<p>Microglia are a population of specialized, tissue-resident immune cells that make up around 10% of total cells in our brain. They actively prune neuronal synapses, engulf cellular debris, and misfolded protein aggregates such as the Alzheimer’s Disease (AD)-associated amyloid-beta (Aβ) by the process of phagocytosis. During AD, microglia are unable to phagocytose Aβ, perhaps due to the several disease-associated changes affecting their normal function. Functional molecules such as lipids and metabolites also influence microglial behavior but have primarily remained uncharacterized to date. The overarching question of this work is, <i>How do microglia become dysfunctional in chronic inflammation</i>? To this end, we developed new chemical tools to better understand and investigate the microglial response to Aβ <i>in vitro</i> and <i>in vivo</i>. Specifically, we introduce three new tools. (1) Recombinant human Aβ was developed via a rapid, refined, and robust method for expressing, purifying, and characterizing the protein. (2) A pH-sensitive fluorophore conjugate of Aβ (called Aβ^pH) was developed to identify and separate Aβ-specific phagocytic and non-phagocytic glial cells <i>ex vivo</i> and <i>in vivo</i>. (3) New lysosomal, mitochondrial, and nuclei-targeting pH-activable fluorescent probes (called LysoShine, MitoShine, and NucShine, respectively) to visualize subcellular organelles in live microglia. Next, we asked, <i>What changes occur to the global lipid and metabolite profiles of microglia in the presence of Aβ in vitro and in vivo</i>? We screened 1500 lipids comprising 10 lipid classes and 700 metabolites in microglia exposed to Aβ. We found significant changes in specific lipid classes with acute and prolonged Aβ exposure. We also identified a lipid-related protein that was differentially regulated due to Aβ <i>in vivo</i>. This new lipid reprogramming mechanism “turned on” in the presence of cellular stress was also present in microglia in the brains of the 5xFAD mouse model, suggesting a generic response to inflammation and toxicity. It is well known that activated microglia induce reactive astrocytes during inflammation. Therefore, we asked, <i>What changes in proteins, lipids, and metabolites occur in astrocytes due to their reactive state? </i>We provide a comprehensive characterization of reactive astrocytes comprising 3660 proteins, 1500 lipids, and 700 metabolites. These microglia and astrocytes datasets will be available to the scientific community as a web application. We propose a final model wherein the molecules secreted by reactive astrocytes may also induce lipid-related changes to the microglial cell state in inflammation. In conclusion, this thesis highlights chemical neuroimmunology as the new frontier of neuroscience propelled by the development of new chemical tools and techniques to characterize glial cell states and function in neurodegeneration.</p>

Efficient control of sea lice is a major challenge for the sustainable production of farmed Atlantic salmon (Salmo salar (Linnaeus, 1758)). These marine ectoparasites feed on mucus, skin and blood of their hosts, thereby reducing the salmon’s growth rate and overall health. In the northern hemisphere, the most prevalent species is Lepeophtheirus salmonis (Krøyer, 1837). In 2006, global costs of sea lice infections are estimated to have exceeded €300 million, with the majority spent on a limited number of chemical delousing agents. Emamectin benzoate (EMB; SLICE®), an avermectin, has been widely used since its introduction in 2000, due to its convenient administration as an in-feed medication and its high efficacy against all parasitic stages of L. salmonis. However, over-reliance on a single or limited range of medicines favours the emergence of drug resistance and, as a result, the efficacy of this compound in treating L. salmonis has decreased in recent years, as reported from e.g. Chile, Norway, Scotland and Canada. Declining efficacy underlines the need for an improved understanding of the molecular mechanisms underlying EMB drug resistance in L. salmonis. Elucidation of these mechanisms would allow for improved monitoring tools, earlier detection of developing resistance, extended usability of current delousing agents and development of new parasiticides. The work described in this thesis sets out to examine the molecular mechanisms underlying EMB resistance in L. salmonis. In earlier studies, research in nematodes and arthropods has linked drug efflux transporters belonging to the family of ATP-binding cassette (ABC) transporters to ivermectin (IVM) resistance, a parasiticide with high chemical similarity to EMB. ABC transporters such as permeability glycoprotein (P-gp), transport a wide range of substrates, including drugs, and have been suggested to provide a potential molecular mechanism through which EMB resistance might be mediated in sea lice. As an example of such mechanisms, increased expression of P-gp is one of the causative factors for drug resistance in human cancer cells and avermectin resistance in nematode parasites such as Caenorhabditis elegans or Haemonchus contortus. Initial research involved screening for novel salmon lice P-gps that might contribute to EMB resistance. A novel P-gp, SL-PGY1, was discovered using a combined bioinformatic and molecular biological approach. The expression was compared in two well-characterised L. salmonis strains differing in their susceptibility to EMB (S = susceptible, R = resistant). Prior to EMB exposure, mRNA levels did not differ from each other, while, after 24 h exposure, a 2.9-fold increase in SL-PGY1 mRNA expression was observed in the R strain. SL-PGY1 appears not to be a major factor contributing to reduced EMB susceptibility, although it could play a role, as expression levels increased upon exposure to EMB. A further four additional drug transporters (ABC C subfamily) were also discovered showing high homology to multidrug-resistance proteins (MRP). The