LOL is a novel secondary metabolite gene cluster associated with the production of loline alkaloids (saturated 1-aminopyrrolizidine alkaloids with an oxygen bridge) exclusively in closely related grass-endophyte species in the genera Epichloë and Neotyphodium. In this study I characterize the LOL cluster in E. festucae, including the presentation of sequence corresponding to 10 individual lol genes as well as defining the boundaries of the cluster and evaluation of the genomic DNA region flanking LOL in E. festucae. In addition to characterizing the LOL cluster in E. festucae, I present LOL sequence from two additional species, Neotyphodium coenophialum and Neotyphodium sp. PauTG-1. Together with two recently published LOL clusters from N. uncinatum, these data allow for a powerful phylogenetic comparison of five clusters from four closely related species. There is a high degree of microsynteny (conserved gene order and orientation) among the five LOL clusters, allowing us to predict potential transcriptional co-regulatory binding motifs in lol promoter regions. The relatedness of LOL clusters is especially interesting in light of the history of interspecific hybridizations that generated the asexual, Neotyphodium lineages. In fact, three of the clusters appear to have been introduced to different Neotyphodium species by the same ancestral Epichloë species, for which present day isolates are no longer able to produce lolines. To address the evolutionary origins of the cluster we have investigated the phylogenetic relationships of particular lol ORFs to their paralogous primary metabolism genes (and gene families) from endophytes, other fungi and even other kingdoms. I present extensive evidence that at least two individual lol genes have evolved from primary metabolism genes within the fungal ancestors of endophytes, rather than being introduced via horizontal gene transfer. I also present complementation studies in Neurospora crassa exploring the functional divergence of one lol gene from its primary metabolism paralog. While it is clear that these insecticidal compounds should convey a selective advantage to the fungus and its host, thus explaining preservation of the trait, this analysis provides an exploration into the evolutionary origin and maintenance of the genes that comprise the LOL and the cluster itself.

novel ozone-assisted aerobic sludge digestion process to stabilize and decrease the amount of excess sludge produced during biological treatment is presented in this study. Excess sludge production is a well known burden for the treatment plants both legally and financially. Moreover, with the arise in the knowledge in recalcitrant compounds it is understood that it can act as a significant secondary pollutant. With the developed pulse ozonation method, waste activated sludge samples from Ankara Tatlar and other Wastewater Treatment Plants (WWTP) were ozonated for different periods in Erlenmeyer flasks once a day on each of four consecutive days. Flasks were continuously aerated between ozone applications on an orbital shaker. The MLVSS, MLSS, COD and OUR parameters were measured routinely during the course of four days of digestion in order to optimize the process. Also pH, CST(capillary suction time) and SVI (sludge volume index) were followed. As a result MLVSS reductions of up to 95% were achieved with an ozone dose of only 0.0056 kg O3/kg-initial MLSS, at the end of the fourth day. In another experimental set, ozone dose was increased on the last day in order to destroy the selected endocrine disrupting compounds, namely diltiazem, carbamazepine, butyl benzyl phthalate and acetaminophen and two natural hormones estrone and progesterone, which accumulated onto the sludge. Over 99% removal of these contaminants were achieved on the fourth day. The analyses were conducted by using LC(ESI) MS/MS after solid phase extraction (SPE). By this process it became possible to save on contact time, as well as achieving a bio-solids digestion far exceeding the standard aerobic process at the expense of a minimum of ozone dose with the additional micropollutants removal. The developed process is deemed superior over side-stream ozonation of activated sludge in that it does not cause any reduction in active biomass amount that should be maintained in the aeration tank.

Per- and polyfluoroalkyl substances are a large group of global environmental contaminants. They can be divided into two sub-groups, 1) perfluoroalkyl acids (PFAAs) and 2) so called precursors, i.e. compounds that can potentially be transformed to form PFAAs. PFAAs are today ubiquitous in wildlife and humans. Food and drinking water are assumed to be the dominant human exposure pathways for PFAAs. The main aim of this doctoral thesis was to develop highly sensitive and fully validated analytical methods for the determination of a range of PFAAs and selected precursors in dietary samples. The methods were based on liquid chromatography coupled to mass spectrometry. Samples were extracted by solvent extraction followed by a cleanup step employing solid phase extraction. The cleanup step could at the same time be used as a fractionation of ionic PFAAs and neutral precursors. Paper I and II describe the development of methods for simultaneous analysis of three groups of PFAAs including perfluoroalkyl phosphonic acids (PFPAs) in drinking water and food. Methyl piperidine was used as ion pairing agent, leading to highly sensitive analysis of PFPAs. A first screening of tap water samples and different food items revealed that human dietary exposure to PFPAs in Europe is currently not of concern. A novel method for simultaneous analysis of perfluoroalkyl carboxylic acids (PFCAs) and polyfluoroalkyl phosphate esters (PAPs) in food and packaging materials is described in paper III. Targeted food samples and their packaging were analyzed. The results showed that PAPs may contribute to human exposure to PFCAs. In paper IV temporal trends (1991-2011) of perfluorooctane sulfonic acid (PFOS) and its precursors in herring were investigated. Rapidly decreasing trends were found for precursors, whereas PFOS did not show a significant change over time. Precursors in fish may have played an important role for human exposure to PFOS in the 1990s but are probably negligible today. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p> / PERFOOD project (KBBE-227525)

Orientador: Isabel Cristina Sales Fontes Jardim / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-04T03:19:20Z (GMT). No. of bitstreams: 1 Faria_LeonardoJardimdaSilva_M.pdf: 547743 bytes, checksum: c807d29f792ecf6c57e17ca59816fdfe (MD5) Previous issue date: 2004 / Mestrado / Quimica Analitica / Mestre em Química

