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).

Cancer cells have been known for some time to have very different metabolismas compared to that of normal non proliferating cells. As metabolism is involvedin almost every aspect of cell function, there has been a recent resurgence ofinterest in inhibiting cancer metabolism as a therapeutic strategy. Inhibitors thatspecifically target altered metabolic components in cancer cells are being developedas antiproliferative agents. However, many such inhibitors have not progressedinto the clinic due to limited efficacy either in vitro or in vivo. In this study weexplore the hypothesis that this is often due to the robustness of the metabolicnetwork and the differences between individual cancer cell lines in their metaboliccharacteristics. We take a systems biology approach. We investigate the cellular bioenergetic profiles of a panel of five non-small celllung cancer cell lines before and after treatment with a novel inhibitor of theglutaminase-1 (GLS1) enzyme. Additionally, we explore the effects of this inhibitoron intracellular metabolism of these cell lines as well as on the uptake and secretionof glucose, lactate and amino acids. To be able to do the latter robustly, wehad to modify the experimental assay considerably from procedures that seemto be standard in the literature; using these earlier procedures the metabolicenvironment of the cells was highly variable, leading to misleading results onthe metabolic effects of the inhibitor. We reduced cell density, altered mediumvolume and changed the time-window of the assay. This led to the cells growingexponentially, appearing indifferent to the few remaining changes. In this newassay, the metabolic effects of the glutaminase inhibitor became robust. One of the most significant results of this study is the metabolic heterogeneitydisplayed across the cell line panel under basal conditions. Differences in themetabolic functioning of the cell lines were observed in terms of both theirbioenergetic and metabolic profile. The amount of respiration attributed tooxidative phosphorylation differed between cell lines and respiratory capacity wasattenuated in most cells. However, the rate of glycolysis was similar betweencell lines in this assay. These results suggest that the Warburg effect arisesthrough a greater diversity of mechanisms than traditionally assumed, involvingvarious combinations of changes in the expression of glycolytic and mitochondrialmetabolic enzymes. The effects of GLS1 inhibition on cellular bioenergetics and metabolism alsodiffered between cell lines, even between resistant cell lines, indicating that theremay also be a diversity of resistance mechanisms. The metabolomic response ofcell lines to treatment suggests potential resistance mechanisms through metabolicadaptation or through the prior differences in the metabolic function of resistantcell lines. Part of the metabolome response to GLS1 inhibition was quite specificfor sensitive cells, with high concentrations of IMP as the strongest marker. Our results suggest that the metabolome is a significant player in what determinesthe response of cells to metabolic inhibitors, that its responses differ between cancercells, that responses are not beyond systems understanding, and that thereforethe metabolome should be taken into account in the design of and therapy withanti-cancer drugs.

<p>Liquid chromatography coupling with mass spectrometry (LC/MS) plays an important role in pharmaceutical characterization because of its ability to separate, identify, and quantify individual compounds from the mixture. Polymer brush layer bonded to the silica surface is designed as a novel stationary phase to improve the LC resolution and MS compatibility. The polymer thickness can be controlled to shield the analyte from interacting with the active silanol on the surface and reduce peak tailing. The functional group of the polymer can be changed to tune the selectivity in different separation modes. </p><p> </p><p>Two projects on LC/MS method development for biomolecule characterization using polymer-shell column are discussed in this work. In the first project, a polymer-shell column is used for disulfide bonds and free thiol subspecies identification, which is a major type of structural heterogeneities in IgG1. Compared with commercial columns, the polymer-shell column is able to resolve the free thiol variants without the presence of trifluoroacetic acid and greatly improve the MS signal. In the second project, a polymer-shell column is used for characterizing the drug-loading profile for antibody-drug-conjugates (ADC) via online LC/MS. The separation employs a mobile phase of 50 mM ammonium acetate to keep the ADC intact, and a gradient of water/isopropanol for ADC elution. MS data show that all ADC species remained intact and native on the column. Positional isomers can be separated and identified with the new method as well. Furthermore, to understand the surface chemistry and protein separation behavior quantitatively, a chromatographic simulation study is performed. The result shows that protein separation in RPLC can be described by a bi-Langmuir adsorption isotherm with mixed-mode retention of strong and weak sites. Smaller fractions and lower equilibrium constant of the strong site, which is the active silanol, give less tailing for protein separation.</p>

Synthetic polymer materials have been a part of human life since the 20th century. A variety of their use had resulted in a growing demand for their mass production, which then led to their increasing accumulation at waste dumps. After their deposition, these materials are exposed to many environmental factors causing their decomposition and subsequent release of the degradation products into the environment. This thesis is focused on the analysis of the photodegradation products from synthetic polymer materials and particularly polyurethane foams modified with the biodegradable fillers. One dimensional GC/MS and two dimensional comprehensive gas chromatography with time-of-flight mass spectrometry (GCxGC/TOF-MS ) were used for the measurements. The most of photodegradation products of polyurethane foams are injurious to health and environment.

