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Environmental toxicants are ubiquitous throughout the environment as a result of human activity. Among these toxicants, environmental estrogens are a category of particular concern due to their environmental prevalence and potency in altering reproductive traits. While many studies have addressed the detrimental effects of environmental estrogens on both aquatic and terrestrial organisms, few have analyzed the potential for these compounds to alter mitochondrial function. Mitochondria are the primary energy-generating system for all eukaryotic life, supporting all aspects of development, metabolism, and growth. Each cell within the body contains many mitochondria which in turn contain multiple copies of their own DNA genome, mitochondrial DNA (mtDNA). Mutations in mtDNA are responsible for a wide range of human diseases such as metabolic syndromes, cancers, and obesity. Among these mitochondrial diseases many are characterized by increased levels of heteroplasmy, multiple mitochondrial DNA haplotypes within an individual. Increased heteroplasmy can alter normal mitochondrial function and influence disease initiation and progression. To date, no studies have investigated the effects of synthetic estrogens on mitochondrial genome stability. Synthetic estrogens have the capacity to bind to estrogen receptors and initiate estrogenic responses through translocation into the mitochondrion. Despite our knowledge about the relationship of heteroplasmy and disease, we still do not have a complete grasp of the mechanisms of heteroplasmic induction. Here we report our analysis of the effects of 17α-ethynylestradiol (EE2) exposure in three studies to investigate its effect on mitochondrial genome stability. Data analysis reveals a statistically significant relationship between EE2 exposure and increased heteroplasmic frequency.

Interferentes endócrinos (IE) são substâncias capazes de afetar o sistema endócrino causando danos à saúde. Os compostos estrogênicos são um tipo de IE que geram resposta biológica semelhante aos hormônios endógenos, chamada atividade estrogênica, são frequentemente encontrados no ambiente devido à poluição de origem antrópica e ineficiência de processos de tratamento de água e esgoto. O objetivo deste trabalho foi avaliar a atividade estrogênica de águas de mananciais e águas tratadas para abastecimento no Estado de São Paulo por meio de bioensaios. Foram utilizados dois métodos, baseados em diferentes linhagens da levedura Saccharomyces cerevisiae capazes de responder a agentes estrogênicos pela presença do gene que codifica para o receptor de estrogênio humano e sistemas de gene repórter da atividade estrogênica. Uma das linhagens contém o gene luc, método denominado neste trabalho de Lesk, enquanto a outra contém genes lux como repórter, denominado método San. Os métodos foram comparados quanto à especificidade e sensibilidade de resposta aos principais interferentes endócrinos de ocorrência em águas e com dados de análises cromatográficas das respectivas amostras ambientais. Foi observada atividade estrogênica expressiva nas águas brutas coletadas em pontos específicos nas cidades de Campinas, Barueri, Cerquilho e em efluente hospitalar e em todas essas amostras foram detectados compostos estrogênicos-alvo. As amostras de água tratada não apresentaram atividade estrogênica nem compostos estrogênicos, com apenas uma exceção. O método San foi mais sensível que o método Lesk, tanto para compostos químicos puros quanto para amostras ambientais. O teor de substâncias detectado por análises cromatográficas não foi suficiente para explicar a atividade estrogênica observada nos bioensaios, indicando que concentrações abaixo dos limites de detecção podem gerar o efeito biológico ou que compostos estrogênicos não estudados e suas misturas podem estar presentes gerando efeitos aumentados. / Endocrine disrupting (ED) chemicals are compounds able to interact with the endocrine system causing health adverse effects. Estrogenic compounds are a type of ED that produce a biological response similar to organism\'s natural hormones, called estrogenic activity; they are frequently found in the environment and are usually associated to the pollution from human activities. The goal of this work was to evaluate the estrogenic activity of rivers raw and treated water around the State of São Paulo Brazil - using bioassays. Two different strains of Saccharomyces cerevisiae able to respond to estrogenic compounds, due to the presence of genes that encodes for the human estrogen receptor, were used. One of the strains has the luc gene (method Lesk), a reporter of estrogenic activity and the other contains lux genes (method San) as reporter of estrogenic activity. Both methods were compared regarding their specificity and sensitivity for chemical substances and environmental samples. The results were also compared with the chemical analysis data of target pure compounds in those waters and with the literature. Estrogenic activity was detected in raw water samples collected in Campinas, Barueri and Cerquilho and also in hospital effluent, for all those samples estrogenic compounds were determined. San method showed to be more sensitive to pure chemical compounds and environmental samples. For treated water no estrogenic activity was found, except for one sample from Barueri that presented a low response. The amounts of compounds detected by chromatographic analysis were not sufficient to explain the observed estrogenic activity when bioassays were used, therefore low compounds concentration or other compounds and their mixtures may be responsible for the high biological effet detected by bioassays.

