Made available in DSpace on 2014-06-11T19:28:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2004-04-15Bitstream added on 2014-06-13T19:16:45Z : No. of bitstreams: 1 shiomi_hf_me_botfca.pdf: 1002815 bytes, checksum: aa3a55c9b04258872a970f0e010f3cdf (MD5) / Universidade Estadual Paulista (UNESP) / Experimentos sob condições controladas e testes de campo com bactérias endofíticas isoladas de folhas e caules de Coffea arabica e Coffea robusta, provenientes das cidades de Pedreira-SP, Mococa-SP e Pindorama-SP, foram realizadas com o objetivo de avaliar a inibição da germinação de urediniosporos de Hemileia vastatrix, raça II e o controle do desenvolvimento da ferrugem alaranjada do cafeeiro, em testes em discos de folhas, folhas destacadas e em plantas da cv. Mundo Novo, pela aplicação de suspensões de bectérias endofíticas 72 e 24 horas antes e após à inoculação de H. vastatrix e concomitantemente à inoculação com o patógeno. Isolados de bactérias endofíticas testados demonstraram eficácia na inibiação da germinação de urediniosporos e/ou no desenvolvimento da ferrugem, com valores acima de 50%, embora os resultados obtidos nos testes de germinação de urediniosporos tenham sido inferiores ao tratamento com o propiconazole. Nos testes em discos de folhas, folhas destacadas e em plantas de cafeeiro, os isolados endofíticos T G4-Ia, T F2-IIc, T F7-Ib e T F9-Ia, demonstraram melhor controle da ferrugem do cafeeiro. Os isolados endofíticos T G4-Ia e T F9-Ia foram identificados como Bacillus lentimorbus e Bacillus cereus, respectivamente. Os resultados indicam a ocorrência de isolados endofíticos eficientes em controlar a ferrugem alaranjada do cafeeiro. Alguns isolados de bactérias edonfíticas podem aumentar a severidade da doença. / Experiments under controlled conditions and field tests with endophytic bacteria from leaves and stem of Coffea arabica and Coffea robusta plants from Pedreira-SP, Mococa-SP and Pindorama-SP, were carried out with th objective to evaluate the inhibition of germination of urediniospores of Hemileia vastatrix, race II and control the development of coffea leaf rust cv. Mundo Novo leaf disk, datached-leaf and coffee plants, by dripping of bacterial suspensions 72 e 24 hours prior and after the H. Vastatrix and also concomitant the aplication of the pathogen. Endophytes tested demonstrated inhibition efficacy of urediniospores germination and/or the development of leaf rust over 50%, althought the results obtained in urediniospores germination was below of propiconazole treatment. In leaf dsk and datached-leaf inoculations, the endophytes strains T G4-Ia, T F2-IIc, T F7-Ib and T F9-Ia demonstrated better control of coffee leaf rust disease. The endophytic strains T G4-Ia and T F9-Ia was identified as Bacillus lentimorbus and Bacillus cereus. The results the ocorrence of efficiente endophytes controlling the coffee leaf rust. Some endophytes can partially improve severity of coffee leaf rust.

Cancer has become a major health problem due to its high rate of morbidity and mortality. Severe side effects associated with most of the available anticancer drugs and the development of the drug resistant cells are the major hurdles limiting their application and therapeutic success. Much attention has thus been focused on natural compounds with minimal or no toxicity in humans and having capacity to suppress proliferation and/or induce apoptosis in cancer cells. Therefore, it is imperative to discover and develop new anticancer drugs. Endophytic fungi though underexploited have remained a rich source of structurally novel and biologically active secondary metabolites. That they are a prolific resource for new compounds in drug discovery is evident from a plethora of reported research findings of the recent past. Those of endophytic fungi especially isolated from medicinal plants with known therapeutic value have especially caught the attention of several research groups worldwide for the production of host associated or novel lead molecules. In this backdrop, the focus of my research work has been centered on exploring the endophytic fungal population associated with Catharanthus roseus in search of production of novel anticancer compounds. Twenty endophytic fungi were isolated from different parts of Catharanthus roseus plant. The ethyl acetate (EA) extracts of three week grown liquid cultures of the individual endophytes were assessed in vitro for their cytotoxic activities in HeLa and HepG2 cancer cells using MTT assay. Of all the isolates thus screened, the EA extract from an isolate identified as Chaetomium nigricolor exhibited significant cytotoxicity in the tested cell lines with a better activity profile against HeLa cells. To understand if the liquid culture filtrate or the mycelium harbored the active cytotoxic principle, the C. nigricolor culture filtrate and mycelia was extracted separately using organic solvents with varying polarity namely ethyl acetate, dichloromethane, chloroform and hexane. These extracts were further tested for cytotoxicity induction in HeLa and MCF-7 cells by MTT assay. The ethyl acetate culture filtrate and mycelial extracts showed best cytotoxic activity on HeLa cells compared to other extracts; therefore, the mycelial and culture filtrate ethyl acetate extracts of C. nigricolor were pooled and was used for further work. Hexane culture filtrate extract of C. nigricolor showed best antiproliferative activity against MCF-7 cells. Thus, the compounds with anticancer potential from both ethyl acetate pooled extracts and hexane culture filtrate extract of C. nigricolor were purified using bioassay guided isolation method. This finally resulted in the isolation of two different anticancer compounds, one each from EA pooled extract and Hexane culture filtrate extracts of C. nigricolor named as compound 1 and compound 2, respectively. Based on various analyses including mass spectrometry (MS and MS-MS) and UV-visible, Fourier transform infrared (FT-IR) and nuclear magnetic resonance (NMR) spectroscopy, the compounds 1 and 2 were identified as 1, 2 Bis (diethylamino) ethene-1, 2 diol (BDED) and Nonacos-4-enoic acid (NA), respectively An improved understanding of the cellular responses to chemotherapeutic agents in cancer cells and the underlying molecular mechanisms gains particular relevance in the efforts to improve the clinical outcome of chemotherapeutic agents. Arresting the