Submitted by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-06-01T14:56:39Z No. of bitstreams: 1 DIS_MAILA_PACHECO_DIAS_COMPLETO.pdf: 1237790 bytes, checksum: 5f0ce43f31e6bb7c9d9d5d922daa8668 (MD5) / Made available in DSpace on 2017-06-01T14:56:39Z (GMT). No. of bitstreams: 1 DIS_MAILA_PACHECO_DIAS_COMPLETO.pdf: 1237790 bytes, checksum: 5f0ce43f31e6bb7c9d9d5d922daa8668 (MD5) Previous issue date: 2016-03-29 / Solanum lycopersicum L., tomato, is an herbaceous plant belonging to the Solanaceae family. Its fruits are consumed worldwide, reaching the world production of 160 million tons per year. In Brazil, it is the second vegetable in economic importance. However, the tomato is attacked by numerous diseases that lead to loss of production and /or poor quality of the fruit, such as the hollow stem, the disease caused by Pectobacterium spp. Due to the large number of diseases caused by plant pathogens, the tomato is a culture in which a significant amount of agrochemicals is used. Therefore, this species is among the vegetables with the greatest amount of residual pesticides. Under these circumstances, it is essential to develop sustainable plant defense techniques in order to reduce the use of agrochemicals. Then, changes in plant metabolism related to defense must be understood so that new strategies and new products can be developed. Disease control using soil microorganisms has been considered as an alternative, since the rhizobacteria, in addition to promoting plant growth, may induce resistance as the result of activation of the natural plant defenses. These, calls plant growth promoting rhizobacteria (PGPR), has been explored for their biofertilizers, biopesticides and phyto-stimulating abilities. The aims of this study were to characterize biochemically the Streptomyces spp. isolates, to determine the antagonism against Pectobacterium carotovorum subsp. brasiliensis (Pcb), to determine the ability of Streptomyces spp. on promoting growth of tomato plants and to evaluate the modulation of the defense-related metabolism of tomato plants when treated with Streptomyces spp. The possible influence of Streptomyces spp. on reducing soft rot disease in tomato plants was also evaluated. Biochemical characterization was evaluated through the ability of Streptomyces spp. on producing siderophores, solubilizing phosphate, and activity of amylase and lipase, as well as volatile organic compounds (VOC) production. Antagonism of Streptomyces spp. against Pcb was determined by dual-culture method and I-plate for VOC effect analysis. Plant growth promotion was evaluated through VOC emission and by direct interaction with Streptomyces spp. isolates (PM1, PM3, PM4, PM5, PM6 e PM9). Enzymes related to plant defense were colorimetric analyzed in plants treated with isolates of Streptomyces spp. Evaluation of soft rot disease was performed on plants treated with Streptomyces spp. and challenged with Pcb through the area under the disease progression curve (AUDPC) and plant mortality. Isolates of Streptomyces spp. displayed characteristics of PGPR and 32 volatile compounds were identified from the different isolates. PM3 was the isolate showing efficient antagonism against Pcb. Most of the isolates promoted increase of root and shoot length of tomato plants by VOC although PM5 was efficient on promoting growth by direct interaction with Streptomyces spp. Treatment with Streptomyces spp. modulated the activity of defense-related enzymes and decrease incidence of soft rot disease. / O tomateiro (Solanum lycopersicum L.) ? uma planta herb?cea pertencente ? fam?lia Solanaceae. Seus frutos s?o consumidos mundialmente, chegando ? produ??o mundial de 160 milh?es de toneladas por ano. No Brasil, ? a segunda hortali?a em import?ncia econ?mica. Contudo, o tomateiro ? alvo de in?meras doen?as que levam ? perda de produ??o e/ou m? qualidade dos frutos, como por exemplo, a doen?a Talo oco causada por Pectobacterium spp. Devido ao elevado n?mero de doen?as causadas por fitopat?genos, o tomateiro ? uma cultura onde se utiliza uma quantidade expressiva de agroqu?micos, estando entre as hortali?as que apresentam maior quantidade de agrot?xicos residuais. Por isso, torna-se imprescind?vel o desenvolvimento de t?cnicas sustent?veis de defesa para o vegetal, a fim de reduzir o uso destes compostos. Para este fim, ? fundamental compreender as altera??es no metabolismo vegetal relacionado ? defesa, para que novas estrat?gias e novos produtos agr?colas possam ser desenvolvidos. O controle de doen?as utilizando microrganismos de solo tem sido considerado uma alternativa, uma vez que as rizobact?rias, al?m de promoverem o crescimento vegetal, podem induzir ? resist?ncia como consequ?ncia da ativa??o da defesa vegetal. Estas, chamadas rizobact?rias promotoras de crescimento vegetal (PGPR), v?m sendo exploradas quanto ? capacidade biofertilizante, fito-estimuladora e biopesticida. Os objetivos deste estudo foram caracterizar seis isolados de Streptomyces spp. como PGPR, determinar o antagonismo contra Pectobacterium carotovorum subsp. brasiliensis (Pcb), determinar a capacidade de isolados de Streptomyces spp. na promo??o do crescimento de plantas de tomate e avaliar a modula??o do metabolismo relacionado ? defesa das plantas de tomate quando tratadas com Streptomyces spp. A poss?vel influ?ncia de Streptomyces spp. na redu??o da doen?a Talo oco em plantas de tomate tamb?m foi avaliada. A caracteriza??o bioqu?mica de isolados de Streptomyces spp. foi realizada por meio da capacidade