1.Adult specimens of Ascaris lumbricoides of similar weights were incubated under nitrogen for 24 hours in a synthetic medium with 1 mM 2,2′-dipyridyl.2.Under these conditions, the viability of the parasites was not affected as evidenced by the amount of ATP in the whole sample and the mobility after mechanical stimulus.3.Incorporation of [14C]proline into non-collagenous proteins in the body wall and cuticle was reproducibly higher in 2,2′-dipyridyl-treated specimens than in untreated worms. Synthesis of collagenous hydroxyproline was inhibited in the cuticle and, to a greater extent, in the muscle layer.4.After transferring the specimens into a fresh medium enriched with 0·1 mM ferrous ions and incubated under 70% oxygen, the muscle collagen remained underhydroxylated. The synthesis of hydroxyproline, however, was almost completely normalized in the cuticle collagen.5.We interpret the data as further evidence of the existence of at least two different enzymes hydroxylating collagenous proline, one located in the subcuticle and the other in the muscle layer of Ascaris lumbricoides.

More human lives have been lost to tuberculosis than to any other disease and despite the availability of effective short course chemotherapy (DOTS) as well as the Bacilli Calmette Guerin (BCG) vaccine, tuberculosis continues to claim more than a million lives annually. Mycobacterium tuberculosis (M. tuberculosis), the causative agent of tuberculosis, is one of the most successful and scientifically challenging pathogens of all time. However in the last two decades, the ability to perform molecular genetic analysis of M. tuberculosis has resulted in powerful new research tools, while the availability of the complete genome sequence has provided us with a wealth of new information and understanding of the biology of this major pathogen. One of the major challenges, however, is to analyze the properties and functions of those genes that are unique to M. tuberculosis genome. The identification and characterization of such genes which impart various survival strategies employed by M. tuberculosis for successful infection will be of particular significance. One of the important outcomes from the complete genome sequence of M. tuberculosis is the discovery of two multigene families designated PE (99 members) and PPE (69 members) named respectively for the Pro-Glu (PE) and Pro-Pro-Glu (PPE) motifs near the N-terminus of their gene products. In addition to these motifs, proteins of the PE family possess highly homologous N-terminal domains of approximately 100 amino acids (PE domain), whereas the PPE proteins possess a highly homologous N-terminal domain of about 180 amino acids (PPE domain). Although the PE and PPE families of mycobacterial proteins are the focus of intense research, no precise function has so far been unraveled for any member of these families. The current study focuses on Rv3097c gene of M. tuberculosis, a PE family gene that was bioinformatically predicted to be a triacylglycerol hydrolase (lipase). In order to decipher the role of the PE domain, we have carried out functional characterization of the Rv3097c gene (also named lipY) as it was, initially, the only known PE protein for which an enzymatic function (i.e. lipase activity) had been predicted. Further, to understand the function of PE family proteins, an important question that needs to be answered is; whether the PE domain of different PE family proteins has similar or different functions? In this context, our studies were focused on studying the functional role of the PE domain in LipY, as outlined below. In general, the in vivo function and subcellular localization of any protein are integrally connected. PE domain has been reported to be essential for cell wall localization of PE_PGRS33, another PE family protein. Therefore we investigated the subcellular localization of LipY and the influence of the PE domain on subcellular localization of LipY. LipY and a truncated form of LipY lacking the PE domain [LipY(ΔPE)] were expressed in mycobacteria(M. smegmatis and M. bovis BCG). Subcellular fractionation and western blot demonstrated that both LipY and LipY(ΔPE) were predominantly detected in the cell wall fraction, indicating that LipY is localized to the cell wall and the PE domain of LipY was not required for translocation of LipY to cell wall. This result is in contrast to the findings for PE_PGRS33, where the absence of the PE domain caused the cell wall associated protein to localize to the cytosol. Furthermore, immuno-electron microscopy of M. bovis BCG expressing LipY(ΔPE) clearly showed a cell surface localization of LipY(ΔPE). These results signify that the function of the PE domain might not always be similar amongst different PE family