As the global population rises to an estimated 9.4bn by 2050, the pressure for food, fuel and freshwater will continue to increase. Current renewable energy technologies are not widely applicable to the transport sector, which requires energy dense liquid fuels that drop into our existing infrastructure. Algal biofuels promise significantly higher yields than plants, without the displacement of valuable agricultural resources and have the potential to meet the global demand for transport fuel. Fossil fuel energy is largely ‘a legacy of algal photosynthesis’, with algae accounting for ~50% of global CO2 fixation today. In addition, these curious organisms show remarkable diversity in form, behaviour and composition. Recently there has been a global resurgence of interest in microalgae as a resource of biomass and novel products. With the present level of technology, knowledge and experience in commercial scale aquaculture, the capital cost and energy investment for algal biomass production is high. Culturing, harvesting and disrupting microalgal cells account for the largest energy inputs with more positive energy balances requiring low energy designs for culture, dewatering and extraction, efficient water and nutrient recycling with minimal waste. Little is known about the variable cell wall of microalgae, which presents a formidable barrier to the extraction of microalgal products. Staining, transmission electron microscopy (TEM) and enzymatic digestion were all utilised in an attempt to visualise, digest and characterise the cell wall of stock strains of Chlorella spp. and Pseudochoricystis ellipsoidea. The presence of algaenan, a highly resistant biopolymer, rendered staining and enzymatic digestion techniques ineffective. TEM revealed that algaenan is present in the outer walls of microalgae in a variety of conformations which appeared to impart strength to cells. A preliminary investigation utilising Fusarium oxysporum f.sp. elaeidis as a novel source of enzymes for the digestion of algaenan has also been described. Methods were developed for the mutagenesis of Chlorella emersonii and P. ellipsoidea using EMS and UV with the intent of generating cell-wall mutants. Although no viable cell wall mutants were produced, a viable pale mutant of C. emersonii was recovered 5 from UV mutagenesis. Growth rates of the pale mutant were significantly slower than the wild type, yet FAME profile was largely unaffected. Fluorescence activated cell sorting (FACS) was also investigated as a means for the rapid screening of mutagenized cells for cell wall mutants. In an attempt to reduce cooling costs of closed-culture systems, temperature tolerant species of microalgae were sought by bioprospecting the thermal waters of the Roman Baths. Numerous methods for isolation and purification of microalgae from the Baths were employed, ultimately yielding seven diverse isolates including cyanobacterial, eukaryotic, filamentous and single celled species. Despite some species possessing an increased tolerance to higher temperatures, none showed marked temperature tolerance coupled with high productivity. Further improvements to the culture conditions may have improved the productivity at higher temperatures. All seven isolates were deposited to the Culture Collection of Algae and Protozoa (CCAP). A variety of extraction methods including soxhlet, beadbeating, sonication and microwaving was investigated for efficacy of extracting fatty acid methyl esters (FAMEs) from C. emersonii. Beadbeating proved most effective in the extraction of FAMEs from C. emersonii. Microwaving showed potential as a rapid method of extraction yet was coupled with degradation of FAMEs, requiring further method development to resolve this issue. Method development has been a significant component of the work described in this thesis.

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.

