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Comparison of in vitro methods for the study of cytotoxicity
Eliášová, Pavla ; Maixnerová, Jana (advisor) ; Bárta, Pavel (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Pavla Eliášová Supervisor: RNDr. Jana Maixnerová, Ph.D. Title of diploma thesis: Comparison of in vitro methods for the study of cytotoxicity People are exposed to a growing number of toxic substances from the environment. Endocrine disrupting chemicals (EDCs) are a broad category of molecules that are thought to cause adverse effects on the endocrine system by interfering with the synthesis, transport, degradation or action of endogenous ligands. One of the aims of this thesis was to determine the in vitro toxicity of 17 selected endocrine disruptors on the human hepatocellular carcinoma HepG2 cell line. Cell viability was determined using the CellTiter96® AQueous One Solution Cell Proliferation Assay colorimetric method, the principle of which is the reduction of MTS to the colored product formazan by mitochondria in viable cells. The cytotoxic potential of the compounds was expressed by using the toxicological parameter IC50, which was measured in three time intervals (6, 12 and 24 hours). For 14 substances: Atrazine, DHEP, Bisphenol A, Carbofuran, 3-hydroxycarbofuran, Cypermethrin, DDE, DES, MEHP, PCB 118, PCB 153, PFOA, PFOS, Propiconazole, IC50 > 100 µM (respectively > 250 µM) was...
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Determination of renal toxicity of antineoplastics in vitro
Zádrapová, Marie ; Maixnerová, Jana (advisor) ; Smutná, Lucie (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Bc. Marie Zádrapová Supervisor: RNDr. Jana Maixnerová, Ph.D. Title of diploma thesis: Determination of renal toxicity of antineoplastics in vitro BRAF inhibitors are important antineoplastics. They work on the principle of inhibition of certain types of protein kinases and turned out to be very efficient for the treatment of melanoma. One of their disadvantages is relatively early onset of resistance; thus, it is important to look for new combinations of drugs that are already in use or work on the development of new structures with similar inhibition efficacy on melanoma cells. Encorafenib and its combination with binimetinib have been shown to be very promising drugs from the group of BRAF inhibitors, however, potential renal toxicity may be a therapeutic limitation. This thesis was focused on the determination of in vitro cytotoxicity of encorafenib on different types of renal cells in three time intervals and on its comparison with two drug standards - amphotericin B and paracetamol. Three types of morphologically and functionally different kidney cells (PODO / TERT256, HK-2 and HEK293) were used for this purpose. The cytotoxic potential was measured by colorimetric method CellTiter 96®...
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Study of substance transport via membrane transporters
Kučerová, Zuzana ; Smutná, Lucie (advisor) ; Maixnerová, Jana (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Zuzana Halamová Supervisor: PharmDr. Lucie Smutná, PhD. Title of diploma thesis: Study of substance transport via membrane transporters Wide range of specialized transport proteins expressed mainly in the kidney proximal tubule ensures the excretion of endogenous and exogenous substances. OCT2 (organic cation transporter 2) is localized on the basolateral membrane of the kidney proximal tubule, the transporter transports broad spectrum of structurally different substances. Drug-drug interactions may occur at the level of this transporter due to its broad substrate specificity. Within this diploma thesis the accumulation of the radiolabeled antiviral agent [3 H]lamivudine and the model hOCT2 substrate [3 H]MPP+ was evaluated using accumulation and transport studies. The canine kidney cells MDCKII or human cell line HeLa were used as model cell lines. Mentioned cell lines expressed the human OCT2 transporter stably or transiently. The antibacterial chemotherapy agent trimethoprim was used as competitive inhibitor of OCT2 transporter. The results indicate the lamivudine accumulation in the cells with OCT2 is low compared to accumulation of MPP+ . Accumulation studies showed that lamivudine had a...
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Determination of toxicity using a cell model.
