No estado do Espírito Santo (ES), uma das principais áreas produtoras de mamão do país, a eliminação sistemática de plantas doentes tem sido aplicada desde a década de 1980 para o controle do mosaico do mamoeiro (Papaya ringspot virus - type P; PRSV-P). O uso permanente dessa prática nos últimos 25 anos levou a uma aparente seleção e predominância de isolados fracos do vírus. Os objetivos deste trabalho foram: investigar a prevalência desses isolados fracos, bem como a estabilidade e o efeito protetor contra isolados severos do vírus; estudar a infecção natural de abobrinha de moita (Cucurbita pepo cv. Caserta) e abóbora moranga (C. maxima cv. Exposição) com o PRSV-P quando plantadas ao lado de mamoeiros infectados e caracterizar um isolado do Alfalfa mosaic virus (AMV) em infecção natural em mamoeiro. A detecção de possíveis isolados fracos do vírus foi realizada por PTAELISA, microscopia eletrônica e RT-PCR. Todos os isolados também foram inoculados mecanicamente em mamoeiro cv. Golden para avaliação de sintomas. Sequências de nucleotídeos e de aminoácidos deduzidos do gene da proteína capsidial de alguns isolados fracos mostraram identidades superiores a 89% e 90%, respectivamente, com isolados do PRSV-P. De 119 amostras de mamoeiros analisadas, 86 estavam infectadas com o PRSV-P, mas somente 75 induziram sintomas fracos em mamoeiros. Quatro isolados fracos foram selecionados ao acaso para estudos de estabilidade, e de proteção em casa de vegetação. Apenas dois isolados fracos induziram sintomas estáveis em mamoeiros até a oitava transferência. A proteção total só foi obtida com plantas premunizadas com dois isolados fracos e desafiados com o isolado PRSV-PES. Plantas de mamoeiros cv. Golden premunizadas com vários isolados fracos do PRSV-P foram expostas em condições de campo na ESALQ/USP, Piracicaba, SP, em dois experimentos independentes. Poucas plantas permaneceram com sintomas fracos de mosaico até o final dos experimentos. Uma terceira exposição foi realizada em Linhares, ES, com mamoeiros cvs. Sunrise Solo e Golden premunizados com oito isolados fracos, coletados nos experimentos em campo na ESALQ/USP. Apenas uma planta premunizada com um isolado fraco permaneceu com sintomas leves da doença até a última avaliação. Tentativas de detectaçao de infecções naturais de cucurbitáceas com o PRSV-P foram realizados em dois plantios de abobrinha de moita e dois de abóbora moranga, na ESALQ/USP, Piracicaba, SP. A detecção do vírus foi feita por meio da inoculação de extratos foliares das cucurbitáceas em mamoeiros cv. Golden. Os mamoerios foram avaliados por meio de sintomas, PTA-ELISA e RT-PCR. Nenhuma planta de mamoeiro inoculada com extratos foliares das duas cucurbitáceas exibiu sintomas de mosaico, embora o gene ci, mas não o cp, tenha sido detectado em uma amostra de folhas de mamoeiro, indicando que ao menos uma planta de abobrinha de moita estava infectada. Finalmente, no decorrer dos ensaios de campo na ESALQ/USP, constatou-se uma planta de mamoeiro apresentando sintomas severos de mosaico amarelo, deformação foliar e necrose sistêmica, diferente daqueles induzidos pelo PRSV-P. Análises biológicas, sorológica e moleculares confirmaram tratar-se do AMV. Este é o primeiro relato de infecção natural de mamoeiro com esse vírus. / Papaya ringspot virus type P (PRSV-P) causes the major disease in Brazilian papaya orchards that result in significant yield losses. In Espírito Santo state systematic rouging of infected plants has been applied since early 1980s for the control of this disease. Its permanent use over the last 25 years has lead to an apparent selection and predominance of mild strains throughout papaya orchards. The objectives of this work were to investigate the prevalence of mild isolates, as well the stability and protective effect against severe isolates of the virus; The aim of this work was to study the natural infecction of zucchini squash (Cucurbita pepo cv. Caserta) and pumpkin (C. maxima cv. Exposição) grown near to papaya trees infected with PRSV-P and characterize an isolate of Alfalfa mosaic virus (AMV) in natural infection in papaya. The detection of possible mild isolates of the virus was performed by PTA-ELISA, electron microscopy and RT-PCR. All isolates were inoculated mechanically in papaya cv. Golden for symptoms evaluation. Nucleotides and deduced amino acids sequences of the coat protein gene of some mild isolates showed identities above 89% and 90%, respectively, with isolates of PRSV-P. Of 119 samples from papaya plants analyzed, 86 were infected with PRSV-P and 75 induced mild symptoms on papaya. Four mild isolates were randomly selected for stability and protection studies under greenhouse. Only two isolates induced mild symptoms on papaya and remained stables until the eighth