de produzir sider?foros, solubilizar fosfato, e da atividade de amilase e lipase, bem como a produ??o de compostos org?nicos vol?teis. O antagonismo de Streptomyces spp. contra Pcb foi determinado pelo m?todo de dupla cultura e placa com barreira para an?lise do efeito de compostos org?nicos vol?teis (VOC). A promo??o do crescimento das plantas foi avaliada por meio de emiss?o de VOC e pela intera??o direta com os isolados de Streptomyces spp. (PM1, PM3, PM4, PM5, PM6 e PM9). Enzimas relacionadas ? resposta de defesa foram analisadas colorimetricamente em plantas tratadas com isolados de Streptomyces spp. A avalia??o da doen?a Talo oco foi realizada em plantas tratadas com Streptomyces spp. e desafiadas com Pcb atrav?s da ?rea sob a curva de progresso da doen?a e da mortalidade das plantas em 24 dias. Os isolados de Streptomyces spp. mostraram caracter?sticas de PGPR e 32 compostos vol?teis foram identificados como produtos dos diferentes isolados. PM3 foi o isolado mais eficiente quanto ao antagonismo contra Pcb. A maioria dos isolados promoveu o aumento do comprimento de raiz e da parte a?rea do tomateiro por VOC, embora PM5 tenha sido tamb?m eficiente na promo??o do crescimento atrav?s da intera??o direta com Streptomyces spp. O tratamento com Streptomyces spp. modulou a atividade de enzimas relacionadas ? defesa e diminuiu a incid?ncia da doen?a Talo oco.

Micro-organismos são profícuos produtores de produtos naturais bioativos. Diversos fármacos inovativos e de extrema importância clínica são de origem microbiana, como antibióticos, antitumorais, imunossupressores e agentes hipolipêmicos. A maioria dos fármacos de origem microbiana é produzida por actinobactérias. A estreita associação de micro-organismos com plantas, insetos, crustáceos e organismos marinhos, pode induzir a produção de substâncias bioativas, que eventualmente conferem vantagens adaptativas e ecológicas ao macro-organismo hospedeiro. Ambientes marinhos e áreas de transição como os manguezais são ainda pouco explorados para estudos químicos e farmacológicos dos micro-organismos presentes nos solos e em simbiose com outros hospedeiros. Neste sentido, o presente trabalho visou o estudo químico monitorado por ensaios de citotoxicidade em células cancerígenas de dois micro-organismos, Streptomyces sp. Act1 e Streptomyces sp. Act2, isolados da casca do caranguejo do maguezal, Ucides cordatus, coletado em Cananéia, SP. Estes micro-organismos apresentaram atividade antagônica frente ao fungo patogênico Aspergillus niger, confirmando o potencial para produção de substâncias bioativas. Os micro-organismos foram cultivados em diferentes meios de cultura para avaliar o potencial de crescimento, perfil químico dos extratos obtidos e atividade citotóxica dos extratos e subfrações. Os meios de cultura escolhidos foram meio líquido ISP-2 e meio sólido (arroz), e ainda, nestas mesmas condições foi avaliado o efeito do uso de água do mar de forma comparativa. Os micro-organismos desenvolveram-se adequadamente nos diferentes meios de cultivo. Os fracionamentos dos extratos obtidos nos experimentos evidenciaram características polares das subfrações obtidas, e neste sentido foram necessárias adaptações das técnicas de separação, incluindo fases estácionárias como sephadex LH-20 e fase reversa. Os ensaios de citotoxicidade frente às linhagens de células tumorais com os extratos obtidos mostraram o grande potencial das linhagens em estudo, em especial a linhagem Streptomyces sp. Act-2 cultivada na presença de água do mar. / Abstract Microorganisms are prolific producers of bioactive natural products. Most of innovative pharmaceuticals that have useful clinical importance have been isolated from from microorganisms, such as antibiotics, anticancer drugs, immunosuppressive drugs and lipid-lowering agents. Most of the drugs of microbial origin are produced by actinomycetes. The close association of micro-organisms with plants, insects, crustaceans or marine organisms may induce the production of bioactive substances. These natural products may confer adaptive advantages to the environment and to the host organism. Marine and transitional areas such as mangroves are still under explored for chemical and pharmacological studies of both soil microorganisms and symbiotic microorganisms. In this sense, this work aimed a chemical study of two actinobacteria strains, Streptomyces sp. Act1 and Streptomyces sp. Act2, guided by cancer cells cytotoxicity assays. Both microorganisms were isolated from the shell of the mangrove crab Ucides cordatus, collected in Cananéia, SP. These microorganisms showed antagonistic activity against the pathogenic fungus Aspergillus niger, confirming the potential for production of bioactive substances. Microorganisms were cultured in different culture media to assess the potential for growth, chemical profile of extracts and cytotoxic activity of extracts and subfractions. The culture media were chosen using liquid (ISP-2) and solid medium (rice). The effect of addition of saline water in the culture media has also been evaluated. The microorganisms developed adequately in different culture media. The fractions of extracts obtained from the experiments showed characteristics of polar subfractions obtained, requiring the use of sephadex LH-20 and reverse phase as chromatographic stationary phases. Cytotoxic assays of the extracts against cancer cell lines showed the great potential of the strains under study, mainly Streptomyces sp. Act2. grown in the presence of seawater.

