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Ames test in the drug development
Klaučová, Martina ; Pávek, Petr (advisor) ; Konečná, Klára (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Martina Klaučová Supervisor: prof. PharmDr. Petr Pávek, Ph.D. Consultant: PharmDr. Ivona Pávková, Ph.D. Diploma thesis title: Ames test in the drug development Background: Thesis objective is the determination of potential genotoxicity of newly developed drugs within primary testing and the introduction of the Ames microfluctuation test which can be used in common laboratory conditions. Methods: I used commercially supplied kit based on the principles of Ames test which detects reverse mutation through colour changes of the samples using bacterial strains S. typhimurium. At first I had to study literary sources and then I could design the procedures of the Ames microfluctuation test, preparation of the chemicals and storage of the strains which are optimal for all laboratories. Results: The drug samples T6445 and T6447 with 30 µM concentration tested by metabolic activation S9 on bacterial strain ST TA 98 show genotoxicity. The sample UOCHB1 with 30 µM concentration tested without activation shows possible genotoxicity on both strains ST TA 98 and ST TA 100. Other samples do not show any toxicity. I used 3 different procedures during the designation of assay. The most suitable version of the...
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Optimization of methods for sensitivity evaluation of biofilm-forming microbial agents towards substances with antimicrobial potential
Roubalová, Jana ; Konečná, Klára (advisor) ; Jílek, Petr (referee)
Charles University Faculty of Pharmacy in Hradec Králové Study program: Pharmacy Candidate: Jana Roubalová Consultant: RNDr. Klára Konečná, Ph.D. Title of thesis: Optimization of methods for sensitivity evaluation of biofilm-forming microbial agents towards substances with antimicrobial potential Background: The aim of this diploma thesis was to optimize the methodical approach to the production of yeast biofilms in vitro on the so-called pegs and the walls of the wells of the 96-well panel. This biofilm formation system should be an analogous approach to the commercially available Calgary Biofilm Device. 10 strains of Candida yeast and 4 different culture media (nutritionally poor / rich) were used to optimize the methodology. Both the reference strains and the clinical isolates were among the yeast strains tested. Methods: The ability to form yeast biofilms by different strains in different culture media was evaluated by the approach of fixation, staining of the formed biofilms by crystal violet and extraction and spectrophotometric measurement of the intensity of the extracted color. Results: Individual strains, after cultivation in different media, were categorized according to their ability to form biofilm. Larger yeast adherence occurs in the wells than on pegs where the yeast adhered very...
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Laboratory diagnostic of mold genus Fusarium
Vitáčková, Petra ; Konečná, Klára (advisor) ; Vejsová, Marcela (referee)
Submitted diploma thesis deals with laboratory diagnostics of filamentous micromycetes (moulds) genus Fusarium in the laboratories of medical mycology. In the first part of the diploma thesis is dedicated space for general familiarization with filamentous microscopic fungi. Next separate chapter describes the genus Fusarium, especially in relation to human medicine. The final part of the diploma thesis summarizes current possibilities of laboratory diagnostics of filamentous micromycetes, focusing on the genus Fusarium. Invasive infections caused by filamentous fungi are increasingly common complication, especially in immunocompromised patients. In most cases they affect the patients with hematologic malignity and patients after hematopoietic stem cell transplantation or solid organ transplantation. The most common etiological agent of such infections is unambiguously the Aspergillus genus. However, in recent years, increasingly cases of infections due to less common filamentous fungi, including Fusarium spp. are described. Invasion fungal infections are associated with very high mortality. The basic requirement thus becomes the speed and accuracy of diagnosis. The most important methods for the laboratory diagnostics of invasive infections caused by filamentous fungi are conventional methods -...
