La tomate (Solanum lycopersicum) est l’espèce modèle pour l’étude des fruits charnus. Le développement du fruit de tomate a été décrit comme une succession de phases de développement distinctes, dans lesquelles les régulations hormonales jouent un rôle crucial. Afin de mieux comprendre la régulation du développement du fruit de tomate, des approches ciblées sur des catégories particulières de protéines régulatrices (F-Box) ainsi que des approches non ciblées de génomique fonctionnelle ont été menées dans le laboratoire. L'objectif de cette thèse est de contribuer à la validation fonctionnelle de gènes candidats mis en évidence dans ces travaux: trois protéines à F-Box (SlFB2, SlFB11 et SlFB24) et deux facteurs de transcription (SlHAT22 et SlTGA2.1). Au cours de cette thèse, la caractérisation de lignées RNAi précédemment générés pour les protéines SlFB2, SlFB11 et SlFB24 a été poursuivie. Ce travail n’a pas permis pas de conclure sur le rôle de ces protéines à F-Box dans le développement du fruit de tomate. Mais elle a permis d'isoler un mutant d'insertion dans une lignée RNAi SlFB2, spécifiquement affecté au niveau des fruits. Ce mutant est caractérisé par l'absence de développement de tissu loculaire, ce qui entraine une altération de la forme des graines, ainsi qu'une augmentation de la fermeté du fruit. Le site d'insertion de l'ADN-T dans ce mutant n'est pas encore identifié. En outre, la caractérisation des lignées RNAi SlFB11, a permis de proposer l'implication de cette protéine dans la régulation des méristèmes apicaux et floraux. La caractérisation fonctionnelle de SlHAT22 et SlTGA2.1 a été initiée par la génération de lignées transgéniques présentant une altération du niveau d'expression de ces facteurs de transcription (sur-expression et RNAi) ou une altération de la fonction du facteur de transcription par utilisation de la technologie CRES-T. La caractérisation phénotypique des lignées transgéniques, ainsi que des analyses métaboliques préliminaires ont révélé que SlTGA2.1 et SlHAT22 sont impliqués dans des processus de régulation au cours du développement précoce du fruit de tomate. En outre, ils ont également suggéré que SlTGA2.1 est impliqué dans la régulation du murissement du fruit de tomate. / Tomato (Solanum lycopersicum) is a model species for the study of fleshy fruits. Tomato fruit development has been described as a sequence of distinct developmental phases where different hormones play crucial regulatory roles. To further gain insight into the regulation of tomato fruit development, targeted approaches focused on particular classes of regulatory proteins (F-Box) as well as global functional genomics approaches were undertaken in the laboratory. The aim of this thesis was to contribute to the functional validation of candidate genes isolated from such approaches: three F-Box proteins (SlFB2, SlFB11 and SlFB24) and two transcription factors (SlHAT22 and SlTGA2.1).During this PhD thesis, the characterization of SlFB2, SlFB11 and SlFB24 RNAi lines previously generated was pursued. This work does not allow to conclude about the role of these F-Box in tomato fruit development. But it allowed to isolate an insertion mutant in a SlFB2 RNAi line, specifically affected at the fruit level. This mutant is characterized by the absence of locular tissue development and subsequent alteration of seed shape, as well as by an increase in fruit firmness. The insertion site of the T-DNA in this mutant is not yet identified. In addition, characterization of SlFB11 RNAi lines, allowed to propose the implication of this protein in the regulation of the shoot apical and floral meristems.Functional characterization of SlHAT22 and SlTGA2.1 was initiated by the generation of transgenic lines carrying an alteration of the transcription factor (TF) expression level (OE and RNAi lines) or with an alteration of TFs function using the CRES-T technology. Phenotypical characterization of the transgenic lines, together with preliminary metabolic analyses revealed that SlTGA2.1 and SlHAT22 are implicated in regulatory processes during tomato early fruit development. In addition, they also suggested that SlTGA2.1 is involved in the regulation of tomato fruit ripening.

