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Quantitation of agents in exhaled breath condensate.
Dobiáš, Jiří ; Jílek, Petr (advisor) ; Konečná, Klára (referee)
Measurement of parameters in exhaled breath condensate constitute (EBC) a rapidly progressing field of research. The collection procedure is simply and comletely noninvasive. EBC contains large number of biomarkers including hydrogen peroxide, nitrite, nitrate, 3-nitrotyrosine, nitrosothiols, adenosine, prostaglandines, thromboxanes, leukotrienes, isoprostanes, cytokines. Use of EBC is based on hypothesis that aerosol particules exhaled in human breath reflect the composition of the bronchoalveolar extracellular lining fluid. The term EBS stricly relates to exhaled samples collected by cooling the exhaled breath. Measurement very low concentrations of some biomarkers requires very senzitive analytical methods. The evaluation of the available studies revealed that asthma, COPD and ARDS compered with healthy control groups lead to a significant increase in hydrogen peroxide concentration. The result of the work indicate that the content of hydrogen peroxide in the breath condensate provides new information for diagnosis and therapy of inflammatory diseases of the respiratory tract when the collection of specimen and the measurement procedure are standardized.
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Expression of SWC7 molecules on the pig B lymphocytes.
Fikrová, Petra ; Konečná, Klára (referee) ; Jílek, Petr (advisor)
The aim of this graduation thesis is to clarify the expression profile of the SWC7 molecule on B cells in swine and eventually to resolve its function during maturation. At present it is possible to define the maturation pathways of porcine B lymphocytes in the periphery on the base of the expression CD2 and CD21 cell surface molecules. The flow cytometry was chosen as a central experimental method for detection of B lymphocytes carrying SWC7 molecule on their surface. The ability of individual B cell subpopulation to produce antibodies was also measured by ELISA. Principal phenotype characterization showed that the SWC7 molecule is found only on the surface of IgM+ and CD21+ B cells. These may be CD2 positive or negative. Based on this finding, the individual subpopulations of IgM+ CD21+ B cells differing in expression of SWC7 and CD2 molecules were purified by flow cytometry. Subpopulations were thereafter cultivated and subsequently analyzed for alterations in their phenotype. Media from cultivated cells were also analyzed for presence of porcine IgM, IgG and IgA antibodies by ELISA. Results indicate that the expression of SWC7 molecule on the surface of B cells may be considered as a maturation marker because loss of SWC7 molecules from the cell surface is irreversible and because only SWC7- B cells are...
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Isolation and proteome analysis of fractions enriched in periplasmic proteins of bacterium Francisella tularensis
Kolářová, Jana ; Vacková, Jana (referee) ; Konečná, Klára (advisor)
Candidate: Bc. Jana Kolářová Supervisor: Mgr. Klára Konečná Supervisor - specialist: PharmDr. Ivona Pávková, Ph.D Title: Isolation and proteome analysis of fractions enriched in periplasmic proteins of bacterium Francisella tularensis Diploma thesis Charles University in Prague, Faculty of Pharmacy in Hradec Králové, Department of Biological and Medical Sciences Study field: Special worker in laboratory methods The aim of the presented diploma thesis was to test several previously published methods for preparation of fractions enriched in periplasmic proteins of bacterium Francisella tularensis. The selected method will be further used in studies aimed at finding potential substrates of hypothetical protein with homology to DsbA as several of them are expected to play a crucial role in Francisella tularensis virulence. DsbA proteins found in periplasm of gramnegative bacteria are in general responsible for disulfide bond introduction and thus for right folding and function of number of extracytosolic proteins, known virulence factors included. Tested methods included the method of cold osmotic shock, the method using chlorofom, polymyxin B sulphate, EDTA in combination with lysozyme and magnesium chloride in combination with lysozyme. The obtained protein samples were separated using...
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Detection of antibodies reactive with F. tularensis in the early phase of infection.
