Bactéria e fungos são as principais fontes de enzimas envolvidas na transformação de compostos chave para o fluxo de carbono em solos de manguezal, caracterizado por alta prevalência de anaerobiose, salinidade e elevado teor de matéria orgânica. A decomposição de plantas ou resíduos de animais nestas condições é muito lenta, devido à pressão seletiva sobre a evolução de enzimas envolvidas nos processos de mineralização de nutrientes. A metagenômica, permiti o acesso a grande maioria da diversidade microbiana no ambiente, por meio da geração de bibliotecas de clones, o que resulta em um cenário promissor para bioprospecção de novas atividades enzimáticas. Neste estudo, foi relatada a descrição e caracterização de uma nova ?-N-acetil-hexosaminidase (EC 3.2.1.52) da família GH3, envolvida na degradação da matéria orgânica em solo de manguezal contaminado por derramamento de óleo localizado no município de Bertioga-SP, por meio de uma triagem de 12.960 clones metagenômicos. O clone positivo para a atividade celulolítica foi sequenciado e um total de 1.175.586 reads foram gerados com tamanho médio de 198 pb. As sequencias foram trimadas com base na qualidade de índice PHRED >= 30.0, e remoção de sequencias do hospedeiro (E. coli) e do vetor (fosmídeo), originando um contig final com 39.586 Kb. Entre as ORF\'s anotadas a partir do contig gerado, uma sequencia de 1.065 nucleotídeos foi identificada como codificante para a enzima ?-N-acetil-hexosaminidase, evidenciando baixa similaridade (32 %) com as demais encontradas no bancos de dados comparativos. A enzima foi expressa e purificada, onde uma banda isolada foi visualizada por SDS-PAGE com massa molecular prevista de 43 kDa. Por fim, as atividades ótimas da enzima (30 °C; pH 5.0; 0.5 M de NaCl; diminuição de atividade após 3hs de incubação) foram caracterizadas por meio do indicador p-nitrophenol (pNP) ligados aos substratos GlNac, GalNac e Glc. A detecção da enzima por meio da metagenômica, evidenciou que os manguezais são reservatórios de novas enzimas com características diferenciadas e altos potenciais de aplicabilidades biotecnológicas / Bacteria and fungi are major sources of enzymes involved in the transformation of key compounds for the carbon fluxes on mangrove soils, characterized by the high prevalence of anaerobiosis salinity and high content of organic matter. The decomposition of plant or animals residues under these conditions is very slow, acting as a selective pressure on the evolution of enzymes involved in the mineralization process of nutrients. Metagenomics has provided access to the vast majority of the microbial diversity in the environment through the generation of fosmid libraries, resulting in a promising scenario for bioprospection enzymatic activities. In this study, we report the description and characterization of a novel ?-N-acetylhexosaminidase (EC 3.2.1.52) of GH3 family, involved in the degradation of organic matter in mangrove soils contaminated by oil spill located in the city of Bertioga-SP through of a screening of 12.960 metagenomic clones. The positive clone for cellulolytic activitie was sequenced and a total of 1.175.586 reads were generated with measuring size 198 bp. The sequences were trimmed based on the index of quality PHRED >= 30.0 and removing the sequences to host (E. coli) and vector (fosmid) resulting in a contig of 39.586 Kb. Between the anoted ORF\'s from generated contig a sequence of 1.065 nucleotides was identified coding for a ?-N-acetylhexosaminidase showing low similatrity (32 %) with the other found in comparatives databases. The enzyme was expressed and purified where an isolated band can be visualized by SDS-PAGE with molecular mass of 43 kDa. Finally, as optimum activity of the enzyme (30 °C; pH 5.0; 0.5M NaCl; decreased activity after 3 h incubation) were characterized by the indicator p-nitrophenol (pNP) linked to the substrates GlNac, GalNac and Glc. The detection of the enzyme through metagenomics indicated that mangroves are reservoirs of novel enzymes with different characteristics and high potential for biotechnological applicability

A biodiversidade microbiana de diferentes ecossistemas tem incentivado estudos químicos e biológicos com micro-organismos dos mais variados habitats, os quais têm conduzido à obtenção de moléculas bioativas com aplicações na medicina, indústria química e agricultura, proporcionando melhorias na qualidade de vida ao homem. O presente trabalho teve como objetivos a bioprospecção por actinobactérias endofíticas e seus metabólitos, além do estudo da via biossintética dos sesquiterpenos aristoloquenos produzidos pelo fungo fitopatogênico Botrytis cinerea. No estudo de bioprospecção foram isoladas 41 linhagens de actinobactérias endofíticas de duas espécies de Asteraceae (Thitonia diversifolia e Lychnophora ericoides). A identificação através do sequenciamento de DNAr indicou predominância do gênero Streptomyces. As linhagens foram cultivadas em meio de arroz e os extratos etanólicos submetidos aos ensaios de citotoxicidade frente a células tumorais e