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Amphibians develop in aquatic environments where they are very susceptible to the effects of pesticides and other environmental contaminants. Glyphosate-based herbicides are widely used and have been shown to affect survival and development of tadpoles under laboratory conditions. The goal my thesis is to determine if agriculturally relevant exposure to Roundup WeatherMax®, a herbicide formulation containing the potassium salt of glyphosate and an undisclosed surfactant, influences the survival and development of wood frogs tadpoles (Lithobates sylvaticus) under both laboratory and field conditions. In the field, experimental wetlands were divided in half using an impermeable curtain so that each wetland contained a treatment and control side. Tadpoles were exposed to two pulses of this herbicide at environmentally realistic concentration (ERC, 0.21 mg acid equivalent (a.e.)/L) and predicted environmental concentrations (PEC, 2.89 mg a.e./L), after which survival, growth, development, and expression of genes involved in metamorphosis were measured. Results indicate that exposure to the PEC is extremely toxic to tadpoles under laboratory conditions but not under field conditions. Results from both experimental conditions show sublethal effects on growth and development, and demonstrate that ERC of glyphosate-based herbicides have the potential to alter hormonal responses during metamorphosis. My secondary objectives were to compare the effects of Roundup WeatherMax® to the well-studied Vision® formulation (containing the isopropylamine (IPA) salt of glyphosate and POEA), and to determine which ingredient(s) are responsible for the sublethal effects on development. Survival, growth and gene expression results indicate that Roundup WeatherMax® has greater toxicity than Vision® formulation. Contrary to my prediction, results suggest that, under realistic exposure scenarios, POEA is not the sole ingredient responsible for the observed developmental effects. However, my results demonstrate that chronic exposure to the POEA surfactant at the PEC (1.43 mg/L) is extremely toxic to wood frog tadpoles in laboratory. As part of the Long-term Experimental Wetlands Area (LEWA) project, this research contributes to overall knowledge of the impacts of glyphosate-based herbicides on aquatic communities.

This study was carried out to evaluate the performance of Soil Aquifer Treatment (SAT) under different loading regimes, using wastewater of much higher strength than usually encountered in SAT systems, and also to investigate the removal of the endocrine disruptors triclocarban (TCC), estrone (E1), 17β-estradiol (E2) and 17α-ethinylestradiol (EE2). SAT was simulated in the laboratory using a series of soil columns under saturated and unsaturated conditions. Investigation of the removal of Chemical Oxygen Demand (COD), Biochemical Oxygen Demand (BOD), Dissolved Organic Carbon (DOC), nitrogen and phosphate in a 2 meter long saturated soil column under a combination of constant hydraulic loading rates (HLRs) and variable COD concentrations as well as variable HLR under constant COD showed that at fixed HLR, a decrease in the influent concentrations of DOC, BOD, total nitrogen and phosphate improved their removal efficiencies. It was found that COD mass loading applied as low COD wastewater infiltrated over short residence times would provide better effluent quality than the same mass applied as a COD with higher concentration at long residence times. On the other hand relatively high concentrations coupled with long residence time gave better removal efficiency for organic nitrogen. Phosphate removal though poor under all experimental conditions, was better at low HLRs. In 1 meter saturated and unsaturated soil columns, E2 was the most easily removed estrogen, while EE2 was the least removed. Reducing the thickness of the unsaturated zone had a negative impact on removal efficiencies of the estrogens whereas increased DOC improved the removal in the saturated columns. Better removal efficiencies were also obtained at lower HLRs and in the presence of silt and clay. Sorption and biodegradation were found to be responsible for TCC removal in a 300 mm long saturated soil column, the latter mechanism however being unsustainable. TCC removal efficiency was dependent on the applied concentration and decreased over time and increased with column depth. Within the duration of the experimental run, TCC negatively impacted on treatment performance, possibly due to its antibacterial property, as evidenced by a reduction in COD removals in the column. COD in the 2 meter column under saturated conditions was modelled successfully with the advection dispersion equation with coupled Monod kinetics. Empirical models were also developed for the removal of TCC and EE2 under saturated and unsaturated conditions respectively. The empirical models predicted the TCC and EE2 removal profiles well. There is however the need for validation of the models developed

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.

