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Interaction of bacterial lectins with human lung epithelium
Vyhnalová, Kateřina ; Hodek, Petr (advisor) ; Nosková, Libuše (referee)
Recessive autosomal disease cystic fibrosis (CF) is caused by a mutation in the CFTR gene ("regulator of cystic fibrosis transmembrane conductance"), which encodes the same named chloride channel. This mutation leads to incorrect ion transport, which causes the formation of an excessively viscous mucus on the surface of the airways and subsequently to the susceptibility to bacterial diseases. This disease mainly affects the respiratory system, where infections are associated with various causes of death in patients with CF. The most common pathogen causing infections is Pseudomonas aeruginosa (PA), which uses many virulence factors, such as pili or adhesins. Lectin PA-IIL, from the group of PA adhesins, is characterized by a high affinity for L-fucose, so it contributes to the adhesion of PA to the low sialylated epithelium of CF patients. In this work the interactions between PA-IIL and lung epithelium were investigated. The cell lines CuFi-1 (CF patient) and NuLi-1 (healthy individual), which were examined ex vivo, were used. A part of these cell lines were exposed to neuraminidase. The PA-IIL lectin was isolated from the E. coli cell line pET25_PAIIL and subsequently fluorescently labeled with DyLight 488. The activity of mentioned lectin was verified by red blood cell agglutination. The...
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Characterization of newly developed fluorescence probes in cellular systems
Kadlecová, Julie ; Hubálek Kalbáčová, Marie (advisor) ; Hendrych, Tomáš (referee)
Nanoparticles (NP) are currently a progressive area of scientific research. The possibility of synthesizing them according to the required parameters opens up possibilities for their wide use also in biomedicine. One example is a nanoparticle that can detect cellular processes, such as pH. We already know that the pH of healthy and cancer cells differs by the opposite gradient on the intracellular and extracellular side of the membrane. In this context, this work deals with the study of fluorescent silicon nanoparticles (SiNP) tested on a human keratinocyte cell line from a healthy donor (HaCaT) and from skin cancer donor (A431). Once found that even the highest concentrations of SiNP used are not cytotoxic, they can be further studied by fluorescence, confocal and super-resolution microscopy. In order to assess the pH detection properties of these SiNPs, a method for measuring intracellular pH with a fluorescent raciometric probe SNARF-1 using fluorescence spectroscopy and flow cytometry was introduced. Since the pH values of the intracellular environment are closely related to cellular metabolism, the metabolism of A431 and HaCaT cells was characterized and compared. To do this, methods for measuring analog glucose consumption (2-NBDG) and another new method for measuring real-time metabolism...
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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 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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The effect of IgY on bacterial adhesion on epithelial cells ex vivo
Vašková, Lucie
0 Abstract Cystic fibrosis is an autosomal recessive disease caused by mutation in CFTR gene coding for a chloride channel in apical membrane of epithelial cells. This disorder leads to the change in ion transport causing the increase in mucus viscosity in airways as well as changes in glycosylation of saccharide structures on the cells. Because of that these cells are the target for bacterial adhesion. Chronic bacterial infections, which lead to gradual decline of lung function and damage of lung tissue, are the major cause of death of patients suffering with cystic fibrosis. Pseudomonas aeruginosa is the main pathogen causing chronic infections in cystic fibrosis patients. This bacterium produces a biofilm protecting them from host immune system and antibiotics. Once the colonization with PA occurs, it is difficult to get rid of this pathogen. The prophylactic treatment with orally administered hen antibodies against the PA virulence structures could be a prevention of chronic PA infections. In this work we tested the antibody against the bacterial lectin PA-IIL, which is suggested to be involved in the adhesion of the pathogen on epithelial cells. First, it was verified that the prepared antibody from egg yolks of a hen immunized with the bacterial lectin PA-IIL recognizes this antigen expressed...
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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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