Os produtos naturais marinhos são apontados com uma das fontes de substâncias bioativas mais importantes para a descoberta de novos fármacos. Neste ambiente, os organismos estão em constante interação ecológica por meio da produção de metabólitos secundários. Fungos endofíticos e cianobactérias representam grupos de micro-organismos que realizam a biossíntese de substâncias com características químicas únicas e atividades biológicas potentes. Entretanto, quando retirados de seu habitat natural, esses seres microbianos geralmente perdem sua capacidade metabólica através de um fenômeno denominado silenciamento gênico, no qual genes biossintéticos deixam de ser transcritos devido a motivos ainda indeterminados. Esse mecanismo genético é intermediado, dentre outros fatores, pelas enzimas DNAmetiltransferase (DNA-MT) e Histona-desacetilase (HDAC). Deste modo, seus inibidores têm sido utilizados com sucesso para promover a eliciação de substâncias que não seriam produzidas em condições laboratoriais. Outra importante abordagem na pesquisa de produtos naturais têm sido a desreplicação baseada na fragmentação (MS/MS) para identificação de substâncias ou análogos. As redes moleculares (molecular networking) constituem uma nova abordagem na qual dados de espectrometria de massas são agrupados de acordo com as semelhanças entre os padrões de fragmentação, formando famílias de moléculas, permitindo a rápida visualização do perfil químico de várias amostras ao mesmo tempo. Deste modo, este trabalho apresenta o isolamento e identificação de metabólitos inéditos a partir de fungos endofíticos e cianobactérias oriundos do ambiente marinho. Para isto, técnicas de eliciação epigenética foram utilizadas em ambos os grupos de organismos, e a desreplicação via redes moleculares foi utilizada em cianobactérias. Fungos endofíticos associados à macroalga vermelha Bostrychia tenella foram alvo de estudos químicos e epigenéticos. As linhagens Xylaria sp. e Nigrospora oryzae foram submetidas ao cultivo em meio sólido arroz, o que resultou no isomento da substância citocalasina D e de um derivado potencialmente inédito da griseofulvina. A linhagem Penicillium decaturense foi cultivada em meio líquido PDB resultando no isolamento da 10,11-deidrocurvularina e possíveis análogos. Experimentos com inibidores epigenéticos (butirato de sódio e procaína) promoveram a modulação do perfil químico desta linhagem, ao estimular a produção de metabólitos não expressos em condições normais de cultivo. Ainda, a linhagem Acremonium sp. produziu várias substâncias quando cultivada em meio de líquido Czapek sob a influência de procaína, sendo uma delas potencialmente inédita e derivada da classe de metabólitos das brevianamidas. Frações orgânicas da cianobactéria Schizothrix sp., coletada no Panamá, foram analisadas em LC-MS/MS e os dados gerados foram utilizados para a criação de redes moleculares. Este estudo resultou na identificação dos metabólitos barbamida, hectoclorina, curacinas A e D, curazole, acetato de malingamida D, dolastatina 10 e carmaficina B. Ainda, análogos das substâncias curazole, dolastatina D e dois análogos inéditos das carmaficinas foram propostos. A cianobactéria Moorea producens JHB, coletada na Jamaica, foi submetida ao cultivo sob influência do ii composto butirato de sódio, e produziu dois metabólitos inéditos, propostos de acordo com os dados de fragmentação, como sendo derivados da jamaicamida e da hectoclorina, num tipo de biossíntese cruzada. Portanto, este trabalho confirma os fungos endofíticos e cianobactérias marinhos como promissores quanto a exploração do metabolismo secundário. / Marine natural products are pointed out as one of the most important sources of bioactive compounds for drug discovery. In this environment, organisms are in constantly interaction ecological through the production of secondary metabolites. Endophytic fungi and cyanobacteria represent groups of microorganisms that perform biosynthesis of substances with unique chemical features and potent biological activities. However, when removed from their natural habitat, these microbial beings generally lose their metabolic capacity through a phenomenon called gene silencing, in which biosynthetic genes are no longer transcribed due to reasons still undetermined. This genetic mechanism is brokered, among other factors, by the enzyme DNA methyltransferase (DNA-MT) and histone deacetylase (HDAC). Thus, their inhibitors have been used successfully to promote the elicitation of substances that would not be produced under laboratory conditions. Another important approach in the natural products research field have been dereplication based on the fragmentation (MS/MS) for the identification of substances or analogues. The molecular networking is a new approach in which data from mass spectrometry are grouped according to the similarities between the patterns of fragmentation, forming families of molecules, allowing rapid visualization of the chemical profile of several samples simultaneously. Thus, this work presents the isolation and identification of novel metabolites from endophytic fungi and cyanobacteria originating from the marine environment. For this propose, epigenetic elicitation techniques were used in both groups of organisms and the molecular networks via dereplication was used in cyanobacteria. Endophytic fungi associated with red seaweed Bostrychia tenella were subjected to chemical and epigenetic studies. Xylaria sp. and Nigrospora oryzae strains were cultured in solid medium rice, resulting in isolation of substance of cytochalasin D and a potentially novel derivative of griseofulvin. Penicillium decaturense strain was grown in PDB liquid medium resulting in the isolation of 10,11- deidrocurvularina and possible analogues. Experiments with epigenetic inhibitors (sodium butyrate and procaine) promoted the modulation of the chemical profile of this strain, to stimulate the production of metabolites not expressed under normal culture conditions. Moreover, Acremonium sp. produced various substances when grown in liquid medium under the influence of Czapek procaine, one of novel and potentially derived from the class of metabolites brevianamides. Organic fractions of the cyanobacteria Schizothrix sp., collected in Panama, were analized by LC-MS/MS and the data generated were used to create molecular networks. This study resulted in the identification of metabolites barbamide, hectochlorin, curacins A and D, curazole malyngamide D acetate, dolastatin 10 and carmaphycin B. Also, analogs of curazole, dolastatin 10 and carmaphycins A and B have been proposed. Cyanobacteria Moorea producens JHB, collected in Jamaica, was grown under the influence of sodium butyrate, and produced two new proposed metabolites in accordance with the fragmentation data as being derived from jamaicamide and hectochlorin, in a sort of crossed biosynthesis. Therefore, this work corroborates marine endophytic fungi and cyanobacteria as promising for exploration of secondary metabolism.

