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Role of disulphide bonds in hA2A subtype adenosine receptor
Zappe, Lukáš ; Matoušková, Petra (advisor) ; Bárta, Pavel (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Lukáš Zappe Supervisor: Ing. Petra Matoušková Ph.D., PD Dr. Anke C. Schiedel Title of diploma thesis: Role of disulphide bonds in hA2A subtype adenosine receptor The adenosine A2A receptor belongs to the G protein coupled receptor family (GPCR). GPCRs are the targets of almost 40% of the drugs on the market. GPCRs are characterized by seven transmembrane helices, which are linked by three extracellular and three intracellular loops (ECL and ICL). The structure of the receptor has been revealed by crystallography, hence we know that ECL1 and ECL2 are connected by several disulphide bonds. The ECL2 is believed to be involved in ligand binding and recognition. In order to understand the relevance of those disulphide bonds involved in this process, four adenosine A2A receptor mutants were generated by one-site direct mutagenesis, in which the cysteine residues were replaced with serine residues (C146S, C159S, C166S and C146S- C159S). These receptor mutants were expressed in the mammalian cell line, CHO K1(Chinese Hamster Ovary) and the receptor expression was tested with ELISA (Enzyme- linked immunosorbent assay). The determination of ligand binding has been carried out by radioligand...
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Role of disulphide bonds in hA2A subtype adenosine receptor
Zappe, Lukáš ; Matoušková, Petra (advisor) ; Bárta, Pavel (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Biochemical Sciences Candidate: Lukáš Zappe Supervisor: Ing. Petra Matoušková Ph.D., PD Dr. Anke C. Schiedel Title of diploma thesis: Role of disulphide bonds in hA2A subtype adenosine receptor The adenosine A2A receptor belongs to the G protein coupled receptor family (GPCR). GPCRs are the targets of almost 40% of the drugs on the market. GPCRs are characterized by seven transmembrane helices, which are linked by three extracellular and three intracellular loops (ECL and ICL). The structure of the receptor has been revealed by crystallography, hence we know that ECL1 and ECL2 are connected by several disulphide bonds. The ECL2 is believed to be involved in ligand binding and recognition. In order to understand the relevance of those disulphide bonds involved in this process, four adenosine A2A receptor mutants were generated by one-site direct mutagenesis, in which the cysteine residues were replaced with serine residues (C146S, C159S, C166S and C146S- C159S). These receptor mutants were expressed in the mammalian cell line, CHO K1(Chinese Hamster Ovary) and the receptor expression was tested with ELISA (Enzyme- linked immunosorbent assay). The determination of ligand binding has been carried out by radioligand...
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Radiolabeling of ramucirumab followed with the study of its internalization in vitro
Gajdoš, Jakub ; Bárta, Pavel (advisor) ; Kuchařová, Monika (referee)
v anglickém jazyce Charles University Faculty of Pharmacy in Hradec Králové Department of Biophysics and Physical Chemistry Student: Bc. Jakub Gajdoš Supervisor: Mgr. Pavel Bárta, Ph.D. Title of diploma thesis: Radiolabeling of ramucirumab followed with the study of its internalization in vitro. The process of angiogenesis ensures the formation of the bloodstream at the site of its increased need. Therefore, it is not surprising that angiogenesis is often included in the tumor production process, because it provides the tumor cells nutrition supply and metabolite removal. The targeting of angiogenesis has become a key topic of some scientific research. The process of tumor blood supply formation provides a family of vascular endothelial factors (VEGFs) and their respective receptors, which have become the target of the angiogenesis attenuation in a cancer treatment. One of many therapeutics is the monoclonal antibody ramucirumab targeted against VEGF receptor type 2 (VEGFR-2). Radioactive labeling of ramucirumab with a suitable radionuclide could bring benefits in either radiotherapy or radiodiagnostics. The aim of this diploma thesis was the indirect radioactive labeling of monoclonal antibody ramucirumab using 99m Tc as radiodiagnostic nuclide via the chelation agent succinimidyl-6-...
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Testing of influence of newly synthetized compounds on viability of cells in vitro
Šebová, Dominika ; Smutná, Lucie (advisor) ; Bárta, Pavel (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Dominika Šebová Supervisor: PharmDr. Lucie Smutná, Ph.D. Title of diploma thesis: Testing of influence of newly synthetized compounds on viability of cells in vitro Research and development of new substances intended for terapeutical use is very difficult and time consuming process. Preclinical and clinical studies are essential part of this process in order to ensure safety, efficacy and quality of a new drug. This thesis is focused on cytotoxicity studies, that form important part of preclinical studies of a new substance. An effect of potential drugs was tested in vitro on a cellular model. Cells of the HepG2 cell line, derived from well-differentiated hepatocellular carcinoma, were used in our experiment. The monitored parameter was cell viability. During the experiment, 9 substances with 9 different concentrations in concentration range 1-1000 µM were tested. Substances, that were tested had antimycobacterial effect and their basic chemical structure was derived from the antituberculotic drug isoniazide. Cell viability was monitored after 24 and 48 hours of incubation of individual substance with HepG2 cells. Cell viability was determined by using colorimetric MTS assay, in which the...
