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Evaluation of antibody quality for Endoglin detection in liver
Kejzlarová, Eliška ; Svobodová, Zuzana (advisor) ; Tripská, Katarína (referee)
The diploma thesis deals with endoglin, a transmembrane glycoprotein that can be cleaved to form so-called soluble endoglin, which then circulates in the blood. The theoretical part of the thesis is devoted to cardiovascular and liver diseases related to changes in the expression or level of endoglin in the blood and summarizes current knowledge about endoglin and its relation to various diseases. The experimental part of the thesis describes the selection of a suitable antibody against endoglin intended for western blot (WB). We have four anti-endoglin primary antibodies available from three manufacturers. The purpose was to test the antibodies and determine, which one had the best ability to detect endoglin in mouse tissue samples such as liver or aorta on WB. Affiblot was used to verify their ability to bind endoglin and also to compare their affinities to this protein. Newly developed patented dot-blot-based device. The binding of the used antibodies to endoglin was verified using the WB method and then the results of both methods were compared. The obtained results showed that the Abcam antibody has the most suitable parameters regarding specificity and sensitivity for the identification of endoglin in mouse tissue on WB. Keywords: endoglin, soluble endoglin, NASH, cirrhosis, non-alcoholic...
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Immunohistochemical analysis of TRC105 effects on the liver fibrosis
Hamerníková, Veronika ; Němečková, Ivana (advisor) ; Svobodová, Zuzana (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department: Department of Biological and Medicinal Sciences Studijní obor: Pharmacy Author: Veronika Hamerníková Supervisor: RNDr. Ivana Němečková, Ph.D. Title of master thesis: Immunohistochemical analysis of TRC105 effects on the liver fibrosis The subject of this diploma thesis was the analysis of the expression of liver fibrosis biomarkers in mice liver. The aim of our work was to test the effect of the drug TRC105 (carotuximab) on a fibrotic liver model after 4 weeks of DDC (3,5-diethoxycarbonyl-1,4-dihydro-2,4,6-colidine) administration. Collagen deposition and expression of α-SMA, GFAP and endoglin were examined and compared. Animals were divided into 3 groups: control group (n=6), DDC group (n=6) and TRC105 group (n=6). The control group was fed a standard diet, while the DDC and the TRC105 groups were fed a diet with 0.1% DDC. Physiological solution was administered to the control and DDC groups while carotuximab was administered to the TRC105 group. The analysis of fibrosis biomarker expression was then performed on fixed liver sections. Collagen expression was evaluated by Picro-Sirius Red staining and α-SMA, GFAP and endoglin expression by immunohistochemistry with fluorescence imaging. Histological staining of collagen...
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Cholesterol depletion using reopheretic and apheretic processes and impact of these methods on immune system
Svrčková, Ellen ; Andrýs, Ctirad (advisor) ; Krejsek, Jan (referee)
LDL-apheresis and haemorheopheresis are the most frequent methods of extracorporeal ellimination methods used for lowering the LDL cholesterol in patients with familial hypercholesterolaemia (FH). In case of failure of conservative therapy (represented by pharmacotherapy and/or dietary regime) these methods reprensent the effective and accessible solution for the clinical status characterized by high morbidity and mortality as well. The lipid components are the most frequent observed markers for the effeciveness of intervention. However, immunity with its effector molecules plays also essential role and there is suppose, that could also reflect the state and progress of atherosclerosis in FH patients. The aim of this thesis was to evaluate the levels of selected immunological markers (plasmatic glykoprotein α-2-macroglobulin, IL-10, soluble endoglin, soluble apoptotic factor sAPO-Fas and soluble form of adhesive P-selectin) and their changes after LDL-apheresis and haemorheopheresis employing enzyme immunoanalysis and immuno nephelometry. Totally, 12 patients were involved in this study, 3 were treated by haemorheopheresis, the rest 9 received LDL-apheresis. As a results, significant decreases in serum levels of α-2- macroglobulin, soluble endoglin and sAPO-1/Fas were recorded. Observed changes of...
