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Proteome analysis of anti-cancer drug effects and characterisation of drug resistance
Hrabáková, Rita ; Kovářová, Hana (advisor) ; Hernychová, Lenka (referee) ; Šulc, Miroslav (referee)
Despite significant progress in the development of anti-cancer drugs, there is still a need for novel therapeutic strategies that would improve the outcome of cancer patients. Using proteomic technologies and cell lines with different phenotype of p53 tumour suppressor, we monitored cancer cell response to anti-cancer treatment with focus on the development of drug resistance. The different levels of metabolic proteins were identified in our study which may help to explain different anti-cancer activity of drugs with only a subtle difference in structure. More importantly, proteins associated with the development of drug resistance were identified and such expression changes have become a focus of interest. Our findings demonstrate a higher protein level of serine hydroxymethyltransferase, serpin B5 and calretinin in cancer cells resistant to Aurora kinase inhibitors. Such proteins promote the tumour growth with no apparent impact of p53 phenotype whilst voltage-dependent anion-selective channel protein 2 contributes to the development of resistance only in cells with functional p53 which is accompanied by the decreased level of elongation factor 2. On the other hand, cancer cells with loss of p53 appear to amplify alternative mechanisms such as protection against oxidative stress. The results...
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Proteome analysis of anti-cancer drug effects and characterisation of drug resistance
Hrabáková, Rita
Despite significant progress in the development of anti-cancer drugs, there is still a need for novel therapeutic strategies that would improve the outcome of cancer patients. Using proteomic technologies and cell lines with different phenotype of p53 tumour suppressor, we monitored cancer cell response to anti-cancer treatment with focus on the development of drug resistance. The different levels of metabolic proteins were identified in our study which may help to explain different anti-cancer activity of drugs with only a subtle difference in structure. More importantly, proteins associated with the development of drug resistance were identified and such expression changes have become a focus of interest. Our findings demonstrate a higher protein level of serine hydroxymethyltransferase, serpin B5 and calretinin in cancer cells resistant to Aurora kinase inhibitors. Such proteins promote the tumour growth with no apparent impact of p53 phenotype whilst voltage-dependent anion-selective channel protein 2 contributes to the development of resistance only in cells with functional p53 which is accompanied by the decreased level of elongation factor 2. On the other hand, cancer cells with loss of p53 appear to amplify alternative mechanisms such as protection against oxidative stress. The results...
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Determination of bilirubine oxidation products
Čepa, Adam ; Smrček, Stanislav (advisor) ; Muchová, Lucie (referee)
This thesis is focused on protective and antioxidant properties of bile pigment bilirubin. Bilirubin is a potent scavenger of free radicals and reactive oxygen species generated by increased oxidative stress. Mildly elevated systemic levels of bilirubin are actually associated with lower incidence of oxidative stress-mediated diseases, such as cardiovascular disease, atherosclerosis, diabetes and certain types of cancer. The main aim of this thesis was to prepare bilirubin oxidation products in vitro, characterize their physical-chemical properties and thein determine biological impact.
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The issue of determining antidepressants
Kočnar, Michal ; Tulková, Tereza (referee) ; Vávrová, Milada (advisor)
The presented bachelor thesis is focused on the issue of antidepressants and the determination of two of their representatives, paroxetine and citalopram, in sewage taken from three municipal waste water treatment plants. In the theoretical part of this study, antidepressants are generally characterized, more specifically two representatives of selective serotonin reuptake inhibitors, paroxetine and citalopram. Subsequently, the methods of determination of these analytes in waste water are presented. The experimental part deals with determination of selected substances in real samples of waste water. The analytes were isolated from the matrix by solid phase extraction and determined by ultra performance liquid chromatography with ultraviolet spectrophotometric detection. Paroxetine was quantified in all samples, citalopram was not able to quantify, in some samples even to detect.
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Artificial sweeteners and their potential impact on the ecosystem
Vavrčíková, Veronika ; Mravcová, Ludmila (referee) ; Zlámalová Gargošová, Helena (advisor)
This diploma thesis deals with the determination and ecotoxicological assessment of artificial sweeteners. The diploma was focused on monitoring the most commonly used sweeteners, which includes acesulfam-K, cyclamate, saccharin and sucralose. These sweeteners were analyzed by liquid chromatography with mass spectrometry, using solid phase extraction for their concentration. The samples for their determination were taken from wastewater treatment plant in Modrice. Artificial sweeteners are synthetically produced substances, which is the main reason for their occurrence in waste water. Some of them are not efficiently removed from the wastewater treatment plant and penetrate into the environment. Analysis of the samples was found, that sucralose penetrates into the environment. The ecotoxicological effect of the sweeteners was evaluated via aquatic organisms, namely on the freshwater crustaceans Thamnocephalus platyurus and Daphnia magna, on the water plant Lemna minor, and the battery of tests was supplemented by a representative of the destructors Vibrio fischeri. For all sweeteners, LC20, EC20 and IC20 values were determined. For some sweeteners, LC50, EC50 and IC50 values were also determined. During testing, saccharin represented the highest toxicity.
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Determination of estrogen pollutants in real water sample by HPLC-UV after solid phase extraction.
