Orientador: Marcos José Salvador / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-26T06:36:31Z (GMT). No. of bitstreams: 1 Correa_WallaceRibeiro_D.pdf: 6338643 bytes, checksum: a9316650e99827e8384a1a7bacfa1c04 (MD5) Previous issue date: 2014 / Resumo: Pfaffias são plantas da família Amaranthaceae, tribo Gomphreneae, conhecidas como ginseng brasileiro e "Paratudo". São plantas com importância na medicina popular, sendo empregadas no tratamento de várias patologias e algumas espécies de Pfaffia apresentam estudos farmacológicos reportando atividade analgésica, antinociceptiva, anti-inflamatória, antimicrobiana e antitumoral, sugerindo potencial para a busca de agentes biologicamente ativos nestas matrizes vegetais. Assim, este estudo teve por objetivo a prospecção de extratos, frações e substâncias bioativas em Pfaffia townsendii Pedersen e Pfaffia tuberosa (Spreng.) Hicken que apresentam poucos estudos documentados na literatura e são espécies praticamente intocadas sob o ponto de vista fitoquímico e de avaliação de atividades biológicas. O estudo foi monitorado pela avaliação da atividade antioxidante (ensaios Folin-Ciocalteu, redução do radical DPPH e ORACFL); antiproliferativa (frente a linhagens de células tumorais e não tumorais); antimicrobiana (frente a bactérias, leveduras e protozoários); anti-inflamatória (in vitro pelo ensaio da desnaturação da albumina bovina (BSA) e in vivo empregando o modelo de edema de pata e pleurisia em camundongos induzida por carragenina). No estudo fitoquímico, primeiramente, obteve-se o perfil químico dos extratos bioativos (CCDC, CG, CLAE e ESI-MS), sendo possível identificar algumas substâncias conhecidas diretamente nos extratos brutos (desreplicação). Os extratos bioativos com substâncias que não puderam ser identificadas na etapa de desreplicação foram submetidos ao fracionamento biomonitorado e as substâncias isoladas foram identificadas utilizando-se métodos espectroscópicos de análise (UV, RMN 1H e RMN 13C) e espectrometria de massas e as substâncias isoladas foram submetidas à avaliação das atividades biológicas. Os extratos brutos e fases de partição dos extratos etanólicos apresentaram considerável atividade antioxidante nos três ensaios realizados, observando correlação positiva da capacidade antioxidante com o conteúdo de fenólicos totais solúveis. Estas amostras mostraram atividade antiproliferativa frente a linhagens de células tumorais, com valores de concentração mínima necessária para inibição total do crescimento celular, TGI, variando entre 0,42 e 250 ?g/mL para as amostras bioativas e não se mostraram citotóxicas para linhagem de células controle, não tumoral, VERO (TGI ? 250 ?g/mL). Ainda, exibiram atividade antimicrobiana frente a bactérias (com valores de concentração biocida mínima, CBM, entre 0,5 e 2,0 mg/mL), frente a fungos (CBM de 1,0 mg/mL) e frente a protozoários com atividade tripanocida moderada para formas tripomastigotas de Trypanosoma cruzi (com % de tripomastigotas viáveis variando de 4,70 a 69,40% para os extratos brutos a 4,0 mg/mL e para as fases de partição a 2,0 mg/mL) e com atividade leishmanicida para formas amastigotas-like de Leishmania (L.) amazonensis (com % de amastigotas viáveis variando de 0 a 8,10% para os extratos brutos a 1,0 mg/mL e para as fases de partição a 0,5 mg/mL). Os flavonoides isolados patuletina 3-O-B-D-glucopiranosídeo e tilirosídeo apresentaram promissora atividade antiprotozoário com efeito na viabilidade celular tanto para tripomastigotas de T. cruzi, quanto frente à amstigotas de L. (L.) amazonensis. Os extratos apresentaram atividade anti-inflamatória in vitro com inibição da desnaturação da proteína BSA (IC50 entre 41,1 e 400 µg/mL) e em modelo in vivo onde as fases etanólicas da partição do extrato etanólico das duas plantas estudadas e os flavonoides isolados patuletina 3-O-B-D-glucopiranosídeo e tilirosídeo inibiram o edema de pata induzido por carragenina e a migração de leucócitos na cavidade intrapleural induzido por carragenina, sendo também mensurado o extravasamento de proteínas pela reação de Bradford. O estudo fitoquímico monitorado pelas atividades biológicas e antioxidantes mostrou-se efetivo, sendo possível identificar cinco esteroides, três triterpenoides, três acetatos de triterpenoides, quatro ácidos orgânicos e três flavonoides nas amostras bioativas. Pode-se também isolar dois flavonoides (Patuletina 3-O-B-D-glucopiranosídeo e Tilirosídeo). Os resultados obtidos justificam, pelo menos em parte, a utilização popular de espécies do gênero Pfaffia e confirma o potencial da família Amaranthaceae como fontes de moléculas bioativas e insumos para a saúde, sendo importante a continuidade dos estudos com as amostras bioativas mais promissoras com vistas a aplicações farmacológicas, biotecnológicas e clínicas / Abstract: Pfaffias are plants of the Amaranthaceae family, Gomphreneae tribe, known as Brazilian ginseng and "paratudo". Plants have great importance in folk medicine, being used in the treatment of various diseases. Some species of Pfaffia exhibit pharmacological studies reporting anti-nociceptive, anti-inflammatory, antimicrobial, analgesic and antitumor activities and suggesting