growth of cancer cells in one way or the other and induction of apoptosis in a drug specific manner is the hallmark of almost all the clinically used anticancer drugs. Towards this end, evaluation of the cytotoxic effects of the newly identified compounds on various human cancer cells was carried out. BDED was examined for in vitro cytotoxicity of against a panel of five human cancer cell lines namely HeLa, A-431, A549, COLO 205, and MCF-7. Among the cell lines screened, HeLa cells were most vulnerable to BDED treatment with an IC50 value of 27 μM. This cytotoxicity was later identified as resulting from apoptosis induction by BDED which was scored by the characteristic events of membrane externalization of phosphatidylserine, cytoplasmic shrinkage and chromatin condensation which were observed in a large majority of the BDED-treated cells. Further detailed studies were performed to delineate the cellular mechanisms of BDED-induced apoptosis in HeLa cells. Analysis of cell cycle progression by propidium iodide (PI) staining revealed BDED-induced cell cycle arrest in the G1 phase of cell cycle. The results indicated that BDED induced a significant ROS generation and a significant loss of mitochondrial membrane potential which were abrogated upon pretreatment of the cells with an antioxidant, N-acetyl cysteine (NAC). These observations suggested the probable involvement of the initial events of BDED-induced ROS production in causing cell death via mitochondria-based intrinsic pathway of apoptosis. In addition, the results from the expression profile of pro- and anti- apoptotic proteins by western blot analysis in the BDED-treated HeLa cells further corroborated this hypothesis. These included an abnormally deregulated cellular abundance of BAX 1 and BCL 2 proteins, elevated levels of APAF-1, and activated cleaved species of procaspases 9 and 3. In addition, a pronounced abundance of cleaved PARP protein was observed in these experiments. Thus, the results suggest that BDED induced apoptosis in HeLa cells via ROS mediated mitochondrial dependent pathway. In parallel, the in vitro cytotoxic effects of NA was also studied, the second antiproliferative compound identified from hexane extracts of C. nigricolor using a panel of four human cancer cell lines - A-431, A549, COLO 205, and MCF 7. NA was thus found to be most potent against MCF 7 (breast cancer) cells. PI staining-based viability assays and microscopic observations showed a dose dependent cytotoxicity of NA on MCF 7 cells. Our data reveled an IC 50 of 40 μM for NA The NA-induced apoptosis was confirmed by flow cytometric detection of membrane externalization of phosphatidylserine using Annexin V FITC/PI dual staining. NA was also observed to induce cytotoxicity best at 72 h this could be attributed to the fact that it is a derivative of a Nonocosane-(a naturally prevalent molecule found in several vegetables). Similar to our results from BDED-treated HeLa cells, ROS generation and loss of mitochondrial membrane potential which were abrogated on pretreatment with NAC, were also observed in NA-treated MCF-7cells. Similar to several other fatty acids, NA was also observed to induce cell cycle arrest in the G1 phase of cell cycle. Further, apoptotic signature of an altered expression of anti-apoptotic BCL-2 and pro-apoptotic BAX, APAF-1 and procaspase 9 and 3 and a cleaved PARP were observed in NA-treated MCF 7 cells. In conclusion, the results indicate that BDED and NA have cytotoxic and apoptotic effects on HeLa (cervical cancer) and MCF 7 (breast cancer) cell lines, respectively. This leaves open further avenues to evaluate their potential application as anticancer agents for treatment of human cervical and breast cancers.

O presente trabalho teve por objetivo avaliar o efeito de três ecossistemas sobre, a ocorrência de fungos rizosféricos, endofíticos e epifíticos. As áreas de estudo estão localizadas em uma propriedade rural, situada na cidade de Venâncio Aires, RS. Os fungos endofíticos e epifíticos foram isolados de amostras de raízes de guajuvira, (Patagonula americana L.), coletadas na mata (área 1), de uva-do-japão (Hovenia dulcis Thunb.), na área intermediária (área 2) e de fumo ou de milho, na lavoura (área 3) e, os fungos rizosféricos isolados de solo coletado junto as raízes dos vegetais citados. As amostras foram coletadas nos meses de janeiro, maio, setembro e novembro de 2004 e 2005. Os fungos foram identificados segundo características morfológicas com auxílio de chaves de identificação. As áreas 2 e 3 foram mais similares com relação aos fungos rizosféricos e epifíticos, enquanto, que, as áreas 1 e 2 foram mais similares com relação aos fungos endofíticos. A diversidade dos fungos isolados das três áreas não diferiu ao longo dos dois anos de coleta. A presença de Fusarium spp., em vegetais sem sintomas de doença indica que, as mesmas podem ser raças avirulentas ou patógenos latentes em equilíbrio com o hospedeiro e o ambiente. A presença de fungos antagonistas, principalmente Trichoderma spp. também, são responsáveis por esse equilíbrio, o qual ocorre nas três áreas. / The present paper aimed at assessing the effect of three ecosystems, on the occurrence of rhizospheric, endophytic and epiphytic fungi. The study areas are located in a farm in Venâncio Aires, Rio Grande do Sul. Samples were collected in the months of January, May, September and November 2004 and 2005. The endophytic and epiphytic fungi were isolated from samples of guajayvi roots (Patagonula americana L.), picked in the forest (area 1), of Japanese raisin tree (Hovenia dulcis Thunb.), in the intermediate area (area 2), and tobacco or corn, in the cultivated area (area 3) and rhizospheric fungi isolated from soil. The fungi were identified according to morphological characteristics, with the help of identification keys. Areas 2 and 3 were more alike concerning rhizospheric and epiphytic fungi; Areas 1 and 2, in turn, were more alike concerning endophytic fungi. The diversity of the isolated fungi from these three areas did not change during the two years of collection. The abundance of fungi with phytopathogenic potential such as the Fusarium spp. and Macrophomina phaseolina, mostly as endophytic in area 3 shows the influence of the host on them. The presence of antagonistic fungi, principally Trichoderma spp. also, are responsable for this equilibrium, which occured in the three areas.