Bacteria produce a range of surface-active compounds called biosurfactants that reduce the surface tension of liquid and exhibit different oil-water behaviours. These are used in various biotechnological applications including agriculture, cosmetics, medical and food. A recent study has predicted a limit to bacterial surface tension-reducing ability. If this limit exists, it has strong negative consequences in surveys for more active compounds. In this work, the aim is to (i) investigate this prediction more robustly by using chemical media and (ii) study the diversity amongst the best-performing surfactants produced by Pseudomonas spp. with the intention of finding novel surfactants that could be used in different biotechnological applications. A total of 251 Pseudomonas spp. were isolated from soil. Strains were first screened for liquid surface tension-reducing ability (LSTRA) using qualitative drop-collapse assay before quantitative surface tension measurement. Of the 58 LSTRA strains, only 46 significantly reduced the surface tension of sterile media. Individual Distribution Identification (IDI) analysis was used to determine the predicted limit for surfactant activity in KB* and M9Glu media, and results were found to be in agreement with earlier studies. To investigate the chemical structural diversity amongst the best performing surfactants, a collection of 25 key strains producing a limited range of very low surface tension in liquid culture media (~24 – 26 mN/m) were examined. Initial phenotypic characterisation including biochemical, metabolic profiling and 16S rDNA sequencing confirmed strains were a diverse collection of Pseudomonas spp. A series of behaviour assays including emulsion formation, foam stabilisation and oil displacement assays to investigate behavioural diversity among surfactants expressed by the key strains were then undertaken. For the oil displacement, diesel, mineral, vegetable, and used lubricating oils were tested with the underlying aqueous layer containing 0 or 200 mM NaCl at pH 6.0 or 8.0 to reflect a range of biotechnological applications and conditions. Analysis of variance of the emulsion indices, foam stabilisation and oil displacement data showed significant difference in surfactant behaviour among the key surfactant-expressing strains (P < 0.001). Moreover, Hierarchical Cluster Analysis (HCA) was used to produce a constellation dendrogram in which isolates were grouped according to similarities in phenotype and surfactant behaviour. Critically, this resulted in more groups (≥ 5 groups) than could be explained by statistically significant differences in mean surface tensions (previously determined by ANOVA and Tukey-Kramer HSD, alpha = 0.05). These findings provide strong evidence that the key strains were expressing structurally more than one type of surfactant with differing air-water and oil-water behaviours. Similarly, in vitro surfactant characterisation within a range of pH and salt concentrations confirmed diversity among strains (P < 0.001). Investigating surfactant potential by a two-way behaviour cluster dendrogram resulted in more diversity among oil types than the conditions used. These findings indicate that bioprospecting surfactants by screening only the more active compounds is likely to reveal a range of functionalities.

Antimicrobial resistant microorganisms are predicted to pose an existential threat to humanity inside of the next 3 decades. Characterisation of novel acting antimicrobial small molecules from microorganisms has historically counteracted this evolutionary arms race, however the bountiful source of pharmaceutically relevant bioactive specialised metabolites discovered in the Golden era of drug discovery has long since dried up. The clinicians' arsenal of useful antimicrobials is diminishing, and a fresh perspective on specialised metabolite discovery is necessary. This call to action is being answered, in part, through advances in genome sequencing, bioinformatics predictions and the development of next generation synthetic biology tools aiming to translate the biological sciences into an engineering discipline. To expedite our route to new pharmaceutically relevant specialised metabolites using the synthetic biology toolbox several bottlenecks need to be addressed, and are tackled here in. Biosynthetic gene clusters (BGCs) represent blueprints to pharmaceuticals, however to date the vast wealth of knowledge about biosynthetic gene clusters is inconsistently reported and sporadically disseminated throughout the literature and databases. To bring the reporting of BGCs in line with engineering principles we designed and built a community supported standard, the Minimum Information about a Biosynthetic Gene cluster (MIBiG), for reporting BGCs in a consistent manner, and centralised this information in an easy to operate and open access repository for rapid retrieval of information, an essential resource for the bioengineer. Prioritisation represents the next bottleneck in specialised metabolite discovery. Bioinformatics tools have predicted a cache of thousands of BGCs within publicly available genome sequences, however high experimental attrition rates drastically slows characterisation of the corresponding specialised metabolite. We designed and built an Output Ordering and Prioritisation System (OOPS), to rank thousands of BGCs in parallel against molecular biology relevant parameters, pairing BGCs with appropriate heterologous expression hosts and facilitating a judicious choice of BGCs for characterisation to reduce experimental attrition. To fully realise the potential of synthetic biology in specialised metabolite discovery a genetically amenable heterologous host, capable of completing rapid design-build-test-learn cycles, is necessary. This cannot be achieved for the pharmaceutically important type II polyketides, as their biosynthetic machinery is largely restricted to Actinobacteria. Using MIBiG datasets, antiSMASH and BLASTP we identify 5 sets of soluble type II polyketide synthases (PKS) in Escherichia coli for the first time. We construct and test the robustness of a plug-and-play scaffold for bioproduction of aromatic polyketides using one PKS in E. coli, yielding anthraquinones, dianthrones and benzoisochromanequinones intermediates. Through bioprospecting for biological 'parts' to expand the chemical diversity of our plug-and-play scaffold we describe a new lineage of type II PKSs predominantly from non-Actinobacteria. The standards, softwares, and plug-and-play scaffold and biosynthetic 'parts' described here-in will act as an engine for rapid and automated bioproduction of existing, and novel, pharmaceutically relevant aromatic polyketides in E. coli using the synthetic biology toolbox.

