Submitted by Cláudia Bueno (claudiamoura18@gmail.com) on 2016-03-10T20:32:55Z No. of bitstreams: 2 Dissertação - Anna Paula Santos Almeida Rotta - 2015.pdf: 1241010 bytes, checksum: 9da763a436471dd9f131621df58acc8c (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-03-14T15:27:32Z (GMT) No. of bitstreams: 2 Dissertação - Anna Paula Santos Almeida Rotta - 2015.pdf: 1241010 bytes, checksum: 9da763a436471dd9f131621df58acc8c (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2016-03-14T15:27:32Z (GMT). No. of bitstreams: 2 Dissertação - Anna Paula Santos Almeida Rotta - 2015.pdf: 1241010 bytes, checksum: 9da763a436471dd9f131621df58acc8c (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2015-10-14 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / In recent decades, the growth of industrial activity and agriculture has been responsible for environmental contamination due to the presence of organic and inorganic substances. Phenol and its derivatives are an important class of environmental contaminants by their presence in many industrial effluents. The seeking of biological alternatives to mitigate the environmental pollution has motivated researches to find microorganisms that combine the capacity to degrade phenol with a sustainable focus. Therefore, bacteras capable of degrading xenobiotics are been used in soil, sediment and water treatment. This study has aimed to evaluate the biodegradation of phenol by bacterial isolates isolated from two Brazilian biomes (Cerrado Goiano and Mangrove of Guarapary, ES), whereupon the influence of pre-adaptation of the bacteria was checked, as well as the effects of growth parameters in different concentrations and the tolerance to this chemical compound. One isolated from Cerrado was identified as Staphylococcus aureus (BF 2.5), and the other one as a gram-positive rod (\BF 2.3.2), and the mangrove bacteria were identified as Bacillus circulans (MF-2) and Bacillus sp. (MF-1). All of the isolates consumed phenol in the approximated of 500mg.L-1 when cultivated in liquid Busnell-Hass (BH) medium and 1.500mg.L-1 in Nutrient Agar medium (NA). The consumption of phenol as carbon source by BF-2.5 isolated was 2,78; 4;79 and 0,35% for concentrations of 100, 200 and 300 mg.L-1 of phenol, respectively. The isolated BF-2.3.2 results were 11, 04; 19,13 and 16,02%, respectively. For the mangrove isolated the results were 22,43; 11,52 and 3.33% to 21,54; 20;54 and 28.85% for the MF-1 and MF-2 isolates, respectively in the same phenol concentrations tested. These results suggest a higher phenol consumption capacity of MF-2 isolated from mangrove. / Nas últimas décadas, a crescente atividade industrial e a agropecuária têm sido as responsáveis pela contaminação do meio ambiente devido à presença de substâncias orgânicas e inorgânicas. O fenol e seus derivados constituem uma importante classe de contaminantes ambientais pela sua presença em muitos efluentes industriais. A busca por alternativas biológicas para combater a poluição ambiental tem motivado pesquisas por micro-organismos que aliem a capacidade de degradar o fenol com enfoque sustentável. Nesta vertente, bactérias degradadoras de xenobióticos, estão sendo utilizadas como inóculo nos diversos tipos de tratamento biológico para a minimização de contaminação de águas, solos e sedimentos. Este trabalho teve como objetivo avaliar a biodegradação do fenol por isolados bacterianos de dois biomas brasileiros (Cerrado Goiano e Manguezal de Guaraparí, ES), no qual, verificou-se a influência da adaptação bacteriana perante as diferentes concentrações de fenol e a tolerância a esse composto químico. Das bactérias de Cerrado uma foi identificada como Staphylococcus aureus (BF 2.5) e a outra somente como bastonete gram-positivo (BF 2.3.2) e entre as bactérias de manguezal uma foi identificada como Bacillus circulans (MF-2) e a outra Bacillus sp. (MF-1) Todos os isolados consumiram fenol na concentração aproximada de 500mg.L-1 quando crescidos em meio de Bushnell-Haas (BH) líquido e em 1.500mg.L-1 quando em meio Ágar Nutriente (AN). Quanto à utilização do fenol como fonte única de carbono observamos que o consumo de fenol pelo isolado BF-2.5 foi de 2,78; 4,79 e 0,35% para as concentrações de 100, 200 e 300mg.L-1 de fenol, respectivamente. Já para o isolado BF-2.3.2 os resultados foram de 11,04; 19,13 e 16,02%, respectivamente. Para os isolados de manguezal os resultados foram 22,43; 11,52 e 3,33% e 21,54; 20,54 e 28,85% para os isolados MF-1 e MF-2, respectivamente nas mesmas concentrações de fenol testadas. Estes isolados sugerem uma maior capacidade de utilização pelo isolados MF-2 de manguezal.

