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Induction of innate immune response against intracellular bacterium Francisella tularensis
Sommerová, Gabriela ; Konečná, Klára (advisor) ; Myslivcová Fučíková, Alena (referee)
Background: The purpose of this bachelor thesis is to describe Francisella tularensis and activation of innate immunity during host infection. Main findings: The main findings of the work include the method of recognition of F. tularensis via Toll-like receptors, the production of cytokines and chemokines and the subsequent involvement of other components of innate immunity, including neutrophils, NK cells, or humoral components of the immune response, which also includes complement. The information is based on a number of studies performed mainly on mouse models. Conclusions: The intracelullar bacterium F. tularensis is known to cause tularemia. Its high infectivity, together with the high risk of death in the lung form, raises great concerns about the misuse of this bacterium as a biological weapon. To date, not all of its mechanisms of pathogenesis are known, which is a major problem in the development of effective vaccines. The induction of innate immunity appears to be very important in the host's defense against F. tularensis. However, despite several decades of research, the mechanisms of F. tularensis involved in host cell manipulation, including the regulation of the induction of immune response to F. tularensis infection, have still not been fully elucidated. Key words: Francisella...
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Taxonomic characterization of new Halorubrum isolates from hypersaline soils.
Horáčková, Markéta ; Jílek, Petr (advisor) ; Konečná, Klára (referee)
Candidate: Markéta Horáčková Title of diploma thesis: Taxonomic characterization of new Halorubrum isolates from hypersaline soils. Charles University, Faculty of Pharmacy in Hradec Králové Department of Biological and Medicinal Sciences University of Sevilla, Faculty of Pharmacy Department of Microbiology and Parasitology Study program: Pharmacy The aim of this work is to continue in taxonomic characterization of new strains of the genus Halorubrum isolated from hypersaline soils from Paraje Natural Marismas del Odiel, Huelva, Spain. These Archaea are micro-organisms that require high salt concentration in their environment. With the characterization started Ana Durán Viseras last year and I participated on continuation of this characterization. To complete phylogenetic study we performed MLSA of rpoB gene of each sample to create phylogenetic trees. Based on results of the phylogenetic study we inferred that studied strains could constitute new species. We determined the guanine plus cytosine content in DNA and also carried out phenotypic studies as important part of the taxonomic characterization of the new species.
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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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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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Proteome analysis of secerned proteins of Francisella tularensis
Konečná, Klára ; Hernychová, Lenka (advisor) ; Krejsek, Jan (referee) ; Bílková, Zuzana (referee)
Title of dissertation thesis: Proteome analysis of secerned proteins of Francisella tularensis The facultative intracellular bacterium Francisella tularensis is the causal agent of the infectious disease called tularaemia. Despite the available wide range of new knowledge focused on bacterium Francisella, till now, mechanisms of tularaemia disease pathogenesis were not completely clarified. The contents of our work was based upon analysis and identification of culture filtrate proteins of bacterium F. tularensis of three strains (LVS, FSC00, SchuS4). Among identified proteins, there were seeked protein candidates for secretion and proteins, which could help with explanation of molecular mechanisms of disease pathogenesis caused by F. tularensis. The best protein candidate for secretion is enzyme acid phosphatase with proven important role in bacterium F. tularensis escape from phagosome. The attention was also focused on the new described mechanism of bacterial secretion, mediated by membrane vesicles. By the help of transmission electron microscopy was demonstrated, that F. tularensis of the strains LVS and FSC200 secretes membrane vesicles into extracellular milieu. Key words: Francisella tularensis, secretion systems, cultivation filtrate proteins, secreted proteins, outer membrane vesicles
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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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