Le mildiou de la pomme de terre, causé par l'agent pathogène Phytophthora infestans est l'une des maladies les plus préjudiciables de la culture. Jusqu'à présent, la lutte chimique reste le moyen de contrôle le plus utilisé pour la maîtriser, classant la pomme de terre au premier rang en termes d'Indices de Fréquence de Traitement en grande culture. Par ailleurs, l'utilisation de variétés résistantes, comportant notamment des gènes de résistances spécifiques, a également démontré son efficacité pour limiter les dégâts engendrés par cette maladie. Mais leur efficacité est peu durable avec une durée moyenne de 4 ans avant l'apparition du phénomène de contournement par les isolats plus virulents. Il est donc nécessaire de développer des stratégies de contrôle de la maladie en combinant un ensemble d'approches génétiques, culturales, physiques, et chimiques afin de satisfaire au mieux les objectifs agronomiques, environnementaux et socio-économiques. Un modèle, nommé SIPPOM (Simulator for Integrated Pathogen POpulation Management), avait été développé dans le cas de la gestion durable du phoma du colza. Le présent travail a consisté à adapter la structure générique de SIPPOM au cas de la gestion intégrée du mildiou de la pomme de terre en développant des modules spécifiques à partir de modèles préexistants ou développés spécifiquement. Un modèle de culture (Spudgro), un modèle épidémiologique (Guntz-Divoux / Milsol), un modèle de nuisibilité (modèle de Shtienberg) et une fonction de dispersion (modèle de Scherm) ont été identifiés dans la littérature, adaptés et intégrés dans la structure générique de SIPPOM. Cet ensemble de modules a donné lieu à la réalisation du premier prototype opérationnel de la version informatisée de SIPPOM-de-terre sur la plate-forme de modélisation RECORD. Un nouveau modèle, appelé VOLPONE, a été développé pour représenter les dynamiques de repousses de pomme de terre dans une parcelle et sur un tas de déchets. Ce modèle permet de représenter les sources d'inoculum primaire à l'échelle du territoire sous l'influence du climat et des pratiques agricoles. La qualité prédictive du modèle de nuisibilité a été estimée à partir d'un jeu de données expérimentales générées au cours du travail de thèse et s'est montrée correcte. Des exemples de résultats issus de simulations réalisées à l'aide du premier prototype montrent la capacité du modèle, en fonction d'une combinaison de pratiques culturales et de conditions climatiques variables, à prédire la dynamique épidémique de la maladie, ainsi que les dégâts et dommages associés à l'échelle de la parcelle ou sur un parcellaire simple. Néanmoins, d'autres tâches restent à accomplir afin de disposer d'une version achevée de SIPPOM-de-terre sur la plate-forme RECORD. Le travail réalisé illustre la généricité du modèle d'origine SIPPOM et contribue à la production de connaissances et de références permettant l'optimisation d'outils existants tels Mileos®. Les avancées réalisées pourront contribuer à la conception de stratégies de gestion intégrée, collectives et durables pour cette maladie. / Potato late blight caused by the pathogen Phytophthora infestans is one of the most dreaded diseases to this culture. So far, chemical control is the most widespread method used against the pathogen. In turn, the amount of fungicides used to control this disease makes potato the crop with the highest Treatment Frequency Index of all arable crops. The use of resistant varieties, including specific resistance genes, has also demonstrated an effective limitation of injuries that this disease can cause. Still, specific resistances lack durability as there is an average span of 4 years before the emergence of a resistance breakdown phenomenon by more virulent strains. It is therefore necessary to develop control strategies that combine a set of methods (genetic, cultural, physical and chemical) to meet agronomic, environmental and socio-economic criteria. A model named SIPPOM (Simulator for Integrated Pathogen POpulation Management), was developed to address these issues for the control of phoma stem canker on oilseed rape. The present work consisted in adapting the generic structure of SIPPOM for the integrated control of potato late blight by designing specific sub-models using existing models or specifically developed models. A crop model (Spudgro), an epidemiologic model (Guntz-Divoux/Milsol), a damage model (Shtienberg's model) and a dispersal function (Scherm's model) were selected in the literature, adapted, and embedded in SIPPOM's generic structure. This set of sub-models led to an operational prototype of SIPPOM-de-terre under the RECORD modelling platform. A new model, named VOLPONE, was designed to simulate potato volunteer dynamics in a field or on a waste pile. This model permits to simulate inoculum sources at the territory scale under the influence of climate and cropping practices. The predictive quality of the damage model was assessed with data generated in specific field experimentations and proved to be good. Simulation examples produced with the first prototype of SIPPOM-de-terre illustrate the capacity of the model to represent epidemiological dynamics at the field scale or at a small regional scale under the influence of cropping practices and climate. However, further work is required before to totally achieve the design and the implementation of SIPPOM-de-terre under the modelling platform RECORD. The conducted study illustrates the genericity of the model SIPPOM and produced knowledge, references and tools for the integrated management of the disease. The outputs of this work will help design integrated, collective and durable control strategies of potato late blight.

La transformation de la paroi de l’ovaire en un péricarpe charnu implique une coordination entre les divisions cellulaires et l’expansion cellulaire. Des données considérables sur le développement et la maturation du fruit de tomate ont été établies, mais la coordination des divisions cellulaires, de l’expansion cellulaire et de l’endoréduplication durant la mise à fruit ainsi que durant la croissance du fruit de tomate reste grossièrement caractérisée au sein du péricarpe et de nombreuses questions ne sont pas résolues : comment ces deux processus sont-ils régulés et coordonnés durant le développement du fruit d’un point de vue cellulaire? Quand commence l’endoréduplication dans les tissus du fruit et quelle est sa fonction? La première partie de ce mémoire concerne la coordination des divisions cellulaires et de l’expansion cellulaire durant la fin du développement de l’ovaire et le début du développement du fruit. Une différenciation précoce des assises cellulaires composant la paroi de l’ovaire puis le péricarpe a été démontrée. Les divisions cellulaires se font principalement au sein de l’épiderme externe et montrent une synchronisation partielle tandis que l’expansion cellulaire se fait principalement dans le mésocarpe. L’endoréduplication semble être initiée avant l’anthèse. La deuxième partie est consacrée à l’analyse du RNA-seq nucléaire en fonction de quatre niveaux de ploïdie (4, 8, 16 et 32C). La majorité des gènes montrent une augmentation proportionnelle de leurs expressions en fonction des niveaux de ploïdie. Cependant, certains gènes révèlent une surexpression ou une sous-expression en fonction des niveaux de ploïdies. / The transformation of the ovary wall into a fleshy pericarp involves a coordinated pattern of cell division and cell expansion. Considerable data have been reported on tomato fruit development and ripening, but the pattern of cell division, cell expansion and endoreduplication at the tomato fruit set and during fruit growth remains grossly appreciated at the whole pericarp level and many questions are not yet resolved: How are cell division and cell expansion coordinated in tomato fruit a cellular level and according to developmental time? When does endoreduplication begin in fruit tissues and what is its function? The first part of this deals with the coordination of cell division and cell expansion during the end of tomato ovary development and the beginning of fruit growth. Evidence for early differentiation of cell layers in the ovary wall and then in fruit pericarp are presented. Cell division happens mainly in the external epidermis and shows partial synchronization, whereas cell expansion happens mostly in mesocarp cell layers. Endoreduplication is initiated as soon as before anthesis. The second part of this work is devoted to RNA-seq based transcriptome profiling of pericarp nuclei which have been sorted according to four ploidy levels (4, 8, 16 and 32C). We demonstrate that the expression of most of the pericarp-expressed genes shows a proportional increase according to ploidy level, on a nuclear basis. However, a significant amount of genes has been identified as over-expressed or under-expressed according to ploidy level.