Os produtos naturais marinhos são apontados com uma das fontes de substâncias bioativas mais importantes para a descoberta de novos fármacos. Neste ambiente, os organismos estão em constante interação ecológica por meio da produção de metabólitos secundários. Fungos endofíticos e cianobactérias representam grupos de micro-organismos que realizam a biossíntese de substâncias com características químicas únicas e atividades biológicas potentes. Entretanto, quando retirados de seu habitat natural, esses seres microbianos geralmente perdem sua capacidade metabólica através de um fenômeno denominado silenciamento gênico, no qual genes biossintéticos deixam de ser transcritos devido a motivos ainda indeterminados. Esse mecanismo genético é intermediado, dentre outros fatores, pelas enzimas DNAmetiltransferase (DNA-MT) e Histona-desacetilase (HDAC). Deste modo, seus inibidores têm sido utilizados com sucesso para promover a eliciação de substâncias que não seriam produzidas em condições laboratoriais. Outra importante abordagem na pesquisa de produtos naturais têm sido a desreplicação baseada na fragmentação (MS/MS) para identificação de substâncias ou análogos. As redes moleculares (molecular networking) constituem uma nova abordagem na qual dados de espectrometria de massas são agrupados de acordo com as semelhanças entre os padrões de fragmentação, formando famílias de moléculas, permitindo a rápida visualização do perfil químico de várias amostras ao mesmo tempo. Deste modo, este trabalho apresenta o isolamento e identificação de metabólitos inéditos a partir de fungos endofíticos e cianobactérias oriundos do ambiente marinho. Para isto, técnicas de eliciação epigenética foram utilizadas em ambos os grupos de organismos, e a desreplicação via redes moleculares foi utilizada em cianobactérias. Fungos endofíticos associados à macroalga vermelha Bostrychia tenella foram alvo de estudos químicos e epigenéticos. As linhagens Xylaria sp. e Nigrospora oryzae foram submetidas ao cultivo em meio sólido arroz, o que resultou no isomento da substância citocalasina D e de um derivado potencialmente inédito da griseofulvina. A linhagem Penicillium decaturense foi cultivada em meio líquido PDB resultando no isolamento da 10,11-deidrocurvularina e possíveis análogos. Experimentos com inibidores epigenéticos (butirato de sódio e procaína) promoveram a modulação do perfil químico desta linhagem, ao estimular a produção de metabólitos não expressos em condições normais de cultivo. Ainda, a linhagem Acremonium sp. produziu várias substâncias quando cultivada em meio de líquido Czapek sob a influência de procaína, sendo uma delas potencialmente inédita e derivada da classe de metabólitos das brevianamidas. Frações orgânicas da cianobactéria Schizothrix sp., coletada no Panamá, foram analisadas em LC-MS/MS e os dados gerados foram utilizados para a criação de redes moleculares. Este estudo resultou na identificação dos metabólitos barbamida, hectoclorina, curacinas A e D, curazole, acetato de malingamida D, dolastatina 10 e carmaficina B. Ainda, análogos das substâncias curazole, dolastatina D e dois análogos inéditos das carmaficinas foram propostos. A cianobactéria Moorea producens JHB, coletada na Jamaica, foi submetida ao cultivo sob influência do ii composto butirato de sódio, e produziu dois metabólitos inéditos, propostos de acordo com os dados de fragmentação, como sendo derivados da jamaicamida e da hectoclorina, num tipo de biossíntese cruzada. Portanto, este trabalho confirma os fungos endofíticos e cianobactérias marinhos como promissores quanto a exploração do metabolismo secundário. / Marine natural products are pointed out as one of the most important sources of bioactive compounds for drug discovery. In this environment, organisms are in constantly interaction ecological through the production of secondary metabolites. Endophytic fungi and cyanobacteria represent groups of microorganisms that perform biosynthesis of substances with unique chemical features and potent biological activities. However, when removed from their natural habitat, these microbial beings generally lose their metabolic capacity through a phenomenon called gene silencing, in which biosynthetic genes are no longer transcribed due to reasons still undetermined. This genetic mechanism is brokered, among other factors, by the enzyme DNA methyltransferase (DNA-MT) and histone deacetylase (HDAC). Thus, their inhibitors have been used successfully to promote the elicitation of substances that would not be produced under laboratory conditions. Another important approach in the natural products research field have been dereplication based on the fragmentation (MS/MS) for the identification of substances or analogues. The molecular networking is a new approach in which data from mass spectrometry are grouped according to the similarities between the patterns of fragmentation, forming families of molecules, allowing rapid visualization of the chemical profile of several samples simultaneously. Thus, this work presents the isolation and identification of novel metabolites from endophytic fungi and cyanobacteria originating from the marine environment. For this propose, epigenetic elicitation techniques were used in both groups of organisms and the molecular networks via dereplication was used in cyanobacteria. Endophytic fungi associated with red seaweed Bostrychia tenella were subjected to chemical and epigenetic studies. Xylaria sp. and Nigrospora oryzae strains were cultured in solid medium rice, resulting in isolation of substance of cytochalasin D and a potentially novel derivative of griseofulvin. Penicillium decaturense strain