transference. Full protection was obtained with preimmunized plants with two mild isolates and challenged with the isolate PRSV-P-ES. Plants of papaya cv. Golden preimmunized with several mild isolates of PRSV-P were exposed under field conditions at ESALQ/USP, Piracicaba,SP, in two independent experiments. Few plants remained with mild mosaic symptoms at the end of the experiments. A third field exposition was held in Linhares,ES, with papaya cvs. Golden and Sunrise Solo preimmunized with eight mild isolates, collected in field experiments at ESALQ/USP. Only one plant preimmunized with a mild isolate remained with mild symptoms of the disease until the last evaluation. Attempts of detection of natural infections of cucurbits with PRSV-P were carried out in two plantations of zucchini squash and pumpkin at ESALQ/USP, Piracicaba,SP. The detection of the virus was made by inoculation of leaf extracts of cucurbits in papaya cv. Golden. The papaya plants were assessed by symptoms, PTAELISA and RT-PCR. None of papaya plants exhibited symptoms of mosaic, while the ci gene, but not the cp, was detected in a sample of leaves of papaya, indicating that at least one clump of zucchini squash plant was infected. Finally, during the field test at ESALQ/USP, a papaya plant was found showing severe symptoms severe yellow leaf mosaic, leaf distortion and systemic necrosis, different from those induced by PRSV-P. Biological, serological and molecular tests confirmed the infection with AMV. This is the first report of natural infection of papaya with this virus.

Le but de notre travail est de développer une stratégie de vectorisation pour conférer la mobilité phloémienne aux produits agrochimiques en appliquant le concept de prodrogue dans l'élaboration de composés phytopharmaceutiques. Le fenpiclonil, fongicide non systémique de la famille des phénylpyrroles, a été choisi comme composé parent modèle et modifié en l'associant à un acide alpha-aminé ou à un monosaccharide.La mobilité phloémienne des conjugués entre le fenpiclonil et les acides L ou D-glutamique ou le D-glucose (D-GFC) a été évaluée chez des plantules de ricin. Le test de systémie a montré que le L-aminoacide était nettement plus favorable à la mobilité phloémienne que l'acide D-aminé ou le D-glucose. Les résultats suggèrent que le conjugué fenpiclonil/L-aminoacide est reconnu et manipulé par un système de transport d'acide aminé stéréospécifique énergisé par la force proton-motrice. Des expériences complémentaires ont montré que le D-GFC était un inhibiteur puissant et sélectif de l'absorption du saccharose par les tissus foliaires et du chargement phloémien du saccharose. En raison de l'inhibition spécifique des transporteurs de saccharose (par exemple AtSUC2), le D-GFC peut être envisagé comme un nouvel outil en phloémologie.Enfin, nous avons exploré l'impact de la modification de structure de l'espaceur afin d'optimiser la stratégie de prodrogue. Après avoir introduit différentes structures d'espaceur entre le fenpiclonil et la fonction acide aminé, le conjugué qui contient un cycle triazole avec l'acide aminé comprenant la chaine la plus courte a montré la meilleure mobilité phloémienne et, par ailleurs, une systémie optimisée par rapport aux dérivés acides du fenpiclonil dans la gamme des valeurs de pH de l'apoplasme foliaire. / The purpose of our work is to develop a vectorization strategy to confer phloem mobility to agrochemicals by applying prodrug concept into agrochemical design. Fenpiclonil, a non-systemic fungicide from the phenylpyrrole family, was selected as a model parent compound and modified by associating it with an amino acid or a monosaccharide.The phloem mobility of L and D-glutamic acid and D-glucose fenpiclonil conjugates (D-GFC) was evaluated in Ricinus seedlings. The systemicity test showed that the L-amino acid promoiety was clearly more favorable to phloem mobility than D-amino acid or D-glucose. The results suggested that the transport of the L-amino acid conjugate is governed by a stereospecific amino acid carrier system energized by the proton motive force. Further investigation indicated that D-GFC was a potent and selective inhibitor of sucrose uptake by leaf tissues and sucrose phloem loading. Due to its specific inhibition of sucrose transporters (e.g. AtSUC2), D-GFC can be a candidate as a new tool in phloemology. Finally, we explored structural modifications of the spacer arm to optimize the prodrug strategy. Different structures of the spacer arm were introduced between fenpiclonil and the amino acid function. The conjugate which contains a triazole ring with the shortest amino acid chain showed the best phloem mobility and a better systemicity than fenpiclonil acidic derivatives within the pH range of the foliar apoplast.