O câncer é a segunda maior causa de morte no Brasil, atrás apenas de doenças cardíacas. Por isto, é evidente que grandes recursos sejam direcionados para a pesquisa no descobrimento de novas opções com a finalidade de erradicar esta doença. Dentre estas opções, a quimioprevenção do câncer tem chamado a atenção já que, mesmo com os imensos avanços no conhecimento sobre os mecanismos da carcinogênese e conseqüente desenvolvimento de novas drogas, os dados estatísticos de mortalidade não se tornaram menores. Somando-se a estes fatos, deve ser considerado que no Brasil o tratamento padrão do câncer não chega a todas as pessoas por ser extremamente caro. Desta forma, a quimioprevenção do câncer com fatores presentes na dieta ou oriundos de fontes consideradas baratas como fitoterápicos, deve ser apreciada. Assim, este trabalho teve como objetivo avaliar os efeitos quimiopreventivos e antineoplásicos de uma planta brasileira, o guaraná (Paullinia cupana Mart var. sorbilis). Foram utilizados alguns experimentos em camundongos como indução genotóxica em fígado pela Dietilnitrosamina (DEN); carcinogênese pulmonar induzida pela 4-(metilnitrosamino)-1-(3-piridil)-1-butanona (NNK), uma nitrosamina presente no tabaco; tumor ascítico de Ehrlich; disseminação hematógena de melanoma B16/f10; e cultivo de células tumorais e não tumorais. Além disso, caracterizou-se o papel da Conexin43 na carcinogênese pulmonar induzida pelo NNK e os efeitos do guaraná sobre o receptor CAR e sua ação quando da administração do ligante do CAR, 1,4-bis[2-(3,5-dichloropiridiloxi)]benzeno (TCPOBOP). Pudemos observar efeitos quimiopreventivos e antineoplásicos do guaraná dependendo do modelo utilizado, demonstrando que seu modo de ação principal é a redução da proliferação celular. Além disso, observamos que os tumores de pulmão dos animais tratados com a planta apresentavam menor tamanho, menor grau maligno, menor índice de proliferação celular e menor ativação do fator de transcrição CREB. Observamos também que a Conexina43 (Cx43) tem importante papel na carcinogênese pulmonar induzida pelo NNK, atuando como supressor tumoral e em fases tardias possivelmente tendo papel inverso, ou seja, como um oncogene. Caracterizamos os efeitos do guaraná sobre a ativação do receptor CAR e demonstramos que, por si só, o guaraná induz a expressão do CAR, além de alterar a expressão de alguns de seus transcritos como a CYP2B10 e CYP3A11. Ao analisarmos os efeitos de extratos de guaraná sobre células de tumor de pulmão (E9) in vitro, verificamos o mesmo efeito antiproliferativo, diminuindo a expressão do PCNA e da Conexina43 de maneira dose-dependente, além de verificar um aumento da expressão do receptor CAR. Ao fim propomos uma hipótese de mecanismo de ação baseando-se nas alterações encontradas oriundas da administração do guaraná. Concluímos que o guaraná apresenta componentes com ação antitumoral em camundongos, tendo efeito quimiopreventivo ou antineoplásico dependendo do modelo utilizado. / Cancer is the second biggest cause of deaths in Brazil, only behind of cardiac diseases. As a result, it is evident that great resources for research will be directed towards the discovery of new options to eradicate this disease. Among these options, cancer chemoprevention has calling for attention since the huge advances in the knowledge of carcinogenesis and development of new drugs did not decrease statistical data on mortality due to cancer. In addition, it must be considered that in Brazil, cancer therapy is not available for all given that it is too expensive. Therefore, cancer chemoprevention with dietary factors or from medicinal plants has got to be treasured. Following these lines, the aim of this work was to evaluate the chemopreventive and antineoplastic effects of a Brazilian plant, Paullinia cupana Mart var. sorbilis, most known as guarana. It was used several experiments in mice and cell culture essays as: protection against DEN-induced DNA damage; NNK-induced lung carcinogenesis; Ehrlich Ascitic Tumor; metastasis of B16/f10 melanoma cells; and cell culture of a tumorigenic and a non-tumorigenic cell lines. Additionally, it was characterized the role of Connexin43 in the NNK-induced lung carcinogenesis and the effects of guarana on the CAR receptor before and after the administration of TCPOBOP. We note a chemopreventive or antineoplastic effect of guarana depending on the model employed and showed that the mode of action responsible for these effects was reduced cell proliferation. Also, the lung tumors of guarana-treated animals were smaller, less aggressive, with decreased cell proliferation and CREB activation. On the other hand, we observed that Connexin43 have an important role on NNK-induced lung carcinogenesis because it may act as a tumor suppressor and in advanced stages as an oncogene. The effects of guarana on the CAR activation were characterized and we showed that guarana induces CAR mRNA expression, altering the levels of its transcripts as CYP2B10 and CYP3A11. We also examined the effects of guarana extracts on a lung tumor cell culture (E9 cells) and demonstrated the same antiproliferative effect observed previously, by decreased PCNA and Connexin43 proteins in a dose-dependent manner along with an increase in CAR protein. At last we hypothesized a mechanism of action for guarana effects basing in our findings. We concluded that guarana presents substances that have antitumoral effects in mice, enclosing a chemopreventive or antineoplastic effect depending on the model studied.