was grown in PDB liquid medium resulting in the isolation of 10,11- deidrocurvularina and possible analogues. Experiments with epigenetic inhibitors (sodium butyrate and procaine) promoted the modulation of the chemical profile of this strain, to stimulate the production of metabolites not expressed under normal culture conditions. Moreover, Acremonium sp. produced various substances when grown in liquid medium under the influence of Czapek procaine, one of novel and potentially derived from the class of metabolites brevianamides. Organic fractions of the cyanobacteria Schizothrix sp., collected in Panama, were analized by LC-MS/MS and the data generated were used to create molecular networks. This study resulted in the identification of metabolites barbamide, hectochlorin, curacins A and D, curazole malyngamide D acetate, dolastatin 10 and carmaphycin B. Also, analogs of curazole, dolastatin 10 and carmaphycins A and B have been proposed. Cyanobacteria Moorea producens JHB, collected in Jamaica, was grown under the influence of sodium butyrate, and produced two new proposed metabolites in accordance with the fragmentation data as being derived from jamaicamide and hectochlorin, in a sort of crossed biosynthesis. Therefore, this work corroborates marine endophytic fungi and cyanobacteria as promising for exploration of secondary metabolism.

In order to continue with the study of natural products from previously isolated endophytes of the Brazilian medicinal plant Lychnophora ericoides, the main goal of this work focused on the metabolic exchange of endophytic microorganisms from this plant. As it has been demonstrated during the last years, elicitation of different molecules is consequence of microbial interactions, mainly due to the need to compete, survive and establish microbial communities in shared environments. Recent mass spectrometry related approaches, such as imaging and molecular networking, in combination with purification and structural elucidation were applied to the current study of the microbial interactions of endophytes. During this study, several chemical families were identified, such as polyene macrocycles, pyrroloindole alkaloids, leupeptins, angucyclines, siderophores, amongst others. Amongst the polyene macrocycles, the well-known antifungal amphotericin B was identified as a biosynthetic product of the endophytic actinobacteria Streptomyces albospinus RLe7. When S. albospinus RLe7 interacted with an endophytic fungus, Coniochaeta sp. FLe4, a red pigmentation was observed. A new fungal compound was detected from microbial interactions. Isolation and structure elucidation of this new compound enabled to demonstrate the elicitation of fungal secondary metabolites by amphotericin B, an actinobacterial metabolite. It was also demonstrated the elicitation of griseofulvin and its analogue dechlorogriseofulvin from another endophytic fungus, FLe9, as a consequence of exposition to amphotericin B. In addition, investigation of the chemical profile of another endophytic actinobacteria, S. mobaraensis RLe3, led to reveal this strain as angucycline-derivatives producer. Besides that, coculturing of this actinobacteria against Coniochaeta sp. FLe4 also demonstrated elicitation of angucycline analogues. In conclusion, this study demonstrated elicitation of natural products from microbial interactions as well as new compounds from endophytes from L. ericoides and contributed to the identification of microbial metabolites / Com o propósito de continuar com os estudos de produtos naturais de microorganismos endofíticos da planta medicinal Brasileira Lychnophora ericoides, o principal objetivo desse trabalho focou-se no estudo da troca metabólica de microorganismos endofíticos dessa planta. Como tem sido demonstrado durante os últimos anos, a elicitação de diferentes compostos é consequência das interações microbianas, principalmente devido à necessidade de competir, sobreviver e estabelecer comunidades microbianas em diversos ambientes naturais. Abordagens recentes de espectrometria de massas, tais como imageamento e molecular networking, junto com purificação e elucidação estrutural foram aplicadas durante o estudo de interações microbianas de endofíticos. Durante este estudo, várias classes químicas foram identificadas, tais como polienos macrocíclicos, alcaloides pirroloindólicos, leupeptinas, anguciclinas, sideróforos, entre outras. Da classe química de polienos macrocíclicos, foi identificada a anfotericina B como produto da biossíntese da actinobactéria endofítica Streptomyces albospinus RLe7. Durante a interação da S. albospinus RLe7 com o fungo endofítico Coniochaeta sp. FLe4, uma pigmentação vermelha foi observada. Um novo composto de origem fúngica foi detectado a partir de interações microbianas. O isolamento e posterior elucidação estrutural do novo composto permitiu demonstrar a eliciação de metabólitos secundários fúngicos pela anfotericina B, metabólito da actinobactéria S. albospinus RLe7. Foi também demonstrada a eliciação de griseofulvina e desclorogriseofulvina a partir de outro fungo endofítico, FLe9, como consequência da exposição à anfotericina B. Adicionalmente, a investigação do perfil químico de outra actinobactéria endofítica, S. mobaraensis RLe3, evidenciou essa linhagem como produtora de compostos da classe das anguciclinas. Além disso, o cocultivo dessa actinobacteria com o fungo endofítico Coniochaeta sp. FLe4 também eliciou análogos de anguciclinas. Em conclusão, neste estudo demonstrou-se a elicitação de produtos naturais a partir das interações microbianas assim como de novos compostos de endofíticos de L. ericoides e contribuiu-se com a identificação de metabólitos microbianos