This present research aimed to evaluate the effects of different endophytic bacteria species regarding the tolerance of sugarcane water stress in the initial growth phase. With that purpose, an experiment was installed, in the greenhouse at the Centro de Ciências Agrárias of Universidade Federal de Alagoas, located in the city of Rio Largo, in the state of Alagoas (9° 28 S, 35° 49 W, altitude 127 m), completely ramdomized on a factorial arrengement 4x2 (endophytic bacteria and levels of water stress in this order), with 8 replications. The levels of the first factor were composed of: non-inoculated plants; inoculated plants with complete inoculant (mix of Gluconacetobacter diazotrophicus, Burkholderia tropica, Herbaspirillum seropedicae, Herbaspirillum rubrisubalbicans e Azospirillum amazonense); inoculated plants with G. diazotrophicus; inoculated plants with B. tropica. The levels of the second factor were: plants kept on the soil with moisture next to field capacity (without stress); plants under water deficit due to irrigation suspension. The morphological and physiological variables analyzed were plant height, stalk diameter, number of green leaves, leaf area, dry mass analyze, leaf water potential (ψwf), potential and effective quantic efficiency and chlorophyll content. The bacteria application didn t interfer in stalk diameter, plants height and estimated leaf area. When the plants average growth was evaluated, it was verified that plants treated without bacteria (control) and with inoculum B. tropica presented showed greater aerial part mass, whereas the bacteria application G. diazotrophicus and the control treatment presented greater root mass. The water stress has significantly affected the potential quantic efficiency (Fv/Fm) on the irrigated plants (control) which the Fv/Fm values were above 0,79 statistically differing of water stress (draught) and rehydratation, wherein the values were below 0,79. Apparently, the damages presented on the photosystem II at midday were recovered during nighttime, showing higher Fv/Fm values at predawn. The presence of bacteria hasn t significantly affected the biometric characteristics of sugarcane plants grown in vases at 114 days after planting (DAP). The leaf area ratio (LAR) utilized the measured leaf area, being greater on control and complete inoculant treatments. On these, the plant developed more leaves and was less efficient on carbon fixation, which refleced on dry mass increase. / A presente pesquisa teve como objetivo avaliar os efeitos de diferentes espécies de bactérias endofíticas sobre a tolerância ao déficit hídrico de cana-de-açúcar em sua fase inicial de crescimento. Para tanto, instalou-se um experimento, em casa de vegetação no Centro de Ciências Agrárias da Universidade Federal de Alagoas, localizado no município de Rio Largo - AL (9° 28 S, 35° 49 W, a 127 m de altitude), em delineamento inteiramente ao acaso em arranjo fatorial, 4 x 2 (tipos de bactérias endofíticas e níveis de déficit hídrico respectivamente), com oito repetições. Os níveis do primeiro fator foram compostos por: plantas não inoculadas; plantas inoculadas com inoculante completo (mistura das Gluconacetobacter diazotrophicus, Burkholderia tropica, Herbaspirillum seropedicae, Herbaspirillum rubrisubalbicans e Azospirillum amazonense); plantas inoculadas com a G. diazotrophicus; plantas inoculadas com a B. tropica. Os níveis do segundo fator foram: plantas mantidas em solo com umidade a próxima da capacidade de campo (sem estresse); plantas submetidas ao déficit hídrico pela suspensão da rega. As variáveis morfológicas e fisiológicas analisadas foram: altura, diâmetro do colmo, número de folhas verdes, área foliar, análise da massa seca, potencial hídrico foliar (ψwf), eficiência quântica potencial e efetiva e estimativa do teor de clorofila. A aplicação de bactéria não afetou o diâmetro do colmo, altura de plantas e área foliar estimada. Quando avaliado o crescimento médio das plantas, verificou-se que plantas tratadas sem bactérias (controle) e com o inoculado B. tropica apresentavam maior massa de parte aérea, enquanto que a aplicação da bactéria G. diazotrophicus e o tratamento controle apresentaram maior massa de raiz. O estresse hídrico afetou significativamente a eficiência quântica potencial (Fv/Fm), nas plantas irrigadas (controle) os valores Fv/Fm estavam acima de 0,79 diferindo estatisticamente do estresse hídrico (seca) e reidratação, em que os valores estavam abaixo de 0,79. Aparentemente os danos apresentados no fotossistema II ao meio-dia foram recuperados durante o período noturno, mostrando valores de Fv/Fm mais elevados na antemanhã. A presença de bactérias não efetou significativamente as características biométricas das plantas de cana-de-açúcar crescidas em vasos aos 114 DAP. A Razão da Área Foliar (RAF) utilizou a AF mensurada, sendo maior nos tratamentos controle e inoculante completo. Nestes, a planta desenvolveu mais folhas e foi menos eficiente na fixação de carbono, refletido no aumento de massa seca.