La thèse aborde différentes questions de chimie prébiotique dans le contexte de l’origine de la vie par une approche de chimie systémique. La première partie est dédiée à l’étude de processus d’activation chimique important non seulement pour la formation de polymères, mais aussi pour alimenter le système en énergie de manière à le maintenir dans un état éloigné de l’équilibre, un prérequis pour l’auto-organisation. Il a été suggéré que les intermédiaires 5(4H)-oxazolones formés par l’activation de l’extrémité C-terminale des peptides pourrait être impliquée dans l’auto-organisation du vivant. Dans ce but, nous avons évalué la réactivité de réactifs pertinents dans un contexte prébiotique et décrits dans la littérature comme capables d’activer des acides α-aminés. Aucun d’entre eux n’a manifesté une activité satisfaisante pour l’activation C-terminale des peptides, montrant qu’une voie possible pour alimenter un protométabolisme des peptides en énergie n’est pas identifiée à ce jour à l’exception notable des N-carboxyanhydrides (NCA) qui peuvent être formé par des voies prébiotiquement plausibles. Nous avons par ailleurs démontré que les carbodiimides sont aussi efficaces pour l’activation des N-carbamoylamino acides que pour celle du carboxyle terminal des peptides en milieu aqueux dilué. La seconde partie du document expose de nouveaux résultats en faveur d’un processus de coévolution peptides-nucléotides. D’abord, une étude de la réactivité d’agents d’aminoacylation de l’extrémité 3’ de l’ARN est présentée. Ensuite, nous évaluons des co-polymères acides α-aminés-nucléotides liés par des enchaînements phosphoramidate et esters comme partenaires éventuels de l’évolution chimique. La pertinence cinétique de ces structures est démontrée ainsi que des voies chimiques permettant leur formation. / The thesis addresses several issues in prebiotic chemistry in the context of the origins of life through a systems chemistry approach. The first part is devoted to the study of chemical activation processes that are not only important in the formation of polymers, but also to feed the system with energy in order that a far from equilibrium state is maintained, a prerequisite for self-organization. It has been suggested that 5(4H)-oxazolones intermediates formed by C-terminus peptide activation could be involved in self-organization of life. To this aim, we have checked the reactivity of relevant prebiotic reagents previously proposed to activate α-amino acids. None of them led to a satisfactory C-terminus activation of peptides, showing that no general process for feeding a protometabolism of peptides with energy is identified yet, with the notable exception of N-carboxyanhydrides (NCAs) that can be formed through prebiotically relevant pathways. Additionally, we demonstrated that carbodiimides reagents are as efficient in the activation of N-carbamoyl amino acids as in that of the C-terminus of peptides in diluted aqueous media. The second part of the dissertation discloses new results in support of a process of coevolution of peptides and nucleotides. Firstly, a study of non-enzymatic aminoacylation reagents of the 3’-terminus of RNA is presented. Secondly, we assessed co-polymers of α-amino acids and nucleotides bound by phosphoramidate and ester linkages as potential players in chemical evolution. The kinetic relevance of these structures was demonstrated as well as potential chemical processes that allow their formation.

Au cours de ma thèse, nous avons utilisé la truite arc-en-ciel, un poisson carnivore et modèle potentiellement pertinent du diabète, pour étudier des mécanismes de régulation du métabolisme intermédiaire hépatique par les nutriments (acides aminés (AA) et le glucose). Nous nous sommes plus particulièrement intéressés aux voies de signalisation de l’insuline et des acides aminés (Akt et mTORC1). Grâce à l’utilisation de rapamycine, un inhibiteur pharmacologique de mTORC1, nous avons montré que l'activation de mTORC1 stimule l'expression de gènes de la lipogenèse, de la glycolyse et du catabolisme des acides aminés, tandis que la voie de signalisation Akt inhibe celle des gènes impliqués dans la néoglucogenèse. Ces études ont été conduites dans le foie de truite ou en culture primaire d’hépatocytes de truite arc-en-ciel. En outre, nous avons démontré lors de stimulations à court terme in vivo et in vitro que l'expression hépatique des gènes de la lipogenèse est plus sensible à l'apport de protéines alimentaires ou d’AA qu’à l'apport de glucides ou de glucose. De plus, nous avons observé que des taux élevés d’AA conduisent, par le biais de l’activation de la voie de signalisation mTORC1, à une augmentation de l'expression des gènes lipogéniques mais surtout à une répression de l’inhibition de l’expression des gènes de la néoglucogenèse induite par l’insuline. Cet effet s’accompagne d’une augmentation de la phosphorylation de IRS-1 sur le résidu Ser302 qui pourrait être responsable de la baisse de phosphorylation d'Akt et par conséquent d’une inhibition de l’action de