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Study of the effect of the amount of chelating agent addition on the result of monoclonal antibody conjugation
Janečková, Adriana ; Bárta, Pavel (advisor) ; Janoušek, Jiří (referee)
v anglickém jazyce Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Mgr. Adriana Janečková Supervisor: Mgr. Pavel Bárta, Ph.D. Title of rigorosum thesis: Study of the effect of the amount of chelating agent addition on the result of monoclonal antibody conjugation Monoclonal antibodies have become the important tool not only for the therapy, but also for the diagnosis of the infectious, autoimmune or cancer diseases in the last few decades. In addition to their own therapeutic effect, monoclonal antibodies are used as vectors for biological substances with therapeutical and diagnostic importance. Besides bacterial and plant toxins, monoclonal antibodies are also effective in their capability to deliver radionuclides to the site of their specific binding, i.e. the antigen against which they were prepared. The so called radioimmunoconjugates are then successfully used in radiodiagnostic and radiotherapeutical field. Radionuclides can be bound to antibody molecule either directly in the case of halogenation or indirectly. The latter method is the most common one due to a greater stability and suitability for more radionuclides, which can be transported by antibodies. Substances that allow the radionuclide biding to the monoclonal antibody...
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The influence of critical condition of patients on DNA damage
Verešpejová, Natália ; Kuchařová, Monika (advisor) ; Bárta, Pavel (referee)
The first cases of patients with pneumonia which grew into an acute respiratory distress syndrome and caused breathing problems began to appear in December 2019. Coronavirus disease 2019 (COVID-19) is the cause of a global pandemic and it is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A complex interplay of factors is responsible for the progression of the disease. Some studies suggest that it promotes oxidative stress and thus may lead to oxidative damage to cells and DNA. The purpose of this study was to observe the relationship between oxidative DNA damage and a critical condition caused by COVID-19 using a comet assay technique. The basic principle of the used method consists in fixation of lymphocytes in an agarose gel, removal of the membrane and cytoplasm of cells, incubation with specific enzymes and electrophoresis. In the process of electrophoresis, negatively charged DNA fragments migrates towards the anode and the cell thus acquires the typical shape of a comet. Comets are visualized using the DNA intercalation dye ethidium bromide. We quantified single - strand breaks and oxidized pyrimidines and purines by using specific enzymes (modification of the method for detecting specific lesions). Results are reported as % tail DNA, thus the percentage of DNA in the...
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Testing of influence of newly synthetized compounds on viability of cells in vitro
Šebová, Dominika ; Smutná, Lucie (advisor) ; Bárta, Pavel (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Dominika Šebová Supervisor: PharmDr. Lucie Smutná, Ph.D. Title of diploma thesis: Testing of influence of newly synthetized compounds on viability of cells in vitro Research and development of new substances intended for terapeutical use is very difficult and time consuming process. Preclinical and clinical studies are essential part of this process in order to ensure safety, efficacy and quality of a new drug. This thesis is focused on cytotoxicity studies, that form important part of preclinical studies of a new substance. An effect of potential drugs was tested in vitro on a cellular model. Cells of the HepG2 cell line, derived from well-differentiated hepatocellular carcinoma, were used in our experiment. The monitored parameter was cell viability. During the experiment, 9 substances with 9 different concentrations in concentration range 1-1000 µM were tested. Substances, that were tested had antimycobacterial effect and their basic chemical structure was derived from the antituberculotic drug isoniazide. Cell viability was monitored after 24 and 48 hours of incubation of individual substance with HepG2 cells. Cell viability was determined by using colorimetric MTS assay, in which the...
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Pharmacological interventions of nuclear receptors in diabettes mellitus
Draský, Jakub ; Pávek, Petr (advisor) ; Bárta, Pavel (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Jakub Draský Supervisor: prof. PharmDr. Petr Pávek, Ph.D. Title of diploma thesis: Pharmacological influence of nuclear receptors in diabetes mellitus therapy Nuclear receptors belong to the superfamily of transcription factors, their main functions include regulating the expression of target genes. In my work I focused mainly on the group of orphan receptors, namely the pregnane X receptor (PXR) and the constitutive androstane receptor (CAR). A common feature of these receptors is their activation by a specific ligand. Both CAR and PXR have an essential function as biological sensors of hydrophobic xenobiotics when they induce enzymes I and II. phase of metabolism. They are also essential in the regulation of gluconeogenesis, insulin response, adipogenesis, cholesterol homeostasis, fatty acids, triglycerides and glycogen. The aim of this experimental work was to introduce a luciferase reporter assay method for two DNA constructs containing the promoter region of the PEPCK and CYP7A1 genes. We used the known agonist rifampicin and the antagonist SPA70 to activate/deactivate PXR. We used CITCO as a CAR receptor agonist. We first verified the functionality of the luciferase reporter gene assay...
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Evaluation of liver toxicity in vitro
Kafuňková, Kateřina ; Maixnerová, Jana (advisor) ; Bárta, Pavel (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Kateřina Kafuňková Supervisor: RNDr. Jana Maixnerová, Ph.D. Title of diploma thesis: Evaluation of liver toxicity in vitro The subject of the diploma thesis is toxicity evaluation of newly synthesised substances on a cellular model representing a hepatocyte. The tested substances have been provided by the Department of Organic and Bioorganic Chemistry at the Faculty of Pharmacy in Hradec Králové, Charles University, as potential antifungal pharmaceuticals and medicine effective against Methicillin-resistant Staphylococcus aureus (MK-NO2-1, MK-NO2-2, DAB-5-K, PABA-Me-5, PABA-Et-5, MK-F-1, PABAN- 3, PABAN-5, MK-F-2, MK-CF3-1, MK-CF3- 2). In order to determine the toxicity we have implemented two methods. The first method is based upon measuring metabolic activity of cells by means of reducing tetrazolium to a colored product. The second method detects the amount of LDH released as a marker of toxicity. The human hepatoma cell line HepG2was used as a model. . The IC50 and EC50 parameters were used to assess the degree of viability and cytotoxicity. The final values obtained from the first method indicated all tested substances showed a certain level of toxicity to the hepatic tissue. MK-CF3-2 is the most...
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