The Major Histocompatibility Complex (MHC) encodes proteins critical to the vertebrate immune response; therefore MHC diversity is an indicator of population health. I have (1) Isolated exon 2 of the class II gene DQA in Tursiops truncatus in the North Indian River Lagoon (IRL) (n=17), South IRL (n=29) and adjacent Atlantic waters (n=20), (2) assessed genetic variability between groups, (3) developed a method to genotype individuals, (4) typed 11 unique alleles in 66 individuals, (5) detected geographic patterns of diversity between estuarine and coastal individuals (FST=0.1255, p<0.05), (6) found evidence of positive selection centered in the binding pockets P1, P6 and P9 of the peptide binding region (w=2.08), (7) found that patterns of polymorphism did not closely match patterns of diversity in neutral markers, (8) performed a pilot study with Orcinus orca. The initial findings highlight the need for further comparative work and suggest that silent mutations are not neutral. / by Tatiana Ferrer. / Thesis (M.S.)--Florida Atlantic University, 2013. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.

Interferentes endócrinos (IE) são substâncias capazes de afetar o sistema endócrino causando danos à saúde. Os compostos estrogênicos são um tipo de IE que geram resposta biológica semelhante aos hormônios endógenos, chamada atividade estrogênica, são frequentemente encontrados no ambiente devido à poluição de origem antrópica e ineficiência de processos de tratamento de água e esgoto. O objetivo deste trabalho foi avaliar a atividade estrogênica de águas de mananciais e águas tratadas para abastecimento no Estado de São Paulo por meio de bioensaios. Foram utilizados dois métodos, baseados em diferentes linhagens da levedura Saccharomyces cerevisiae capazes de responder a agentes estrogênicos pela presença do gene que codifica para o receptor de estrogênio humano e sistemas de gene repórter da atividade estrogênica. Uma das linhagens contém o gene luc, método denominado neste trabalho de Lesk, enquanto a outra contém genes lux como repórter, denominado método San. Os métodos foram comparados quanto à especificidade e sensibilidade de resposta aos principais interferentes endócrinos de ocorrência em águas e com dados de análises cromatográficas das respectivas amostras ambientais. Foi observada atividade estrogênica expressiva nas águas brutas coletadas em pontos específicos nas cidades de Campinas, Barueri, Cerquilho e em efluente hospitalar e em todas essas amostras foram detectados compostos estrogênicos-alvo. As amostras de água tratada não apresentaram atividade estrogênica nem compostos estrogênicos, com apenas uma exceção. O método San foi mais sensível que o método Lesk, tanto para compostos químicos puros quanto para amostras ambientais. O teor de substâncias detectado por análises cromatográficas não foi suficiente para explicar a atividade estrogênica observada nos bioensaios, indicando que concentrações abaixo dos limites de detecção podem gerar o efeito biológico ou que compostos estrogênicos não estudados e suas misturas podem estar presentes gerando efeitos aumentados. / Endocrine disrupting (ED) chemicals are compounds able to interact with the endocrine system causing health adverse effects. Estrogenic compounds are a type of ED that produce a biological response similar to organism\'s natural hormones, called estrogenic activity; they are frequently found in the environment and are usually associated to the pollution from human activities. The goal of this work was to evaluate the estrogenic activity of rivers raw and treated water around the State of São Paulo Brazil - using bioassays. Two different strains of Saccharomyces cerevisiae able to respond to estrogenic compounds, due to the presence of genes that encodes for the human estrogen receptor, were used. One of the strains has the luc gene (method Lesk), a reporter of estrogenic activity and the other contains lux genes (method San) as reporter of estrogenic activity. Both methods were compared regarding their specificity and sensitivity for chemical substances and environmental samples. The results were also compared with the chemical analysis data of target pure compounds in those waters and with the literature. Estrogenic activity was detected in raw water samples collected in Campinas, Barueri and Cerquilho and also in hospital effluent, for all those samples estrogenic compounds were determined. San method showed to be more sensitive to pure chemical compounds and environmental samples. For treated water no estrogenic activity was found, except for one sample from Barueri that presented a low response. The amounts of compounds detected by chromatographic analysis were not sufficient to explain the observed estrogenic activity when bioassays were used, therefore low compounds concentration or other compounds and their mixtures may be responsible for the high biological effet detected by bioassays.

