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Preparation of recombinant forms of the extracellular part of mouse leukocyte receptors from NKR-P1 family.
Adámek, David
Mouse NK cell receptors belonging to NKR-P1 family plays role in activation, inhibition and cytokine secretion by these cells. Aim of this thesis is preparation of extracellular parts of C57BL/6 mouse strain activating receptors mNKR-P1A and mNKR-P1C. Production vectors with coding sequences of both proteins were prepared. Next, optimization of production in E. coli was done and appropriate in vitro refolding and purification protocol were developed. Purified proteins were characterized by mass spectrometry and labeled by a fluorescent dye. Primary screening for potential ligand was performed. Further work will involve structural characterization of the receptors and identification of their ligands. These data may help to clarify the function of NK cells.
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Applications of flow cytometry in the study of microbial subpopulations.
Hřebíček, Ondřej ; Lichá, Irena (advisor) ; Sudzinová, Petra (referee)
This work reviews common flow cytometric methods and applications for the study of bacterial organisms. Flow cytometry is fluorescent method capable of both quantitative and qualitative analysis at the single cell level. It can offer insights about bacterial population dynamics, phenotypic heterogeneity and more. This work features a basic introduction to flow cytometry and presents some of the commonly measured variables, such as viability or membrane potential with an emphasis on the fluorescent probes used to visualize them. The difficulties of adapting flow cytometry to bacterial physiology are discussed, as well as the advantages and disadvantages of the particular probes and methods. Finally, this work seeks to demonstrate the flexibility as well as the shortcomings of flow cytometry using examples of practical applications in basic research, environmental microbiology, biotechnology, clinical practice. Keywords: flow cytometry, microbial subpopulations, fluorescent labelling, bacterial physiology, bacterial viability
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Selectively substituted cyclodextrins for analytical and pharmaceutical applications
Benkovics, Gábor ; Jindřich, Jindřich (advisor) ; Lhoták, Pavel (referee) ; Šindelář, Vladimír (referee)
3 Selectively substituted cyclodextrins for analytical and pharmaceutical applications Abstract This thesis is focused on the selective modification of cyclodextrins, and its primary aim is the preparation and characterization of mono- and persubstituted derivatives of cyclodextrins in a regioselective and straightforward manner. The work is divided into two main parts describing synthetic strategies and applications of modified cyclodextrins with one or several substituents, respectively. The first section deals with the introduction of a single chromophoric moiety on the cyclodextrin scaffold such as cinnamyl, rhodaminyl, fluoresceinyl and eosinyl groups. The complete set of monocinnamyl-α-cyclodextrin regioisomers has been prepared by direct alkylation, and the self-assembling properties of the corresponding regioisomers were thoroughly investigated by dynamic light scattering and NMR experiments. These investigations revealed that the different isomers (mono-6-O-, mono-2-O- and mono-3-O- cinnamyl-α-cyclodextrin) form distinct supramolecular species through intermolecular association. A fast method for the unambiguous identification of the pure regioisomers has also been developed based on a series of 2D NMR measurements. Xanthene-modified β-cyclodextrins, other representatives of monosubstituted...
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Development of a model system to study adhesion of Burkholderia cenocepacia to epithelial lung cells of patients with cystic fibrosis
Volejníková, Anna ; Bořek Dohalská, Lucie (advisor) ; Kubíčková, Božena (referee)
Cystic fibrosis (CF) is a common autosomal recessive disorder that primarily affects epithelial cells in respiratory system. As a consequence of lung damage the patients are chronically colonized by a number of pathogenic microorganisms such as Pseudomonas aeruginosa or Burkholderia cepacia komplex. Due to the seriousness of these bacterial infection and its primary resistance to commonly used antibiotics the suitable therapeutic alternatives are being sought. The passive immunization with yolk antibodies seems to be a suitable alternative. This thesis studied the bacterial adhesion of Burkholderia cepacia, the opportunistic human pathogen, to lung epithelial cells of healthy individuals and patients with CF, respectively. The aim of this study was to develop a model system suitable for this research. Two types of immortalised cell lines have been used for this purpose. CuFi-1 is the cell line derived from patient with CF caused by ∆F508 mutation , NuLi-1 is a healthy epithelial cell line. The strains of B. cenocepacia (ST28 a ST32) were fluorescently labeled with fluorescent cell linker PKH26. Furthermore, the way of fluorescent labeling of cell lines CuFi-1 and NuLi-1 using PKH67 dye was optimized. Using this model system, the adhesion of bacteria to cell line CuFi-1 was up to three times higher...
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The effect of IgY on bacterial adhesion on epithelial cells ex vivo
Vašková, Lucie ; Hodek, Petr (advisor) ; Nosková, Libuše (referee)
0 Abstract Cystic fibrosis is an autosomal recessive disease caused by mutation in CFTR gene coding for a chloride channel in apical membrane of epithelial cells. This disorder leads to the change in ion transport causing the increase in mucus viscosity in airways as well as changes in glycosylation of saccharide structures on the cells. Because of that these cells are the target for bacterial adhesion. Chronic bacterial infections, which lead to gradual decline of lung function and damage of lung tissue, are the major cause of death of patients suffering with cystic fibrosis. Pseudomonas aeruginosa is the main pathogen causing chronic infections in cystic fibrosis patients. This bacterium produces a biofilm protecting them from host immune system and antibiotics. Once the colonization with PA occurs, it is difficult to get rid of this pathogen. The prophylactic treatment with orally administered hen antibodies against the PA virulence structures could be a prevention of chronic PA infections. In this work we tested the antibody against the bacterial lectin PA-IIL, which is suggested to be involved in the adhesion of the pathogen on epithelial cells. First, it was verified that the prepared antibody from egg yolks of a hen immunized with the bacterial lectin PA-IIL recognizes this antigen expressed...