Submitted by MAIARA STEFANINI BORGES null (borges.maiara@iq.unesp.br) on 2016-03-22T21:06:41Z No. of bitstreams: 1 MAIARA STEFANINI BORGES.pdf: 2921643 bytes, checksum: 71bed0595a70fef8c32665cc82adc905 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-03-23T13:30:07Z (GMT) No. of bitstreams: 1 borges_ms_me_araiq.pdf: 2921643 bytes, checksum: 71bed0595a70fef8c32665cc82adc905 (MD5) / Made available in DSpace on 2016-03-23T13:30:07Z (GMT). No. of bitstreams: 1 borges_ms_me_araiq.pdf: 2921643 bytes, checksum: 71bed0595a70fef8c32665cc82adc905 (MD5) Previous issue date: 2016-03-09 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Micro-organismos endofíticos, geralmente fungos e bactérias, vivem sistematicamente no interior das plantas sem causar, aparentemente, danos a seus hospedeiros. Fungos endofíticos representam um importante grupo de fungos com potencial fármaco e biotecnológico e podem ser isolados de diversas plantas medicinais. A espécie vegetal Eugenia jambolana Lam. tem sido intensamente explorada química e biologicamente devido a seus usos populares e por apresentar diversas atividades biológicas. Logo, o estudo de fungos endofíticos, associados a esse espécie vegetal, apresenta-se como uma estratégia promissora na obtenção e identificação de substâncias potencialmente bioativas. Dentro deste contexto, este trabalho teve como objetivo principal a avaliação química do fungo endofítico Saccharicola sp. isolado de Eugenia jambolana Lam. Este endófito foi cultivado em pequena escala nos meio de cultura liquido (MDB) e sólido (Milho) e em escala ampliada no meio Czapek. O caldo foi separado dos micélios após 28 dias (meios líquidos) e 21 dias (meio sólido) por filtração e o filtrado foi submetido à partição líquido/líquido com AcOEt fornecendo, após a evaporação do solvente, os respectivos extratos brutos. Os extratos foram avaliados frente a produção metabólica por CCDC, CLAE-DAD e RMN de 1H e também avaliados frente aos ensaios biológicos. Portanto para os extratos MDB, Milho Fr.CH3CN, Milho Fr. aquosa e Milho Fr.Hexânica, foram realizados ensaios antifúngico, antioxidante e anticolinesterásico, onde estes extratos demonstraram ser promissores. O extrato obtido em escala ampliada (Czapek) foi fracionado em cartucho SPE (extração em fase sólida), resultando em seis frações (Ss.C.Fr1- Ss.C.Fr6). Do estudo da fração Ss.C.Fr1 foi possível o isolamento de uma substância o Ácido fusárico (1) e a fração Ss.C.Fr3 resultou no isolamento de 6,7 dihidroxi- 4,6 dimetil octanoato de metila (2), substância inédita. O extrato obtido em escala reduzida (Milho Fr.CH3CN) foi submetido a fracionamento em coluna de sílica gel de fase reversa (C18), resultando em oito frações (Ss.M. CH3CN Fr1 - Ss.M. CH3CN Fr8). O estudo da fração Ss.M. CH3CN Fr5 resultou no isolamento da substância 4-hidroximeleina (3). O estudo da fração Milho Fr.Aquosa-XAD resultou no isolamento de duas substâncias Timidina (4) e Adenosina (5). Portanto, o estudo químico com o fungo endofítico Saccharicola sp. resultou no isolamento de cinco substâncias puras. Sendo assim, este trabalho visou isolar substâncias potencialmente bioativas através da investigação dos extratos e frações de Saccharicola sp.. / Endophytic microorganisms, often bacteria and fungi systematically live within the plant without apparent harm their hosts. Endophytic fungi represent an important group of fungi with biotechnological potential and may be isolated from various medicinal plants. The plant species Eugenia jambolana Lam. has been intensively explored chemically and biologically due to its popular uses and it has several biological activities. Thus, the study of endophytic fungi associated with this plant species, presents itself as a promising strategy in obtaining or identifying potentially bioactive substances. Within this context, this study aimed to evaluate the chemical endophytic fungus Saccharicola sp. isolated from Eugenia jambolana Lam., encoded as Ej-c04. This endophyte was cultivated on a small scale to a liquid culture medium PDB solid Corn and enlarged scale for the Czapek medium. The broth was separated from the mycelium after 28 days (liquid media) and 21 days (solid medium) by filtration and the filtrate was subjected to liquid / liquid partition with ethyl acetate providing after evaporation of the solvent, their crude extracts. The extracts were evaluated against metabolic production by TLC, CLAE-DAD and 1H NMR also evaluated and compared to the bioassay. So for MDB extracts, Fr.CH3CN Corn, aqueous Fr. Corn and Corn Fr.Hexânica, tests were performed antifungal, antioxidant and anticholinesterase where these extracts have shown promise.The extract obtained in enlarged scale (Czapek) was fractionated on SPE cartridge (solid phase extraction), resulting in six fractions (Ss.C.Fr1- Ss.C.Fr6). From Ss.C.Fr1 fraction was possible to isolate a substance fusaric acid (1) and Ss.C.Fr3 fraction resulted in the isolation of (2E, 4E) - 6,7 dihydroxy-4,6-dimethyl octanoate (2) novel substance. The extract obtained in small scale (Corn Fr.CH3CN) was subjected to fractionation column of silica gel reversed phase (C18) resulting in eight fractions (Ss.M. CH3CN- Fr1 - CH3CN Ss.M. Fr8). The study fraction Ss.M. CH3CN Fr5 resulted in the isolation of 4-hidroximeleina substance (3). The study of fraction Corn Fr. Aqueous -XAD resulted in the isolation of two substances Thymidine (4), adenosine (5). Therefore the chemical study of the endophytic fungus Saccharicola sp. resulted in the isolation of five pure substances Thus, this study aimed to isolate potentially bioactive substances through the investigation of extracts and fractions of Saccharicola sp..