antimicrobiano. Um total de 58,5% dos extratos apresentou atividade em pelo menos um dos ensaios realizados. Foram selecionadas as linhagens Streptomyces cattleya RLe 4 e Streptomyces sp. RLe 8 para cultivo em escala ampliada, isolamento e identificação de metabólitos bioativos. O isolamento dos compostos foi realizado através de diferentes técnicas cromatográficas e a identificação estrutural foi baseada em dados de ressonância magnética nuclear de 1H e 13C e espectrometria de massas. De S. cattleya RLe 4 foram isolados quatro compostos: 2-hidroxibenzamida, desferrioxamina E, 1-(3\',4\'-dimetoxifenil)-1-propanona e 1-(3\',4\'-dimetoxifenil)-1-etanona. Dos extratos de Streptomyces sp. RLe 8 foram isolados dez compostos: benzamida, 3- hidroxibenzamida, 3-hidróxi-4-metoxibenzamida, 4-hidróxi-3-metoxibenzamida, 3,4- dimetoxibenzamida, 2-fenilacetamida, dois isômeros de 3,4-diidro-3,4,6,8-tetraidróxi-1(2H)- naftalenona, 2,3-diidro-2,2-dimetil-4(1H)-quinazolinona e desferrioxamina B. O composto 2,3-diidro-2,2-dimetil-4(1H)-quinazolinona apresentou elevada atividade frente as células de câncer de cólon (HCT-8) e glioblastoma (SF295), com 93,9 % e 87.0 % de inibição, respectivamente. O outro enfoque da tese envolveu a otimização da produção de sesquiterpenos aristoloquenos por linhagens de B. cinerea, seguido de estudo biossintético destes produtos naturais através de experimentos de incorporação de precursores isotopicamente enriquecidos com 2H (deutério) e 13C (carbono treze). As análises dos dados obtidos de RMN de 2H e de 13C do sesquiterpeno majoritário indicaram que a biossíntese desta substância ocorre pela via do mevalonato (MVA). Os resultados também sugeriram o possível envolvimento da via do metil-eritritolfosfato ou 1-desoxi-D-xilulose-fosfato (MEP/DPX) na biossíntese deste sequiterpeno. Estes resultados podem contribuir para o planejamento racional de fungicidas seletivos com aplicação na agricultura. O trabalho desenvolvido mostrou o grande potencial de actinobactérias endofíticas para a obtenção de moléculas bioativas e que estudos usando precursores isotopicamente marcados fornecem informações precisas acerca da origem biossintética de produtos naturais. / The microbial biodiversity from different ecosystems has incited chemical and biological studies with microorganisms from several habitats, leading to the isolation of bioactive natural products with applications in medicine, chemical industry and agriculture, and thus contributing to a better quality of life. This thesis aimed the biopropecting on endophytic actinobacteria and their natural products, and also the biosynthetic study of aristolochene sesquiterpenes in the phytopathogenic fungus Botrytis cinerea. A total of 41 actinobacterial strains were isolated of two Asteraceae species (Thitonia diversifolia and Lychnophora ericoides) for the bioprospecting study. The rDNA sequencing showed predominancy of Streptomyces genus. All the strains were cultured on rice medium, and the ethanolic extracts were screened in cytotoxity and antimicrobial assays. As a result, 58.5% of the extracts showed activity in al least one bioassay. The strains Streptomyces cattleya RLe 4 and Streptomyces sp. RLe 8 were selected for scale up cultures, isolation and identification of bioactive compounds. Different chromatographic methods were applied for the isolation of compounds, and structural analysis were based on 1H and 13C nuclear magnetic resonance and mass spectrometry data. Four compounds were isolated from S. cattleya RLe 4: 2- hydroxybenzamide, desferrioxamine E, 1-(3\',4\'-dimethoxyphenyl)-1-propanone, and 1-(3\',4\'- dimethoxyphenyl)-1-etanone. Ten compounds were isolated from Streptomyces sp. Rle 8: benzamide, 3-hydroxybenzamide, 3-hydroxy-4-methoxybenzamide, 4-hydroxy-3- methoxybenzamide, 2-phenylacetamide, two isomers of 3,4-dihydro-3,4,6,8-tetrahydroxy- 1(2H)-naphthalenone, 2,3-dihydro-2,2-dimethyl-4(1H)-quinazolinone, and desferrioxamine B. Compound 2,3-dihydro-2,2-dimethyl-4(1H)-quinazolinone showed high antiproliferative activity against colon cancer cells (HCT-8) and glioblastoma cells (SF295), with 93.9 and 87.0% of inhibition, respectively. The second focus of the thesis involved the optimization of aristolochene sesquiterpenes production by two strains of B. cinerea, followed by the biosynthetic study through feeding experiments with 2H (deuterium) and 13C isotopically labeled precursors. The 2H and 13C NMR obtained data showed that the biosynthesis of the sesquiterpene proceeds by the mevalonate pathway (MVA). The results also suggested the possible participation of the non mevalonate pathway, methylerytritol phosphate ou 1-deoxy- D-xylulose phosphate (MEP/DXP), in the biosynthesis. These results might contribute to the rational design of selective fungides with application in agriculture. This thesis showed the endophytic actinobacteria as promising sources of bioactive natural products, and also showed that the isotopically labeled feeding experiments give reliable information about the natural products biosynthetic pathways.