O fungo Moniliophthora perniciosa, fitopatógeno responsável pela doença vassoura-de-bruxa no cacaueiro, é responsável por reduzir a produtividade de frutos de cacau nas Américas. Visto que os métodos atuais de controle desta doença não são eficientes, faz-se necessário a busca por compostos para seu controle químico. Os produtos naturais têm contribuído na síntese de novos pesticidas e a família Solanaceae é reconhecida como uma fonte promissora de compostos antifúngicos. Neste contexto, este trabalho teve por objetivo explorar o potencial biológico e químico de metabólitos secundários produzidos por plantas da família Solanaceae com atividade antifúngica à M. perniciosa. Para tanto, extratos aquosos de folhas de dez plantas foram avaliados quanto sua atividade inibitória a M. perniciosa e, observou-se maior atividade antifúngica pelos extratos de Cestrum nocturnum, Solanum seaforthianum e Brunfelsia uniflora. Destas, as plantas S. seaforthianum e B. uniflora mostram-se como promissoras e foram selecionadas para isolamento de seus compostos ativos. A partir do extrato aquoso de S. seaforthianum obteve-se duas frações constituídas por saponinas furostanas, a mistura (25R)-karatavioside C / (25R)-purpureagitoside, com CI50 de 40,9 &mu;g mL-1, e a mistura (25R)-timosaponin H1/ (25R)-uttroside B, com CI50 de 22,3 &mu;g mL-1 ao crescimento micelial. E, a partir do extrato aquoso de B. uniflora obteve-se três compostos da classe das saponinas espirostanas, o composto karatavioside A e os compostos parcialmente identificados BuM8i4, com aglicona identificada como (25R)-yucagenina, e BuM8i6, com aglicona identificada como (25R)-diosgenina, todos com CI50.< 15,63 &mu;g mL-1 ao crescimento micelial de M. perniciosa. Todos os compostos caracterizados neste trabalho estão sendo relatados pela primeira vez a partir das plantas estudas e também para atividade antifúngica. / The fungal phytopathogen Moniliophthora perniciosa is the causal agent of Witches\' Broom disease and the main responsible by limiting cacao production in Americas. The current disease control methods are not efficient and new antifungal compounds are necessary to the chemical management. Natural products has contributed to the development of natural product-based pesticides and the Solanaceae plants are known as a promising source of antifungal compounds. In this context, this work aimed to explore the biological and chemical potential of secondary metabolites produced by Solanaceae plants with antifungal activity against M. perniciosa. For this, antifungal activity of water extracts from leaves of ten plants was evaluated against M. perniciosa and the best results were observed by using extracts from Cestrum nocturnum, Solanum seaforthianum e Brunfelsia uniflora. Among these plants, S. seaforthianum and B. uniflora were selected for active compounds isolation due to their promising characteristics. From water extract of S. seaforthianum two furostan saponin fractions were obtained, the mixture (25R)-karatavioside C / (25R)- purpureagitoside, with IC50 of 40,9 &mu;g mL-1, and the mixture (25R)-timosaponin H1/ (25R)-uttroside B, with IC50 of 22,3 &mu;g mL-1 to the mycelial growth. From water extract of B. uniflora three spirostan saponin compounds were obtained, the compound karatavioside A and the compounds partially identified BuM8i4, with aglycone identified as (25R)-yucagenina, and BuM8i6, with aglycone identified as (25R)- diosgenina, all these compounds showed IC50< 15,63 &mu;g mL-1 to the mycelial growth of M. perniciosa. All compounds characterized in this study were obtained for the first time from these plants and also described about their antifungal activity.

