Os agricultores tradicionais da região sul do Estado de São Paulo têm se mostrado mantenedores de um grande repositório de diversidade genética e de conhecimento a respeito das peculiaridades de manejo desta diversidade. A cultura do inhame (Dioscorea spp.) é mantida e manejada nesta região em roças que empregam o sistema de coivara. Esta pesquisa teve como objetivos coletar etnovariedades de inhame nas roças/quintais dos agricultores tradicionais do Vale do Ribeira, visando manter um banco de conservação ex situ na Escola Superior de Agricultura “Luiz de Queiroz” em Piracicaba – SP e estimar a diversidade morfológica e isoenzimática de inhames mantidos em sistemas agrícolas autóctones. Visitou-se 91 agricultores que ainda praticam a agricultura tradicional no Vale do Ribeira dos quais 45 cultivavam o inhame. Desses, 31% o cultivavam em roças itinerantes e 69% em quintais. Quatro espécies de inhame foram encontradas: D. trifida, D. bulbifera, D. alata e D. cayenensis. As etnovariedades foram caracterizadas por meio de marcadores isoenzimáticos, utilizando géis de poliacrilamida (seis sistemas) e amido (um sistema), e por marcadores morfológicos, num total de 24 caracteres. Para ambos os marcadores, foram calculados o índice de similaridade de Jaccard entre pares de indivíduos. Análises de agrupamento foram realizadas para variedades, roças e comunidades, a partir dos índices de similaridade de Jaccard e do critério aglomerativo UPGMA. Outro parâmetro analisado foi a correlação entre as matrizes de distância genética (isoenzimática e morfológica) e geográfica pela correlação de Pearson (r) e teste de Mantel, para verificar se a diversidade genética encontrava-se estruturada no espaço. Além disso, realizou-se a análise de variância molecular (AMOVA) para se verificar a distribuição da variabilidade nos diferentes níveis hierárquicos: entre e dentro das unidades evolutivas biológicas (roças) e culturais (comunidades). Os resultados revelaram que os agricultores tradicionais do Vale do Ribeira manejam grande diversidade genética, tanto isoenzimática como morfológica, em suas roças e que essa diversidade não está estruturada no espaço para todas as espécies cultivadas. A AMOVA indicou que para as espécies D. trifida, D. bulbifera e D. cayenensis a distribuição da variabilidade concentra-se entre roças dentro de comunidades, enquanto que para D. alata concentra-se dentro de roças para os marcadores isoenzimáticos. Para os marcadores morfológicos, a maior parte da variabilidade concentra-se entre roças dentro de comunidades para D. bulbifera e D. cayenensis e dentro de roças para D. alata e D. trifida, ressaltando-se a seleção humana para os aspectos visuais. Conclui-se que os agricultores tradicionais do Vale do Ribeira cultivam grande diversidade de etnovariedades e espécies de Dioscorea, além de possuírem grande conhecimento a respeito das peculiaridades de manejo desta cultura. Contudo, as pressões que esses agricultores estão sofrendo poderá no futuro provocar perda de diversidade genética. Recomenda-se novos estudos na região para verificar os danos causados por essas pressões aos processos evolutivos presentes nas roças dos agricultores tradicionais do Vale do Ribeira. / Traditional agriculturists of the south region of the State of São Paulo have been maintainers of a great repository of genetic diversity and knowledge regarding the management peculiarities of this diversity. The yam (Dioscorea spp.) culture is maintained and cultivated in this region in swiddens that use the system of “coivara”. This research had the objective of collecting ethnovarieties of yam in the swiddens/home gardens of the traditional agriculturists of the Ribeira River Valley, aiming at maintaining a germplasm bank for ex situ conservation at the Escola Superior de Agricultura "Luiz de Queiroz" in Piracicaba, SP and estimating the morphological and isoenzymatic diversity of yams kept in authoctone agricultural systems. Ninety-one agriculturists that still practice traditional agriculture were visited in the Ribeira River Valley, of which 45 cultivated yam. Of these, 31% cultivated this crop on itinerant swiddens and 69% in home gardens. Four yam species were found: D. trifida, D. bulbifera, D. cayenensis and D. alata. The ethnovarieties of each species were characterized with isoenzymatic markers, using polyacrilamide gels (six systems) and starch gels (one system), and with morphological markers, in a total of 24 characters. For both markers, the Jaccard similarity indices between pairs of individuals were obtained. Cluster analyses were conducted for varieties, swiddens and communities using the Jaccard similarity indices and the UPGMA method. Another parameter analyzed was the correlation between the genetic distances (isoenzymatic and morphological) and the geographic distance matrices using the Pearson correlation and Mantel test, to verify if the genetic diversity was structured in space. An analysis of molecular variance (AMOVA) was carried out to verify the distribution of the variability in the different hierarchic levels: within and between the biological evolutionary units (swiddens) and cultural units (communities). Results showed that the traditional agriculturists of the Ribeira River Valley maintain a great genetic diversity, both isoenzymatic and morphological, in their swiddens and home gardens, and that this diversity is not structured in space for the four species cultivated. The AMOVA results for D. trifida, D. bulbifera and D. cayenensis showed that the isoenzymatic variability is mainly concentrated among swiddens within communities, and within swiddens for D. alata. For the morphological markers, where visual aspects are an important issue in human selection, most of the variability is concentrated among swiddens within communities for D. bulbifera and D. cayenensis, and within swiddens for D. alata and D. trifida. It is concluded that the traditional agriculturists of the Ribeira River Valley cultivate great diversity of ethnovarieties and species of Dioscorea, also possessing great knowledge concerning the management peculiarities of this crop. However, the pressures that these agriculturists are suffering may cause a loss of genetic diversity in the future. Further studies are recommended in this region to verity the damage caused by these pressures in the evolutionary processes that occur in the swiddens of the Ribeira River Valley traditional agriculturists.