relative expression levels of each MRP was compared in the strains S and R, before and after exposure to EMB. No significant changes were found in their expression patterns. If ABC drug transporters mediate the efflux of EMB and thereby reduce the intracellular concentrations of the drug in exposed animals, the inhibition of those ABC drug transporters was expected to lead to higher intracellular levels of EMB. This could result in an enhanced toxic effect when EMB is co-administered with an inhibitor. Two known inhibitors of human P-gps and MRPs, cyclosporin A (CSA) and verapamil (VER), were co-administered with EMB. CSA increased the toxic effect of EMB in both tested strains, implying that the targets of CSA are expressed at comparable levels and that they may be part of the mechanism conferring EMB resistance. VER increased the toxic effect of EMB in the R strain, but had no significant effects on the S strain. This implies that the expression of factors inhibited by VER differs between the two L. salmonis strains. It is hypothesised that a number of ABC transporters with distinct, yet overlapping patterns of inhibitor specificity are affected by those inhibitors. The search for drug-resistance conferring genes was complemented with a systematic, genome-wide survey of ABC transporters in L. salmonis to find additional members of this important gene family. Next-generation high-throughput RNA sequencing (RNA-seq) was employed to assemble a reference transcriptome from pooled total RNA of salmon lice at different development stages. The transcriptome was assembled against the L. salmonis genome and annotated. Thirty-nine putative ABC transporters were found. Of further interest were transcripts of the subfamily B, C and G, as they contain drug-transporting ABC proteins. For the ABC B subfamily, one full (SL-PGY1) and three half transporter transcripts were found. Only full transporters are known to transport drugs and SL-PGY1 is apparently not a major factor contributing to EMB resistance. Fourteen ABCC sequences were found – 11 MRPs and 3 homologues to sulfonylurea receptors. Of interest are MRPs, as they contribute to drug detoxification in humans and invertebrates. Four MRPs had been identified previously and their expression ratios did not differ between S and R strain parasites. Seven sequences belonging to ABCG subfamily were found. However, none of the L. salmonis ABCG transcripts identified showed sufficient homology to known drug transporters in other species. With the currently limited understanding of the mechanisms conferring EMB resistance, monitoring the susceptibility of L. salmonis subpopulations is essential. Dose-response bioassays are currently widely used. Tests with pre-adult II or adult parasites requires relatively large numbers of parasites (~150) to conduct this type of bioassay, which may not always be available. Addressing this issue, we tested the feasibility of a single-dose bioassay (requiring fewer test animals than dose-response bioassays) to discriminate between L. salmonis strains with differing EMB susceptibility. This alternative approach uses time-course toxicity analysis, where the toxic effect of EMB is monitored over time. After clearly defining the effect criteria, we found that it is possible to discriminate between those L. salmonis strains. However, while requiring fewer test animals, time course toxicity analysis is more labour-intensive, but the alternative design can be suitable under certain circumstances. The work reported here has provided new knowledge concerning the mechanisms of EMB resistance in sea lice. Several novel putative drug transporters have been identified, an important first step toward unravelling the complex interactions of genes involved in EMB resistance in this commercially important parasite.

M.Sc. (Aquatic Health) / Sediments can contain a variety of organic and inorganic contaminants. These contaminants accumulate, resulting in extremely high concentrations even once the overlying water concentrations are at or below acceptable water quality guidelines. Any changes in the physical parameters'of the overlying water can cause these pollutants to be released back into solution. Accumulated contaminants can be released at even higher concentrations than previously detected. In recent years, sediment contamination has highlighted the need to monitor these previously overlooked pollutant sources that have accumulated in aquatic ecosystems. South Africa does not currently have standardised methods to assess sediment toxicity. Although international methods exist, they are largely untested in South Africa and the organisms needed to conduct these tests are not readily available. Over the years numerous culture methods have been develop globally for culturing organism to be used for water and sediment toxicity tests. In South Africa, the focus has mainly been on culturing organisms for water. toxicity testing. Sediment toxicity testing with indigenous organism however, was not developed. Established international culture methods from the United States Environmental Protection Agency, the Organisation for Economic Cooperation and Development, and Environment Canada were taken into consideration when developing the laboratory culture method for two (2)of the selected organisms (Chironomus spp. & Hydra sp.) from this study. A preliminary culture method was also developed for the third selected organism, Melanoides tuberculata (gastropod). The organisms cultured in this study were selected based on their extent of contact with the substrate, ease of handling, availability, culture maintenance as well as their reproductive cycle. The Hydra, Chironomids and M. tuberculata cultures were successfully breeding under laboratory conditions and remained stable. The Chironomus sp. and M. tuberculata maintain contact with the sediment making them suitable as ecologically relevant organisms for use in whole sediment toxicity testing in South Africa.