a potential for biologically active agents in these vegetable matrices. Thus, this study aimed to explore for extracts, fractions and bioactive compounds in Pfaffia townsendii Pedersen and Pfaffia tuberosa (Spreng.) Hicken, which show little documented data in the literature studies and are species virtually untouched from the point of view of phytochemical evaluation of biological activities. The study was monitored by the evaluation of the activities: antioxidant (Folin-Ciocalteu assays, reducing the DPPH and ORAC-FL); antiproliferative (against strains of tumor and non-tumor cells); antimicrobial (against bacteria, yeasts and protozoa); anti-inflammatory (in vitro assay for denaturation of bovine serum albumin (BSA) and in vivo study using the model of paw edema and carrageenan-induced pleurisy in mice). In the first step, the phytochemical, it was obtained the chemical profile of the bioactive extracts (TLC, GC, HPLC and ESI-MS), and it was possible to identify some known substances directly from crude extracts (dereplication). The bioactive extracts with substances that could not be identified in the dereplication step were subjected to bioassay-guided fractionation and isolated substances were identified using spectroscopic analysis methods (UV, 1H and 13C NMR) and mass spectrometry and isolated compounds were subjected to evaluation of bioactivity. Extracts and phases of partition of ethanol extracts showed significant antioxidant activity in the three trials, observing positive correlation between antioxidant capacities with the phenolic content. These samples showed antiproliferative activity against tumor cell lines with minimum concentration values required for complete inhibition of cell growth, TGI, ranging between 0.42 and 250 ug/mL for bioactive samples and were not cytotoxic to the cell line control, non-tumor VERO (TGI ? 250 ug/mL). They also showed antimicrobial activity against bacteria (with values of Minimum Biocidal Concentration, MBC, between 0.5 and 2.0 mg / ml), against fungi (MBC 1.0 mg mL) and trypanocidal moderate activity against protozoans such as trypomastigote forms of Trypanosoma cruzi (% viable with trypomastigotes ranging from 4.70 to 69.40% for the gross 4.0 mg/mL and extract phases partition to 2.0mg/mL), leishmanicidal activity for axenic amastigotes of Leishmania (L.) amazonensis (% viable with amastigotes ranging from 0 to 8.10% for the crude at 1,0 and 0.5 mg/mL extracts). The flavonoids isolated patuletin 3-O-B-D-glucopyranoside and tiliroside showed promising antiprotozoal activity and effect on cell viability for both T. cruzi tipomastigotas, and amastigotas of L. (L.) amazonensis. The extracts exhibited anti-inflammatory activity in vitro with inhibition of denaturation of BSA protein (IC50 between 41.1 and 400 ug/mL), where the ethanolic phases for the ethanol extract partition and flavonoids isolated patuletin 3-O-B-D-glucopyranoside and tiliroside inhibited in vivo the paw edema induced by carrageenan and the migration of leukocytes in the pleural cavity induced by carrageenan. The extravasation of proteins were also measured by Bradford reaction. The phytochemical study was monitored by biological and antioxidants activities, and was possible to identify five steroids, three triterpenoids, three acetates triterpenoids, four phenolic acids and three flavonoids in bioactive samples. It is as well possible to isolate two flavonoids (patuletin 3-O-B-D-glucopyranoside and tiliroside). The results obtained in this study justify, at least in part, the popular use of the genus Pfaffia and confirm the potential of the Amaranthaceae family as a source of bioactive molecules and its inputs to health. It is important to continue on the studies with the most promising bioactive samples, with goals on the pharmaceutical, biotechnological and clinical applications / Doutorado / Fármacos, Medicamentos e Insumos para Saúde / Doutor em Biociências e Tecnologia de Produtos Bioativos

Photorhabdus are motile Gram-negative bacteria that have a mutualistic association with entomopathogenic Heterorhabditis nematodes. Nematodes vector the bacteria from one insect host to another, while the bacterial symbiont produces toxins and secondary metabolites that kill that the insect host. In this study, we characterize the bacterial symbiont of Heterorhabditis sonorensis, recently discovered in the Sonoran desert. Biochemical and molecular methods including sequence data from five genes: 16s rDNA, gyrB, recA, gltX, dnaN were considered. Evolutionary relationships of this new Photorhabdus subsp. were inferred considering maximum parsimony and Bayesian analyses. We also surveyed for secondary metabolites (SM) produced by this microorganism, considering HPLC and mass spectrometry analyses. SM crude extracts showed activity against the corn ear worm Helicoverpa zea, the root-knot nematode (Meloidogyne incognita), the bacterium Pseudomonas syringae, and the fungus Fusarium oxysporum; and were more toxic that those produced by related species. Results from these studies showed that Photorhabdus l. sonorensis' secondary metabolites have potent antagonistic activity against these plant pathogens.

