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The Use of Separation Methods for the Study of Selected Drugs and Pesticides in Water
Burešová, Jitka ; Hajšlová, Jana (referee) ; Chýlková, Jaromíra (referee) ; Kráčmar, Stanislav (referee) ; Vávrová, Milada (advisor)
This work is focused on the study of penetration of pesticides and pharmaceuticals in the aquatic environment. The aim was to develop and verify the optimal method for long-term monitoring of the occurrence of selected pesticides and pharmaceuticals in surface waters. Two frequently used analgesics ibuprofen and diclofenac, and carbamazepine that is used to treat epilepsy, were chosen from the group of drugs. A group of 41 pesticides were selected for monitoring too. The attention was focused on a group of urea pesticides and phenoxyacetic acids, as well as on selected pesticides falling to the group of priority substances or identified as environmental quality standards. Some of pesticides were selected because their consumption in the Czech Republic exceeds 30 tons a year. Liquid chromatography with tandem mass spectrometry (LC / MS / MS) was used for the determination of selected analytes in surface waters. Two optimized analytical method were developed. Ten analytes were determined in negative ion mode and 34 analytes in positive ion mode. Solid phase extraction (SPE) was used for purification and concentration of the samples. Developed methodology was validated and used for monitoring of selected compounds in samples of surface waters from the Morava River basin. Samples were collected on the rivers Morava and Svratka monthly for two years. Samples from the river Blata and stream Nivnička were collected for one year. Surface water samples were also collected from the river Litava. Four samples were taken throughout the length of the flow during one day. Smaller streams were chosen for monitoring because of the probability of occurrence of pesticides. They flow abundantly agriculturally cultivated areas. Drugs ibuprofen and diclofenac were detected in all taken samples, carbamazepine mostly. Among the most commonly occurring pesticides were bentazon, diuron, isoproturon, carbendazim, MCPA, MCPP, propiconazole, tebuconazole and 2,4-D.
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The Usage of Separation Methods for Research of Biologically Active Substances in Waters
Vydrová, Lucie ; Friedl, Zdeněk (referee) ; Opatřilová, Radka (referee) ; Chýlková,, Jaromíra (referee) ; Vávrová, Milada (advisor)
Pharmaceuticals are biological active compounds with different functional groups, physico-chemical and biological properties. These chemical compounds are called as “new contaminants” which cumulate in various environmental components. These contaminants input to environment from industrial processes, hospitals and health care institutions or household sources and these pollutants can to negatively interact with environmental components. Pharmaceuticals are separated according to structure and their effects to live organisms. On the basis of drug consumption in Czech Republic the non-steroidal anti-inflammatory drugs are the most using pharmaceuticals, because these drugs can be obtain without prescriptions. The choosing analgesics for study were monitored in waste water from waste waters treatment plant (WWTP Brno – Modřice) and in surface water from two sampling sites of river Kretinka. Pharmaceuticals were monitored in surface waters in Czech Republic, in Scotland (river Thurso) and in Taiwan (river Erren, Agongdian, Yanshuei and canal of Tainan) Solid phase extraction (SPE) is used for extraction of study pharmaceutical from water system. The high performance liquid chromatography (HPLC) with diode array detection or mass spectrometry was used for determination of drugs in surface waters from Czech Republic. The water samples from Scotland and Taiwan were analysed and the high performance liquid chromatography with mass spectrometry (HPLC/MS) was used for determination of pharmaceuticals. The method of HPLC/MS enables the determination of study compounds in the very low range of concentrations (ng ? l-1). All monitored pharmaceuticals were identified and quantified in water samples from river Kretinka in Czech Republic, river Thurso (Scotland) and rivers Erren, Agongdian, Yanshuei and canal of Tainan in Taiwan and the obtained data were compared.
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Multidisciplinary characterization of interactions between oestrogen hormones and their receptors during sperm capacitation.
Bosáková, Tereza
This doctoral thesis was focused on the multidisciplinary characterizatrion of interactions between hormones and their receptors during sperm capacitation, which is the final step in sperm maturation in mammals and the key event to achieve egg fertilization. Three oestrogenic hormones (17β-estradiol (E2), 17α-estradiol (αE2), 17α-ethynylestradiol (EE2)), which are part of environmental pollution, were selected for the study. These hormones are called endocrine disruptors as they can cause disorders of the hormonal system in animals, thereby adversely affecting physiological processes, including reproduction. A new analytical HPLC-MS/MS method was developed for monitoring of concentration changes of oestrogens (E2, αE2 and EE2) during time-dependent capacitation of mouse and rat sperm in vitro. For individual oestrogens, the concentration dependences of free, unbound hormone on the capacitation time were measured for three concentrations of individual hormones (200, 20 and 2 μg/L). The obtained concentration data were converted into values of relative concentrations, which were subjected to kinetic analysis. Kinetic models were proposed for the interaction of individual hormones with sperm, which were described by a system of differential equations with optimization of rate constants and molar...
