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La réduction de l’expression de deux gènes codant pour la L-galactono-lactone-1,4-déshydrogénase (GalLDH) et de la GDP-D-mannose-3’,5’-épimérase (GME), enzymes de la voie de biosynthèse de l’ascorbate, a permis de mieux comprendre le rôle physiologique de ces enzymes chez la tomate. D’une part, l’étude de la GalLDH a mis en évidence la régulation complexe du métabolisme de l’ascorbate et la fonction essentielle de cette protéine au sein de la chaîne de transport des électrons au niveau mitochondrial. D’autre part, ce travail a révélé le rôle central de la GME à la fois pour la biosynthèse de l’ascorbate et la biosynthèse des polysaccharides pariétaux, notamment les mannanes et le rhamnogalacturonane II. Chez les plantes sous-exprimant la GME, nous avons pu noter l’incidence de perturbations de la structure pariétale sur les propriétés mécaniques des tiges et des fruits ainsi que sur la fécondation. Ces modifications ont notamment engendré une fragilité accrue des tiges et une stérilité partielle. La GME est donc déterminante pour la qualité nutritionnelle et organoleptique du fruit de tomate. Enfin, dans le cadre d’une approche de biologie intégrative, nos résultats associés aux données issues de plantes sous-exprimant des gènes codant pour des enzymes de la voie de recyclage de l’ascorbate chez la tomate ouvrent des perspectives originales pour l’approfondissement des connaissances sur la régulation et sur l’intégration du métabolisme de l’ascorbate dans le fonctionnement de la cellule. / Down-regulation of two genes encoding the L-galactono-1,4-lactone dehydrogenase (GalLDH) and the GDP-D-mannose-3',5'-epimerase (GME), enzymes of ascorbate biosynthesis pathway, led to a better understanding of the physiological role of these enzymes in tomato plants. On one hand, the study of GalLDH highlighted the complex regulation of ascorbate metabolism and the essential function of this protein in mitochondrial electron transport chain. Moreover, this work revealed the central role of the GME for both the ascorbate biosynthesis and the biosynthesis of cell wall polysaccharides, including mannans and rhamnogalacturonan II. In the GME-silenced plants, we found that modifications of the cell wall structure change the mechanical properties of stems and fruit as well as the fertilization. These changes led to an increase of stem fragility and to an increase of sterility. Therefore, GME plays a crucial role regarding the nutritional and organoleptic quality of tomato fruit. Finally, within the context of a systems biology approach, our results associated to datas obtained with plants silenced for recycling pathway related genes lead to the prospect to unravel the knowledges on the regulation and the integration of ascorbate metabolism in cell functions.

Les spirocétals sont des sous-unités présentes dans de nombreuses molécules naturelles d'intérêt biologique. Pour accéder à ces structures, la spirocyclisation d'éthers d'énol en milieu acide est une méthode de choix. L'application de la réaction de Julia-Kocienski à des lactones a permis d'obtenir exo-glycals et éthers d'énol exo-cycliques tri- et tétrasubstitués. Selon l'hétérocycle porté parla sulfone engagée, l'un ou l'autre des diastéréoisomères de l'éther d'énol peut être obtenu préférentiellement. La spirocyclisation des produits formés, si elle est réalisée dans des conditions thermodynamiques, mène au [6.6]-spirocétal le plus stable. Des conditions permettant d'obtenir le diastéréoisomère cinétique ont également été étudiées. Les spirocétals ainsi préparés ont été utilisés en synthèse totale. Par exemple, le fragment spirocétal de la Broussonetine H, ainsi que l'unité iminosucre, ont été obtenus efficacement de façon énantiopure. Enfin, les spirocétals diversement substitués ont permis de préparer plusieurs analogues du Bistramide A. Ce métabolite marin est un agent anticancéreux puissant qui se lie à l'actine pour bloquer la division cellulaire mais des interactions avec PKC-TM, notamment impliquant l'apoptose, sont à l'étude.