A utilização de produtos naturais para a terapêutica do câncer foi iniciada com a actinomycina D, obtida a partir de culturas de Streptomyces e desde então, a busca por compostos bioativos de origem natural constitui uma importante linha de pesquisa. Estima-se que aproximadamente 60% dos agentes antineoplásicos, introduzidos para a terapia do câncer nas últimas décadas, tem origem vegetal ou microbiana. Dentre os micro-organismos proeminentes para produção de compostos ativos, as actinobactérias se destacam pela versatilidade metabólica, praticidade para cultivo in vitro e eficiência para produção de compostos com atividade anticâncer. Em seu último relatório, a Organização Mundial da Saúde reportou 8,8 milhões de mortes em decorrência de câncer, no ano de 2017. O índice representa um em cada seis óbitos em todo o mundo, sendo mais expressivo em países de média e baixa renda. Vale ressaltar que avanços significativos foram alcançados nos últimos anos para o tratamento de leucemia aguda infantil e tumores derivados de células germinais. Contudo, tumores sólidos de pulmão, próstata, mama e cólon ainda representam altos índices de mortalidade. Frente a isso, torna-se evidente a necessidade de identificar e desenvolver estratégias para o tratamento da doença. Com intuito de acessar novos recursos microbianos com potencial biotecnológico, a prospecção avança para áreas pouco exploradas, como por exemplo, o Continente Antártico. A Antártica foi o último dos continentes a ser acessado pelo homem e apresenta características edafoclimáticas favoráveis ao endemismo. Em vista da problemática e da potencialidade do Continente Antártico, os recursos microbiológicos associados à rizosfera de Deschampsia antarctica Desv. foram acessados e avaliados para a produção de compostos com propriedade antitumoral. Em resultado foram obtidos 42.528 clones metagenômicos e 72 linhagens de actinobactérias, dentre as quais Streptomyces sp. CMAA 1527, que apresentou pronunciada atividade antiproliferativa in vitro, para tumores de mama, pulmão, rim e sistema nervoso central, através da produção de cinerubina B. A análise taxonômica das actinobactérias isoladas revelou a presença de linhagens com baixo índice de similaridade, com as linhagens tipo conhecidas, o que pode significar a presença de novas espécies para os gêneros Nocardia, Rhodococcus e Streptomyces, reconhecidos pela capacidade de produzir metabólitos ativos e enzimas de interesse biotecnológico. A análise taxonômica polifásica da linhagem CMAA 1533 possibilitou a descrição da espécie Rhodococcus psychrotolerans sp. nov. (TaxoNumber TA00191; NRRL B-65465T = DSM 104532T), grupo bacteriano promissor como agente de biorremediação e produção de compostos bioativos. Com isso, o Continente Antártico foi considerado um ambiente promissor para a busca de novos micro-organismos, dentre eles actinobactérias, eficientes na produção de compostos antitumorais e outras substâncias com potencial biotecnológico. / The use of natural products for cancer therapy was initiated with the actinomycin D, obtained from Streptomyces. Since then, the search of bioactive from natural sources represent an essential line of research. It is estimated that approximately 60% of the antineoplasic agents inserted for the cancer therapy in recent decades have vegetal and microbial origin. Among the prominent microorganisms used to produce active compounds, actinobacterias are known by their metabolical versatility, convenience related to in vitro culture, and efficiency on the production of anticancer compounds. The Health World Organization, on its last review, reported 8.8 million of deaths in 2017, caused by cancer. Those numbers represent one out of six deaths worldwide, being more expressive in middle and low income countries. It is worth pointing out that meaningful advances were established in recent years for the treatment of childhood acute leukemia and germ cell-derived tumors. However, solid tumors of the lung, prostate, breast and colon still represent high mortality rates. For this reason, it is necessary to identify and develop strategies for the treatment of the disease. With the aim of accessing new microbial resources that contain biological potential, the prospection advance to areas barely explored, such as the Antarctic Continent. Antarctica was the last of the continents to be accessed by man and presents edaphoclimatic characteristics favorable to endemism. In light of the problematic and the potentiality of the Antarctic Continent, the microbiological resources associated with the rhizosphere of Deschampsia antarctica Desv. were accessed and evaluated for the production of compounds with antitumor properties. The results obtained had shown 42,528 metagenomic clones and 72 strains of actinobacteria, among them Streptomyces sp. CMAA 1527, which had presented anti-proliferative activity in vitro to breast, lung, kidney and central nervous system tumors, through the production of cinerubin B. The taxonomic analysis of the actinobacteria isolated revealed the presence of strains with low rate of similarity, with known type strains, which may mean the presence of new species for the genera Nocardia, Rhodococcus and Streptomyces, recognized for the ability to produce active metabolites and enzymes of biotechnological interest. The polyphasic approach of the CMAA 1533 strain made possible the description of the species Rhodococcus psychrotolerans sp. nov. (TaxoNumber TA00191; NRRL B-65465T = DSM 104532T), promising bacterial group as a bioremediation agent and production of bioactive compounds. As a result, the Antarctic Continent was considered a promising environment to search new microorganisms, among them, the actinobacteria, which is efficient on the production of antitumor compounds and other substances with biotechnological potential.