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Similarity search in Mass Spectra Databases
Novák, Jiří ; Skopal, Tomáš (advisor) ; Svozil, Daniel (referee) ; Nahnsen, Sven (referee)
Shotgun proteomics is a widely known technique for identification of protein and peptide sequences from an "in vitro" sample. A tandem mass spectrometer generates tens of thousands of mass spectra which must be annotated with peptide sequences. For this purpose, the similarity search in a database of theoretical spectra generated from a database of known protein sequences can be utilized. Since the sizes of databases grow rapidly in recent years, there is a demand for utilization of various database indexing techniques. We investigate the capabilities of (non)metric access methods as the database indexing techniques for fast and approximate similarity retrieval in mass spectra databases. We show that the method for peptide sequences identification is more than 100x faster than a sequential scan over the entire database while more than 90% of spectra are correctly annotated with peptide sequences. Since the method is currently suitable for small mixtures of proteins, we also utilize a precursor mass filter as the database indexing technique for complex mixtures of proteins. The precursor mass filter followed by ranking of spectra by a modification of the parametrized Hausdorff distance outperforms state-of-the-art tools in the number of identified peptide sequences and the speed of search. The...
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2-DE analysis of hepatocyte secretome under hyperhomocysteinemia
Chrudinová, Martina ; Ryšlavá, Helena (advisor) ; Novák, Petr (referee)
Homocysteine is a plasmatic, sulphur-containing amino acid derived from methionin. It is an amino acid not used in protein synthesis and it's role is to serve as an intermediate in numerous metabolic pathaways. Elevated concentrations of homocysteine in blood are associated with many pathologies, as cardiovascular and neurodegenerative diseases, diabetes or osteoporosis, thus homocysteine has been a common subject of many biochemical experiments in last two decades. For this bachelor thesis, the proteomic comparison of proteins secreted by primary human hepatocytes under 2mM homocysteine and primary human hepatocytes with no homocysteine added to the incubation medium was made, using large-gel two-dimensional electrophoresis. Hepatocytes were incubated in serum free medium for 48 hours. Proteins secreted to medium were precipitated by acetonitrile. Two dimensional electrophoresis was made. Protein maps were analysed by PDQuest Advanced 8.0.1 2D Gel Analysis Software (Bio-Rad, USA). Search criteria for differentially secreted proteins were three-fold change at the signicance level 95 %, Student's t-test. These proteins were cut off the preparative gels and identified by tandem mass spectrometry. In total, there were 17 spots equal to 15 proteins found as differentially exprimed proteins. Among these...
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Analysis of biologically active compounds using high performance separation methods
Adamusová, Hana ; Bosáková, Zuzana (advisor) ; Jelínek, Ivan (referee) ; Tůma, Petr (referee)
In the first part of this doctoral thesis, a new analytical HPLC-MS/MS method for monitoring of concentration changes of 17β-estradiol (βE2) during in vitro mouse sperm capacitation was developed. Capacitation was performed for three initial concentrations of βE2 (200, 20 and 2 μg/L). For all the concentrations a similar trend for the total unbound βE2 was observed. In general, the βE2 concentration decreased to reach its minimum and then increased again. The position of the minimum differed for the individual tested βE2 concentrations. Experimentally obtained results were subjected to the kinetic analysis. The curves fitted through the experimentally determined points displayed an autocatalytic character. For the agreement between the curves obtained by fitting through the experimental points and the theoretical calculated curves, it is necessary to assume that the first step is adsorption of βE2 onto the surface of the sperm controlled by Langmuir isotherm. The kinetic study was also used to study the effects of fluorides and aluminium fluoride complexes on the capacitation of mouse sperm. The experimental points were in very good agreement with the shape of the theoretical curves and this fact verifies the mechanism of the mouse sperm capacitation kinetics. In the second part of this work, two...
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Multidisciplinary characterization of interactions between oestrogen hormones and their receptors during sperm capacitation.