Cell-free protein synthesis (CFPS) is a powerful protein expression platform where protein synthesis machinery is borrowed from living organisms. Target proteins are synthesized in a reaction tube together with cell extract, amino acids, energy source, and DNA. This reaction is versatile, and dynamic optimizations of the reaction conditions can be performed. The "œopen" nature of CFPS makes it a compelling candidate for many technologies and applications. This dissertation reports new and innovative applications of CFPS including 1) enzyme encapsulation in a virus-like particle, 2) detection of endocrine disrupting chemicals in the presence of blood and urine, and 3) expression of a multi-disulfide bond therapeutic protein. Two major limitations of enzymes are their instability and recycling difficulty. To overcome these limitations, we report the first enzyme encapsulation in the CFPS by immobilizing in a virus-like particle using an RNA aptamer. This technique allows simple and fast enzyme production and encapsulation We demonstrate, for the first time, the Rapid Adaptable Portable In vitro Detection biosensor platform (RAPID) for detecting endocrine disrupting chemicals (EDCs) in human blood and urine samples. Current living cell-based assays can take a week to detect EDCs, but RAPID requires only 2 hours. It utilizes the versatile nature of CFPS for biosensor protein complex production and EDC detection. Biotherapeutic protein expression in E. coli suffers from inclusion body formation, insolubility, and mis-folding. Since CFPS is not restricted by a cell wall, dynamic optimization can take place during the protein synthesis process. We report the first expression of full-length tissue plasminogen activator (tPA) using CFPS. These research works demonstrate the powerful and versatile nature of the CFPS.

Orientador: Ricardo de Lima Isaac / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Civil, Arquitetura e Urbanismo / Made available in DSpace on 2018-08-18T23:00:14Z (GMT). No. of bitstreams: 1 Peres_MarianaRodrigues_M.pdf: 4938252 bytes, checksum: e65c217dee0ac585f070316b2ec3fbda (MD5) Previous issue date: 2011 / Resumo: Nas últimas décadas, verifica-se um crescente interesse científico a respeito dos potenciais adversos associados à exposição humana e animal aos interferentes endócrinos. A ocorrência de interferentes endócrinos em efluente de Estação de Tratamento de Esgoto (ETE) e em águas superficiais constitui um crescente interesse científico devido à probabilidade de que esses micropoluentes não sejam removidos nos processos físico-químicos empregados em Estação de Tratamento de Água (ETA) convencional. A adsorção em carvão ativado tem sido apontada com uma opção para a remoção de interferentes endócrinos em ETA. O presente trabalho avaliou a adsorção do estrógeno natural 17?-estradiol (E2), do estrógeno sintético 17?-etinilestradiol (EE2) e do xenoestrogênio 4-nonilfenol (NP) por carvão ativado em pó (CAP) em água deionizada e água bruta do rio Atibaia. Os procedimentos analíticos empregados foram extração em fase sólida e análise por cromatografia gasosa acoplada a detector de massas (CG-EM). O modelo de Freundlich foi o que melhor representou a adsorção dos compostos nos carvões ativados usualmente aplicados nas estações de tratamento de água de Campinas-SP. Em água deionizada, a remoção mais significante dos compostos foi observada na dosagem de CAP equivalente a 15 mg/L, alcançando níveis de remoção da ordem de 95%. Os resultados obtidos nos ensaios com água bruta demonstraram que a dosagem de 10 mg/L apresenta eficiência superior a 77% na remoção dos interferentes endócrinos da água do manancial. Observou-se que o CAP com número de iodo superior a 800 mg/g apresentou, de modo geral, as melhores eficiências de remoção, favorecendo a adsorção dos compostos nos microporos do carvão. Considera-se que, embora ainda não exista legislação em vigor no Brasil que limite a presença desses micropoluentes na água de abastecimento público, recomenda-se que a adição de CAP seja realizada pelo princípio da precaução / Abstract: In the last decades, a growing scientific concern has been raised over the potential adverse effects associates to human and animal exposure to endocrine disrupting chemicals (EDC). The occurrence of these compounds in wastewater treatment plant (WWTP) effluent and surface waters has been an interesting research field due to the probability of these micropollutants to break through water treatment plant (WTP). Adsorption on activated carbon has been recognized as an option for the removal of EDC. Present paper evaluated the adsorption of the natural estrogen 17?