A utilização de produtos naturais para a terapêutica do câncer foi iniciada com a actinomycina D, obtida a partir de culturas de Streptomyces e desde então, a busca por compostos bioativos de origem natural constitui uma importante linha de pesquisa. Estima-se que aproximadamente 60% dos agentes antineoplásicos, introduzidos para a terapia do câncer nas últimas décadas, tem origem vegetal ou microbiana. Dentre os micro-organismos proeminentes para produção de compostos ativos, as actinobactérias se destacam pela versatilidade metabólica, praticidade para cultivo in vitro e eficiência para produção de compostos com atividade anticâncer. Em seu último relatório, a Organização Mundial da Saúde reportou 8,8 milhões de mortes em decorrência de câncer, no ano de 2017. O índice representa um em cada seis óbitos em todo o mundo, sendo mais expressivo em países de média e baixa renda. Vale ressaltar que avanços significativos foram alcançados nos últimos anos para o tratamento de leucemia aguda infantil e tumores derivados de células germinais. Contudo, tumores sólidos de pulmão, próstata, mama e cólon ainda representam altos índices de mortalidade. Frente a isso, torna-se evidente a necessidade de identificar e desenvolver estratégias para o tratamento da doença. Com intuito de acessar novos recursos microbianos com potencial biotecnológico, a prospecção avança para áreas pouco exploradas, como por exemplo, o Continente Antártico. A Antártica foi o último dos continentes a ser acessado pelo homem e apresenta características edafoclimáticas favoráveis ao endemismo. Em vista da problemática e da potencialidade do Continente Antártico, os recursos microbiológicos associados à rizosfera de Deschampsia antarctica Desv. foram acessados e avaliados para a produção de compostos com propriedade antitumoral. Em resultado foram obtidos 42.528 clones metagenômicos e 72 linhagens de actinobactérias, dentre as quais Streptomyces sp. CMAA 1527, que apresentou pronunciada atividade antiproliferativa in vitro, para tumores de mama, pulmão, rim e sistema nervoso central, através da produção de cinerubina B. A análise taxonômica das actinobactérias isoladas revelou a presença de linhagens com baixo índice de similaridade, com as linhagens tipo conhecidas, o que pode significar a presença de novas espécies para os gêneros Nocardia, Rhodococcus e Streptomyces, reconhecidos pela capacidade de produzir metabólitos ativos e enzimas de interesse biotecnológico. A análise taxonômica polifásica da linhagem CMAA 1533 possibilitou a descrição da espécie Rhodococcus psychrotolerans sp. nov. (TaxoNumber TA00191; NRRL B-65465T = DSM 104532T), grupo bacteriano promissor como agente de biorremediação e produção de compostos bioativos. Com isso, o Continente Antártico foi considerado um ambiente promissor para a busca de novos micro-organismos, dentre eles actinobactérias, eficientes na produção de compostos antitumorais e outras substâncias com potencial biotecnológico. / The use of natural products for cancer therapy was initiated with the actinomycin D, obtained from Streptomyces. Since then, the search of bioactive from natural sources represent an essential line of research. It is estimated that approximately 60% of the antineoplasic agents inserted for the cancer therapy in recent decades have vegetal and microbial origin. Among the prominent microorganisms used to produce active compounds, actinobacterias are known by their metabolical versatility, convenience related to in vitro culture, and efficiency on the production of anticancer compounds. The Health World Organization, on its last review, reported 8.8 million of deaths in 2017, caused by cancer. Those numbers represent one out of six deaths worldwide, being more expressive in middle and low income countries. It is worth pointing out that meaningful advances were established in recent years for the treatment of childhood acute leukemia and germ cell-derived tumors. However, solid tumors of the lung, prostate, breast and colon still represent high mortality rates. For this reason, it is necessary to identify and develop strategies for the treatment of the disease. With the aim of accessing new microbial resources that contain biological potential, the prospection advance to areas barely explored, such as the Antarctic Continent. Antarctica was the last of the continents to be accessed by man and presents edaphoclimatic characteristics favorable to endemism. In light of the problematic and the potentiality of the Antarctic Continent, the microbiological resources associated with the rhizosphere of Deschampsia antarctica Desv. were accessed and evaluated for the production of compounds with antitumor properties. The results obtained had shown 42,528 metagenomic clones and 72 strains of actinobacteria, among them Streptomyces sp. CMAA 1527, which had presented anti-proliferative activity in vitro to breast, lung, kidney and central nervous system tumors, through the production of cinerubin B. The taxonomic