Fojkesová, Karolína ; Maixnerová, Jana (advisor) ; Bárta, Pavel (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Karolína Fojkesová Supervisor: RNDr. Jana Maixnerová, Ph.D. Title of diploma thesis: Determination of toxicity using a cell model The subject of the diploma thesis was measure and evaluate the cytotoxicity in vitro of 22 newly synthesized substances with potential use for their antifungal, antibacterial and antimycobacterial activity on the standard HepG2 cell line. The tested substances were synthetized at the Department of Organic and Bioorganic Chemistry at the Faculty of Pharmacy in Hradec Králové, Charles University and their chemical structure is mafenide derivative. The compounds are MAF, MAF-4, MAF-8, MAF-9, MAF-10, MAF-11, MAF-12, MAF-13, MAF- 14, MAF-16, A-5-F, A-3,5-F2, A-3-Br-5-Cl, A-3,5-Cl2, A-3,5-Br2, A-5-Br, A-THIOF, A-SA, A-3,5-I2, A-5-Cl, A-3-I-5-Cl, A-5-I. Cytotoxicity has been measured using the CellTiter 96® AQueous One Solution Cell Proliferation Assay, which is a colorimetric method based on the reduction of the tetrazolium salt of MTS to the colored soluble formazan, and substances have been compared and evaluated according to the IC50 parameter. In case of 9 substances, specifically MAF, MAF-8, MAF-11, MAF-12, MAF-16, A-5- F, A-3,5-F2, A-SA, A-5-Cl, the IC50 was greater than...
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Determination of potential cytotoxic effect of experimental substances in vitro.
Osinková, Barbora ; Maixnerová, Jana (advisor) ; Smutná, Lucie (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Barbora Osinková Supervisor: RNDr. Jana Maixnerová Ph.D. Title of diploma thesis: Determination of potential cytotoxic effect of experimental substances in vitro The subject of this master's thesis was to evaluate the cytotoxic potential of newly synthesized substances in vitro in the cell model HepG2, which is cell line obtained from a human liver carcinoma. These substances were synthesized within the Department of Organic and Bioorganic Chemistry at the Faculty of Pharmacy in Hradec Králové, Charles University (GUAM, GUAM-NH2, THIOSEMIK, GUAM-2, GUAM-6, GUAM- 11, GUAM-12, GUAM-18, GUAM-19, GUAM-2-1, GUAM-6-3). These substances are being investigated for assumed antimicrobial activity against G+ bacteria and fungi or for their ability to inhibit cholinesterases. The commercial colorimetric method CellTiter 96® AQueous One Solution Cell Proliferation Assay was used for in vitro determination of cytotoxicity, the principle of which is to measure the metabolic activity of cells based on the reduction of the tetrazolium salt of MTS to coloured formazan. The half-maximal inhibitory concentration IC50 was used to determine the number of viable cells and for evaluation of cytotoxic potential of...
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Determination of the stoichiometry of the coopper complexes with 7,8-dihydroxycoumarins
Mocová, Klára ; Mladěnka, Přemysl (advisor) ; Maixnerová, Jana (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate: Klára Mocová, MSc. Supervisor: Assoc. Prof. Přemysl Mladěnka, Pharm.D., Ph.D. Title of Thesis: Determination of the stoichiometry of the copper complexes with 7,8-dihydroxycoumarins Copper is an important element, which is necessary for right functioning of the organism because of its presence in wide range of enzymes. However, increased level of this element can cause various health complications. Coumarins are natural or synthetic substances, which contain 1,2-benzopyrone in their chemical structure. These compounds are known, among others, by their antioxidant activity, which includes the ability to chelate metals and hence form complexes with them. The stoichiometry of the complex is one of the important characteristics of metal complexes. The aim of this in vitro study was to assess the stoichiometry of 7,8- dihydroxycoumarins complexes with copper. We have been dealing with chelating activity of 7,8-dihydroxy-4-methylcoumarin and 7,8-dihydroxycoumarin with Cu+ and Cu2+ ions. Direct UV-Vis spectrophotometry was used to measure the complex formation at four physiologically or pathophysiologically relevant pH conditions (4,5; 5,5; 6,8 a 7,5). Both tested coumarins chelated Cu2+ ions in...
