Quillaja brasiliensis (A. St.-Hil. &Tul.) Mart. é uma espécie nativa do Rio Grande do Sul, conhecida popularmente como pau-sabão, devido à capacidade de suas folhas e cascas formarem abundante espuma em água. A espécie congênere chilena, Q. saponaria, é uma das principais fontes industriais de saponinas, as quais são utilizadas, entre outros, como adjuvantes em vacinas. Tendo em vista a presença de saponinas em Q. brasiliensis, métodos por cromatografia em camada delgada (CCD) e cromatografia líquida de alta eficiência (CLAE) foram desenvolvidos para a caracterização e o doseamento de fração purificada de saponinas, a partir do extrato aquoso de folhas, denominada QB-90. Ensaios para verificar a toxicidade subcutânea e o perfil dose-dependente para atividade adjuvante também foram realizados com esta fração. Para o doseamento de QB-90 no extrato aquoso foi desenvolvido e validado um método por CLAE empregando coluna de fase reversa C8, sistema isocrático acetonitrila:água, fluxo de 0,8 ml/min e detecção a 214 nm. Na validação do método, foram avaliados os parâmetros de linearidade e intervalo de variação, precisão, exatidão, limite de detecção, limite de quantificação e robustez. Em relação à toxicidade subcutânea de QB-90 em camundongos, não foram observados efeitos sistêmicos no intervalo de doses de 50 a 400 μg. Vacinas experimentais preparadas com herpesvírus bovino tipo 1 (BoHV-1) como antígeno e QB-90 (50-200 μg) foram capazes de aumentar a resposta imunológica em camundongos de modo comparável às saponinas de Q. saponaria (QUIL-A®, 100 μg). Com vistas à potencial utilização sustentável da espécie brasileira na obtenção de saponinas de interesse industrial, protocolos básicos de obtenção de plantas de Q. brasiliensis por micropropagação e por germinação de sementes foram desenvolvidos. Para melhor compreender o perfil de acúmulo de QB-90, seu conteúdo foi investigado em diferentes órgãos vegetais, em diferentes estações do ano e durante o desenvolvimento de plântulas, assim como em resposta a fatores de estresses bióticos e abióticos. O conteúdo de QB-90 não foi afetado pela aplicação de ácido salicílico exógeno (5 mM). No entanto, foi aumentado pela aplicação exógena de ácido jasmônico (40 μM e 400 μM), bem como pela exposição à radiação UVC. Tendência de aumento no teor de QB-90 foi observada com a aplicação de dano mecânico controlado e com a exposição à radiação UVB. A distribuição órgão-específica de QB-90 foi avaliada, detectando-se maiorconcentração nas folhas do que em raízes e caules de plantas propagadas em laboratório. O teor de QB-90 também foi analisado nas diferentes estações climáticas ao longo de dois anos, indicando que a redução da insolação, geralmente associada a períodos de baixa pluviosidade, está associada à maior produção de QB-90. / Quillaja brasiliensis (A. St.-Hil. & Tul.) Mart. is a native tree of Rio Grande do Sul, the Southern state of Brazil, commonly known as soap tree due to the capacity of their leaves and barks to produce abundant foam in water. The related Chilean species, Q. saponaria, is one of the main sources of industrial saponins which are used as adjuvant formulation for vaccines. Considering the presence of saponins in Q. brasiliensis, thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) methods were developed to characterize and quantify the purified saponin fraction named QB-90, obtained from the aqueous extract of leaves. Additionally, the subcutaneous toxicity and dose-response profile to adjuvant activity of QB-90 were evaluated in mice. An HPLC method was developed and validated to quantify QB-90 content in aqueous extract, employing RP-8 column, mobile phase acetonitrile:water, flow rate of 0.8 ml/min and detection at 214 nm. The validation parameters evaluated were linearity and range, precision, accuracy, detection limit, quantitation limit and robustness. In relation to QB-90 subcutaneous toxicity in mice, systemic effects were not observed in doses ranging from 50-400 μg. Experimental vaccines prepared with bovine herpesvirus type I (BoHV-1) antigen and QB-90 (50- 200 μg) were able to enhance the immune responses of mice in a comparable manner to saponins from Q. saponaria (QuilA, 100 μg). Considering the potential sustainable utilization of the Brazilian species as a source of saponins of industrial use, basic protocols for obtaining Q. brasiliensis plants by micropropagation and seed germination were developed. To better understand the accumulation patterns of QB-90, we investigated its content in different plant organs; throughout the seasons and during seedling development, as well as in response to potential biotic and abiotic stress factors. Content of QB-90 wasn’t affected by exogenous application of salicylic acid (5mM). However, it was increased by exogenous application of jasmonic acid (40 μM and 400 μM), as well as by exposure to UVC. Trends toward increase in QB-90 content were observed with exposure to UVB and wounding. The organ-specific QB-90 distribution was evaluated and higher amounts were observed in leaves than roots and stems of plants propagated in the laboratory. Variations inQB-90 content in different seasons for two years showed that lower insolation, generally combined with low precipitation periods, were associated with higher QB- 90 content.