Endophytic microorganisms were screened for hemicellulases production using plate assays and liquid cultivations. Two strains were selected and used in further studies. Aspergillus niger DR02 strain and Annulohypoxylon stigyum DR47. In A.niger fed-batch submerged cultivation approaches were developed using liquor from hydrothermal sugar cane pretreatment, and maximum xylanase activities obtained were 458.1 U/mL for constant fed-batch mode. For A. stygium DR47 media optimization and bioreactor cultivation using citrus bagasse and soybean bran were explored and revealed a maximum production of 6.26 U/mL of pectinase and 10.13 U/mL of β-glucosidase. Improved hemicellulase production was also done by genetic modifications at carbon catabolic repression levels in A. niger. Mass spectrometric studies were done in enzymatic extracts produced and contributed to understand the enzymatic supplementation of Celluclast 1.5L.In addition the extracts produced were tested for an enzyme formulation and that was able to increase sugar cane bagasse hydrolysis. / Microrganismos endofíticos foram selecionados para a produção de hemicelulases através de testes em placa e de cultivo líquido. Duas linhagens foram selecionadas para posteriores cultivos, Aspergillus niger DR02 e Annulohypoxylon stigyum DR47. Para o fungo A.niger batelada alimentada em cultivo submerso com licor do tratamento hidrotérmico do bagaço de cana de açúcar permitiram obtenção de máxima atividade de xilanase de 458,1 U/mL. Para a linhagem A. stygium DR47 otimização do cultivo utilizando bagaço cítrico e farelo de soja foram explorados e revelaram produção de 6,26 U/mL de pectinase e 10,13 U/mL de β-glucosidase. Melhoramento da produção de hemicelulase foi realizado com modificações genéticas ao nível de repressão catabólica de carbono em A. niger. Estudos de espectrometria de massas foram realizados nos extratos enzimáticos produzidos e contribuíram para entender a suplementação da Celluclast 1.5L. Além disso, os extratos produzidos foram testados para uma formulação que foi capaz de aumentar a hidrólise do bagaço de cana de açúcar.

O agroresíduo mais importante gerado no Brasil é o bagaço da cana de açúcar e seu excedente pode ser utilizado para a produção de etanol celulósico. O objetivo desse trabalho foi bioprospectar e identificar genes que codificam para enzimas lignocelulósicas. Para tanto, aproximadamente 42 linhagens de actinomicetos endofíticos e de solo, passaram por dois tipos de triagem (meio sólido e molecular). Como resultado, a maioria das linhagens apresentou bom desempenho em teste em meio sólido. Nenhuma linhagem foi capaz de hidrolisar igualmente todos os substratos testados. Na triagem molecular, os resultados indicam que os genes para as enzimas estão presentes. Como conclusão pode-se considerar que os actinomicetos são potencialmente bons produtores de enzimas lignocelulolíticas, O material biológico é relativamente pouco estudado e não foram encontrados dados na literatura sobre o tema, mas são interessantes para estudos de obtenção de bioetanol e outros segmentos biotecnológicos. / The most important agro waste generated from Brazil\'s sugarcane bagasse and its surplus can be used to produce cellulose ethanol. The aim of this study was bioprospected and identify genes encoding for lignocellulosic enzymes. Therefore, a batch of about 42 strains of endophytic actinomycetes and soil, passed through two types of screening (soild medium and molecular). The results demonstrated that the majority of the analysed strains presented good results when tested on solid medium. The strains presented better results for a restricted number of substrates, but no strain was able to hydrolyse all substrates. With regards to the results of molecular screening, strains had amplicons, indicating that the genes for the enzymes are present. As a conclusion, it can be considered that the actinomycetes are potentially good producers of lignocellulolytic enzymes. The biological material is relatively poor studied and no literature data e regarding the subject were found, but they can be considered appealing to be studied in order to obtain bioethanol and other biotech segments.