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No. of bitstreams: 1 silva_ad_me_araiq.pdf: 4535490 bytes, checksum: 8bb674faf58fdd954d9ebfabaecc42b1 (MD5) Previous issue date: 2016-03-18 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O fungo Hypoxylon investiens ocorre em ambientes terrestres e aquáticos, sendo que a maior parte dos estudos já realizados com o gênero Hypoxylon refere-se aos fungos terrestres. Diversas substâncias e atividades biológicas foram determinadas para o gênero e o presente estudo é o primeiro a realizar a bioprospecção de H. investiens associado à alga marinha. Neste trabalho, foram estudados os constituintes químicos produzidos pelo fungo H. investiens e avaliadas algumas atividades biológicas promissoras (antifúngica e citotóxica). O extrato bruto dos metabólitos produzidos em meio líquido de PDB obtido em escala ampliada apresentou forte atividade frente aos fungos fitopatogênicos Cladosporium cladosporioides e Cladosporium sphaerospermum e mostrou-se ativo (75,7% de inibição) frente à linhagem tumoral HCT-116 (carcinoma de cólon). O seu fracionamento por CC em Sephadex LH-20 eluída com metanol resultou em 6 frações (G1 a G6): a fração G4 mostrou forte atividade antifúngica contra linhagens de C. cladosporioides e C. sphaerospermum, além de alto potencial de inibição (97,3%) de células tumorais HCT-116. As frações G5 e G6 também se mostraram ativas frente à linhagem tumoral HCT-116 apresentando, respectivamente, 104,8% e 99,4% de inibição dessa linhagem de células tumorais. A fração G5 apresentou ainda atividade (inibição de 84,5%) contra a linhagem MCF-7 (adenocarcinoma de mama metastático). No entanto, as frações G4 e G6 mostraram-se inativas contra esta linhagem, o que evidenciou a seletividade dessas frações frente às linhagens tumorais testadas. Estes resultados indicaram elevado potencial de produção de metabólitos bioativos por H. investiens. Os procedimentos cromatográficos levaram ao isolamento de 14 substâncias da fração G5, 3 substâncias da fração G6 e de outras 2 substâncias da fração inativa G3. A análise estrutural preliminar por RMN das 17 substâncias isoladas das frações bioativas G5 e G6 indica que devam se tratar de compostos inéditos, derivados do BNT (4,4´,5,5’-tetraidroxi-1,1´-binaftila), sendo que três delas, isoladas da fração G5, tiveram seus esqueletos carbônicos completamente caracterizados, utilizando-se experimentos uni e bidimensionais de RMN e espectrometria de massas de alta resolução. Experimentos adicionais estão em andamento para se determinar a estereoquímica dos centros de quiralidade das substâncias elucidadas, e as demais estruturas estão sob processo de investigação estrutural. Uma das substâncias isoladas da fração não-ativa G3 foi o ácido 3-nitropropanóico, uma toxina mitocondrial produzida por fungos para sua defesa e que foi produzido com alto rendimento (304,5 mg a partir de 605,4 mg da fração G3) por H. investiens. Os resultados obtidos foram promissores, evidenciando a destacada quimiodiversidade de fungos obtidos do ambiente marinho e elevado potencial de bioatividade de seus extratos e frações. Sinalizam ainda para a necessidade de estudos mais aprofundados da química e das propriedades biológicas do fungo H. investiens contribuindo para a exploração sustentável da biodiversidade marinha brasileira. / The fungus Hypoxylon investiens occurs in terrestrial and aquatic environments, and most studies carried out so far on genus Hypoxylon refer to the terrestrial fungal strains. Many compounds and biological activities were determined for the genus and the present study is the first to carry out the bioprospection of H. investiens associated to marine algae. In this work, the chemical constituents produced by a fungal strain of H. investiens were investigated and some promising biological activities were evaluated (antifungal and cytotoxicity). The crude extract of the metabolites produced in PDB liquid medium by H. investiens was obtained in large scale and showed strong activity against the phytopathogenic fungi Cladosporium cladosporioides and C. sphaerospermum. It also showed inhibitory activity (75.7% of inhibition) on colon carcinoma tumor cell line HCT-116. Its fractionation by column chromatography over Sephadex LH-20 eluted with methanol afforded 6 fractions, (G1-G6). Fraction G4 showed strong antifungal activity against C. cladosporioides and C. sphaerospermum strains, in addition to high inibitory potential (97,3%,) towards tumor cells HTC-116. Fractions G5 and G6 were also active against tumor cell line HCT-116, showing 104,8% and 99,4% inhibition, respectively. Additionally, fraction G5 inhibited metastatic breast adenocarcinoma cell line MVF-7 whereas fractions G4 and G6 were inactive, which evidenced selectivity of such fractions towards the tested tumor cells lines. These results evidenced a high potential of production of bioactive metabolites by H. investiens. The chromatographic procedures led to the isolation of 14 compounds from fraction G5, 3 compounds from fraction G6 and 2 compounds from the non-active fraction G3. The preliminary analysis of the 17 isolated compounds from the bioactive fraction G5 and G6 by NMR spectroscopy indicated they are novel compounds structurally related to BNT (4,5,4',5'-tetrahydroxy-1,1'-BiNaphThyl). From those, three compounds were isolated from fraction G5 and had their carbon skeletons fully characterized and consistent with uni and bidimensional 1H and 13C NMR spectral data and high-resolution mass spectrometry. Additional experiments are being developed to determine the stereochemistry of asymmetric centers in the elucidated structures. Additionally, the non-active fraction G3 afforded high yield (304,5 mg from 605,4 mg of G3 fraction) of 3-nitropropanoic acid, a mitochondrial toxin wide spread among fungi and produced for their own defense. The results obtained were promising, pointing out the need for further in-depth investigations on the chemistry and biological properties of the fungus H. investiens. Such promising results evidence the outstanding chemodiversity of marine-derived fungi and highlight the bioactivity potencial of their extracts and fractions. They also point out the need for further studies of H. investiens chemistry and biological properties, which may contribute for the sustainable exploration of Brazilian marine biodiversity.