Microrganismos endofíticos apresentam grande diversidade genética e metabólica, com enorme potencial biotecnológico para aplicações em diferentes áreas. A bioprospecção desses microrganismos possibilita a obtenção de novos bioprodutos agrícolas. Endofíticos podem ser aplicados, por exemplo, para a biorremediação de áreas contaminadas ou para o controle de insetos pragas pela produção de compostos entomotóxicos. Apesar de sua importância, ainda existem poucos estudos voltados à bioprospecção da microbiota endofítica em regiões tropicais. Assim, o presente trabalho pretendeu verificar a diversidade de microrganismos endofíticos cultiváveis em milho e a comparar a diversidade de metabólitos desses endofíticos ao de isolados de Citrus. Também foi explorado o potencial dos diferentes endófitos em degradar os principais inseticidas (lufenuron, spinosad, chlorpyrifos ethyl, lambda-cyhalothrin e deltamethrin) utilizados em lavouras de milho para o controle de lagartas de Spodoptera frugiperda (J.E. Smith, 1797) (Lepidoptera, Noctuidae), para exploração dos mesmos em programas de biorremediação. Finalmente, foi avaliado o potencial entomotoxida dos extratos orgânicos dos endofíticos estudados até a identificação das moléculas ativas. Foram isolados 30 microrganismos endofíticos de milho (27 bactérias e 3 fungos), os quais foram comparados aos endófitos de citros do acervo do Laboratório de Genética de Microrganismos \"Prof. João Lúcio de Azevedo\". Dos isolados obtidos, sete apresentaram capacidade de metabolização dos cinco inseticidas testados, sendo a maioria deles capaz de metabolizar pelo menos um dos inseticidas testados. Ensaios de bioatividade dos extratos orgânicos do cultivo dos endofíticos contra lagartas de S. frugiperda, levaram à identificação de extratos que induziram 100% de mortalidade de lagartas do terceiro ínstar. O isolamento e a caracterização da molécula ativa do extrato produzido pelo isolado LGM-CR-JM-02 - Streptomyces badius, permitiu a identificação da valinomicina (CL50 = 4,38 Mi g/mL) como molécula ativa. Assim, demonstramos que a comunidade de endofíticos pode reunir grande diversidade biológica e, principalmente, metabólica, sendo uma excelente oportunidade para exploração de microrganismos com potencial para biorremediação e produção de compostos com atividade inseticida / Endophytes are microorganisms with great genetic and metabolic diversity, and with a huge biotechnological potential for applications in different areas. The bioprospection of these microorganisms makes possible to obtain new agricultural bioproducts. Endophytes may be applied, for example, to bioremediation of contaminated areas or to the control of pest insects with entomotoxic compounds. Despite its importance, there are few studies focused on the bioprospection of endophytic microbiota in tropical regions. Thus, we investigated the biological diversity of culturable endophytes from maize and compared the diversity of metabolites endophytes from maize and Citrus produced. We also explored the potential of different endophytes to grow in minumum medium containing one of the main insecticides (lufenuron, spinosad, chlorpyrifos ethyl, lambda-cyhalothrin and deltamethrin) applied in maize crops to control the caterpillars Spodoptera frugiperda (J.E. Smith, 1797) (Lepidoptera, Noctuidae) as the only source of carbon in order to evaluate their potential use in bioremediation programs. Finally, we evaluated the entomotoxic potential of the organic extracts of the endophytes and the fractions up to the identification of the active molecules. In total, thirty maize endophytes (27 bacteria and 3 fungi) and five endophytes isolated from citrus that were deposited at the collection of the Laboratory of Genetics of Microorganisms \"Prof. João Lúcio de Azevedo\" were investigated. Seven out of 35 endophytes tested metabolized the five insecticides used, and most of them were able to metabolize at least one of the tested insecticides. Bioactivity assays of the organic extracts produced from cultured endophytes were fed to third instars of S. frugiperda causing 100% mortality. The isolation and characterization of the active molecule produced by one of these isolates (LGM-CR-JM-02 - Streptomyces badius) led to the identification of valinomycin (LC50 = 4.38 Mi g / mL) as the active molecule. Thus, we demonstrated that endophytes can gather great biological and metabolic diversities, demonstrating endophytes are an excellent opportunity for the identification of microorganisms with potential for bioremediation and production of compounds with insecticidal activity

Palm Oil is the highest produced edible oil globally, with over 66 million tonnes produced annually. It has been estimated that up to 50% of all products sold in the supermarket contain palm oil in some form. Palm oil has attractive properties such as a high melting point and texture due to a balanced ratio of unsaturated and saturated fatty acids. It contains approximately 40% oleic acid (monounsaturated fatty acid), 10% linoleic acid (polyunsaturated fatty acid), 45% palmitic acid and 5% stearic acid (saturated fatty acid), that results in an edible oil that is suitable for use in a variety of food, detergent and cosmetics products. In addition, palm oil is the least expensive oil produced due to its high productivity and extensive production. Due to the high demand for the product, vast amounts of rainforest have been cleared to make way for more plantations, reducing biodiversity and releasing huge levels of carbon dioxide into the atmosphere. There is a clear need for an alternative lipid that can match palm oils properties but can be produced sustainably. Recent work suggests that some yeasts are capable of producing a similar oil to palm oil and can be grown on waste resources. In this thesis a novel bioprospecting protocol was developed to isolate yeasts that can survive the harsh conditions necessary for industrial biotechnology. In