Les espèces du genre Colletotrichum sont majoritairement des agents pathogènes de nombreuses plantes et provoquent des dégâts importants aux cultures partout dans le monde. Colletotrichum higginsianum est un champignon phytopathogène hémibiotrophe capable d’infecter de nombreuses Brassicaceae dont la plante modèle Arabidopsis thaliana. L’intérêt du pathosystème C. higginsianum – A. thaliana réside dans la possibilité de manipuler génétiquement les deux partenaires. Par ailleurs, la disponibilité de très nombreuses lignées transgéniques et d’outils génétiques aide à disséquer les mécanismes de résistance et de sensibilité de la plante. Nous avons re-séquencé (technologie PacBio) le génome de C. higginsianum afin d’obtenir les séquences complètes des 12 chromosomes. L’analyse détaillée de cette seconde version du génome a permis de mettre en évidence l’un des plus grands arsenaux fongiques de gènes du MS et de résoudre les problèmes de fragmentation de la première version du génome. Afin d’identifier les métabolites secondaires que C. higginsianum produit au cours de l’infection de la plante, nous avons supprimé des répresseurs du MS qui modifient l’état de la chromatine (Kmt1, Kmt6, Hep1, CclA) et sur-exprimé des inducteurs du MS (LaeA, Sge1) décrits chez d’autres mycètes. Grâce à des analyses de transcriptomique (RNA-Seq), des gènes du MS différentiellement exprimés dans ces mutants ont pu être identifiés. Ensuite, par des analyses de chimie analytique nous avons pu identifier et caractériser deux familles de métabolites secondaires. Enfin, nous avons mis en évidence des activités inédites pour ces deux familles de composés. Ces résultats ouvrent de nouvelles perspectives pour aborder le déterminisme du pouvoir pathogène de C. higginsianum. / Colletotrichum spp. are major plant pathogens of numerous crops worldwide. Colletotrichum higginsianum uses a hemibiotrophic lifestyle to infect many members of the Brassicaceae including the model plant Arabidopsis thaliana. The C. higginsianum – A. thaliana pathosystem facilitates dissection of the mechanisms of plant resistance and susceptibility and the traits underlying fungal pathogenicity. Both partners can be genetically manipulated and powerful genetic tools and transgenic lines are available on the plant side. With a view to obtain a more complete inventory of C. higginsianum secondary metabolism (SM) genes, we re-sequenced the genome de novo using PacBio technology, providing 12 complete chromosome sequences. The in-depth analysis of this second version of the genome revealed one of the largest SM repertoires described to date for any ascomycete and solved numerous fragmentation problems in the first genome assembly. In order to identify secondary metabolites produced by C. higginsianum during plant infection, we deleted negative SM regulators that modify chromatin status (Kmt1, Kmt6, Hep1, CclA), and over-expressed positive SM regulators (LaeA, Sge1), all of which were well-described in other fungi. Using transcriptomics (RNASeq), we identified SM genes differentially expressed in these mutants. Using metabolite profiling, we also identified and characterised two families of secondary metabolites. Finally we describe novel biological activities for these compound families. These results provide new insights into the molecular basis of pathogenicity in C. higginsianum.

In the first part of this research, we constructed a Genome scale Metabolic Model (GEM) of Taxus cuspidata, a medicinal plant used to produce paclitaxel (Taxol®). The construction of the T. cuspidata GEM was predicated on recent acquisition of a transcriptome of T. cuspidata metabolism under methyl jasmonate (MJ) elicited conditions (when paclitaxel is produced) and unelicited conditions (when paclitaxel is not produced). Construction of the draft model, in which transcriptomic data from elicited and unelicited conditions were included, utilized tools including the ModelSEED developed by Argonne National Laboratory. Although a model was successfully created and gapfilled by ModelSEED using their software, we were not able to reproduce their results using COBRA, a widely accepted FBA software package. Further work needs to be done to figure out how to run ModelSEED models on commonly available software. In the second part of this research, we modeled the MJ elicited/defense response phenotype in Arabidopsis thaliana. Previously published models of A. thaliana were tested for suitability in modeling the MJ elicited phenotype using publicly available computation tools. MJ elicited and unelicited datasets were compared to ascertain differences in metabolism between these two phenotypes. The MJ elicited and unelicited datasets were significantly different in many respects, including the expression levels of many genes associated with secondary metabolism. However, it was found that the expression of genes related to growth and central metabolism were not generally significantly different for the MJ+ and MJ- datasets, the pathways associated with secondary metabolism were incomplete and could not be modeled, and FBA methods did not show the difference in growth that was expected. These results suggest that behavior associated with the MJ+ phenotype such as slow growth and secondary metabolite production may be controlled by factors not easily modeled with transcriptome data alone. Additional research was performed in the area of cryosectioning and immunostaining of fixed Taxus aggregates. Protocols developed for this work can be found in Appendix B.

