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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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Morphological characteristic of alterations in the striatum induced by neurodegenerative process in the brain
Němečková, Ivana
("Morphological characteristics of alterations in the striatum induced by neurodegenerative process in the brain") Huntington's disease (HD) is an inherited neurodegenerative disorder. Although the cause of HD, i.e. the production of the mutant form of unstable protein huntingtin (mhtt) which contains 40 and more CAG repeats is known, the effective therapy is not yet available. Therefore, the use of animal models is crucial for the study of the pathogenesis of this fatal disorder. To date, there is no suitable experimental model simulating the neurodegenerative process (NDP) developing in the striatum of the human HD brain. Most of rodent models of HD fall into two broad categories - the neurotoxic lesions and genetically engineered models. The primary aim of our study was a comprehensive morphological description of the development of NDP of HD phenotype in the striatum of the rat brain. We compared the progression of NDP in the lesion induced by intrastriatal injection of quinolinic acid (QA) and in rats transgenic for HD. The groups of male rats surviving for 3, 6-7, 14 days, 1, 3, 6, 9 and 12 months after the QA lesion were compared with 2-, 6-, 12-, 18-, 22-24-month-old tgHD rats and age-matched control (intact) counterparts in both groups. The primary morphological feature of the NDP of HD...
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Copper reducin properties of a series of xanthe-3-ones
Kopková, Nikola ; Mladěnka, Přemysl (advisor) ; Němečková, Ivana (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Candidate: Nikola Kopková, MSc Consultant: Assoc. Prof. Přemysl Mladěnka, PharmD., Ph.D. Title of diploma thesis: Copper reducing properties of a series of xanthen-3-ones Key words: copper, reduction, xanthene-3-one, homeostasis Copper is present as a trace element in all tissues and is essential in cellular respiration, in the biosynthesis of neurotransmitters, for the scavening of reactive oxygen species, and is also a cofactor for various enzymes. Disruption of homeostasis can lead to liver and CNS damage, and tumor formation. Typical examples of an imbalance in copper homeostasis represents Wilson and Menkes disease. Xanthene derivatives are biologically active compounds with a possible broad therapeutic spectrum. Some of these derivatives have antitumor, antipyretic, immunomodulatory, antioxidant and other positive biological activities. The aim of this work was to determine whether 2,6,7-trihydroxyxanthen-3-one derivatives have the ability to reduce cupric ions and possibly to detect the effect of various substituents on this ability. As a methodology, a spectrophotometric method based on bathocuproindisulfonic acid as an indicator was used. Ten compounds were tested at different pH values. All...
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Effect of glucose treatment on endoglin and biomarkers of endothelial dysfunction in endothelial cells
Adamcová, Adriana ; Nachtigal, Petr (advisor) ; Němečková, Ivana (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Title of Diploma Thesis: Effect of glucose treatment on endoglin and biomarkers of endothelial dysfunction in endothelial cells Author: Adriana Adamcová Supervisor of Diploma Thesis: prof. PharmDr. Petr Nachtigal, Ph.D. Background: The aim of this thesis was to evaluate the expression of endoglin and biomarkers of endothelial dysfunction (eNOS, VCAM-1) in aortic endothelial cells exposed to oscillating glucose. Methods: Human aortic endothelial cells (HAECs) were treated with alternating normal (5 mM) and high (25mM) glucose for 7 days. The control group, was treated with a constant level of normal glucose. Gene expression of endoglin, eNOS, VCAM-1, HIF1α, KLF6, RELA and LXR was measured by quantitative real-time PCR. Flow cytometry was used to detect endoglin and VCAM-1 protein levels. Results: Treatment with high levels of glucose resulted in significantly increased expression of endoglin, eNOS, VCAM-1, HIF1α, RELA and LXR by means of PCR. The flow cytometry method showed a significant difference in protein levels in both endoglin and VCAM-1. Conclusions: The results of this diploma thesis showed that oscillating glucose increases the expression of endoglin and other biomarkers of endothelial...
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Copper reducin properties of a series of xanthe-3-ones
Kopková, Nikola ; Mladěnka, Přemysl (advisor) ; Němečková, Ivana (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Candidate: Nikola Kopková, MSc Consultant: Assoc. Prof. Přemysl Mladěnka, PharmD., Ph.D. Title of diploma thesis: Copper reducing properties of a series of xanthen-3-ones Key words: copper, reduction, xanthene-3-one, homeostasis Copper is present as a trace element in all tissues and is essential in cellular respiration, in the biosynthesis of neurotransmitters, for the scavening of reactive oxygen species, and is also a cofactor for various enzymes. Disruption of homeostasis can lead to liver and CNS damage, and tumor formation. Typical examples of an imbalance in copper homeostasis represents Wilson and Menkes disease. Xanthene derivatives are biologically active compounds with a possible broad therapeutic spectrum. Some of these derivatives have antitumor, antipyretic, immunomodulatory, antioxidant and other positive biological activities. The aim of this work was to determine whether 2,6,7-trihydroxyxanthen-3-one derivatives have the ability to reduce cupric ions and possibly to detect the effect of various substituents on this ability. As a methodology, a spectrophotometric method based on bathocuproindisulfonic acid as an indicator was used. Ten compounds were tested at different pH values. All...
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