Straková, Kristina ; Janovská, Sylva (referee) ; Konečná, Klára (advisor)
Kristina Straková Detection of antibodies reactive with F. tularensis in early phase of infection Thesis Charles University in Prague, Faculty Of Pharmacy in Hradec Králové Study program Pharmacy Because of its high virulence and mortality Francisella tularensis is considered to be a dangerous potential biological weapon and therefore this etiological agent is a suitable object of the primary research. Problems of an antibody response to the infection with intracellular pathogens are currently gaining in importance. Opinions and conclusions of various research teams differ, so this topic is up-to-date. The aim of this study was to find whether F. tularensis induces the antibody immune reaction in an early phase of infection (after 24 and 48 hours). The virulent strain of F. tularensis was used. We also intended to validate a feasibility of chosen methodology. Methods: We prepared a whole-cell bacteria lysate of F. tularensis holarctica FSC200. This lysate was separated by 2D electrophoresis with subsequent Western blotting. Proteins were marked by immunodetection using serum of BALB/c mice. Mice sera were collected 24 and 48 hours post challenge with F. tularensis. The serum of uninfected mice was utilized as control. Using comparative analysis, we tried to find proteins reacting only with the...
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tudy of the interaction of newly synthesized compounds with bacterial agents.
Petlánová, Tereza ; Konečná, Klára (advisor) ; Voxová, Barbora (referee)
Charles University Faculty of Pharmacy in Hradec Králové Study program: Pharmacy Candidate: Mgr. Tereza Petlánová Consultant: RNDr. Klára Konečná, Ph.D. Title of thesis: Study of the interaction of newly synthesized compounds with bacterial agents. Background: The aim of this thesis was to test antibiotic activity of substances produced by the Department of Inorganic and Organic Chemistry, Faculty of Pharmacy of Charles University in Hradec Králové. Methods: Substances were tested by using microdilution broth method on eight strains of bacteria: Staphylococcus aureus, Staphylococcus aureus methicilin resistant, Staphylococcus epidermidis, Enterococcus sp., Escherichia coli, Klebsiella pneumoniae, Klebsiella pneumoniae ESBL positive and Pseudomonas aeruginosa. Results: Substances were divided into five groups according to associated structural features. The most effective was the group of salicilanilid derivates. In this group, the bacterial effect of all 28 tested substances were shown. Conclusion: The antibacterial effect was demonstrated in 50 from the total number of 73 tested compounds. The most susceptible strains were Staphylococcus aureus, Staphylococcus aureus methicilin resistant, Enterococcus sp. and Staphylococcus epidermidis. On the contrary, none of the tested compounds showed efficacy...
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Study of interspecies differences in plasma protein binding of 18F-labeled radiopharmaceuticals
Pašková, Barbora ; Konečná, Klára (advisor) ; Jílek, Petr (referee)
Mgr. Barbora Pašková The study of antimycotic activity of newly synthetized substances Rigorous thesis Charles University in Prague, Faculty of Pharmacy in Hradec Králové Study program: Pharmacy Background: The aim of this rigorous thesis was to evaluace the antimycotic activity of 77 substances prepared at the Department of Inorganic and Organic Chemistry, Faculty of Pharmacy in Hradec Králové of Charles University in Prague. Method: The substances were tested by the microdilution broth method and all substances were tested at eight strains of yeasts and filamentous fungi. The tested substances were divided into five groups according to their chemical structure to salicylanilide carbamates, sulfoneamide ureas - derivates of sulphamethoxazole, sulphonamide imidazolidinetriones - derivates of sulphamethoxazole, derivates of isoniazide and substances with disunited structure. Results: The highest antimycotic activity was discovered in group of salicylanilide carbamates. There were tested 28 derivates of this group, from which 18 of them showed antimycotic activity. On the other hand, substances from group of derivates of isoniazide showed no antimycotic activity. The most susceptible strain was Trichophyton mentagrophytes and the most resistant was strain Aspergillus fumigatus. Conclusions: To...
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