this way a vineyard and the local area was sampled for yeasts which were then cultured under extremes of pH, multiple sugars and inhibitors caused from the breakdown of lignocellulose. The wild yeast were cultured in four stages: minimal medium with Lysine; minimal medium with inhibitors; minimal medium with xylose as sole carbon-source; and lastly minimal medium with only arabinose and cellobiose as carbon-sources. Only strains that survived each stage were taken forward to the next, to isolate species that were truly suited to these conditions. Out of the estimated 1000s of strains screened this resulted in 12 strains of yeast, mostly in the Metschnikowia pulcherrima, group being able to cope with the conditions. The 12 strains were further analyzed by culturing them in an array of 4 different model lignocellulosic feedstocks namely wheat straw, corn Stover, sugarcane bagasse, and palm kernel cake hydrolysates. Other conditions incorporated in these analysis were a range of pH from pH 1.5 to pH 7.0; four levels of a mixture of 5 inhibitors; and two different temperatures. All of the 12 strains showed similar behaviour where inhibitor tolerance was only marked at higher pH, and at low pH the strains could not grow at all. Though all strains were able to grow on the hydrolysate models, even those with little glucose and/or xylose content. The lipid profile of the strains was also assessed and proved to be similar to most terrestrial crops, with suitable lipid profiles for a rapeseed oil, and in some cases palm oil substitute. Lastly, to further evaluate the accurate identification of the strains as there are some ambiguity in the Metschnikowia pulcherrima group, we applied an approach only widely used for Pathogenic Bacteria/Yeast identification, Multilocus Sequence Typing (MLST). Using 25 strains (7 of this collection), 6 type species and some isolates from the original culture collection in Bath. Sequences of 6 genes was analysed using the Bayesian statistical method. The result showed grouping of M. pulcherrima into 3-4 groups 9 different for each gene. M. Corniflorae being the outgroup. In all 3 genes successfully sequenced: M. Fruticola; R6; Mp DAH 3; and ICS48 were consistently shown to be clonal. The work presented here demonstrates a new method for bioprospecting strains capable of isolating strains for industrial biotechnology, and for characterisation of the yeast in the Metschnikowia genus. Some of the yeasts identified were oleaginous, and could potentially be used as a novel source of palm oil substitute.

Submitted by Programa de Pós-graduação em Biotecnologia (mebiotec.ufba@gmail.com) on 2017-04-04T13:16:42Z No. of bitstreams: 1 DISSERTAÇÃO Final - LUISA CARVALHO ANDRADE.pdf: 1905703 bytes, checksum: d26c7953ce5663f0aca0a866c5701328 (MD5) / Approved for entry into archive by Delba Rosa (delba@ufba.br) on 2017-07-06T12:52:48Z (GMT) No. of bitstreams: 1 DISSERTAÇÃO Final - LUISA CARVALHO ANDRADE.pdf: 1905703 bytes, checksum: d26c7953ce5663f0aca0a866c5701328 (MD5) / Made available in DSpace on 2017-07-06T12:52:48Z (GMT). No. of bitstreams: 1 DISSERTAÇÃO Final - LUISA CARVALHO ANDRADE.pdf: 1905703 bytes, checksum: d26c7953ce5663f0aca0a866c5701328 (MD5) / FAPESB / A Baía de Todos os Santos (BTS), segunda maior baía do Brasil, abriga grande diversidade de ambiente, flora e fauna, porém pouco se sabe sobre sua diversidade microbiológica e seu potencial de bioprospecção. Este trabalho teve por objetivo a busca de novas substâncias antimicrobianas, capazes de inibir o crescimento de bactérias patogênicashumanas e de animais marinhos, isolados do solo de cinco praias da BTS. No total foram isoladas 107 bactérias das quais dez apresentaram produção de substâncias antimicrobianas. Após o sequenciamento verificou-se que dos dez isolados, somente oito foram de fato identificadas molecularmente, sendo sete destes membros da família Bacillaceae, grupo ao qual pertencem diversas produtoras de substâncias antimicrobianas. Entre os isolado que apresentaram produção de substâncias inibidoras do crescimento constatou-se que os isolados 21, 50 e 54 tinham maior poder de inibição. O extrato destes três isolados foi confeccionado através da acidificação do sobrenadante livre de células, isolado após o período de incubação. Os extratos do isolado 21 e do isolado 50 foram capazes de inibir a bactéria Klebsiella rizophyla, e o extrato 21 conseguiu ainda inibir a Vibrio vulnificus. Após verificação molecular através de primers específicos de sete substâncias antimicrobianas produzidas por Bacillus, constatou-se que os isolados 21 e 50 são produtores da substância ericina. / The All Saints´s Bay (BTS), the second largest bay in Brazil, where can be founded great diversity of flora and fauna including diversity of landscapes, but unknown about their microbial diversity and potential to bioprospecting. This study aimed to search for new antimicrobial substances that can be able to inhibit the growth of human pathogenic bacterias and marine animals, isolated from sediment from five BTS beaches. Was isolated a total of 107 bacterias where 10 showed antimicrobial substances production. After 16S sequencing, was found that from the ten isolates, only eight were identified by amplification PCR and seuqencing, seven members of the Bacillaceae family, where antimicrobial substances belonging this group are known. It was found that the isolates numbers 21, 50 and 54 had a greater power of inhibition. The extract of these three strains was made by acidification of the cell-free supernatant, isolated after the incubation period. The extracts of the isolate 21 and 50 were able to inhibit the bacteria Kocuria rhizophyla , and the same extract could also inhibit Vibrio vulnificus. In advance, was realized a molecular bioprospecting using specific molecular primers from conserved regions belongs to seven antimicrobial substances produced by Bacillus. It was found that the isolates 21 and 50 are Erycina producing.