A lesão granulomatosa é caracterizada como um agregado compacto de fagócitos maduros formado em resposta à um estímulo persistente. Os mediadores pró-inflamatórios da família da IL-1, ao promoverem a ativação da imunidade inata e o remodelamento tecidual descontrolados, geram a fisiopatologia da paracoccidioidomicose, doença pulmonar granulomatosa causada pelo fungo P. brasiliensis. A principal via inflamatória envolvida na secreção de IL-1?, IL-18 e IL-1? é a ativação dos inflammasomas, complexos protéicos conhecidos pela sua capacidade de ativar proteoliticamente a enzima caspase-1. Neste estudo abordamos os mecanismos subjacentes às vias canônica e não canônica do inflamassoma, avaliando a importância funcional das caspases 1, 11 e 8 na resistência do hospedeiro durante a infecção por P. brasiliensis. Demonstramos que a resposta imunológica protetora mediada pelo IFN-?, após a liberação de IL-18 pela via da caspase-1, é o principal mecanismo responsável pelo controle fúngico quando o dano celular desencadeado pelo fungo é reconhecido pelo inflamassoma de NLRP3. No entanto, apesar do papel importante da caspase-1 na maturação da IL-1?, a falta de caspase-1 apenas reduz parcialmente os níveis de IL-1? durante a infecção por P. brasiliensis. Ao contrário do esperado, a deficiência de caspase-11 não prejudicou a produção de IL-1?, mas em vez disso, preveniu, na célula, a morte por piroptose e a secreção IL-1?, citocina importante para restringir o crescimento fúngico através da síntese de IL-17. Por fim, observamos que a ativação de caspase-8 pela sinalização de dectina-1 / Syk, além de mediar a maturação da IL-1?, que acontece de maneira independente de caspase-1 e 11, é necessária também para o funcionamento eficiente da via canônica de caspase-1, demonstrando uma rede interligada entre as vias canônica de caspse-1 e não canônica de caspase-8 para coordenar o processamento da IL-1?. Em conjunto, nossos resultados mostram contribuições distintas das vias canônica e não canônica do inflamassoma na produção de citocinas da família da IL-1, enfatizam a versatilidade desta plataforma em recrutar várias proteínas efetoras para adequar a resposta imunológica antifúngica e evidenciam a complexidade envolvida nas interações patógeno-hospedeiro. / Granuloma is a lesion characterized by a compact aggregate of mature phagocytes arising in response to a persistent stimulus. By driving uncontrolled innate immunity and tissue remodelling, IL-1 family pro-inflammatory mediators govern the pathophysiology of paracoccidioidomycosis, a granulomatous lung disorders caused by Paracocccidioides brasiliensis. A major inflammatory pathway involved in IL-1?, IL-18 e IL-1? secretion is the activation of inflammasomes, large multimolecular complexes best known for their ability to control activation of the proteolytic enzyme caspase-1. In this study we addressed the mechanisms that underlie canonical and non-canonical inflammasome pathways, assessing the functional importance of caspase-1, caspase-11 and caspase-8 in the regulation of inflammasome-mediated host resistance during P. brasiliensis infection. We found that IFN-?-mediated protective immune response following by caspase-1- dependent IL-18 release after is the key mechanism responsible for the fungal control after P. brasiliensis-induced cell damage recognition by NLRP3 canonical inflammasome pathway. Nonetheless, despite the important role of caspase-1 in the IL-1? maturation, the lack of caspase-1 only partially reduced IL-1? levels during P. brasiliensis infection. Unlike caspase-1, caspase-11 deficiency did not impair IL-1? production, cytokine strictly secreted by canonical caspase-1 inflammasome pathway. Instead, P. brasiliensis-triggered caspase-11 activation in an ill-defined manner leads to a rapid pore-mediated cell lysis and is required for IL-1? production during P. brasiliensis infection. IL-1?, in turn, is important for promoting the restriction of fungal growth trough IL-17-based inflammation. Finally, the caspase-8 induction by dectin-1/Syk signaling besides playing a role in mediating the caspase-1/11-independent IL-1? maturation is also required to efficient canonical caspase-1 inflammasome pathway, demonstrating a connected network between non-canonical caspase-8 and canonical caspase-1 inflammasome pathways to coordinate IL-1?. Taken together, our results revealed distincts contributions of both canonical and non-canonical inflammasome pathways in IL-1 family cytokine production and emphasizes the versatility of this platform to recruit several effector proteins to tailor the antifungal immune response, sheding new light on the complexity of this hostpathogen interaction.