O presente trabalho teve por objetivo avaliar o efeito de três ecossistemas sobre, a ocorrência de fungos rizosféricos, endofíticos e epifíticos. As áreas de estudo estão localizadas em uma propriedade rural, situada na cidade de Venâncio Aires, RS. Os fungos endofíticos e epifíticos foram isolados de amostras de raízes de guajuvira, (Patagonula americana L.), coletadas na mata (área 1), de uva-do-japão (Hovenia dulcis Thunb.), na área intermediária (área 2) e de fumo ou de milho, na lavoura (área 3) e, os fungos rizosféricos isolados de solo coletado junto as raízes dos vegetais citados. As amostras foram coletadas nos meses de janeiro, maio, setembro e novembro de 2004 e 2005. Os fungos foram identificados segundo características morfológicas com auxílio de chaves de identificação. As áreas 2 e 3 foram mais similares com relação aos fungos rizosféricos e epifíticos, enquanto, que, as áreas 1 e 2 foram mais similares com relação aos fungos endofíticos. A diversidade dos fungos isolados das três áreas não diferiu ao longo dos dois anos de coleta. A presença de Fusarium spp., em vegetais sem sintomas de doença indica que, as mesmas podem ser raças avirulentas ou patógenos latentes em equilíbrio com o hospedeiro e o ambiente. A presença de fungos antagonistas, principalmente Trichoderma spp. também, são responsáveis por esse equilíbrio, o qual ocorre nas três áreas. / The present paper aimed at assessing the effect of three ecosystems, on the occurrence of rhizospheric, endophytic and epiphytic fungi. The study areas are located in a farm in Venâncio Aires, Rio Grande do Sul. Samples were collected in the months of January, May, September and November 2004 and 2005. The endophytic and epiphytic fungi were isolated from samples of guajayvi roots (Patagonula americana L.), picked in the forest (area 1), of Japanese raisin tree (Hovenia dulcis Thunb.), in the intermediate area (area 2), and tobacco or corn, in the cultivated area (area 3) and rhizospheric fungi isolated from soil. The fungi were identified according to morphological characteristics, with the help of identification keys. Areas 2 and 3 were more alike concerning rhizospheric and epiphytic fungi; Areas 1 and 2, in turn, were more alike concerning endophytic fungi. The diversity of the isolated fungi from these three areas did not change during the two years of collection. The abundance of fungi with phytopathogenic potential such as the Fusarium spp. and Macrophomina phaseolina, mostly as endophytic in area 3 shows the influence of the host on them. The presence of antagonistic fungi, principally Trichoderma spp. also, are responsable for this equilibrium, which occured in the three areas.

Orientador: Ângela Regina Araujo / Banca: Silvia Noeli López / Banca: Mônica Tallarico Pupo / Banca: Maria da Conceição Farreira de Oliveira / Banca: Luce Maria Brandão Torres / Resumo: Este trabalho, descreve a busca de substâncias bioativas a partir do estudo químico e biológico dos extratos brutos produzidos pelos fungos endofíticos AM-01 identificado como Penicillium sp1, AM-02 e AM-03 não identificados e AM-04, também pertencente ao gênero Penicillium, que foram isolados das folhas de Alibertia macrophylla. Os fungos endofíticos isolados, foram cultivados em meio líquido MBD, e sólido (milho) a 25oC, sob agitação, e no modo estático, respectivamente. Para o meio líquido, o caldo foi separado do micélio e submetido à partição com AcOEt, fornecendo os extratos brutos após a evaporação do solvente. Para o meio sólido, foi realizado uma extração com metanol, seguido de filtração e evaporação do solvente, originando os extratos brutos. Após análises por RMN de ¹H e CLAE-DAD, os extratos brutos obtidos em milho, foram fracionados por cromatografia em coluna, seguida de separação via Cromatografia Líquida de Alta Eficiência (CLAEprep.). O extrato bruto de AM-01 (Penicillium sp1) forneceu seis substâncias da classe das isocumarinas, sendo duas inéditas. O fracionamento cromatográfico do extrato bruto de AM-02, conduziu ao isolamento de três sesquiterpenos inéditos identificados como Xylarenonas C, D e E. Também do extrato bruto de AM-02 foi isolado um composto furânico identificado como (E)-5-(1- hidroxialil)-4-(pent-1-enil)-2,5-diidrofurano-3-ácido carboxílico. O fracionamento cromatográfico de AM-04, permitiu o isolamento de um benzenóide, identificado como orcinol. O estudo químico dos extratos brutos obtidos em MBD, de AM-03 e AM-04, permitiram o isolamento das dicetopiperazinas ciclo (Pro-Try) e ciclo (L-Pro- L-Val), além da uracila. As substâncias isoladas foram identificadas por análises de RMN 1D e 2D e espectrometria de massas, bem como por comparação com dados da literatura... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: This work describes the search for bioactive substances from the chemical/biological of the crude extracts produced by endophytic fungi AM-01 identified as Penicillium sp1, AM-02, AM-03 and AM-04 also belonging to the genus Penicillium isolated from the leaves of Alibertia macrophylla. The endophytic fungal isolates were grown in liquid medium and solid MBD to 25 °C under shaking and static mode, respectively. For the liquid medium the mycelium was separated from the broth and subjected to partition with AcOEt, giving the crude extracts after evaporation of the solvent. For solid medium, the extraction was performed with MeOH, followed by filtration and evaporation of the solvent yielding the crude extracts. After analysis by the ¹H NMR and HPLC-DAD, the crude extracts from maize were fractionated by chromatography on a column followed by separation using High Performance Liquid Chromatography (HPLCprep.). The crude extract of AM-01 (Penicillium sp1) provided six isocoumarins, two news. Chromatographic fractionation of the crude extract of AM-02 led to the isolation of three novel sesquiterpenes identified as Xylarenonas C, D and E and a furanic compound identified as 2,5-dihydro-5-(1-hydroxyallyl)-4-(E)-pent-1-enyl)furan-3-carboxylic acid. Chromatographic fractionation of AM-04 allowed the isolation of a benzenoid, identified as orcinol. The chemical study of AM-03 crude extracts and AM-04 cultivated in MDB allowed the isolation of diketopiperazines cyclo (Pro-Try), cyclo (L-Pro-L-Val), and uracil. The substances isolated were identified through analysis of 1H NMR, mass spectrometry and by comparison with literature data. The crude extracts and pure substances were submited for trials for antifungal agents, antioxidants, anticholinesterasic and inhibition of protease. All substances showed biological activities. / Doutor