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Enzyme kinetic evaluation of several potential inhibitors of certain human cysteine and serine proteases
Hympánová, Michaela ; Konečná, Klára (advisor) ; Janďourek, Ondřej (referee)
IN ENGLISH Charles University Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Supervisors: prof. Dr. Michael Gütschow RNDr. Klára Konečná, Ph.D. Candidate: Michaela Hympánová Title of the diploma thesis: Enzyme kinetic evaluation of several potential inhibitors of certain human cysteine and serine proteases Background Cysteine and serine proteases are enzymes involved in many physiological processes. The imbalance between them and their endogenous inhibitors is associated with various diseases such as cancer and osteoporosis. Synthetic inactivators could be useful in the treatment of these enzyme-mediated pathological conditions. Therefore, there are ongoing attempts to develop low-molecular weight inactivators for therapeutically relevant cysteine and serine proteases. In the course of this thesis, compounds synthesized in prof. Gütschow's group were investigated as potential inhibitors of selected human proteases. They belong to imidazole compounds derived from N-protected cyclohexylalanine, 2-phenyl-7,8-dihydroimidazo[1,2- a]pyrazin-6(5H)-one derivatives, ,-unsaturated peptidomimetic compounds, carbamates, an N,N-dibenzylcrotonamide derivatives and peptoides. Aims This diploma thesis has been focused on the evaluation of new potential inhibitors against...
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Attitude of mothers to compulsory vaccination in children
Červinková, Pavla ; Rathouská, Jana (advisor) ; Konečná, Klára (referee)
Bc. Pavla Červinková Attitude of mothers to compulsory vaccination in children Charles University, Faculty of Pharmacy in Hradec Králové Specialist in laboratory methods The most effective prevention of serious infectious diseases is vaccination. In the Czech Republic, there is a compulsory vaccination against nine diseases, which have not been eradicated in the world yet. The topic of compulsory vaccination is currently a very topical and discussed issue. Objectives: Theoretical overview of the issue of compulsory vaccination in children, individual diseases, the possible risks of vaccination and available vaccines. The aim of the questionnaire study is to determine the percentage of mothers with a negative attitude to this issue and their reasons for their attitude. Methods: Questionnaire study within the Czech Republic. The questionnaire was intended for women - mothers of different age categories. Results: In the general public, there is a wide range of views on this issue. Most of mothers have a positive attitude towards compulsory vaccination. A significant part of mothers point to a bad legislation in the Czech Republic. They particularly criticize the vaccination scheme and the obligation of using combined vaccines, in which mothers often observe side effects. Conclusion: The questionnaire...
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Biofilms and human body
Chlupáčová, Adéla ; Jílek, Petr (advisor) ; Konečná, Klára (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Author: Adéla Chlupáčová Supervisor: PharmDr. Petr Jílek, CSc. Supervisor specialist: doc. MUDr. Pavel Čermák, CSc. Title of Diploma thesis: Biofilms and human body Subtitle: The occurrence of biofilm producers on central venous catheters and the effect of xanthohumol on the biofilm of selected clinical isolates Background: The aim of the work was to show the frequency of appearance of biofilm on the catheters, which leads to serious health complications. In the second part of the thesis we investigated the biofilm resistance of common biofilm agents such as Staphylococcus aureus and Enterococcus faecalis to natural substances, namely xanthohumol. Methods: Part of the thesis which monitors the occurrence of biofilm producers on catheters was processed using tables and charts in Microsoft Excel. To testing xanthohumol were used laboratory techniques such biofilm staining with crystal violet in microtiter plate, the minimal inhibitory concentrations of xanthohumol were determined using the broth microdilution method, the minimal bactericidal concentrations were determined on Columbia blood agar, inhibition of bacterial release from biofilm was monitored in microtiter plates with...
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In vitro screening of novel potentially active antibacterial compounds
Davidová, Lucie ; Janďourek, Ondřej (advisor) ; Konečná, Klára (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Study program: Specialist in Laboratory Methods Candidate: Bc. Lucie Davidová Supervisor: PharmDr. Ondřej Janďourek, Ph.D. Title of diploma thesis: In vitro screening of novel potentially active antibacterial compounds. The diploma thesis deals with the antibiotics policy and is focused on resistance and mechanism of action of the antibiotics. A great attention is also paid to novel substances testing in terms of their impact on the selected bacteria. Theoretical part of thesis describes the system of antibiotic policy including its structure and organization in the Czech Republic. It also briefly summarizes this issue globally as well as individual organizations involved in the organization of the antibiotic policy. The theoretical part is also concerned with the history, classification, effects and significance of the antibiotics. A particular attention is paid to the chapter describing the macromolecular mechanism of action of 2 selected groups of substances (antibiotics) on bacteria, including physiological functions of some bacterial structures. And, last but not least, the theoretical part summarizes principles of bacterial resistance including its origin and development. The diploma thesis...
