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Introduction of an invertebrate animal model, Galleria Galleria mellonellamellonella, to study the in vivo efficacy of existing and newly synthesized, candidate antituberculotics
Kubíček, Jiří ; Konečná, Klára (advisor) ; Němečková, Ivana (referee)
Charles university, Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Study program: Pharmacy Author: Jiří Kubíček Supervisor: RNDr. Klára Konečná, Ph.D. Title of thesis: Introduction of an invertebrate animal model, Galleria mellonella, to study the in vivo efficacy of existing and newly synthesized, candidate antituberculotics. Background: The aim of this diploma thesis has been to present an invertebrate organism, Galleria mellonella (G. mellonella), Pyralidae, as an alternative, more economically advantegous and morally more acceptable model for in vivo studies of an antituberculotics (anti-TB) efficacy. In the experimental part of this work, attention was paid to the optimization of methodological steps, that should lead to a higher reproducibility and validity of the obtained data. The effect of the disaggregating supplement, tyloxapol, on the growth of Mycobacterium aurum (M. aurum) was evaluated. The growth of this microbial strain in the culture medium, Middlebrook 7H9, was also mapped to determine the exponential growth phase. In the second part of the experiments, G. mellonella larvae were infected with a M. aurum bacterial strain, followed by an administration of the selected, commercially available anti-TB. Methods: To map the exponential phase of M....
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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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Endothelial dysfunction markers and soluble endoglin
Pešlová, Lenka ; Nachtigal, Petr (advisor) ; Němečková, Ivana (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Kralove Department of Biological and Medical Sciences Consultant: Doc. PharmDr. Petr Nachtigal, PhD. Student: Lenka Pešlová Title of Thesis: Endothelial dysfunction markers and soluble endoglin Background: To analyze possible changes in histological structure in myocardium of mice with low or high levels of soluble endoglin. We focused on description of general histology, observation of ligament changes and possible development of inflammatory reactions using immunohistochemical analysis. Method: In the study we used genetically modified female mice, strain CBAxC57BL/6J, with low or high levels of soluble endoglin (Sol-Eng+). Six-month- old female mice were fed for three months using a high fat rodent diet containing 1,25% of cholesterol and 40% of fat. Then their myocardia were resected. For identification of endoglin expression we used immunohistochemical analysis called En Vision-CD3-T-lymfocyty (rabbit anti mouse). In histological analysis we used hematoxylin-eosin and green trichrome staining. Results: Histological analysis did not reveal any pathological accumulation of collagen in any of animal myocardium we focused on. There were any changes in staining neither of nuclei or cytoplasm, nor any infiltration of leucocytes....
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Immunohistochemical detection of macrophages in atherosclerotic lesions brachiocephalic artery I
Škodová, Sabina ; Nachtigal, Petr (advisor) ; Němečková, Ivana (referee)
Sabina Škodová Immunohistochemical detection of macrophages in atherosclerotic lesions brachiocephalic artery. Bachelor thesis Charles University in Prague, Faculty of Pharmacy in Hradec Králové Medical Laboratory Technician Background: The aim of this bachelor thesis was to set imunohistochemic methods for detection of the macrophages in brachiocephalic artery apoE/LDLR receptor deficient mice. This thesis also deals with quantification of expression macrophages in group of mice which were fed with chow diet and in group of mice which were fed with low carbohydrate high protein diet. Methods: For this study were used apoE/LDLR deficient female mice which were separated into two groups (in each group were 6 mice). The first group was fed with chow diet (control group) and the second one was fed with low carbohydrate high protein diet (LCHP). This diet was administered to the mice for 8 weeks. After that the imunohistochemical analysis was performed on the cuts of brachiocephalic artery which uses Immpress method with diaminobenzidin (DAB) for better visibility of macrophages. Afterwards was performed biochemical and stereological analysis. Results: Biochemical analysis showed increased levels of total cholesterol in mice with LCHP diet. Immunohistochemical analysis showed presence of macrophages in...
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The study oxidative stress markers in animal model of chronic colitis in large intestine.
Hocková, Erika ; Nachtigal, Petr (advisor) ; Němečková, Ivana (referee)
Crohn's disease and ulcerative colitis are both entities of inflammatory bowel disease (IBD). The nuclear transcription factor kappaB (NFκB) family consists of five different members, which are namely p65 (RelA), c-Rel, RelB, p50 and p52. NFκB has ability to promote the expression of various proinflammatory genes, to influence the course of mucosal inflammation and to mediate different cell-type specific effects. Bilirubin is an endogenous substance with anti-oxidative and anti-inflammatory effects, which influences the process of inflammation and has protective effect. This diploma thesis was aimed at conduction of methods of NFκB detection on paraffine tissue slices and observation of NFκB expression in large intestine walls of normobilirubin and hyperbilirubin rats (Gunn's variety) after chronic imunohistochemically- induced inflammation. Eight Gunn's variety rats (G+ ) and eight Wistar's variety rats (K+ ) were used in this study, all after long time administration of dextrane-sulfate (DSS). For seven following days, DSS (in concentration of 2%w/v) was added into drinkable water for these rats, followed by 14 days of clear drinkable water (without DSS) administered to rats. This three-weeks long cycle was repeated twice (3 cycles in total were completed). Six rats of both varieties (6 G+ and 6...