Actinomycetes and fungi were successfully isolated from both New Zealand and Malaysian marine invertebrates and classified as facultatively marine based on their ability to grow on both sea water and non-sea water media. Most of the extracts obtained from selected isolates were cytotoxic. A clear preference of the actinomycetes for solid-state fermentation was observed, however, for fungi no significant preference was seen. Three isolates of Streptomyces spp., four Penicillium spp. and two Paecilomyces spp. whose extracts showed good cytotoxicity were selected for further investigation. A small-scale extract obtained from a solid culture of Streptomyces sp. (LA3L2) showed good cytotoxicity and a new cytotoxic metabolite was isolated from a large-scale extract of Streptomyces sp. (LA3L2). This metabolite was characterized as S-methyl 2,4-dihydroxy-6-isopropyl-3,5-dimethylbenzothioate (5.15) and is only the third compound reported to contain the S-methyl benzothioate group. Two known compounds, montagnetol (5.16) and erythrin (5.18), were isolated from a further large-scale cultivation of Streptomyces sp. (LA3L2) and is the first reported actinomycete to produce these lichen-related compounds. In addition, two known inactive metabolites (bohemamine (5.1) and bohemamine B (5.2)) were identified from the small-scale extract. Streptomyces sp. (LA3L2) was also investigated for the effect of temperature and salinity on growth and cytotoxicity and shown to produce bohemamine only at 20 - 28℃ and 4% sea salt concentration on solid media. This isolate gave a low yield of active metabolite under all conditions. Small-scale extracts of two other Streptomyces spp. yielded three known cytotoxic metabolites. These were thiazostatin B (7.14) from Streptomyces sp. (LA5L4) and chromomycin A2 (7.1), chromomycin A3 (7.2) and chromomycin 02-3D (7.3) from Streptomyces sp. (LA3L1). All four Penicillium spp. produced known metabolites. Penicillium sp. (LY1L5) yielded two known metabolites, cycloaspeptide A (7.4) and α-cyclopiazonic acid (7.5). α-Cyclopiazonic acid (7.5) and three other known metabolites (roquefortine A (7.6), cyclopeptin (7.7) and viridicatin (7.8)) were isolated from Penicillum sp. (KK3T23). Penicillium sp. (KK3T8) produced brefeldin A (7.10), while mycophenolic acid (7.12) and brevianamide A (7.11) were produced by Penicillium sp. (KK4T14b). The effect of salinity on growth and cytotoxicity was investigated for the two Penicillium isolates producing the cytotoxic metabolite, α-cyclopiazonic acid (7.5). Saline conditions were not required for growth but metabolite production differed between the two isolates with respect to salinity. Isolate LY1L5 required saline conditions for α-cyclopiazonic production whereas isolate KK3T23 produced the metabolite under non-saline conditions and in concentrations of sea salt up to 6%. Three known compounds, indole-3-carboxylic acid (7.15), indole-3-carboxylate (7.17) and 5-carboxymellein (7.16) were identified from Paecilomyces sp. (PR5L9). Investigation of a small-scale extract obtained from a solid culture of another Paecilomyces sp. (PR10T2) resulted in the isolation and characterization of a unique structure of a symmetrical cyclic depsipeptide, epi-angolide (NAM 6-1). NAM 6-1 was considered as a new compound based on four homoisomeric configurations (A1, A2, A3 and A4). The value of dereplication procedures with respect to the rapid identification of metabolites and enhancement of in-house metabolite libraries is discussed. Structural elucidation of nine known metabolites (7.1, 7.2, 7.3, 7.5, 7.6, 7.7, 7.8, 7.10 and 7.11) was greatly aided by the in-house dereplication techniques using LC-MS-UV and AntiMarin database. A significant advantage was gained by the use of the CapNMR which enabled NMR characterization of very small quantities of metabolites (<20 µg). Approximately <5 µg of materials were required to perform 1D proton NMR experiments for the dereplication of seven known compounds; bohemamine (5.1), bohemamine B (5.2), thiazostatin B (7.14), indole-3-carboxylate (7.17) and 5-carboxymellein (7.16). Approximately 20 µg of materials were needed to acquire 1D and 2D (HSQC, HMBC and NOE) NMR spectra for structural elucidation of the new metabolite, S-methyl 2,4-dihydroxy-6-isopropyl-3,5-dimethylbenzothioate (5.15). Some 8 µg of materials were sufficient to perform 1D and 2D (COSY, HSQC and HMBC) NMR experiments for complete structural characterization of two known metabolites, montagnetol (5.16) and erythrin (5.18). Approximately 10 µg of materials were needed to acquire 1D and 2D NMR (COSY, HSQC and HMBC) experiments for structural elucidation of the new compound, epi-angolide NAM 6-1 (A1, A2, A3 and A4). Rapid identification of known fungal metabolites enabled the in-house HPLC-UV/Rt library to be enhanced by eight metabolites (7.5, 7.6, 7.7, 7.8, 7.10, 7.11, 7.17 and 7.16). An HPLC-UV/Rt library for actinomycete metabolites was successfully established with the insertion of eight known metabolites (5.1, 5.2, 5.16, 5.18, 7.1, 7.2, 7.3 and 7.14).