Kozlík, Petr ; Coufal, Pavel (referee) ; Bosáková, Zuzana (advisor)
4 Abstract Estrogens are considered to belong to chemicals that negatively affect the endocrine system, even if present at very low concentrations. They are discharged into environment as a result of an increasing application of drugs etc. This work is focused on the separation and quantification of five estrogens, namely estrone (E1), 17β-estradiol (βE2), 17α-estradiol (αE2), 17α-ethynylestradiol (EE2) and estriol (E3) in natural water samples by HPLC-UV method. The chromatographic system consisted of a C18 stationary phase (SunFire® C18, 150 x 4.6 mm, octadecyl bounded to silica gel, particle size 5 µm) and binary mobile phase of acetonitrile/water in various ratios in isocratic separation mode. The effect of acetonitrile content in the mobile phase and flow rate of the mobile phase on retention and separation parameters was tested. Under the optimized separation conditions (acetonitrile/water 40/60 (v/v), 1.3 ml/min), all the compounds were baseline resolved and eluted within 15 min. These experimental conditions were applied to the calibration measurements which were carried out within the concentration range from 0.001 to 1 mg/ml. Limits of detection (LOD) and limits of quantification (LOQ) for the individual estrogens and their mixture (standards dissolved in methanol) were determinated. The detection...
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Portable and Miniaturized Separation Techniques Applicable for Food and Biotechnology Analysis
Dvořák, Miloš ; Pospíchal, Jan (referee) ; Bednář,, Petr (referee) ; Rittich, Bohuslav (advisor)
Capillary electrophoresis was used for determination of 6 fractions of caseins. Those fractions were measured in 144 samples of cow’s milk originated from the feeding experiment focused on explanation the influence of the feeding onto casein productions. In this work were separated 6 fraction of caseins first time with total resolution of the peaks. Capillary electrophoresis was applied for determination of short-chain organic acids during fermentation of wine must. It was compared the fermentation of must fermented by different yeast. The difference of profile short-chain organic acids during fermentation were not statistically significant. The once difference was in the utilisation of the malic acid and production of the lactic acid. A portable miniaturized system for medium pressure liquid chromatography was developed. The components were tested and system was used for the isocratic and gradient elution of various analytes (food dyes, parabens). New line of electroluminescent diodes (LEDs) for deep-UV areas of wavelength based on a different materials substrate was characterised. The new line was compared with old line LEDs. The new line LEDs was incorporated in deep-UV absorbance detectors. Detectors were characterised and tested for a detection various analytes in modes flow injection analysis and chromatography separation. First time was characterised this new line of the LEDs and the origin of the parasitic emission band produced by deep-UV LEDs light sources was explained. This origin is given by disturbances of a materials substrates. This work is a contribution for an advance of low-cost and portable systems and detection devices in the field of analytical chemistry.
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Surfactants in Surface and Waste Water
Štefka, Michal ; Vávrová, Milada (referee) ; Chýlková, Jaromíra (referee) ; Kráčmar, Stanislav (referee) ; Čáslavský, Josef (advisor)
This work is focused on the occurrence of surface-active substances in the environment. It deals with the development and optimization of methods for the determination of selected surfactants in samples from rivers and from influent and effluent of wastewater treatment plants. Representatives of anionic, nonionic and zwitterionic surfactants were selected as target compounds. Based on optimization of analytical methods and their pilot testing abroad on samples of surface water, the selection of analytes for the conditions in the Czech Republic were corrected and operatively expanded. For the final analysis liquid chromatography with mass spectrometry was used. Analysis of cationic surfactants was realized using liquid chromatograph with UV-VIS detector. This optimized method for analysis of anionic surfactants was applied to real samples. Samples of surface running water were from the River Thurso in Scotland and then from watercourses in the catchment area of Moravia river were collected. In addition to the grab sampling also continuous weekly sampling of water from Tvaroženský potok and Litava was realized. Waste water was collected at inflow and outflow of three wastewater treatment plants (WWTP) in South Moravia (Brno – Modřice, Břeclav and Hodonín).
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Vývoj, optimalizace a validace analytické metody na stanovení neurotoxinu \recke{beta}-N-methylamino-L-alaninu ve vodě pomocí LC/MS
HOŘEJŠÍ, Karel
This bachelor thesis deals with the development, optimization and validation of an analytical method for determination of neurotoxin \recke{beta}-N-methylamino-L-alanine in water using LC/MS. At first, the development and optimization of the analytical method was performed. For the optimization of the analytical method following parameters were selected: temperature of HESI and MS capillary, gases flow rate, voltage on S-lens, F-lens and capillary, collision energy. In addition, mobile phase composition, flow rate of mobile phase and injection volume of sample were optimized too. Then, the analytical method was successfully validated. Finally, the method was applied to the analysis of real samples of surface water.
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Probiotics and prebiotics and their co-encapsulation
Šnajdarová, Karolína ; Bokrová, Jitka (referee) ; Márová, Ivana (advisor)
This bachelor thesis is focused on study of probiotic cultures and options of coencapsulation with specific substrates called prebiotics. Lactobacillus acidophilus and Bifidobacterium breve were used as encapsulated microorganisms. As the specific substrates inulin, psyllium, apple fiber and hemp fiber were tested. Prebiotics was added to probiotics both in untreated form and in hydrolyzed form. The stability and viability of the cells have been studied by microscopy for the period of 6 weeks. Particles were stored in the enviroment of 2.5% citric acid. As the best substrate for cell growth the hemp fiber was found. The hemp fiber exhibited the highest increase of cells count. During observation of long-term stability in the model digestive conditions, particles with hemp fiber were destroyed in instestinal juices. For that reason, particles with hemp fiber could be used in food supplements with targeted transport. The bachalor thesis is also focused on characterisation of prebiotics components. The highest amount of total sugars in the group of non-hydrolyzed prebiotics exhibited psyllium. The highest content of reducing sugars both in the groups of non-hydrolyzed and hydrolyzed prebiotic had hemp fiber. By the method of thin-layer chromatography was found out that the main components of hydrolyzed prebiotics are glucose and fructose. Further, some disaccharides and oligosaccharides were obtained by TLC analysis. For characterisation of prebiotics by HPLC the column Rezex exhibited better sensitivity. HPLC analysis confirmed that the main components of prebiotics are glucose and fructose, some more complex carbohydrates were found too.
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