Solanum lycocarpum A. St.-Hil. (Solanaceae Solanum), popular lobeira, é espécie arbustiva nativa e característica do Cerrado brasileiro. Seus frutos são empregados na medicina caseira como diurética, calmante, anti-espasmódica, antiofídica e antiepilética. As espécies do gênero Solanum são produtoras de heterosídeos alcaloídicos, os quais possuem atividade antitumoral, incluindo-se anticâncer de pele. O câncer de pele tem preocupado as autoridades no mundo com os crescentes índices atuais e, por esse motivo, a busca por novos ativos anticâncer é primordial. Dos frutos da lobeira, pode-se produzir extrato alcaloídico, o qual se constitui de uma mistura de glicoalcalóides composta majoritariamente de solasonina e solamargina. Tais compostos têm sido estudados quanto ao potencial anticancerígeno, são relativamente seletivos para células tumorais e, em consequência, apresentam baixa toxicidade às células sadias. Por estes motivos, o potencial antitumoral desta espécie deve ser investigado. Para isso, os frutos foram coletados, secos e triturados. A droga vegetal resultante foi submetida à extração tipo ácido-base para obtenção do extrato alcaloídico. O método analítico empregado para quantificação dos heterosídeos alcaloídicos foi desenvolvida e validada em CLAE-UV. A partir do extrato alcaloídico padronizado, foram desenvolvidas formulações para uso tópico. Estas formulações foram avaliadas por meio de ensaios de permeação e retenção dérmica in vitro, cujo método analítico para quantificação de solasonina e solamargina foi validado. Para escolha da formulação mais promissora para ensaio anticâncer in vivo, as formulações de melhor desempenho in vitro foram avaliadas quanto à retenção dérmica in vivo em camundongos hairless. A formulação B, contendo 1% de extrato alcaloídico, 5% de monoleína (monoleato de glicerol), 5% de propilenoglicol em base de gel de Natrosol (pH 6,5), apresentou retenção dérmica total adequada para futuros ensaios anticâncer de pele in vivo. / Solanum lycocarpum A. St.-Hil. (Solanaceae - Solanum), popular wolf-fruit is a native shrub species and characteristic of the Brazilian Cerrado vegetation. Its fruits are used in folk medicine as diuretic, sedative, anti-spasmodic, antiepileptic and antiophidic. This species belongs to the genus Solanum, known to produce alkaloid heterosides, which possess antitumor activity, including skin anticancer. Skin cancer has concerned the authorities in the world because of its growing rates. Therefore, the search for new active anticancer is paramount. The wolf-fruit furnish an alkaloidic extract, which is rich in a mixture of heteroside alkaloids and composed mostly of solasonine and solamargine. Such compounds have been studied as potential anticancer, because they are relatively selective for tumor cells and, consequently, have low toxicity to health cell. For these reasons, the antitumor potential of this species should be investigated. For that, the fruits were collected, dried and crushed. The dried plant biomass was submitted to acid-base extraction to obtain the alkaloidic extract. The analytical method for the quantitation of the alkaloids in both crude alkaloid extract and plant biomass was developed and validated by HPLC-UV. From the alkaloidic extract, formulations were developed for topical use. These formulations were evaluated by in vitro tests of skin permeation and retention in Franz diffusion cell, for which an analytical method for quantifying solasonine and solamargine was developed, as well. The selection of the most promising formulation for anticancer assay in vivo was based in both, the permeation performance of the formulation in vitro and its skin retention in vivo in hairless health mice. The formulation B, containing crude alkaloid extract 1%, Monoolein 5%, propyleneglycol 5% in Natrosol gel (pH 6.5), showed total dermal retention, which is suitable for future trials of skin anticancer in animal models.

O trabalho teve como objetivo avaliar a resposta à produção total, comercial e por classes de tamanho; ao teor de matéria seca e aos defeitos fisiológicos mancha-chocolate, coração-oco e coração-negro dos tubérculos do clone de batata IAC 2.5, submetido a três doses dos fertilizantes minerais 04-14-08 (N-P2O5-K2O) e sulfato de amônio, complementadas ou não com um composto orgânico de marca comercial Provaso. O experimento foi conduzido de outubro de 2012 a fevereiro de 2013, na Unidade de Pesquisa e Desenvolvimento de Itararé, da Agência Paulista de Tecnologia dos Agronegócios (APTA), em Itararé, SP, nas coordenadas geográficas 24º16\'06\"S e 49º12\'19\"W e altitude de 1200 m. Foi adotado o delineamento em blocos ao acaso em arranjo fatorial 3x2, com quatro repetições. Os tratamentos consistiram em três doses de 04-14-08 no plantio e de sulfato de amônio em cobertura, sendo respectivamente: 2150 kg ha-1 + 200 kg ha-1; 3225 kg ha-1 + 100 kg ha-1; e 4300 kg ha-1 + 0 kg ha-1, com (5000 kg ha-1) ou sem composto orgânico. As doses de 04-14-08 foram calculadas com base na análise de solo e recomendação de 100, 150 e 200% em P2O5 para a cultura da batata, e para a adubação em cobertura, adotou-se respectivamente as doses 40, 20 e 0 kg ha-1 de N e 44, 22 e 0 kg ha-1 de S. A parcela experimental foi composta por quatro fileiras de 3,5 m cada, totalizando 40 plantas. Como parcela útil, foram consideradas as 16 plantas das duas fileiras centrais, excetuando as plantas das extremidades, consideradas bordaduras de cabeceira. O espaçamento foi de 0,35 m entre plantas na fileira e de 0,8 m entre as fileiras. Os dados foram submetidos à análise da variância e as médias foram comparadas pelo teste de Tukey (p < 0,10), utilizando o programa estatístico Sisvar. Nas condições em que o experimento foi conduzido, a alta produtividade comercial de tubérculos (52,4 t ha-1) do clone IAC 2.5 apresentada pelo tratamento com 3225 kg ha-1 de 04-14-08 + 5000 kg