Submitted by (lucia.rodrigues@ufrpe.br) on 2016-06-09T11:49:13Z No. of bitstreams: 1 Marcia Nieves Carneiro da Cunha.pdf: 873639 bytes, checksum: 110419134a2cc87e1cbcfafbb7b86801 (MD5) / Made available in DSpace on 2016-06-09T11:49:13Z (GMT). No. of bitstreams: 1 Marcia Nieves Carneiro da Cunha.pdf: 873639 bytes, checksum: 110419134a2cc87e1cbcfafbb7b86801 (MD5) Previous issue date: 2010-02-25 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Bovine Mastitis is one of the main diseases that affect the dairy herds in the world and is considered one of the greatest obstacles to their operation profitable. The greatest losses from mastitis are due to the decrease in milk production, depreciation in nutritional quality, treatment cost, cost of veterinary care and laboratory losses in genetic potential. Several microorganisms are cited as agents of bovine mastitis and the species most frequently isolated are Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis and Escherichia coli. The β-lactam antibiotics are important antibacterial agents, but sensitive to β-lactamase enzymes capable of cleaving the β-lactam ring, rendering them inactive. Inhibitors of β-lactamases are an alternative for diseases treatment caused by resistant-penicillin bacteria. This study evaluated the ability to produce β-lactamase inhibitors by 71 actinomycetes isolated from lichens of the Amazonian and its antimicrobial activity using Staphylococcus aureus, penicillin-resistant isolates of bovine mastitis. The selection of actinomycetes that produced β-lactamases inhibitors was performed using the agar block technique against Klebsiella pneumoniae ATCC 29665 and selected actinomycetes were tested against Staphylococcus aureus penicillin resistant. The best producers of β-lactamase inhibitors were Streptomyces sp. DPUA 1542 and Nocardia sp DPUA 1571, which were subjected to submerged cultivation in the study of the growth curve, pH and antimicrobial activity. The largest inhibition zones using S. aureus, were obtained after 96 hours of culture for both Nocardia sp (13.5 and 12.0 mm) and for Streptomyces sp. (8.0 and 14.0 mm) with diffusion-tests on the discs and wells, respectively. Nocardia sp DPUA 1571 was used in the production of inhibitors of β-lactamases by factorial design where the influence of three variables: concentrations of carbon source (0.5, 1.0 and 1.0%), concentrations of nitrogen source (1.0, 2.5 and 4.0%) and pH (6.0, 6.5 and 7.0) were evaluated. The production carried out in 250mL flasks containing 100mL of culture medium MS-2 modified according to the experimental design, the flasks were incubated in an orbital shaker (200 rpm) at 28 ° C for 120 hours and each 24 hours, aliquots of 5 mL were withdrawn and centrifuged at 5000 rpm for 20 minutes at 4 ° C for the study of biological activity and dry weight. The biological activity of the liquid assay was performed using the diffusion-tests on the discs and wells, the best cultive conditions (test 3) with concentration of carbon source of 1.0% concentration of the soy bean flour 1% and pH 6,0, were obtained from the inhibition of up to 22mm diameter test against microorganisms used. The results showed that the actinomycetes are a promising source of β-lactamase inhibitors, demonstrated the potential application to pharmaceutical industry. / A mastite bovina é uma das principais enfermidades que acometem os rebanhos leiteiros no mundo, sendo considerada um obstáculo à sua exploração lucrativa. As maiores perdas causadas pela mastite são devidas à diminuição na produção de leite, depreciação na qualidade nutritiva, custo de tratamento, custo de atendimento veterinário e laboratorial e perdas no potencial genético. Vários microorganismos são citados como agentes etiológicos da mastite bovina sendo que as espécies mais frequentemente isoladas são Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis e Escherichia coli. Os antibióticos β-lactâmicos são importantes agentes antibacterianos, porém sensíveis às enzimas β-lactamases, capazes de clivar o anel β-lactâmico, tornando-os inativos. Os inibidores de β-lactamases são uma alternativa para o tratamento de doenças causadas por bactérias resitentes à penicilina. Neste estudo foi avaliada a capacidade de produzir inibidores de β-lactamases por 71 actinomicetos isolados de liquens da Região Amazônica e sua atividade