Submitted by Carolina Rabal Biasetto null (carolinarabal@yahoo.com.br) on 2016-05-18T19:36:08Z No. of bitstreams: 1 Tese_Doutorado_Carolina_Rabal_Biasetto_2016.pdf: 15348096 bytes, checksum: 59245cf961f6aaf1a999156f53853b5d (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-05-20T18:01:57Z (GMT) No. of bitstreams: 1 biasetto_cr_dr_araiq_par.pdf: 1896284 bytes, checksum: 8b4dcb25761fe239f657abc24e10c1e7 (MD5) / Made available in DSpace on 2016-05-20T18:01:57Z (GMT). No. of bitstreams: 1 biasetto_cr_dr_araiq_par.pdf: 1896284 bytes, checksum: 8b4dcb25761fe239f657abc24e10c1e7 (MD5) Previous issue date: 2016-04-27 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / A capacidade biossintética dos fungos endofíticos, aliado aos estudos químico e biológicos relatados para Eugenia brasiliensis, motivou a idealização do projeto, a prospecção química, biológica e enzimática em fungos endofíticos associados a folhas, caules e frutos saudáveis de Eugenia brasiliensis e a avaliação do co-cultivo e epigenética em Xylaria cubensis, Diaporthe sp. e Colletotrichum sp., na obtenção de novas substâncias. O isolamento dos fungos endofíticos, resultou em dezessete linhagens de fungos endofíticos, sendo estes cultivados em escala reduzida em meio líquido de batata e dextrose (PDB) e Czapek, a 25 oC, sob modo estático para obtenção dos respectivos extratos brutos em AcOEt. A avaliação metabólica destes extratos foi realizada por CCDC, HPLC-DAD e RMN de 1H, como também a potencialidade enzimática e biológica pela avaliação das atividades antifúngica, anticolinesterásica e citotóxica, sendo que estes extratos demonstraram ser promissores. A prospecção inicial conduziu a seleção de três fungos endofíticos identificados como Xylaria cubensis (Eb_caH_5), Diaporthe sp. (Eb_caS_4), e Colletotrichum sp. (Eb_frmH_1), os quais foram cultivados (escala ampliada) em PDB para isolamento e determinação/elucidação estrutural dos metabólitos secundários. O estudo de Xylaria cubensis, resultou no isolamento de 8 substâncias, sendo da classe dos nucleosídeos, dicetopirazinas, isocumarinas e citocalasinas. De Diaporthe sp. foi isolado e identificado 8 substâncias: duas dicetopiperazinas, ácido nitropropiônico, uracila, tirosol, zygosporina D, pirrolidona (inédita) e alternariol. Colletotrichum sp. resultou no isolamento de 6 substâncias, sendo três dicetopiperazinas, além das substâncias N-(2-feniletil)acetamida, N-acetiltriptamina e metanoato de 2-hidroxibutila 3-indol (inédita). Todas as classes de substâncias produzidas por estes fungos endofíticos apresentam diversas atividades biológicas relatadas na literatura. Destaca-se a atividade fitotóxica da citocalasina D frente a coleóptilos de trigo superior ao herbicida comercial GOAL®. Para verificar a influência na produção metabólica de Xylaria cubensis, Diaporthe sp. e Colletotrichum sp., utilizou-se estratégias como o co-cultivo em meio sólido (PDA) e líquido (PDB) e a epigenética (escala reduzida), nas quais os endófitos mostraram produção metabólica diferente em relação à produção das monoculturas e na ausência do modulador epigenético ácido hidroxâmico suberoilanilida (SAHA), respectivamente. As ferramentas estatísticas, como PCA (Análise de componentes principais) e PLS-DA (Análise discriminante com calibração multivariada por mínimos quadrados parciais) permitiram uma rápida identificação e localização dos perfis metabólicos dos co-cultivos comparados às monoculturas. Estes dados contribuem para o conhecimento dos perfis metabólicos, biológicos e enzimáticos dos fungos endofíticos isolados de Eugenia brasiliensis, bem como suas interações com o hospedeiro. / The biosynthetic capacity of endophytic fungi, allied chemical and biological studies reported to Eugenia brasiliensis motivated the idealization of the project, prospection chemical, biological and enzymatic in endophytic fungi associated with healthy leaves, stems and fruits from Eugenia brasiliensis and evaluation co-culture and epigenetic in Xylaria cubensis, Diaporthe sp. and Colletotrichum sp., in obtaining new substances. The isolation of endophytic fungi resulted in seventeen endophytics fungi, these being cultivated in small scale on potato dextrose broth (PDB) and Czapek at 25 °C under static mode to obtain the corresponding crude extracts in AcOEt. The metabolic evaluation of these extracts was performed by CCDC, HPLC-DAD and 1H NMR, also enzymatic and biological potential were performed by antifungal, cytotoxic, and anticholinesterase activities, and these extracts have shown promise. The preliminary prospection led to the selection of three endophytic fungi identified as Xylaria cubensis (Eb_caH_5), Diaporthe sp. (Eb_caS_4) and Colletotrichum sp. (Eb_frmad_1) which were cultivated (larger scale) in PDB for isolation and determination or structural elucidation of secondary metabolites. The study of Xylaria cubensis resulted in the isolation of eight substances, as follows: nucleoside, diketopiperazines, isocoumarins and cytochalasins class. From Diaporthe sp. was isolated and identified eight substances, as follows: two diketopiperazines, nitropropionic acid, uracil, tyrosol, zygosporin D, pyrrolidone (unpublished) and alternariol. Colletotrichum sp. resulted