ha-1 de composto orgânico no plantio e 100 kg ha-1 de sulfato de amônio em cobertura sugere a importância da complementação da matéria orgânica no aumento da eficiência da adubação mineral. / The study aimed to evaluate the response to total production, marketable production and production by tuber size classes; dry matter content and internal physiological disorders internal brown spot, hollow heart and black heart of potato tubers clone IAC 2.5 subjected to three rates of mineral fertilizers 04-14-08 (N-P2O5- K2O) and ammonium sulphate, supplemented or not with an organic compost trademark Provaso. The trial was conducted from October 2012 to February 2013, at the Research & Development Unity of Agência Paulista de Tecnologia dos Agronegócios (APTA), in Itararé, SP, Brazil (lat. 24º16\'06\'\'S; long. 49º12\'19\'\'W), and altitude of 1200 m. The treatments were arranged in a 3x2 factorial scheme in a completely randomized block design, with four replications. The treatments consisted of three rates of 04-14-08 at planting and ammonium sulphate in dressing fertilization, follow respectively: 2150 kg ha-1 + 200 kg ha-1; 3225 kg ha-1 + 100 kg ha-1; e 4300 kg ha-1 + 0 kg ha-1, with (5000 kg ha-1) or without organic compost. 04-14-08 rates were calculated based on the soil analysis and 100, 150 and 200% on P2O5 recommendation for the potato crop, and for dressing fertilization, was adopted quantities respectively 40, 20 and 0 kg ha-1 of N and 44, 22 and 0 kg ha-1 of S. The experimental plot consisted of four rows of 3.5 m each, for a total of 40 plants. Useful plot was the 16 plants from central rows. The spacing was 0.35 m between plants in the row and 0.8 m between rows. The data were submitted to analysis of variance and treatment means were compared by Tukey test (p < 0,10) using the statistical program Sisvar. Taking in account the conditions in wich the trial was conducted, a high yield of marketable tubers (52.4 t ha-1) for the clone IAC 2.5 by 3225 kg ha-1 of 04-14-08 + 5000 kg ha-1 of organic compost in planting and 100 kg ha-1 of ammonium sulphate in dressing fertilization suggests the importance of organic matter complementing in increasing the efficiency of mineral fertilization.

O fungo Moniliophthora perniciosa, fitopatógeno responsável pela doença vassoura-de-bruxa no cacaueiro, é responsável por reduzir a produtividade de frutos de cacau nas Américas. Visto que os métodos atuais de controle desta doença não são eficientes, faz-se necessário a busca por compostos para seu controle químico. Os produtos naturais têm contribuído na síntese de novos pesticidas e a família Solanaceae é reconhecida como uma fonte promissora de compostos antifúngicos. Neste contexto, este trabalho teve por objetivo explorar o potencial biológico e químico de metabólitos secundários produzidos por plantas da família Solanaceae com atividade antifúngica à M. perniciosa. Para tanto, extratos aquosos de folhas de dez plantas foram avaliados quanto sua atividade inibitória a M. perniciosa e, observou-se maior atividade antifúngica pelos extratos de Cestrum nocturnum, Solanum seaforthianum e Brunfelsia uniflora. Destas, as plantas S. seaforthianum e B. uniflora mostram-se como promissoras e foram selecionadas para isolamento de seus compostos ativos. A partir do extrato aquoso de S. seaforthianum obteve-se duas frações constituídas por saponinas furostanas, a mistura (25R)-karatavioside C / (25R)-purpureagitoside, com CI50 de 40,9 &mu;g mL-1, e a mistura (25R)-timosaponin H1/ (25R)-uttroside B, com CI50 de 22,3 &mu;g mL-1 ao crescimento micelial. E, a partir do extrato aquoso de B. uniflora obteve-se três compostos da classe das saponinas espirostanas, o composto karatavioside A e os compostos parcialmente identificados BuM8i4, com aglicona identificada como (25R)-yucagenina, e BuM8i6, com aglicona identificada como (25R)-diosgenina, todos com CI50.< 15,63 &mu;g mL-1 ao crescimento micelial de M. perniciosa. Todos os compostos caracterizados neste trabalho estão sendo relatados pela primeira vez a partir das plantas estudas e também para atividade antifúngica. / The fungal phytopathogen Moniliophthora perniciosa is the causal agent of Witches\' Broom disease and the main responsible by limiting cacao production in Americas. The current disease control methods are not efficient and new antifungal compounds are necessary to the chemical management. Natural products has contributed to the development of natural product-based pesticides and the Solanaceae plants are known as a promising source of antifungal compounds. In this context, this work aimed to explore the biological and chemical potential of secondary metabolites produced by Solanaceae plants with antifungal activity against M. perniciosa. For this, antifungal activity of water extracts from leaves of ten plants was evaluated against M. perniciosa and the best results were observed by using extracts from Cestrum nocturnum, Solanum seaforthianum e Brunfelsia uniflora. Among these plants, S. seaforthianum and B. uniflora were selected for active compounds isolation due to their promising characteristics. From water extract of S. seaforthianum two furostan saponin fractions were obtained, the mixture (25R)-karatavioside C / (25R)- purpureagitoside, with IC50 of 40,9 &mu;g mL-1, and the mixture (25R)-timosaponin H1/ (25R)-uttroside B, with IC50 of 22,3 &mu;g mL-1 to the mycelial growth. From water extract of B. uniflora three spirostan saponin compounds were obtained, the compound karatavioside A and the compounds partially identified BuM8i4, with aglycone identified as (25R)-yucagenina, and BuM8i6, with aglycone identified as (25R)- diosgenina, all these compounds showed IC50< 15,63 &mu;g mL-1 to the mycelial growth of M. perniciosa. All compounds characterized in this study were obtained for the first time from these plants and also described about their antifungal activity.