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Evaluation of endoglin and VCAM-1 expression and co-expression in aortas of apoE-deficient mice
Minaříková, Lucie ; Rathouská, Jana (advisor) ; Fikrová, Petra (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Evaluatiton of the expresion and co-expresion of endoglin and VCAM-1 in the aorta on apoE- deficient mice Diploma thesis Lucie Minaříková Supervisor: Mgr. Jana Rathouská Background: The aim of this work was to monitor the expression and a possible co-expression of endoglin (TGF-β receptor III) and VCAM-1 in mouse aortic endothelium. As an experimental model, we used the mouse strain C57BL/6J, that was genetically modified, and is characteristic by a deficit of apolipoprotein E. Methodes: In the study, we focused on testing the mouse strain C57BL/6J with gene knockout for apolipoprotein E in different stages of the atherosclerotic process. 10 weeks old female mice were divided into three groups and fed diets with different content of cholesterol. One experimental group was fed a standard diet (called "chow type diet") for a period of two months. The other two groups were fed a diet containing 21% fat (called "Western type diet") for a period of two and four months. For the determination of the levels of total cholesterol, a biochemical analysis of blood was performed. Obtained parts of ascending aorta were analyzed by ImmPRESSTM immunohistochemical method with the detection reagent DAB....
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Evaluation of endoglin and P-selectin expression and co-expression in aortas of apoE-deficient mice
Brlicová, Monika ; Rathouská, Jana (advisor) ; Kovařík, Miroslav (referee)
Charles University in Prague Faculty of Pharmacy in HradecKrlov Department of Biological and Medical Sciences Evaluation of endoglin and P-selectin expression and co-expression in aortas of apoE-deficient mice Diploma thesis MonikaBrlicov Supervisor: Mgr.JanaRathousk Background: We observed the expression and the reciprocal co-expression of endoglin (receptor III for TGF- cytokine) and P-selectin (adhesion molecule and marker of endothelial dysfunction) in ascending aortas of apoE-deficient mice which were fed by standard diet for rodents and Western type diet (high-cholesterol diet) for achieving of different phases of the atherosclerotic process. The changes of total cholesterol levels in mice after administration of both types of diets were also evaluated. Methods: The modified strain C57BL/6J of mice with a deficiency of apolipoprotein E, which is prone to aterogenesis was used for this diploma thesis. Mice were divided into three groups. The first group was fed by standard diet (so-called "chow" diet) for a period of two months and the second two groups were fed by Western type diet for a period of two and four months. The levels of total cholesterol in the blood were biochemically determinated and then we statistically evaluated this levels in all groups. Immunohistochemical...
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Endoglin and livers
Jozefčeková, Nikola ; Doleželová, Eva (advisor) ; Prašnická, Alena (referee)
Author: Nikola Jozefčeková Title: Endoglin and liver Form: Diploma Thesis University: Charles University, Faculty of Pharmacy in Hradec Králové Degree: Pharmacy This diploma thesis concludes the available information about endoglin, its isoforms in the liver and its impact on the liver during various pathological conditions. In the first part of the thesis describes morphology and physiology of the liver, its structure, histology and metabolic functions. Second part contains an information about endoglin, its isoforms and role in TGF-β signaling complex, expression of endoglin and regulation of the expression. In the third part are described liver diseases during which is expression of endoglin changed. This part deals with the significance of endoglin as a diagnostic and prognostic marker. Endoglin (CD105, TGF-β receptor III) is a homodimeric transmembrane glycoprotein and a membrane co-receptor TGF-β with high expression in endothelial cells. Endoglin plays an important role in the vascular development. Endoglin is involved in the processes of angiogenesis, vascular homeostasis and TGF-β signalization. It affects activity of TGF-βRII, ALK1 and ALK5 receptors. Due to the interactions with TGF-β complex, modulation of activity of ALK receptors and Smads, endoglin controls fibrotic and anti-...
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Morphological characteristics of alterations in lungs in a mouse model of atherosclerosis.
Vyskočilová, Martina ; Němečková, Ivana (advisor) ; Blažíčková, Kateřina (referee)
Bachelor thesis Morphological characteristics of alterations in lungs in a mouse model of atherosclerosis Author: Martina Vyskočilová Study course: Medical laboratory technician Charles University Faculty of Pharmacy in Hradec Králové Background: Atherosclerosis is a multifactorial disease caused by deposition of blood lipids into the intima layer of blood vessels. Due to the fact that process of atherogenesis is associated with immune reactions, disease is considered as an inflammatory process. Atherosclerotic plaques rarely affect pulmonary arteries, where this condition is accompanied by an increase of pulmonary pressure, which is very likely the trigger of disease. Aim of this study is to find out whether there are morphological changes in the lungs of ApoE/LDLr-/- mouse model of atherosclerosis, characteristic for pulmonary atherosclerosis, compared to lungs of healthy mice. Methods: Lungs of ApoE/LDLr-/- and healthy C57BL/6J control mice have been fixed in 4% neutral formaldehyde, embedded in paraffin and sliced into 4 μm thin sections. One of the sections has been stained with hematoxylin-eosin, the other one with green trichrome. Staining with hematoxylin-eosin is primary staining used for basic imaging of histological structure of tissues, staining with green trichrome is used for imaging...