by Tsui Tsz Ki, Martin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 119-138). / Abstracts in English and Chinese. / Acknowledgements --- p.I / Abstract --- p.III / Table of Contents --- p.VII / List of Tables --- p.XII / List of Figures --- p.XIV / Abbreviations --- p.XVI / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Research Background --- p.1 / Chapter 1.1.1 --- General description of glyphosate --- p.1 / Chapter 1.1.2 --- Physical and chemical properties of glyphosate --- p.2 / Chapter 1.1.3 --- Commercial formulations based on glyphosate --- p.3 / Chapter 1.1.4 --- Overview of ecotoxicological studies of glyphosate-based formulations --- p.4 / Chapter 1.1.4.1 --- Aquatic toxicity of glyphosate-based formulations --- p.4 / Chapter 1.1.4.2 --- Environmental fate of glyphosate-based formulations in waters --- p.12 / Chapter 1.1.5 --- Interaction of glyphosate and other substances --- p.14 / Chapter 1.2 --- Overview of Aquatic and Sediment Toxicology --- p.16 / Chapter 1.2.1 --- Aquatic toxicology --- p.16 / Chapter 1.2.2 --- Introduction to sediment toxicology --- p.19 / Chapter 1.3 --- "Significance, Outline and Objectives of the Present Study" --- p.20 / Chapter 1.3.1 --- Significance of the research --- p.20 / Chapter 1.3.2 --- Thesis outlines and research objectives --- p.22 / Chapter Chapter 2 --- Aquatic Toxicity of Glyphosate-based Herbicides to Different Organisms and the Effects of Environmental Factors / Chapter 2.1 --- Introduction --- p.25 / Chapter 2.2 --- Materials and Methods --- p.26 / Chapter 2.2.1 --- Test organisms --- p.26 / Chapter 2.2.2 --- Test chemicals --- p.27 / Chapter 2.2.3 --- Comparison between different organisms --- p.27 / Chapter 2.2.4 --- Environmental factors in modifying Roundup® toxicity --- p.30 / Chapter 2.2.5 --- Analysis of glyphosate concentration --- p.31 / Chapter 2.2.6 --- Validity of tests and statistical analyses --- p.32 / Chapter 2.3 --- Results --- p.32 / Chapter 2.3.1 --- Comparison between different groups of organisms --- p.32 / Chapter 2.3.2 --- Environmental factors in modifying Roundup® toxicity to C.dubia --- p.35 / Chapter 2.4 --- Discussion --- p.36 / Chapter 2.4.1 --- Toxicity of glyphosate to photo synthetic organisms --- p.36 / Chapter 2.4.2 --- pH-associated toxicity of glyphosate --- p.37 / Chapter 2.4.3 --- High potency of surfactant --- p.38 / Chapter 2.4.4 --- Effects of environmental factors on Roundup® toxicity --- p.38 / Chapter 2.5 --- Conclusions --- p.39 / Chapter Chapter 3 --- "Toxicity of Rodeo®, Roundup® Biactive and Roundup® to Water-column and Benthic Organisms and the Effect of Organic Carbon on Sediment Toxicity" / Chapter 3.1 --- Introduction --- p.41 / Chapter 3.2 --- Materials and Methods --- p.43 / Chapter 3.2.1 --- Test chemicals --- p.43 / Chapter 3.2.2 --- Test organisms --- p.43 / Chapter 3.2.3 --- Toxicities to water-column and benthic organisms --- p.44 / Chapter 3.2.4 --- Effect of sediment organic carbon --- p.45 / Chapter 3.2.5 --- Statistical analyses --- p.48 / Chapter 3.3 --- Results --- p.48 / Chapter 3.3.1 --- Toxicities to water-column and benthic organisms --- p.48 / Chapter 3.3.2 --- Effect of sediment organic carbon --- p.49 / Chapter 3.4 --- Discussion --- p.54 / Chapter 3.4.1 --- Different sensitivities between water-column and bethic animals --- p.54 / Chapter 3.4.2 --- Relative toxicities of three herbicides --- p.56 / Chapter 3.4.3 --- Route of exposure of herbicides in sediment to organisms --- p.57 / Chapter 3.4.4 --- Sediment toxicity of glyphosate-based formulations --- p.58 / Chapter 3.4.5 --- Effect of organic carbon on partitioning and toxicity --- p.60 / Chapter 3.5 --- Conclusions --- p.61 / Chapter Chapter 4 --- Joint Toxicity of Glyphosate and Several Selected Environmental Pollutants to Ceriodaphnia dubia / Chapter 4.1 --- Introduction --- p.63 / Chapter 4.2 --- Materials and Methods --- p.65 / Chapter 4.2.1 --- Test organisms and toxicity tests --- p.65 / Chapter 4.2.2 --- Test chemicals --- p.66 / Chapter 4.2.3 --- Experiment I: Joint acute toxicity of Roundup® and nine toxicants --- p.66 / Chapter 4.2.4 --- Experiment II： Effect of IPA salt of glyphosate alone at EEC on toxicities of heavy metals --- p.67 / Chapter 4.2.5 --- Basic water properties and chemical analyses --- p.69 / Chapter 4.2.6 --- Statistical analyses --- p.70 / Chapter 4.3 --- Results --- p.70 / Chapter 4.3.1 --- General conditions and recovery for spiked chemicals --- p.70 / Chapter 4.3.2 --- Experiment I: Joint acute toxicity of Roundup® and nine toxicants --- p.71 / Chapter 4.3.3 --- Experiment II: Effect of IPA salt of glyphosate alone at EEC on toxicities of heavy metals --- p.73 / Chapter 4.4 --- Discussion --- p.75 / Chapter 4.4.1 --- Interactions of Roundup® and other toxicants --- p.75 / Chapter 4.4.2 --- Joint toxicity of dissimilar chemicals --- p.77 / Chapter 4.4.3 --- Complexation of glyphosate with metals interactions between liquid/solid phases --- p.79 / Chapter 4.5 --- Conclusions --- p.83 / Chapter Chapter 5 --- Environmental Fate of Glyphosate and its Nontarget Impact: a Case Study in Hong Kong / Chapter 5.1 --- Introduction --- p.85 / Chapter 5.2 --- Materials and Methods --- p.87 / Chapter 5.2.1 --- Description of study sites --- p.87 / Chapter 5.2.2 --- Physicochemical characteristics of different matrices --- p.88 / Chapter 5.2.3 --- Continuous