proteins. In order to further investigate the role of the PE domain in LipY, we studied the lipase activity of LipY and the influence of the PE domain on lipase activity. Bioinformatic analysis confirmed the presence of a lipase domain containing a GDSAG active site motif characteristic of lipases. Overexpression of LipY in mycobacteria (M. smegmatis and M. bovis BCG) resulted in a significant reduction in the pool of triacylglycerols (TAG), consistent with the lipase activity of this enzyme. Interestingly, this reduction was more pronounced in mycobacteria overexpressing LipY(ΔPE), suggesting that the presence of the PE domain diminishes the lipase activity of LipY. In vitro lipase assays also confirmed LipY(ΔPE) as a more efficient lipase compared to the wild-type LipY. Together these results suggest that the PE domain of LipY might be involved in the modulation of lipase activity. Surprisingly, M. marinum, another pathogenic mycobacteria, possesses a protein homologous to LipY, termed LipYmar, in which the PE domain is substituted by a PPE domain. The overexpression of LipYmar in M. smegmatis significantly reduced the TAG pool suggesting that it is a triacylglycerol hydrolase/lipase. Interestingly, similar to the removal of the PE domain of LipY, this reduction in the TAG pool was further pronounced when the PPE domain of LipYmar was removed. This suggests that PE and PPE domains might share similar functional roles in modulating the enzymatic activities of these lipase homologs. In order to assess the in vivo relevance of LipY expression during M. tuberculosis infection, we examined the humoral immune responses against LipY in sera derived from various clinical categories of tuberculosis patients. The presence of specific antibodies against any protein is suggestive of expression of the protein during infection and could potentially be used to differentiate between healthy individuals and infected patients (serodiagnosis of tuberculosis). The cell wall localization suggested that LipY may be accessible for interaction with the host immune system during infection. Moreover, humoral responses were observed against LipY in mice immunized with DNA constructs expressing LipY, indicating that LipY could be an effective B-cell antigen. Accordingly, a strong humoral response against LipY and LipY(ΔPE) was observed in tuberculosis patients compared to healthy individuals, suggesting that LipY is expressed during infection by clinical strains of M. tuberculosis and might represent an immunodominant antigen of M. tuberculosis with potential use in serodiagnosis of tuberculosis.

L'organocatalyse asymétrique est un domaine en plein développement. L'immobilisation de ce type de catalyseurs pourrait présenter de multiples avantages. Ces travaux de thèse s'intéressent à la préparation de silices hybrides organiques/inorganiques par voie sol-gel et aux applications de ces matériaux en organocatalyse asymétrique. La première partie du manuscrit est consacrée à une présentation bibliographique du sujet. Dans la deuxième partie, l'utilisation de la L-proline comme modèle est décrite. Des matériaux contenant un fragment L-proline ont été préparés et leurs performances catalytiques évaluées pour une réaction d'aldolisation asymétrique. Les processus à l'origine des propriétés catalytiques modérées de ces catalyseurs supportés sont discutés. La troisième partie porte sur le catalyseur de Takemoto, organocatalyseur bifonctionnel contenant un groupement donneur de liaisons hydrogène et une fonction amine tertiaire. Les différentes stratégies envisagées pour préparer des dérivés silylés de ce catalyseur sont exposées. Enfin, la nanostructuration de silsesquioxanes par le biais de liaisons hydrogène entre fonctions thiourée est présentée. / Asymmetric organocatalysis is a blossoming area of research. Immobilisation of this kind of catalysts could present numerous advantages. This thesis deals with the sol-gel synthesis of organic/inorganic hybrid silicas and their applications in asymmetric organocatalysis. The first part of this work is dedicated to a bibliographic presentation of this area of research. In the second part, the use of L-proline as a model is described. Hybrid materials containing a L-proline component were prepared and their catalytic performances were evaluated in an asymmetric aldolisation reaction. The processes accounting for the moderate performances of these materials are discussed. The third part relates the synthetic strategies used to prepare silylated derivatives of the Takemoto catalyst, a bifunctional catalyst containig a H-bond donnor and a tertiary amine. Finally the nanostructuring of bridged silsesquioxanes through H-bonding interactions between thiourea cross-linkers is presented.