O presente projeto de pesquisa teve por objetivo a descoberta de metabólitos secundários potencialmente bioativos contra atividade parasitária e como agentes anti-virais e antibióticos, usando desreplicação como metodologia essencial na seleção e priorização de extratos brutos oriundos de invertebrados, para a descoberta de produtos naturais estruturalmente inéditos.<br /><br />Foram preparados diversos extratos de invertebrados marinhos a partir de um fracionamento inicial. O desenvolvimento e a aplicação de uma metodologia de desreplicação permitiu a seleção de três organismos de estudo, Iotrochota birotulata, Amathia verticillata, e Tedania brasiliensis. Os extratos destes organismos foram submetidos a diferentes metodologias de fracionamento, isolamento e purificação de substâncias, dependendo dos resultados de desreplicação para cada organismo.<br /><br />Como resultado do isolamento e identificação de substancias destes organismos, obteve-se dois compostos já conhecidos na literatura e dezesseis compostos inéditos, dos quais dois são derivados sintéticos. De todos os compostos isolados, a pseudoceratidina, as desbromopseudoceratidinas e as tedamidas A e B apresentaram um índice de seletividade de 243.1, 217.1 e 89.3 respectivamente para Leishmania (L.) infantum, e o composto N12-acetilpseudoceratidina de 23.2 para Trypanosoma cruzi, sendo estes resultados muito promissores para a utilização das estruturas destas moléculas como potenciais modelos para o desenvolvimento de fármacos. / This research project aimed at the discovery of potentially bioactive secondary metabolites against parasitic activity and as antiviral agents and antibiotics, using dereplication as an essential methodology in the selection and prioritization of crude extracts derived from invertebrates, to the discovery of natural products structurally unpublished.<br /><br />Various extracts of marine invertebrates from an initial fractionation were prepared. The development and application of a dereplication methodology allowed the selection of three study organisms, Iotrochota birotulata, Amathia verticillata, and Tedania brasiliensis. The extracts of these organisms were subjected to different methods of fractionation, isolation and purification of substances, depending on the results of dereplication for each organism.<br /><br /> As a result of the isolation and identification of substances of these organisms, it obtained two compounds already known in the literature and unpublished sixteen compounds, two of which are synthetic derivatives. In all isolated compounds, pseudoceratidina, the desbromopseudoceratidinas and tedamidas A and B showed an index of selectivity 243.1, 217.1 and 89.3 respectively for Leishmania (L.) infantum, and the compound N12-acetilpseudoceratidina 23.2 for Trypanosoma cruzi, which are very promising results for the use of these molecules as structures potential models for drug development.

Dans un contexte de changements climatiques, le stress hydrique et la gestion de l'eau sont considérés comme une contrainte importante pour le secteur agricole. Ainsi la sélection pour la tolérance à la sécheresse est devenue un objectif majeur pour de nombreux programmes de sélection. La vulnérabilité à la cavitation est considérée comme un trait d'intérêt pour la sélection à une sécheresse extrême, en particulier pour les plantes ligneuses. Pourtant, l'étendue de sa variabilité et sa relation avec la tolérance à la sécheresse sont mal documentées à l’échelle intra-spécifique, et en particulier dans les espèces cultivées. Dans cette étude, la variabilité génétique de la vulnérabilité à la cavitation a été étudiée sur trois dispositifs expérimentaux différents avec trois arbres d’intérêt agronomique qui sont menacés par le risque de sécheresse: noyer, hévéa et pommier. Une faible ou aucune, variation de la vulnérabilité à la cavitation a été trouvée dans les espèces étudiées ainsi qu'entre deux espèces de noyers malgré les différences de traits précédemment rapportés. Ces résultats suggèrent une canalisation de la résistance à la cavitation dans les organes critiques (branches, tiges). Chez le pommier, le porte-greffe est soupçonné de provoquer de la plus étroite résistance à la cavitation sur le greffon. Par contre, des différences ont bien étés trouvées sur d’autres traits de réponse à la sécheresse comme la régulation stomatique, la chute des feuilles ou encore la vulnérabilité à la cavitation dans le pétiole. Ainsi, la vulnérabilité à la cavitation des organes critiques n’est pas un paramètre pertinent pour la sélection de la tolérance à la sécheresse, et les programmes de sélection sur les espèces étudiées ne semblent pas avoir affecté ce paramètre. / In a context of climatic changes, drought