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Study on cytotoxicity of compounds in vitro.
Vašková, Lucie ; Maixnerová, Jana (advisor) ; Kuchařová, Monika (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Studentka: Lucie Vašková Školitel: RNDr. Jana Maixnerová, Ph.D. Název diplomové práce: Study on cytotoxicity of compounds in vitro The subject of this diploma thesis was to assess the effect of newly synthesized antimycobacterial substances on the viability of human hepatocellular carcinoma (HepG2) cells. The tested substances were esters (HE-nMe, HE-4PHOPH, HE-KARVA, HE-2NAFT, HE-METRO, HE-CH2PY, HE-8CHIN) and thioesters (HES-4H, HES- nETH) of antituberculotic isoniazid. Experiments performed with these substances have shown, that like isoniazid, the substances inhibit InhA enzyme in mycobacteria and therefore interfere with cell wall biosynthesis. Isoniazid is a drug standardly used in the first line of TB treatment. Together with other first-line antituberculotics, some hepatotoxic potential has been reported during treatment. To assess the possible cytotoxic effect of the tested isoniazid derivatives, the standard human hepatocyte cell line HepG2 was chosen as the cell model. Cell viability was assessed by a colorimetric method that measures the metabolic activity of cells based on the reduction of the tetrazolium compound MTS. Obtained values were quantitatively compared using the toxicological...
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Interaction of human immune cells with ultrasmall nanoparticles
Javorová, Pavlína ; Hubálek Kalbáčová, Marie (advisor) ; Krulová, Magdaléna (referee)
The application of nanoparticles in the field of theranostics requires knowledge of the specific interaction of nanoparticles with the immune system. One of the first cells with which nanoparticles interact when given to the body are cells of the mononuclear phagocytic system. The aim of this diploma thesis is to prepare an in vitro study that describes the effect of two types of gold and three types of silicon ultra-small nanoparticles on immune cells. Immune cells are presented in the form of primary PBMCs isolated from whole blood , and cells of monocytic cell line THP-1 in the form of monocytes and differentiated macrophages. During the experiments with primary cells, emphasis is placed on maintaining the concept of personalized protein corona. After characterization of the immune cells used, cells are subsequently stimulated with ultra-small nanoparticles and the influence of these nanoparticles on cell metabolism, viability, degree of differentiation and secretion of pro- and antiinflammatory cytokines is monitored. The outcome takes into account further use of the tested nanoparticles in the field of biomedicine. Key words: primary monocytes, cell lines, differentiation, macrophages, cytokines, cytotoxicity
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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 organ toxicity of BRAF inhibitors in vitro.
Miškovčíková, Zuzana ; Maixnerová, Jana (advisor) ; Smutná, Lucie (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Zuzana Miškovčíková Supervisor: RNDr. Jana Maixnerová, PhD. Title of diploma thesis: Determination of organ toxicity of BRAF inhibitors in vitro. Malignant melanoma is one of the most serious skin diseases today. Therapy of advanced melanoma is difficult and often ineffective. BRAF inhibitors (dabrafenib and vemurafenib) have dramatically changed the results of melanoma treatment in the last few years. BRAF inhibitors are one of the most effective drugs against melanoma, but their clinical application is largely limited by drug resistance. Available clinical studies have shown an adverse nephrotoxic effect of BRAF inhibitors, but information on its mechanism is limited. Published studies further suggest that the toxic effect of BRAF inhibitors is primarily directed to podocytes located in the glomerular membrane. Thus, the aim of our study was to assess the cytotoxic effect of BRAF inhibitors on selected model renal cells in vitro in order to confirm the renal target toxicity. The main objective of the study was to analyse whether the nephrotoxic effect of BRAF inhibitors is specifically limited to podocytes or whether it can damage other renal cells. The experiments were performed on human cell lines...
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Determination of organ toxicity of BRAF inhibitors in vitro.
Miškovčíková, Zuzana ; Maixnerová, Jana (advisor) ; Hyršová, Lucie (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Zuzana Miškovčíková Supervisor: RNDr. Jana Maixnerová, PhD. Title of diploma thesis: Determination of organ toxicity of BRAF inhibitors in vitro. Malignant melanoma is one of the most serious skin diseases today. Therapy of advanced melanoma is difficult and often ineffective. BRAF inhibitors (dabrafenib and vemurafenib) have dramatically changed the results of melanoma treatment in the last few years. BRAF inhibitors are one of the most effective drugs against melanoma, but their clinical application is largely limited by drug resistance. Available clinical studies have shown an adverse nephrotoxic effect of BRAF inhibitors, but information on its mechanism is limited. Published studies further suggest that the toxic effect of BRAF inhibitors is primarily directed to podocytes located in the glomerular membrane. Thus, the aim of our study was to assess the cytotoxic effect of BRAF inhibitors on selected model renal cells in vitro in order to confirm the renal target toxicity. The main objective of the study was to analyse whether the nephrotoxic effect of BRAF inhibitors is specifically limited to podocytes or whether it can damage other renal cells. The experiments were performed on human cell lines...