Os produtos naturais são considerados excelentes fontes de princípios ativos. O gênero Passiflora L. pertencente à família Passifloraceae, possui cerca de 500 espécies e, devido à sua ampla distribuição pela América do Sul, faz do Brasil uma região importante de sua ocorrência. Desde o século XVIII, há estudos científicos voltados ao conhecimento desse gênero, e suas espécies são tradicionalmente utilizadas como sedativas. Quanto à sua composição química, sabe-se que seus metabólitos majoritários são pertencentes à classe dos flavonoides, principalmente os do tipo C-glicosilados. Apesar da grande importância do gênero, os trabalhos concentram-se nas espécies de uso tradicional, P. incarnata; P. alata e P. edulis. Esse fato gera a necessidade da extensão dos estudos a espécies de menor conhecimento cientifico. P. cincinnata é uma espécie nativa do Brasil, com ampla distribuição geográfica, sendo encontrada facilmente no estado da Bahia, entretanto há poucas informações quanto à sua composição química. O avanço da espectrometria de massas, juntamente com a eficiência da CLAE, faz dessas técnicas hifenadas a principal ferramenta para o estudo da química de produtos naturais. Recentemente a estratégia de integração da espectrometria de massa com rede molecular criada pelo GNPS alterou os métodos convencionais de estudo da química de produtos naturais, auxiliando na desreplicação de compostos conhecidos e na identificação de novos metabólitos. Visando o melhor conhecimento da química de espécies de Passiflora, realizou-se um estudo fitoquímico de P. cincinnata, isolando e identificando os seus compostos majoritários: isoorientina; isovitexina; isoscoparina; isovitexina-2\"-O-?-glicopiranosídeo; isovitexina-2\"-O-?-xilopiranosídeo e isoorientina-2-O-?-xilopiranosídeo, bem como a determinação do perfil fitoquímico dos principais metabólitos presentes em suas folhas e caules por CLAE-DAD-IES-ITEMn, que levou à identificação de mais 19 metabólitos pertencentes à classe dos flavonoides e dos derivados do ácido clorogênico. Utilizando os dados de EM/EM das substâncias isoladas e das identificadas em P. cincinnata, foram geradas redes moleculares pelo GNPS, identificando mais 16 flavonoides e obtendo um modelo que auxiliou no estudo de desreplicação de 46 espécies mantidas em herbário. A obtenção dos perfis cromatográficos por CLAE-DAD-IES-IT-EM/EM dessas espécies e sua integração com redes moleculares viabilizou a realização de uma proposta de identificação de mais de 100 derivados fenólicos, pertencentes às classes dos flavonoides, proantocianidinas e derivados do ácido clorogênico, gerando maior conhecimento sobre a composição química dessas espécies, abrindo, assim, fronteira para novos estudos e confirmando o potencial da combinação dessas ferramentas no estudo da química de produtos naturais. / Natural products historically have been considered excellent source of active principles. The genus Passiflora L. belonging to the Passifloraceae family, has about 500 species and due to its wide distribution in South America, makes Brazil an important region of its occurrence. Since the 18th century, there are scientific studies aimed at the knowledge of this genus and its species have traditionally been used as sedatives. Regarding its chemical composition, it is well known that its major metabolites belong to the class of flavonoids, especially C-glycosylated type. Despite the great importance of the genus, the research are focused on species of traditional use, P. incarnata; P. alata and P. edulis. This fact generates the need to extend the studies to species with less scientific knowledge. P. cincinnata is a native species of Brazil, with a large geographic distribution, being easily found in the State of Bahia, however, there is little information about its chemical composition. The advance of mass spectrometry together with the efficiency of the HPLC makes these hyphenated techniques the main tool for the study of the chemistry of natural products. Recently the strategy of integrating mass spectrometry with molecular networking created by GNPS has changed the conventional methods of studying the chemistry of natural products, assisting the dereplication of known compounds and the identification of new metabolites. Aiming to expand the knowledge of the chemistry of Passiflora species, the phytochemical study of the species P. cincinnata was carried out, isolating and identifying its major compounds, isoorientin, isoorientin-(1\'\"->2\")-O-?- xyloside, isovitexin-(1\'\"->2\")-O-?-xyloside, isovitexin, isovitexin-(1\'\"->2\")-O-?- glucopyranoside and isoscoparin, as well as determinating the phytochemical profile of the main metabolites present in its leaves and stems by HPLC-DAD-ESI-IT-MSn, the identification of 19 more metabolites belonging to the class of flavonoids and derivatives of chlorogenic acid. Using the MS/MS data of the isolated and identified substances in P. cincinnata, molecular networking were generated by the GNPS identifying 16 flavonoids and obtaining a model that assisted in the study of the dereplication of another 46 species from the herbarium. The chromatographic profile obtained by HPLC-DAD-ESI-IT-MS/MS of these species and the integration of them with molecular networking enabled a proposal to identify about 100 phenolic derivatives belonging to the classes of flavonoids, proanthocyanidins and chlorogenic acid derivatives, generating a greater knowledge about the chemical composition of these species, opening up for new studies and confirming the potential of the combination of these tools in the study of the chemistry of natural products.

