A citricultura brasileira é a maior produtora e exportadora de citros e tem sido afetada por doenças que causam sérios prejuízos a produção e a qualidade dos frutos. No entanto, a cultura apresenta grandes problemas, entre eles, os fatores fitossanitários, que vem dizimando milhares de plantas e afetando a produtividade e a competitividade do setor. Atualmente, o huanglongbing (HLB), associado às bactérias de floema Candidatus Liberibacter spp., é considerado uma das mais destrutivas doenças de citros. A inexistência de cultivares de laranja doce resistentes ao HLB torna a transformação genética de citros uma ferramenta importante no controle desta doença. Para se defender do ataque de pragas e patógenos as plantas desenvolveram, durante o processo evolutivo, uma série de mecanismos de defesa, no qual pode-se incluir a produção de peptídeos com atividade antimicrobiana. As defensinas vegetais são peptídeos pequenos relacionadas à patogênese (PR), que possuem atividade antimicrobiana associada aos mecanismos de defesa das plantas. Assim, o objetivo deste trabalho foi a obtenção de plantas transgênicas de laranja doce (Citrus sinensis L.) cvs. \'Hamlin\', \'Natal\', \'Valência\' e \'Pera\', via Agrobacterium tumefaciens, superexpressando o gene codificador de defensina (def), isolado de Citrus sinensis cv. \'Valência\', dirigido pelo promotor com expressão preferencial no floema AtSUC2 (transportador de sacarose, clonado de Arabidopsis thaliana) ou pelo promotor constitutivo CaMV 35S (clonado do vírus do mosaico da couve-flor). Os explantes utilizados na transformação genética foram segmentos de epicótilo obtidos de plantas germinadas in vitro. A identificação das plantas transgênicas foi realizada por meio da análise da PCR, utilizando-se primers para a detecção do fragmento do gene de seleção nptII. As plantas PCR+ foram aclimatizadas e transferidas para casa-de-vegetação. A análise de Southern blot confirmou a integração do transgene em 36 plantas. Foram obtidas 7 plantas transgênicas da cultivar \'Hamlin\', 9 da cultivar \'Natal\', 1 da cultivar \'Pera\' e 9 da cultivar \'Valência\' contendo a construção gênica pC35S/def, e 3 plantas transgênicas da cultivar \'Hamlin\', 6 da cultivar \'Natal\' e 1 da cultivar \'Valência\' contendo a construção gênica pcAtSUC2/def. Os resultados obtidos neste trabalho serão importantes para futura avaliação e estudo visando o controle de Candidatus Liberibacter spp.. / The Brazilian citrus industry is the world\'s largest producer and exporter of citrus, however, it has been affected by diseases that cause serious production losses and damages to fruit quality. However, the culture faces problems, namely phytosanitary issues that have been damaging thousands of plants, affecting yield and competitiveness of the sector. Currently, Huanglongbing (HLB), associated with phloem bacteria Candidatus Liberibacter spp., is considered one of the most destructive citrus diseases. The lack of sweet orange cultivars resistant to HLB makes genetic transformation an important tool in the disease control. To defend from pest and pathogen attack, plants developed a series of defense mechanisms during the evolutionary process, which may include the production of peptides with antimicrobial activity. Plant defensins are small peptides related to pathogenesis (PR) which have antimicrobial activities, associated with plant defense mechanisms . The objective of this study was to obtain transgenic plants of sweet orange (Citrus sinensis L.) cultivars \'Hamlin\', \'Natal\', \'Valência\' and \'Pera\' with Agrobacterium tumefaciens overexpressing the defensin gene (def), isolated from Citrus sinensis cv. \'Valência\', controlled by the promoter with preferential expression in the phloem AtSUC2, (sucrose transporter, cloned from Arabidopsis thaliana) or by the constitutive promoter CaMV 35S (cloned from the mosaic virus of cauliflower). The explants used in genetic transformation were epicotyl segments obtained from germinated plants in vitro. The identification of transgenic plants was accomplished by PCR analysis using primers for the detection of nptII gene fragment. The PCR+ plants were acclimatized and transferred to greenhouse. The analysis of Southern blot confirmed the transgene integration in 36 plants. Seven transgenic plants were obtained for the cultivar \'Hamlin\', nine for \'Natal\', one for \'Pera\' and nine for \'Valência\' containing the gene construct pC35S/def and three transgenic plants for \'Hamlin\', six for \'Natal\' and one for \'Valência\' containing the gene construct pcAtSUC2/def. The results obtained in this work are important for future evaluation of the plants for resistance to Candidatus Liberibacter spp..