Orientador: Rogério Lopes Vieites / Resumo: A pitaya vermelha de polpa branca é uma fruta que vem conquistando o mercado devido a sua alta aceitação pelos consumidores e seus benefícios para a saúde. Objetivou-se avaliar a conservação pós-colheita dos frutos de pitaya vermelha de polpa branca durante o armazenamento refrigerado, após serem submetidos a diferentes doses de irradiação gama, visando a preservação da qualidade, bem como verificar as alterações no metabolismo secundário quanto aos compostos bioativos, e também identificar o melhor solvente extrator e o melhor peso da amostra, na extração de compostos bioativos e atividade antioxidante dos frutos. Os frutos foram colhidos em um pomar comercial localizado no município de Presidente Prudente-SP, e em seguida transportados para o Departamento de Horticultura da FCA-UNESP. Os frutos embalados foram transportados para o Instituto de Pesquisas Energéticas e Nucleares, onde foram submetidos as doses de irradiação gama. Após, foram armazenados em câmara fria no Departamento de Horticultura, onde foram realizadas as análises físico-químicas da polpa e bioquímicas da casca e polpa dos frutos. O delineamento experimental foi inteiramente casualizado em esquema fatorial 5 x 5, cinco doses de irradiação (0 KGy, 0,3 KGy, 0,6 KGy, 0,9 KGy e 1,2 KGy) e cinco dias de avaliação (zero, 5, 10, 15 e 20 dias). O delineamento para otimização da extração de compostos bioativos e atividade antioxidante foi o inteiramente casualisado com fator único e três repetições. A utilização da... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Red pitaya with white pulp is a fruit that has been conquering the market due to its high acceptance by consumers and its health benefits. The objective was to evaluate the postharvest conservation period of the fruits of the red white pulpy pitaya after being subjected to different doses of gamma irradiation, aiming at the preservation of the quality, as well as verifying the alterations in the secondary metabolism regarding the bioactive compounds, and also identify the best extractor solvent and the best weight of the sample, in the extraction of bioactive compounds and antioxidant activity of the fruits. The fruits were harvested in a commercial orchard located in the municipality of Presidente Prudente-SP, and then transported to the Horticulture Department of FCAUNESP. The packaged fruits were transported to the Energy and Nuclear Research Institute, where they were subjected to gamma irradiation doses. Afterwards, they were stored in a cold chamber at the Horticulture Department, where the physical-chemical analyzes of the pulp and biochemical analyzes of the skin and fruit pulp were carried out. The experimental design was completely randomized in a 5 x 5 factorial scheme, five irradiation doses (0 KGy, 0.3 KGy, 0.6 KGy, 0.9 KGy and 1.2 KGy) and five evaluation days (zero, 5, 10, 15 and 20 days). The design for optimizing the extraction of bioactive compounds and antioxidant activity was completely randomized with a single factor and three replications. The use of gam... (Complete abstract click electronic access below) / Mestre

Les Streptomyces sont des bactéries filamenteuses à Gram positif que l’on retrouve dans les couches superficielles du sol. La souche modèle S. coelicolor M145 est capable de produire des antibiotiques et accumuler de faibles quantités de triacylglycérol lorsqu’elle est mise en culture avec le glucose ou le glycérol comme source de carbone. La souche produit de l’actinorhodine seulement en culture avec le glycérol comme source de carbone, et nous assistons à une accumulation d’acides gras, notamment sous forme d’esters d’acide oléique avec l’une ou l’autre source de carbone. Ces accumulations d’acides gras ne durent que