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Evaluation of activity of potentional antimycotic substances through the use of microdilution broth method III
Zavřelová, Alena ; Konečná, Klára (advisor) ; Němečková, Ivana (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Title of thesis: Evalution of activity of potentional antimycotic substances through the use of microdilution broth method III Autor of thesis: Alena Zavřelová Supervisor: Mgr. Klára Konečná, Ph.D. Background: The aim of this thesis, due to the growing incidence of fungal diseases and the growing incidence of resistance, was to determine the activity of potentially antimycotic active substances. We tested newly synthesized compoused, structurally based on the substance of pyrazinamide which is significant antituberculotic. Methods: Testing of substances was carried out using a broth microdilution method on eight strains of pathogenic fungi consisting of five yeast strains: Candida albicans, Candida tropicalis, Candida krusei, Candida glabrata, Trichosporon asahii and three strains of filamentous fungi: Aspergillus fumigatus, Absidia corymbifera, Trichophyton mentagrophytes. Results: From the range of tested concentrations and from the total number of the thirty tested substances, the most noticeable antifungal aktivity was revealed in the substance of 5-methyl-6-(2-phenylhydrazinyl)pyrazine-2,3- dicarbonitrile. All strains, except Trichophyton mentagrophytes strain, showed a relatively...
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Optimalisation of methods for determination of CD44 receptor activation
Skalická, Nela ; Němečková, Ivana (advisor) ; Konečná, Klára (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Title of diploma thesis: Optimalisation of methods for determination of CD44 receptor activation. CD44 is a transmembrane glycoprotein present in most cell types which is involved in many physiological and pathological processes and it represents the main hyaluronic acid (HA) receptor. HA is a nonsulfated glycosaminoglycan that is present extensively in extracellular matrix, where it provides cell hydratation. HA is involved in cancer, affects cell migration, supports an angiogenesis and participates in wound healing. In this study, we focused on the optimization of methods for the evaluation of activation, the amount and cleavage of CD44 receptor in HT-29 cell line. We observed the total amount of the receptor and its cleaved domains using methods Western Blot, ELISA and flow cytometry. We used HA molecular weight 71 kDa and PMA (Phorbol- 12-Myristate-13-Acetate) for activation. Time-dependent changes appeared in the amount of total CD44 and its fragments after activation. We also observed cell death depending on the presence of HA and PMA. We demonstrated no significant effects of these substances on cell viability. We used fluorescent and confocal microscopy for visual evaluation of...
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C-reactive protein effects on aortic endothelium in spontaneously hypertensive rats
Kosán, Aleš ; Němečková, Ivana (advisor) ; Doleželová, Eva (referee)
C-REACTIVE PROTEIN EFFECTS ON AORTIC ENDOTHELIUM IN SPONTANEOUSLY HYPERTENSIVE RATS Diploma thesis Author: Aleš Kosán Study program: Pharmacy Charles University in Prague Faculty of Pharmacy in Hradec Králové Endothelial dysfunction and chronic inflammation are factors figuring in pathogenesis of metabolic syndrome. This paper is outlined as a pilot study, which represents basis for further research in the question of C-reactive protein influence on endothelium. The expression of four proteins previously associated with the development of endothelial dysfunction, was studied using the Western blot method: endothelin-1, heme oxygenase-1, endoglin and phosphorylated nitric oxide synthase. Analysis was carried out on the samples of homogenized rat aortas of transgenic spontaneously hypertensive rats with expression of human C-reactive protein. A standard spontaneously hypertensive rat strain was used as a control group. The detection was made by chemiluminescence substrate on the X-ray films and data were semiquantitatively evaluated by densitometry. We found increased expression in endoglin levels, valued at 186.3 % contrary to the control group. The expression of phosphorylated nitric oxide synthase was 325.6 % in comparison with the control group. The increase of expression was minimal (109.0 %) in...
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Soluble endoglin effects on endothelial dysfunction markers in mouse aorta
Kalmanová, Lenka ; Nachtigal, Petr (advisor) ; Němečková, Ivana (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Title of Diploma Thesis: Soluble endoglin effects on endothelial dysfunction markers in mouse aorta Author of Diploma Thesis: Lenka Kalmanová Supervisor of Diploma Thesis: prof. PharmDr. Petr Nachtigal, Ph.D. Background: The aim of the presented diploma thesis was to identify and subsequently evaluate the expression of endothelial dysfunction markers (ICAM-1, VCAM-1, P- selectin) in aortas of two groups of transgenic mice. The difference between control and experimental group was in the level of human soluble endoglin. Methods: 12 months old male mice of transgenic strain CBAxC57BL/6J with inserted genetic information for the expression of human soluble endoglin were utilized for the experiment. The mice were divided into two groups - the experimental group (high levels of sEng) and the control group (low levels of sEng) by ELISA analysis of levels of sEng in plasma. Both groups of mice were fed with standard laboratory rodent diet. Western blot analysis of adhesion molecules ICAM-1, VCAM-1 and P-selectin in mouse aorta and subsequently ELISA analysis of plasma levels of sVCAM-1 molecule were performed. Total cholesterol and triacylglycerol levels were demonstrated by biochemical analysis....