stress and water management are regarded as one of the most important constraints for agricultural sector. Thus the selection for drought tolerance became a main objective for many breeding programs. Vulnerability to cavitation is considered a trait of interest for the selection for extreme drought stress, especially for woody species. However, the extent of its variability and its relation to drought tolerance are poorly documented on intraspecific level, particularly for cultivated species. In this study the genetic variability of vulnerability to cavitation was studied on three different experimental devices with three trees of agronomic interest that are threatened by the risk of drought: walnut, rubber and apple trees. Low or no variation in xylem vulnerability to cavitation was found in the studied species and between two species of walnuts despite differences previously reported features. These results suggested a canalization of cavitation resistance on critical organs (branches and stems). In apple tree, the rootstock was suspected to cause the narrow resistance to cavitation on the scion. On the contrary, differences on other traits in response to drought stress such as stomatal regulation, leaf shedding or vulnerability to cavitation on petiole were found. Therefore, vulnerability to cavitation of critical organs was not a relevant parameter for the selection of drought tolerance and breeding programs on the species studied did not appear to affect this parameter.

Pour d'évidentes raisons agronomiques et environnementales, être en mesure de concevoir et mettre en place des systèmes de culture dans lesquels les plantes accèdent aux ressources de manière optimale revêt une importance cruciale pour tous les intervenants impliqués dans la production agricole. Les techniques d'intensification telles que la mise en place de cultures d'inter-rang et l'agro-foresterie visent à accroître la productivité globale des terres tout en assurant la durabilité des agro-écosystèmes, via une optimisation de l'utilisation des ressources environnementales (lumière, eau et nutriments) par les plantes et une préservation des cycles géochimiques. En théorie, les moyens d'atteindre ces objectifs sont nombreux mais en pratique, les interactions souterraines sont complexes et difficiles à mesurer, de sorte que les progrès réalisés dans la conception d'agro-écosystèmes améliorés et durables demeurent modestes. Dans ce contexte, l'objectif de ce travail a été d'évaluer, au travers de mesures racinaires detaillées en rhizotron et au champ, les effets des cultures d'inter-rang sur la croissance des jeunes hévéas. La dynamique des interactions souterraines a été étudiée, tant au niveau de la racine individuelle qu'à celui du système racinaire entier, sur la base, notamment, de descriptions numériques détaillées. Une telle approche a permis de proposer, en outre, une voie novatrice pour l'analyse de la dynamique racinaire à l'echelle du systeme racinaire entier, à savoir l'analyse des trajectoires de croissance. Dans le cas de l'association maïs-hévéa, les expérimentations en rhizotron ont permis de mettre en evidence que les interactions souterraines entre ces deux plantes peuvent induire des modifications de la croissance de leurs racines, à la fois à l'échelle de la racine individuelle et à celle du système racinaire entier. Toutefois, une telle coordination des dynamiques racinaires des plantes associées n'a pas pu être confirmée dans le cas des traitements manioc-hévéa et arachide– hévéa. Les expérimentations au terrain ont fourni, de manière assez prévisible, une image complexe des interactions souterraines entre hévéa et cultures d’inter-rang. Toutefois, un premier résultat obtenu par le biais de la mise en place de 'pièges à racines' dans un traitement niébé-hévéa, a été de montrer que ces deux plantes n'avaient pas un comportement compétitif marqué l'une vis-à-vis de l'autre. Il est également apparu que les hévéas paraissent ‘investir’ dans des racines ‘coûteuses’, car de faible longueur spécifique, probablement pour assurer une certaine durabilité de ces organes, tandis que les cultures d’inter-rang favorisent l'allocation des assimilâts vers des racines de longueur spécifique élevée, de construction moins ‘coûteuses’, probablement en réponse a un impératif de croissance plus rapide (suggéré par les taux d’élongation racinaire mesurés au cours des expérimentations en rhizotron). Enfin, excepté le cas du manioc, l’introduction de cultures d’inter-rang telles que le maïs et l'arachide n'a pas eu d'impact significatif sur le développement des jeunes hévéas, comme en attestent l'évolution de leur circonférence, hauteur et développement foliaire. Ce résultat de terrain est compatible avec les