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No. of bitstreams: 2 2a_Dissertação Cristine - Correção final (1).pdf: 2629972 bytes, checksum: 213b057f595c42869e0c693389458afc (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-07-26 / As agriculturas de base ecológica têm como pressupostos o conhecimento, o uso e a preservação da biodiversidade local. O Brasil é um país formado por seis Biomas distintos e detentor de ampla biodiversidade e, assim, é fundamental considerar a influência dos fatores ambientais na composição fitoquímica dos óleos essenciais. Não só como um apelo comercial, mas também como uma estratégia de sustentabilidade dos agroecossistemas, o cultivo de plantas aromáticas para extração de óleo essencial ou uso como técnicas de manejo em sistemas de produção de base ecológica constitui importante estratégia para o desenvolvimento rural sustentável. Neste sentido, o estudo objetivou caracterizar as fases fenológicas de Tagetes minuta L., conforme a época de plantio, correlacionando os fatores edafoclimáticos ao desenvolvimento e composição fitoquímica do óleo essencial da espécie. Para tanto, a pesquisa foi realizada em duas etapas distintas, a primeira referiu-se à bioprospecção de populações de Tagetes minuta L. em dez municípios da região sul do Rio Grande do Sul, onde o método utilizado para extração do óleo essencial foi a destilação água-vapor. A segunda etapa tratou da instalação, condução e avaliação do experimento realizado na Embrapa Clima Temperado, Estação Experimental Cascata, Pelotas, RS (31°37’15” S; 52°31’24” O; 180 m a.n.m.). O plantio foi realizado em quatro épocas distintas para determinação das fases fenológicas da espécie e a extração do óleo essencial realizada pelo método de hidrodestilação. O óleo essencial oriundo de plantas coletadas e cultivadas caracterizou-se pela presença dos compostos majoritários cis-tagetona (37,47%), cis-β-ocimeno (35,76%), dihidrotagetona (14,19%), trans-tagetona (3,24%) e limoneno (3,16%). O ciclo de desenvolvimento da época 2 foi 29 dias mais curto que a época 1. As duas primeiras épocas de plantio 20 de dezembro e 18 de janeiro alcançaram o estadio de desenvolvimento reprodutivo no mesmo período. Considerando o alto rendimento de óleo essencial e o tempo de duração do ciclo, a melhor época para o cultivo de Tagetes minuta L. foi 18 de janeiro. / Ecological-based farms have as their presuppositions the knowledge, use and preservation of local biodiversity. Brazil is a country made up of six distinct Biomes and has a wide biodiversity and, therefore, it is fundamental to consider the influence of environmental factors on the phytochemical composition of essential oils. Not only as a commercial appeal but also as a sustainability strategy for agroecosystems, the cultivation of aromatic plants for extraction of essential oil or use as management techniques in ecologically based production systems is an important strategy for sustainable rural development. In this sense, the study aimed to characterize the phenological phases of Tagetes minuta L., according to the planting season, correlating the edaphoclimatic factors to the development and phytochemical composition of the essential oil of the species. In order to do so, the research was carried out in two distinct stages, the first one referring to the bioprospection of Tagetes minuta L. populations in ten municipalities in the southern region of Rio Grande do Sul, where the method used to extract the essential oil was distillation water vapor. The second stage dealt with the installation, conduction and evaluation of the experiment performed at Embrapa Temperate Climate, Cascata Experimental Station, Pelotas, RS (31 ° 37'15 "S, 52 ° 31'24" W, 180 m a.m.). The planting was carried out in four distinct seasons to determine the phenological phases of the species and the extraction of essential oil by the hydrodistillation method. The essential oil from the collected and cultivated plants was characterized by the presence of cis-tagetone (37.47%), cis-β- ocimene (35.76%), dihydrotagetone (14.19%), trans-tagetone (3.24%) and limonene (3.16%). The development cycle of season 2 was 29 days shorter than season 1. The first two planting seasons December 20 and January 18 reached the stage of reproductive development in the same period. Considering the high yield of essential oil and the duration of the cycle, the best time for the cultivation of Tagetes minuta L. was January 18.

The economics of algae biofuels and bioproducts would be improved by increased biomass productivity. Two studies on this potential are described in this thesis – one on a locally isolated filamentous yellow-green alga and the other on a planktonic strain genetically improved via selective enrichment. Polycultures have been viewed as productive, stable, and, in some cases, harvestable by natural bioflocculation. Local native strains might have higher productivity than culture collection strains because they are already adapted to local outdoor conditions. In this study, the filamentous yellow-green alga Tribonema minus was isolated from a local volunteer polyculture. Its productivity as a monoculture was compared to a volunteer polyculture in a year of thrice-weekly samples. The study was conducted in duplicate 1,000-L, 3.5-m2 outdoor raceway ponds fed with nitrified and filtered reclaimed wastewater. T. minus monocultures were more productive (17.6 ± 0.5 g/m2-d; mean ± range) than the polyculture (13.3 ± 0.4 g/m2-d). The T. minus monocultures were stable, growing for an average of 38 days before significant contamination with other algae genera, at which point the cultures were restarted. The annual average biochemical composition, in percent of ash-free dry-weight, of the T. minus cultures was 28.3 ± 0.4% (mean ± std. dev.) carbohydrates, 37.6 ± 0.7% proteins, and 6.1 ± 0.3% lipids. Eicosapentaenoic acid, a valuable nutritional omega-3 fatty acid, comprised 0.3% to 4% of the ash-free dry-weight and was the predominant fatty acid methyl ester measured. In summary, an alga isolated from a volunteer polyculture was more productive as a monoculture than the originating polyculture. The monoculture biomass contained a valuable nutritional fatty acid. Scenedesmus obliquus was subjected to UV mutagenesis followed by cultivation in benchtop bubble columns at high dilution rates to select for cultures (cultigens) that grew faster than the wild-type. Fast growing cultigens were transferred to 1,350-L outdoor raceways ponds for productivity measurement. Cultigen and wild-type cultivation was conducted on reclaimed wastewater media in coastal central California for seven months. One cultigen, MBE 501, had 23% higher productivity than the S. obliquus wild-type (11.5 ± 0.02 vs. 9.4 ± 0.6 g/m2-d) during July 28-December 30, 2019. MBE 501 had been subjected to 1:400 and 1:200 dilutions twice per week for the first two months and last five months of selection, respectively, and went through 289 generations in the lab. Compared to a volunteer polyculture (14.4 ± 1.3 g/m2-d), MBE 501 was not as productive on average. This study demonstrated that high dilution rates in lab cultures can select for cells that are more productive in outdoor raceways. Genetic comparison of MBE 501 and its wild-type are pending.