L'astringence résulte de l'interaction des tanins, polyphénols abondants dans les végétaux, avec les protéines salivaires et plus particuliÈrement les protéines salivaires riches en proline (PRP), appartenant à la famille des protéines peu structurées. Les tanins participent aux mécanismes de défense des végétaux et présentent des effets antinutritionnels dus à leur capacitè à inhiber les enzymes digestives. La synthÈse de PRP salivaires constitue un mècanisme d'adaptation à la consommation d'aliments riches en tanins. Ce travail vise à caractèriser les complexes ètablis en solution entre les PRP et les tanins, par une approche basèe sur la spectromètrie de masse (MS). Pour cela, les protèines salivaires humaines IB5, PRP basique, et II-1, PRP glycosylèe, ont ètè produites par voie hètèrologue. AprÈs purification, les deux protèines ont ètè caractèrisèes par MS avec une source d'electronèbullisation (ESI-MS) et avec une source MALDI (Matrix-Assisted Laser Desorption/Ionisation). L'ètude des interactions par ESI-MS a confirmè la prèsence en solution de complexes non-covalents IB5tanin et permis de prèciser leurs stchiomètries. Des expèriences de compètition entre diffèrents tanins et de dissociation des complexes IB5tanin ont mis en èvidence l'influence des principales caractèristiques structurales des tanins sur cette interaction. L'ètude structurale des èdifices IB5tanin, par diffèrentes techniques de MS/MS (Collision Induced Dissociation, Electron Capture Dissociation et photodissociation) et par mobilitè ionique couplèe à la MS, a mis en èvidence la prèsence de plusieurs sites d'interaction sur IB5 ainsi que des changements conformationnels liès à l'interaction / Astringency is an important organoleptic property of plant-based food. It is attributed to interactions of tannins, which are polyphenolic compounds, with salivary proteins and especially proline rich proteins (PRPs), which belong to the group of intrinsically unstructured protein (IUP). Tannins play an important part in plant defence mechanisms. Indeed, they have an antinutritional effect as they inhibit digestive enzymes. Production of salivary PRP is thus an adaptation process to tannin-rich diets. The purpose of this work is to provide a closer look at PRPtannin supramolecular edifices in solution, using a mass spectrometry (MS) approach. The human salivary proteins IB5, a basic PRP, and II-1, a glycosylated PRP, have been produced by heterologous expression. After purification, both proteins have been characterized by MS using electrospray (ESI) and Matrix-Assisted Laser Desorption/Ionisation (MALDI) sources. The study of the interaction between IB5 and model tannins by ESI-MS confirmed the presence of IB5tannin non covalent complexes in solution and provided new information on their stoichiometries. Competitive interaction experiments between IB5 and two tannins, along with IB5tannin complexes dissociation studies revealed the impact of the main tannin chemical features on this interaction. Structural studies performed on IB5tanin edifices by Collision induced dissociation (CID), Electron Capture Dissociation (ECD) and photodissociation MS/MS experiments and by ion mobility coupled with MS showed the presence of several interaction sites on IB5 and conformational changes arising from the interaction.