Bosáková, Tereza ; Komrsková, Kateřina (advisor) ; Smrček, Stanislav (referee) ; Postlerová, Pavla (referee)
This doctoral thesis was focused on the multidisciplinary characterizatrion of interactions between hormones and their receptors during sperm capacitation, which is the final step in sperm maturation in mammals and the key event to achieve egg fertilization. Three oestrogenic hormones (17β-estradiol (E2), 17α-estradiol (αE2), 17α-ethynylestradiol (EE2)), which are part of environmental pollution, were selected for the study. These hormones are called endocrine disruptors as they can cause disorders of the hormonal system in animals, thereby adversely affecting physiological processes, including reproduction. A new analytical HPLC-MS/MS method was developed for monitoring of concentration changes of oestrogens (E2, αE2 and EE2) during time-dependent capacitation of mouse and rat sperm in vitro. For individual oestrogens, the concentration dependences of free, unbound hormone on the capacitation time were measured for three concentrations of individual hormones (200, 20 and 2 μg/L). The obtained concentration data were converted into values of relative concentrations, which were subjected to kinetic analysis. Kinetic models were proposed for the interaction of individual hormones with sperm, which were described by a system of differential equations with optimization of rate constants and molar...
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HPLC and LC-MS/MS methods for diagnosis of inherited metabolic diseases
Bártl, Josef
This dissertation thesis is focused on development and optimization of high- performance liquid chromatography (HPLC) and tandem mass spectrometry (LC-MS/MS) methods, and its utility for diagnosis of inherited metabolic diseases. The first thematic part describes a comprehensive laboratory approach to diagnostics of patients with hereditary xanthinuria by determination of specific markers and enzyme activity. For this purpose HPLC method with diode array detection for measurement of hypoxanthine, xanthine, allopurinol and oxypurinol in urine and plasma and HPLC method with fluorimetric detection for analysis of pterin and isoxanthopterin in plasma were employed. These methods were successfully applied in clinical practice to ascertain two patients with hereditary xanthinuria type I. The second thematic part aims at developing and clinical application of new LC-MS/MS method for simultaneous determination of total homocysteine (tHcy), methionine (Met) and cystathionine (Cysta) in dried blood spots (DBS) and plasma. The results demonstrated the clinical utility of this method for detection of patients with homocystinuria and possibility to distinguish between defects in the remethylation and transsulfuration pathways of homocysteine metabolism. Due to ease of DBS collection and sample transportation...
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Tandemová hmotnostní spektrometrie sfingolipidů s aplikací pro metabolické studie a diagnostiku sfingolipidos
Kuchař, Ladislav
In recent years, mass spectrometry (MS) become the dominant technology in lipidomic analysis and widely influenced research and diagnosis of diseases of lipid metabolism, e.g. lysosomal storage disorders (LSD) characterized by impairment of the lysosomal functions. Defects in lysosomal processing of sphingolipids SFL belong to the category of sphingolipidoses. This condition has severe and even fatal clinical outcome. The primary aim of this work was to establish quantitative and qualitative methods of SFL analysis useful for research and diagnosis of LSD. At first, semisynthesis of mass labeled lipid standards utilizing immobilized sphingolipid ceramide N-deacylase was performed. Established methods of quantitative analysis were then used to prove the increased excretion of urinary SFL in LSD with characteristic storage in the kidney. Determination of excreted urinary SFL was found useful for differential diagnosis of prosaposin and saposin B deficiences for which routine enzymology is failing. MS also enabled monitoring of individual molecular species (isoforms) of SFL, which led to the finding that their urinary pattern is changing in some LSD. This resulted in the development of new screening method in dry urinary samples based on isoform profile evaluation. Another MS application referred to...
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Pathology and physiology of de novo purine synthesis.
Krijt, Matyáš ; Zikánová, Marie (advisor) ; Šebesta, Ivan (referee) ; Čajka, Tomáš (referee)
Purines are organic compounds with miscellaneous functions that are found in all living organisms in complex molecules such as nucleotides, nucleosides or as purine bases. The natural balance of purine levels is maintained by their synthesis, recycling and degradation. Excess purines are excreted in the urine as uric acid. Purine nucleotides may be recycled by salvage pathways catalysing the reaction of purine base with phosphoribosyl pyrophosphate. A completely new central molecule of purine metabolism, inosine monophosphate, can be synthesized from precursors during the de novo purine synthesis (DNPS). DNPS involves ten steps catalysed by six enzymes that form a multienzymatic complex, the purinosome, enabling substrate channelling through the pathway. DNPS is activated under conditions involving a high purine demand such as organism development. Currently, three DNPS-disrupting disorders have been described: ADSL deficiency, AICA-ribosiduria and PAICS deficiency. All three disorders are caused by genetic mutations leading to the impaired function of particular enzyme causing insufficient activity of respective DNPS step, manifested biochemically by accumulation of substrate of deficient enzyme, biologically by disruption of purinosome formation and clinically by unspecific neurological features,...
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