<p>  </p> <p>Actinobacteria are a large and diverse group of bacteria that are known to produce a wide range of secondary metabolites, many of which have important biological activities, including antibiotics, anti-cancer agents, and immunosuppressants. The biosynthesis of these compounds is often highly regulated, with many natural products being produced at very low levels in laboratory settings. Environmental factors, such as small molecule elicitors, can induce the production of secondary metabolites. These elicitors can be natural products, including antibiotics or hormones, or synthetic compounds. The use of small molecule elicitors to induce the production of secondary metabolites has several advantages. First, addition of elicitors to fermentation media can result in increased titers of known natural products. Second, elicitors can enable the discovery of novel natural products typically produced at undetectable levels. In recent years, there has been a growing interest in the use of small molecule elicitors to induce the production of secondary metabolites from actinobacteria, especially for the discovery of “silent” natural products. In this work, we sought to expand on the method of chemical induction by utilizing oxytetracycline at a sub-MIC concentration to induce secondary metabolite production in Streptomyces. We have shown that translation-inhibiting antibiotics, specifically oxytetracycline, have a profound effect on the production of coeliomycin P1, actinorhodin, and calcium-dependent antibiotics (CDAs) in S. coelicolor and S. lividans. The expression of actinorhodin in S. lividans under these conditions is unique, unlike its counterpart, S. coelicolor, which can produce actinorhodin under standard conditions. In addition to the increased production of known secondary metabolites, we have also demonstrated the induction of BGCs in several other strains of Streptomyces, which were observed via LC-MS. </p> <p>In addition to exploring antibiotics as elicitors we have explored the traditional approach of natural product discovery by taking an bioactivity guided approach. Several strain that we isolated from soil collect of Hawaii were screened for activity against several pathogenic strains primarily looking for which strain will inhibit the growth of a. baumannii, which is an intriguing target because the rate of resistance to common antibacterial medication is rising and it’s membrane composition is vastly different compared to other gram negative bacterium like E.coli. From this preliminary screening 1 strain (Streptomyces sp. CS62) out of the 8 that tested exhibited the desired biological activity. The supernatant of Streptomyces sp .CS62 was processed and screen by LC-MS to gain insight on the type of molecules that Streptomyces CS62 could produce. Upon our initial screening process none of the masses observed in the mass spec were matched to knowns. However, after 2D NMR analysis and genomic analysis it was unveiled that Streptomyces sp. CS62 produces factumycin a known antibacterial agent that targets A.baumannii .This unfourtunate turn of events illustrates the issues with natural product discovery and the need to improve natural product databases.</p> <p>In conjunction to discovering a novel producer of factumycin we are also investigating the production of antifungal compounds from Staphylococcus lugdunensis  a commensal strain that modulates the microbiome by producing lugdunin. The supernatant collected of Staphylococcus lugdunensis  is exclusively being test against Candida auris due to the immense health risk it possess to society because of its innate resistance to many antifungal drugs and its ability to rapidly gain resistance to other classes of antifungals.</p> <p>In addition to exploring the influence of antibiotics on secondary metabolite production and using bioactivity as a guide to discovering antibiotics. We are evaluating the soils collected from unique environments as potential sources for novel natural products. Specifically, we are evaluating the biosynthetic potential of bacteria from ore-forming environments, specifically fluorspar and topaz mines. Soils from ore-forming environments tend have low pH, high saline content, low water holding capacity, and poor nutrient availability. Therefore, ore-forming environments pose a hostile environment for life. To date, no one has explored the natural product potential, or the bacterial diversity, exhibited in these harsh environments. To assess the bacterial diversity, bacteria were isolated from various ore-forming environments using a procedure that is selective for actinobacteria. Following bacterial isolation, genomic DNA was isolated and 16s rRNA gene sequencing was performed to gauge the type of bacteria that were isolated. To stimulate secondary metabolite production, bacteria were then subjected to 7 different media conditions. The supernatant collected from these media conditions were tested against ESKAPE pathogens utilizing the CTSI broth microdilution assay. LC-MS MS analysis was performed for samples exhibiting biological activity. GNPS molecular networking was then utilized to determine potential molecules present in each sample.  Through this process we were able to identify one strain, which we named Streptomyces sp. S1A that exhibited a board range of biological activity (anticancer and antibacterial) and possess a wide array of biosynthetic gene clusters ranging complex macrolides (PKS and NRPS) to terpenes. </p> <p>In summary this multifaced approach to natural product discovery may lead to the discovery of novel antibiotics, enable us to increase production of known or unknown antibiotics through chemical induction, and the characterization of metabolites from Streptomyces sp. S1A will shed insight on the biochemical potential of organisms that inhabit ore-forming environments </p>