Desreguladores endócrinos são compostos que podem alterar o sistema endócrino dos indivíduos, bem como sua fertilidade e por em risco a saúde dos mesmos. Muitas questões relacionadas a esses compostos ainda não foram elucidadas, apesar de serem muito estudados recentemente. Diversas pesquisas têm sido feitas investigando-se o emprego de processos oxidativos avançados na degradação dos desreguladores endócrinos em águas naturais e residuárias. O presente trabalho teve como objetivo estudar a remoção do hormônio 17\'beta\'-estradiol em soluções aquosas por oxidação química. A pesquisa foi dividida em duas etapas, sendo que a primeira etapa consistiu na investigação do uso de brometo de prata/dióxido de titânio (\'AG\'BR\'/\'TI\'O IND.2\') como catalisador no processo de fotocatálise com luz visível. Para isso, foram avaliadas a influência da concentração de \'AG\'BR\'/\'TI\'O IND.2\' (0,020; 0,025; 0,050; 0,070 e 0,100 g/L) e a intensidade de luz visível incidente (6,6 x \'10 POT.5\' e 1,3 x \'10 POT.6\' Ix) na eficiência de degradação do 17\'beta\'-estradiol. Na segunda etapa avaliou-se o emprego de ozônio na degradação de 17\'beta\'-estradiol em soluções aquosas, variando-se as dosagens de ozônio (1,7; 3,3, 5,0 e 7,5 mg \'0 IND.3\'/L) aplicadas e o pH (3; 5; 7; 9 e 11) das amostras. Na primeira etapa, os resultados demonstram que E2 foi bastante sensível aos processos oxidativos empregados. A fotocatálise foi eficiente para oxidar 17\'beta\'-estradiol, demandando tempo de exposição pequeno, em torno de 4 minutos para obtenção de remoção da ordem de 98%, quando aplicada concentração de \'AG\'BR\'/\'TI\'O IND.2\' de 0,100 g/L e a intensidade de luz visível incidente de 1,3 x \'10 POT.6\' Ix. Na segunda etapa, observou-se que o aumento do pH promoveu maior rapidez na degradação do E2 e o aumento da massa de ozônio aplicada promoveu maior eficiência de degradação do 17\'beta\'-estradiol. / Endocrine disrupting chemicals are compounds that can alter the endocrine system of individuals and their fertility and threaten their health. Many issues related to these compounds have not been elucidated, although much studied recently. Many researchers have investigated the use of advanced oxidative processes in the degradation of endocrine disrupting compounds in water and wastewater. This work is a study of the removal of 17\'beta\'-estradiol in aqueous solutions by chemical oxidation. This work was divided into two stages. In the first one, the use of bromide of silver/titanium dioxide (\'AG\'BR\'/\'TI\'O IND.2\') was investigated as catalyst in the photocatalysis activity under visible light irradiation. It was evaluated the influence of the concentration of \'AG\'BR\'/\'TI\'O IND.2\' (0.020; 0.025; 0.050; 0.070 and 0.100 g/L) and the incident light intensity (6.6 x \'10 POT.5\' and 1.3 x \'10 POT.6\' Ix) in the efficiency of degradation of 17\'beta\'-estradiol. In the second step, it was evaluated the use of ozone in the degradation of 17\'beta\'-estradiol in aqueous solutions, ranging up the masses of applied ozone (1.7; 3.3; 5.0 and 7.5 mg \'0 IND.3\'/L) and the pH (3, 5, 7, 9 and 11) of the samples. In the first stage, the results show that E2 was very sensitive to oxidative processes applyed. The photocatalysis was efficient to oxidize the 17\'beta\'-estradiol, requiring short exposure time, around 4 minutes, to obtain the removal of 98% (0.100 g/L of \'AG\'BR\'/\'TI\'O IND.2\' and incident visible light intensity of 1.3 x \'10 POT.6\'. In the second step, it was observed that the raise in pH promoted faster degradation of E2 and the increase of the mass of applied ozone promoted high efficiency of degradation of 17\'beta\'-estradiol.

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.