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New pharmacological interventions influencing food intake focused on effects of CART (cocaine- and amphetamine-regulated transcript) peptide and prolactin-releasing peptide
Maixnerová, Jana ; Maletínská, Lenka (advisor) ; Zorad, Štefan (referee) ; Skálová, Lenka (referee)
The thesis was focused on characterization of biological activities of two recently discovered anorexigenic neuropeptides: CART (cocaine- and amphetamine-regulated transcript) peptide and prolactin-releasing peptide (PrRP). In order to find a pharmacophore of CART peptide, shorter fragments of CART(61- 102) peptide were tested for binding to PC12 cells and inhibition of food intake in fasted mice. The results showed that a compact structure of CART peptide containing three disulphide bridges is necessary for preservation of its full biological activity. In the second part of the thesis, synergistic and long-lasting effect of centrally administered peptide CART and peripherally administered cholecystokinin (CCK) is described. In fasted C57BL/6 mice, the anorexigenic effect of CART was enhanced by a subthreshold dose of CCK, while CCK1 receptor antagonist devazepide blocked the effect of CART peptide on food intake. In the third part of the thesis, food intake in fed lean and MSG (monosodiumglutamate treated) obese male mice with lesions in nucleus arcuatus (ARC) was followed. Anorexigenic action of CART peptide was preserved but satiety effect of CCK was completely lost in MSG obese mice and therefore, effective leptin signaling in ARC is necessary for satiety effect of CCK. Finally, the PrRP...
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Development of insecticides inhibiting acetylcholineseterase
Mányová, Brigita ; Vopršalová, Marie (advisor) ; Maixnerová, Jana (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Brigita Mányová Supervisor: PharmDr. Marie Vopršalová, CSc. Supervisor-specialist: PharmDr. Vendula Hepnarová, Ph.D. Title of diploma thesis: Development of insecticides inhibiting acetylcholineseterase Pest insects cause problems and damage all over the world. They are vectors of serious diseases such as malaria, dengue fever, yellow fever, Zika virus disease and chikungunya disease. They cause huge damage to agricultural crops and are annoying to everyday life in living spaces. The aim of this diploma thesis was in vitro testing of selected compounds from the group of bis-isoquinoline and bispyridinium acetylcholinesterase inhibitors as potential insecticides. Another goal was also to create relationships between structure and effect. The ability of these compounds to inhibit both human (hAChE) and fly acetylcholinesterase (MdAChE) was evaluated. The modified Ellman spectrophotometric method was used. The half inhibitory concentration (IC50) values were obtained for both enzymes and the selectivity indexes (SI) were then calculated. Compounds having IC50s in micromolar or nanomolar range and exhibiting selectivity for MdAChE were most desirable. During the testing of these inhibitors, three...
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Preparation of genetically modified cell line as a model for accumulation studies
Rückelová, Alexandra ; Smutná, Lucie (advisor) ; Maixnerová, Jana (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Alexandra Rückelová Supervisor: PharmDr. Lucie Hyršová, Ph.D. Title of diploma thesis: Preparation of genetically modified cell line as a model for accumulation studies The studied megalin transporter belongs to the group of lipoprotein receptors, namely low density lipoprotein receptor-related protein 2 (LRP2). It is a transporter capable of transporting large organic molecules into the cell through the receptor - mediated endocytosis. Huge variety of peptide compounds including albumin, hemoglobin, vitamins, hormones as well as the amoniglycoside antibiotics (e.g. gentamicine), polymixin B, cadmium and other substrates belong among its substrates. This theses is focused on the creation of cell models for the study of the transport of large organic molecules across the cell membranes. I am dealing with reducing megalin expression in two model cell lines using siRNA against LRP2 gene. The inhibition of expression was determined using the RT-PCR method and subsequently verified on the functional level using known megalin substrate gentamicine. It is an aminoglycoside antibiotic with nephrotoxic action. The principle of these validation experiments was to reduce gentamicine toxic effects in cells...
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