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Optimalization of Leishmania antigen formulations for the diagnosis of canine leishmaniasis.
Danková, Barbora ; Němečková, Ivana (advisor) ; Vokřál, Ivan (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Student: Barbora Danková Supervisor of diploma thesis: RNDr. Ivana Němečková, Ph.D. Specialized Supervisor: Prof. Anabela Cordeiro da Silva Title of diploma thesis: Optimalization of Leishmania antigen formulations for the diagnosis of canine leishmaniasis Leishmaniasis is a vector-borne disease caused by a protozoan parasite of the genus Leishmania and is prevalent in 98 countries worldwide. Reliable and accurate tests are necessary for laboratory diagnosis of Leishmania infection because of the wide spectrum of clinical characteristics and symptoms and high rate of asymptomatic infections that may occure. Serological diagnosis of canine leishmaniasis (CanL), especially enzyme-linked immunosorbent assay (ELISA), proved to be usefull tool. Data obtained from ELISA assay for the detection of antibodies against selected recombinant A, B, C, D, E and F proteins were evaluated in infected canine sera from Portugal and Brazil. This work presents the best formulation of recombinant proteins for serological diagnosis. We found that antigen C showed high level of sensitivity and specificity in recognition of positive and asymptomatic sera infected with CanL. Key words: canine leishmaniasis,...
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Alteration of transport proteins expression during obstructive cholestasis in rats II
Skořepová, Zuzana ; Doleželová, Eva (advisor) ; Němečková, Ivana (referee)
5 Abstract Zuzana Skořepová Alteration of transport proteins expression during obstructive cholestasis in rats II Diploma thesis Charles University in Prague, Faculty of Pharmacy in Hradec Králové Pharmacy Background: The aim of the diploma thesis was the verification of cholestatic liver impairment induced by bile duct obstruction lasting for 28 days in rats, by analysis of mRNA and protein expression of liver efflux transport proteins (Bsep, Bcrp, Mrp2, Mdr1, Mdr2, Mrp3, Mrp4). Methods: Wistar rats (n = 6, in each group; weighing 280 to 320 g) were divided into two groups: control rats were sham-operated (Sham) and BDO group (bile duct obstruction lasting for 28 days). mRNA and protein expression changes of the transporters were perfomed by qRT-PCR and Western blot. Results: As compared to Sham group, BDO group showed increase of mRNA expression of Mdr2 transporter to 303%, Mdr1b to 340% and Mrp3 to 9900%. Mrp2 mRNA expression was decreased to 42%. Obstructive cholestasis led to significant increase of protein levels - Bcrp to 160%, Mdr1 to 1010%, Mrp3 to 940% and Mrp4 to 140%. Mrp2, Bsep and Mdr2 protein levels were increased to 31%, 59% and 82%, respectively. Conclusion: Obstructive cholestasis led to decrease of canalicular liver transporters expression and increase of basolateral liver transporters...
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Screening for systemic amyloidoses in endoscopic biopsy samples.
Květoň, Martin ; Nachtigal, Petr (advisor) ; Němečková, Ivana (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Diploma thesis Candidate: Bc. Martin Květoň Branch of study: Specialist in Laboratory Methods Supervisor: prof. PharmDr. Petr Nachtigal, Ph.D. Title: Screening for systemic amyloidoses in endoscopic biopsy samples BACKGROUND Systemic amyloidoses constitute a complex and heterogeneous group of serious diseases that result from protein misfolding and whose common feature is the deposition of pathological protein substances (amyloid) in the extracellular space of tissues. Diagnostics of amyloidoses is based on histopathological examination of tissue samples and direct demonstration of the presence of amyloid deposits. The overall purpose of this diploma thesis was to create, apply and subsequently evaluate a screening program focused on systemic amyloidoses. METHODS In the first part of the experimental study, available staining techniques used for the detection of amyloid deposits in the tissue were compared. The second part of the experimental study consisted of performing screening examinations on endoscopic biopsy samples of the gastrointestinal tract. RESULTS Based on the results of comparison, a method employing the Sirius red F3B dye was chosen as a suitable screening technique....
