirmed the important role of bilirubin, product of HMOX reaction, in protection of hepatocytes against oxidative damage caused by high concentrations of...
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