Effluents with high concentration of organic matter and xenobiotic compounds, especially in the case of the agrochemicals, cause environmental concern. In the present work, the adaptative ability of two filamentous fungi, which cause white rot (Geotrichum candidum Link & Pers. and Phanerochaete chrysosporium Burds.), to tolerate high concentration of xenobiotic substances was analyzed. In solid medium, P. chrysosporium and G. candidum presented excellent development under aerated and not aerated conditions (30ºC and dark), in potato-dextrose-agar and malt extract-agar, respectively. In liquid medium, the same was observed in broth-potato-dextrose and malt extract. The fungi also developed well in liquid media deficient in proteins and carbohydrates, but with high concentrations (10 and 25 mg/L) of the agrochemicals Boral, Decanote, Gliphosate, Paraquat and Plenum - all used in fields of sugar-cane, as the ones of the "S.A Usina Coruripe Açúcar e Álcool" (Coruripe - AL). These chemicals are classified as persistent, belong to the toxicological classes II and III, and have different solubilities. The fungi presented tanninolitic activity when inoculated in samples of the crude effluent with supplement of nutrients, under aeration or not. The activity of the laccase of G. candidum in the same medium was higher in the 3rd day of the growth without aeration, occurring a reduction of that in the 5th day. On the other hand, the fungus P. chrysosporium presented a higher activity of laccase in the 7th day of the growth, also without aeration. Both fungi did not interfere in the color, turbidity, concentration of nitrates and nitrites of this medium. G.candidum revealed equal efficiency in the reduction of the concentration of phosphates of the crude effluent, under aeration (78.18%) and not aeration (79.33%) conditions. However, it did not reduce the COD. P. chrysosporium was efficient in the reduction of this COD, and the difference between the aerated growth (26.77%) and the not aerated one (29.55%) was very small. It did not reduce the total tanning bark concentration, but it diminished the concentration of phosphates under aerated (55.55%) and under not aerated (61.65%) condition. The addition of nutrients in the medium of diluted effluent (2 X, 4 X and 8 X) showed that there is a direct effect of the concentration of the COD on its percentage of removal by the inoculated fungi. There is also a direct effect on the remotion of total phenolic compounds and reduced glycids. The conversion of COD in the diluted effluent (8X) was higher in the growth of P. chrysosporium (65.66%) than in the one of G.candidum (57%). The same it can be said regarding to the best rates of phenol degradation (59.66% and 50.33%, respectively). The pH measured in the media supplemented with nutrients was always higher (7,56-7,8) than the initial one (5,5). In the higher dilution of the effluent (8X) plus the supplement of nutrients, the maximum removal of COD and total phenols after the treatment occurred in 7th day of incubation of both fungi. In all the cases of diluted samples plus supplement of nutrients, the discoloration drastically increased after the inoculation of the fungi. Therefore, the use of these microorganisms in the bioremediation of the effluent of sugar-alcohol industry is recommended only after a correction of the ratio C: N: P to about 100:1,6:1. / Efluentes com alta concentração de matéria orgânica e compostos xenobióticos, especialmente no caso dos agroquímicos, causam preocupação ambiental. No presente trabalho, dois fungos filamentosos causadores de podridão branca (Geotrichum candidum Link e Pers. e Phanerochaete chrysosporium Burds.) mostraram-se capazes de se adaptar a meios com elevados teores de matéria orgânica xenobiótica ou não. Em meio sólido, verificou-se que P. chrysosporium e G. candidum, apresentaram ótimo desenvolvimento em condições aeradas e não aeradas, a 30ºC e escuro, em meios batata-dextrose-ágar e extrato de malte-ágar, respectivamente. Em meio líquido, o mesmo foi observado em caldo-batatadextrose e meio extrato de malte. Os fungos também desenvolveram-se bem em cultivos líquidos deficientes em proteínas e carboidratos, e contendo altas concentrações (10 e 25 mg/L) dos agroquímicos Boral, Decanote, Glifosato, Paraquat e Plenum, todos utilizados nas lavouras de cana de açúcar da S.A Usina Coruripe Açúcar e Álcool (Coruripe AL), classificados como persistentes, de classes toxicológicas II e III, e com diferentes solubilidades. Ambos os fungos apresentaram atividade taninolítica