pendant une certaine période de temps, à l’issu de laquelle l’accumulation s’arrête, et on assiste à la production de métabolites secondaires qui peut s’accompagner de la consommation des acides gras précédemment accumulés. Ces observations suggèrent d’une part que la source de carbone est un effecteur différentiel dans la production de métabolites secondaire, et que d’autre part le métabolisme des lipides est lié au métabolisme secondaire. Ce travail de thèse va tenter d’établir les liens entre le métabolisme primaire et le métabolisme secondaire chez S.coelicolor via le métabolisme des lipides. Pour se faire, nous avons dans un premier temps défini un milieu synthètique, ainsi que des stratégies d’alimentation de type fed-batch en bioréacteur, pour permettre d’obtenir d’une part une phase de croissance exponentielle, et d’autre part une phase de croissance non exponentielle et imposée par une limitation en azote. Cette limitation nutritionnelle est classiquement utilisée à la fois lors des études de l’accumulation de lipides chez les microorganismes oléagineux tels que Yarrowia lipilytica ou Rhodotorula glutinis, et aussi dans le cas d’études de la production d’antibiotiques par les Streptomyces. L’étude du métabolome couplée à l’étude du protéome nous a permis d’établir la direction générale des flux globaux de carbone, d’énergie et de pouvoir réducteur lors des différentes phases de croissances. Ces travaux démontrent une hétérogénéité métabolique dans la population bactérienne totale, avec la présence de 2 à 3 sous-populations différentes pouvant coexister. Les évolutions temporelles du protéome et notamment des régulateurs transcritptomiques montrent des réactions tardives, très certainement dues à la présence d’effecteurs intracellulaires accumulés, ainsi qu’au dynamisme de la culture. / Streptomyces are filamentous Gram positive bacteria found in the soil upper layers. The model strain S. coelicolor M145 can produce antiobiotics and accumulate low levels of triacylglycerol when cultivated with glucose or glycerol as carbon source. This strain produces actinorhodin only when glycerol is used as carbone source, but the fatty acids accumulation as esterified oleic acid occurs with both carbon sources. However, the fatty acids accumulation only last for a short period of time, and afterward, a production of secondary metabolites is observed and the previously accumulated fatty acids are consummed. Those results suggest that the carbon source act as an effector for the production of secondary metabolites and the lipids metabolism is some ways linked to the secondary metabolism. With this work, we will try to establish the links between the primary and secondary metabolism via the lipids metabolism with S. coelicolor. First, in order to obtain cultures with an exponential growth phase and a non-exponential growth phase under a nitrogen limitation, a synthetic media and fed-batch feeding strategies have been designed. The nitrogen limitation is usually used to study lipids accumulation with oleaginous microorganisms such as Yarrowia lipilytica or Rhodotorula glutinis, and also during studies of antibiotics production with Streptomyces. The metabolomics study paired with the proteomics study made possible to establish the global directions of carbon, energy and reduced power fluxes during all the obtained growth phases. This work also showed that the microbial population is heterogeneous and 2 to 3 subpopulations can coexist with both carbon sources. The proteomics temporal changes and in particular of transciptional regulators show some late reactions to the nitrogen limitation especially when glycerol is used, probably because of the intracellular accumulation of effector compounds over time, and because of growth kinetics.