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Entry of bacteria Mycobacterium bovis BCG into B lymphocytes
Šamajová, Marianna ; Konečná, Klára (advisor) ; Kubelková, Klára (referee)
Charles University Faculty of Pharmacy in Hradec Králové Study program: Pharmacy Author: Marianna Šamajová Supervisor: RNDr. Klára Konečná, Ph.D. Consultant: plk. gšt. doc. RNDr. Zuzana Kročová, Ph.D. Title of diploma thesis: Entry of bacteria Mycobacterium bovis BCG into B lymphocytes Background: The objective of this work was to evaluate the entry of bacterium Mycobacterium bovis BCG into B lymphocytes and the role of selected receptors in this process. Methods: Peritoneal cell suspensions with unblocked and/or blocked receptors on BALB/c mouse B lymphocytes we infected by bacterium M. bovis BCG-GFP unopsonized and/or opsonized by fresh murine serum ("complement") or immune serum ("antibodies"). Using flow cytometry we evaluated the entry of bacterium M. bovis BCG-GFP into B lymphocytes and their subpopulations B1a, B1b and B2. Results: M. bovis BCG-GFP actively enters into B lymphocytes. Depending on the subpopulation, it most infects B1a, less B1b and at least B2 lymphocytes. Only the subpopulation B2 responds significantly to the opsonization by complement. Opsonization by antibodies had no significant effect on the infection. Entry into CD19+ cells is mediated through the BCR receptor, especially in subpopulations B1a and B1b. Under the opsonized conditions, the CR1/2 complement receptor is...
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Detection of Sap2 in the secretome of Candida albicans cell wall and secretory mutants
Kollárová, Nikola ; Janďourek, Ondřej (advisor) ; Konečná, Klára (referee)
Candidate: Nikola Kollárová Title of diploma thesis: Detection of Sap2 in the secretome of Candida albicans cell wall and secretory mutants Charles University, Faculty of Pharmacy in Hradec Králové, Department of Biological and Medicinal Sciences Complutense University of Madrid, Faculty of Pharmacy, Department of Microbiology II Study program: Pharmacy Backgound: The aim of this diploma thesis was to search for C. albicans proteins involved in the secretion of the secreted aspartyl proteinase 2 enzyme (Sap2) evaluating the ability to degrade BSA (bovine serum albumin) as a source of nitrogen in several cell wall and secretory mutants of C. albicans. The work was carried out at the Department of Microbiology II, Faculty of Pharmacy, Complutense University of Madrid. Methods: The supernatant samples of several Candida albicans mutants were tested by SDS-PAGE electrophoresis and stained. Bands corresponding to BSA were observed and compared to controls. The other method was counted with 96-well plate. Results: The correlation between optical density and degradation of BSA was observed. Some mutants with disability to degrade BSA were found in a pilot screening of the ability to degrade BSA using 96-well plate method. That fact was confirmed by SDS-PAGE electrophoresis. C. albicans mutants showing...
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Detection of Sap2 in the secretome of Candida albicans cell wall and secretory mutants
Kollárová, Nikola ; Janďourek, Ondřej (advisor) ; Konečná, Klára (referee)
Candidate: Nikola Kollárová Title of diploma thesis: Detection of Sap2 in the secretome of Candida albicans cell wall and secretory mutants Charles University, Faculty of Pharmacy in Hradec Králové, Department of Biological and Medicinal Sciences Complutense University of Madrid, Faculty of Pharmacy, Department of Microbiology II Study program: Pharmacy Backgound: The aim of this diploma thesis was to search for C. albicans proteins involved in the secretion of the secreted aspartyl proteinase 2 enzyme (Sap2) evaluating the ability to degrade BSA (bovine serum albumin) as a source of nitrogen in several cell wall and secretory mutants of C. albicans. The work was carried out at the Department of Microbiology II, Faculty of Pharmacy, Complutense University of Madrid. Methods: The supernatant samples of several Candida albicans mutants were tested by SDS-PAGE electrophoresis and stained. Bands corresponding to BSA were observed and compared to controls. The other method was counted with 96-well plate. Results: The correlation between optical density and degradation of BSA was observed. Some mutants with disability to degrade BSA were found in a pilot screening of the ability to degrade BSA using 96-well plate method. That fact was confirmed by SDS-PAGE electrophoresis. C. albicans mutants showing...
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