Orientador: Bastiaan Philip Reydon / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Economia / Made available in DSpace on 2018-08-22T16:46:03Z (GMT). No. of bitstreams: 1 Pereira_AndreiaMara_D.pdf: 3044162 bytes, checksum: 5b4e661c49e0f4646dca3b29bc6c1898 (MD5) Previous issue date: 2013 / Resumo: A presente tese de doutorado teve como foco apresentar uma proposta de criação de uma instituição gestora para o uso da biodiversidade e dos conhecimentos tradicionais associados à biodiversidade, para as atividades de bioprospecção no Brasil. Para tanto, identificaram-se alguns dos elementos que fazem parte das práticas de bioprospecção, ressaltando as especificidades dos biomas brasileiros e apresentando as peculiaridades dos povos dos conhecimentos tradicionais, indígenas e não indígenas. A tese abordou algumas distinções entre o conhecimento tradicional e o conhecimento científico, tendo em vista que as práticas de bioprospecção envolvem a gestão de acordos que utilizam recursos da biodiversidade, mas também agentes com interesses nem sempre convergentes. Para melhor visualização deste framework, foram apresentadas as características das atividades econômicas da bioprospecção e foram analisados estudos de casos internacionais e do Brasil, com base na Economia dos Custos de Transação aplicados a estes acordos de bioprospecção. Dentro dos casos analisados, identificou-se que há necessidade da criação de instituições intermediárias e de mecanismos pelo governo federal para facilitar a interação entre os agentes. Neste sentido, como resultado, o estudo propõe o desenho de um modelo institucional para a biodiversidade e conhecimentos tradicionais associados para as práticas de bioprospecção no Brasil / Abstract: This doctoral thesis focused on presenting a proposal to establish an institution for managing the use of biodiversity and traditional knowledge associated with biodiversity, bioprospecting activities in Brazil. For this, we identified some of the elements that compose the bioprospecting practices, emphasizing the specificities of Brazilian biomes and explaining the singularities of traditional indigenous and non-indigenous peoples and knowledge. We have also addressed some distinctive features between traditional knowledge and scientific knowledge. All considering that bioprospecting practices involve the management of agreements, which use biodiversity resources, but also agents who not always show converging interests. To provide a better understanding of this framework, we present the characteristics of economic activities related to bioprospection and analyze national and international case studies based on the Transaction Cost Economics applied to these bioprospecting agreements. Based on the cases analyzed, we realized that the Federal Government should create intermediate institutions and mechanisms to facilitate the interaction between the agents. In this regard, and as a result, the study proposes the design of an institutional model for the use of biodiversity and associated traditional knowledge with bioprospecting practices in Brazil / Doutorado / Desenvolvimento Economico, Espaço e Meio Ambiente / Doutora em Desenvolvimento Econômico

In this thesis, I draw attention to how representations of indigenous peoples and knowledges in the rhetoric of bioprospecting weave the people into multiple coordinates of discursive control. Bioprospecting, or the exploration of biological resources in search of valuable genetic and chemical material for commercial use, is portrayed by proponents as an ideal project which benefit all of its stakeholders. I challenge such perception by exposing the power relationships underlying bioprospecting proposals as well as the various interests built into their rhetoric. My particular interest lies in exploring the implications for indigenous peoples whose appearances in bioprospecting proposals are less than voluntary. I make three claims: (1) that the representation of indigenous peoples as stewards of the environment is a role assigned to them, which is then circulated and mobilized within the bioprospecting rhetoric in order to support its arguments concerning biodiversity conservation; (2) that indigenous knowledges of the environment, of medicinal plants in particular, are taken out of their original socio-cultural contexts, utilized, appropriated, and valorized by bioprospectors who construct the rhetoric; (3) that the visibility of indigenous peoples and knowledges, which was heightened as a result of the increased interest taken in controlling them, opens up new opportunities for the people to resist misappropriation and struggle for self-definition. In short, this project takes indigenous peoples and knowledges as the intersection of forces and interests comprising an intricate web of power relationships, within which any participant can attempt to empower oneself either by resisting or manipulating the control to which one is exposed. / Master of Science