quando inoculados em amostras de efluente de agroindústria sucro-alcooleira não diluídas e não acrescida de nutrientes, sob aeração ou não. A atividade da lacase do fungo G. candidum, nesse mesmo meio, foi maior no 3o dia de cultivo sem aeração, ocorrendo uma diminuição no 5o dia. Já o fungo P. chrysosporium, apresentou maior atividade da lacase no 7o dia de cultivo sem aeração. Em relação à cor, turbidez, concentração de nitratos e nitritos, não houve variação desses parâmetros nas amostras inoculadas por ambos os fungos. G. candidum mostrou-se igualmente eficiente na redução da concentração de fosfatos, em cultivos aerados (78,18%) e não aerados (79,33%) do efluente bruto. Todavia, não reduziu a DQO do mesmo. P. chrysosporium mostrou-se eficiente na redução da DQO, sendo pequena a diferença entre os cultivos aerados (26,77%) com relação aos não aerados (29,55%), e não reduziu o teor de taninos totais, mas diminuiu a concentração de fosfatos sob aeração (55,55%) e não aerado (61,65%). A adição de nutrientes no cultivo dos fungos em efluente agroindustrial diluído (2X, 4X e 8X), mostrou que houve um efeito direto da concentração da DQO sobre sua porcentagem de remoção e sobre a redução de fenóis totais e glicídeos redutores. A conversão da DQO em efluente diluído (8X) foi maior no cultivo de P. chrysosporium (65,66%) do que no de G. candidum (57%). O mesmo pode ser dito quanto às melhores taxas de degradação de fenóis (59,66% e 50,33%, respectivamente). Quanto ao pH mensurado nos cultivos suplementados com nutrientes, foi sempre maior (7,56-7,8) que o inicial (5,5). Já nos cultivos utilizando a maior diluição (8X) e o suplemento de nutrientes, a máxima remoção de DQO e de fenóis totais após o tratamento ocorreu no 7º dia do cultivo para ambos os fungos. Em todos os casos de amostras diluídas e acrescidas de nutrientes, a descoloração aumentou drasticamente após o cultivo dos fungos estudados. Portanto, recomenda-se a utilização desses microrganismos na biorremediação de efluentes sucro-alcooleiros desde que haja uma correção da proporção C:N:P de cerca de 100:1,6:1.

Submitted by (edna.saturno@ufrpe.br) on 2016-12-14T17:45:59Z No. of bitstreams: 1 Elica Santos Rios.pdf: 736943 bytes, checksum: 973f89d16d74c3e4d0210b33228d1a9c (MD5) / Made available in DSpace on 2016-12-14T17:45:59Z (GMT). No. of bitstreams: 1 Elica Santos Rios.pdf: 736943 bytes, checksum: 973f89d16d74c3e4d0210b33228d1a9c (MD5) Previous issue date: 2013-02-19 / The palm is recognized as a major forage resources for animal production in the semiarid. However, in recent years, most of palmais North East began to be compromised by the cochineal carmine (Dactylopius opuntiae Cockerell), being necessary control measures that are efficient and that do not affect the activity of the microbiota present in the soil. Thus, the present study aimed to evaluate the effect of xenobiotics applied in the culture of palm infested by D. opuntiae on natural enemies encountered in culture and soil microbes. The study was conducted in an area with palm cv. gigantic (Opuntia ficus-indica) scale-infested cochineal carmine in the region of Caétes – PE. The experimental design was a randomized block with 5 treatments: control (water), detergent + bleach, oil neem, methomyl and thiamethoxam lambda-cyhalothrin, and 3 repetitions. Before application of xenobiotics were conducted eight data collections, to observe the growth of the population of cochineal carmine and distribution of natural enemies. After application of the products, three samples were conducted to quantify the efficiency of xenobiotics and their effect on natural enemies and soil microbes by respirometry microbial, bacterial count and genetic variability of bacteria. For this, were collected soils (0-20cm), the respirometry assessed by monitoring the release of CO2 by the microorganisms for a period of 30 days. In microbiology, was evaluated population density of the bacteria after 24, 48 and 72h of incubation, morphological variability and by staining. Subsequently, we assessed the genetic variability, by molecular techniques, using BOX-PCR. There was increase in the number of colonies of cochineal carmine even after application of xenobiotics, which are not significantly affected natural enemies (IN) present in the culture, as these insects occurred even at low densities, independent of the application of the products. The predatory species of cochineal carmine were observed Zagreus bimaculosus, Cybocephalus sp. and larvae Salpingogaster cochenillivorus, and Z. bimaculosus, considered