En milieu marin, toute surface immergée est soumise à une colonisation par de nombreux organismes (biofouling). Le développement de biofilms est une étape clé du phénomène. Les systèmes de communication y sont contrôlés par le biais de signaux chimiques. Dans ce travail, l’étude de la signature métabolique de biofilms naturels formé in situ a été réalisée selon un gradient de pollution en contaminants métalliques dans la rade de Toulon et selon la nature du revêtement de la surface immergée. De nettes variations chimiques des biofilms prélevés sont observées et sont corrélées avec des variations en termes de communauté microbiennes. L’étude in vitro de 4 souches bactériennes issues de biofilms naturels a permis, après optimisation des méthodologies d’analyse, une discrimination selon leur profil métabolique. Des biomarqueurs ont été mis en évidence, avec notamment la production de lipides ornithine par la souche Pseudoalteromonas lipolytica. La réponse biologique de cette souche en fonction de son phénotype et face à un stress cuprique a été étudiée par métabolomique et protéomique révélant d’importantes modulations de certaines voies biosynthétiques. / In the marine environment, any immersed surface is subjected to colonization by many organisms (biofouling). The biofilms development is a key stage of this phenomenon. Communication systems are controlled in these structures by chemical signals. In this work, the study of the chemical signature of natural biofilms formed in situ was carried out among a gradient of contamination of metal contaminants in the bay of Toulon and according to the nature of the coating on the immersed surface. Clear chemical variations of the biofilms collected were observed and were correlated with variations in microbial community. The in vitro study of 4 bacterial strains harvested from natural biofilms allowed, after optimization of the analysis methodologies, their discrimination according to their metabolic profile. Biomarkers were highlited, particularly ornithine lipids production by the Pseudoalteromonas lipolytica strain. The biological response of this strain depending on its phenotype and face to copper stres was studied by metabolomics and proteomics revealing important modulations of certain biosynthetic patways.

Dans le but d'identifier de nouveaux inhibiteurs du chikungunya (CHIKV), de la dengue (DENV) et de la zika (ZIKV), une étude systématique portant sur 339 extraits d'Euphorbiaceae tropicales a été réalisée grâce à un test cellulaire d'inhibition du CHIKV et à des tests d'inhibition de l'ARN-polymérase de DENV et ZIKV.Sandwithia guyanensis et Sagotia racemosa, deux espèces provenant de Guyane française, dont les extraits d'écorce présentaient une forte activité anti-CHIKV importante, ont d'abord été étudiées. À la suite d'un processus d'isolement bioguidé classique, plus de 20 nouveaux diterpènes ont été caractérisés, mais aucun d'entre eux ne présentait une activité antivirale significative. Pour résoudre ce problème, des réseaux moléculaires (MN) ont été construits grâce aux données de LC-MS/MS obtenues à partir des fractions chromatographiques des deux extraits. L'annotation des MN, a permis l'identification d'analogues du phorbol, connus pour être de puissants inhibiteurs du CHIKV. Présents à l'état de traces, ces composés fournissent une explication plausible à l'activité anti-CHIKV des deux extraits.Dans une deuxième étude, une stratégie de priorisation des extraits de plantes basée sur la fusion de données taxonomiques et d'essais biologiques au sein d'un MN a conduit à l'isolement et à la caractérisation ciblée de plusieurs inhibiteurs doubles de Codiaeum peltatum. Les deux études illustrent comment les MN et les progrès récents en matière d'annotation des données de LC-MS/MS peuvent être mis en œuvre dans les études phytochimiques pour améliorer le processus découverte de composés bioactifs. / In order to identify new inhibitors of chikungunya (CHIKV), dengue fever (DENV) and zika (ZIKV), systematic study with 339 extracts from tropical Euphorbiaceae species was performed in a virus-cell-based assay for CHIKV and DENV and ZIKA NS5 inhibition assaysThe French Guianese species Sandwithia guyanensis and Sagotia racemosa, from which bark extracts exhibited significant anti-CHIKV activities, were first investigated. Following a classical bioguided isolation workflow, more than 20 new diterpenes were characterized but none of them showed significant antiviral activity. To adress this issue, molecular networks (MN) were built from the LC-MS/MS data acquired from the chromatographic fractions of both extracts. The annotation of the MNs led to the identification of phorbol analogues, known to be potent CHIKV inhibitors. Present at trace levels, these compounds provide a plausible explanation for the anti-CHIKV activity of both extracts.In a second study, a strategy to prioritize plant extracts based on the fusion of taxonomic data and bioassays within a MN led to the isolation and targeted characterization of several dual inhibitors from Codiaeum peltatum. Both studies illustrate how MN and recent advances in LC-MS/MS data annotation can be implemented in phytochemical studies to improve the bioactive compounds discovery process.