weather monitoring --- p.89 / Chapter 5.2.4 --- Herbicide applications --- p.89 / Chapter 5.2.5 --- Experimental designs --- p.90 / Chapter 5.2.5.1 --- Estuarine enclosure experiment --- p.90 / Chapter 5.2.5.2 --- Freshwater pond experiment --- p.92 / Chapter 5.2.6 --- Schedule of sample collection and sample storage --- p.92 / Chapter 5.2.7 --- Sample preparation --- p.94 / Chapter 5.2.7.1 --- Water samples --- p.94 / Chapter 5.2.7.2 --- Sediment samples --- p.94 / Chapter 5.2.8 --- Sample determination --- p.95 / Chapter 5.2.8.1 --- Pre-column derivatization --- p.95 / Chapter 5.2.8.2 --- High performance liquid chromatography analyses --- p.95 / Chapter 5.2.8.3 --- Calibration of glyphosate and AMPA --- p.95 / Chapter 5.2.8.4 --- Recovery of glyphosate in spiked samples --- p.96 / Chapter 5.2.9 --- Statistical analyses --- p.96 / Chapter 5.3 --- Results --- p.96 / Chapter 5.3.1 --- Site characteristics --- p.96 / Chapter 5.3.2 --- Weather conditions during herbicide application --- p.99 / Chapter 5.3.3 --- Chemical analyses --- p.100 / Chapter 5.3.4 --- In-situ toxicity tests --- p.104 / Chapter 5.4 --- Discussion --- p.106 / Chapter 5.4.1 --- Site-specific factor affecting the environmental fate --- p.106 / Chapter 5.4.1 --- Site-specific factor affecting the environmental fate of glyphosate --- p.106 / Chapter 5.4.2 --- Glyphosate in water and sediment --- p.106 / Chapter 5.4.3 --- Homogeneity of glyphosate in surface water and sediment --- p.109 / Chapter 5.4.4 --- Effect of weather conditions on environmental fate of glyphosate --- p.109 / Chapter 5.4.5 --- Biological impact of Roundup® --- p.110 / Chapter 5.5 --- Conclusions --- p.112 / Chapter Chapter 6 --- General Conclusions --- p.113 / References --- p.119

Submitted by Biblioteca de Pós-Graduação em Geoquímica BGQ (bgq@ndc.uff.br) on 2017-09-18T18:23:01Z No. of bitstreams: 1 DISSERTAÇÃO DE MESTRADO - THIAGO DIAS LOPES DA ROSA.pdf: 1856868 bytes, checksum: 24f79a23cf37a8161ea8b1930ad99eb2 (MD5) / Made available in DSpace on 2017-09-18T18:23:01Z (GMT). No. of bitstreams: 1 DISSERTAÇÃO DE MESTRADO - THIAGO DIAS LOPES DA ROSA.pdf: 1856868 bytes, checksum: 24f79a23cf37a8161ea8b1930ad99eb2 (MD5) / Universidade Federal Fluminense. Instituto de Química. Programa de Pós-Graduação em Geoquímica, Niterói, RJ / Atualmente, pouco se sabe sobre as possíveis relações entre as concentrações das formas biodisponíveis de metais e os biomarcadores microbiológicos de toxicidade. Este estudo teve como objetivo avaliar a relação entre os indicadores geoquímicos e microbiológicos de biodisponibilidade e toxicidade de metais em sedimentos da região do Saco do Engenho (Baía de Sepetiba, RJ). O fracionamento geoquímico de Cd, Zn, Pb e Cu, através de uma extração seqüencial (BCR de 3 etapas), e 3 indicadores microbiológicos de toxicidade (biomarcadores enzimáticos, demanda energética bacteriana e biomassa) foram avaliados em amostras de sedimentos coletados na Baía de Sepetiba, na região próxima ao Porto de Itaguaí e ao Saco do Engenho. Um bioensaio de ressuspensão de sedimento também foi realizado no qual foram avaliados os mesmos parâmetros microbiológicos além do pH, oxigênio dissolvido, temperatura e a concentração de metais dissolvidos. Nas amostras de sedimento, em geral, as concentrações de metais estavam predominantemente na forma oxidada, associados hidróxidos de Fe e Mn. As concentrações máximas de metais obtidas pela extração seqüencial foram observadas próximas ao Saco do Engenho, atingindo 22 mg.kg-1 de Cd, 3855 mg.kg-1 de Zn, 20 mg.kg-1 de Pb e 18 mg.kg-1 de Cu. Esta região ainda possui uma das principais fontes de Cd e Zn da baía. O total (somatório) das concentrações extraídas pelo método BCR de 3 etapas e a demanda energética bacteriana correlacionaram-se significativamente e foram considerados como os melhores indicadores da biodisponibilidade e toxicidade de metais utilizados. No bioensaio a maior degradação da matéria orgânica, via enzimas esterases, pela atividade bacteriana e as maiores concentrações de metais dissolvidos coincidiram em 192 horas, sugerindo que a comunidade bacteriana influenciou mais a remobilização de metais para fase dissolvida do que as variações físico-químicas do meio. Neste experimento, os indicadores microbiológicos de toxicidade foram limitados quando as concentrações de metais biodisponiveis atingiram níveis letais. Desta forma foi possível concluir que marcadores geoquímicos e microbiológicos são limitados quando aplicados individualmente na avaliação de impactos causados pela contaminação ambiental por metais. / The possible relations between metal bioavailable forms and microbiological indicartors of toxicity are currently poorly understood. This study aims to evaluate the relationship between geochemical and microbiological indicators of metal toxicity in sediments from Saco do Engenho (Sepetiba Bay, Brazil). The geochemical partitioning of Cd, Zn, Pb and Cu (following the BCR method) and toxicity indicators (enzymatic biomarkers, bacterial energetic demand and bacterial biomass) were evaluated in bottom sediments and in a sediment resuspension bioassay. In the bioassay, these variables were also evaluated, besides pH, dissolved oxygen, temperature and dissolved metal concentrations. Sediment samples generally presented trace metals in the oxidized form (associated with Fe and Mn