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Preparation of glutamate carboxypeptidase III using mammalian expression system
Šimonová, Lenka ; Konvalinka, Jan (advisor) ; Obšil, Tomáš (referee)
The role of glutamate carboxypeptidase II in mammalian organism is already known quite well but only little is known about its homologue glutamate carboxypeptidase III. For structural and functional characterization of any protein, a large amount of protein is required. Protein could be obtained by expression in tissue culture. Properties of the protein may be affected by post translational modifications, where different organisms create different modifications. Therefore, we set to develop a system for recombinant expression of GCPIII in mammalian cells. First the mammalian expression system HEK 293-6E was introduced as a substitute for the current insect expression system. The advantage of this mammalian expression system is its option of transient transfection and that it is easy to cultivate cells under suspension conditions. Further, transfection conditions for this system were optimized by green fluorescent protein expression, for easy detection by flow cytometry. DNA encoding the extracellular part of mouse GCPIII (mEXSTII) was cloned into five expression plasmids with His or Fc tags attached to N- or C-termini. Cells were transfected with prepared plasmids. The presence of mEXSTII in media was tested using Western blot and subsequently the activity of GCPIII was tested by cleaving its...
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Interaction of a surface marker of immune cells with low-molecular weight ligands and their polymer conjugates
Šimonová, Lenka ; Konvalinka, Jan (advisor) ; Obšil, Tomáš (referee)
Millions of people worldwide die of cancer every year. In the last decade, im- munotherapy offered new treatment options achieving long-lasting remissions in a number of patients. Several new immunotherapy-based drugs have been ap- proved by Food and Drug Administration. However, majority of patients either do not respond or soon relapse. Combination of therapies as well as exploring new immune checkpoints seems promising. This thesis focuses on the new immunotherapeutic target CD73. CD73 is membrane ectonucleotidase, widely expressed on the regulatory leukocytes and on cancer cells. The enzymatically active CD73 contributes to the tumour mi- croenvironment by production of immunosuppressive adenosine. This novel im- mune checkpoint is being intensively studied. This thesis aims on development of new approaches for targeting and inhibition of CD73. Soluble recombinant CD73 (rhCD73) was prepared in mammalian expression system and transfectants stably expressing membrane-bound CD73 were prepared as well. Inhibitors necessary for both of my goals have been designed based on published inhibitor of CD73. Development and evaluation of novel antibody mimetic for CD73 characteri- sation was done. The so-called iBody, HPMA polymer conjugate decorated with CD73 inhibitor for targeting, fluorophore for...
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Interaction of a surface marker of immune cells with low-molecular weight ligands and their polymer conjugates
Šimonová, Lenka ; Konvalinka, Jan (advisor) ; Obšil, Tomáš (referee)
Millions of people worldwide die of cancer every year. In the last decade, im- munotherapy offered new treatment options achieving long-lasting remissions in a number of patients. Several new immunotherapy-based drugs have been ap- proved by Food and Drug Administration. However, majority of patients either do not respond or soon relapse. Combination of therapies as well as exploring new immune checkpoints seems promising. This thesis focuses on the new immunotherapeutic target CD73. CD73 is membrane ectonucleotidase, widely expressed on the regulatory leukocytes and on cancer cells. The enzymatically active CD73 contributes to the tumour mi- croenvironment by production of immunosuppressive adenosine. This novel im- mune checkpoint is being intensively studied. This thesis aims on development of new approaches for targeting and inhibition of CD73. Soluble recombinant CD73 (rhCD73) was prepared in mammalian expression system and transfectants stably expressing membrane-bound CD73 were prepared as well. Inhibitors necessary for both of my goals have been designed based on published inhibitor of CD73. Development and evaluation of novel antibody mimetic for CD73 characteri- sation was done. The so-called iBody, HPMA polymer conjugate decorated with CD73 inhibitor for targeting, fluorophore for...
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Preparation of glutamate carboxypeptidase III using mammalian expression system
Šimonová, Lenka ; Konvalinka, Jan (advisor) ; Obšil, Tomáš (referee)
The role of glutamate carboxypeptidase II in mammalian organism is already known quite well but only little is known about its homologue glutamate carboxypeptidase III. For structural and functional characterization of any protein, a large amount of protein is required. Protein could be obtained by expression in tissue culture. Properties of the protein may be affected by post translational modifications, where different organisms create different modifications. Therefore, we set to develop a system for recombinant expression of GCPIII in mammalian cells. First the mammalian expression system HEK 293-6E was introduced as a substitute for the current insect expression system. The advantage of this mammalian expression system is its option of transient transfection and that it is easy to cultivate cells under suspension conditions. Further, transfection conditions for this system were optimized by green fluorescent protein expression, for easy detection by flow cytometry. DNA encoding the extracellular part of mouse GCPIII (mEXSTII) was cloned into five expression plasmids with His or Fc tags attached to N- or C-termini. Cells were transfected with prepared plasmids. The presence of mEXSTII in media was tested using Western blot and subsequently the activity of GCPIII was tested by cleaving its...
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Economic analysis of selected company
Šimonová, Lenka ; Mach, Jiří (advisor) ; Kateřina, Kateřina (referee)
The thesis Economic analysis of a chosen company presents economic performance assessment of the company Rabbit Trhový Štěpánov a. s. during five closed accounting period from 2010 to 2014.
The first part of the thesis is focused on the theoretical definition individual methods of economic analysis. Followed the practical part, in this are utilized in processing data emerging from company results of individual analysis. The thesis conclusion offers result data summary and evaluation and provides a baseline for recommendations on future company development.
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