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.

Made available in DSpace on 2015-03-26T13:52:00Z (GMT). No. of bitstreams: 1 texto completo.pdf: 996773 bytes, checksum: 7322b4697c096a6f17a4388ca498fede (MD5) Previous issue date: 2014-02-17 / Fundação de Amparo a Pesquisa do Estado de Minas Gerais / The L-ascorbic acid (ALA) is produced naturally by plants from D-glucose. Yeasts synthesize a similar metabolite, D-eritroascorbic acid (ADEA). Although this compound does not show activity against scurvy, it contains an antioxidant function, but it is produced in low concentrations by the microorganism. Recently, in order to make yeasts are capable of converting D-galactose D-lactose from cheese whey into ALA, the wild strain Kluyveromyces lactis CBS2359 has been transformed with genes that integrate the L-galactose biosynthesis pathway, isolated from Arabidopsis thaliana. In the presence of intermediate L-galactose, ALA can be synthesized by yeast with enzymes responsible for ADEA production. It has been demonstrated that the engineered strain JVC 1-56 was able to synthesize ALA compared to the wild type, but the yield was still low. In this regard, studies to assist the increased of ALA production are relevant. Once the engineered ALA pathway and native cell wall formation pathway of K. lactis have a common intermediate, GDP-D-mannose, we need to evaluate the effect of uncoupling of vitamin C synthesis route of the growth that requires constant wall synthesis. Moreover, considering the antioxidant properties of ALA, the expectation is that the production of this metabolite increase when JVC 1-56 is exposed to a oxidative stress condition by menadione. In this context, this study investigated the both physiological conditions, using the media Yeast Galactose Base (YGB) and ultrafiltered cheese whey (SQU - byproduct of cheese industry that predominates lactose as carbon source). It was analyzed that Kluyveromyces lactis JVC 1-56 produces higher yields of ALA when it is grown for 96 hours in batch culture in the middle YPGal. Cultivation at low growth rates (0.04 h-1) predisposes the cells to synthesize more ALA per unit cell mass (0.80 mg/mg). However, the yield of continuous culture operated as quimostato, limiting the substrate nitrogen, still proved to be lower than expected in the batch (1.94 mg/mg). Oxidative stress by 12.5 mM menadione on K. lactis JVC1 -56, in the conditions applied, was not effective in the increasing the ALA production (1.47 mg/mg). Still, ADEA yield was higher than ALA in two strategies: 1.37 mg/mg at 0.21 h-1 in culture under steady state, 8.52 mg/mg in batch under oxidative stress and 10.33 mg/mg in the batch in the absence of oxidizing agent, indicating that ADEA may be in a more stable configuration than vitamin C. Finally, the permeate cheese whey remains a prospect for the conversion of lactose into a biotechnological product of higher value aggregate. / O ácido L-ascórbico (ALA) é produzido naturalmente por plantas a partir de D-glicose. Leveduras sintetizam um metabólito semelhante, o ácido D-eritroascórbico (ADEA). Embora este composto não mostre atividade contra o escorbuto, ele contém uma função antioxidante, mas é produzido pelo micro-organismo em baixas concentrações. Recentemente, com a finalidade de fazer as leveduras serem capazes de converter o componente D-galactose da D-lactose do soro de queijo em ALA, a linhagem selvagem de Kluyveromyces lactis CBS2359 foi transformada com genes, que integram a via de biossíntese de L-galactose, isolados de Arabidopsis thaliana. Na presença do intermediário L-galactose, ALA pode então ser sintetizado pela levedura com as enzimas responsáveis pela produção de ADEA. Foi demonstrado que a linhagem engenheirada JVC 1-56 foi capaz de sintetizar ALA em comparação com a selvagem, mas com baixo rendimento. Neste sentido, estudos que auxiliem o aumento da produção de ALA são relevantes. Uma vez que a via engenheirada da síntese de ALA e a via nativa da formação da parede celular de K. lactis possuem um intermediário comum, GDP-D-manose, faz-se necessário avaliar o efeito do desacoplamento da produção de ALA do crescimento que requer constante síntese da parede. Além disso, considerando a propriedade antioxidante do ALA, a expectativa é de que a produção deste metabólito aumentasse quando JVC 1-56 fosse exposta a uma condição de estresse oxidativo por menadiona. Neste contexto, este trabalho investigou as condições fisiológicas mencionadas utilizando os meios Yeast Galactose Base (YGB) e soro de queijo ultrafiltrado (SQU - subproduto de indústrias de queijo em que predomina lactose como fonte de carbono). Foi averiguado que Kluyveromyces lactis JVC 1-56 produz ALA com rendimentos maiores quando cultivada por 96 horas em batelada no meio YPGal. O cultivo em baixas velocidades de crescimento (0,04 h -1) predispõe as células a sintetizar mais ALA por unidade de massa celular (0,80 mg/mg). Entretanto, o rendimento da cultura contínua operada como quimostato, tendo nitrogênio como substrato limitante, ainda mostrou ser inferior ao previsto na batelada (1,94 mg/mg). Já o estresse oxidativo por 12,5 μM de menadiona sobre K. lactis JVC1- 56, nas condições aplicadas, não foi eficaz no aumento da produção de ALA (1,47 mg/mg). Ainda, o rendimento de ADEA foi superior ao de ALA nas duas estratégias: 1,37 mg/mg a 0,21 h-1 na cultura sob regime permanente, 8,52 mg/mg na batelada sob estresse oxidativo e 10,33 mg/mg na batelada na ausência do agente oxidante, indicando que ele pode estar em uma configuração mais estável do que a vitamina C. Finalmente, o permeado do soro de queijo continua sendo uma perspectiva para a conversão da lactose em um produto biotecnológico de maior valor agregado.