La levure Saccharomyces cerevisiae est capable d’utiliser une grande variété de sucres comme source de carbone et d’énergie. La plupart des enzymes impliquées dans l’utilisation de ces sucres sont codées par des gènes issus de familles multigéniques. C’est le cas de la famille IMA identifiée comme impliquée dans l’utilisation de l’isomaltose. Cette famille comprend cinq gènes qui codent pour quatre isomaltases partageant une forte identité de séquence (de 65% à 100 %). Dans ce travail , la diversitéfonctionnelle de la famille IMA a été étudiée, en caractérisant de façon exhaustive in vitro leurs propriétés biochimiques et enzymologiques. Ima1p et Ima2p possèdent des propriétés biochimiques identiques (pH, température, et thermostabilité) mais Ima3p se distingue par rapport à ces deux protéines bien que n’ayant que trois acides de différence avec Ima2p (thermostabilité plus faible). Ima5p quant à elle, est la protéine la plus dissemblable (température optimale plus faible et demi-vie basse dès 37°C). Les quatre isomaltases sont cependant très sensibles au Tris et aux ions Fe3+. Les quatre isoenzymes présentent une préférence pour les disaccharides liés en α-1,6 (isomaltose et palatinose), avec une cinétique de type Michaëlis-Menten et une inhibition par le substrat à une concentration élevée. Les isomaltases Imap sont cependant aussi capables d'hydrolyser les disaccharides α-1,2, α-1,3 et α-1,5 ainsi que les trisaccharides portant une liaison α-1,6, ce qui met en évidence leur ambiguïté de substrat .Nos résultats ont toutefois montré de nombreuses singularités dans cette famille de protéines. Alors que Ima1p et Ima2p présentent des propriétés très semblables, l’activité catalytique de Ima3p est globalement très faible malgré sa forte ressemblance avec Ima2p. Le variant Ima3p_R279Q retrouve des niveaux d'activité proches de ceux d’Ima2p, tandis que la substitution d’une leucine par une proline à la position 240 a permis d’augmenter de manière significative la stabilité d’Ima3p confirmant le rôle des prolines dans la thermostabilité des protéines. L’hydrolyse de l’isomaltose par Ima5p réfute lesconclusions précédemment publiées sur l'exigence d'acides aminés spécifiques pour déterminer la spécificité de α-1,6 puisque le variant IMA5-MQH ne permet pas de restaurer une activité semblable à Ima1p malgré la présence des trois résidus MQH. Nous avons également trouvé qu’Ima5p est inhibé par le maltose suivant une inhibition mixte tandis qu’Ima1p est inhibée de façon compétitive à faible concentration et de manière incompétitive à forte concentration en isomaltose / Most enzymatic systems for sugar uptake and assimilation rely on multigene families in theyeast Saccharomyces cerevisiae. The IMA / MAL family has been used as a model system to study themolecular mechanisms that govern evolution of duplicated genes. The five IMA multigene familymembers encode four isomaltases sharing high sequence identity from 65% to 99%, of which IMA3and IMA4 are 100% identical to encode the same isomaltase. In this work, the functional diversity ofIMA family was further explored, with exhaustive in-vitro characterization of their biochemical andenzymological properties.Ima1p and Ima2p were similar to biochemical properties; Ima3p showed some differences fromthe two proteins; amongst them, Ima5p was the most distant protein. The four isomaltases were highlysensitive to Tris and Fe3+, but were unaffected by the addition or the removal of Ca2+ despiteconservation of the calcium binding site. Besides, four isoenzymes exhibited a preference for the α-(1,6)disaccharides isomaltose and palatinose, with Michaelis-Menten kinetics and inhibition at highsubstrates concentration. They were also able to hydrolyse trisaccharides bearing an α-(1,6) linkage,but also α-(1,2), α-(1,3) and α-(1,5) disaccharides including sucrose, highlighting their substrateambiguity. While Ima1p and Ima2p presented almost identical characteristics, the results neverthelessshowed many singularities within this protein family. In particular, Ima3p presented lower activitiesthan Ima2p despite only 3 different amino acids between these two isoforms. The Ima3p_R279Qvariant recovered activity levels of Ima2p, while the Leu-to-Pro substitution at position 240significantly increased the stability of Ima3p and supported the role of prolines inthermostability.Ima5p presented the lower optimal temperature and was also extremely sensitive to temperature. Isomaltose hydrolysis by Ima5p challenged previous conclusions about the requirement of specificamino acids for determining the specificity for α-(1,6) substrates. We finally found a mixed inhibitionby maltose for Ima5p while, contrary to a previous work, Ima1p inhibition by maltose was competitiveat very low isomaltose concentrations and uncompetitive as the substrate concentration increased.The presented Ph.D’s work provided preliminary insights into determining structural factorswithin this family, exemplifying for example the role of proline residues for thermosability. Moreover,it was illustrated that a gene family encoding proteins with strong sequence similarities can lead toenzyme with notable differences in biochemical and enzymological properties.

