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Zjišťování potenciálu chemických látek používaných k ochraně proti ultrafialovému záření (UV filtrů) způsobovat anti-progestagenní aktivitu ve vodním prostředí
SMĚŠNÁ, Tereza
The substances used for protection against UV radiation (UV filters) are widely used in industry and also in personal care products. It has been reported from many countries around the world that they enter the aquatic environment. The presence of UV filters in surface waters can pose a risk to aquatic organisms. The aim of this thesis was to test a range of substances from the group of UV filters for anti-progestagenic activity, detect this activity in the aquatic environment, detect UV filters in the aquatic environment, and based on this information, evaluate the potential of organic UV filters to cause anti-progestagenic activity in the aquatic environment. In this thesis, 20 organic UV filters were tested with in vitro bioassay anti-PR-CALUX. In this bioassay cells were exposed to pure substances (UV filters) and reference substance mifepristone. Samples were taken by grab (active) sampling from wastewater treatment plant in Tábor and from surface waters (outdoor swimming pools and Lužnice River). Samples were extracted using solid phase extraction. These extracts from waste and surface waters were tested for progestagenic and anti-progestagenic activity with the (anti-)PR-CALUX bioassay and the concentrations of UV filters were measured using mass spectrometry in the extracts. Samples were also tested for cytotoxicity with the resazurin reduction assay. Nine organic UV filters have shown anti-progestagenic activity, but compared to the reference substance, the activities exhibited by UV filters were several orders of magnitude lower. Ethylhexyl methoxycinnamate, benzophenone-3, and octocrylene were the most potent anti-progestagens. Progestagenic activity was not found at any of the sites. Anti-progestagenic activity was found in 60% of the environmental extracts, and the concentrations ranged from 2.6 to 4.5 ng/L equivalents of mifepristone. Anti-progestagenic activity was detected in the influent of wastewater into the treatment plant, in the recipient of the Lužnice River, and in ponds used for fish farming and also as outdoor swimming pools, namely the Podroužek and Štilec ponds. Thirteen organic UV filters were identified by chemical analysis at the tested sites. The UV filters octocrylene and ethylhexyl methoxycinnamate were found in the highest concentrations, these two UV filters contributed the most to the overall anti-progestagenic activity. At the wastewater treatment plant influent, UV filters contributed 49% to the total anti-progestagenic activity. In the recipient of the Lužnice River and outdoor swimming pools, the contribution of UV filters to this activity was very small, which means that this activity in surface waters will be caused by the occurrence of other substances than in wastewater. In ponds, it can be assumed that wastewater treatment plants will not be the source of these substances. Further research should be aimed at detecting the impact of the occurrence of organic UV filters in the aquatic environment on organisms, but also at identifying substances causing anti-progestagenic activity in the aquatic environment.
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Studium dynamiky živých lidských buněk v odezvě na běžné kontaminanty vod s cílem vývoje citlivého specifického analytického postupu
KOŠEK, Jan
The bachelor thesis is devoted to testing the toxicity of chemical substances. In the current chemical and pharmaceutical industry, thousands of new substances are produced annually, which brings about the need to ensure sufficient consumer safety and environmental protection. In vitro testing is becoming an increasingly important research tool in this issue. This work verifies the theoretical foundations and proposes an algorithm developed by Lonhus et al. from the Laboratory of experimental complex systems in Nové Hrady and its possible future applications in in vitro testing. This new approach uses the principles of microrheology and tracks Speed-Up Robust Features (SURF) in a digital image and converts them into vectors of trajectories of intracellular mass particles. Microrheology is a relatively new field of research in life sciences and deals with the study of flow and deformation of materials at the microscopic level. The algorithm separates two main components of the movements inside the cells: direct movement of organelles as larger particles and random Brownian motion. The advantage of this method is its potential of easy use and sensitivity. The method makes it possible to obtain important primary information about the effect of chemical substances on cell cultures.