L'induction du métabolisme secondaire fait partie du système de défense des plantes lors d’une attaque par un microorganisme pathogène. Les propriétés antimicrobiennes ou de signalisation des métabolites secondaires (MS) peuvent être régulées par un processus majeur et efficace, la glycosylation. Cette réaction est catalysée par les glycosyltransférases (UGTs) qui assurent le transfert d’une molécule de sucre sur un MS. Mon travail de thèse a porté sur l’étude de trois UGTs d’Arabidopsis thaliana (Arabidopsis), UGT76E12 d’une part, et UGT73B3 et UGT73B5 d’autre part, et a montré que 1) UGT76E12 est impliquée dans la mise en place de la résistance basale à la souche virulente de la bactérie Pseudomonas syringae pv. tomato, Pst DC3000, et participe au dialogue entre les voies de signalisation hormonale de l’acide jasmonique (JA) et de l’acide salicylique (SA). Des analyses par clustering hiérarchique ont révélé une co-expression forte entre le gène UGT76E12, et deux gènes codant une terpène synthase (TPS4) et un cytochrome P450 (CYP82G1), impliqués dans la biosynthèse d’un diterpène, le TMTT ((E,E)-4,8,12-triméthyletridéca-1,3,7,11-tetraène), suggérant un rôle pour UGT76E12 dans la régulation de la voie de biosynthèse des terpènes ; 2) UGT73B3 et UGT73B5 sont impliquées dans la mise en place de la résistance spécifique d’Arabidopsis à la souche avirulente Pst DC3000-AvrRpm1, notamment par la régulation de MS intervenant dans le maintien de l’état redox cellulaire au cours de la réaction d’hypersensibilité (HR). Ces deux gènes appartiennent à la classe des « SA-early induced-genes » et présentent un profil co-expression avec le gène TOLB-related et deux gènes codant des glutathion S-transférases (GSTU7 et GSTU24) impliqués dans la réponse au stress oxydant et la détoxication des MS. Nos résultats suggèrent un rôle pour UGT73B3 et UGT73B5 dans la glycosylation de métabolites oxydants associés à la mise en place de la HR. / Plant secondary metabolism induction is part of an integrated defense system after pathogen infection. Signalling and antimicrobial properties of secondary metabolites (SM) can be regulated by an efficient process named, glycosylation. This reaction is ensured by glycosyltransferases (UGTs) which catalyze the transfer of a sugar moiety on a SM. My PhD work was divided in two parts, the first on UGT76E12 and the second part on UGT73B3 and UGT73B5, three UGTs of Arabidopsis thaliana (Arabidopsis) and it demonstrated that 1) UGT76E12 is involved in basal resistance establishment during plant challenge with the virulent strain of Pseudomonas syringae pv. tomato (Pst DC3000), and participates to the hormonal crosstalk between jasmonic acid (JA) and salicylic acid (SA). Clustering analyses revealed a strong co-expression between UGT76E12 and two genes encoding a terpene synthase (TPS4) and a cytochrome P450 (CYP82G1), both involved in the biosynthesis of a diterpene named TMTT ((E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene), which suggests a role for UGT76E12 in terpene biosynthesis pathway regulation. 2) UGT73B5 and UGT73B3 are involved in the establishment of Arabidopsis specific resistance against the avirulent strain of Pst DC3000-AvrRpm1, especially in the regulation of MS playing a role in redox homeostasis status during the hypersensitive response (HR). These two genes are part of SA-early induced-genes and showed co-expression patterns with the TOLB-related gene and two genes encoding glutathione S-transferases (GSTU7 and GSTU24) involved in oxidative stress responses and in MS detoxification. Our results suggest a role for UGT73B3 and UGT73B5 in the glycosylation of oxidant MS associated with HR establishment.

Natural product drug discovery has traditionally been the corner stone of medicine having provided cures to many of today's most common diseases. In particular, antibiotics have revolutionised healthcare and extended human lifespan. However, since their introduction into the clinic, resistance to these drugs has arisen. With the number of new antibiotics being discovered in recent years declining, and fewer drugs making it past clinical trials, we have reached the point where antibiotic resistant infections have become common place and a serious threat to health and society. There is now an urgent requirement for the discovery of new antibiotics and in particular those with unexploited mode of action. This thesis details the different areas of natural product drug development from discovery through to analogue generation. In Chapter one, the history of natural products as therapeutics is explored with a particular focus on antibiotics and how resistance arises against these agents. It outlines why the discovery of new antibiotics is so important and new methods used to facilitate this search. Chapter two follows with the development of a screening platform for antibiotic induction, using the model Streptomyces; Streptomyces coleiolor M145. A variety of culture additives are explored for their ability to induce secondary metabolism production. Chapter three then details the sampling and identification of microbes from a pseudo-marine environment and their screening for their ability to produce secondary metabolites with antibiotic properties. The second half of this thesis centres on the non-ribosomal peptide echinomycin. Collaborators Aquapharm supplied the marine derived strain AQP-4895, capable of producing echinomycin. Chapter four details the establishment of AQP-4895 culturing conditions and the shift observed in production profile. Next Chapter five looks at producing echinomycin analogues through precursor directed biosynthesis. A range of halogenated quinoxaline carboxylic acids are synthesised and fed to AQP-4895, and the respective echinomycin analogues monitored by LC-MS. Chapter Six then aims to direct biosynthesis of the halogenated analogues, using mutasynthesis. Due to the lack of genetic data available surrounding the strain, an unusual approach was taken, using iPCR to create a template for homologous recombination.