-estradiol (E2), the synthetic estrogen 17?-ethinylestradiol (EE2) and the xenostrogen 4-nonylphenol (NP) by powdered activated carbon (PAC) in ultra-pure water and raw water from Atibaia River, at Campinas City, São Paulo State, Brazil. The analytic technique used was solid phase extraction (SPE) and gas chromatography tandem mass spectrometry (GC-MS). Freundlich's model resulted more adequate to describe the adsorption of the compounds on PAC usually applied at local WTP. In ultra-pure water it was observed more significant removal for dosages equal to 15 mg PAC/L, reaching 95% of removal. The results obtained for raw water showed that 10 mg/L PAC dosage promotes a removal of the endocrine disruptor from river water higher than 77%. It was observed that PAC with iodine number greater than 800 mg/g presented, in general, the highest removal efficiencies, supporting the adsorption of compounds in the activated carbon micropores. Although there is no current legislation in Brazil that regulates the presence of these micropollutants at water supply systems, the addition of PAC must be performed by the precautionary principle / Mestrado / Saneamento e Ambiente / Mestre em Engenharia Civil

L'objectif de cette étude était le développement de particules colloïdales magnétiques possédants un cœur magnétisable et une écorce polymère, capables d'éliminer les métaux lourds, les colorants cationiques et les perturbateurs endocriniens. Ces adsorbants pourraient être séparés magnétiquement permettant ainsi de remplacer les techniques lourdes comme la centrifugation, la sédimentation, et la filtration. Les résultats des analyses, en termes de taille, de morphologie, de composition chimique, de propriétés magnétiques et de potentiel Zeta, confirme bien l'encapsulation de cœur magnétique par une écorce en polymère, et la fonctionnalisation de la surface de ces particules. Nous avons pris comme exemple l'élimination les métaux lourds (Cu2+, Pb2+, Zn2+ et HCrO4-), le bleu de méthylène ainsi le Bisphenol A, en solutions aqueuse. La fixation de divers polluants considérés se fait via l'adsorption à la surface des particules. Divers paramètres physico-chimiques influent le phénomène d'adsorption; l'effet de la quantité d'adsorbant magnétique élaboré, l'effet pH initial du milieu, l'effet du temps de contact, ainsi l'effet de la concentration initiale des polluants sont considérés. Cette étude a montré que l'adsorption est rapide et l'équilibre est atteint au bout de 30 min. Le processus d'adsorption est fortement dépendant du pH initial du milieu. La capacité d'adsorption de Latex des particules magnétiques élaborées vis-à-vis des éléments étudiés s'avère très satisfaisante comparé aux différents adsorbants magnétiques étudiés dans la littérature. La cinétique d'adsorption pour tous les systèmes étudiés pourrait être considérée comme pseudo-deuxième ordre et le processus d'adsorption de ces éléments par les particules magnétiques suit le modèle monocouche de Langmuir. Nous nous sommes intéressé à décontaminer les effluents industriels chargés en métaux lourds, issus des bains de traitement de l'Entreprise SOPAL. Les résultats obtenus nous ont permis de déduire que les particules magnétiques sont efficaces pour la décontamination. Les latex magnétiques pourraient être recommandés comme des adsorbants rapides, efficaces, et réutilisables pour l'élimination et la récupération des métaux lourds des eaux usées / The aim of this study was to prepare magnetic latex particles being magnetic core-polymer shell, able to remove heavy metals, cationic dyes and endocrine disrupting chemicals, by batch adsorption. These adsorbents were magnetically separated allows replacement of the heavy techniques such as centrifugation, sedimentation, and filtration by applying magnetic field.The results analysis in terms of colloidal and surface properties, transmission electron microscopy, hydrodynamic particle size, thermogravimetric analysis, and zeta-potential measurements, confirms the encapsulation of the magnetic core and the polymer shell, the surface functionalization of these particles, and the good colloidal stability. Heavy metals (Cu2+, Pb2+, Zn2+ and HCrO4-), methylene blue and Bisphenol A were taken as model of contaminants. Various