the most frequent. As to respirometry, there is, in relative terms, the amount of CO2 released from the soil samples was higher in the plots with the application of the insecticide thiamethoxam + lambda-cyhalothrin, relative to the control, not differing from other treatments. Soils treated with water alone (control) and detergent + bleachshowed the largest populations (0.96 and 0.94 x 102 UFC.g-1, respectively). It was observed that soils with water application (control) and detergent + bleach, showed bacteria with slower growth, and the other treatments, with accelerated growth occurred 24h, decreasing from 48 h of incubation. In morphological characteristic (coloration) bacterial colonies predominated white colonies, demonstrating visually low genetic variability. However, the application of molecular techniques (BOX-PCR), revealed high genetic variability between colonies of white coloration analyzed. Demonstrating that given the importance of cactus for semiarid region is of great importance more detailed studies on the effects of xenobiotics on the microbiota and associated insects. / A palma é reconhecida como um dos principais recursos forrageiros para a produção animal no semiárido. Entretanto, nos últimos anos, grande parte dos palmais do Nordeste passaram a ser comprometidos pela cochonilha do carmim (Dactylopius opuntiae Cockerell), sendo necessárias medidas de controle que sejam eficientes e que não afetem a atividade da microbiota presente no solo. Diante disso, o presente trabalho teve como objetivo avaliar o efeito dos xenobióticos aplicados na cultura da palma infestada por D. opuntiae sobre os inimigos naturais encontrados na cultura e na microbiota do solo. A pesquisa foi conduzida numa área com palma cv. gigante (Opuntia ficus-indica) infestada pela cochonilha do carmim na região de Caetés - PE. O delineamento experimental foi em blocos casualizados, com 5 tratamentos: controle (água); detergente + água sanitária; óleo de nim; metomil e tiametoxam lambda-cialotrina, e 3 repetições. Antes da aplicação dos xenobióticos foram realizadas 8 coletas de dados, para observar o crescimento da população da cochonilha do carmim e distribuição dos inimigos naturais. Após a aplicação dos produtos, foram realizadas 3 coletas para quantificar a eficiência dos xenobióticos e seu efeito sobre os inimigos naturais e a microbiota do solo, através da respirometria microbiana, quantificação bacteriana e variabilidade genética de bactérias. Para isso, foram coletados solos (0-20cm), sendo a respirometria avaliada monitorando-se a liberação de CO2 pelos micro-organismos, por um período de 30 dias. Na microbiologia, foi avaliada a densidade populacional das bactérias, após 24, 48 e 72h de incubação, e a variabilidade morfológica, através da coloração. Posteriormente, avaliou-se a variabilidade genética, através de técnicas moleculares, utilizando-se BOX-PCR. Houve aumento do número de colônias da cochonilha do carmim mesmo após a aplicação dos xenobióticos, os quais também não afetaram de maneira significativa os inimigos naturais (IN) presentes na cultura, já que estes insetos ocorreram, mesmo que em baixas densidades, independente da aplicação dos produtos. As espécies predadoras da cochonilha do carmim observadas foram Zagreus bimaculosus, Cybocephalus sp. e larvas de Salpingogaster cochenillivorus, sendo Z. bimaculosus, considerado a mais frequente. Quanto à respirometria, verifica-se, em termos relativos, que a quantidade de CO2 liberado nas amostras de solo foi maior nas parcelas com a aplicação do inseticida tiametoxam+lambda-cialotrina, em relação ao controle, não diferindo dos demais tratamentos. Os solos tratados com apenas água (controle) e detergente + água sanitária apresentaram as maiores populações (0,96 e 0,94 x 102 UFC.g-1, respectivamente). Observou-se que solos com aplicação de água (controle) e detergente + água sanitária, apresentaram bactérias com crescimento mais lento, e nos demais tratamentos, ocorreram crescimento mais acelerado com 24h, diminuindo a partir de 48h de incubação. Na característica morfológica (coloração), as colônias bacterianas houve predomínio de colônias brancas, demostrando visualmente baixa variabilidade genética. Entretanto, a aplicação da técnica molecular (BOX-PCR), revelou alta variabilidade genética entre as colônias de coloração brancas analisadas. Demostrando que diante da importância da palma forrageira para o semiárido é de suma importância estudos mais detalhados sobre os efeitos dos xenobióticos sobre a microbiota e os insetos associados.