La progression maligne de l’adénocarcinome canalaire pancréatique (ADKP) s'accompagne d'une profonde réaction desmoplasique, limitant la vascularisation de la tumeur et de fait privant les cellules tumorales en nutriments, forçant les cellules tumorales à adapter leur métabolisme. L’objectif de thèse était de définir les changements métaboliques relatifs à l’ADKP. Par une analyse transcriptomique des tumeurs pancréatiques développées de manière spontanée chez les souris, nous avons établi le profil métabolique des ADKPs lié aux acides aminés au cours de leur progression. Ainsi, nous avons montré que les voies métaboliques de la proline et des acides aminés à chaînes branchées, en particulier le catabolisme de la leucine, sont celles étant les plus dérégulées dans l’ADKP. Concernant le métabolisme de la proline, nous avons montré que les cellules tumorales privées en nutriments capturent et utilisent le collagène, produit par les fibroblastes du stroma tumoral grâce à la macropinocytose, de façon le dégrader en proline. Aussi, l’inhibition de la dégradation de la proline entraine une diminution de la prolifération tumorale in vitro et in vivo. Concernant la leucine, nous montrons que l’élément clé de ce métabolisme est un de ces produits de dégradation finaux à savoir le β-hydroxybutyrate (βOHB) dont la production repose sur une enzyme cruciale : HMGCL. Dans nos travaux, nous démontrons que la suppression d’HMGCL dans les cellules d’ADKP humains entrave leurs capacités oncogéniques et métastatiques in vitro et in vivo. De plus, nous montrons in vivo que le βOHB augmente la croissance tumorale ainsi que la formation de métastases. / The malignant progression of pancreatic ductal adenocarcinoma (PDAC) is accompanied by a profound desmoplasia, depriving tumor cells from oxygen and nutrients, which forces tumor cells to adapt their metabolism to proliferate. The thesis purpose is to define the metabolic changes related to ADKP. Using a transcriptomic analysis of PDAC from mice model, we established the PDAC metabolic profile. Focusing on amino acid metabolic pathways, we identified the metabolic pathways of proline and the branched-chain amino acid, especially the leucine catabolism, as the most deregulated in ADKP compared to the normal pancreas. We demonstrated that tumor cells take up collagen-derived fibroblasts, thanks macropinocytosis, when they are nutrient deprived. Once collagen is internalized, its subsequent digestion supplies TCA with proline. Also, inhibition of proline degradation leads to a decrease in tumor proliferation in vitro and in vivo. We have shown leucine catabolism is specific to tumor cells and the final degradation products: the β-hydroxybutyrate (βOHB) appears as a key element of this metabolism. To produce βOHB, tumor cells use HMGCL, a crucial enzyme involved in leucine degradation. In our work we demonstrated that HMGCL suppression in PDAC cells decreases their oncogenic and metastatic capacities in vitro and in vivo. In addition, we have demonstrated in vivo that βOHB increases tumor growth and metastasis formation. Thus, our works show 1/ the metabolic plasticity of cells, 2/the influence of microenvironment on tumor cell metabolism, 3/ the importance to study tumor metabolism for the finding of new therapeutic targets.

Os nossos resultados demonstraram a atividade enzimática prolina desidrogenase (PRODH) do produto do gene anotado como codificante de uma prolina oxidase na base dados do genoma de Trypanosoma cruzi. A atividade da proteína codificada por esse gene foi avaliada inicialmente por complementação de uma linhagem de S. cerevisiae deficiente na expressão funcional da PRODH endógena, e posteriormente mediante a obtenção da enzima recombinante ativa expressa em um sistema bacteriano. Nos estudos feitos tanto com leveduras complementadas com o gene PRODH, quanto com as formas epimastigotas de T. cruzi, observamos que nesses organismos há uma correlação positiva entre o nível intracelular de prolina livre e a resistência ao estresse oxidativo. Através dos ensaios de RT-PCR quantitativo observamos que o RNAm do gene da PRODH é regulado ao longo do ciclo de vida do T. cruzi, com maior nível de expressão no estágio epimastigota intracelular, perfil apresentado também em experimentos de Western Blotting, o que é concordante com o papel fundamental desse aminoácido na diferenciação dessa forma para as forma tripomastigota. Nos diferentes ensaios de localização subcelular observou-se que a proteína PRODH está presente na mitocôndria. A seqüência da PRODH apresenta um sítio EF-hand de ligação a Ca2+. Mostramos experimentalmente a funcionalidade desse sítio e a sua função na regulação da atividade por Ca2+. Em ensaios de respiração mitocondrial, utilizando