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The impact of synthetic chelators from the group of 4-acylpyrazole-5-ones on alcohol dehydrogenase
Sobolová, Zuzana ; Mladěnka, Přemysl (advisor) ; Jirkovský, Eduard (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Zuzana Sobolová Supervisor: Prof. Přemysl Mladěnka, Pharm.D., Ph.D. Consultant: Marcel Hrubša, MS.c. Title of diploma thesis: The impact of synthetic chelators from the group of 4-acylpyrazole-5-ones on alcohol dehydrogenase Inhibitors of alcohol dehydrogenase play a key role in the treatment of poisoning caused by methanol, ethanol, ethylene glycol, and their toxic metabolites. Such treatment can therefore be considered as life-saving. In clinical practice, a single substance, fomepizole, is used. This thesis is aimed at investigation of the effect of eleven experimental chelators from the 4- acylpyrazol-5-ones group on equine and yeast alcohol dehydrogenases, and the possible definition of structural features that would be the most suitable for interaction with this enzyme. Due to the ability of these substances to chelate zinc ions, it is believed that they could inhibit an enzyme that has a zinc molecule in the structure. The measurement itself is based on principle of increase in absorbance during the enzymatic reaction, which is caused by the formation of NADH. This measurement is relatively simple, efficient and, due to used kinetic method, also less susceptible to be affected by...
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Výskyt (anti-)thyroidní aktivity ve vodním prostředí České republiky
SMĚŠNÁ, Tereza
Xenobiotics present in the aquatic environment can exhibit hormonal activity and thus they have potential to disrupt endocrine system of aquatic organisms. The aim of this study was to perform the first monitoring of the occurrence of (anti-)thyroid activity in samples from surface waters from the Czech Republic and subsequently identify potential hot-spots of the occurrence of (anti-)thyroid activity. Samples were taken using passive samplers of the POCIS type from 21 localities in 17 rivers from the Czech Republic. Detection of (anti-)thyroid activity was performed using the (anti-)TRbeta-CALUX in vitro bioassay in which cells were exposed to reference substances and dilutions of extracts from passive samplers. Triiodothyronine was used as reference compound for thyroid activity, while deoxynivalenol was used as reference compound for anti-thyroid activity. Thyroid activity was not found in any of the surface water samples. Anti-thyroid activity was found in 38 % of the tested samples and ranged from 89 to 310 micrograms/l equivalents of deoxynivalenol. In this study, eight localities with anti-thyroid activity were identified in surface waters of the Czech Republic. The strongest anti-thyroid activities were found in the rivers Bílina, Odra a Svratka. In addition, samples from Texas (USA) were tested in this study. Samples were collected using passive samplers of the POCIS type from 12 localities in 4 rivers from Texas. Neither thyroid nor anti-thyroid activity was found in any of the surface water samples. Further research is needed to determine the compounds responsible for the detected anti-thyroid activities in Czech surface waters and to assess potential risk that the compounds responsible for these activities may pose to aquatic organisms.
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Coculture of cells in vitro for bone regeneration
Sloupová, Lenka ; Filová, Eva (advisor) ; Tlapáková, Tereza (referee)
Cocultivation of two (or more) cell types in vitro leading to the formation of functioning bone tissue, later inserted into the damaged area, could be a solution for patients for whom the current methods (e.g. use of human bone grafts) are insufficient. In coculture, one cell type is used to accomplish osteogenesis, while the other is used for angiogenesis, because the limit of diffusion of O2 and essential nutrients is only 200 μm, which means that establishing a vascular network in vitro should prevent the new bone tissue from dying after implantation. Creation and understanding of a functioning coculture in vitro are crucial for developing a coculture successful in vivo. This work summarises and compares information about the influence of in vitro cocultivation on proliferation, osteogenesis and angiogenesis in coculture which uses osteoblasts (or osteoprogenitors), bone marrow mesenchymal stem cells (BMSC) or adipose derived mesenchymal stem cells (ADSC) as it's osteogenic cell type combined with various endothelial cell types. In order to understand the impact of cocultivation on these processes, one chapter deals with interactions between cocultured cell types. Keywords coculture, osteogenesis, angiogenesis, in vitro, osteoblasts, BMSC, ADSC
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In vitro cultures of medicinal plants - XI.
