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Evaluation of the effectiveness of selected methodical approaches for disagregation of staphylococcal biofilm biomass
Nosková, Martina ; Konečná, Klára (advisor) ; Jílek, Petr (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Subject of study: Bioanalytical laboratory diagnostics in healtcare Student: Bc. Martina Nosková Supervisor: RNDr. Klára Konečná, Ph.D. Title: Evalution of the effectiveness of selected methodical approaches for disagregation of staphylococcal biofilm biomass Background: Evaluation of the impact of selected biochemical and physical approaches on the disaggregation of the biofilm community formed by the bacterium Staphylococcus aureus. For the biochemical approach was chosen enzyme proteinase K, the impact of sonication, shaking with/without glass beads was studied within the physical approaches. Methods: Staphylococcal biofilm was formed in vitro in two methodical configurations - in the wells of a microtiter plate and on pegs (analogy to the Calgary Biofilm Device). To evaluate the degree of consistency of the obtained data, which reflects the homogenity of the biofilm biomass disaggeragation, the "recovery" method, and the method of metabolic activity evaluation, using the Alamar Blue indicator. To express the degree of effectiveness of the chosen approaches on the overall disaggregation of the biofilm community, the method of quantification of biofilm biomass by crystal violet staining was...
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Evaluation of the effectiveness of selected methodical approaches for disagregation of staphylococcal biofilm biomass
Nosková, Martina ; Konečná, Klára (advisor) ; Jílek, Petr (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biological and Medical Sciences Subject of study: Bioanalytical laboratory diagnostics in healtcare Student: Bc. Martina Nosková Supervisor: RNDr. Klára Konečná, Ph.D. Title: Evalution of the effectiveness of selected methodical approaches for disagregation of staphylococcal biofilm biomass Background: Evaluation of the impact of selected biochemical and physical approaches on the disaggregation of the biofilm community formed by the bacterium Staphylococcus aureus. For the biochemical approach was chosen enzyme proteinase K, the impact of sonication, shaking with/without glass beads was studied within the physical approaches. Methods: Staphylococcal biofilm was formed in vitro in two methodical configurations - in the wells of a microtiter plate and on pegs (analogy to the Calgary Biofilm Device). To evaluate the degree of consistency of the obtained data, which reflects the homogenity of the biofilm biomass disaggeragation, the "recovery" method, and the method of metabolic activity evaluation, using the Alamar Blue indicator. To express the degree of effectiveness of the chosen approaches on the overall disaggregation of the biofilm community, the method of quantification of biofilm biomass by crystal violet staining was...
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Specific prion protein antibodies characterisation and use in diagnostic
Šafaříková, Eva ; Holada, Karel (advisor) ; Mareš, Michael (referee) ; Živný, Jan (referee)
Transmissive spongiform encephalopathies (TSEs) are neurodegenerative diseases characterized by depositions of abnormally folded prion protein (PrPTSE ) in brain. PrPTSE is at present the only specific biochemical marker of human and animal TSEs. Diagnostic tests are based on the detection of PrPres after proteinase K digestion of brain homogenate using Western blot or on the immunohistochemistry of fixed brain tissue, which are both difficult and time consuming. In this work we focused on development of a new type of tests based on PrP detection without need of proteinase K digestion. As deposits of PrPTSE remain in the body for a long time, there is a substantial chance of them being nonenzymatically modified by glycation. The detection of glycated PrPTSE may have a potential to serve as a diagnostic marker. We prepared monoclonal antibodies specific for carboxymethyl lysine/arginine modified prion protein. Bacterially expressed and purified recombinant human prion protein (rhPrP) was modified by glyoxylic acid that introduces carboxymethyl groups on lysine and arginine residues present within the molecule of the protein. Modified rhPrP (rhPrP-CML) was used for immunization of laboratory mice and hybridoma cells were prepared. Screening of cell supernatants resulted in the selection of 4...
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Specific prion protein antibodies characterisation and use in diagnostic
Šafaříková, Eva ; Holada, Karel (advisor) ; Mareš, Michael (referee) ; Živný, Jan (referee)
Transmissive spongiform encephalopathies (TSEs) are neurodegenerative diseases characterized by depositions of abnormally folded prion protein (PrPTSE ) in brain. PrPTSE is at present the only specific biochemical marker of human and animal TSEs. Diagnostic tests are based on the detection of PrPres after proteinase K digestion of brain homogenate using Western blot or on the immunohistochemistry of fixed brain tissue, which are both difficult and time consuming. In this work we focused on development of a new type of tests based on PrP detection without need of proteinase K digestion. As deposits of PrPTSE remain in the body for a long time, there is a substantial chance of them being nonenzymatically modified by glycation. The detection of glycated PrPTSE may have a potential to serve as a diagnostic marker. We prepared monoclonal antibodies specific for carboxymethyl lysine/arginine modified prion protein. Bacterially expressed and purified recombinant human prion protein (rhPrP) was modified by glyoxylic acid that introduces carboxymethyl groups on lysine and arginine residues present within the molecule of the protein. Modified rhPrP (rhPrP-CML) was used for immunization of laboratory mice and hybridoma cells were prepared. Screening of cell supernatants resulted in the selection of 4...
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