Les travaux présentés dans ce mémoire contribuent d'une part, à la diversification de l'offre PPAM en région Corse en caractérisant les plantes par l'étude détaillée de la composition chimique des huiles essentielles et d'extraits et d'autre part, au développement de la RMN du 13C en tant qu'outil d'analyse des mélanges complexes naturels. Ainsi, nous avons étudié par CPG (IR), CPG-SM et RMN du 13C la composition chimique d'huiles essentielles et d'extraits (hydrolat et micro-ondes) obtenus à partir de quatre espèces de menthe poussant à l'état spontané en Corse ainsi que de cinq espèces du genre Fortunella cultivés à la station INRA-GEQA de San Ghjulianu. La majorité des huiles essentielles de M. aquatica, M. pulegium et M. suaveolens ssp. suaveolens possèdent des compositions chimiques fréquemment décrites dans la littérature. En revanche, certaines huiles essentielles de M. suaveolens ssp. suaveolens présentent une composition jamais décrite, caractérisée par la présence majoritaire de la pipériténone (73,5 %). De la même manière, les huiles essentielles et extraits d'hydrolat de la sous-espèce insularis, endémique corso-sarde, possèdent des compositions chimiques originales, caractérisées par d'importantes teneurs en pulégone (respectivement 44,4 et 14,8 %) mais surtout en cis-cis-p-menthénolide (respectivement 27,3 et 67,3 %). Cette lactone p-menthanique, possédant le motif -méthylene--butyrolactone, n'avait jamais été identifiée à partir d'une source naturelle. Si la différenciation botanique des deux sous-espèces M. suaveolens ssp. suaveolens et ssp. insularis apparait délicate, la prise en compte du critère présence (ssp. insularis)/absence (ssp. suaveolens) de la cis-cis-p-menthénolide, constitue un outil fiable pour la détermination de la sous espèce. Les huiles essentielles de fruits du genre Fortunella présentent des similitudes avec celles du genre Citrus avec des teneurs en limonène voisines de 90%. Par contre, les d'huiles essentielles et extraits (hydrolat et micro-ondes) de feuilles de Fortunella japonica présentent des compositions chimiques, très originales caractérisés par des fortes teneurs en sesquiterpènes. Le germacrène D et le -élémol sont les composés majoritaires. L'utilisation de la RMN du 13C nous a permis d'identifier de nombreux sesquiterpènes oxygénés peu communs tels que le germacra-1(10),5-dièn-4-ol, l'amorph-4-èn-10-ol, l'amorph-4-èn-10-ol et le trans-guai-6-èn-10-ol. L'étude des huiles essentielles de fruits et de feuilles ainsi que des extraits micro-ondes de cinq espèces du genre Fortunella (F. japonica, F. margarita, F. obovata, F. crassifolia et F. hindsii) montre que seules des variations quantitatives peuvent permettre de différencier ces espèces.

In this study, we have synthesized two families of new carbohydrate-based amphiphilic derivatives: a series of alkyl glucoside ethers varying in terms of chain length and position on the sugar, and a series of glucosteroids varying in terms of alkyl spacer and, for the disutibstuted systems, in terms of alkyl side chain length. By the means of analytical methods, such as NMR spectroscopy, mass spectroscopy and elementary analysis, the structure of all the compounds was carefully established, as well as their purity. Their liquid crystalline behaviors were studied by the means of transmission light microscopy and differential scanning calorimetry. The two families of compounds which have been studied illustrate how much the behavior can be essentially related to polar interactions (H-bonding), therefore to the sugar moiety, for the ether series, or to hydrophobic interactions (lipid-lipid) in the glucosteroid series. In this latter series, preference for either steroid-stroid or steroid alkyl packing appears as an insight in understanding the behavior of complex lipids, showing potentially more than one conformational structure with important consequences on the supramolecular level, therefore to their potential biological role. This could be regarded as "lipid denaturation" by analogy to the protein