analysis of the actinobacteria isolated revealed the presence of strains with low rate of similarity, with known type strains, which may mean the presence of new species for the genera Nocardia, Rhodococcus and Streptomyces, recognized for the ability to produce active metabolites and enzymes of biotechnological interest. The polyphasic approach of the CMAA 1533 strain made possible the description of the species Rhodococcus psychrotolerans sp. nov. (TaxoNumber TA00191; NRRL B-65465T = DSM 104532T), promising bacterial group as a bioremediation agent and production of bioactive compounds. As a result, the Antarctic Continent was considered a promising environment to search new microorganisms, among them, the actinobacteria, which is efficient on the production of antitumor compounds and other substances with biotechnological potential.

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

This thesis critically explores the issue of access to biological resources and Indigenous knowledge Historically, biological resources collected and documented, and knowledge associated with their use, have been considered the 'common heritage of mankind' The Convention on Biological Diversity (CBD) changed this understanding to tights of states over biological resources, but also gave rise to issues of equity and justice, especially with regard to Indigenous Nations encapsulated within First World states-so-called 'Fourth World Nations', A central concern of Fourth World Peoples is their marginalisation within access negotiations, despite their claims of connate (birth) rights to r esou.r ces and knowledge they identify as their own. Increasing global Indigenous activism over their concerns, has in turn raised an increasingly important policy gap that is becoming recognised in fora and processes with regard to access to biological resources. My thesis addresses this policy gap. I explore some of the complex historical, political and cultural dimensions that led to the emergence and resilience of this policy problem The failure to address the concerns of Indigenous peoples, and Fourth World Nations in particular, is more important and problematic now because of contemporary biotechnological developments and the emergence of bioprospecting. Bioprospecthg refers to the practice of appropriating biological resources, and Indigenous knowledge of those resources, and incorporating them into biopharmaceutical processes. Literature on bioprospecting as a problematic issue for Third World States has been emerging steadily over the last decade under the impact of the commercialisation of biodiversity, which has become big business for biopha.rmaceutical companies. The unique interests and experiences of Fourth World Nations are not recognised within this literature as significantly different to that of the Third World, and of their encapsulating states.. This study has addressed this significant gap by utilising and developing an analytical approach that uses Fourth World theory, synthesised with elements of Foucault's analytics of power. When combined, these two theoretical approaches provide a new and rich under standing of how dominant 'ways of knowing' and 'ways of owning' have been privileged, while other knowledge and ownership systems have been, and continue to be, marginalised, Eoucault's understanding of discursive power as having the capability to be either, or both, dominant and resistant is important to my analysis, as it accommodates the Fourth World as a discursive site of resistance to dominant power. I posit that richer insights are gained through the development and application of this theoretical framework to the issue of fair and equitable access to biological resources, than other approaches offer. I demonstrate the framework's utility by applying it to a case study on bioprospecting in Australia. Important findings have emerged while tracking the activities of Fourth World peoples on the international stage, and their attempts to challenge dominant power/knowledge structures within political institutions For example, participation at the international level has enabled Fomth World peoples to apply pressure on their encapsulating states to accommodate their interests. This has been furthered through forming alliances with, for example, environmentalists, and through the adoption of the language of effective participation within international fora.. Overall, however, the study found that the participation of Eourth World peoples within international, central state and local state policy processes is not always empowering in challenging dominant interests Instead, the more accurate impression is that at this stage of the discursive policy terrain, it may only create an illusion of participation that actually serves to entrench their disempowerment. This places pressule on policy processes to address and resolve this access issue equitably if social turbulence is to subside, justice be served, and certainty provided for all.