in the isolation of six substances, three diketopiperazines, besides the substances N-(2-phenylethyl) acetamide, Nβ-acetyltryptamine and 3-hydroxybutan-2-yl-1H-indol-3-ylacetate (unpublished). All classes of substances produced by these endophytic fungi present several biological activities reported in the literature. Noteworthy is the phytotoxic activity of cytochalasin D against wheat coleoptile higher than commercial herbicide GOAL®. To verify the influence on the metabolic production of Xylaria cubensis, Diaporthe sp. and Colletotrichum sp., used strategies such as the co-culture on solid (PDA) and liquid medium (PDB) and epigenetic (small scale), in which endophytes showed an interesting and different metabolic production when compared with the production of monocultures and the absence of epigenetic modulator SAHA, respectively. The statistical tools, such as PCA (Principal component analysis) and PLS-DA (discriminant analysis with multivariate calibration partial least squares) allowed quick identification and location of the metabolic profiles of the co-culture compared monocultures. These data will contribute to the knowledge of the metabolic profiles, biological and enzymatic of endophytic fungi isolated from Eugenia brasiliensis and their interactions with host. / CNPq: 140980/2012-1

Made available in DSpace on 2016-06-02T19:02:45Z (GMT). No. of bitstreams: 1 6576.pdf: 2966508 bytes, checksum: 60d9666ae228bda42b9fc4a21dae76c6 (MD5) Previous issue date: 2014-11-28 / Universidade Federal de Sao Carlos / Microorganisms, such as those belonging to the groups of Penicillium, Bacillus, Paenibacillus, represent represent an important source for the production of bioactive metabolites with varied applications. The production of these substances is directly related to the culture media, as well as the available carbon and nitrogen sources, conditions of pH, temperature and agitation (gas exchange). Among the microbial groups most studied about the production of various interest metabolites are the fungi and bacteria that inhabit the interior of plants, the endophytics. Thus, the present study aimed to isolate and identify endophytics microorganisms from leaves of four species of Tabebuia spp. and Hymenaea courbaril and study the production of interest metabolites by the lineages found. The microbial activities were studied: antibiotic capability against Gram positive and Gram negative bacteria, antifungal, antibiotic resistance, enzyme production, antitumor activity and pigment production. Microorganisms that stood out due to production of antibiotics, cytotoxicity against tumor cells and pigment production. This study selected microorganisms based on their most relevant production, in this case, it was the antibiotics production, pigments production and cytotoxicity, they were cultured in larger scale aiming at the improvement of the production of metabolite chosen according to the results. The selected microorganisms were a bacterium identified as Paenibacillus terrae and a fungus identified as Penicillium minioluteum/Talaromyces minioluteus complex. The P. terrae proved producing bioactive metabolites against Staphylococcus aureus, Escherichia coli and Candida albicans, in addition to novel cytotoxic activity against HTC, and OVCAR8 SF295 tumor cells. This bacterium also showed unprecedented anti-Leishimania activity, acting against the promastigote form of the parasite. The isolated fungus proved producing a red pigment, in large quantities without cytotoxic, antifungal or antibacterial activity, favoring its industrials applications. Both selected microorganisms demonstrated potential for biotechnological and industrial applications, suggesting the viability of promising continuity of the work performed. / Os microrganismos, como os pertencentes ao grupo dos Penicillium, Bacillus, Paenibacillus, representam uma fonte importante para produção de metabólitos bioativos com diversas aplicações. A produção destas substâncias está diretamente relacionada ao cultivo em meios de cultura assim como as fontes de carbono e nitrogênio disponíveis, condições de pH, temperatura e trocas gasosas. Dentre os grupos microbianos que mais se destacam nas pesquisas para produção de diversos metabólitos encontram-se os fungos e bactérias que habitam o interior de plantas, os microrganismos endofíticos. Desta forma, o presente trabalho teve como objetivo isolar e identificar microrganismos endofíticos do cerrado a partir de folhas de quatro espécies de Tabebuia spp. e um exemplar de Hymenaea courbaril e estudar a produção de metabólitos de interesse biotecnológico das linhagens encontradas. As atividades microbianas estudadas foram: capacidade antibiótica contra bactérias Gram positivas e Gram negativas, atividade antifúngica, resistência a antibióticos, produção enzimática, atividade antitumoral e produção de pigmentos. Os microrganismos que se destacaram devido a produção de antibióticos, citotoxicidade contra células tumorais e produção de pigmentos foram selecionados e cultivadas em maior escala objetivando o melhoramento da produção do metabólito escolhido por testes bioguiados. Selecionou-se Paenibacillus terrae e Penicillium complexo minioluteum/Talaromyces complexo minioluteus. O P. terrae mostrou-se produtor de metabólitos bioativos contra bactérias Gram positivas, Gram negativas e fungos leveduriformes, além da inédita atividade citotóxica contra células HTC, SF295 e OVCAR8. Esta bactéria apresentou também inédita atividade anti-Leishimania, atuando contra a forma promastigota do parasita. O fungo produziu pigmento de coloração vermelha em grande quantidade e foi selecionado devido à ausência de atividade citotóxica, antifúngica e antibacteriana. Ambos microrganismos selecionados demonstraram potencial de aplicações biotecnológicas e industriais, sugerindo a viabilidade promissora da continuidade dos trabalhos desenvolvidos.