O câncer é a segunda maior causa de morte no Brasil, atrás apenas de doenças cardíacas. Por isto, é evidente que grandes recursos sejam direcionados para a pesquisa no descobrimento de novas opções com a finalidade de erradicar esta doença. Dentre estas opções, a quimioprevenção do câncer tem chamado a atenção já que, mesmo com os imensos avanços no conhecimento sobre os mecanismos da carcinogênese e conseqüente desenvolvimento de novas drogas, os dados estatísticos de mortalidade não se tornaram menores. Somando-se a estes fatos, deve ser considerado que no Brasil o tratamento padrão do câncer não chega a todas as pessoas por ser extremamente caro. Desta forma, a quimioprevenção do câncer com fatores presentes na dieta ou oriundos de fontes consideradas baratas como fitoterápicos, deve ser apreciada. Assim, este trabalho teve como objetivo avaliar os efeitos quimiopreventivos e antineoplásicos de uma planta brasileira, o guaraná (Paullinia cupana Mart var. sorbilis). Foram utilizados alguns experimentos em camundongos como indução genotóxica em fígado pela Dietilnitrosamina (DEN); carcinogênese pulmonar induzida pela 4-(metilnitrosamino)-1-(3-piridil)-1-butanona (NNK), uma nitrosamina presente no tabaco; tumor ascítico de Ehrlich; disseminação hematógena de melanoma B16/f10; e cultivo de células tumorais e não tumorais. Além disso, caracterizou-se o papel da Conexin43 na carcinogênese pulmonar induzida pelo NNK e os efeitos do guaraná sobre o receptor CAR e sua ação quando da administração do ligante do CAR, 1,4-bis[2-(3,5-dichloropiridiloxi)]benzeno (TCPOBOP). Pudemos observar efeitos quimiopreventivos e antineoplásicos do guaraná dependendo do modelo utilizado, demonstrando que seu modo de ação principal é a redução da proliferação celular. Além disso, observamos que os tumores de pulmão dos animais tratados com a planta apresentavam menor tamanho, menor grau maligno, menor índice de proliferação celular e menor ativação do fator de transcrição CREB. Observamos também que a Conexina43 (Cx43) tem importante papel na carcinogênese pulmonar induzida pelo NNK, atuando como supressor tumoral e em fases tardias possivelmente tendo papel inverso, ou seja, como um oncogene. Caracterizamos os efeitos do guaraná sobre a ativação do receptor CAR e demonstramos que, por si só, o guaraná induz a expressão do CAR, além de alterar a expressão de alguns de seus transcritos como a CYP2B10 e CYP3A11. Ao analisarmos os efeitos de extratos de guaraná sobre células de tumor de pulmão (E9) in vitro, verificamos o mesmo efeito antiproliferativo, diminuindo a expressão do PCNA e da Conexina43 de maneira dose-dependente, além de verificar um aumento da expressão do receptor CAR. Ao fim propomos uma hipótese de mecanismo de ação baseando-se nas alterações encontradas oriundas da administração do guaraná. Concluímos que o guaraná apresenta componentes com ação antitumoral em camundongos, tendo efeito quimiopreventivo ou antineoplásico dependendo do modelo utilizado. / Cancer is the second biggest cause of deaths in Brazil, only behind of cardiac diseases. As a result, it is evident that great resources for research will be directed towards the discovery of new options to eradicate this disease. Among these options, cancer chemoprevention has calling for attention since the huge advances in the knowledge of carcinogenesis and development of new drugs did not decrease statistical data on mortality due to cancer. In addition, it must be considered that in Brazil, cancer therapy is not available for all given that it is too expensive. Therefore, cancer chemoprevention with dietary factors or from medicinal plants has got to be treasured. Following these lines, the aim of this work was to evaluate the chemopreventive and antineoplastic effects of a Brazilian plant, Paullinia cupana Mart var. sorbilis, most known as guarana. It was used several experiments in mice and cell culture essays as: protection against DEN-induced DNA damage; NNK-induced lung carcinogenesis; Ehrlich Ascitic Tumor; metastasis of B16/f10 melanoma cells; and cell culture of a tumorigenic and a non-tumorigenic cell lines. Additionally, it was characterized the role of Connexin43 in the NNK-induced lung carcinogenesis and the effects of guarana on the