antimicrobiana utilizando Staphylococcus aureus, resistentes à penicilina, isolados de mastite bovina. A seleção dos actinomicetos que produziram inibidores de β-lactamases foi realizada através da técnica de bloco de gelose contra Klebsiella pneumoniae ATCC 29665 e os actinomicetos selecionados foram testados contra Staphylococcus aureus resistentes. Os melhores produtores de inibidores de β-lactamases foram Streptomyces sp. DPUA 1542 e Nocardia sp DPUA 1571, os quais foram submetidos a cultivo submerso para o estudo da curva de crescimento, pH e atividade antimicrobiana. Os maiores halos de inibição, utilizando S. aureus, foram obtidos após 96 horas de cultivo tanto para Nocardia sp (13,5 e 12,0 mm) como para Streptomyces sp. (8,0 e 14,0 mm) com os testes-difusão nos discos e poços, respectivamente. Nocardia sp DPUA 1571 foi utilizado na produção de inibidores de β-lactamases por planejamento fatorial onde a influencia de três variáveis foi avaliada: concentrações da fonte de carbono (0,5, 1,0 e 1,0%), concentrações de fonte de nitrogênio (1,0, 2,5 e 4,0%) e pH (6,0, 6,5 e 7,0). A produção ocorreu em Erlenmeyers de 250mL contendo 100mL do meio de cultivo MS-2 modificado de acordo o modelo experimental, os frascos foram incubados em agitador orbital (200 rpm) a 28°C por um período de 120 horas e a cada 24 horas, alíquotas de 5mL foram retiradas e centrifugadas a 5.000 rpm por 20 minutos a 4ºC, para a determinação da atividade biológica e peso seco. A atividade biológica do líquido metabólico foi realizada através da técnica de difusão em disco e poço, nas melhores condições de cultivo (ensaio 3) com concentração da fonte de carbono de 1,0%, concentração do filtrado de soja de 1% e o pH 6,0, foram obtidos halos de inibição de até 22mm de diâmetro frente aos microrganismos teste utilizados. Os resultados permitiram concluir, que os actinomicetos isolados de liquens da Região Amazônica são uma fonte promissora de inibidores de β-lactamases, com potencial para aplicação futura na indústria farmacêutica.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2016-10-06T10:55:44Z No. of bitstreams: 2 Dissertação - Aline Rodrigues Gama - 2016.pdf: 2281415 bytes, checksum: a79af0203dd2a1cfd381c59e3e9d6d7b (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2016-10-06T11:22:35Z (GMT) No. of bitstreams: 2 Dissertação - Aline Rodrigues Gama - 2016.pdf: 2281415 bytes, checksum: a79af0203dd2a1cfd381c59e3e9d6d7b (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2016-10-06T11:22:35Z (GMT). No. of bitstreams: 2 Dissertação - Aline Rodrigues Gama - 2016.pdf: 2281415 bytes, checksum: a79af0203dd2a1cfd381c59e3e9d6d7b (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-09-14 / The use of carbohydrates derived from agricultural waste is important in the industry of food, textiles, paper, detergents, animal feed and in the production of bioethanol. Some filamentous bacteria are used in the enzymatic degradation processes, such as Streptomyces thermocerradoensis I3, which was isolated from soil and this work was selected for the production of cellulases and xylanases by growing in semi-solid fermentation (SSF) in medium supplemented with wheat bran (WB) as carbon source. S. thermocerradoensis I3 it was maintained for 4 days at 37 ° C and showed a higher production of Endoglucanase (2,92 U/mL) and Xylanase (12,34 U/mL) after 72 hours of cultivation and β-glucosidase (0,023 U/mL) and FPase (2,82 U/mL) after 96 hours of cultivation. The 96-hour extract was concentrated. The enzyme present in the concentrated extract presented molecular mass of 17 kDa. It showed activity for cellulase and xylanases confirmed by zimograms and enzymatic activities. The crude extract (EB) and the concentrated extract (EC) were analyzed for pH and temperature optima for activity of cellulases and xylanases. The results showed that the highest activity of the total cellulase (FPase) was at pH 6.0 at 60 °C (EB) and pH in the range 3.0 to 6.0 at 80 °C (EC); the endoglucanase has higher activity at pH 6.0 at 55 °C (EB and EC); xylanase showed higher activity at pH 8.0 at 70 °C (EB and EC). The xylanase activity of EB and EC showed thermostability 60% after 2 hours of incubation at 60 °C and the endoglucanase activity of EB and EC remained above 50% after 4 hours of incubation at 50 °C, 60 °C and 70 °C. Qualitative analysis observed by TLC (Thin layer