Oil spills, especially in water have happened throughout the years. The consequences from this kind of incident have always been a threat to marine life and take a lot of money and time to clean up. The forensic investigations of oil usually focus on oil spill in water, however, this kind of investigation can be applied on more areas, such as in sexual offense cases and arson. The oil from the crime scene and the oil from the suspected source are analyzed and compared to each other to see if the compositions match. The aim of this thesis was to optimize the clean-up process of heavy fuel oil (HFO) using solid-phase extraction (SPE) according to the European Committee for standardization (CEN) 15522-2 method. HFOs are black oils, containing particles and soot which will decrease injector performance. Thus, a cleanup procedure is attractive when analyzing such samples. The three factors of optimization in this study were elution volumes, solvents, and SPE sorbents.The SPE method used in this study was to use the cartridges as a filter; therefore, the samples were both loaded and eluted with an organic solvent, in this case dichloromethane (DCM) or dichloromethane/heptane (DCM/Hp). The gas chromatography mass spectrometry (GC-MS) analysis method used is described in Annex B in the CEN 15522-2 method.Concluding, increasing the eluent volume did not indicate improvement on the extraction method. In terms of the solvents, both dichloromethane and dichloromethane/heptane showed similar results. The percentage weathering plots (PW-plots) showed that the SPE sorbents (dual layer florisil/Na2SO4, florisil and silica gel sorbents) worked similar. The overall conclusion from this thesis work was that further optimization must be made before implementing the method.

This thesis deals with optimization of conditions for the determination of platinum by electrothermal atomic absorption spectrometry. The theoretical part describes the occurrence of platinum in different part of the environment, its physical and chemical properties, transport and bioavailability. There is also evaluated appropriate method for determination and preconcentration of platinum. The experimental part describes advance of optimize the measurement conditions for ETA-AAS, further is describes optimization of condition for SPE and work with real samples on which are applied this conditions.

This diploma thesis in the theoretical part analyses the characteristic feature of individual platinum group elements, their toxicity, anthropogenic sources and the presence of platinum group elements in the environment of the Czech Republic. In addition, there are specified types of bioindicators focusing mainly on lichens, mosses and needles. There are described individual bioindication techniques and pre-treatment of samples for platinum metals determination in bioindicators. The subject of the experimental part is to verify the sorption efficiency and ability of platinum and palladium using the SPE method and to determine the actual platinum metal contamination in selected localities in the city of Brno. There was also the additionally subject to verify the sorption potential of selected bioindicators and determination of the environmental pollution by platinum metals using the bag technique. In the experimental part, the bioindicators of moss Pleurozium schreberi, lichen Xanthoria parietina, lichen Flavoparmelia caperata and needles Pinus nigra were used.

Polychlorinated biphenyls (PCBs) are a class of important organic pollutants which undergo very slow degradation and tend to persist for a long time in the environment. PCBs have various negative effects on living organisms, human health and the environment in general. A method for determining PCB levels in aqueous matrices was developed consisting of solid-phase extraction (SPE) and GC/MS determination. The average recoveries of PCB congeners from artificially spiked deionized water were around 85%. The PCB concentration of 3.3±0.1 μg·l-1 (a sum of selected congeners) was detected in contaminated groundwater originated from the area of a former asphalt concrete producing plant. The most abundant PCBs were trichlorinated congeners which together comprised about 80% of the total PCB content. The optimised SPE method was further applied together with accelerated solvent extraction (ASE) to assess the degradation potential of oyster mushroom (Pleurotus ostreatus) which shows high degradation efficiency of various organic pollutants including PCBs. P. ostreatus belongs to ligninolytic (white-rot) fungi, which is a very promising group of microorganisms frequently studied due to their bioremediation potential. P. ostreatus, strain KRYOS was able to remove 41±8% of the initial amount (50 μg) of the...

5 Abstract Protein lipidation occupies an important place in the post-translational modification group. It plays an important role in the processes of cell differentiation or synaptic transmission. Changes in the presence of lipidated proteins may in some cases indicate the appearance of human diseases such as Huntington's disease, schizophrenia, or cancer. For this reason, their analysis has become desirable, but also problematic due to its specific characteristics. This work is focused on the development of conditions for measurement of lipopeptides produced by cleavage of proteins containing lipomodification. Two separation procedures were tested, in which the aim of work was to enrich the lipopeptides and remove as many other molecules as possible from the samples. The first test method - phase interface separation, using water and chloroform, was able to concentrate the lipopeptides on the phase interface and separate some of the added unmodified peptides from the mixture. The second method of separation was a solid phase using a C18 stationary phase. As a more suitable solid phase extraction variant, elution with solutions containing acetonitrile with trifluoroacetic acid was chosen as compared to solutions containing isopropyl alcohol. With the modified elution procedure, it was possible to separate...

Although Lake Victoria is threatened by pollution there is a lack of knowledge about pharmaceuticals contaminants drained into the lake from large cities bordering the lake. Hence, the purpose of this project was to develop, validate and apply a method for analysis of pharmaceutical compounds accumulating in the Lake Victoria sediments. A simple quantitative method for 11 pharmaceuticals combining accelerated solvent extraction, solid phase extraction, trimethylsilylation derivatization, and gas chromatography mass spectrometry was developed, partly validated, and applied to 18 surface sediments and a sediment core dated using the 210Pb method. The results showed the presence of the pharmaceuticals estriol, gemfibrozil, metoprolol, ketoprofen, naproxen, 17α-ethinylestradiol and estrone concentrated around the regions Napoleon Gulf and Thurston Bay with accumulation rates decreasing towards the top of the sediment core. Nonetheless, a randomness in the distribution of these compounds behooves a systematic assessment investigating not only the provenance of these compounds but also further investigations to errors meaning that this study should be treated as a preliminary investigation.