Over the last decade, the innate immune system has been the subject of extensive research. Often overlooked by the robustness and specificity of the adaptive immune system, the innate immune system is proving to be just as complex. The identification of several families of pattern recognition receptors (PRRs) has revealed an ancient yet multifaceted system of proteins that are responsible for initiating host defense. A wide array of pathogens, from virus to bacteria, is detected using this assortment of receptors. One such family, the Toll-like receptors (TLRs), has been at the forefront of this research. To date, 10 TLRs have been described in the human genome. Activation of TLRs leads to the induction of immune-related genes that ultimately control the response of the host. However, the signaling pathways emanating from activated TLRs and other PRRs are not fully understood. In particular, the pathway leading to the activation of interferon regulatory factor 3 (IRF3), a transcription factor crucial for the induction of type I interferon, remains undefined. IRF3 activation occurs as the consequence of viral infection and through the activation of TLRs 3 and 4 by dsRNA and lipopolysaccharide (LPS), respectively. The focus of this research is to describe components of the IRF3 activation pathway, partly through the analysis of TLR signal transduction. IRF3 normally resides in the cytoplasm of cells. Upon infection with certain viruses and bacteria, IRF3 is activated though phosphorylation at its C-terminus. Phosphorylated IRF3 homodimerizes and associates with co-activators CBP-p300. After translocating to the nucleus, the activate IRF3 complex induces the activation of type 1 interferon and interferon related genes. Little is known about the pathways that lead to the activation of IRF3, especially the kinases involved. In this study we report that the non-canonical IкB kinase homologues, IкB kinase epsilon (IKKε) and TANK-binding kinase-1 (TBK1), which were previously implicated in NF-кB activation, are also essential components of the IRF3 signaling pathway. In particular, mouse embryonic fibroblasts from TBK1 deficient mice fail to activate IRF3 in response to both viral infection and stimulation with LPS or poly (IC), a dsRNA analog. Thus, both IKKε and TBK1 play a critical role in innate immunity and host defense. In addition to viral infection, IRF3 activation also occurs via the activation of TLR3 and 4. TLRs signal through a subfamily of Toll-IL-1-Resistance (TIR) domain containing adapter molecules. One such adapter, MyD88, is crucial for all TLRs, with the exception of TLR3. MyD88 participates in a signal transduction pathway culminating in the activation of the transcription factor NF-кB. Studies from MyD88-deficient mice reveal that both TLR3 and 4 still are capable of activating NF-кB, although with slightly delayed kinetics. Another aspect of the MyD88-independent signal transduction pathway is the activation of IRF3. A second TIR domain containing adapter molecule called Mal/Tirap was discovered and originally thought to mediate the MyD88-independent pathway. However, Mal-deficient mice were found to be defective in both TLR2 and 4 mediated NF-кB activation. We hypothesized that other TIR domain containing adapters could mediate this MyD88-independent pathway of TLR3 and 4 leading to the activation of IRF3. Two additional TIR adapters were discovered, TRIF and TRAM. TRIF was shown to mediate TLR3 signal transduction. In this study, we report that both TRIF and TRAM mediate the activation of the MyD88-independent pathway in response to LPS/TLR4 activation. Unlike any of the other known TIR domain containing adapters, TRAM appears to be restricted to the LPS/TLR4 activation pathway while TRIF plays a role in both TLR3 and TLR4 pathways leading to IRF3 target gene expression. Our studies revealed that TRAM could be acting upstream of TRIF in the LPS/TLR4 pathway. To this end, we sought to determine the localization of TRAM within the cell. We found that TRAM localizes to the plasma membrane. TRAM localization is the result of myristoylation since mutation of the predicted myristoylation site (G2A) resulted in the re-distribution of TRAM from the membrane into the cytoplasm. Reconstitution of TRAM-deficient macrophages with TRAM G2A is unable to rescue LPS/TLR4 signal transduction. Thus, myristoylation and membrane association of TRAM are critical for LPS/TLR4 signal transduction. The data generated in this dissertation extends our understanding of the signaling pathways of the innate immune system. Indeed, the molecules and pathways described herein could prove to be beneficial targets for ameliorating symptoms of disease, both autoimmune and pathogen-associated. Finally, the research described here will spur further insight into the complex signaling pathways of a once ignored arm of the immune system.

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