oxides). Maximum trace metal concentrations were found near Saco do Engenho (22 mg kg-1 Cd, 3855 mg kg-1 Zn, 20 mg kg-1 Pb and 18 mg kg-1 Cu), indicating that it still is a major source of Cd and Zn to the bay. The sums of BCR method phases extracted for each metal and the bacterial energetic demand presented a significant positive correlation and were considered as the better indicators of metal bioavailability and toxicity. In the bioassay, the higher activity of the esterase enzyme and metal concentration peaks coincided at 192 h of resuspension, suggesting that bacterial activity influenced metal mobilization to dissolved phase in a greater extent than physicochemical variability. In such experiment, the microbiological indicators were limited when bioavailable metals reach lethal levels. Therefore, it was concluded that geochemical and microbiological markers are limited when applied individually to evaluate metal pollution impacts

Submitted by Biblioteca de Pós-Graduação em Geoquímica BGQ (bgq@ndc.uff.br) on 2017-09-26T17:16:29Z No. of bitstreams: 1 dissertação mestrado-Savergnini.pdf: 1862145 bytes, checksum: e45f0963fc6b204e533d27e1e8f57ffb (MD5) / Made available in DSpace on 2017-09-26T17:16:29Z (GMT). No. of bitstreams: 1 dissertação mestrado-Savergnini.pdf: 1862145 bytes, checksum: e45f0963fc6b204e533d27e1e8f57ffb (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Universidade Federal Fluminense. Instituto de Química. Programa de Pós-Graduação em Geoquímica, Niterói, RJ / drenagem da bacia hidrográfica e de lançamentos pontuais de efluentes do processamento da cana, como o vinhoto. Este efluente consiste em um líquido com alta carga orgânica, demanda de oxigênio e sólidos em suspensão. Para caracterizar a matéria orgânica proveniente do impacto da agroindústria da cana, ferramentas geoquímicas podem ser utilizadas, determinando sua origem e o estado trófico ambiental. Análises microbiológicas também são importantes na medida em que microrganismos degradam a matéria orgânica, estabelecendo o equilíbrio dinâmico do ecossistema. Este trabalho apresentou como objetivo a caracterização do impacto ambiental e degradação microbiana do vinhoto no Baixo Rio Paraíba do Sul. As análises foram realizadas na água e no sedimento ao longo da zona de mistura fluvial no início (Campanhas I – 07/2006 e III – 07/2007) e final da safra (Campanha II – 10/2006). No laboratório, foram realizados bioensaios para se estimar o potencial de biodegradação do vinhoto. As estações se localizaram a montante e a jusante do lançamento do efluente em até cerca de 200 metros de distância, também sendo realizadas coletas na foz do rio como referência. Os sedimentos foram fracionados em finos (<63 μm) e grossos (>63 μm). Na água, valores elevados de temperatura e oxigênio dissolvido juntamente com baixos valores de pH foram bons indicadores da presença de vinhoto e a alta concentração de potássio foi essencial para a identificação de seu lançamento no efluente industrial. No sedimento, as diferenças encontradas entre as concentrações de fósforo orgânico e inorgânico, e as altas concentrações de carbono orgânico, razão C:N e potássio foram eficientes na caracterização do impacto do efluente e da agricultura. Concentrações semelhantes de fósforo orgânico e inorgânico, juntamente com razões C:N em torno de 12 demonstraram que a foz do rio é influenciada pela agricultura de cana, sugerindo que a matéria orgânica sedimentada é tanto de origem fitoplanctônica como de vegetais superiores. A fração fina correlacionou-se significativamente com a maior parte dos parâmetros e se mostrou útil na avaliação do impacto, pois o principal componente do vinhoto, o potássio, se concentrou preferencialmente nos grãos menores que 63 μm. As análises microbiológicas revelaram que a entrada dos efluentes influencia a comunidade bacteriana, aumentando a biomassa e o ganho energético (analisado através da ETSA) na água e no sedimento no início da safra. Entretanto, o acúmulo de carga orgânica no sedimento do final da safra, inibiu a respiração aeróbia e diminuiu a capacidade de auto-depuração ambiental. Os microrganismos isolados do sedimento foram capazes de crescer e utilizar o vinhoto como única fonte de energia nos bioensaios, demonstrando seu possível uso em processos de biorremediação. Observou-se que o número de células por cm3 deve ser no mínimo da ordem de 108 a 109 para uma eficiente remoção de biopolímeros (37%) e percebeu-se que o fosfato é consumido eficientemente (99%) somente quando em concentrações próximas a 3 mg L-1, observando-se uma baixa eficiência de consumo quando em concentrações próximas a 9 mg L-1.. A eficiência de degradação foi alta nos primeiros 6 dias do bioensaio, não sendo observado reduções eficientes nos demais dias de análise. / The pollution in rivers caused by sugar cane agriculture and industry is a result of runoff and point source discharge of sugar cane effluents, mainly vinasse with high organic content, oxygen biological demand and suspended solids. Geochemical tools can be used to characterize the organic matter from effluents, like determining origin and environmental trophic state. Microbiological analyses are also important, as microorganisms degraded organic matter and establish the ecosystem dynamic equilibrium. The aim of this work was to characterize the impact and microbial