Since the soils nutrient composition along with the associated biotic and abiotic factors direct the diversity of the contained microbiome and its potential to produce bioactive compounds, many studies have been focused on sediment types with unique features characteristic of extreme environments. Red Sea lagoon ecosystems are environments with such unique features as they are highly saline. However, not much is known about the potential of their microbiomes to produce bioactive compounds. Here, we explored sediment types such as mangrove mud, microbial mat, and barren soil collected from Rabigh harbor lagoon (RHL) and Al-Kharrar lagoon (AKL) as sources for antibiotic bioprospecting. Our antibiotic bioprospecting process started with a metagenomic study that provides a more precise view of the microbial community inhabiting these sites and serves as a preliminary screen for potential antibiotics. Taking the outcomes of the metagenomic screening into account, the next step we established a library of culturable strains from the analyzed samples. We screened each strain from that library for antibiotic activity against four target strains (Staphylococcus aureus ATCC 25923, Escherichia coli dh5 α, Pseudomonas syringae pv. tomato dc3000 and Salmonella typhimurium dt2) and for the presence of polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) genes known to support synthesis of secondary metabolites that act like antimicrobial agents. The metagenomics study showed a shift in dominant phyla consistent with a historical exposure to hydrocarbon contamination and that AKL unexpectedly displayed more contamination than RHL. This may be due to dominant phyla in AKL being consistent with early hydrocarbon exposure (when contamination levels are still high) and the dominant phyla in RHL being consistent with late hydrocarbon exposure (when contamination levels are lower as a result of an extended period of hydrocarbon degradation). Additionally, RHL samples showed a higher percentage of enzymes associated with antibiotic synthesis, PKS and NRPS. When considering sediment type, mangrove mud samples showed a higher percentage of enzymes associated with antibiotic synthesis than microbial mat samples. Taken together, RHL was shown to be the better location with an increased probability of successful antibiotic bioprospecting, while the best sediment type in RHL for this purpose is microbial mat. Moreover, the phylum Actinobacteria tends to be the common target for research when it comes to antibiotic bioprospecting. However this culture-independent metagenomic study suggests the tremendous potential of Proteobacteria, Bacteroidetes, Cyanobacteria and Firmicutes for this purpose. Taking into account that the metagenomic screen suggests other phyla beyond Actinobacteria for antibiotic bioprospecting, the culture-dependent experiments were not designed to target actinobacteria alone. A total of 251 bacterial strains were isolated from the three collected sediments. Phylogenetic characterization of 251 bacterial isolates, based on 16S rRNA gene sequencing, supported their assignment to five different phyla: Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, and Planctomycetes. Fifteen putative novel species were identified based on a 16S rRNA gene sequence similarity of ≤ 98 % to other strain sequences in the NCBI database. We demonstrate that 52 of the 251 isolates exhibit the potential to produce an antimicrobial effect. Additionally, at least one type of biosynthetic gene sequence, responsible for the synthesis of secondary metabolites, was recovered from 25 of the 52 isolates. Moreover, 10 of the isolates had a growth inhibition effect towards all target strains. In conclusion, this study demonstrated the significant microbial diversity associated with Red Sea harbor/lagoon systems and their potential to produce antimicrobial compounds and novel secondary metabolites. To the best of our knowledge, this is the first study that has analyzed the microbiomes in Red Sea lagoons for antibiotic bioprospecting.

O filo Cyanobacteria constitui um grupo filogeneticamente coerente, embora, apresente grande diversidade morfológica, sendo sua sistemática constantemente revisada. Esses micro-organismos são, ainda, alvos de estudos biotecnológicos em razão da produção de toxinas e na busca por substâncias de interesse farmacológico. Dentre as linhagens analisadas neste estudo, sete sequências do gene rRNA 16S foram geradas e avaliadas com sequências previamente obtidas. Ao menos dois grupos podem representar novos gêneros de cianobactérias, sendo que um grupo demonstra ser endêmico de manguezais brasileiros. Os genes de inibidores de proteases, aeuruginosina, cianopeptolina e microviridina, foram detectados e a produção de aeruginosina foi confirmada por LC-MS nos gêneros Cyanobium e Nostoc. Sequências de aminoácidos do precursor de microviridina indicaram a produção de três novas variantes em quinze linhagens de cianobactérias dos gêneros Cyanobium, Synechococcus, Cyanobacterium, Nodosilinea e Nostoc. O potencial genético para produção de cilindrospermopsina (cyrJ) foi confirmado em vinte e seis linhagens. Em cinco linhagens dos gêneros Cyanobium e Nostoc foram encontrados os genes mcyD, mcyE e mcyG, envolvidos na biossíntese de microcistina. A sequência McyG da linhagem Nostoc sp. CENA175 agrupou-se filogeneticamente com outras de linhagens produtoras de microcistina. Os genes sxtA e sxtI, envolvidos na biossíntese de saxitoxina, foram encontrados em nove linhagens dos gêneros Cyanobium, Oxynema, Leptolyngbya, Nodosilinea e Nostoc. A sequência de SxtI da linhagen Leptolyngbya sp. CENA134 apresentou similaridade >= 70 % com proteínas hipotéticas enquanto as de Nostoc sp. CENA159 e Nostoc sp. CENA160 apresentaram similaridade >= 82 % com O-carbamoiltransferase. Na análise filogenética, a sequência de SxtI da linhagem Nostoc sp. 160 agrupou-se com sequências de linhagens produtoras de saxitoxina. Nas análises químicas, a fração 3 do extrato da linhagem Oxynema sp. CENA135 revelou uma substância com características de ácidos graxos poli-insaturados e a fração 2 do extrato da linhagem Nostoc sp. CENA175 apresentou uma estrutura aromática ligada a uma cadeia alifática. Outros três extratos, obtidos das linhagens Cyanobium sp. CENA157, Nodosilinea