Les alcaloïdes indolo-monoterpéniques (AIMs) constituent une classe de molécules naturelles très étudiée en raison d’un fort potentiel pharmacologique et thérapeutique et d’une grande diversité structurale. D’autre part, les techniques de déréplication par chromatographie liquide couplée à la spectrométrie de masse tandem ont évolué récemment, avec l’émergence de l’approche par réseaux moléculaires (molecular networking). Dans ce contexte, nous avons créé une base de données de spectres de masse tandem moyennés de 172 AIMs témoins, en collaboration avec plusieurs équipes de pharmacognosie dans le monde. Cette base de données, nommée MIADB (Monoterpene Indole Alkaloids DataBase), rendue publique, peut être utilisée comme référence dans des stratégies de déréplication fondées sur l’utilisation des réseaux moléculaires. Nous avons ensuite exploité la MIADB pour l’étude phytochimique de deux Apocynaceae : Geissospermum laeve (Vell.) Miers et Alstonia balansae Guillaumin. Dans un premier temps, l’annotation par la MIADB d’un extrait alcaloïdique des écorces de G. laeve a permis l’isolement et l’élucidation structurale de 3 nouveaux AIMs, dont deux à motif butyrolactone. Par la suite, notre approche a été améliorée par l’emploi du nouvel outil d’annotation in silico MetWork, fondé sur une prédiction métabolique et la modélisation de spectres de masse tandem. C’est ainsi que l’exploration de l’espace chimique d’un extrait alcaloïdique des feuilles d’A. balansae a permis l’identification, puis l’isolement, de 5 nouveaux AIMs du type N-oxyde-sarpagane. La stéréochimie des nouveaux composés a pu être déterminée par l’exploitation de spectres prédits et expérimentaux de dichroïsme circulaire éléctronique.Ce manuscrit décrit, après une introduction consacrée notamment aux emplois du molecular networking pour le ciblage et la découverte de petites molécules naturelles puis aux interconnections biosynthétiques en série AIM, la création puis l’évolution de la MIADB, puis son utilisation dans un workflow de déréplication efficace et de ciblage de nouveaux composés dans des mélanges complexes issus d’Apocynaceae. / Monoterpene indole alkaloids (MIAs) constitute a class of natural products that has been extensively studied due to its important pharmacological and therapeutic potentials, and to its large structural diversity. Dereplication techniques based on liquid chromatography coupled to tandem mass spectrometry have recently evolved, with the implementation of molecular networking-based approaches.In this context, we have created a spectral database that encompasses the averaged tandem mass spectra of 172 reference MIAs, in collaboration with several pharmacognosy research teams around the world. This database, named MIADB (standing for Monoterpene Indole Alkaloids DataBase), was made publicly available and can be used as a reference in the application of molecular networking as a dereplication strategy. Thereafter, we used the MIADB to carry out the phytochemical investigation of two Apocynaceae species: Geissospermum laeve (Vell.) Miers and Alstonia balansae Guillaumin. As a first application, the MIADB-based annotation of an alkaloid extract of the barks of G. laeve led to the isolation and the structural elucidation of three new MIAs, two having a butyrolactone moiety. Afterwards, this approach was improved by the application of a new tool for in silico annotation called MetWork, which is based on metabolic prediction and on the generation of predicted tandem mass spectra. Following this approach, the exploration of the chemical space of an alkaloid extract of the leaves of A. balansae allowed the anticipation and further isolation of five novel MIAs of the N-oxide-sarpagine type. The stereochemistry of all the new molecules could be determined on the basis of experimental and predicted electronic circular dichroism spectra.In the introduction of this manuscript the multiple uses of molecular networking for the identification of small natural molecules are described, as well as the biosynthetic interconnections in the MIAs group. The creation and evolution of the MIADB are then presented, followed by its utilization in efficient dereplication workflows for the targeting of new natural products within complex mixtures from Apocynaceae species.

O ambiente marinho apresenta uma rica biodiversidade e, por ser ainda pouco explorado configura-se como uma fonte potencial de novos organismos da flora e fauna marinhas, o que possibilita a descoberta de estruturas distintas e biologicamente ativas. Microrganismos marinhos como cianobactérias e fungos possuem um metabolismo secundário rico, o qual produz substâncias bioativas. Em virtude do exposto, um dos objetivos deste trabalho foi avaliar química e biologicamente as linhagens de cianobactérias marinhas Cyanobium sp. CENA178, Cyanobium sp. CENA181 e Oxynema sp. CENA135 provenientes de manguezais da Ilha do Cardoso, do Estado de São Paulo. Foram identificados os aminoácidos alanina, treonina e valina presentes na fração ACN:H2O (40:60) da linhagem Oxynema sp. CENA135. Por meio das análises realizadas em CG-EM, a fração n-hexano/acetato de etila (9:1) da linhagem Cyanobium sp. CENA178 apresentou os componentes: 2,4-bis (1,1-dimetiletil) fenol, (Z)-7-hexadeceno 6,10,14-trimetil-2-pentadecanona e eicosano; a fração n-hexano da linhagem Cyanobium sp. CENA181 apresentou como componente majoritário o neoftadieno, o qual também foi encontrado na fração acetato de etila da linhagem Oxynema sp. CENA135, juntamente com os componentes majoritários: heptadecano, [R-[R*,R*-(E)]]- 3,7,11,15-tetrametil-2-hexadeceno e octadecino. Com relação à triagem biológica, as