résultats des expérimentations en rhizotron qui n'ont démontré aucun effet inhibiteur des cultures d’inter-rang sur le développement de la partie aérienne des hévéas. Bien que les travaux présentés dans ce rapport, ne permettent pas, à eux seuls de conclure de manière définitive sur la façon dont les espèces cultivées en association peuvent se compléter mutuellement sur le plan fonctionnel, ils apportent des éléments de réponse préliminaires à cette question complexe ainsi que des méthodes permettant de les obtenir. Au total, ce travail représente donc une contribution à la conception des agro-écosystèmes durables qui deviennent de plus en plus indispensables dans le contexte d'une demande mondiale croissante en produits alimentaires et matières premières. En outre, certains des résultats obtenus dans le cadre de cette thèse ouvrent des perspectives pour des recherches plus approfondies, avec une finalité agronomique appliquée / For obvious agronomic and environmental reasons, being able to design and implement agro-ecosystems in which crops have optimal access to resources is of pivotal importance to all stakeholders involved in agricultural production. Intensification techniques such as agro-forestry or the introduction of inter-crops aim to increase land productivity while conserving geochemical cycles, to ensure the sustainability of agro-ecosystems through an optimized use of environmental resources (light, water and nutrients). In theory, there are many ways of achieving such a goal, but in practice, below-ground interactions between plants are complex and difficult to measure, so that progress with the development of sustainable agro-ecosystems has been slow and remains modest. In this context, the objective of this work was to assess the effects of inter-crops on the growth of young rubber trees, based on a detailed analysis of below-ground interactions between the associated plants. The dynamics of below-ground interactions has been studied in rhizoboxes, at both the scale of individual roots and that of the whole root system, using detailed numerical descriptions of root architecture. Such an approach resulted in the design of an innovative method for the analysis of the entire root system dynamics, namely, the analysis of growth trajectories. In the case of the maize-rubber tree association, the experiments in rhizoboxes showed that the below-ground interactions between these two plants can induce changes in root growth, at both the individual root and the whole root system levels. However, such a coordination of rooting patterns could not be confirmed in the case of the cassava-rubber tree and groundnut-rubber tree associations. Not unexpectedly, field experiments provided a rather complex picture of the underground interactions between rubber trees and inter-crops. However, initial results obtained using 'root traps' in a cowpea-rubber tree treatment indicated that these two plants were unlikely to have a marked underground competitive behavior relative to each other. Results of field experiments also indicated that, in general, rubber trees seem to 'invest' in 'expensive roots' of low specific root length, presumably to confer some degree of durability to these organs, while inter-crops favoured the allocation of assimilates to 'cheaper' roots, i.e. roots of much higher specific length, probably in response to a 'fast growth imperative' (an hypothesis supported by the root elongation rate values measured during the rhizobox experiments). Finally, to the possible exception of cassava, inter-crops were found to have no significant impact on the development of young rubber trees, as evidenced by measured changes in tree circumference, height and leaf development. This result is consistent with results from the rhizobox experiments which also showed no inhibitory effect of inter-crops on the above-ground development of rubber trees. Although the work presented in this report does not allow, in itself, to conclude firmly on the issue of the functional complementarity between plants grown in association in inter-cropping systems, they provide preliminary answers to this complex issue together with methods to obtain such answers. Overall, this work therefore represents a contribution to the design of sustainable agro-ecosystems which are becoming increasingly needed in the context of a growing global demand for food and raw materials. In addition, some of the results generated by this work open up prospects for future research for the development of sustainable agro-ecosystems