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The effect of atorvastatin on the expression of markers of the cascade transforming growth factor beta in experimental atherogenesis.
Frnochová, Lenka ; Nachtigal, Petr (advisor) ; Němečková, Ivana (referee)
Lenka Frnochová The effect of atorvastatin on the expression of markers of the cascade transforming growth factor beta in experimental atherogenesis. Diploma thesis Charles Univerzity in Prague, Faculty of Pharmacy in Hradec Králové Pharmacy Background: The aim of this diploma thesis was to observed the expression of ALK-1 and VEGF in atherosclerotic plaques in ApoE and LDL receptor- deficient mice with regard to the use of atorvastatin. Methods: We used for research female C57BL/6J mice with double deficiency of apolipoprotein E and LDL receptor. Mice were 8 weeks old. Mice were divided into two groups: atorvastatin and control group. Both groups were fed cholesterol diet for 8 weeks and in atorvastatin group was also fed with atorvastatin. Then biochemical analysis of blood was performed. For detection of lipids in atherosclerotic lesions we used histological staining by oil red. Immunohistochemical analysis was performed in 1cm aortic sinus and part of aortic arch. The method used to detect the expression of ALK-1 and VEGF was the avidin-biotin method with detection using DAB. Results: Biochemical analysis showed that atorvastatin reduced levels of total cholesterol. After the staining by oil red atherosclerotic lesions were seen in both groups. Atorvastatin reduced the size of atherosclerotic...
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Sunitinib Effects on the Expression of ICAM-1 in Normotensive and Hypertensive Rats
Frnochová, Lenka ; Nachtigal, Petr (advisor) ; Němečková, Ivana (referee)
Mgr. Lenka Frnochová Sunitinib effects on the expression of ICAM-1 in normotensive and hypertensive rats Thesis Charles University in Prague, Faculty of Pharmacy in Hradec Králové Pharmacy Background: We quantified the expression of ICAM-1 in aorta of normotensive Wistar Kyoto rats and spontaneously hypertensive rats during use of sunitinib. For quantification we applied stereological and immunohistochemical methods. Methods: We used inbred male of Wistar Kyoto rats and spontaneously hypertensive rats for our research. Rats of both strains were divided into two groups - a sunitinib group and a control group. The experimental group was receiving drinking water with sunitinib of 10 mg/kg per day and rats in the control group was receiving only drinking water. Spontaneously hypertensive rats were receiving sunitinib for 8 weeks and after 5 days of regeneration they got sunitinib into the water back for another 8 weeks. Wistar Kyoto rats were receiving sunitinib in water for 8 weeks, but after 5 days of regeneration, these rats were receiving sunitinib only for 2 weeks due to toxicity and weight loss. Immunohistochemical analysis was applied in rat aorta. For detection of the ICAM-1 expression was used En Vision method with DAB detection. Results: Immunohistochemical staining showed that expression of...
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Sunitinib effects on endothelial expression of hemeoxygenase-I in normotensive rats
Svobodová, Barbora ; Nachtigal, Petr (advisor) ; Němečková, Ivana (referee)
Barbora Svobodová Sunitinib effects on endothelial expression of hemeoxygenase-I in normotensive rats. Bachelor thesis Charles University in Prague, Faculty of Pharmacy in Hradec Králové Medical Laboratory Technician Background: The aim of this bachelor thesis was to define and describe the expression of hemeoxygenase-1(HO-1) in the aortas of normotensive Wistar Kyoto (WKY) rats and evaluate possible effects of sunitinib treatment by means of immunohistochemical and stereological analysis. Methods: Two groups of WKY rats were used for the experiment (6 animals in each of them). Sunitinib was administered to the first group. The other one (the control one) received water only. Application of sunitinib proceeded by following scheme: 8 weeks of using/ 5 weeks rest period/ 2 weeks of using. After that we performed the immunohistochemical analysis on 60 preparations of rat's aortas which observed the expression of HO-1 by methodology En Vision with DAB detection. Then the stereological evaluation was performed. Results: The immunohistochemical analysis showed the expression of HO-1 in the endothelium only and it was noticed just in several animals especially from the group with sunitinib treatment. Conclusion: The immunohistochemical and stereological analysis demonstrated a significant increase of the...
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