chromatography) showed the production of oligosaccharides and xilooligômeros from the hydrolysis of different substrates. S. thermocerradoensis I3 successfully used the WB in SSF producing enzymes that have the characteristics necessary for their industrial application. / A utilização de carboidratos oriundos dos resíduos agrícolas faz-se importante nas indústrias de alimentos, tecidos, papeis, detergentes, ração animal e ainda na produção de bioetanol. Algumas bactérias filamentosas são utilizadas nos processos de hidrólise enzimática, como Streptomyces thermocerradoensis I3, o qual foi isolado do solo e, neste trabalho, selecionado para produção de celulases e xilanases através de cultivo em fermentação semi-sólida (FSS) em Meio Mínimo suplementado com farelo de trigo (FT) como fonte de carbono. S. thermocerradoensis I3 foi cultivado por 4 dias à 37 °C e apresentou maior produção de Endoglucanase (2,92 U/mL) e Xilanase (12,34 U/mL) após 72 horas de cultivo e de β-glicosidase (0,023 U/mL) e FPase (2,82 U/mL) após 96 horas de cultivo. O extrato de 96 horas foi concentrado e a enzima presente no extrato concentrado apresentou Massa Molecular de 17 kDa. Ela apresentou atividade de celulase e xilanase, as quais foram confirmadas por zimogramas e determinação das atividades enzimáticas. O extrato bruto (EB) e o extrato concentrado (EC) foram analisados quanto ao pH e temperatura ótimos para a atividade de celulases e xilanase. Os resultados obtidos demonstraram que a maior atividade de celulases totais (FPase) foi em pH 6,0 à 60 °C (EB) e pH na faixa de 3,0 e 6,0 à 80 °C (EC); a enduglucanase apresentou maior atividade na faixa de pH 6,0 à 55 °C (EB e EC); a xilanase apresentou maior atividade em pH 8,0 à 70 °C (EB e EC). A atividade de xilanase do EB e da EC apresentou termoestabilidade de 60% após 2 horas de incubação à 60 °C e a atividade de endoglucanase do EB e do EC permaneceu acima de 50% após 4 horas de incubação à 50, 60 e 70 °C. As análises qualitativas observadas por TLC (Thin Layer Chromatography) revelaram a liberação de oligossacarídeos e xilooligômeros a partir da hidrólise de diferentes substratos. S. thermocerradoensis I3 utilizou com sucesso o FT em FSS produzindo enzimas que apresentam as características necessárias para sua aplicação industrial.

O agroresíduo mais importante gerado no Brasil é o bagaço da cana de açúcar e seu excedente pode ser utilizado para a produção de etanol celulósico. O objetivo desse trabalho foi bioprospectar e identificar genes que codificam para enzimas lignocelulósicas. Para tanto, aproximadamente 42 linhagens de actinomicetos endofíticos e de solo, passaram por dois tipos de triagem (meio sólido e molecular). Como resultado, a maioria das linhagens apresentou bom desempenho em teste em meio sólido. Nenhuma linhagem foi capaz de hidrolisar igualmente todos os substratos testados. Na triagem molecular, os resultados indicam que os genes para as enzimas estão presentes. Como conclusão pode-se considerar que os actinomicetos são potencialmente bons produtores de enzimas lignocelulolíticas, O material biológico é relativamente pouco estudado e não foram encontrados dados na literatura sobre o tema, mas são interessantes para estudos de obtenção de bioetanol e outros segmentos biotecnológicos. / The most important agro waste generated from Brazil\'s sugarcane bagasse and its surplus can be used to produce cellulose ethanol. The aim of this study was bioprospected and identify genes encoding for lignocellulosic enzymes. Therefore, a batch of about 42 strains of endophytic actinomycetes and soil, passed through two types of screening (soild medium and molecular). The results demonstrated that the majority of the analysed strains presented good results when tested on solid medium. The strains presented better results for a restricted number of substrates, but no strain was able to hydrolyse all substrates. With regards to the results of molecular screening, strains had amplicons, indicating that the genes for the enzymes are present. As a conclusion, it can be considered that the actinomycetes are potentially good producers of lignocellulolytic enzymes. The biological material is relatively poor studied and no literature data e regarding the subject were found, but they can be considered appealing to be studied in order to obtain bioethanol and other biotech segments.