O presente projeto de pesquisa teve por objetivo a descoberta de metabólitos secundários potencialmente bioativos contra atividade parasitária e como agentes anti-virais e antibióticos, usando desreplicação como metodologia essencial na seleção e priorização de extratos brutos oriundos de invertebrados, para a descoberta de produtos naturais estruturalmente inéditos.<br /><br />Foram preparados diversos extratos de invertebrados marinhos a partir de um fracionamento inicial. O desenvolvimento e a aplicação de uma metodologia de desreplicação permitiu a seleção de três organismos de estudo, Iotrochota birotulata, Amathia verticillata, e Tedania brasiliensis. Os extratos destes organismos foram submetidos a diferentes metodologias de fracionamento, isolamento e purificação de substâncias, dependendo dos resultados de desreplicação para cada organismo.<br /><br />Como resultado do isolamento e identificação de substancias destes organismos, obteve-se dois compostos já conhecidos na literatura e dezesseis compostos inéditos, dos quais dois são derivados sintéticos. De todos os compostos isolados, a pseudoceratidina, as desbromopseudoceratidinas e as tedamidas A e B apresentaram um índice de seletividade de 243.1, 217.1 e 89.3 respectivamente para Leishmania (L.) infantum, e o composto N12-acetilpseudoceratidina de 23.2 para Trypanosoma cruzi, sendo estes resultados muito promissores para a utilização das estruturas destas moléculas como potenciais modelos para o desenvolvimento de fármacos. / This research project aimed at the discovery of potentially bioactive secondary metabolites against parasitic activity and as antiviral agents and antibiotics, using dereplication as an essential methodology in the selection and prioritization of crude extracts derived from invertebrates, to the discovery of natural products structurally unpublished.<br /><br />Various extracts of marine invertebrates from an initial fractionation were prepared. The development and application of a dereplication methodology allowed the selection of three study organisms, Iotrochota birotulata, Amathia verticillata, and Tedania brasiliensis. The extracts of these organisms were subjected to different methods of fractionation, isolation and purification of substances, depending on the results of dereplication for each organism.<br /><br /> As a result of the isolation and identification of substances of these organisms, it obtained two compounds already known in the literature and unpublished sixteen compounds, two of which are synthetic derivatives. In all isolated compounds, pseudoceratidina, the desbromopseudoceratidinas and tedamidas A and B showed an index of selectivity 243.1, 217.1 and 89.3 respectively for Leishmania (L.) infantum, and the compound N12-acetilpseudoceratidina 23.2 for Trypanosoma cruzi, which are very promising results for the use of these molecules as structures potential models for drug development.

Menace potentielle pour l'hévéaculture mondiale, la maladie sud-américaine des feuilles de l'hévéa provoquée par le champignon Microcyclus ulei est aussi responsable du faible développement de cette culture en Amérique latine. La sélection de variétés résistantes est privilégiée pour anticiper une apparition accidentelle de la maladie dans les pays encore épargnés et pour développer la culture de l'hévéa dans les zones infestées. La principale source de résistance décrite jusqu'à présent n'est pas fonctionnelle face à des isolats très agressifs du champignon. Une autre source de résistance identifiée chez un cultivar originaire du Pérou se maintient depuis plus de trente ans en conditions de forte infestation. La cartographie génétique réalisée sur la descendance de ce cultivar en croisement révèle l'existence de deux gènes majeurs de résistance, l'un situé sur le groupe de liaison g15 et efficace contre les isolats de Guyane et l'autre sur le groupe de liaison g13 efficace vis-à-vis des isolats de l'état de Bahia. L'analyse de la diversité des populations naturelles du sud-ouest Amazonien fait apparaître une structure en trois groupes principaux recouvrant les états brésiliens de l'Acre, du Rondônia et du Mato Grosso. La population Madre de Dios au Pérou est rattachée au groupe de l'Acre. La différentiation entre les populations est expliquée principalement par l'isolement par la distance et secondairement par l'existence de bassins hydrographiques. Le déséquilibre de liaison entre marqueurs liés est plus étendu dans le groupe Acre que dans le groupe Rondônia, mais reste faible dans les deux cas. Une association avec le caractère de résistance à M. ulei est détectée avec un marqueur microsatellite situé à proximité du gène majeur de résistance du groupe de liaison g15. Une autre association est détectée dans une portion du génome où aucun locus de résistance n'était identifié jusqu'à présent. L'importance de ces résultats pour la sélection de nouveaux cultivars résistants à M. ulei est discutée.