degradation of vinasse in the Low River Paraíba do Sul. Analyses were performed in water and sediment along the fluvial mixture zone in the beginning (Campaign I -07/2006 e III – 07/2007) and at the end of crop (Campaign III – 10/2006). In laboratory were performed bioassays to estimate the vinasse potential biodegradation. The samples were collected upstream and downstream ranging in 200 m from the effluent discharge source, as well as at the mouth of the river how reference. The sediment samples were fractionated in fine (<63 μm) and coarse (>63 μm). In water, high values of temperature and dissolved oxygen with low values of pH were indicators of vinasse discharge and high concentration of potassium was fundamental to identify the presence of vinasse in the effluent. In sediments, differences were found between organic and inorganic phosphorus concentration, and the high values of organic carbon, C:N ratio and potassium were efficient descriptors of effluent and sugar cane agriculture. Similar concentrations of organic and inorganic phosphorus, and the C:N ratios around 12 demonstrated that the mouth of river was influenced by agriculture, suggested that the sedimentary organic matter originates from phytoplankton and also vacular plants. The fine fraction was significant correlated with the most part of parameters and was useful in the impact evaluation, as the main component of vinasse, the element potassium, was concentrated in grains smaller than 63 μm. The microbiological analyses revealed that the effluent influence in the bacterial community enhancing biomass, energetic gain (analyzed by ETSA) in both water and sediment at beginning of crop. However the organic fraction accumulated in the sediment inhibited aerobic respiration and reduced the capacity of environmental auto-depuration at the end of harvest period. Microorganisms isolated from local sediment were capable to grow and to use vinasse as a single source of energy during bioassays, demonstrating possible uses in bioremediation. It was observed that the number of cells/cm-3 has be in the minimum of the order 108 a 109 to an efficient remove of biopolymers (37%) and realized the fosfate is consumed efficiently (99%) only when in concentrations near of 3 mg L-1 , observing low efficient of consume when concentrations are nearly of 9 mg L-1 . The efficiency of degradation was high in the first 6 days of bioassay, not being observed efficient reductions in the others days of analyses.

Large numbers of samples often need to be prepared and analysed in surveys of organic pollutants in the environment, but while the methods commonly used in such surveys can provide abundant detail they are generally costly, time-consuming and require large amounts of resources, so there is a need for simpler techniques. The work underlying this thesis assessed the potential utility of more convenient sample preparation and bioanalytical techniques for rapidly screening various environmental matrices that could be useful complements to higher resolution methods. Initially, the utility of a simplified extraction technique followed by an enzyme-linked immunosorbent assay (ELISA) for detecting polycyclic aromatic hydrocarbons (PAHs) in authentic (i.e. unspiked) contaminated soils was explored. The results showed that there are relationships between the structure and cross-reactivity among compounds that often co-occur with target PAHs. However, their potential contribution to deviations between estimates of total PAH contents of soils obtained using ELISA and gas chromatography-mass spectrometry (GC-MS) based reference methods were limited. Instead, the cross-reactivity of target PAHs and the failure to extract all of the PAHs prior to the ELISA determinations were the main reasons for these deviations. Polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) were detected in food and feed matrices, as well as in authentic contaminated soils using different bioanalytical techniques – ELISA and two cell-based bioassays: CAFLUX and CALUX (chemically activated fluorescent/luciferase gene expression) assays. In addition, enhanced sample preparation techniques based on accelerated solvent extraction (ASE) were developed. ASE with integrated carbon fractionation (ASE-C) in combination with CAFLUX produced estimates of PCDD and PCDF contents in fish oil and fish meal that agreed well with results obtained using reference methods. Furthermore, results from ELISA and GC-high resolution MS analyses of extracts of PCDD- and PCDF-contaminated soil samples obtained using an adjusted ASE-C technique were strongly correlated. Finally, the thesis reports the first experiments in which the results of CAFLUX, CALUX, and ELISA determinations of PCDDs and PCDFs in extracts of authentic contaminated soil samples were evaluated and compared to those obtained using a reference method. All of the bioanalytical techniques were found to be sufficiently sensitive, selective, and accurate for use in screening in compliance with soil quality assessment criteria. Overall, the improved sample preparation and bioanalytical techniques examined proved to be useful potential complements to conventional methods, enhancing the analytical framework for PAHs, PCDDs, and PCDFs. However, further validation has to be undertaken before they are applied on a large-scale.