Micro-organismos são profícuos produtores de produtos naturais bioativos. Diversos fármacos inovativos e de extrema importância clínica são de origem microbiana, como antibióticos, antitumorais, imunossupressores e agentes hipolipêmicos. A maioria dos fármacos de origem microbiana é produzida por actinobactérias. A estreita associação de micro-organismos com plantas, insetos, crustáceos e organismos marinhos, pode induzir a produção de substâncias bioativas, que eventualmente conferem vantagens adaptativas e ecológicas ao macro-organismo hospedeiro. Ambientes marinhos e áreas de transição como os manguezais são ainda pouco explorados para estudos químicos e farmacológicos dos micro-organismos presentes nos solos e em simbiose com outros hospedeiros. Neste sentido, o presente trabalho visou o estudo químico monitorado por ensaios de citotoxicidade em células cancerígenas de dois micro-organismos, Streptomyces sp. Act1 e Streptomyces sp. Act2, isolados da casca do caranguejo do maguezal, Ucides cordatus, coletado em Cananéia, SP. Estes micro-organismos apresentaram atividade antagônica frente ao fungo patogênico Aspergillus niger, confirmando o potencial para produção de substâncias bioativas. Os micro-organismos foram cultivados em diferentes meios de cultura para avaliar o potencial de crescimento, perfil químico dos extratos obtidos e atividade citotóxica dos extratos e subfrações. Os meios de cultura escolhidos foram meio líquido ISP-2 e meio sólido (arroz), e ainda, nestas mesmas condições foi avaliado o efeito do uso de água do mar de forma comparativa. Os micro-organismos desenvolveram-se adequadamente nos diferentes meios de cultivo. Os fracionamentos dos extratos obtidos nos experimentos evidenciaram características polares das subfrações obtidas, e neste sentido foram necessárias adaptações das técnicas de separação, incluindo fases estácionárias como sephadex LH-20 e fase reversa. Os ensaios de citotoxicidade frente às linhagens de células tumorais com os extratos obtidos mostraram o grande potencial das linhagens em estudo, em especial a linhagem Streptomyces sp. Act-2 cultivada na presença de água do mar. / Abstract Microorganisms are prolific producers of bioactive natural products. Most of innovative pharmaceuticals that have useful clinical importance have been isolated from from microorganisms, such as antibiotics, anticancer drugs, immunosuppressive drugs and lipid-lowering agents. Most of the drugs of microbial origin are produced by actinomycetes. The close association of micro-organisms with plants, insects, crustaceans or marine organisms may induce the production of bioactive substances. These natural products may confer adaptive advantages to the environment and to the host organism. Marine and transitional areas such as mangroves are still under explored for chemical and pharmacological studies of both soil microorganisms and symbiotic microorganisms. In this sense, this work aimed a chemical study of two actinobacteria strains, Streptomyces sp. Act1 and Streptomyces sp. Act2, guided by cancer cells cytotoxicity assays. Both microorganisms were isolated from the shell of the mangrove crab Ucides cordatus, collected in Cananéia, SP. These microorganisms showed antagonistic activity against the pathogenic fungus Aspergillus niger, confirming the potential for production of bioactive substances. Microorganisms were cultured in different culture media to assess the potential for growth, chemical profile of extracts and cytotoxic activity of extracts and subfractions. The culture media were chosen using liquid (ISP-2) and solid medium (rice). The effect of addition of saline water in the culture media has also been evaluated. The microorganisms developed adequately in different culture media. The fractions of extracts obtained from the experiments showed characteristics of polar subfractions obtained, requiring the use of sephadex LH-20 and reverse phase as chromatographic stationary phases. Cytotoxic assays of the extracts against cancer cell lines showed the great potential of the strains under study, mainly Streptomyces sp. Act2. grown in the presence of seawater.

A utilização de técnicas moleculares têm facilitado o estudo de comunidades bacterianas complexas no ambiente. O presente trabalho teve como objetivo utilizar a técnica DGGE para avaliação dos efeitos do aumento da temperatura, causado pela queima de um canavial, na estrutura da comunidade bacteriana de solo, com ênfase ao grupo dos actinomicetos. Foram coletadas amostras de solo em diferentes profundidades, antes e depois da queima, e dados físico-químicos e climáticos associados. O DNA da comunidade bacteriana foi amplificado utilizando conjunto de primers específicos para o Domínio Bacteria e para o grupo de actinomicetos, e os produtos de amplificação analisados por DGGE. Resultados obtidos para as populações de actinomicetos não foram conclusivos. Apesar da variação dos parâmetros físico-químicos do solo provocadas pela queima, os padrões de bandas obtidos com os primers para o Domínio Bacteria, apresentaram-se uniformes. Sendo assim, nas condições de estudo deste trabalho, os resultados obtidos não revelaram alterações na estrutura da comunidade bacteriana do solo de canavial depois da queima. / The utilization of molecular techniques has facilitated the study of complex bacterial communities in the environment. The present study aimed at using DGGE technique to evaluate the effect of temperature variation, caused by sugar-cane plantation burn, in the soil bacterial community structure emphasizing the actinomycete group. Soil samples from different depths, were collected before and after the bum, as well as physical-chemical and climatic associated data. The bacterial community DNA was amplified using a specific primer set and the amplification products analyzed by DGGE. The results obtained for the actinomycete populations were not conclusive. Despite the variation of the soil parameters caused by the burn, the band patterns obtained used in this study were uniform. Therefore, under the conditions used in this study, the results obtained did not show any alteration in the structure of soil bacterial community associated with sugar-cane plantation after the burn.