Solanum lycocarpum é uma espécie brasileira conhecida por sua diversidade em glicoalcalóides (GA). Os GA solasonina (SS) e solamargina (SM), extraídos dos frutos desta planta, apresentaram propriedades antioxidantes e anticancerígenas atuando em diferentes mecanismos moleculares para a terapia do câncer. No entanto, a baixa solubilidade em água destas moléculas e a sua toxicidade limita a aplicação destes glicoalcalóides. Desta forma, o desenvolvimento de uma formulação nanoestruturada contendo GA pode possibilitar a sua aplicação clínica na terapia do câncer de bexiga (CB). Assim, este projeto visa o desenvolvimento e caracterização de carreadores lipídicos nanoestruturados (CLN) como sistemas de carreamento do extrato alcaloídico de S. Lycocarpum (EA), rico em GA, e avaliação da citotoxidade in vitro em células de câncer de bexiga. Os CLN foram preparadas pelo método de emulsão à quente e sonicação e otimizados utilizando um planejamento fatorial 23. No planejamento avaliou-se a influência de três variáveis independentes: tipo e quantidade de lipídio e tipo de surfactante nos fatores de resposta diâmetro e índice de polidispersão (PdI). CLNs foram caracterizadas de acordo com diâmetro médio, distribuição de tamanho e potencial zeta por espalhamento dinâmico de luz (DLS), índice de recristalização (IR) por calorimetria exploratória diferencial (DSC), porcentagem da eficiência de encapsulamento (EE%) por LC-MS/MS, avaliação morfológica por microscopia de força atômica (AFM), distribuição e concentração de nanopartículas por análise de rastreamento de nanopartículas (NTA), perfil de liberação do EA encapsulado em CLN pelo ensaio de perfil de liberação in vitro em células de Franz, e estabilidade ao longo do tempo. A citotoxidade dos glicoalcalóides foi avaliada pelo ensaio de captura do corante de vermelho neutro utilizando células de câncer de bexiga (RT4). As células foram tratadas com EA livre e encapsulado no CLN nas concentrações de 5 ?g/mL à 20 ?g/mL, assim como CLN vazia no período de 24h e 48h e foram calculadas suas respectivas concentrações inibitórias médias (IC50). O CLN desenvolvido apresentou tamanho inferior a 80 nm e potencial zeta negativo. O PdI foi inferior a 0,2 indicando uma baixa polidispersão. O CLN foi estável por 60 dias em relação ao tamanho, PdI e potencial zeta. O índice de recristalização (IR) do CLN com EA foi 47,23%, indicando uma estrutura menos cristalina do lipídio no CLN que propicia uma alta eficiência de encapsulamento de SS (86%) e SM (89,1%) e evita a expulsão dos ativos durante a estocagem. Os CLNs apresentaram-se semiesféricas como observado por AFM. A concentração de partículas, medida por NTA, foi de 9,74x 1011 partículas/mL e o diâmetro médio foi de 125 nm. Os glicoalcalóides presentes no EA encapsulado em CLN apresentam perfil de liberação in vitro sustentado após 36 h de liberação em condição \"sink\". O mecanismo de liberação dos GA foram diferentes, sendo que para o SS foi não-fickano enquanto que o SM foi fickano. O EA livre e encapsulado (CLN-EA) foi citotóxico frente células RT4, sendo que o CLN-EA foi mais citotóxico que o EA livre após 48h de tratamento. O IC50 das CLN-EA, após 24h de tratamento, foi de 15,94 ?g.mL-1 e 12,35 ?g.mL-1 após 48 h de tratamento. Esta redução no valor do IC50 está relacionada ao perfil de liberação dos glicoalcalóides SS e SM das nanopartículas. Os resultados demonstraram que a CLN desenvolvida é um sistema interessante para encapsular EA com potencial para futura terapia do câncer de bexiga / Solanum lycocarpum is a native species of the Brazilian savannah known for its diversity in glycoalkaloids (GA). The GA solasonine (SS) and solamargine (SM), extracted from the fruits of their plant, have antioxidant and antitumoral activity; acting on different molecular mechanisms interesting for cancer therapy. However, the low water solubility of these molecules and their toxicity limits their application. Thus, the development of a nanostructured formulation containing these glycoalkaloids may allow their clinical application in bladder cancer therapy (BC). Thus, this project aims at the development and characterization of nanostructured solid lipid (NLC) as carrier systems of alkaloidic extract of S. lycocarpum (AE), rich in GA, and their cytotoxic evaluation in order to obtain a nanostructured system for future intravesical therapy of bladder cancer. The NLC were prepared by the emulsion and sonication method and optimized using a factorial design 23. The influence of three independent variables was evaluated as type and amount of lipid and type of surfactant in response factors diameter and polydispersity index (PdI). NLCs were characterized according to average diameter, size distribution and zeta potential by dynamic light scattering (DLS), recrystallization index (RI) by differential scanning calorimetry (DSC), percentage of encapsulation efficiency (EE%) by LC MS/MS, morphological analysis by atomic force microscopy (AFM), distribution and concentration of nanoparticles by nanoparticle tracking analysis (NTA), the in vitro release profile of GA from NLC was done by Franz\'s cells diffusion, and stability over time by DLS. Cytotoxicity assay was evaluated by neutral red uptake assay in bladder cancer cells (RT4). Cells were treated with different concentrations of GA free and encapsulated in NLC (5-40 ?g/mL) and empty NLC for 24h, 48h and 72h. The NLC developed showed diameter smaller than 80 nm and zeta potential of -9.26 mV. The PdI was less than 