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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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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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Structural study of the DCL-1 receptor using mass spectrometry
Růžičková, Barbora ; Kavan, Daniel (advisor) ; Mrázek, Hynek (referee)
DCL-1 (CD302) is a type I transmembrane C-type lectin receptor, which is expressed on monocytes, macrophages, granulocytes and dendritic cells. However, its extracellular domain lacks the amino acids motives essential for carbohydrate binding in the presence of calcium ions, suggesting that it does not have the classic binding capacity found in other C-type lectin receptors such as the mannose receptor. No exogenous or endogenous ligands have been identified yet, though. Due to internal colocalization with F-actin we can assume, that this unconventional lectin receptor plays a role not only in endocytosis and phagocytosis but also in the cell adhesion and migration. The receptor DCL-1 was first identified as a genetic fusion partner of human DEC-205 multilectin receptor in Hodgkin's lymphoma cell lines. The experimental part of this thesis deals with the characterization of disulfide bonds and data acquisition for validation of DCL-1 crystal structure. First the production and refolding conditions were optimized to obtain the highest amount of DCL-1 protein, precisely its extracellular domain. These optimal conditions were used to prepare the protein for in-gel digestion using specific endopeptidases in the presence of cystamine followed by LC-MS analysis. DCL-1 disulfide bonds were determined by comparing...
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