l'insuline. Enfin, en réalisant un test de tolérance au glucose chez des truites préalablement traitées avec de la rapamycine, nous avons conclu que la néoglucogenèse hépatique joue un rôle probablement majeur dans le contrôle de l'homéostasie glucidique chez la truite. Ainsi, une absence d’inhibition de la néoglucogenèse pourrait contribuer au maintien de l'hyperglycémie prolongée et au phénotype d’intolérance au glucose caractéristique des poissons carnivores. Cette thèse met en avant le rôle des protéines/AA dans la régulation du métabolisme intermédiaire de la truite et identifie certaines voies de signalisation cellulaire sollicités par les acides aminés pour réguler le métabolisme. Elle permet ainsi d’éclaircir certaines particularités nutritionnelles de la truite. / During my doctoral study, we used rainbow trout, a representative carnivorous fish and relevant diabetic model, to study the mechanisms underlying the regulation of hepatic intermediary metabolism by nutrients (amino acids (AAs) and glucose), and determine the potential involvement of insulin/Akt and mTORC1 signaling pathways in these regulations. Using acute administration of rapamycin, a pharmacological inhibitor of TOR, we first identified that mTORC1 activation promotes the expression of genes related to fatty acid biosynthesis, glycolysis and amino acid catabolism, while Akt negatively regulates gluconeogenic gene expression in rainbow trout liver and primary hepatocytes. Furthermore, we demonstrated hepatic fatty acid biosynthetic gene expression is more responsive to dietary protein intake/AAs than dietary carbohydrate intake/glucose during acute stimulations in vivo and in vitro. Moreover, we further showed that high levels of AAs up-regulate hepatic fatty acid biosynthetic gene expression through an mTORC1-dependent manner, while excessive AAs attenuate insulin-mediated repression of gluconeogenesis through elevating IRS-1 Ser302 phosphorylation, which in turn impairs Akt phosphorylation and dampens insulin action. Finally, using glucose tolerance test and acute inhibition of rapamycin, we concluded that hepatic gluconeogenesis probably plays a major role in controlling glucose homeostasis, which maybe account for the prolonged hyperglycemia and glucose intolerance phenotype of carnivorous fish. The present thesis brings forward our understandings about the roles of protein/AAs in the regulation of hepatic intermediary metabolism in trout and identifies relevant cellular signaling pathways mediating the action of amino acids on metabolism. It also clarifies some nutritional characteristics of the trout.

Les acides α-aminés non protéinogènes sont des composés riches d’applications et peuvent donner accès à des composés possédant des propriétés biologiques intéressantes ou à des analogues de peptides. Ainsi, de nombreuses méthodes de synthèse asymétrique ont été développées. Parmi celles-ci, seulement quelques méthodes utilisent la chiralité d’acides α -aminés tertiaires naturels pour accéder à des acides α -aminés quaternaires et il n’existe que peu de synthèses asymétriques absolues d’acides α -aminés tertiaires.Notre équipe a précédemment développé une méthode de synthèse d’acides α-aminés quaternaires reposant sur le concept de la mémoire de chiralité. Cette méthode utilise la chiralité axiale d’amides aromatiques tertiaires pour l’alkylation stéréoselective d’énolates d’acides aminés. Lors de ma thèse, nous avons souhaité appliquer cette méthode d’alkylation stéréosélective d’énolate à d’autres types de réaction tels que la réaction d’aldolisation (utilisant un aldéhyde comme électrophile, et dans ce cas il est nécessaire de contrôler un second centre asymétrique), la réaction d’arylation (utilisant un sel de diaryliodonium comme électrophile) ou à la synthèse totale des composés biologiques intéressants. Des résultats préliminaires encourageants obtenus dans des réactions d’aldolisation (dans le cas avec du benzaldéhyde), d’arylation ainsi que dans la synthèse du précurseur de la L-Méthyl DOPA seront présentés.D’autre part, nous exposerons également la stratégie de synthèse des dérivés d’acides α-aminés tertiaires et des aminoalcools, qui utilise le concept de la chiralité gelée. Cette méthode repose sur l’emploi de la chiralité axiale dynamique des amides aromatiques tertiaires, qui est gelée dans un cristal chiral, et une réaction d’alkylation stéréosélective d’énolate conduisant à des acides aminés énantiomériquement enrichis. En effet, en partant de la glycine, nous avons réussi à trouver un composé de départ qui cristallise dans un groupe d’espace chiral, qui donne donc des cristaux chiraux après la cristallisation. Après avoir trouvé des conditions optimales pour la réaction d’allylation, plusieurs autres électrophiles ont été employés avec succès. Les produits alkylés sont obtenus avec des rendements allant jusqu’à 80% et des excès énantiomériques allant jusqu’à 96% sans utiliser d’autre source de chiralité externe que celle du cristal. L’ouverture de ces composés conduit à la formation des acides