denaturation. Also, when we see that compounds like the glycosteroids having an long chain ester -CAG, BbGL-I, are found to exist in Nature, and how much glycolipid-cholesterol interactions were recently shown to be critical in some biological processes, it is hoped that our observations can provide a new vision angle for the study of complex lipids and glycolipids. As a start to develop new probes targeting the "lipid raft" microdomain in membranes, we also explored a sequence towards carbohydrate laurdan hybrids. Further development of this strategy and evaluation of the biological properties is programmed within new collaborative projects.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-07-04T14:51:48Z No. of bitstreams: 1 jordanadamascenogitiranadesantana.pdf: 3160925 bytes, checksum: 910c9f83a7dacd60918a4704f0d0fdeb (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-08-08T13:39:58Z (GMT) No. of bitstreams: 1 jordanadamascenogitiranadesantana.pdf: 3160925 bytes, checksum: 910c9f83a7dacd60918a4704f0d0fdeb (MD5) / Made available in DSpace on 2017-08-08T13:39:58Z (GMT). No. of bitstreams: 1 jordanadamascenogitiranadesantana.pdf: 3160925 bytes, checksum: 910c9f83a7dacd60918a4704f0d0fdeb (MD5) Previous issue date: 2015-07-30 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / Vernonia polyanthes Less. (Asteraceae), popularmente conhecida como assa-peixe, é uma espécie vegetal nativa da América do Sul, principalmente do Brasil. Esta planta medicinal é tradicionalmente utilizada em casos de gripes, resfriados, tosses, febre, bronquite, contusões, hemorroidas, infecções do útero e no tratamento de malária. Farmacologicamente, V. polyanthes tem sido relatada como detentora de propriedades anti-hipertensiva, antinociceptiva, anti-ulcerogênica, anti-inflamatória, leishmanicida e antimicrobiana. Estudos fitoquímicos focando esta espécie, disponibilizados na literatura, relatam a presença de metabólitos secundários pertencentes, principalmente, às classes dos flavonoides e terpenos, especialmente lactonas sesquiterpênicas. O presente estudo descreve a avaliação da atividade antibacteriana do extrato de lavagem foliar (extrato VP) e da lactona sesquiterpênica glaucolídeo A isolada de V. polyanthes, no intuito de corroborar cientificamente com o uso tradicional dessa espécie vegetal e de verificar a potencialidade do extrato VP e desta molécula como prováveis fitoterápico e protótipo de um agente antibacteriano, respectivamente. O extrato VP foi obtido por meio da lavagem das folhas íntegras e secas de V. polyanthes com diclorometano e o glaucolídeo A isolado a partir do fracionamento desse extrato por métodos de cromatografia em coluna. A atividade antibacteriana foi investigada por meio da determinação da Concentração Inibitória Mínima (CIM) pelo método de microdiluição em caldo, segundo recomendações do Clinical Laboratory Standards Institute (CLSI). Em seguida, procedeu-se ao estabelecimento da Concentração Bactericida Mínima (CBM) e à classificação do efeito farmacológico bactericida ou bacteriostático. Staphylococcus aureus ATCC® 6538™/ATCC® 29213™, Escherichia coli ATCC® 10536™/ ATCC® 25922™, Salmonella enterica subsp. enterica serovar Choleraesuis ATCC® 10708™, Salmonella enterica subsp. enterica serovar Thyphimurium ATCC®13311™ e Pseudomonas aeruginosa ATCC® 9027™/ATCC® 27853™ foram usadas como cepas de referência. O extrato VP revelou atividade antibacteriana frente a 5 das 8 cepas testadas, sendo mais ativo contra S. aureus ATCC® 6538™ e ATCC® 29213, com valores de CIM iguais a 0,625 mg/mL, refletindo efeito bacteriostático e CBM de 2,5 mg/mL e 1,25 mg/mL, respectivamente. O glaucolídeo A apresentou atividade antibacteriana frente às referidas cepas de S. aureus, com valores de CIM de 250 µg/mL e 500 µg/mL, efeito bacteriostático e CBM de 500 µg/mL e > 500 µg/mL, nessa ordem, sendo inativo contra as demais cepas nas concentrações testadas. Os resultados do presente estudo sugerem que V. polyanthes e glaucolídeo A apresentam potencial antibacteriano, especialmente frente