CAR receptor before and after the administration of TCPOBOP. We note a chemopreventive or antineoplastic effect of guarana depending on the model employed and showed that the mode of action responsible for these effects was reduced cell proliferation. Also, the lung tumors of guarana-treated animals were smaller, less aggressive, with decreased cell proliferation and CREB activation. On the other hand, we observed that Connexin43 have an important role on NNK-induced lung carcinogenesis because it may act as a tumor suppressor and in advanced stages as an oncogene. The effects of guarana on the CAR activation were characterized and we showed that guarana induces CAR mRNA expression, altering the levels of its transcripts as CYP2B10 and CYP3A11. We also examined the effects of guarana extracts on a lung tumor cell culture (E9 cells) and demonstrated the same antiproliferative effect observed previously, by decreased PCNA and Connexin43 proteins in a dose-dependent manner along with an increase in CAR protein. At last we hypothesized a mechanism of action for guarana effects basing in our findings. We concluded that guarana presents substances that have antitumoral effects in mice, enclosing a chemopreventive or antineoplastic effect depending on the model studied.

Abstract The evaluation of the effects and levels of contaminants in wildlife is an essential part of assessing risks for chemical exposure in the environment. Although the circumstances are not as controlled as in laboratory, wildlife studies offer the concept of environmental exposure in its entirety, with all the natural variation. In the present study, two wild vole species, bank vole (Myodes glareolus) and field vole (Microtus agrestis), were used in assessing environmental levels of dioxins. The effects of dioxin exposure on tooth and bone development were studied in order to determine whether they could be used as biomarkers for environmental exposure. Xenobiotic metabolism activity after dioxin exposure – both natural and experimental – was studied by quantifying selected cytochrome P-450 (CYP) enzymes. The results confirmed the fact that dioxins are ubiquitous in the environment, also in areas far from contaminant sources and human activity. The development of the third molar in bank vole was found to be a sensitive biomarker for dioxin exposure. The two vole species under study do not respond similarly to environmental concentrations of dioxins; there were significant differences in body burdens and activity levels of xenobiotic metabolizing enzymes. / Tiivistelmä Haitallisten kemikaalien tason ja vaikutusten arviointi ympäristössä on olennainen osa kemikaalien riskin arviointia. Vaikka laboratoriossa olosuhteita kontrolloidaan ja tutkimukseen vaikuttava variaatio on paremmin hallittavissa, luonnonvaraisten lajien tutkiminen luo kokonaisvaltaisen ja todenmukaisen kuvan ympäristön kemikaalialtistuksesta kaikkine todellisine vaihteluineen. Tässä väitöskirjassa tarkastellaan kahden luonnonvaraisen pikkunisäkkään, metsämyyrän (Myodes glareolus) ja peltomyyrän (Microtus agrestis), käyttöä ympäristön kemikaalitason arvioinnissa. Pääpaino on dioksiinien kaltaisissa yhdisteissä. Työssä tutkitaan yhdisteiden kertymistä myyriin kahdessa ympäristössä: voimakkaasti dioksiineilla saastuneella maa-alueella sekä kaukana ihmistoiminnasta sijaitsevassa erämaassa. Herkiksi tiedettyjä vasteita – hampaiden ja luiden kehitystä – käytetään dioksiinialtistuksen indikaattoreina. Vierasainemetaboliasta vastaavien entsyymien (sytokromi P450 eli CYP) aktiivisuutta kartoitetaan molemmilla myyrälajeilla, jotta saadaan tietoa entsyymien indusoinnista luonnonvaraisilla myyrillä yleensä ja selvitetään havaittuja lajien välisiä eroja dioksiinivasteissa. Tulokset vahvistavat, että dioksiinit ovat laajalle levinneitä yhdisteitä, joita löytyy paitsi läheltä päästölähdettä myös kaukana ihmistoiminnasta olevilta alueilta. Metsämyyrällä kolmannen poskihampaan kehitys osoittautuu herkäksi dioksiinialtistuksen biomarkkeriksi. Samasta elinympäristöstä huolimatta tutkituista myyrälajeista mitatut dioksiinipitoisuudet eroavat huomattavasti toisistaan, samoin kuin vierasainemetaboliasta vastaavien entsyymien aktiivisuus ja niiden induktio TCDD-altistuksen jälkeen.