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Introduction of cellular NFkappa-B model
Čečrle, Michal ; Pávek, Petr (advisor) ; Červený, Lukáš (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Michal Čečrle Supervisor: Prof. PharmDr. Petr Pávek, Ph.D. Title of diploma thesis: Introduction of cellular NF-κB model NF-κB is the most important transcription factor involved in cell signaling of inflammatory processes. It participates in the inflammatory reaction in the distinct compartments of the living organism. As a transcription factor, it controls the gene expression of many genes, especially cytokines (tumor necrosis factor alfa, interleukins: IL-1β, IL-2, IL-6, IL-12; chemokines etc.). NF-κB is also a key factor in the activation of monocytes and macrophages In this diploma thesis I focused on the role of NF-κB in the monocyte cell line THP-1. This line is an important model of human macrophages in which the THP-1 line can be differentiated. Using available literature, I summarized all the available knowledge on this issue. At the same time, I conducted several experiments on NF-κB activation in the THP- 1 line as a potential model in the research and development of therapeutic intervention in NF-κB signaling to suppress inflammation.
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Study of bacterial adherence on lung epithelia of Cystic fibrosis patients
Vašková, Lucie ; Hodek, Petr (advisor) ; Nosková, Libuše (referee)
A b stra ct C ystic fibrosis is an autosom alrecessive disease that is one of the m ost com m on hereditary disorders. T he disease is caused by the m utation in the gene encoding C F T R chloride channe l w hich leads to the failure of ion transportand a significant increase in viscosity o f m ucus,affecting especially the respiratory system .T he increase of saltand the presence of thick m ucus in the lungs suppress the antibacterialeffects of the im m une system .B acterial infections are the m ost com m on cause of death in patients w ith C F, especially the P seudom onas aerugino sa infection. A ntibiotics are used for treatm ent of the infection by this pathogen,butthe treatm ent is frequently com plicated by developing resistance.D ue to this fact, new w ays of treatm ent have been searched for. T he m ethod of passive im m unization of patients w ith yolk IgY antibodies seem s to be prom ising. T o exam ine the effect of these antibodies in in vivo m odel system , lung epithelial cells of a healthy subject (N uL i-1) and of a C F patient (C uF i-1) w ere exposed to 3 strains of P seudom onas aerugino sa. A ntibodies against the lectin of P seudom onas aerugino sa P A - IIL , w hich is an im portant adhesion structure of this bacterium , prepared from tw o im m unized anim als w ere com pared. T o...
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Study of inflammation using epithelial cells
Majerová, Barbora ; Hodek, Petr (advisor) ; Koblihová, Jitka (referee)
The human respiratory system is in constant contact with heterogeneous agents from the environment. There must be effective mechanical lung barriers and sufficient immune protection due to continuous deposition of various substances in the respiratory system. The mutual balance between the mechanisms of natural and adaptive immunity of the lungs is essential for destruction of infectious agents without initiation of inflammatory response. Overreaction of the immune system of the lungs may lead to the production of various inflammatory mediators and cytokines such as interleukins IL-1, IL-6 and IL-18. When determining the immunogenicity of a substance, it has to be exposed to lung epithelial cells, and then the concentration of cytokines produced is measured. To determine the immunogenicity of mammalian immunoglobulin G and chicken immunoglobulin Y the subsequent twenty-four hour exposure to A549 lung cancer cell line was made. Concentration measurement of cytokines IL-1 and IL-6 was performed using Luminex method, which pointed out the immunogenicity of goat immunoglobulin G and certain chicken immunoglobulins Y.
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Study of interaction of antimicrobial peptides with cells in culture
Kroupová, Hilda ; Stiborová, Marie (advisor) ; Votruba, Ivan (referee)
In English The thesis deals with research of novel antimicrobial peptides (AMP) Halictines (HAL-1, GMWSKILGHLIR-NH2 a HAL-2, GKWMSLLKHILK-NH2) and their structural analogs isolated from the venom of the wild bee Halictus sexcinctus. The structure and antimicrobial activity of these peptides had been described earlier [1]. The goal of this diploma thesis is to find peptide which is strongly toxic only for cancer cells and nontoxic for normal cells. Using of the fluorescent marked peptides we aimed to acquire the information about mechanism of action of the studied peptides on the cells. Using the MTT test (determination of valuation IC50), the toxicity of HAL-1 and HAL-2 and their analogs against 2 normal cell lines (Human umbilical vein endothelial cells, HUVEC, and normal rat intestinal cells, IEC) and against 2 cancer cell lines (cancer cells of suppository uterine, HeLa-S3 and cancer cells of human colorectal carcinoma, CRC SW 480) was determined. First we tested antimicrobial peptides with antimicrobial activity and low hemolytic activity. For verification the toxicity of less active analogs was also determined. We found out that the HeLa-S3 cells are the most sensitive to these peptides. The most toxic peptides (HAL-1/9, HAL-1/18, HAL-2/2) kill 50% of cells in the concentration 2,5 - 10 µM. To obtain...
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Cytotoxicity of the filamentous cyanobacteria in relation to its ecology
TOMEK, Petr
The aims of this study was to screen a cytotoxic activity in 53 strains of the genera Nostoc and Anabaena and to compare occurence of active isolates within different ecological groups. Selected cytotoxic strains were subjected to HPLC/MS analysis to identify their extracts composition. Isolates with strong cytotoxic effect containing unknown substances were fractionated to obtain pure fraction of this compound. Finally, the selected strains toxicity dependence on time and concentration was evaluated.
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