0.2 indicating dispersion with low polydispersity. The NLC was stable for 60 days regarding to size, PdI and zeta potential. Recrystallization index (RI) of NLC-AE was 47.23%, indicating a less crystalline structure of the lipid in the particle, which provides high encapsulation efficiency of SS (86%) and SM (89.1%). This low RI prevents the expulsion of GA from NLC during the storage. NLCs exhibited semi-spherical form as observed by AFM. The particles concentration, measured by NTA, was 9.74 x 1011 particles/mL and the diameter was about 125 nm. The GA present in NLC-AE showed an in vitro sustained release profile after 36 h of release in a sink condition. The release mechanisms of SS and SM were different. The release mechanism of SS was non-Fickan while SM was Fickan. The AE free and NLC-AE was cytotoxic to RT4 cells, and the NLC-AE was more cytotoxic than the AE free after 48 hours of treatment. The IC50 of NLC-AE, after 24 hours of treatment, was 15.94 ?g.mL-1 and 12.35 ?g.mL-1 after 48 h of treatment. This reduction in IC50 value can be related to the release profile of the GA (SS and SM) of the nanoparticles. The results demonstrate that NLC developed is an interesting system to encapsulate AE with potential for future therapy of bladder cancer

Embora o cultivo de tomate seja muito antigo e amplamente distribuído, ainda enfrenta desafios para o melhoramento dos níveis de produção e, da qualidade para o processamento e consumo fresco. A grande maioria das características de interesse agronômico está determinada por loci de caracteres quantitativos (QTL), dificultando ainda mais a identificação e transferência gênica. Diversas características tornam o tomateiro um bom modelo para estudos de dissecação dos determinantes genéticos de QTL. Primeiro, a disponibilidade de fontes de germoplasma selvagens ainda inexploradas que podem aumentar a variabilidade genética, somada à possibilidade de cruzamento entre espécies não simpátricas e à autogamia. Segundo, a grande quantidade de informação genética como mapas, coleções de ESTs, QTL, e populações de mapeamento. Terceiro, o genoma de S. lycopersicum está completamente sequenciado. Por fim, devido a suas diferenças morfogenéticas em relação à espécie modelo Arabidopsis thaliana, o tomate se torna uma alternativa para estudos de eudicotiledonias, principalmente em estudos relacionadas a metabolismo de frutos carnosos. A partir da abundante plataforma de dados disponível e por meio de ferramentas de genômica, e genética reversa, este trabalho aborda o estudo de duas regiões do genoma do tomateiro envolvidas no metabolismo de aminoácidos e na determinação da estrutura da parede celular. O estudo de genômica comparativa de uma região do cromossomo 7 permitiu revelar a perfeita sintenia existente entre S. lycopersicum e a espécies selvagem S. pennellii e estimar o tempo de divergência em 2,7 MAA. Complementarmente, foi possível determinar que as diferenças fenotípicas entre as espécies são maiormente devidas a mudanças nas regiões regulatórias e à presença de SNPs. O estudo funcional do gene LFP (Low Free Putrescine) permitiu caracterizar uma proteína plastidial, até o momento desconhecida em tomateiro, que participa do metabolismo de poliaminas. O silenciamento de LFP resultou na redução da disponibilidade de putrescina livre e no aumento da biomassa vegetativa. Por outro lado, os resultados obtidos do estudo do gene GAUT4 (galacturonosiltransferase 4) demonstraram que a enzima codificada se localiza em Golgi e participa do metabolismo das pectinas. Em frutos, a redução dos níveis de GAUT4 resultou na diminuição de pectina e na alteração da sua composição, porém estes se apresentaram mais firmes. Adicionalmente, o silenciamento do gene GAUT4 modificou o particionamento de açúcares provocando o aumento de massa vegetativa em detrimento do índice de colheita, revelando assim um mecanismo regulatório que comunica o metabolismo da parede celular ao controle da relação fonte-dreno. Desta forma, os resultados obtidos aportam dados fundamentais para a melhor compreensão de caracteres de interesse agronômico, assim como de processos fisiológicos complexos e pouco explorados até o momento no tomateiro / Although tomato is an old and widely distributed culture, it still faces challenges to improve production levels and quality for processing and consumption. The vast majority of agronomical important characteristics are determine by quantitative trait loci (QTL), further hindering the gene identification and transfer. Several features make tomato a good model for studying the genetic determinants underneath QTL. First, the availability of unexploited sources of wild germplasm that can increase genetic variability, coupled with the possibility of interbreeding between no sympatric species and autogamy. Second, the large amount of available genetic information as maps, EST collections, QTL, and an extensive collection of mapping populations. Third, the genome of S. lycopersicum is completely sequenced. Finally, due to their morphogenetic differences in relation to model species Arabidopsis thaliana, tomato becomes an alternative to eudicotyledons studies, especially in studies related to fleshy fruits metabolism. From the abundant data platform available and by means of genomic tools, and reverse genetics, this work addresses the study of two tomato genomic regions involved in amino acids and cell wall metabolisms. The comparative genomic study in a region of chromosome 7 has revealed the perfect