Im Rahmen der EU-Richtlinie REACH müssen industrierelevante Chemikalien bezüglich des Risikos für Mensch und Umwelt (neu) untersucht und bewertet werden, wobei die Anzahl an Tierversuchen zu minimieren ist und neue toxikologische Prüfmethoden, Bewertungsstrategien sowie alternative Testsysteme entwickelt und optimiert werden sollen. Ubiquitär vorkommende Testorganismen, welche ähnliche Eigenschaften und eine vergleichbare Sensitivität bezüglich äußerer Einflüsse wie komplexere Organismen (Fisch, Säugetier, Mensch) besitzen, sind hier besonders gefragt um die Belastung abzuschätzen und Trendaussagen über die Wirkung zu formulieren. Durch den Einsatz vieler dieser Testorganismen sowie durch die Ermittlung der chemischen Reaktivität und der Strukturmerkmale einer Verbindung kann ein globales Bild über die Wirkung erhalten werden. Ein Organismus, der diese Charakteristika aufweist, ist der eukaryotische Einzeller Tetrahymena pyriformis GL. In der vorliegenden Dissertation wird zum einen die jeweilige Toxizität elektrophiler Substanzen unterschiedlicher Stoffklassen und Reaktions-mechanismen (α,β-ungesättigte Carbonyl- und Carboxylverbindungen, heterozyklische Drei- und Vierringe, α-halogenierte Carbonyle, Harnstoffe und Thioharnstoffe, aromatische Disulfide sowie aliphatische und aromatische Nitroverbindungen) nach den Expositionszeiten 24 h, 48 h und 72 h mit Hilfe eines etablierten Wachstumshemmtests (Müller, 2001) bestimmt und Struktur-Toxizitäts-Beziehungen bzw. Strukturalarme abgeleitet. Neben der methodischen Optimierung werden die substanzspezifischen Eigenschaften, Flüchtigkeit und Sorptionsfähigkeit, quantitativ erfasst und durch neu aufgestellten Modellgleichungen für jede Substanz ermittelt. Mit der Bestimmung der Toxizitätserhöhung, Te, welche auch als ein Maß für die Reaktivität angesehen werden kann, werden Organismen-spezifische Narkose-Basis-Geraden generiert, exzesstoxische Verbindungen identifiziert und Strukturalarme für die jeweiligen Stoffklassen aufgestellt. Zum anderen werden binäre Mischungen untersucht, welche aus den Einzelstoffen bestehen, deren Toxizität im ersten Abschnitt der Arbeit ermittelt wurden und bei denen theoretische Annahmen über die Reaktionsmechanismen bestehen. Dieser Abschnitt identifiziert, ob gleiche bzw. unähnliche primäre Wechselwirkungen zweier Substanzen mit den biologischen Targets vorliegen und welcher Mechanismus, der reaktive Mechanismus exzesstoxischer Substanzen oder die Wechselwirkungen mit Membran-Bestandteilen, dominierend ist. Für die Auswertung der Mischungsergebnisse werden die etablierten biometrischen Modelle Konzentrations-Additivität und Unabhängigen Wirkung verwendet. Es wird dargestellt, dass die Mischungstoxizitäten der binären Mischungen ähnlich wirkender Substanzen zwar relativ genau mithilfe beider Modelle berechnet werden, jedoch keine eindeutige Charakterisierung der Mechanismen möglich ist. Die Ergebnisse der untersuchten binären Mischungen unterschiedlich wirkender Substanzen zeigen dagegen, dass die Mischungstoxizitäten unähnlicher Substanzen relativ genau mithilfe des Modells der Unabhängigen Wirkung berechnet werden und eine Charakterisierung der Mechanismen möglich ist.