linhagens Cyanobium sp. CENA178 e Cyanobium sp. CENA181, apresentaram atividade anticolinesterásica moderada e fraca, respectivamente. Concomitantemente foi realizada uma co-cultura entre a linhagem de cianobactéria Cyanobium sp. CENA181 e o fungo endofítico marinho Penicillium decaturense, onde foi isolada do extrato bruto do fungo P. decaturense a substância 10,11-deidrocurvularina e como produto da co-cultura foi obtida a substância curvularina. Deste modo, foi demonstrado que houve interação química entre os microrganismos devido a produção de diferentes metabólitos secundários. Adicionalmente, foram investigadas nove linhagens de cianobactérias do gênero Symploca quanto à análise química e biológica, provenientes do Panamá, Samoa Americana e uma da coleção de cultura de Pasteur (PCC8002). Por meio da análise de redes moleculares (molecular networking) foram identificadas três substâncias conhecidas: apratoxina A; palmiramida A e curacina D. Das frações A2143 E e F foi isolada uma substância inédita denominada de caracolamida A. Da fração G foi isolada uma substância conhecida pertencente à classe dos polimetoxi-alquenos isotáticos denominada de 4,6,8,10,12,14,16,18,20,22-decametóxi-heptacos-1-eno. A substância inédita caracolamida A foi comparada com o padrão feniletilamina e ambas demonstraram ter atividade neuromoduladora em concentrações subnanomolares, possuindo efeitos similares na oscilação e frequência dos canais de Ca2+. Os resultados apresentados neste trabalho acrescentam dados químicos e biológicos às espécies estudadas e enriquecem a área de Produtos Naturais Marinhos. / The marine environment contains a rich biodiversity and, since it is relatively underexplored, is a great source for finding new organisms including marine flora and fauna. This enables the discovery of chemicals with distinct and structures and biological activity. Marine microorganisms, such as cyanobacteria and fungi, have rich secondary metabolism, which yield biologically active molecules. Accordingly, one aim of this work was to evaluate the chemical profiles and biological activities of the compounds isolated from marine cyanobacteria strains Cyanobium sp. CENA178, Cyanobium sp. CENA181 and Oxynema sp. CENA135 from Ilha do Cardoso mangrove, State of São Paulo. The results indicate that it would help to adopt different laboratory cultivation methods for growing cyanobacteria strains to mimic the natural habitat and increase the opportunity to obtain new secondary metabolites. Aminoacids alanine, threonine and valine were identified of the fraction ACN:H2O (40:60) of the strain Oxynema sp. CENA135. Through the analyzes performed in CG-MS, the hexane/ethyl acetate (9:1) fraction of the strain Cyanobium sp. CENA178 showed the components: 2,4-bis (1,1-dimethylethyl) phenol, (Z)-7-hexadecene, 6,10,14-trimethyl-2-pentadecanone and eicosane. From the hexane fraction of Cyanobium sp. CENA181, neophytadiene was observed as the major component, and this was also found in the ethyl acetate fraction of Oxynema sp. CENA135 strain along with heptadecane, 2-hexadecene, 3,7,11,15-tetramethyl-, [R-[R*,R*-(E)]] and 1-octadecyne. Cyanobium sp. CENA178 and Cyanobium sp. CENA181 showed moderate and weak anticholinesterase activity, respectively. Simultaneously, a co-culture was performed using Cyanobium sp. CENA181 and the marine endophytic fungus, Penicillium decaturense. The compound 10,11-dehydrocurvularin was isolated from the crude extract of P. decaturense, and curvularin from only the co-culture. Therefore, it was clear that the microorganisms exhibited an interaction leading to the different production of secondary metabolites. Nine species of cyanobacteria of the genus Symploca from Panama, American Samoa and one from Pasteur culture collection (PCC8002) were investigated to yield new natural products. Through molecular networking analysis three known compounds were identified: apratoxin A; palmiramide A and curacin D. Caracolamide A is a new compound isolated from fractions A2143 E and F. A known compound isolated from the same organism is called 4,6,8,10,12,14,16,18,20,22-decamethoxy-heptacos-1-ene, and it is an isotactic polymethoxy-1-alkene. The new compound caracolamide A demonstrated neuromodulatory activity at subnanomolar concentrations, and displayed similar effects as a phenylethylamine standard on the oscillation amplitude and frequency in Ca2+ channels. The results presented in this work provide chemical and biological information about the species studied, and enrich marine natural products research

Cette thèse initie et développe un programme ayant pour but de définir une stratégie de priorisation efficace pour accélérer la découverte de molécules bioactives au sein d’extraits végétaux. Dans le cadre de ce projet, plusieurs criblages biologiques ont été menés sur une collection de 292 extraits d'Euphorbiaceae.Afin d’identifier et cibler au sein de ces mélanges complexes les structures d’intérêt biologique tout en écartant les molécules connues ou présentant un intérêt structural limité, les profils métabolomiques des extraits ont été acquis par spectrométrie de masse tandem. Pour exploiter au mieux la quantité d'information générée par ces analyses, les spectres MS2 ont ensuite été organisés sous forme de réseaux moléculaires. Ces réseaux permettent de lier les ions détectés en fonction de la similarité de leurs voies de fragmentation et donc de leur proximité structurale. Les informations taxonomiques et d’activités biologiques ont été croisées avec les données spectrales au sein de cette carte moléculaire multi-informative, offrant ainsi une approche nouvelle pour accélérer la