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Orientador: Ana Marisa Fusco Almeida / Coorientador: Fernando Rogério Pavan / Banca: Katiany Rizzieri Caleffi Ferracioli / Banca: Luís Otávio Regasini / Banca: Maria José Soares Mendes Giannini / Banca: Marlus Chorilli / Resumo: As infecções pulmonares são um problema global e estão classificadas em terceiro lugar entre as principais causas de morte. Entre as infecções pulmonares crônicas estão a tuberculose (TB) e a paracoccidioidomicose (PCM). A TB é uma doença cujo principal agente etiológico é o Mycobacterium tuberculosis e a PCM tem como agente etiológico o complexo Paracoccidioides spp. As duas doenças podem ocorrer na forma de coinfecção, com incidência aproximada de 2,8 a 5,5% dos casos nos países da América do Sul e 10% no Brasil. Ambos os microrganismos já foram descritos na forma de biofilme monoespécie e o tratamento de ambas as doenças tem merecido atenção, devido a casos de resistência e as poucas opções terapêuticas. Diante do exposto, este trabalho visa mostrar, pela primeira vez, a capacidade de M. tuberculosis e P. brasiliensis se associarem por meio do crescimento na forma de um biofilme misto in vitro, a fim de elaborar um novo protótipo de 3'chalcona carreada numa nanoemulsão com atividade para as formas planctônica e de biofilme. Para isso, inicialmente foi padronizada e caracterizada a formação dos biofilmes monoespécie da linhagem ATCC M. tuberculosis (H37Rv), da linhagem clínica (Rv40) e da linhagem de P. brasiliensis (Pb18) e dos biofilmes mistos das associações de H37Rv+Pb18 e Rv40+ Pb18 (nesta ordem de crescimento), ambas as associações mostraram um aparente sinergismo entre elas. Na formação do biofilme misto inverso denominado assim pela ordem de associação, onde primeir... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Pulmonary infections are a global problem and rank third among the leading causes of death. Among the chronic lung infections are tuberculosis (TB) and paracoccidioidomycosis (PCM). The TB is a disease whose main etiologic agent is Mycobacterium tuberculosis and PCM has the etiological agent Paracoccidioides spp. The two diseases may occur in the form of coinfection, with an approximate incidence of 2.8 to 5.5% of the cases in the countries of South America and 10% in Brazil. Both microorganisms have already been described in the form of monospecies biofilm and the treatment of both diseases has deserved attention, due to cases of resistance and the few therapeutic options. In view of the above, this research aims to show, for the first time, the ability of M. tuberculosis and P. brasiliensis to associate by means of growth in the form of a mixed biofilm in vitro, in order to elaborate a new prototype in a nanoemulsion with activity for planktonic and biofilm forms. For this purpose, it was initially standardized and characterized the formation of monospecies biofilms of the ATCC strain M. tuberculosis (H37Rv), the clinical lineage (Rv40) and the P. brasiliensis (Pb18) lineage and the mixed biofilms of the associations of H37Rv + Pb18 and Rv40 + Pb18 (in this order of growth), both associations showed an apparent synergism between them. In order to establish the mixed biofilm (Pb18 + H37Rv), it was first proposed the association of P. brasiliensis (Pb18), followed by ATCC M. tuberculosis (H37Rv) with order of inverse growth, in this association a apparent competition between the microorganisms was observed. All biofilms, monospecies and mixed, were consolidated from 30 to 45 days... (Complete abstract click electronic access below) / Doutor

O Characifome dourado, Salminus brasiliensis, tem despertado crescente interesse dos piscicultores por apresentar rápido crescimento e ótima qualidade de carne. Porém, a falta de conhecimento sobre as exigências nutricionais e valor biológico dos alimentos para a espécie têm dificultado a formulação de dietas que permitam o máximo desempenho e, por conseqüência, sua produção intensiva. Para determinação da exigência nutricional em energia e proteína, juvenis de dourado (5,29 ± 0,21 g) foram distribuídos em 75 gaiolas (60 L) alojadas em tanques de 1.000 L e alimentados por 60 dias com dietas contendo níveis de proteína bruta (PB) entre 35 e 51% (incremento de 4%) e teores de energia bruta (EB) de 4.200 a 5.000 kcal/kg de alimento (incremento de 200 kcal/kg). O experimento foi conduzido em um delineamento inteiramente aleatorizado em esquema fatorial 5 × 5 (cinco níveis de EB e cinco níveis de PB, com três repetições). Foram avaliados parâmetros de ganho de peso (GP), ganho de peso relativo (GPR), consumo alimentar diário (CAD), índice de conversão alimentar (ICA), taxa de crescimento específico (TCE) e retenção de nutrientes. Não houve interação significativa (P>0,05) entre os níveis de proteína e energia para os parâmetros avaliados. Os valores de CAD, GP, GPR, CA e TCE e de retenção de nutrientes foram