à S. aureus, sendo a atividade do extrato VP resultante, provavelmente, de um sinergismo entre essa lactona sesquiterpênica e outros metabólitos secundários presentes nessa planta medicinal. / Vernonia polyanthes Less. (Asteraceae), popularly known as "assa-peixe", is a native species of South America, especially in Brazil. This medicinal plant is traditionally used in cases of cold, flu, cough, fever, bronchitis, bruise, hemorrhoid, uterus infection and malaria's treatment. Pharmacologically, V. polyanthes has been reported as anti-hypertensive, antinociceptive, anti-ulcerogenic, anti-inflammatory, leishmanicidal and antimicrobial. Phytochemical studies focusing this plant, available in the literature, report the presence of secondary metabolites mainly belonging to the flavonoids and terpene classes, especially sesquiterpene lactones. The present study investigated the antibacterial activity of the leaf rinse extract (VP extract) and the sesquiterpene lactone glaucolide A isolated from V. polyanthes in order to scientifically corroborate with the traditional use of this plant species and to verify the potential of VP extract and this molecule as probable herbal and an antibacterial agent prototype, respectively. VP extract was obtained by washing the dried intact leaves of V. polyanthes with dichloromethane and the glaucolide A, isolated from the VP extract fractionation by column chromatography methods. Antibacterial activity was established by the determination of the Minimal Inhibitory Concentration (MIC) using the microdilution method according to the Clinical Laboratory Standards Institute (CLSI) guidelines followed by the Minimal Bactericidal Concentration (MBC), classifying the pharmacological effect as bacteriostatic or bactericidal. Staphylococcus aureus ATCC® 6538™/ATCC® 29213™, Escherichia coli ATCC® 10536™/ATCC® 25922™, Salmonella enterica subsp. enterica serovar Choleraesuis ATCC® 10708™, Salmonella enterica subsp. enterica serovar Thyphimurium ATCC® 13311™ and Pseudomonas aeruginosa ATCC® 9027™/ATCC® 27853™ were used as reference bacterial strains. The VP extract revealed antibacterial activity against 5 of 8 tested strains, being more active against S. aureus ATCC® 6538™ and ATCC® 29213™ with MIC values of 0.625 mg/mL, demonstrating bacteriostatic effect and MBC of 2.5 mg/mL and 1.25 mg/mL, respectively. Glaucolide A showed antibacterial activity against S. aureus with MIC values of 250 µg/mL and 500 µg/mL and, revealing bacteriostatic effect and CBM of 500 µg/mL and > 500 µg/mL, in this order, being inactive against the other strains at the concentrations tested. Our results suggest the antimicrobial potential of V. polyanthes and glaucolide A, notably against S. aureus, and the VP extract activity is probably due to the synergism between this sesquiterpene lactone and other secondary metabolites of this medicinal plant.

The development of selective SmI2-H2O-mediated mono-reductions of cyclic-1,3-diesters to the corresponding 3-hydroxy acids is described. The reaction proceeds with complete selectivity for cyclic-1,3-diesters over acyclic esters. Sequential one-pot conjugate reduction-ester reduction of alkylidene cyclic-1,3-diesters is also reported. Furthermore, we describe the exploitation of the unusual ketyl radical intermediates formed via single electron reduction of the ester carbonyl in unprecedented 5-exo-trig cyclisations providing access to highly substituted, stereo-defined, cyclopentanols and cyclopentanones. Also described is the use of a silicon control element to direct the stereochemical outcome of the SmI2-MeOH-mediated conjugate reduction-intramolecular aldol cyclisations of α,β-unsaturated lactones. These cyclisations generate two contiguous quaternary centres with complete diastereocontrol; the utility of the silicon-directing group as a synthetic handle for derivatisation of the cyclisation products has also been demonstrated. These cyclisations have been applied in a model approach to the anti-mitotic natural product pseudolaric acid B.