α-aminés tertiaires énantiomériquement enrichis. Il s’agit donc d’une synthèse asymétrique absolue d’acides α-aminés. / Non proteinogenic α-amino acids can lead to compounds which exhibit interesting biological properties, or peptides analogues. Numerous methods for asymmetric synthesis of these compounds have been developed. However, few examples have used the chirality of natural tertiary α-amino acids for the synthesis of quaternary α-amino acids, and few examples of asymmetric absolute synthesis to access to tertiary α -amino acids have been described so far. Our research group has previously developed a synthesis of enantioenriched quaternary α-amino acids, based on memory of chirality and using the axial chirality of tertiary aromatic amides for stereoselective alkylation of an enolate of an amino acid.This thesis focuses on expending this methodology to other type of reactions, for example, aldolisation reactions (using an aldehyde as electrophile, in this case it is necessary to control the second asymmetric center), arylation reactions (using a diaryliodonium salt as electrophile) or to the the total synthesis of compounds exhibiting interesting biological properties. Herein, we will show our preliminary results in aldolisation reactions (with benzaldehyde), in arylation reactions and also in the total synthesis of L-Methyl DOPA.On the other hand, we will also present an enantioselective synthesis of tertiary α-amino acids derivatives and of amino alcohols based on the principle of frozen chirality. The strategy uses the dynamic axial chirality of tertiary aromatic amides, which is frozen in chiral crystal, and a stereoselective alkylation reaction of enolate leads to enantioenriched α-amino acids. A compound synthesized from glycine has been finally selected to optimise the asymmetric allylation reaction. These optimales conditions were then successfully employed with various electrophiles. Alkylated products were obtained in yield up to 80% and enantiomeric excesses up to 96% using only chirality of crystal. The deprotection of alkylated products leads to the formation of enantienriched α-amino acids.

No estado do Espírito Santo (ES), uma das principais áreas produtoras de mamão do país, a eliminação sistemática de plantas doentes tem sido aplicada desde a década de 1980 para o controle do mosaico do mamoeiro (Papaya ringspot virus - type P; PRSV-P). O uso permanente dessa prática nos últimos 25 anos levou a uma aparente seleção e predominância de isolados fracos do vírus. Os objetivos deste trabalho foram: investigar a prevalência desses isolados fracos, bem como a estabilidade e o efeito protetor contra isolados severos do vírus; estudar a infecção natural de abobrinha de moita (Cucurbita pepo cv. Caserta) e abóbora moranga (C. maxima cv. Exposição) com o PRSV-P quando plantadas ao lado de mamoeiros infectados e caracterizar um isolado do Alfalfa mosaic virus (AMV) em infecção natural em mamoeiro. A detecção de possíveis isolados fracos do vírus foi realizada por PTAELISA, microscopia eletrônica e RT-PCR. Todos os isolados também foram inoculados mecanicamente em mamoeiro cv. Golden para avaliação de sintomas. Sequências de nucleotídeos e de aminoácidos deduzidos do gene da proteína capsidial de alguns isolados fracos mostraram identidades superiores a 89% e 90%, respectivamente, com isolados do PRSV-P. De 119 amostras de mamoeiros analisadas, 86 estavam infectadas com o PRSV-P, mas somente 75 induziram sintomas fracos em mamoeiros. Quatro isolados fracos foram selecionados ao acaso para estudos de estabilidade, e de proteção em casa de vegetação. Apenas dois isolados fracos induziram sintomas estáveis em mamoeiros até a oitava transferência. A proteção total só foi obtida com plantas premunizadas com dois isolados fracos e desafiados com o isolado PRSV-PES. Plantas de mamoeiros cv. Golden premunizadas com vários isolados fracos do PRSV-P foram expostas em condições de campo na ESALQ/USP, Piracicaba, SP, em dois experimentos independentes. Poucas plantas permaneceram com sintomas fracos de mosaico até o final dos experimentos. Uma terceira exposição foi realizada em Linhares, ES, com mamoeiros cvs. Sunrise Solo e Golden premunizados com oito isolados fracos, coletados nos experimentos em campo na ESALQ/USP. Apenas uma planta premunizada com um isolado fraco permaneceu com sintomas leves da doença até a última avaliação. Tentativas de detectaçao de infecções naturais de cucurbitáceas com o PRSV-P foram realizados em dois plantios de abobrinha de moita e dois de abóbora moranga, na ESALQ/USP, Piracicaba, SP. A detecção do vírus foi feita por meio da inoculação de extratos foliares das cucurbitáceas em mamoeiros cv. Golden. Os mamoerios foram avaliados por meio de sintomas, PTA-ELISA e RT-PCR. Nenhuma planta de mamoeiro inoculada com extratos foliares das duas cucurbitáceas exibiu sintomas de mosaico, embora o gene ci, mas não o cp, tenha sido detectado em uma amostra de folhas de mamoeiro, indicando que ao menos uma planta de abobrinha de moita estava infectada. Finalmente, no