Abstract: Indigenous communities have worked for centuries to develop systems of knowledge pertaining to their local environments. Much of the knowledge that has been directly acquired or passed down over generations is of marketable use to corporations, especially in the pharmaceutical industry. Upon gaining the necessary information to convert traditional knowledge into a marketable entity, the corporation will place a patent on the product of their research and development and reap the monetary benefits under the protection of intellectual property legislation. Without appropriate benefit sharing, indigenous communities are robbed of their cumulative innovation and development and denied access to the very medicines that they assisted in development. This study will examine the efforts made by indigenous communities to develop benefit-sharing agreements under national ‘sui generis’ legislation and the international legislation of the Agreement on Trade-Related Aspects of Intellectual Property Rights (TRIPS) and the Convention on Biological Diversity (CBD).

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Os conhecimentos produzidos por comunidades tradicionais, quilombolas e povos indígenas são cada vez mais reconhecidos como importante fonte de informação para as atividades de bioprospecção, especialmente àqueles conhecimentos associados à biodiversidade. Em uma sociedade denominada por muitos autores como a sociedade do conhecimento, torna-se urgente a discussão sobre a titularidade e a forma de proteção de tais conhecimentos tradicionais, que geram lucros de forma direta ou indireta às empresas do setor biotecnológico sem, contudo possuírem ainda sistemas eficazes de proteção. A presente tese analisa a forma de produção desses conhecimentos tradicionais associados à biodiversidade e à bioprospecção, a fim de estudar os mecanismos legais regulatórios incidentes sobre a sua proteção, acesso e repartição de suas informações, conforme estabelecido pela Convenção sobre a Diversidade Biológica. O objetivo central é contribuir ao estabelecimento de marco jurídico, através da discussão sobre a viabilidade, os benefícios e as limitações para a sua elaboração. Através de uma metodologia qualitativa são estabelecidas inicialmente a importância, as características e as diversas espécies dos conhecimentos tradicionais. Em seguida é abordada a interface desses conhecimentos com os conhecimentos científicos, os direitos de propriedade intelectual e a natureza jurídica dos direitos sobre tais conhecimentos que passam a ser denominados COTABIOs. Após essa etapa cognitiva, é apresentado o arcabouço institucional-legal da repartição de benefícios, acesso e proteção dos COTABIOs em diversos fóruns internacionais e em alguns países, com especial ênfase ao panorama nacional brasileiro. Ao término desse processo são formuladas propostas de medidas especificas que poderão contribuir para a proteção legal dos COTABIOs. A tese é concluída com considerações gerais sobre as propostas formuladas, a fim de contribuir para o preenchimento da lacuna existente em decorrência da esparsa legislação nacional e internacional sobre a repartição de benefícios, acesso e proteção dos COTABIOs sem, contudo ter a pretensão de esgotar o tema e sim contribuir para a reflexão e discussão sobre a necessidade de mudanças nesse setor / The knowledge generated by traditional communities, quilombolas and indígenous people, have been even more recognized as an important source of information for bioprospecting activities, in special those associated with biodiversity knowledge. In a knowledges society, as understood for several authors, it is mandatory to discuss about the ownership and the way of protection of such traditional knowledges, that are giving profits to biotechnologic sector corporations, however without any effective protection mecanism. This thesis examines these traditional knowledges associated with biodiversity and bioprospecting are produced, studying the legal mechanisms usually applyied for protecting them, and accessing the available informations as preconized by Biologic Diversity Convention. Our main objective is to make a contribution to the establishment of legal framework, by discussion about its viability, benefits and limitations. Adopting a qualitative methodology, it establish first the traditional knowledges importance, characterístics and the categories envolved. Afterwards it is approached the connection among traditional and cientific knowledges, the rights about the intellectuak property and the legal nature will be called here after as COTABIOs. After this cognitive stage, it is submitted the legal and institutional framework of benefit sharing, access and protection from COTABIOs on many international foruns and some countries, emphasizing mainly the national brazilian panorama. Subsequently are proposed specific mechanisms that could contribute to the legal COTABIOs protection. The thesis is concluded doing general considerations about the proposals presented, believing to be contributing to close the gap resulting from the termous national and international legislation about benefit sharing, access and protection from COTABIOs, without however understanding the discussion is been finalized, but only a new horizon for reflexion and discussion about changes on this area was proposed