Activation of mammalian stress responsive pathways by plant secondary metabolites may contribute to the protection against certain chronic diseases afforded by fruit and vegetable consumption. This work focuses on the identification of plant compounds that activate the stress-responsive enzyme quinone reductase (QR) by stabilizing the transcription factor NF-E2 related factor-2 (Nrf2). Screening methanolic extracts of plants from Western Canada for QR induction in a mouse hepatoma cell line (Hepa-1c1c7) led to the identification of twenty-one extracts capable of doubling the activity of QR. Bioassay-guided fractionation of six extracts led to the identification of novel classes of compounds with QR-inducing activity including fatty-acid derived polyacetylenes, phthalides, and cannabinoids. Studies using low molecular weight thiols and the recombinantly expressed protein Keap1, the principal negative regulator of Nrf2, supported a mechanism of QR activation involving covalent modification of Keap1 cysteines for the polyacetylenes and phthalides. Analysis of transcriptional changes in response to treatment with a panel of QR-inducing compounds provided strong support for Nrf2 activation by the polyacetylene (3S,8S)-falcarindiol and the isothiocyanate (R)-sulforaphane and weaker support for the compounds (3R,8S)-falcarindiol, 6-isovaleryl-umbelliferone (6-IVU) and (Z)-ligustilide. Additionally, transcript level analyses supported a role for the aryl-hydrocarbon receptor in QR-activation by (3R,8S)-falcarindiol, (Z)-ligustilide, (R)-sulforaphane, 6-IVU and cannabidiol and suggested that treatment with polyacetylenes with a (3R)-configuration, (Z)-ligustilide and 6-IVU causes substantial changes in the expression of genes associated with lipid homeostasis and energy metabolism. As a whole, this work provides evidence that compounds that activate QR (and Nrf2) are widely distributed in the Canadian flora. However, of these QR activators, few are active at concentrations that are expected to be achieved through dietary consumption. Nevertheless, the most exceptional compounds isolated in this work, the compounds (3S,8S)-falcarindiol and epoxyfalcarindiol are highly potent and appear to be or are expected to be specific for activating Nrf2 and thus warrant attention with respect to dietary implications and as drug candidate leads.

Botulinum toxin type A (BTTA) is a polypeptide with 1296 amino acids and its used in some pathologies like hyperhidrosis and migraine, but the cosmetic area is the most important application. BTTA is produced from broth-culture synthesized by the Clostridium botulinum as a single peptide with 150 kDa. Reversed-phase liquid chromatography (RP-LC) method was developed and validated for the assessment of botulinum toxin type A in biopharmaceutical formulations. A RP-LC method was carried out on a Zorbax 300 SB C18 column (150 mm x 4.6 mm i.d.), maintained at 45 ºC. The mobile phase A consisted of 0.05 M sodium sulphate buffer, pH 2.8, and the mobile phase B was acetonitrile, run isocratically at flow rate 0.3 mL/min. Cromatographic separation was obtained with retention time of 11.4 min, and was linear over the concentration range of 0.2-100 IU/mL (r2 = 0.9999) with photodiode array (PDA) detection at 214 nm. The limits of detection and quantitation were 0.04 and 0.16 IU/mL, respectively. Specificity was established in degradation studies, which also showed that there was no interference of the excipients. Equally, the accuracy was 100.31% with bias lower than 0.80%. The method was correlated with in vivo and in vitro bioassays. Moreover, the in vitro cytotoxicity test of related proteins and higher molecular weight forms showed significant differences (p <0.05) compared to intact molecule. The validated methods were applied for the determination of BTTA in biopharmaceutical-derived products, giving mean values of the estimated content/potencies 1.16% lower compared to the in vivo bioassay. It is concluded that represents a contribution to establish new alternatives to monitor stability, quality control and thereby assure efficacy of the biotechnology-derived product. / A toxina botulínica tipo A (BTTA) é um polipeptídio constituído de 1296 aminoácidos e é recomendada para patologias, como hiperhidrose e enxaqueca, mas é especialmente usada como cosmético. A toxina botulínica é produzida através do processo de fermentação bacteriana e após sucessivas etapas de purificação resulta em uma proteína com 150 kDa responsável pelo seu efeito biológico. No presente trabalho foi desenvolvido e validado método por cromatografia líquida em fase reversa (CL-FR) para a avaliação de potência de BTTA em formulações de produtos biofarmacêuticos. No método foi utilizada coluna Zorbax 300 SB C18 (150 mm x 4,6 mm d.i.), mantida a 45 ºC. A fase móvel A foi constituída por tampão sulfato de sódio 0,05 M, pH 2,8, e a fase móvel B por acetonitrila, eluídas em vazão isocrática de 0,3 mL/min. O método utilizou detector de arranjo de diodos (DAD) a 214 nm. A separação cromatográfica foi obtida em 11,4 min, sendo linear na faixa de concentração de 0,2-100 UI/mL (r2 = 0,9999). Os limites de detecção e quantificação foram 0,04 e 0,16 UI/mL, respectivamente. A especificidade foi avaliada em estudos de degradação, que também demonstraram que não houve interferência dos excipientes. A exatidão foi 100,31 com bias inferior a 0,80. O método validado foi correlacionado com bioensaios in vivo e in vitro. Além disso, realizou-se o teste de citotoxicidade in vitro das formas degradadas, as quais apresentaram diferença significativa em relação a forma intacta (p <0,05). O método proposto foi aplicado para avaliação da potência de BTTA em formulações biofarmacêuticas, e os resultados foram comparados com o bioensaio in vivo, observando-se diferença das médias de teor/potência de 1,16% inferior para o método cromatográfico. Contribuíu-se assim para estabelecer procedimentos que aprimoram a caracterização ou o controle da qualidade, garantindo a segurança e eficácia do produto biotecnológico.