decorrer dos ensaios de campo na ESALQ/USP, constatou-se uma planta de mamoeiro apresentando sintomas severos de mosaico amarelo, deformação foliar e necrose sistêmica, diferente daqueles induzidos pelo PRSV-P. Análises biológicas, sorológica e moleculares confirmaram tratar-se do AMV. Este é o primeiro relato de infecção natural de mamoeiro com esse vírus. / Papaya ringspot virus type P (PRSV-P) causes the major disease in Brazilian papaya orchards that result in significant yield losses. In Espírito Santo state systematic rouging of infected plants has been applied since early 1980s for the control of this disease. Its permanent use over the last 25 years has lead to an apparent selection and predominance of mild strains throughout papaya orchards. The objectives of this work were to investigate the prevalence of mild isolates, as well the stability and protective effect against severe isolates of the virus; The aim of this work was to study the natural infecction of zucchini squash (Cucurbita pepo cv. Caserta) and pumpkin (C. maxima cv. Exposição) grown near to papaya trees infected with PRSV-P and characterize an isolate of Alfalfa mosaic virus (AMV) in natural infection in papaya. The detection of possible mild isolates of the virus was performed by PTA-ELISA, electron microscopy and RT-PCR. All isolates were inoculated mechanically in papaya cv. Golden for symptoms evaluation. Nucleotides and deduced amino acids sequences of the coat protein gene of some mild isolates showed identities above 89% and 90%, respectively, with isolates of PRSV-P. Of 119 samples from papaya plants analyzed, 86 were infected with PRSV-P and 75 induced mild symptoms on papaya. Four mild isolates were randomly selected for stability and protection studies under greenhouse. Only two isolates induced mild symptoms on papaya and remained stables until the eighth transference. Full protection was obtained with preimmunized plants with two mild isolates and challenged with the isolate PRSV-P-ES. Plants of papaya cv. Golden preimmunized with several mild isolates of PRSV-P were exposed under field conditions at ESALQ/USP, Piracicaba,SP, in two independent experiments. Few plants remained with mild mosaic symptoms at the end of the experiments. A third field exposition was held in Linhares,ES, with papaya cvs. Golden and Sunrise Solo preimmunized with eight mild isolates, collected in field experiments at ESALQ/USP. Only one plant preimmunized with a mild isolate remained with mild symptoms of the disease until the last evaluation. Attempts of detection of natural infections of cucurbits with PRSV-P were carried out in two plantations of zucchini squash and pumpkin at ESALQ/USP, Piracicaba,SP. The detection of the virus was made by inoculation of leaf extracts of cucurbits in papaya cv. Golden. The papaya plants were assessed by symptoms, PTAELISA and RT-PCR. None of papaya plants exhibited symptoms of mosaic, while the ci gene, but not the cp, was detected in a sample of leaves of papaya, indicating that at least one clump of zucchini squash plant was infected. Finally, during the field test at ESALQ/USP, a papaya plant was found showing severe symptoms severe yellow leaf mosaic, leaf distortion and systemic necrosis, different from those induced by PRSV-P. Biological, serological and molecular tests confirmed the infection with AMV. This is the first report of natural infection of papaya with this virus.

Flavonoids are secondary metabolites that are important in plant defense, protection and human health. Most naturally-occurring flavonoids are found in glucosylated form. Glucosyltransferases (GTs) are enzymes that catalyze the transfer of glucose from a high energy sugar donor to an acceptor molecule. A flavonol-specific 3-O-GT enzyme has been identified and cloned from leaf tissues of grapefruit. The enzyme shows rigid substrate specificity and regiospecificity. F3-O-GTs from grape (Vitis vinifera) and grapefruit (Citrus paradisi) were modeled against F7-O-GTs from Crocus sativus and Scrutellaria biacalensis, and several non-conservative amino acid differences were identified that may impact regioselectivity. This research is designed to test the hypothesis that specific amino acid residues impart the regiospecificity of the grapefruit enzyme. Site-directed mutagenesis was performed on three potentially key amino acid residues within the grapefruit F3-O-GT that were identified through homology modeling. Analyses of the enzyme activity of the mutant F3-O-GT proteins revealed that the single point mutations of serine 20 to leucine (S20L) and proline 297 to phenylalanine (P297F) rendered the recombinant enzyme inactive with flavonol substrates. Mutation of glycine 392 to glutamate (G392E) was active at 80% relative to the wild type. The mutant enzyme also did not show broadened acceptor specificity as it also favored flavonols as the preferred acceptor substrate. The glucosylation products of the active mutant enzyme will be analyzed to determine if this resulted in a change in regiospecificity.