sp. CENA183 e Nostoc sp. CENA184 mostraram-se promissores quanto à presença de substâncias nitrogenadas. Os ensaios de bioatividade revelaram que 48 % dos extratos metanólicos inibiram o crescimento de ao menos um isolado de bactéria e/ou levedura. Os extratos das linhagens Cyanobium sp. CENA142 e Cyanobacterium sp. CENA169 foram eficientes contra o crescimento de seis bactérias patogênicas. Nos ensaios de inibição de células tumorais, o extrato de DCM da linhagem Cyanobium sp. CENA154 (100 ?gomL-1) inibiu moderadamente culturas de células 3LL. Os extratos etanólicos de Oxynema sp. CENA135 (20 ?gomL-1) e Cyanobium sp. CENA154 (100 ?gomL-1) inibiram as células CT-26. Em ensaios conduzidos com linhagens de células de glioma (U251), câncer de mama (MCF-7) e câncer de pulmão (NCI-H460), o extrato de DCM da linhagem Cyanobium sp. CENA136 inibiu 50 % do crescimento das respectivas células tumorais nas concentrações 7,8; 27,1 e 14,0 ?gomL-1. Desta forma, além de filogeneticamente diversas, as cianobactérias isoladas de ambiente marinho do Estado de São Paulo constituem fonte promissora de inibidores de proteases, cianotoxinas e substância bioativas com ação antibacteriana, antifúngica e antitumoral. / The phylum Cyanobacteria is a phylogenetically coherent group, although presenting great diversity, and its systematic have been constantly reviewed. These microorganisms are also targets of biotechnological studies due to the production of toxins and the search for novel substances of pharmacological interest. Among the strains analyzed in this study, sequences of the 16S rRNA gene were generated for seven and, than, analyzed with sequences previously obtained. At least two groups may represent new cyanobacterial genera, while a group of Cyanobium proves to be endemic of Brazilian mangroves. Genes of the proteases inhibitors, aeuruginosin, cyanopeptolin and microviridin, were detected and the production of aeruginosin was confirmed by LC-MS for Nostoc and Cyanobium. The amino acid sequences of microviridin precursor indicated the production of three new variants in fifteen cyanobacterial strains of the genera Cyanobium, Synechococcus, Cyanobacterium, Nostoc and Nodosilinea. The genetic potential for production of cylindrospermopsin (cyrJ) was confirmed in twenty-six strains. In five strains of the genera Nostoc and Cyanobium the mcyD, mcyE and mcyG genes, which are involved in the microcystin biosynthesis, were found. The McyG sequence of Nostoc sp. CENA175 was phylogenetically grouped with sequences of microcystin-producing strains. The sxtA and sxtI genes, from saxitoxin biosynthesis, were found in nine strains of the genera Cyanobium, Oxynema, Leptolyngbya, Nodosilinea and Nostoc. The SxtI sequence of Leptolyngbya sp. CENA134 showed similarity >= 70 % with hypothetical proteins, while the sequences of Nostoc sp. CENA159 and Nostoc sp. CENA160 showed similarity >= 82% with O-carbamoyltransferase. In the phylogenetic analysis, the SxtI sequence of Nostoc sp. CENA160 grouped with sequences of strains that produce saxitoxin. In chemical analysis, the fraction 3 of the Oxynema sp. CENA135 extract revealed a substance with poly-unsaturated fatty acids characteristics and the fraction 2 of Nostoc sp. CENA175 extract indicated an aromatic structure, attached to an aliphatic chain. Other three extracts obtained from Cyanobium sp. CENA157, Nodosilinea sp. CENA183 and Nostoc sp. CENA184 were promising for the presence of nitrogenous substances. Bioactivity assays revealed that 48 % of the methanolic extracts inhibited the growth of at least one isolate of bacteria and/or yeast. The extracts of Cyanobium sp. CENA142 and Cyanobacterium sp. CENA169 were efficient against six pathogenic bacteria. In the inhibition assays of tumor cells, the DCM extract of Cyanobium sp. CENA154 (100 mgomL-1) moderately inhibited the growth of 3LL cells. Ethanol extracts of Oxynema sp. CENA135 (20 mgomL-1) and Cyanobium sp. CENA154 (100 mgomL-1) were able to inhibit cultures of CT- 26 cells. In tests conducted with glioma cell lines (U251), breast cancer (MCF-7) and lung cancer (NCI-H460), the DCM extract of Cyanobium sp. CENA136 caused 50 % of growth inhibition, respectively, when used at concentrations of 7.8, 27.1 and 14.0 mgomL-1. Thus, besides their phylogenetically diversity, the cyanobacteria strains from marine environment of the São Paulo state are a promising source of protease inhibitors, cyanotoxins and bioactive compounds with antibacterial, antifungal and antitumor activities.

O filo Cyanobacteria constitui um grupo filogeneticamente coerente, embora, apresente grande diversidade morfológica, sendo sua sistemática constantemente revisada. Esses micro-organismos são, ainda, alvos de estudos biotecnológicos em razão da produção de toxinas e na busca por substâncias de interesse farmacológico. Dentre as linhagens analisadas neste estudo, sete sequências do gene rRNA 16S foram geradas e avaliadas com sequências previamente obtidas. Ao menos dois grupos podem representar novos gêneros de cianobactérias, sendo que um grupo demonstra ser endêmico de manguezais brasileiros. Os genes de inibidores de proteases, aeuruginosina, cianopeptolina e microviridina, foram detectados e a produção de aeruginosina foi confirmada por LC-MS nos gêneros Cyanobium e Nostoc. Sequências de aminoácidos do precursor de microviridina indicaram a produção de três novas variantes em quinze linhagens de cianobactérias dos gêneros Cyanobium, Synechococcus, Cyanobacterium, Nodosilinea e Nostoc. O potencial genético para produção de cilindrospermopsina (cyrJ) foi confirmado em vinte e seis linhagens. Em cinco linhagens dos