L'environnement marin est constitué d'écosystèmes nombreux et variés, qui sont habités par des organismes appartenant aux trois domaines du vivant dont celui des bactéries. La coopération bactérienne est basée sur un mécanisme moléculaire appelé quorum sensing (QS). Ce système est dépendant de la densité cellulaire de la communauté, ce qui permet aux bactéries de synchroniser leur expression génétique, pour coordonner certaines de leurs activités physiologiques. Les bactéries produisent plusieurs types de molécules signal dont les acyl-homosérines lactones (AHLs ou AI-1) et le furanosyl diester borate (AI-2). La majorité des recherches dans le domaine du QS s'est portée sur des modèles bactériens d'intérêt biomédical ou agronomique. Cependant, l'importance du QS chez les bactéries marines reste mal connu. Ce travail de thèse a pour question: Quelle est la prévalence des AI chez les bactéries marines ? Pour aborder cette problématique : trois axes : Le criblage de la collection de bactéries marines MOLA, qui est basé sur des techniques de chimie des substances naturelles, et a permis de mettre en exergue des souches bactériennes originales comme Maribius sp MOLA 401, qui produisent un très grand nombre d'AHLs avec de nouvelles structures. Le second axe s'appuie sur des méthodes de bio-informatique pour réaliser le criblage du métagénome marin du Global Ocean Sampling pour la recherche de gènes luxI, ainS, hdtS et luxS. L'ensemble de ces données sur la diversité, l'abondance et la répartition biogéographique des AI-1 dans l'écosystème marin suggère une forte importance des mécanismes du QS à l'¿uvre dans les communautés bactériennes marines naturelles. / The marine environment is composed of many and varied ecosystems, which are inhabited by organisms belonging to the three domains of life including the bacteria. Bacterial cooperation is based on a molecular mechanism called quorum sensing (QS). This system is dependent on the cell density of the community, which allows bacteria to synchronize their gene expression, to coordinate some of their physiological activities. Bacteria produce several types of signal molecules with acyl-homoserines lactones (AHLs or AI-1) and the furanosyl borate diester (AI-2). The majority of research in the field of QS has focused on bacterial models for biomedical and agricultural interest. However, the importance of QS in marine bacteria remains unclear. This work has the question: What is the prevalence of AI in marine bacteria? To address this problem two axes: Screening the collection of marine bacteria MOLA, which is based on chemistry techniques of natural substances, and allowed to highlight original bacterial strains as Maribius sp MOLA 401, which produce very large number of AHLs with new structures. The second axis is based on the methods of bioinformatics to perform screening of metagenome marine Global Ocean Sampling for research luxI, ainS, luxS and hdtS genes,. All these data on diversity, abundance and biogeographical distribution of AI-1 in the marine ecosystem suggests a strong importance of QS mechanisms at work in the natural marine bacterial communities.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Biotechnology is inserted within the search for new products and processes, thus becoming an important tool in the development of biotechnological natural aroma production processes. Flavors compounds obtained from these processes represent a very promising and viable way of production for industries mainly due to the increase of consumer preference for natural food additives and products. Many microorganisms isolated from various environments have potential to synthesize flavors compounds when cultivated in an appropriate culture medium. Fermentation processes allow the use of agro-industrial wastes in which disposal on the environment may cause serious pollution issues. The residues composition, mostly the carbon source, allows the growth of microorganisms and the production of higher aggregate value compounds. The aim of this work is the to produce and quantify the 6-pentyl-á-pyrone lactone, responsible for the coconut-like aroma, by using agro-industrial waste as support and the filamentous fungus Trichoderma harzianum. Initially three agro-industrial wastes (sugarcane bagasse, coconut shell powder and cassava bagasse) were tested concerning the aroma production. Then the gas chromatography coupled to headspace extraction technique provided the quantitative and qualitative analysis of the lactone. Through the chromatogram area, the 4040 strain of Trichoderma fungus and the cassava