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The overall hypothesis of this work is that the physiological microbial stress response could serve as a rapid, sensitive, and mechanistically-based indicator of process upset in biological wastewater treatment systems that receive sporadic shock loads of toxic chemicals. The microbial stress response is a set of conserved and unique biochemical mechanisms that an organism activates or induces under adverse conditions, specifically for the protection of cellular components or the repair of damaged macromolecules. Using traditional immunochemical analysis techniques, the heat shock protein, GroEL, was found to be induced in activated sludge cultures exposed to perturbations of chemicals at all concentrations tested (cadmium, pentachlorophenol, and acetone) or heat stress. As total cadmium concentrations increased above 5 mg/L, there was a significant and consistent increase in effluent volatile suspended solids concentrations from activated sludge sequencing batch reactors relative to unstressed controls, but there was no additional increase in GroEL levels. Stress proteins may serve as sensitive and rapid indicators of mixed liquor toxicity which can adversely impact treatment process performance, but GroEL may not be a good candidate protein for this purpose due to the lack of a dose/response relationship. Additionally, production of stress proteins did not explain the significant deflocculation upsets that were characteristic of many of the industrially-relevant chemicals tested, including pentachlorophenol and cadmium. Although the purpose of stress response mechanisms is protective at the cellular level, the effect may be disruptive at the macroscopic level in engineered bioreactor systems. The goal of the second research phase was to determine whether the bacterial glutathione-gated, electrophile-induced potassium efflux system is responsible for deflocculation observed due to shock loads of toxic electrophilic (thiol reactive) chemicals. The results indicate significant K+ efflux from the activated sludge floc structure to the bulk liquid in response to shock loads of 1-chloro-2,4-dinitrobenzene (CDNB), N-ethylmaleimide (NEM), 2,4-dinitrotoluene (DNT), 1,4-benzoquinone (BQ), and Cd2+ to a bench-scale sequencing batch reactor (SBR) system. In most cases, the stressor chemicals caused significant deflocculation, as measured by an increase in effluent volatile suspended solids (VSS), at concentrations much less than that required to reduce the maximum specific oxygen uptake rate by 50% (IC50). This suggests that electrophile-induced activated sludge deflocculation is caused by a protective bacterial stress mechanism (as hypothesized) and that the upset event may not be detectable by aerobic respirometry. More importantly, the amount of K+ efflux appeared to correlate well with the degree of deflocculation. The transport of other cations including sodium, calcium, magnesium, iron, and aluminum, either to or from the floc structure, was negligible as compared to K+ efflux. In bench-scale SBRs, it was also determined that the K+ efflux occurred immediately (within minutes) after toxin addition and then was followed by an increase in effluent turbidity. K+ efflux and deflocculation responses were similar for bench-scale SBRs and continuous-flow reactor systems, indicating that the periods of elevated exogenous substrate levels typical in SBR systems are not required to activate electrophile-induced K+ efflux or deflocculation. This also suggests that the initial and rapid efflux of K+ immediately following electrophile addition is the factor that leads to deflocculation, not the increase in bulk liquid K+. Sphingomonas capsulata, a bacterium consistent with that found in biological wastewater treatment systems, Escherichia coli K-12, and activated sludge cultures exhibited very similar dynamic efflux/uptake/efflux responses due to the electrophilic stressors, NEM and CDNB, and the thiol reducing agent, dithiothreitol (DTT). The polyether ionophore antibiotic, nigericin, was used to artificially stimulate K+ efflux from S. capsulata and activated sludge cultures. Thus, glutathione-gated K+ efflux (GGKE) activity may cause K+ release from the cytoplasm of activated sludge bacteria into the floc structure and extracellular polymeric substances (EPS) and then diffusion-limited transport into the bulk liquid. It was not possible to resolve the effect of the GGKE system on changes in bulk liquid or floc-associated pH. However, calculations indicate that the localized K+ concentration within the floc structure immediately after chemical stress is consistent with that known to induce floc disruption as a result of KCl addition. Using alkaline phosphatase as a periplasmic marker as well as fluorescent membrane-permeable and impermeable nucleic acid stains, it was determined that a negligible amount of the K+ efflux response was due to lysis of activated sludge microorganisms. The current results are very promising and are the first to suggest that activated sludge upset (i.e. deflocculation) may be caused by a specific protective stress response in bacteria. / Ph. D.