synteny between S. lycopersicum and the wild species S. pennellii, and estimated the time of divergence between both species in 2.7 MYA. Additionally, it was possible to determine that the phenotypic differences between species are mostly due to changes in regulatory regions and the presence of SNPs. The functional study of LFP gene (Low Free Putrescine) allowed us to characterize a plastid protein, yet unknown in tomato, which participates in the metabolism of polyamines. The LFP silencing resulted in reduced availability of free putrescine and increased vegetative biomass. Furthermore, the functional characterization of the GAUT4 (galacturonosyltransferase 4) gene demonstrated that the encoded enzyme is located in the Golgi apparatus and participates in the pectin metabolism. In fruits, the reduced levels of GAUT4 resulted in decreased pectin and in the change of its composition. Additionally, GAUT4 gene silencing modified sugars partitioning leading to an increased vegetative biomass together with a drastic reduction of the harvest index. Thus, revealing a regulatory mechanism that communicates the cell wall metabolism to source-sink relationship control. Concluding, the results obtained contribute to a better understanding of agronomical important traits, as well as of complex physiological processes little explored in tomato so far

Nous avons réalisé une enquête ethnobotanique sur le nom vernaculaire bwa kaka en Martinique. Durant l’enquête, nous avons constaté que la génération de jeunes avait une plus grande connaissance d’usages pour les plantes médicinales que les personnes plus âgées interrogées. De plus, les utilisations proposées selon le sexe de l'interrogé étaient significativement différentes. Les hommes ont indiqué le bwa kaka principalement pour le traitement des maladies de l'appareil digestif, tandis que pour les femmes l'utilisation principale est magico-religieuse. Les enquêtes ethnobotaniques publiées jusqu’à présent en Martinique n’ont pas examiné ce résultat en fonction du sexe. L’utilisation du bwa kaka, qui était une plante très demandée par le passé, semble avoir aujourd’hui régressé. L’explication est peut-être liée à sa vocation magico-religieuse, cette utilisation ayant quasiment disparu dans la société moderne martiniquaise. Six espèces sont dénommées bwa kaka localement. Nous avons recueilli les utilisations thérapeutiques qui y ont été associées. Cependant, nous avons choisi d’évaluer le potentiel thérapeutique de Solanum triste pour une utilisation qui n’a été citée que pour cette espèce : le traitement des lithiases urinaires. L’espèce a d’abord fait l’objet d’une étude phytochimique qui a permis d’identifier plusieurs composés (acides phénoliques, flavonoïdes, glycoalcaloïdes) par LC-DAD-ESI-MS2. La structure complète du glycoalcaloïde majoritaire intéressant a été obtenue: le 22S, 25S 3-beta-spirosol-5-ene -O- α -L-rhamnopyranosyl (1→2)beta-D-xylopyranosyl-O(1→3)alpha-L-rhamnopyranosyl O(1→4) glucopyranose. Afin d’évaluer le potentiel antilithiasique de l’extrait aqueux de S. triste, trois tests in vitro ont été réalisés : l’évaluation de sa capacité antioxydante, son effet sur la cristallisation de l’oxalate, et sur les calculs déjà formés. Le résultat le plus significatif est le pouvoir préventif in vitro des extraits de S. triste sur la lithiase urinaire ce qui n’a jamais été mis en évidence pour cette espèce et qui nécessite d’être conforté dans des travaux ultérieurs. / We have performed an ethnobotanical survey in Martinique on the common name “bwa kaka”. During this survey, we observed that the younger generation interviewed knew more about the uses of medicinal plants than the older generation. Moreover, the suggested uses for this plant were significantly different and depended on the gender of the person interviewed. Men indicated that “bwa kaka” was mainly used for diseases of the digestive system whereas women indicated that the main use was linked to magicoreligious rites. The ethnobotany surveys published up until now in Martinique did not investigate this result based on gender. The use of “bwa kaka”, which was a plant in great demand in the past, seems to have declined nowadays. This could be explained by its link to magico-religious rites, a practice which has practically disappeared in modern Martinique society. Six species are locally called “bwa kaka”. We have documented the therapeutic uses associated with this plant. However, we have chosen to evaluate the therapeutic potential of Solanum triste for a use that has only been associated with this species, namely the treatment of urinary lithiasis. This plant species was first used in a phytochemical study which made it possible to identify several compounds such as phenolic acids, flavonoids and glycoalkaloids by LC-DAD-ESI-MS2. The complete structure of the predominant glycoalkaloid has been obtained, i.e. 22S, 25S 3-beta-spirosol-5-ene -O- α -L-rhamnopyranosyl (1→2)beta-Dxylopyranosyl-O(1→3)alpha-L-rhamnopyranosyl O(1→4) glucopyranose. In order to evaluate the antilithiasis capacity of an aqueous extract of S. triste, three in vitro tests were performed: the evaluation of its anti-oxydant capacity, its action on the crystallization of oxalate, and on urinary stones already formed. The most significant result is the in vitro preventive capacity of extracts of S. triste on urinary lithiasis, a result which has never been previously demonstrated for this species and which needs to be confirmed in subsequent experiments.