No description available.

Foi realizada uma investigação fitoquímica envolvendo diversos órgãos de plantas adultas (raízes, caules, folhas, inflorescências e sementes), plântulas (cultivadas in vivo e in vitro) e suspensões celulares de P. solmsianum. Dos órgãos estudados de plantas adultas, as raízes apresentaram a maior complexidade e diversidade química da planta, abrangendo ácidos benzóicos e benzaldeídos substituídos, esteróides, fenilpropanóides, lignanas tetraidrofurânicas e aristolactamas, enquanto que os outros órgãos acumulam principalmente fenilpropanóides e lignanas tetraidrofurânicas. As raízes de plântulas apresentaram semelhança qualitativa de metabólitos secundários quando comparadas às raízes de plantas adultas. No entanto, as partes aéreas apresentaram diferentes compostos como farnesol, fitol e α-tocoferol. Os extratos de plântulas separadas por tamanho foram comparados em relação à diversidade dos metabólitos encontrados, não sendo observada uma variação qualitativa muito significativa. Os extratos obtidos das células de suspensões celulares de P. solmsianum indicaram prevalência de ácidos graxos e esteróides, enquanto que o extrato obtido do meio de cultura apresentou como componente majoritário o ácido salicílico. / The phytochemical investigation was carried out to describe the composition in organs of adult plants (roots, steams, leaves, inflorescences and seeds), plantlets (in vivo and in vitro) and cell suspensions of Piper solmsianum. The roots of adult plants presented highest chemical diversity including benzoic acids, benzaldehydes, sterols, phenylpropanoids, tetrahydrofuran lignans and aristolactams, whereas aerial organs accumulate mainly phenylpropanoids and tetrahydrofuran lignans. The roots from plantlets presented qualitative similarity of secondary metabolites when compared to the roots of adult plants. However, the aerial parts from plantlets presented different composition including farnesol, phytol and α-tocopherol in addition to phenylpropanoids, but the plantlets at different developmental stages showed no significant qualitative variation. The extracts of cell suspensions of P. solmsianum indicated a suppressed phenylpropanoid metabolism with fatty acids and sterols in the cells and salicylic acid as major excreted compound in the culture medium.

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No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) tese_diego_severo_fraga.pdf: 1548587 bytes, checksum: c109de985c27f806698e98816b90240d (MD5) Previous issue date: 2016-03-22 / Sem bolsa / A rotação da cultura da soja ao arroz irrigado e sucessão com azevém pode ser considerado opção para manejo de plantas daninhas em área de terras baixas, principalmente o arroz-vermelho. Além disso, o surgimento de novas tecnologias de soja resistente a herbicidas do grupo das imidazolinonas como a Cultivance® pode auxiliar o produtor de sementes no controle de plantas daninhas quando associadas a sistemas de manejo como “plante-aplique” e “aplique-plante”. Dessa forma, os objetivos da pesquisa foram avaliar a fitotoxicidade, os componentes da produtividade e a qualidade fisiológica das sementes de cultivares de soja contendo as tecnologias Cultivance®, tolerância a sulfonilureias e Roundup Ready®, frente ao resíduo de doses crescentes da mistura dos herbicidas imazapyr e imazapic, aplicado no manejo da cultura do arroz irrigado; avaliar a permanência do resíduo do herbicida no solo em virtude do acúmulo de massa da matéria seca da parte aérea de plantas de azevém, semeadas em sucessão com a cultura da soja; quantificar a atividade da enzima ALS em cultivares de soja resistentes ou suscetíveis ao herbicida imazapic; avaliar as alterações nos metabólitos secundários de plantas de soja contendo essas tecnologias, submetidas a mistura dos herbicidas imazapyr e imazapic nos sistemas de manejo “plante-aplique e “aplique-plante”. Para isso, foram realizados experimentos em campo, casa-de-vegetação e laboratório da Universidade Federal de Pelotas, Embrapa Clima Temperado e Embrapa Trigo, utilizando diferentes cultivares de soja submetidas ao herbicida imazapyr+imazapic. Os resultados demonstraram que a cultivar de soja contendo a tecnologia Cultivance® é resistente a mistura dos herbicidas imazapyr e imazapic aplicado no manejo do arroz irrigado, porém, as demais cultivares são suscetíveis. O resíduo do herbicida afeta a produtividade e a qualidade fisiológica da semente das cultivares estudadas. A massa da matéria seca da parte aérea do azevém, é reduzida na presença do resíduo do herbicida. O KM (concentração de piruvato) da enzima ALS das cultivares de soja são semelhantes, contudo o Vmáx (velocidade máxima da reação) da cultivar resistente é menor, comparado às demais, supondo-se que a resistência da cultivar contendo a tecnologia Cultivance® pode ser decorrente da insensibilidade da enzima ALS ao herbicida imazapic. As cultivares suscetíveis apresentam, em geral, maior teor de clorofila e sofrem maior ação das espécies reativas de oxigênio, comparadas a resistente. A elevação da dose da mistura dos herbicidas imazapyr e imazapic aumenta a ação do sistema antioxidante enzimático, por meio das enzimas superóxido dismutase, catalase e ascorbato peroxidase, independente do sistema de manejo. / Soybean crop rotation with irrigated rice and sucession with ryegrass may be considered an option to weed management in flooded area, mainly for red rice. Besides that new technologies appearance of soybean herbicide resistant of imidazolinone group such as Cultivance® may help seed farmers in the weed control when associated to management systems such as “plant-apply” and “apply-plant”. Thus the reseach objectives were evaluate phytotoxicity, yield components and physiological quality of soybean seeds’ cultivars containing Cultivance®, sulfonylurea tolerant and Roundup Ready® technologies, as affected by carryover of increasing doses of mixture of herbicides imazapyr and imazapic spraying in management of irrigated rice crop; evaluate herbicide carryover in soil due ryegrass shoot dry matter, seeded in sucession with soybean crop; quantify ALS enzyme activity in soybean cultivars resistants or susceptibles to imazapic herbicide; evaluate the changes on secondary metabolism of soybean plants containing these technologies, submmited to mixture of herbicides imazapyr and imazapic in “plant-apply” and “apply-plant” management systems. Therefore experiments were performed on field, greenhouse and laboratories in the Federal University of Pelotas, Embrapa Temperate Climate and Embrapa Wheat, using differents soybean cultivars submmited to imazapyr+imazapic herbicide. The results showed that soybean cultivar containing Cultivance® technology is resistant to mixture of herbicides imazapyr and imazapic sprayed in management of irrigated rice, but the others cultivars are susceptibles. The herbicide carryover affect the crop yield and physiological quality of seed of the studied cultivars. Ryegrass shoot dry matter is affected by herbicide carryover. ALS enzyme KM (pyruvate concentration) of soybean cultivars are similar, however the Vmáx (maximum reaction speed) of resistant cultivar is minor, compared with the other cultivars, supposing that the resistance of the cultivar containing Cultivance® technology may be due insensibility ALS enzyme to imazapic herbicide. The susceptibles cultivars show, in general, more chlorophyll and suffer more action of reactives oxygen species, compared to resistant. The increasing dose of mixture of herbicides imazapyr and imazapic enhance the action of enzymatic antioxidant system, by means of superoxide dismutase, catalase and ascorbate peroxidase enzymes, independent of management system.

Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro / A biodiversidade brasileira abrange plantas de importância medicinal que podem ser utilizadas na formulação de novos fármacos. Contudo, tem sido reduzida em velocidade alarmante, em função de diferentes ações antrópicas. A cultura de tecidos vegetais propicia a conservação e uso do germoplasma permitindo a obtenção de substâncias de importância medicinal. As leishmanioses são consideradas um problema de saúde pública mundial sendo a espécie Leishmania braziliensis de maior importância epidemiológica no Brasil. Recentemente tem-se registrado aumento da resistência à linha de tratamento usual. Do mesmo modo, o uso indiscriminado de antibióticos levou ao aumento de bactérias multirresistentes, que representam sério risco de infecção. A espécie Annona mucosa (Jacq.) possui substâncias, como acetogeninas e alcaloides, que apresentam atividades antiparasitária e antimicrobiana. Nesse sentido, o objetivo do trabalho foi avaliar o potencial leishmanicida e antibacteriano de extratos de A. mucosa de material produzido in vitro e in vivo. Foi proposto um protocolo de germinação in vitro, ainda não reportada para a espécie, com vistas à obtenção de plântulas axênicas. Em meio WPM foram cultivados explantes hipocotiledonares e foliares em meio MS, suplementados com PIC e diferentes concentrações de KIN, BAP ou TDZ. Os calos obtidos foram cultivados em meio líquido de mesma composição para a produção de suspensões celulares. Os materiais foram submetidos à extração metanólica e posterior fracionamento em hexano e diclorometano. Para a avaliação da atividade dos extratos sobre L. braziliensis foi usado o modelo in vitro, com a forma promastigota, e in vivo na forma amastigota, a partir do tratamento de macrófagos peritoneais de camundongos infectados com o parasito. Ambas as formas foram tratadas com os extratos por 96 e 48h, respectivamente. A atividade antimicrobiana foi avaliada por macrodiluição do extrato em Mueller-Hinton, sendo avaliado o crescimento das cepas após 16h de incubação a 48C. A germinação in vitro da espécie foi alcançada em substrato vermiculita estéril umedecido com solução de sais do meio MS, com taxa média de 85%. A maior produção de calos friáveis foi obtida em meios contendo KIN, com potencial uso para cultivo em suspensões celulares. Os extratos do material in situ e in vitro apresentaram atividade leishmanicida, apesar da toxicidade para macrófagos. Culturas de células em suspensão apresentaram potencial leishmanicida in vitro e redução da infecção em macrófagos. Os extratos do material avaliado apresentaram atividade antimicrobiana seletiva, com inibição do crescimento de Streptococcus pyogenes e Bacillus thurigiensis em diferentes concentrações avaliadas. Os métodos biotecnológicos empregados permitiram a obtenção de materiais com propriedades medicinais para as atividades leishmanicida e antibacteriana, assim como o material in vivo, constituindo este estudo o primeiro relato para as atividades propostas em A. mucosa. / The Brazilian flora encompasses medicinal importance species that can be used to develop new drugs. However, plants have been reduced in alarming rate, due to various human activities. Plant tissue culture promotes the conservation and use of species allowing for the production of bioactive substances for medicinal products. Leishmaniasis is considered a worldwide health problem, where Leishmania braziliensis is the most epidemiological importance specie in Brazil. Recently it has been registered increased resistance to treatment line. Similarly, the widespread use of antibiotics has led to an increase in multidrug-resistant bacteria, which represent a serious risk of infection. Annona mucosa have substances, such as acetogenins and alkaloids that exhibit antiparasitic and antimicrobial activities. In view of the problem, the aim of this work was to evaluate the leishmanicidal and antibacterial potential of extracts of A. mucosa obtained in vitro and in vivo. We proposed a protocol of in vitro germination, not yet reported for the species, aiming to achieve axenic seedlings. Hypocotyls explants was cultivated in WPM and leaf explants on MS medium supplemented with PIC and different concentrations of KIN, BAP or TDZ. The formed callus was cultured in liquid medium of the same composition for the production of cell suspensions. The materials were subjected to methanolic extraction and subsequent fractionation in hexane and dichloromethane. The activity of extracts on L. braziliensis was evaluated in vitro in the form promastigote and in vivo in amastigote form, in infected mice macrophages. Both, were treated with the extract per 96h and 48h, respectively. The evaluation of antimicrobial activity was performed by macrodilution assay in Mueller-Hinton, analyzing the growth of the strains after 16h of incubation at 48C. The in vitro germination of A. mucosa was achieved in sterile vermiculite as substrate and has a 85% germination rate. The higher callus formation and friability was obtained in media containing KIN, that may be cultured in suspension. The extracts of the material in situ and in vitro showed leishmanicidal activity, despite the toxicity in macrophages. Cell suspension cultures showed antileishmanial potential in vitro and reduction of infection in macrophages. The evaluated extracts showed selective antimicrobial activity, with growth inhibition of Streptococcus pyogenes and Bacillus thuringiensis in different tested concentrations. It was concluded that the biotechnological techniques allowed to use plant materials regarding pharmacological properties for antileishmanial and antibacterial activities.