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Made available in DSpace on 2015-04-22T22:02:16Z (GMT). No. of bitstreams: 1 Joelma Moreira.pdf: 4509931 bytes, checksum: bba95bccb3eff996782f9bce07ed8f0a (MD5) Previous issue date: 2009-10-28 / Fundação de Amparo à Pesquisa do Estado do Amazonas / The Amazon is one of the largest possessors of plant biodiversity on the planet. The constituents of the flora have enormous potential in the pharmaceutical, cosmetics, perfumes and essences, among others. This study aimed to search for Amazonian species of the Lauraceae potentially useful both therapeutically and commercially, holding alkaloidal composition or aromatic potential. The selected species were collected in Adolpho Ducke Forest Reserve, for this study were: Aniba panurensis, Aniba rosaeodora, Dicypellium manausense, Endlicheria chalisea, Licaria cannella angustata, Licaria martiniana, Mezilaurus duckei, Mezilaurus itauba, Ocotea nigrescens, Ocotea splendens, Rhodostemonodaphne negrensis, Rhodostemonodaphne parvifolia, Pleurothyrium vasquezii e Sextonia rubra. Of the species selected the ones with some chemical study are A. rosaeodora, A. panurensis and M. itauba. The essential oils were obtained by hydrodistillation and analyzed for their chemical composition (GC-FID, GC-MS and 1H NMR), where the -caryophyllene was the major constituent of most oils: leaves of A. panurensis (33.5%), D. manausense (39.5%), M. itauba (51.8%), O. nigrescens (37.9%) and O. splendens (51.0%); leaves and branches of L. martiniana (41.7% and 21.4%, respectively) and R. parvifolia (41.3% and 16.2%, respectively). For the other major oils constituents were caryophyllene epoxide, santalenone, -eudesmol, -pinene, germacrene D, p-cymene, -elemene, -copaene and linalool (89.4%) which is the main substance found in species A. rosaeodora. When testing inhibitory activity of free radicals, all essential oils showed inhibition of free radical DPPH qualitatively. However, quantitative analyses of active concentrations were very high, exceeding 700 g/mL. Only the essential oils of branches of L. martiniana and leaves of M. duckei and R. parvifolia showed inhibitory activity of acetylcholinesterase. Tests of platelet inhibition resulted in little potential antiplatelet agent with inhibition below 20%. Extracts of the genus Aniba, Licaria, Mezilaurus, Ocotea and Rhodostemonodaphne obtained by extraction with ethanol 100%, were subjected to phytochemical screening in which the presence of phenols, tannins, flavanones, saponins, steroids and alkaloids were shown to be frequent. The ethanol extracts of the species of A. panurensis, S. rubra and L. martiniana showed the highest antioxidant activity against DPPH free radical. The acetylcholinesterase inhibitory activity was higher in extracts of the Aniba and Licaria genus. Extracts of the Licaria genus were showed better activities against tumor cells SF-295 (human glioblastoma), HCT-8 (human colon) and MDAMB-435 (human breast). And for the bioassays against Leishmania amazonensis and Trypanosoma cruzi extract of leaves of A. panurensis showed the best activity (67 g/mL e 79 g/mL, respectively). The extracts that showed better biological activities should be fractionated for the isolation of active principles and retested / A Amazônia é uma das maiores detentora da biodiversidade vegetal do planeta. Os constituintes de sua flora apresentam enorme potencial nas indústrias farmacêuticas, de cosméticos, perfumes e essências, dentre outras. O presente trabalho teve por objetivo a pesquisa de espécies amazônicas da família Lauraceae potencialmente úteis tanto terapeuticamente quanto comercialmente, que possuam composição alcaloídica ou potencial aromático. As espécies selecionadas, coletadas na Reserva Florestal Adolpho Ducke, para este estudo foram: Aniba panurensis, Aniba rosaeodora, Dicypellium manausense, Endlicheria chalisea, Licaria cannella angustata, Licaria martiniana, Mezilaurus duckei, Mezilaurus itauba, Ocotea nigrescens, Ocotea splendens, Rhodostemonodaphne negrensis, Rhodostemonodaphne parvifolia, Pleurothyrium vasquezii e Sextonia rubra. Das espécies selecionadas as únicas que possuem algum estudo químico são A. rosaeodora, A. panurensis e M. itauba. Os óleos essenciais foram obtidos através de hidrodestilação e analisados quanto suas composições químicas (CG-DIC, CG-EM e RMN de 1H), em que o -cariofileno foi o constituinte majoritário da maioria dos óleos: folhas de A. panurensis (33,5%), D. manausense (39,5%), M. itauba (51,8%), O. nigrescens (37,9%) e O. splendens (51,0%); folhas e galhos de L. martiniana (41,7% e 21,4%, respectivamente) e R. parvifolia (41,3% e 16,2%, respectivamente). Para os demais óleos os principais constituintes foram epóxido de cariofileno, santalenona, -eudesmol, -pineno, germacreno D, p-cimeno, -elemeno, -copaeno, e o linalol (89,4%) que é a principal substância encontrada na espécie A. rosaeodora. Nos ensaios de atividade inibidora de radicais livres, todos os óleos essenciais apresentaram inibição do radical livre DPPH qualitativamente. Entretanto, na análise quantitativa as concentrações ativas foram muito altas, superiores a 700 g/mL. Apenas os óleos essenciais dos galhos de L. martiniana e das folhas de M. duckei e R. parvifolia apresentaram atividade