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The effect of IgY on bacterial adhesion on epithelial cells ex vivo
Vašková, Lucie
0 Abstract Cystic fibrosis is an autosomal recessive disease caused by mutation in CFTR gene coding for a chloride channel in apical membrane of epithelial cells. This disorder leads to the change in ion transport causing the increase in mucus viscosity in airways as well as changes in glycosylation of saccharide structures on the cells. Because of that these cells are the target for bacterial adhesion. Chronic bacterial infections, which lead to gradual decline of lung function and damage of lung tissue, are the major cause of death of patients suffering with cystic fibrosis. Pseudomonas aeruginosa is the main pathogen causing chronic infections in cystic fibrosis patients. This bacterium produces a biofilm protecting them from host immune system and antibiotics. Once the colonization with PA occurs, it is difficult to get rid of this pathogen. The prophylactic treatment with orally administered hen antibodies against the PA virulence structures could be a prevention of chronic PA infections. In this work we tested the antibody against the bacterial lectin PA-IIL, which is suggested to be involved in the adhesion of the pathogen on epithelial cells. First, it was verified that the prepared antibody from egg yolks of a hen immunized with the bacterial lectin PA-IIL recognizes this antigen expressed...
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Studies on interactions between natural killer cell lectin receptors and their protein ligands.
Hernychová, Lucie ; Novák, Petr (advisor) ; Drbal, Karel (referee)
NK cells are innate lymphocytes which constitute the first line of organism's defence against infections through their receptor system. These cells represent an important part of antiviral and antitumor immunity, they also play a role in transplant immunity, autoimmunity and reproduction. This diploma thesis inquires into the structure of the transmembrane receptor NKR-P1B of mouse NK cells and the interaction with its ligand Clr-b. The aim was to prepare the expression vector coding the ligand-binding and whole extracellular region of the receptor NKR-P1B and to optimize its production and refolding in vitro. Purified protein samples were analyzed by size-exclusion chromatography, electrophoresis and mass spectrometry. Interaction between NKR-P1B and Clr-b proteins was tested using biophysical (size-exclusion chromatography and surface plasmon resonance) and biological methods (labelling of cellular sample with NKR-P1B proteins marked with fluorescent dye). In vitro binding experiments have not confirmed mutual interaction between NKR-P1B and Clr-b despite the prepared proteins binding to the bone marrow cells.
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Effect of binding of a fluorescent label on the protein structure and function
Petrovová, Gabriela ; Kavan, Daniel (advisor) ; Martínek, Václav (referee)
Fluorescent labeling is a method used for visualization of various types of biomolecules including proteins and protein complexes. However, the effect of protein labeling on protein structure and functions has not been investigated so far. The goal of the diploma thesis was to examine an influence of NHS-fluorescein binding on structure and function of human carbonic anhydrase I (hCA-I). The particular aims of this work were to prepare recombinant 15N-hCA-I which was used for NMR structure analysis of carbonic anhydrase upon fluorescent labeling. Furthermore, enzyme activity was measured in order to find out a correlation between the concentration of NHS- fluorescein and protein function. In addition, the reaction mixtures were systematically analyzed by ESI FT-ICR mass spectrometry. The analysis revealed experimental conditions for fluorescent labeling of human carbonic anhydrase I with minimal effect on protein structure and function. The results of this study show that the calculation of molar excess of NHS-fluorescein cannot rely on a simple procedure provided by manufacturer. However, due to decrease of enzyme activity upon fluorescent labeling, it is better to take into count the influence of NHS-fluorescein concentration on the relative enzymatic activity. Moreover, the calculation of molar...
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Preparation of recombinant forms of the extracellular part of mouse leukocyte receptors from NKR-P1 family.
Adámek, David
Mouse NK cell receptors belonging to NKR-P1 family plays role in activation, inhibition and cytokine secretion by these cells. Aim of this thesis is preparation of extracellular parts of C57BL/6 mouse strain activating receptors mNKR-P1A and mNKR-P1C. Production vectors with coding sequences of both proteins were prepared. Next, optimization of production in E. coli was done and appropriate in vitro refolding and purification protocol were developed. Purified proteins were characterized by mass spectrometry and labeled by a fluorescent dye. Primary screening for potential ligand was performed. Further work will involve structural characterization of the receptors and identification of their ligands. These data may help to clarify the function of NK cells.
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Preparation of recombinant forms of the extracellular part of mouse leukocyte receptors from NKR-P1 family.
Adámek, David ; Man, Petr (advisor) ; Brdička, Tomáš (referee)
Mouse NK cell receptors belonging to NKR-P1 family plays role in activation, inhibition and cytokine secretion by these cells. Aim of this thesis is preparation of extracellular parts of C57BL/6 mouse strain activating receptors mNKR-P1A and mNKR-P1C. Production vectors with coding sequences of both proteins were prepared. Next, optimization of production in E. coli was done and appropriate in vitro refolding and purification protocol were developed. Purified proteins were characterized by mass spectrometry and labeled by a fluorescent dye. Primary screening for potential ligand was performed. Further work will involve structural characterization of the receptors and identification of their ligands. These data may help to clarify the function of NK cells.
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