bagasse were selected as culture medium. The latter was studied by the physical chemistry aspect. The tests done in order to evaluate the microorganism lactone toxic level did not show a complete inhibition in cell growth, only a few spores. The total factorial design in two levels was applied to verify the influence of the nutrients concentration (C/N) and temperature in the production of 6-pentyl-á-pyrone. The largest production of aroma compound resulted in 3.78 mg/g MS at the 7th culture day. The linear and quadratic terms related to temperature were important to the proposed model at a 95% trust level for aroma maximum concentration, by setting values between 25 and 28°C. The C/N ratio effect and the interaction between these two variables were not statistically meaningful. / A biotecnologia se insere na busca de novos produtos e processos, tornando-se uma ferramenta importante no desenvolvimento de processos de produção de aromas naturais biotecnológicos. Compostos de aroma obtidos por estes processos representam uma alternativa de produção bastante promissora e viável para as indústrias, principalmente devido ao aumento da preferência dos consumidores por aditivos alimentícios e produtos naturais. Muitos microrganismos, isolados dos mais variados ambientes, possuem potencial para sintetizar compostos de aroma quando cultivados em meios de cultura adequados. Os processos fermentativos possibilitam o aproveitamento de resíduos agroindustriais, cuja disposição no meio ambiente gera sérios problemas de poluição. A composição dos resíduos, principalmente a fonte de carbono, permite o crescimento dos microrganismos e produção de compostos de maior valor agregado. O presente trabalho tem como objetivo produzir e quantificar a lactona 6-pentil-á-pirona, responsável pelo aroma característico de coco, utilizando resíduo agroindustrial como suporte e fungo filamentoso Trichoderma harzianum. Inicialmente, três resíduos agroindustriais (bagaço de cana, pó da casca de coco e bagaço de mandioca) e cinco microrganismos foram testados quanto à produção do aroma. A técnica de cromatografia gasosa acoplada à extração em headspace permitiu as análises qualitativas e quantitativas da lactona. Através da área do cromatograma, a linhagem 4040 do fungo Trichoderma e o suporte bagaço de cana foram selecionados como meio de cultivo. O resíduo bagaço de cana foi caracterizado sob o aspecto físico-químico. Os testes realizados para avaliar o nível de toxidez da lactona sobre o microrganismo não apresentaram inibição total no crescimento celular, apenas pouca presença de esporos. O planejamento fatorial completo em dois níveis foi empregado para avaliar a influência da concentração de nutrientes (C/N) e da temperatura sobre a produção do 6-pentil-á-pirona. A maior produção do composto de aroma resultou em 3,78 mg/g MS no 7º dia de cultivo. Os termos lineares e quadráticos relacionados à temperatura foram significativos no modelo proposto a um nível de confiança de 95% para a máxima concentração do aroma, sendo direcionadas para valores de 25 a 28ºC. O efeito da razão C/N e de interação entre as duas variáveis não foram estatisticamente significativos.

Furo[3,4-c] pyranone is a unique bicyclic molecular structure found in bioactive sesquiterpene isobolivianine and in artificial cytotoxic stilbene derivatives. The structure of furo[3,4-c] pyranone is analogous to cyclopenta[c] pyran structure found in potent cytotoxic iridoids like catapol. Chiral substrates for intramolecular hetero-Diels-Alder (IHDA) reaction have been synthesized in yields ranging from 39% to 81%. These compounds undergo [4+2] cycloaddition via ambimodal/ bispericyclic process to give a mixture of furo[3,4-c] pyranone in yields ranging 40-70% and aryl tetralin lactone derivatives. Density functional theory (DFT) calculations have been performed to gain insight into the mechanism leading to the formation of these compounds. Podophyllotoxins are aryl tetralin lignans with great potential as lead compounds for anti-cancer and antibiotic agents. The bioactivity of these compounds is attributed to their unique stereochemistry that is not easy to synthesize. Chiral substrates for intramolecular dehydro-Diels-Alder (IDDA) reaction have been synthesized in moderate yields. These novel compounds have been used to synthesize aryl tetralin lactone cores by IDDA reaction. This work demonstrates the potential utility of asymmetric IHDA and IDDA reactions in the total synthesis of bioactive compounds featuring furo[3,4-c] pyranone core and aryl tetralin lactones found in anticancer podophyllotoxins.