physicochemical parameters influencing the adsorption phenomenon, which we have studied; the effect of adsorbents amount, the initial pH medium, the contact time, and the effect of the initial concentration of the pollutants. This study showed that the adsorption is fast and equilibrium is achieved within 30 min. The adsorption process is highly dependent on the initial pH. Adsorption capacities of the elaborated magnetic latex particles are very satisfactory when compared with different magnetic adsorbents reported in the literature. The adsorption kinetics for all the studied systems could be considered pseudo-second order model and the adsorption process of these elements by magnetic latex particles follows the Langmuir monolayer model. SOPAL wastewater sample load in heavy metals was analyzed by the prepared magnetic adsorbent. The results have enabled us to deduce that magnetic latex particles are effective for decontamination of real waste water. The prepared magnetic latex particles in this research can be recommended as fast, effective, and reusable for removal and recovery of metal ions from wastewater effluents

Sewage effluents disposed into the marine environment from De Beers Marine Namibia diamond mining vessels have the potential to cause endocrine disruptive effects in marine organisms. Endocrine disruption refers to the alteration of the normal functioning of the endocrine system and various chemicals have the ability to mimic hormones, effecting endogenous hormone synthesis, transport, receptor interaction and intracellular signaling. The potential endocrine disruptive effects, caused by the release of different types of sewage effluents into the ocean, on fish species is a concern due to the commercial importance of fish species found in the mining area e.g. hake, sole, horse mackerel. Increased awareness of marine environmental degradation due to the presence of chemical contaminants has resulted in research being done on early warning systems, in the form of biomarkers. Cytochrome P450 monooxygenase 1A (CYP1A) and vitellogenin (vtg) are important proteins found in fish liver and blood, that have been used as biomarkers for the detection of pollutants in fish. CYP1A is a subfamily of the P450 superfamily of enzymes and catalyzes the oxidation, hydrolysis and reduction of exogenous and endogenous compounds (phase I reactions) and thus has the capacity to regulate the metabolism of several organic contaminants. CYP1A expression is altered by exposure to planar xenobiotic compounds e.g. polyaromatic hydrocarbons. Vtg is an important precursor for egg yolk proteins and plays a role in the growth and development of an oocyte. Expression of this protein is altered upon exposure to estrogenic compounds. The aim of this project was to isolate CYP1A from fish liver by differential centrifugation and optimize conditions for the CYP1A-mediated ethoxyresorufin-Odeethylase (EROD) assay and western blot analysis (to assess CYP1A expression). Another aim of this study was to evaluate the potential effects of biologically disruptive chemicals from sewage effluents, discharged into the marine environment, on the expression of CYP1A in two species of hake, Merluccius capensis and M. paradoxus (Cape hake). CYP1A in Cape hake is approximately a 60 kDa protein and the highest EROD activity was detected in the microsomal fraction after differential centrifugation. Optimal EROD assay conditions were observed at pH 7.5, a temperature of 25 °C, 10 μl of sample and a reaction time of 30 seconds. Enzyme stability assays indicated a drastic decrease in enzyme activity after 30 seconds. The EROD assay was not NADPH dependent but was limited by NADPH supply, with an increase of 300% in EROD activity being observed with the addition of 0.1 M exogenous NADPH. The addition of dicumarol (40 μM), a phase II enzyme inhibitor, showed a 232% increase in EROD activity. This is because dicumarol inhibited enzymes with the capacity to metabolize the product (resorufin) of the EROD reaction. With regard to western blot analysis, the optimal primary (rabbit antifish CYP1A peptide) and secondary (anti-mouse/rabbit antibody-horseradish peroxidase conjugate (POD)) antibody dilutions were determined to be 1:1000 and 1:5000, respectively. The comparison of CYP1A expression in Cape hake samples from De Beers Marine mining area and reference sites showed higher EROD activity (16.29 ± 0.91 pmol/min) in fish samples