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Evaluation of endoglin and P-selectin expression and co-expression in aortas of apoE-deficient mice
Brlicová, Monika ; Rathouská, Jana (advisor) ; Kovařík, Miroslav (referee)
Charles University in Prague Faculty of Pharmacy in HradecKrlov Department of Biological and Medical Sciences Evaluation of endoglin and P-selectin expression and co-expression in aortas of apoE-deficient mice Diploma thesis MonikaBrlicov Supervisor: Mgr.JanaRathousk Background: We observed the expression and the reciprocal co-expression of endoglin (receptor III for TGF- cytokine) and P-selectin (adhesion molecule and marker of endothelial dysfunction) in ascending aortas of apoE-deficient mice which were fed by standard diet for rodents and Western type diet (high-cholesterol diet) for achieving of different phases of the atherosclerotic process. The changes of total cholesterol levels in mice after administration of both types of diets were also evaluated. Methods: The modified strain C57BL/6J of mice with a deficiency of apolipoprotein E, which is prone to aterogenesis was used for this diploma thesis. Mice were divided into three groups. The first group was fed by standard diet (so-called "chow" diet) for a period of two months and the second two groups were fed by Western type diet for a period of two and four months. The levels of total cholesterol in the blood were biochemically determinated and then we statistically evaluated this levels in all groups. Immunohistochemical...
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Evaluation of endoglin and VCAM-1 expression and co-expression in aortas of apoE-deficient mice
Minaříková, Lucie ; Rathouská, Jana (advisor) ; Fikrová, Petra (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Evaluatiton of the expresion and co-expresion of endoglin and VCAM-1 in the aorta on apoE- deficient mice Diploma thesis Lucie Minaříková Supervisor: Mgr. Jana Rathouská Background: The aim of this work was to monitor the expression and a possible co-expression of endoglin (TGF-β receptor III) and VCAM-1 in mouse aortic endothelium. As an experimental model, we used the mouse strain C57BL/6J, that was genetically modified, and is characteristic by a deficit of apolipoprotein E. Methodes: In the study, we focused on testing the mouse strain C57BL/6J with gene knockout for apolipoprotein E in different stages of the atherosclerotic process. 10 weeks old female mice were divided into three groups and fed diets with different content of cholesterol. One experimental group was fed a standard diet (called "chow type diet") for a period of two months. The other two groups were fed a diet containing 21% fat (called "Western type diet") for a period of two and four months. For the determination of the levels of total cholesterol, a biochemical analysis of blood was performed. Obtained parts of ascending aorta were analyzed by ImmPRESSTM immunohistochemical method with the detection reagent DAB....
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Cholesterol depletion using reopheretic and apheretic processes and impact of these methods on immune system
Svrčková, Ellen ; Andrýs, Ctirad (advisor) ; Krejsek, Jan (referee)
LDL-apheresis and haemorheopheresis are the most frequent methods of extracorporeal ellimination methods used for lowering the LDL cholesterol in patients with familial hypercholesterolaemia (FH). In case of failure of conservative therapy (represented by pharmacotherapy and/or dietary regime) these methods reprensent the effective and accessible solution for the clinical status characterized by high morbidity and mortality as well. The lipid components are the most frequent observed markers for the effeciveness of intervention. However, immunity with its effector molecules plays also essential role and there is suppose, that could also reflect the state and progress of atherosclerosis in FH patients. The aim of this thesis was to evaluate the levels of selected immunological markers (plasmatic glykoprotein α-2-macroglobulin, IL-10, soluble endoglin, soluble apoptotic factor sAPO-Fas and soluble form of adhesive P-selectin) and their changes after LDL-apheresis and haemorheopheresis employing enzyme immunoanalysis and immuno nephelometry. Totally, 12 patients were involved in this study, 3 were treated by haemorheopheresis, the rest 9 received LDL-apheresis. As a results, significant decreases in serum levels of α-2- macroglobulin, soluble endoglin and sAPO-1/Fas were recorded. Observed changes of...
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