influenciados (P<0,05) pelos níveis de proteína e de energia da dieta. O CAD diminuiu com o aumento do nível energético das dietas. As relações lipo-somáticas e víscero-somáticas foram afetadas (P<0,05) pela energia dietética, aumentando com o aumento dos níveis de energia. Por meio do uso da regressão segmentada estimou-se a exigência protéica e energética para juvenis de dourado como sendo de 45,08% de PB e 4.600,0 kcal EB/kg, e uma relação energia:proteína variando entre 10,20 e 10,65 kcal de EB/g de PB, como ideal para ótimo desempenho e retenção de nutrientes e energia. Para avaliação do valor nutricional dos alimentos, foi determinado o coeficiente de digestibilidade aparente (CDA) da energia e nutrientes de fontes protéicas de origem animal e vegetal em dietas práticas para o dourado (19,49 ± 5,04 g). Os peixes, foram confinados em gaiolas de polipropileno (80 L; 40 peixes/gaiola) e alimentados com dietas teste granuladas que continham 30% do ingrediente teste e 70% de uma ração referência (RR) prática contendo 48,14% de PB, 4.453,2 kcal de EB/kg e 0,1% de óxido de crômio III. Após a última alimentação, as gaiolas eram transferidas para aquários cilindro-cônicos (200 L) onde as fezes eram coletadas por sedimentação em recipientes refrigerados. Houve diferença (P<0,01) para os CDAs dos nutrientes e energia das diferentes fontes protéicas. Foram obtidos valores de CDA de: 94,25; 91,26; 93,05 e 93,47% para a PB, 91,03; 90,33; 87,83 e 88,78% para a EB, 92,06; 84,49; 80,58 e 79,25% para matéria mineral, 83,90; 80,26; 84,25 e 84,55% para matéria seca e 97,38; 96,74; 93,32 e 91,46% para os lipídios, para a farinha de peixe (FP), farinha de vísceras (FV), farelo de soja (FS) e farelo de glúten de milho (FGM), respectivamente. O CDA médio dos aminoácidos foi de: 93,60; 89,95; 92,07 e 92,45% para FP, FV, FS e FGM, respectivamente. Todos os ingredientes testados foram eficientemente aproveitados e podem substituir parcialmente a FP em rações para o dourado. / The dourado, Salminus brasiliensis, presents excellent farming characteristics, such as fast growth and excellent flesh quality. However, the lack of the knowledge on the species nutritional requirements and biological value of feedstuffs hamper the formulation of diets for optimized performance and, consequently, the species\' intensive farming. To determine de species nutritional requirements, juveniles dourado (5.29 ± 0.21 g) were stocked in 75 cages (60-L) set up in 1,000-L containers, and fed for 60 days diets containing levels of crude protein (CP) between 35 to 51% (4% increments) and gross energy (GE) varying of 4,200 to 5,000 kcal/kg feed (200 kcal increments). The trial was set up in a completely randomized design, 5 × 5 factorial scheme (five levels of GE and five levels of CP, with three replications). Data on weight gain (WG), percentage weight gain (PWG), daily feed consumption (DFC), food conversion ratio (FCR), specific growth rate (SGR) and nutrient retention were recorded. No interaction (P>0.05) between dietary energy and protein levels for all parameters was detected. The values of DFC, WG, RWG, FCR, SGR and nutrient retention were affected (P<0.05) by the dietary protein and energy levels. DFC decreased with increasing energy levels. The liposomatic and viscerosomatic indexes were affected (P<0.05) by dietary energy levels, and increased with increasing energy levels. Through broken line analysis technique, the requirements estimated for optimum performance and nutrient and energy retention of dourado were 45.08% CP, 4,600.0 kcal GE/kg, and a energy:protein ratio varying between 10.20 and 10.65 kcal of GE/g of CP. Apparent digestibility is one of the main tools to evaluate the potential of an ingredient to be used in aquafeeds. To evaluate the apparent digestibility coefficient (ADC) of energy and nutrients of animal and plant protein sources in practical diets for the dourado (19.49 ± 5.04 g), fish were stocked in plastic cages (80-L) and fed pelleted test diets containing 30% of test ingredients plus 70% of a reference diet (RR) containing 48.14% CP, 4,453.2 kcal GE/kg, and 0.1% of chromium oxide III. After the last daily meal, cages were transferred to cylindrical conical-bottomed aquaria (200-L) where feces were collected by sedimentation in refrigerated containers. ADCs of nutrients and energy of selected protein sources differed (P<0.01); ADC values were: 94.25; 91.26; 93.05 and 93.47% for CP, 91.03; 90.33; 87.83 and 88.78% for GE, 92.06; 84.49; 80.58 and 79.25% for ash, 83.90; 80.26; 84.25 and 84.55% for dry matter, 97.38; 96.74; 93.32 and 91.46% for lipid for fish meal (FM), poultry by-product meal (PBM), soybean meal (SBM), and corn gluten meal (CGM), respectively. The average ADC of amino acids was: 93.60; 89.95; 92.07 and 92.45% of FM, PBM, SBM and CGM, respectively. All tested ingredients were efficiently used and can partially replace FM in diets for carnivorous dourado.