prolina como substrato, foi mostrado que a prolina fornece elétrons à cadeia respiratória através da PRODH, transferindo elétrons e gerando FADH2 no mesmo nível e com a mesma eficiência que o complexo II (succinato desidrogenase). O análogo de prolina T4C, inibidor do transporte de prolina, causa uma diminuição do conteúdo de prolina livre intracelular, o que foi relacionado com uma diminuição significativa da sobrevivência do parasito em combinação com o estresse nutricional e oxidativo, mostrando a participação do metabolismo de prolina na resistência a esses estresses. Todos esses dados sugerem ademais que o transportador de prolina do parasita pode ser um alvo para drogas terapêuticas de interesse quando combinada com outras drogas que agem via geração de espécies reativas de oxigênio. / In the present work, we demonstrated the proline dehydrogenase enzymatic activity (PRODH) for the protein encoded by a gene annotated as a proline oxidase in the T. cruzi genome data base. This activity was shown firstly through complementation of a S. cerevisiae lineage lacking its endogenous PRODH gene. The PRODH gene was also expressed in a bacterial system and the active recombinant protein was obtained. Experiments performed with both, complemented yeasts and T. cruzi epimastigotes, showed a correlation between the intracellular free proline levels and the oxidative stress resistance. Quantitative RT-PCR assay revealed that the PRODH gene is differentially expressed across T. cruzi life cycle, being the highest expression level shown by the intracellular epimastigote form, this result was confirmed by Western blotting. Both results are in accordance with the fact that proline is essential for the differentiation of the intracellular epimastigote into trypomastigote. Subcellular localization assays showed that PRODH is present preferentially in the mitochondria. In silico analyses of the PRODH peptidic sequence indicated the presence of an EF-hand domain, wich is, usually, involved in Ca2+ binding. In fact, our results confirm not only the ability of such domain of binding Ca2+ but also its function in the activity regulation. Mitochondrial respiration assays using proline as substrate showed that PRODH transfers electrons and generates FADH2, with an eficience comparable to that of the complex II (Succinate dehydrogenase). Experiments using the T4C, an analogue of proline that inhibits the proline uptake, caused the depletion of the intracellular free proline, which was followed by the significantly decrement of the cellular viability of the parasites under nutritional and oxidative stresses. Taken together, this data suggest that proline transporters are promising drug targets when combined with other drugs that act by generating reactive oxygen species.

Ce travail de thèse porte dans un premier temps sur la synthèse de nouveaux sels d’azolinium chiraux précurseurs de carbènes N-hétérocycliques. Deux nouvelles familles de sels ont été préparées à partir de la L-proline. Nous avons pu caractériser les NHC issus de ces sels, par la formation des dimères, des thiones et des complexes de rhodium correspondants. Dans un second temps, ces nouveaux sels d’azolinium ont été évalués dans la réaction d’addition conjuguée de réactifs de Grignard sur des cétones α,β insaturées. Les résultats ont montré de très bonnes activités catalytiques, et de très bonnes régiosélectivités. Les énantiosélectivités obtenues sont encourageantes. Ensuite, ces catalyseurs ont été engagés dans la réaction de substitution allylique. Une bonne activité catalytique a aussi été observée malgré des régiosélectivités et des énantiosélectivités modérées. Enfin, ces sels ont été employés comme précurseurs de NHC pour la réduction asymétrique de cétones aromatiques par transfert d’hydrogène. Les complexes formés se sont montrés actifs mais non énantiosélectifs. / This work deals with, at first, the synthesis of new chiral azolinium precursors to N-heterocyclic carbenes. Two families have been prepared from the L-proline. The structures of some NHC dimmers, thiones and [Rh-NHC] complexes were characterized and confirmed by different analysis methods.Secondly, these new salts were evaluated in the conjugate addition reaction of Grignard reagents to α,β-unsaturated ketones. The results obtained shown very good catalytic activity, excellent regioselectivity with moderate enantioselectivity. They were also involved in the allylic substitution reaction. Good catalytic activity was observed despite moderate regioselectivity and enantioselectivity. Finally, these azolinium salts were employed as NHC precursors for the asymmetric transfer hydrogenation of aromatic ketones. The complexes proved to be active, but non enantioselective.