The aim of the diploma thesis is the determination of macrolide antibiotics in drinking water by using of high performance liquid chromatography. Erythromycin and clarithromycin were selected such as representative macrolides due to frequently prescribed pharmaceuticals in this time. Solid phase extraction (SPE) by using of Oasis HLB cartridges was applied for pre concentration and purification of chosen analytes in real samples of drinking water. Optimalization of method and analysis were performed by using of high performance liquid chromatography with mass spectrometry detection (HPLC-MS). The suitable method was selected for determination of macrolides in real samples taken from two sources of drinking water, the interception of water in Litovel and Černovír Olomouc.

Sulfur amino acids have critical function as intracellular redox buffers and maintain homeostasis in the external milieu by combating oxidative stress. Synthesis of glutathione (GSH) is regulated at a substrate level by cysteine, which is synthesized by homocysteine via the transsulfuration pathway. Oxidative stress and diminished glutathione pools play a sustained role in the pathogenesis of acute pancreatitis. One of the aims of this study was to experimentally address the temporal relationship between plasma sulfur amino acid levels in patients suffering from acute pancreatitis. The data indicated low concentration of cysteine initially, at levels similar to those of healthy controls. Glutathione was found reduced whilst cysteinyl-glycine and ¿- glutamyl transpeptidase activity were increased in both mild and severe attacks. As the disease progressed, glutathione and cysteinyl-glycine were further increased in mild attacks and cysteine levels correlated with homocysteine and ¿-glutamyl transpeptidase activity. The progress of severe attacks was associated with glutathione depletion, reduced ¿-glutamyl transpeptidase activity and increased cysteinyl-glycine, that correlated with glutathione depletion. The corollary that ample supply of cysteine and cysteinly-glycine does not contribute towards glutathione synthesis in acute pancreatitis poses an important issue that merits resolution. Heightened oxidative stress and depletion of glutathione rationalized the progression of disease in severe attacks. An upsurge that reactive oxygen species can shift redox state of cells is determined by the ratio of the abundant redox couples reduced and oxidized glutathione (GSH: GSSG) in cell. The study reported a novel methodology for quantification of total oxidized glutathione (tGSSG) and total glutathione (tGSH) in whole blood using reverse phase high performance liquid chromatography. The novelty of the method is ascertained by the use of a mercaptan scavenger 1, methyl-2-vinyl-pyridinium trifluromethanesulfonate for the total oxidized glutathione determination. The results reported permit quantitation of tGSSG and tGSH and was applied to a control group. Finally, the study was also focussed in developing a liquid chromatography-mass spectrometric method to evaluate free and conjugated bile acids in patients suffering from various degrees of cholestatic-hepatobiliary disorders. The study reported low levels of ursodeoxycholic acid (UDCA) and slightly high levels of lithocholic acid (LCA). All the primary bile acids seem to be conjugated with glycine and taurine amino acid.

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Polar organic compounds (POCs) are chemicals with polar functional groups in their structure. The functional groups make the compounds hydrophilic and less prone to partition with biota. However, the knowledge of their fate is limited due to difficulties associated with their measurements. Although, the persistence of POCs in the environment is generally low, they are considered to be semi-persistent compounds due to their continuous introduction to the environment via wastewater. Studies have shown that complex mixtures of POCs of different classes may have synergistic toxic effects on biota at environmental concentration levels. Therefore, it is important to develop analytical methods in order to establish the occurrence and fate of POCs in aquatic environments. In Study I, a positive correlation between the sorption of a novel poly(ethylene-co-vinyl acetate-co-carbon monoxide) (PEVAC) material and the theoretical logarithmic dissociation partition coefficient (Log D) for seven POCs was observed. The PEVAC material showed an enhanced sorption of the POCs compared to the silicone material. Study II, demonstrated that the PEVAC sampler assess the freely dissolved concentration of POCs in aquatic environments. The results showed that the PEVAC polymer is an attractive alternative to silicone for mimicing the biological uptake of POCs in aquatic environments. Additionally, Study II showed that total extraction is appropriate for determination of the freely dissolved concentration of uncharged POCs with Log KOW &lt; 2.67 in natural water. In study III, a novel bag-solid phase extraction (bag-SPE) technique was compared to a conventional SPE-technique. Despite that the extraction efficiencies for POCs in wastewater were lower using the bag-SPE method, the two methods showed similar detection limits due to the lower ion-suppression experienced with the bag-SPE. In study IV the bag-SPE method was further developed with the aim of lowering the detection limits for POCs. Detection limits (LOD) below 13 ng/L showed that the bag-SPE method was suitable for determination of POCs in surface sea water. / This research was financially supported by European Union (European Commission, FP6 Contract No. 003956) “Novel Methods for Integrated Risk Assessment of Cumulative Stressors in the Environment” (NoMiracle) and by the Swedish research council Formas.