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Immunochemical Detection of PECAM-1 Expression in Hypertensive Rats
Hocková, Erika ; Nachtigal, Petr (advisor) ; Němečková, Ivana (referee)
6 ABSTRACT Charles University in Prague, Faculty of Pharmacy in Hradec Kralove Department of Pharmacology and Toxicology Candidate: Mgr. Erika Hocková Consultant: Doc. PharmDr. Petr Nachtigal, Ph.D. Title of Thesis: Immunochemical detection of PECAM-1 expression in hypertensive rats Hypertension - abnormally high blood pressure - is a lifestyle disease, which belongs to the most frequent cardiovascular diseases. Hypertension seems to be a non-infectious epidemy with 25 to 30 % prevalence in adult population and presents a serious health problem. PECAM-1 is an adhesive signal molecule expressed on hematopoetic and endothelial cells, creating the main component of the vascular tissue. Regarding its structure, it belongs to the immunoglobuline super-family of adhesive molecules. Sunitinib is a biological treatment molecule of the group of tyrosine-kinase inhibitors used for treatment of generalised renal carcinoma and gastrointestinal stromal tumours. Farmacologically, sunitinib is antineoplastic substance - protein-kinase inhibitor. The main adverse effect of sunitinib is hypertension. The aim of this rigorous thesis was to define and to describe the expression of endothelial PECAM-1 in the aorta of spontaneously hypertensive rats (SHR) and their normotensive control group (Wistar - Kyoto rats - WKY)...
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Influence of fixation time and fixation type on immunohistochemical analysis in the detection of stem cells in endometrial tissue.
Macháčová, Dominika ; Nachtigal, Petr (advisor) ; Němečková, Ivana (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Title of Diploma Thesis: Influence of fixation time and type of fixative solution on immunohistochemical analysis in the detection of stem cells in endometrial tissue Author: Bc. Dominika Macháčová Supervisor of Diploma Thesis: prof. PharmDr. Petr Nachtigal, Ph.D. Backgroud: The aim of this diploma thesis was to investigate the effect of the type of fixative solution and the length of fixation on immunohistochemistry. Materials and methods: A total of five porcine and five bovine endometria obtained from hysterectomy specimens were included in this study. Immunohistochemical staining with progesterone receptors and SOX2 in endometrium fixed in five fixative solutions (Formol, Antigenfix, Greenfix, Bouin, Methacarn) were performed after 1,5; 8,5; 15,5; 29,5; 64,5 and 189,5 days of fixation. An automated immunostainer Ventana Benchmark Ultra was used for all immunohistochemical analysis. Results: Expression of progesterone receptors was demonstrated in 92-87 % of glandular cells after 1,5-64,5 days and in 59 % of cells after 189,5 days of formalin fixation. The intensity of staining was strong, but after 189,5 days it was evaluated as being weak. In Antigenfix-fixed tissues, strong progesterone...
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Immunohistochemical analysis of endoglin and cell adhesion molecules expression in NASH experimental model
Vašinová, Martina ; Nachtigal, Petr (advisor) ; Němečková, Ivana (referee)
Nonalcoholic steatohepatitis (NASH) is an aggressive form of nonalcoholic fatty liver disease, which is characterized by liver inflammation and fibrosis. It was shown that membrane endoglin (ENG) participates on fibrosis and plasma concentrations of soluble endoglin were increased in patients with type II diabetes mellitus, and hypercholesterolemia. The purpose of this study was to analyze the expression of ENG, biomarker of fibrosis (αSMA/alpha smooth muscle actin), and biomarkers of inflammation (ICAM-1, VCAM-1) in liver in mice. Wild type mice were divided into two groups (n=8 in each group), control group was fed with chow diet and experimental/NASH group was fed with high fat diet with fructose and glucose in drinking water (NASH diet) for 6 months. Expression of selected proteins was evaluated by means of immunohistochemistry (ImmPRESSTM and VECTASTAIN® ELITE® ABC kit) on formalin-fixed, paraffin-embedded liver sections. The results of this study showed that the expression of VCAM-1 and ICAM-1 was detected in liver sinusoids and it was higher in NASH group. Similarly, we showed higher expression of ENG in NASH group in comparison with control. Moreover, αSMA was found not only in blood vessels, but also in hepatic stellate cells in NASH group compared to control suggesting fibrotic process...
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