Les tanins jouent un rôle clé dans les qualités organoleptiques du vin rouge. Ils sont à l’origine de l'Astringence, une sensation de sècheresse perçue en bouche lors de la dégustation. Cette sensation est la conséquence d'une interaction spécifique entre des tanins et des protéines salivaire, principalement les Protéines Riches en Prolines (PRPs). Une première partie de ce travail a consisté à étudier l'influence de divers sucres sur le processus d'auto-association des tanins ainsi que sur les interactions tanins-protéines. Le comportement colloïdal d’un tanin (l’Epigallocatechine Gallate – EGCG), ainsi que son interaction avec un peptide modèle IB9-14 représentatif des PRPs, ont été étudiés en présence de différents sucres simples et polysaccharides. Les paramètres de l’interaction ont été déterminé pour l’ensemble des systèmes, mettant en évidence l’existence d’une interaction entre EGCG et sucres dont l’affinité semble dépendre du degré de polymérisation du sucre. Cette interaction ne perturbe pas, dans les conditions expérimentales testées, l’association entre les tanins et le peptide. Une deuxième partie de ce travail correspond à la synthèse complète de la protéine IB9 incluant la séquence peptidique d’IB9-14, et à l’étude de son interaction avec deux procyanidines : l’EGCG et le dimère B3. Cette étude a permis d’observer et de confirmer une influence de la longueur de la chaîne peptidique sur les interactions avec les tanins. / Tannins play a key role in the organoleptic qualities of red wine. They are responsible for wine astringency, a dry, rought and puker sensation perceived in the mouth while tasting. This sensation is the consequence of a specific interaction between tannins and saliva proteins, mainly Proline Rich Protein (PRPs). The first part of this work was to study the influence of various sugars on the self-association of tannins process as well as tannins - proteins interactions. The colloidal behavior of a tannin (the epigallocatechin gallate - EGCG), as well as its interaction with a representative peptide IB9-14 of PRPs was studied in the presence of various simple sugars and polysaccharides. The parameters of the interaction were determined for all systems, highlighting the existence of an interaction between EGCG and sugars whose affinity seems to depend on the sugar polymerization degree. This interaction does not interfere, under the experimental conditions tested, on the association between tannins and peptide. The second part of this work was to realize the full synthesis of the protein IB9 including the peptidic sequence of IB9-14, and to study its interaction with two procyanidins: EGCG and dimer B3. The results show and confirm the influence of the length of the peptide chain interactions with tannins.

L’hyperphosphorylation de la protéine Tau, à l’origine de la formation de dégénérescences neurofibrillaires et de la mort cellulaire observée, chez les patients atteints de la maladie d’Alzheimer, est causée par un déséquilibre entre l’activité des kinases et des phosphatases. Cette modification post traductionnelle touche de nombreux sites dans la Région Riche en Proline (RRP) de Tau. Or, des études RMN, réalisées au sein du laboratoire, ont montré que les polyphénols sont capables d’interagir avec des fragments de la protéine issues de la RRP avec des affinités de l’ordre de grandeur de celles décrites pour les kinases. Dans ce contexte, il a été envisagé de synthétiser une bibliothèque de polyphénols et de mettre au point une réaction de phosphorylation sur des peptides modèles afin d’étudier, par spectrométrie de masse, la capacité de ces composés à protéger Tau contre l’attaque des kinases. / Hyperphosphorylation of Tau protein, which leads to their abnormal aggregation into neurofibrillary tangles and to neuronal loss observed in patients with Alzheimer disease, is regulated by a disequilibrium between kinases and phosphatases activities. This post traductional modification affects several residues, in particular in the Proline Rich Region of Tau (PRR). But, NMR studies, realized in our laboratory, have shown that polyphenols are able to interact with peptide Tau models issued from the PRR and the affinity are in the same range that those described for the kinases. In this context, we have envisaged to synthetize several polyphenols and to develop a phosphorylation reaction of peptide models in order to study, by mass spectrometry, the ability of these compounds to protect Tau against kinase attack.