Glucosyltransferases, or GTs, are enzymes which perform glucosylation reactions. These glucosylation reactions involve attaching a UDP-activated glucose molecule to acceptor molecules specific to the enzyme. The products of these reactions are observed to have a myriad of effects on metabolic processes, including stabilization of structures, solubility modification, and regulation of compound bioavailability. The enzyme which our lab focuses its research on is a flavonol-specific 3-O-GT found in Citrus paradisi, or grapefruit. This enzyme is part of the class of enzymes known as flavonoid GTs, which are responsible for, among other things, the formation of compounds which can affect the taste of citrus. Our lab focuses its research on performing site-directed mutagenesis on Citrus paradisi 3-O-GT in an attempt to modify its substrate specificity and regiospecificity. In this poster, we report our findings thus far concerning the addition of specific residues to the 3-O-GT's amino acid sequence based on an alignment with the sequence of a putative flavonoid GT found in Citrus sinensis.

Les objectifs de cette thèse étaient d'apporter une percée conceptuelle pour une compréhension en profondeur des mécanismes moléculaires des protéines intrinsèquement désordonnées (IDPs) et de leurs rôles dans la physiologie cellulaire de Chlamydomonas reinhardtii. La combinaison d’approches expérimentale et bioinformatique m’a permis d’identifier 682 protéines thermorésistantes chez C. reinhardtii. Parmi celles-ci, 299 protéines sont systématiquement prédites comme potentielles IDP par quatre algorithmes de prédiction de désordre. Nos résultats indiquent que le pourcentage désordonné moyen de ces protéines prédites comme étant des IDPs est d'environ 20%, et la plupart d'entre elles (~70%) sont adressées à d'autres compartiments que la mitochondrie et le chloroplaste. Leur composition en acides aminés est biaisée par rapport à d'autres IDPs de la base de données de protéines désordonnées (DisProt). Ces IDPs potentielles jouent des fonctions moléculaires diverses, et 54% d'entre elles sont des cibles de phosphorylation.Notre travail a également augmenté l’état des connaissances sur l'adénylate kinase 3 (ADK3), une enzyme contenant une région intrinsèquement désordonnée (IDR). Cette enzyme a été isolée par notre approche globale pour caractériser les IDPs de l’algue verte. L’extension C-terminale désordonnée (CTE) de cette enzyme lui confère de nouvelles fonctions comme par exemple, la formation d’un complexe bi-enzymatique avec la glycéraldéhyde-3-phosphate déshydrogénase (GAPDH), la régulation (négative) de l'activité GAPDH avec le NADPH comme cofacteur, et le rôle de chaperon pour la GAPDH en la protégeant de la dénaturation par traitement thermique et de l’agrégation. / The objectives of this work were to bring a conceptual breakthrough for an in-depth understanding of the molecular mechanisms of intrinsically disordered proteins (IDPs) and their roles in the cellular physiology of Chlamydomonas reinhardtii. Using experimental approaches, 682 heat-resistant proteins were identified as putative IDPs. Among them, 299 proteins were consistently predicted as IDPs by all four disordered predictors. The mean percentage of disordered residues content of these IDPs is about 20%, and most of them (~70%) are addressed to other compartments than mitochondrion and chloroplast. These newly identified IDPs from C. reinhardtii have a biased amino acid composition as regard to other IDPs from the Database of protein disorder (DisProt). Furthermore, they play diverse molecular functions, and 54% of them are the targets for phosphorylation. Our work also revealed more knowledge of the IDR-containing protein adenylate kinase 3 (ADK3) that was extracted by heat-treatment. Its disordered C-terminal extension (CTE) brought new functions to this protein. For instance, via its CTE, ADK3 can form a bi-enzyme complex with glyceraldehyde-3-phosphate dehydrogenase (GAPDH), down-regulates the NADPH-dependent GAPDH activity, and behaves as a chaperone for GAPDH against its aggregation and inactivation under heat-treatment.