inibidora de acetilcolinesterase. Os ensaios de inibição plaquetária resultaram em pouco potencial antiagregante plaquetário, com inibição inferior a 20%. Os extratos brutos, dos gêneros Aniba, Licaria, Mezilaurus, Ocotea e Rhodostemonodaphne, obtidos por extração com etanol 100%, foram submetidos a uma triagem fitoquímica na qual a presença de fenóis, taninos condensados, flavanonas, saponinas, esteróides e alcalóides mostraram-se frequente. Os extratos etanólicos das espécies de A. panurensis, S. rubra e L. martiniana foram os que mostraram maior atividade antioxidante frente ao radical livre DPPH. A atividade inibidora de acetilcolinesterase foi maior nos extratos dos gêneros Aniba e Licaria. Os extratos do gênero Licaria foram os que apresentaram melhores atividades contras as células tumorais SF-295 (glioblastoma humano), HCT-8 (cólon humano) e MDAMB-435 (mama humana). E para os bioensaios contra Leishmania amazonensis e Trypanosoma cruzi o extrato das folhas de A. panurensis foi o que apresentou melhor atividade (67 g/mL e 79 g/mL, respectivamente). Os extratos que apresentaram melhores atividades biológicas deverão ser fracionados para o isolamento dos princípios ativos e novamente testados

Submitted by (edna.saturno@ufrpe.br) on 2016-12-13T15:12:46Z No. of bitstreams: 1 Joao Tiago Correia Oliveira.pdf: 2100029 bytes, checksum: 18f980542ee8d080c1ef8dc66665d64f (MD5) / Made available in DSpace on 2016-12-13T15:12:46Z (GMT). No. of bitstreams: 1 Joao Tiago Correia Oliveira.pdf: 2100029 bytes, checksum: 18f980542ee8d080c1ef8dc66665d64f (MD5) Previous issue date: 2012-07-31 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The genus Brachiaria grasses are widely used throughout Brazil, enduring a series of restrictive limits and conditions of use, which corrobora para that is widely cultivated throughout the country. The changes in agricultural systems, mostly aiming at the improvement of environmental quality, in order to know the microbial diversity associated with these systems has been useful for ecosystem sustainability and improvement of production, where the combination of grasses with diazotrophs can provide plant growth and represents a promising alternative for the better development and plant performance. Given the above, the objective of this study was to isolate diazotrophic bacteria potentially associated grasses B. decumbens and B. humidicola cultivated in Agreste region of Pernambuco State, niches in the rhizosphere and endophytic root, evaluating functional and genetic variability. After isolation on semi selective fixation of atmospheric nitrogen (NFB semisolid medium) and in a non-functional selection was the selection of bacterial isolates for characteristics involved with the promotion of plant growth and genetic variability, using the technique of BOX-PCR and PCR-DGGE of nifH. The phenotypic characteristics were analyzed: biological nitrogen fixation (BNF), production of indole acetic acid (IAA) by different biochemical pathways, inorganic phosphate solubilization, production of extracellular enzymes and the quorum sensing molecule. The results obtained evidenced the high and variable functionality of the bacteria associated with these grasses in both media (especially the ones from the middle NFB, with the highest frequency of positive isolates for the evaluated tests), plant species and niches evaluated. In bacteria associated with the grass was observed fixation of atmospheric nitrogen in different salt concentrations in the culture medium, producing different biochemical pathways by EIA with and without the presence of precursor L-tryptophan, solubilization of inorganic phosphate in the presence of different sources of carbon production of extracellular enzymes (cellulase, pectinase and amylase) and quorum sensing molecule. Regardless of the technique used to study the genetic variability was observed high genetic diversity for isolates from both the root and the rhizoplane of grasses B. decumbens and B. humidicola. Thus we conclude that this association bacteria-plant-soil can provide several benefits to the plant, not just the biological nitrogen fixation. Therefore, investments in research using the association grasses/bacteria are essential for the high value they represent in animal feed. / As gramíneas do gênero Brachiaria são largamente utilizadas por todo o Brasil, tolerando uma série de limites e condições restritivas de utilização, o que corrobora para que seja largamente cultivada em todo território nacional. As mudanças ocorridas nos sistemas agrícolas, em sua maioria, têm por finalidade a melhoria da qualidade ambiental, neste sentido conhecer a diversidade microbiana associada a estes sistemas tem sido útil para sustentabilidade dos ecossistemas e o aperfeiçoamento da produção, onde a associação de gramíneas com bactérias diazotróficas pode proporcionar crescimento vegetal e representa uma alternativa promissora para o melhor desenvolvimento e desempenho vegetal. Diante do exposto, o objetivo deste trabalho foi isolar bactérias potencialmente diazotróficas associadas as gramíneas B. decumbens e B. humidicola cultivadas na Região Agreste do Estado de Pernambuco, nos nichos rizosfera e endofítico de raiz, avaliando-se características funcionais e variabilidade genética. Após o isolamento em meio semi seletivo para fixação de nitrogênio atmosférico (meio NFb semi-sólido) e em meio não seletivo ocorreu a seleção funcional dos isolados bacterianos quanto a características envolvidas com a promoção de crescimento vegetal, e a variabilidade genética, através da