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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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Determination of selected biomarkers of nephrotoxicity in urine and plasma.
Pražáková, Aneta ; Maixnerová, Jana (advisor) ; Bárta, Pavel (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Aneta Pražáková Supervisor: RNDr. Jana Maixnerová, Ph.D. Title of diploma thesis: Determination of selected biomarkers of nephrotoxicity in urine and Plasma The discovery and development of novel biomarkers, that can be used for diagnosis of kidney damage earlier and more accurately, are needed for the effective prediction of drug-induced nephrotoxicity. Mechanisms of drug-induced nephrotoxicity include changes in glomerular hemodynamics, tubular cell cytotoxicity, inflammation, crystalline nephropathy, etc. Detection at initial stage of damage using sensitive and specific biomarkers belongs between one of the most important strategies in the treatment of acute kidney injury and renal failure. Although some these biomarkers do not show specificity and sensitivity, several promising biomarker candidates have been established recently to evaluate nephrotoxicity, e.g. selected KIM-1, cystatin C and NGAL. The advantages of these biomarkers compared to traditionally used biomarkers are higher sensitivity, specificity, just mentioned early diagnosis and non-invasiveness (the possibility of determination levels of the biomarkers from blood or urine). The aim of this diploma thesis was to determine...
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Study of cytotoxicity of newly prepared bronchodilators in vitro
Havelková, Michaela ; Smutná, Lucie (advisor) ; Bárta, Pavel (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Michaela Havelková Supervisor: PharmDr. Lucie Hyršová, PhD. Title of diploma thesis: Study of cytotoxicity of newly prepared bronchodilators in vitro This diploma thesis is focused on the determination the influence of developmental bronchodilatory active substances on viability of model tumor cell lines of human liver cells HepG2 and human lung cells HCC827. These potential drugs are theoretically suitable for the treatment of asthma bronchiale and COPD. The tested compounds (VN014a, VN015a, VN045b, VN122c) were derived from structure of the quinazoline alkaloids vasicine and vasicinone, which are substances contained in the Justicia adhatoda plant. The plant originated in India is widely used in traditional Ayurvedic medicine besides others to treat respiratory problems. The combination of both alkaloids showed significant bronchodilation activity. Cytotoxic activity of compounds was tested using colorimetric method by measuring cell metabolic activity based on reduction of tetrazolium compound. The IC50 value was determined for each studied compound on both used cell lines to assess the influence on cell viability. Overall, the tested developmental bronchodilators proved cytotoxic effect...
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Study of transport of protein molecules across cell membranes
Hornová, Aneta ; Smutná, Lucie (advisor) ; Bárta, Pavel (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Aneta Hornová Supervisor: PharmDr. Lucie Hyršová, Ph.D. Title of diploma thesis: Study of transport of protein molecules across cell membranes Kidney transporters are found mainly in the proximal tubules, they participate in the secretion and reabsorption of endogenous and exogenous substances. Plenty of kidney transporters have been identified, however this diploma thesis is focused on megalin. Specifically, we dealed with determination of transport of peptides via this renal transporter in HK-2 and JEG-3 cell lines. Megalin is a glycoprotein whose main function is tubular reabsorption of albumin filtered in glomerules and other high molecular weight substances. Another endogenous substrates of megalin are e.g. transcoblamine - vitamin B12, insulin or light chains of immunoglobulins. It is also able to transport some drugs like aminoglycoside and polypeptide antibiotics. Within this work, we investigated the influence of reduced expression of megalin on the transport of albumin and anti-VEGFR2 aptamer. Reduction of expression was achieved by transfection of cells with siRNA against the LRP2 gene. Lower expression of LRP2 led to reduction of transport of albumin and anti-VEGFR2 aptamer into...
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In vitro saturation study of gallium-67 and zirconium-89 labelled monoclonal antibody ramucirumab on SKOV-3 cell line
Holodňáková, Nikola ; Bárta, Pavel (advisor) ; Smutná, Lucie (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Biophysics and Physical Chemistry Student: Nikola Holodňáková Supervisor: Mgr. Pavel Bárta, Ph.D. Name of the work: In vitro saturation study of gallium-67 and zirconium-89 labelled monoclonal antibody ramucirumab on SKOV-3 cell line. Targeted biological treatment becomes more and more important with the development of a new therapy in oncology. It stimulates immune system to eliminate cancer cells. Significant progress has been made since the introduction of monoclonal antibodies. They represent one of the newest possibility used in diagnosis and treatment of tumours. The ability of the monoclonal antibody ramucirumab is to recognize and bind specifically to tumour cell antigens such as the VEGF type 2 receptor (VEGFR-2) and thus to supress angiogenic process. Anti-angiogenic ramucirumab inhibits this receptor via blocking of VEGF binding sites, which prevents the growth of tumours. It is possible to increase antitumor effect of monoclonal antibodies by their combination with other molecules like radionuclides, toxins and cytostatics when forming the so called conjugates. Prepared immunoconjugates serve as diagnostic and therapeutic tools also in Nuclear Medicine. The aim of the experimental work in was the preparation of...
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