The thesis entitled ‘Studies of NIS Mediated Cyclopropane Ring Opening Reactions in Carbohydrate Chemistry’ is divided into four chapters. Chapter 1: Section 1: Efficient Synthesis of Fused Perhydrofuro[2,3-b]pyrans (and furans) by Ring Opening of 1,2-Cyclopropanated Sugar Derivatives. In this section a general and efficient methodology for the synthesis of carbohydrate derived perhydrofuro[2,3-b]pyrans (and furans) from the corresponding 1,2-cyclopropane carboxylates has been discussed. A wide range of linear-fused perhydrofuro[2,3-b]pyran or furan ring systems are encountered in a number of biologically active natural products. A few approaches are available for the construction of this kind of fused motifs which involve harsh reaction conditions and lengthy reaction sequence. The methodology utilizes the potential ability of cyclopropanated sugars to undergo N-iodosuccinimide (NIS) mediated electrophilic ring opening assisted by the pyran ring oxygen followed by intramolecular trapping of oxonium intermediate to generate the furan ring system. Cyclopropantion of tribenzyl glucal using methyl diazoacetate and catalytic amount of dirhodiumtetracetate furnished corresponding exo-1,2-cyclopropane carboxylate exclusively. To generate a nucleophile, cyclopropane carboxylate ester was reduced to the corresponding alcohol which upon treatment with NIS in CH3CN underwent ring opening followed by intramolecular ring closure to give the corresponding perhydro[2,3-b]furopyran along with an oxidized product. After various modifications we found that using CH2Cl2 as a solvent gave the expected perhydrofuropyran as the sole product in good yield (Scheme I). The stereochemistry of the product was established on the basis of 1H-1H NOESY experiment. There are many natural products that contain the perhydrofuro[2,3-b]furyl glycal core such as clerodin, jodrelline B and caryoptin, which show insect anti-feedant properties. With this in mind, the methodology has been successfully extended to the cyclopropanated tetrahydrofuran derivatives resulting in the synthesis of furofuryl glycal moiety (Scheme II). Scheme II Chapter 1: Section 2: Synthesis of Carbohydrate Derived Fused Perhydrofuro/pyrano[2,3-b]-γ-butyrolactones. In this section a general and efficient methodology for the synthesis of carbohydrate derived perhydrofuro/pyrano[2,3-b]-γ-butyrolactones has been discussed. The fusion of the γ-butyrolactone onto a substituted tetrahydrofuran/pyran ring makes a distinctive class natural diterpenoids. Representative members of this family include the marine diterpenoids norrisilide and miniolutelide A. In this chapter we describe a neutral and general method for the construction of perhydrofuro/pyrano[2,3-b]-γ-butyrolactones by NIS mediated ring opening of carbohydrate derived 1,2-cyclopropane carboxylic acids (Scheme III). Scheme III The present strategy is complementary to the existing methods and it is useful since it incorporates an additional chiral center in the molecule under milder conditions, which can be used for further transformations. Chapter 2: Ring Opening of Activated Cyclopropanes with NIS/NaN3: One-pot Synthesis of C-1 Linked Pseudo Disaccharides. Ring opening reactions of activated cyclopropanes have been widely used in organic synthesis. But they are restricted to only selected nucleophiles such as alcohol/ water, as most of the ring opening reactions need acidic activation. This chapter deals with studies of reactivity of various activated cyclopropanes with NIS as a neutral activator and sodium azide as a source of nitrogen nucleophile (Scheme IV). Scheme IV We have clearly demonstrated not only the importance of the donor-acceptor feature in the cyclopropanes in the electrophilic ring opening reaction, but also the selectivity in its functionality. Scheme V This methodology has been successfully utilized in a one-pot synthesis of C-1 linked pseudo-disaccharides from carbohydrate derived 1,2-cyclopropane carboxylates (Scheme V). Chapter 3: Synthesis of Unnatural C-2 Amino Acid Nucleosides Using NIS Mediated Ring Opening of 1,2-Cyclopropane Carboxylated Sugar Derivatives. In this chapter, we have efficiently demonstrated the utility of NIS mediated regioselective ring opening of carbohydrate derived donor-acceptor cyclopropanes for the synthesis of C-2 amino acid nucleosides. This leads to a new class of analogs of peptidyl nucleosides (Scheme VI). Scheme VI One of the advantageous factors is the attachment of nucleobase as well as generation of amino acid precursor in the same reaction which avoids lengthy reaction sequence. We have also shown the synthetic utility of our methodology to pyrimidine based furanosyl C-2 amino acid nucleosides which are of interest, since polyoxins having similar structural core exhibit antifungal activity (Scheme VII). Chapter 4: Attempts Towards the Synthesis of Carbohydrate Derived Spiro-perhydrofuropyrans Using NIS Mediated Cyclopropane Ring Opening Reaction. In this chapter we present various attempts to synthesize spiro-perhydrofuropyran/furans by ring opening of spiro-cylopropane derivatives and attempts towards stereoselective synthesis of spiro-cyclopropane carboxylates. Spiroacetal can be synthesized from the corresponding exo-cyclopropyl methanol, which can be obtained from the corresponding exo- cyclopropane carboxylate. The cyclopropyl carboxylate can be obtained from an exo- vinyl ether. Cyclopropanation of carbohydrate derived exo-glycal failed to give any selectivity under a variety of reaction conditions (Scheme VIII). Carbohydrate derived C1-unsaturated ester on cyclopropanation reaction using standard conditions (Pd(OAc)2/CH2N2) was found to be inert. The reaction under Simmons-Smith cyclopropanation conditions also gave similar results. Reduction of the ester part of the molecule to the corresponding alcohol was found to be helpful in the Simmon-Smith cyclopropanation reaction (CH2I2, Et2Zn) to obtain the corresponding exo-cyclopropane, but disappointingly without any selectivity (Scheme IX). In order to get exo-cyclopropane carboxylate with high stereoselectivity, we decided to use one of the hydroxyl group present in the molecule, as a chiral auxiliary. All the established methods for the diazoester formation failed to attach diazo ester at C-4 position (Scheme X). Scheme X (For structural formula pl see the pdf file)