Sojková, Kristýna ; Tůmová, Lenka (advisor) ; Kašparová, Marie (referee)
In vitro cultures of medicinal plants - XI. Elicitation is one of the few strategies that can be used in enhancement of secondary metabolites production from explant cultures. The effect of abiotic elicitor (silver nitrate) on flavonolignan and flavonoid taxifolin production in suspension culture of Silybum marianum L. (Gaertn.) and on isoflavones production in suspension culture of Genista tinctoria L. was tested. Silver nitrate in various concentrations (5.887.10-3 mol/l; 5.887.10-4 mol/l; 5.887.10-5 mol/l) was used as elicitor. Content of secondary metabolites in suspension cultures was determinated by high performance liquid chromatography (HPLC). The samples were taken after 6, 12, 24, 48, 72 and 168 hours after elicitor treatment. The highest content of taxifolin production (0.02 %) in suspension culture of Silybum marianum L. (Gaertn.) after silver nitrate (5.887.10-4 mol/l) treatment and 72 h sampling was detected. The highest content of genistin (0.05 %) in suspension culture of Genista tinctoria L. was found after silver nitrate (5.887.10-4 mol/l) treatment and 48 h sampling. The highest content of daidzein (0.09 %) was detected after elicitor (5.887.10-4 mol/l) treatment and 168 h sampling.
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Ecophysiological relevance of extreme sensitivity of orchids to nitrates
Figura, Tomáš ; Ponert, Jan (advisor) ; Tylová, Edita (referee)
Many orchid species are seriously endangered at present. Reasons for their disappearing from natural habitats remain often unclear. Orchids depend on mycorrhizal symbiosis in nature, however only little is known about this symbiosis. Seeds of some species do not germinate in vitro, making their cultivation for scientific and rescue purposes impossible. We found that seed germination of one of such reluctant species, Pseudorchis albida, is strongly inhibited by nitrates even at extremely low concentrations. As this species prefers oligotrophic mountain meadows, nitrate-induced inhibition probably take place in natural conditions. Surprisingly, we found similar but slightly weaker inhibitory effect also in typical mesophillic species and even in slightly eutrophic ones. The sensitivity to nitrates correlates with trophy level of species canopy. This inhibitory effect of nitrates could be weakened by application of a range of growth regulators, including auxins, cytokinins and gibberellins, and also by mycorrhizal fungi. The action of nitratereductase is essential for this inhibitory effect of nitrates. Experiments with NO donors, scavengers, and NO quantification are pointing right at NO as compound which perhaps mediates nitrate inhibitory effect. Based on these results, the nitrate inhibitory...
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Preparation and characterization of diamond-based nanocarriers for transfection of siRNA
Majer, Jan ; Cígler, Petr (advisor) ; Fišer, Radovan (referee)
Although nanodiamonds were discovered and produced tens of years ago, they have been utilized in medical and biological fields just recently, particularly in drug and gene delivery into a cell and in bioimaging methods. Nanodiamonds can be modified with specific positively charged moieties for complexation with negatively charged nucleic acids. These complexes afterwards overcome extracellular and intracellular barriers and transport the nucleic acid either into cytosol or into the nucleus. Owing to fluorescence centres nitrogen- vacancy, which can be formed in the nanodiamonds, nanodiamonds exhibit excelling optical properties, as they emit stable fluorescence without "photoblinking" or "photobleaching". This thesis reviews properties, synthesis and modifications of nanodiamonds and other selected nanoparticles and their in vitro applications. This thesis also compares their cytotoxicity and gene knockdown efficiency.
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Ability of copper chelators to interact with iron and zinc
Hanuščinová, Lucia ; Mladěnka, Přemysl (advisor) ; Čonka, Patrik (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Student: Lucia Hanuščinová Supervisor: Assoc. Prof. Přemysl Mladěnka, PharmDr., PhD. Title of diploma thesis: Ability of copper chelators to interact with iron and zinc Copper plays in the human organism a role of an element with indispensable significance, whose biological influence and effects depend on its quantity. With elevated concentrations in the human body, copper becomes toxic, resulting in pathological conditions. The most well-known diseases is the Wilson's disease, whose treatment consists of oral administration of chelators, i.e. chemical compounds, which are capable of binding copper ions in various proportions and eliminating them from the organism. Chelation therapy is currently the first choice after confirmation of the diagnosis. Chelation toxicity results from several factors, e.g. inhibition of copper dependent enzymes or low selectivity to metals. And precisely the selectivity of chelators is being discussed in this diploma thesis. An ideal chelator should not interact with any of the other physiological ions, that are necessary for the proper functioning of the organism. Five of the most frequently therapeutically or experimentally used substances /trientine, D-penicillamine,...
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