découverte de nouveaux ligands des cibles biologiques étudiées et pour une sélection plus pertinente des extraits à forte diversité structurale.Si l’outil des réseaux moléculaires représente une méthode innovante et particulièrement instructive pour le phytochimiste, il présente cependant quelques défauts qui limitent son spectre d'utilisation et ses capacités en métabolomique. Une deuxième partie de cette thèse a donc été consacrée à l’implémentation d’une étape de prétraitement pour améliorer la fiabilité des réseaux et au développement de MetGem, un logiciel dédié à la génération de réseaux moléculaires permettant d’optimiser la gestion et l’analyse des matrices de scores de similarité spectrale. / This thesis initiates and develops a program seeking to accelerate the discovery of new therapeutic molecules using an efficient prioritization strategy. As part of this project, a collection of 292 Euphorbiaceae extracts was screened over several biological targets.To focus on unknown bioactive chemicals and to avoid the isolation of known or inactive molecules, the acquisition of high resolution tandem mass spectrometry profiles of these extracts was performed. To highlight relevant information within these data, MS2 spectra were organized as molecular networks. It consists in visualizing tandem mass spectrometry data by detecting related MS2 spectra and representing them in a same spectral space. Taxonomical details and bioassay screening results were merged with the network visualization to generate a comprehensive multi-informative molecular map, which offers a radically novel outlook to target novel bioactive scaffolds and select extracts with high structural diversity. Although very instructive for the phytochemist, the molecular networking tool has some imperfections that limit its potential in metabolomics. Therefore, the second part of this thesis was dedicated to the introduction of a data preprocessing step to enhance the networks reliability and to the development of MetGem, a software dedicated to the generation of molecular networks to improve the way matrices of similarity scores are managed and analyzed.

L’étude chimique de 11 plantes du Gabon, dont certaines utilisées en médecine traditionnelle, a été réalisée à la recherche de composés antiplasmodiaux de structures nouvelles. Deux Apocynaceae (Pleiocarpa mutica Benth., Callichilia inaequalis Stapf) et une Gelsemiaceae (Mostuea brunonis Didr.) ont été plus spécifiquement étudiées, via une stratégie de déréplication fondée sur des réseaux moléculaires générés à partir de données CLHP-MS/MS (Molecular Networking) et annotés avec une base de données d’alcaloïdes indolomonoterpéniques mise au point au laboratoire (MIADB). Cette investigation a guidé un travail de fractionnement et d’isolement, qui a permis l’obtention d’alcaloïdes indolomonoterpéniques très originaux, en termes de décorations ou de modes d’assemblage.Des écorces de tiges de P. mutica, 7 dimères indolomonoterpéniques non décrits dans la littérature ont été obtenus : 5 sont des bis-aspidofractanes dont 4 sont reliés par un pont méthylène (pléiokomenines A-D) ; deux sont des dimères du type aspidofractane-éburnamine, analogues de la pléiomutine.Les tiges et les racines de C. inaequalis ont livré 2 bis-indoles nouveaux, analogues de la criophylline, dont le premier porteur d’un sulfate dans cette classe d’alcaloïdes, ainsi que l’inaequalisine A, premier indolomonoterpène monomérique lié à un reste phénylpropène via une liaison C-C.L’étude des tiges et des feuilles de M. brunonis a conduit à l’isolement de quatre nouveaux composés : un monomère de type sarpagine (16-epi-méthylester-panarine) et 3 dimères bis-vobasines inédits à pont sulfide (théionbrunonines A-C). Des molécules connues, mais pas toujours identifiées dans les genres étudiés, ont également été isolées. Plusieurs des composés nouveaux présentent une activité antiplasmodiale de l’ordre du µM in vitro sur une souche de Plasmodium falciparum chloroquino-résistante. / The chemical study of 11 Gabonese plant species, some being used in traditional medicine, was performed in search of antiplasmodial compounds with new structures. Two Apocynaceae (Pleiocarpa mutica Benth., Callichilia inaequalis Stapf) and a Gelsemiaceae (Mostuea brunonis Didr.) were more specifically investigated, using LC-MS/MS data in a dereplicative approach based on the “molecular networking” strategy, with an annotation performed using an “in-house” monoterpene indole alkaloids database (MIADB). This approach was used to guide the isolation of original alkaloids, in terms of substitution patterns or of linkage. From the stem bark of P. mutica, 7 previously undescribed bis-indoles were obtained: five are bis-aspidofractanes, four of them being linked by a methylene bridge (pleiokomenines A-D); Two are aspidofractane-eburnane dimers analogous to pleiomutine. The twigs and roots of C. inaequalis yielded 2 new bis-indoles analogous to criophylline, among which one is the first natural sulfate-bearing indole monoterpene. Inaequalisine A, the first monomeric indole monoterpene linked to a phenylpropene moiety by a C-C linkage was also obtained.The study of the twigs and leaves of M. brunonis lead to 4 new compounds: A monomeric sarpagine (16-epi-methylester-panarine) and 3 unknown bis-vobasines which constituting monomers are linked by a sulfide bridge (theionbrunonines A-C).Known compounds were also isolated, some of which were previously undescribed in the genera studied here. Several of the new molecules exhibited antiplasmodial in vitro activity in the micromolar range against a chloroquine-resistant strain of P. falciparum.