técnica de BOX-PCR e PCR-DGGE do gene nifH. As características fenotípicas analisadas foram: fixação biológica de nitrogênio (FBN), produção de ácido indol acético (AIA) por diferentes rotas bioquímicas, solubilização de fosfato inorgânico, produção de enzimas extracelulares e da molécula quorum sensing. Nos resultados obtidos ficou evidente a elevada e variável funcionalidade das bactérias associadas a estas gramíneas em ambos os meios (com destaque para os isolados provenientes do meio NFb, que apresentou maior frequência de isolados positivos para os testes avaliados), espécies vegetais e nichos avaliados. Nas bactérias associadas as gramíneas se observou fixação de nitrogênio atmosférico em diferentes concentrações de sal no meio de cultura, produção de AIA por diferentes rotas bioquímicas com e sem a presença do precursor L-triptofano, solubilização de fosfato inorgânico com a presença de diferentes fontes de carbono, produção de enzimas extracelulares (celulase, pectinase e amilase) e molécula quorum sensing. Independente da técnica utilizada para o estudo da variabilidade genética foi observado elevada diversidade genética para os isolados tanto da raiz como do rizoplano das gramíneas B. decumbens e B. humidicola. Desta forma é possível concluir que tal associação bactéria-planta-solo pode proporcionar vários benefícios à planta, não apenas a fixação biológica do nitrogênio. Portanto, investimentos em pesquisas utilizando a associação gramíneas forrageiras/bactérias são de fundamental importância pelo elevado valor que estas representam na alimentação animal.

Made available in DSpace on 2014-08-20T13:32:55Z (GMT). No. of bitstreams: 1 dissertacao_claudio_fernando_oliveira.pdf: 4911756 bytes, checksum: 14504fcb4c0f3469413049df6651ec64 (MD5) Previous issue date: 2010-07-22 / Factors like the frequent worry with the environmental impact cause by the discard of petrochemical plastic waste in the environment due to its low degradability and the constant increase of petrol price took to a world search for innovations with the production of bioplastics. They are the polymers produced by microorganisms from the renewable raw material with properties similar plastic derived from petrol, such as polypropylene and polyethylene. The more promising bioplastics are the polyhydorxyalkanoates, which comprehend a great polyester family accumulated in the form of intracellular granules by microorganisms like bacteria. The most common type is the poli(3 hydroxybutyrate) P(3HB); this polymer brings great interest for being biocompatible, biodegradable and thermoplastic. The objective of this study was to realize a bioprospecting between Beijerinckia sp. strains from the collection of microorganism culture from CDTec UFPel for the selection of possible producer strains of P (3HB). Initially, 7 strains were cultivated in solid mean, YM and YDC, and colored cell with Sudan Black for Optic Microscopy visualization, when 3 strain were selected for further analysis through Transmission Electronic Transmission; the data revealed a strong evidence of granules P(3HB) production by strain C31 in the cellular multiplication phase. The biopolymer production was evaluated in 3 means, the best result was obtained with F4 mean, being a concentration of 0,93 gl of polymer and content of 34%. The polymer was identified, and measured, by Gas Chromatography as P(3HB). The polymer identity was confirmed for Fourier transform infrared spectroscopy (FT-IR). The spectra revealed characteristic bands of P(3HB), confirming the production of this polymer by C31 strain, which was considered with great power for the industrial production / Fatores como a crescente preocupação com o impacto ambiental causado pelo descarte dos resíduos plásticos petroquímicos no meio ambiente devido sua baixa degradabilidade e o constante aumento do preço do petróleo levaram a uma busca mundial por inovações como a produção dos bioplásticos. Os bioplásticos são polímeros produzidos por microrganismos a partir de matérias-primas renováveis com propriedades semelhantes aos plásticos derivados do petróleo, como polipropileno e polietileno. Os bioplásticos mais promissores são os polihidroxialcanoatos, que compreendem uma grande família de poliésteres acumulados na forma de grânulos intracelulares por microrganismos como as bactérias. O tipo mais comum é o poli(3-hidroxibutirato) (P(3HB); este polímero desperta grande interesse por ser biocompatível, biodegradável e termoplástico. O objetivo deste trabalho foi realizar uma bioprospecção entre cepas de Beijerinckia sp. da coleção de cultura de microrganismos do CDTec UFPel para seleção de cepas possivelmente produtoras de P(3HB). Inicialmente, foram cultivadas 7 cepas em meio sólido, YM e YDC, e as células coradas com Negro de Sudão B para visualização por Microscopia Óptica, sendo selecionadas 3 cepas para posterior análise por Microscopia Eletrônica de Transmissão; os dados revelaram um forte indício da produção de grânulos de P(3HB) pela cepa C31 já na fase de multiplicação celular. Foi avaliada a produção de biopolímero em 3 meios; o melhor resultado foi obtido com o meio F4, sendo uma concentração de 0,93gL-1 de polímero e conteúdo de 34 %. O polímero foi identificado, e dosado, por Cromatografia Gasosa como P(3HB). A identidade do polímero foi confirmada por espectroscopia na região do infravermelho com transformada de Fourier (FT-IR). Os espectros revelaram bandas características de P(3HB), confirmando a produção deste polímero pela cepa C31, que foi considerada com potencialidade para produção industrial.