from the mining area in comparison to the reference site (10.42 ± 2.65 pmol/min). Western blot analysis was in agreement with the EROD assay results and a higher CYP1A expression was observed in fish from the mining sites. The increased CYP1A expression observed in fish from the mining area is not definitively an indication of a pollutant effect in the environment, as several environmental and biological factors (e.g. photoperiod and age) must also be considered before reaching this conclusion. Another aim of this study was to purify vtg from Cape hake blood samples. Cape hake vtg was purified from fish plasma by selective precipitation with MgCl2 and EDTA. Precipitated sample was subjected to anion exchange chromatography using fast protein liquid chromatography (FPLC). Vtg eluted as two broad peaks and had a molecular weight above 200 kDa. SDS-PAGE analysis also resolved smaller molecular weight proteins below 70 kDa, which were thought to be vitellogenin cleavage proteins, lipovitellin and phosphovitins. Western blot analysis was performed; however, it did not produce any conclusive results. The purification of vtg enables further studies in characterizing this protein and developing assay aimed at detecting estrogenic pollutants in the marine environment

Les méthodes actuellement disponibles pour la surveillance de la qualité du milieu se réfèrent au suivi de composés définis par les réglementations et ne permettent pas d’appréhender l’(éco)toxicité réelle de tous les composés présents dans l’environnement (e.g. composés inconnus, produits de transformation, effets de mélange…). Dans ce contexte, une méthode d’analyse pluridisciplinaire intégrative, l’analyse dirigée par l’effet (EDA), a été développée. Cette approche bio-analytique vise à établir un lien de causalité entre une exposition aux contaminants et l’effet biologique observé. La démarche est basée sur la simplification séquentielle d’un échantillon guidée par les bio-essais afin d’individualiser puis identifier, par des techniques analytiques chimiques performantes, les composés biologiquement actifs. Les objectifs de cette thèse ont porté sur (1) le développement et la validation d’une méthodologie, allant du pré-traitement jusqu’au fractionnement, adaptée à différentes matrices environnementales ; (2) la mise en place d’une stratégie d’identification moléculaire par spectrométrie de masse haute résolution ; (3) l’application de la démarche EDA à plusieurs échantillons afin d’illustrer l’adaptabilité et l’apport d’une telle méthode à des problématiques environnementales. Ces travaux de thèse permettent de disposer aujourd’hui d’une démarche bio-analytique complète de la préparation d’échantillon à l’identification de molécules, et adaptée à tous types d’échantillons. Les différentes études environnementales réalisées au cours de ces travaux ont permis, entre autre, d’établir une liste de composés diverses non recherchés pouvant représenter un risque pour l’environnement. / To date, environmental risk assessment is based on targeted chemical analyses. These analyses allow the detection of known active compounds and this preselected set does not often explain the observed toxic effects in complex environmental samples (e.g. unknown compounds, transformation product, cocktail effect...). For an integrative contamination assessment, a multidisciplinary approach, Effect-Directed Analysis (EDA) was developed. This approach combining biological tools and chemical analyses allows determining active compounds and identitying them in such environmental complex mixtures. EDA aims at the establishment of cause-effect relationships by sequential reduction of the complexity of environmental mixtures, eventually to individual toxicants. The aims of this thesis were (1) to develop and to validate the EDA-based strategy on various environmental complex samples; (2) to set up an identification strategy for identifying non targeted or unknown molecules using high resolution mass spectrometry; (3) to prove the usefulness of EDA approach for the identification of active compounds in environnemental complex samples. This thesis work provides to a complet bio-analytical approach from the preparation step of samples to the identification of molecules. The potential of this tool was fully confirmed on various environmental studies. These investigations allowed establishing a list of non-target compounds which can represent a risk for the environment.