La vaccination est l’un des moyens les plus efficaces de la médecine moderne dans le combat contre les maladies infectieuses. L’amélioration de l’efficacité des vaccins requiert la mise au point d’adjuvants permettant d’accroître la qualité de la réponse immunitaire. À titre d’exemple, les nanoparticules (NP) de poly(acide lactique) (PLA) constituent un système efficace pour la délivrance d’antigènes. Afin de renforcer leur potentiel vaccinal, ce travail de recherche a eu pour objectif d’élaborer des NP de PLA décorées en surface par des molécules immunostimulantes, le D-mannose ou un peptide dérivé de l’interleukine-Beta, et au cœur, par l’imiquimod. Notre stratégie repose sur l’utilisation d’un tensioactif macromoléculaire composé d’un bloc de PLA et d’un bloc de poly(N-acryloxysuccinimide-co-N-vinylpyrrolidone) (P(NAS-co-NVP)), dont les fonctions ester de N-succinimidyle (NS) permettent le couplage de biomolécules. Ce copolymère a été synthétisé par combinaison de la polymérisation par ouverture de cycle et de la polymérisation radicalaire contrôlée par les nitroxydes (NMP). Après l’étude de la copolymérisation du NAS et de la NVP par NMP à partir d’une alcoxyamine modèle (MAMA-SG1), leur copolymérisation a été réalisée à partir de la macro-alcoxyamine PLA-SG1, conduisant au copolymère PLA-b-P(NAS-co-NVP) désiré. Des NP de PLA ont alors été préparées par nanoprécipitation et diafiltration en présence du copolymère, conduisant à des tailles respectives de 150 et 500 nm. Des études de potentiel zêta et de spectrométrie UV ont démontré la présence des esters de NS à la surface des NP (2.4 fonctions.nm-2), disponibles pour le couplage des biomolécules. Des micelles de copolymère ont été également préparées, après substitution des esters de NS par des sucres, et permettent une encapsulation efficace de l’imiquimod, contrairement aux NP de PLA. Ces systèmes constituent une plateforme flexible d’adjuvants potentiels comme alternative aux adjuvants non biodégradables actuellement utilisés. / Vaccination represents one of the most powerful tools of medicine for the fight against infectious diseases. The improvement of vaccine efficiency needs the development of adjuvants able to increase the quality of the immune response. Poly(lactic acid) (PLA) nanoparticles (NPs) represent an efficient system for antigen delivery. In order to improve their vaccine potential, the goal of this research work was to elaborate PLA NPs decorated at the surface with immunostimulatory molecules, D-mannose or peptide derived from interleukine-Beta, and into the core with imiquimod. Our strategy relies on the use of a macromolecular surfactant composed of a PLA block and a poly(N- acryloxysuccinimide-co-N-vinylpyrrolidone) (P(NAS-co-NVP)) block, whose N-succinimidyl (NS) activated esters allow the coupling of biomolecules. This diblock copolymer was synthesized by the combination of ring opening polymerization and nitroxide mediated polymerization (NMP). After the study of the copolymerization of NAS and NVP by NMP from the MAMA-SG1 model alkoxyamine, their copolymerization was performed from the macro-alkoxyamine PLA-SG1, leading to the desired copolymer PLA-b-P(NAS-co-NVP). PLA NPs were then prepared by nanoprecipitation and diafiltration, in the presence of the copolymer, leading to 150 nm and 500 nm sized particles, respectively. Studies of zeta potential and UV spectrometry demonstrated the presence of NS-activated esters at the NP surface (2.4 functions.nm-2), available for the coupling of biomolecules. Micelles from copolymer were also prepared, after substitution of esters with carbohydrates, and allowed an efficient encapsulation of imiquimod, contrary to PLA NPs. These systems represent a flexible platform of potential adjuvants as an alternative to non-biodegradable adjuvants currently used.