gêneros Cyanobium e Nostoc foram encontrados os genes mcyD, mcyE e mcyG, envolvidos na biossíntese de microcistina. A sequência McyG da linhagem Nostoc sp. CENA175 agrupou-se filogeneticamente com outras de linhagens produtoras de microcistina. Os genes sxtA e sxtI, envolvidos na biossíntese de saxitoxina, foram encontrados em nove linhagens dos gêneros Cyanobium, Oxynema, Leptolyngbya, Nodosilinea e Nostoc. A sequência de SxtI da linhagen Leptolyngbya sp. CENA134 apresentou similaridade >= 70 % com proteínas hipotéticas enquanto as de Nostoc sp. CENA159 e Nostoc sp. CENA160 apresentaram similaridade >= 82 % com O-carbamoiltransferase. Na análise filogenética, a sequência de SxtI da linhagem Nostoc sp. 160 agrupou-se com sequências de linhagens produtoras de saxitoxina. Nas análises químicas, a fração 3 do extrato da linhagem Oxynema sp. CENA135 revelou uma substância com características de ácidos graxos poli-insaturados e a fração 2 do extrato da linhagem Nostoc sp. CENA175 apresentou uma estrutura aromática ligada a uma cadeia alifática. Outros três extratos, obtidos das linhagens Cyanobium sp. CENA157, Nodosilinea sp. CENA183 e Nostoc sp. CENA184 mostraram-se promissores quanto à presença de substâncias nitrogenadas. Os ensaios de bioatividade revelaram que 48 % dos extratos metanólicos inibiram o crescimento de ao menos um isolado de bactéria e/ou levedura. Os extratos das linhagens Cyanobium sp. CENA142 e Cyanobacterium sp. CENA169 foram eficientes contra o crescimento de seis bactérias patogênicas. Nos ensaios de inibição de células tumorais, o extrato de DCM da linhagem Cyanobium sp. CENA154 (100 ?gomL-1) inibiu moderadamente culturas de células 3LL. Os extratos etanólicos de Oxynema sp. CENA135 (20 ?gomL-1) e Cyanobium sp. CENA154 (100 ?gomL-1) inibiram as células CT-26. Em ensaios conduzidos com linhagens de células de glioma (U251), câncer de mama (MCF-7) e câncer de pulmão (NCI-H460), o extrato de DCM da linhagem Cyanobium sp. CENA136 inibiu 50 % do crescimento das respectivas células tumorais nas concentrações 7,8; 27,1 e 14,0 ?gomL-1. Desta forma, além de filogeneticamente diversas, as cianobactérias isoladas de ambiente marinho do Estado de São Paulo constituem fonte promissora de inibidores de proteases, cianotoxinas e substância bioativas com ação antibacteriana, antifúngica e antitumoral. / The phylum Cyanobacteria is a phylogenetically coherent group, although presenting great diversity, and its systematic have been constantly reviewed. These microorganisms are also targets of biotechnological studies due to the production of toxins and the search for novel substances of pharmacological interest. Among the strains analyzed in this study, sequences of the 16S rRNA gene were generated for seven and, than, analyzed with sequences previously obtained. At least two groups may represent new cyanobacterial genera, while a group of Cyanobium proves to be endemic of Brazilian mangroves. Genes of the proteases inhibitors, aeuruginosin, cyanopeptolin and microviridin, were detected and the production of aeruginosin was confirmed by LC-MS for Nostoc and Cyanobium. The amino acid sequences of microviridin precursor indicated the production of three new variants in fifteen cyanobacterial strains of the genera Cyanobium, Synechococcus, Cyanobacterium, Nostoc and Nodosilinea. The genetic potential for production of cylindrospermopsin (cyrJ) was confirmed in twenty-six strains. In five strains of the genera Nostoc and Cyanobium the mcyD, mcyE and mcyG genes, which are involved in the microcystin biosynthesis, were found. The McyG sequence of Nostoc sp. CENA175 was phylogenetically grouped with sequences of microcystin-producing strains. The sxtA and sxtI genes, from saxitoxin biosynthesis, were found in nine strains of the genera Cyanobium, Oxynema, Leptolyngbya, Nodosilinea and Nostoc. The SxtI sequence of Leptolyngbya sp. CENA134 showed similarity >= 70 % with hypothetical proteins, while the sequences of Nostoc sp. CENA159 and Nostoc sp. CENA160 showed similarity >= 82% with O-carbamoyltransferase. In the phylogenetic analysis, the SxtI sequence of Nostoc sp. CENA160 grouped with sequences of strains that produce saxitoxin. In chemical analysis, the fraction 3 of the Oxynema sp. CENA135 extract revealed a substance with poly-unsaturated fatty acids characteristics and the fraction 2 of Nostoc sp. CENA175 extract indicated an aromatic structure, attached to an aliphatic chain. Other three extracts obtained from Cyanobium sp. CENA157, Nodosilinea sp. CENA183 and Nostoc sp. CENA184 were promising for the presence of nitrogenous substances. Bioactivity assays revealed that 48 % of the methanolic extracts inhibited the growth of at least one isolate of bacteria and/or yeast. The extracts of Cyanobium sp. CENA142 and Cyanobacterium sp. CENA169 were efficient against six pathogenic bacteria. In the inhibition assays of tumor cells, the DCM extract of Cyanobium sp. CENA154 (100 mgomL-1) moderately inhibited the growth of 3LL cells. Ethanol extracts of Oxynema sp. CENA135 (20 mgomL-1) and Cyanobium sp. CENA154 (100 mgomL-1) were able to inhibit cultures of CT- 26 cells. In tests conducted with glioma cell lines (U251), breast cancer (MCF-7) and lung cancer (NCI-H460), the DCM extract of Cyanobium sp. CENA136 caused 50 % of growth inhibition, respectively, when used at concentrations of 7.8, 27.1 and 14.0 mgomL-1. Thus, besides their phylogenetically diversity, the cyanobacteria strains from marine environment of the São Paulo state are a promising source of protease inhibitors, cyanotoxins and bioactive compounds with antibacterial, antifungal and antitumor activities.