Por meio do processo conhecido como bioprospecção, propõe-se a aplicação dos lucros obtidos com a comercialização de produtos, como fármacos, em ações para conservação biológica. O objetivo deste trabalho é verificar a importância deste instrumento para a conservação a partir de estudo de caso focado na experiência do Instituto Nacional de Biodiversidade (INBio), na Costa Rica. Os procedimentos metodológicos estão baseados nas estratégias de pesquisa de: a) estudo de caso; b) documental e c) pesquisa bibliográfica. O INBio, uma associação sem fins lucrativos, se empenhou na elaboração do inventário nacional da biodiversidade e firmou diversos contratos de bioprospecção, entre eles o comentado acordo com a indústria farmacêutica Merck. Alguns pontos positivos devem ser destacados a partir dessa experiência: geração de conhecimento sobre os recursos biológicos locais em função do inventário; criação de sistema de controle ao acesso e identificação dos materiais coletados; o aparato jurídico desenvolvido; aprimoramento da capacidade de negociação; disseminação de informação em publicações científicas; atividades voltadas à Educação Ambiental e apoio a pequenas e médias empresas nacionais no desenvolvimento de produtos baseados em recursos biológicos do país. Por outro lado, os dados analisados mostram que tem sido baixo o retorno financeiro do Instituto; os pagamentos não têm sido suficientes para cobrir os gastos. Esta questão está ligada ao fato de que, ainda que tenha começado o recebimento pela comercialização de alguns produtos desenvolvidos internamente, ainda não há produtos desenvolvidos resultantes de contratos com grandes companhias e, portanto, ainda não foram correspondidas as expectativas com relação ao recebimento de royalties. Há deficiência com relação ao controle administrativo dessas atividades por parte do Estado, o que reflete na falta de transparência das fontes disponíveis de informações. A análise dos recursos financeiros obtidos versus repasse de verbas para fins de conservação fica comprometida em razão de informações incompletas ou pela falta de padronização na apresentação dos dados no decorrer dos anos. O desmatamento tem apresentado queda contínua, mas não é possível relacionar este fato com os contratos de bioprospecção, devido ao sistema variado de fontes de contribuições e pagamentos por serviços destinados à conservação biológica naquele país. Ademais, os conhecimentos tradicionais das populações locais estão sendo ignoradas em todo esse processo conduzido pelo INBio. Portanto, até o momento, o que acontece na Costa Rica está mais próximo de representar uma evolução no sentido de empreender medidas em direção ao uso e conservação dos recursos biológicos, porém ainda não representa a solução ou ajuda significativa à conservação biológica naquele país. / Using the process known as bioprospecting, it is proposed the use of the profits obtained with the commercialization of products, such as pharmaceuticals, in biological conservation activities. The objective of this work is to verify the importance of this instrument for conservation, by means of a case study focused in the experience of the National Biodiversity Institute (Instituto Nacional de Biodiversidade) (INBio), in Costa Rica. The methodology is based in the strategies of researching: a) case study; b) documents and c) bibliography research. The National Biodiversity Institute (INBio), a non-profit association which has endeavored to elaborate a national biodiversity inventory and signed various bioprospecting contracts, among them the renowned agreement with Merck pharmaceutical company. Some positive points should be emphasized from this experience: generation of knowledge about local biological resources through inventory; creation of a control system for accessing and identifying the collected materials; the development of a legal apparatus; the improvement of negotiation skills; the dissemination of information through scientific publications, activities oriented to Environmental Education and supporting national small and medium firms in the development of products based on the country\'s biological resources. On the other hand, the data analyzed show low financial return, in spite of the fact that the income for the commercialization of some internally-developed products has begun. Payments have not been enough to cover the Institute\'s expenditures and there are still no developed products resulting from contracts with big companies and, therefore, the expectations relative to the receiving of royalties were still not fulfilled. There is a deficiency relative to the administrative control of these activities by the State, which reflects the lack of transparency of the available sources of information. The analysis of the obtained resources versus the transfer of funds for conservation is jeopardized due to incomplete information or lack of standardization in the presentation of data through the years. Deforestation has declined continuously, but it is impossible to relate this fact to the bioprospecting contracts, due to the diverse system of contribution and payment sources for services destined to the conservation of that country\'s natural resources. Besides, the traditional knowledge of the local population has been ignored in the entire process conducted by INBio. Therefore, for the moment, what happens in Costa Rica is closer to represent an evolution in the sense of taking steps in the direction of using and conserving biological resources, but it still does not represent the solution or significant aid to the biological conservation in Costa Rica.