Verticillium is a soil-borne fungus that is one of the world's foremost plant pathogens. Commercial plant grafting suggests that resistance occurs in the root; this conflicts with decades of research indicating that resistance occurs in the stem. The goal of this thesis work was to use an alternative approach to determine the location of resistance by expressing the Ve1 gene using organ-specific promoters. Promoter sequences for the stem-specific gene, Ribulose 1,5-bisphosphate carboxylase oxygenase small chain 2A (Rbsc2A), and root-specific gene, Tobacco Mosaic Virus Induced (TMVi) were taken from the Sol Genomics Network (SGN) database, cloned into constructs with the Ve1 gene and susceptible tomato germplasm was transformed using Agrobacterium tumefaciens. Preliminary results suggest that resistance may not be localized and expression of the Ve1 gene in either the root or the stem is sufficient to develop whole plant resistance to the Verticillium pathogen.

A citricultura brasileira é a maior produtora e exportadora de citros e tem sido afetada por doenças que causam sérios prejuízos a produção e a qualidade dos frutos. No entanto, a cultura apresenta grandes problemas, entre eles, os fatores fitossanitários, que vem dizimando milhares de plantas e afetando a produtividade e a competitividade do setor. Atualmente, o huanglongbing (HLB), associado às bactérias de floema Candidatus Liberibacter spp., é considerado uma das mais destrutivas doenças de citros. A inexistência de cultivares de laranja doce resistentes ao HLB torna a transformação genética de citros uma ferramenta importante no controle desta doença. Para se defender do ataque de pragas e patógenos as plantas desenvolveram, durante o processo evolutivo, uma série de mecanismos de defesa, no qual pode-se incluir a produção de peptídeos com atividade antimicrobiana. As defensinas vegetais são peptídeos pequenos relacionadas à patogênese (PR), que possuem atividade antimicrobiana associada aos mecanismos de defesa das plantas. Assim, o objetivo deste trabalho foi a obtenção de plantas transgênicas de laranja doce (Citrus sinensis L.) cvs. \'Hamlin\', \'Natal\', \'Valência\' e \'Pera\', via Agrobacterium tumefaciens, superexpressando o gene codificador de defensina (def), isolado de Citrus sinensis cv. \'Valência\', dirigido pelo promotor com expressão preferencial no floema AtSUC2 (transportador de sacarose, clonado de Arabidopsis thaliana) ou pelo promotor constitutivo CaMV 35S (clonado do vírus do mosaico da couve-flor). Os explantes utilizados na transformação genética foram segmentos de epicótilo obtidos de plantas germinadas in vitro. A identificação das plantas transgênicas foi realizada por meio da análise da PCR, utilizando-se primers para a detecção do fragmento do gene de seleção nptII. As plantas PCR+ foram aclimatizadas e transferidas para casa-de-vegetação. A análise de Southern blot confirmou a integração do transgene em 36 plantas. Foram obtidas 7 plantas transgênicas da cultivar \'Hamlin\', 9 da cultivar \'Natal\', 1 da cultivar \'Pera\' e 9 da cultivar \'Valência\' contendo a construção gênica pC35S/def, e 3 plantas transgênicas da cultivar \'Hamlin\', 6 da cultivar \'Natal\' e 1 da cultivar \'Valência\' contendo a construção gênica pcAtSUC2/def. Os resultados obtidos neste trabalho serão importantes para futura avaliação e estudo visando o controle de Candidatus Liberibacter spp.. / The Brazilian citrus industry is the world\'s largest producer and exporter of citrus, however, it has been affected by diseases that cause serious production losses and damages to fruit quality. However, the culture faces problems, namely phytosanitary issues that have been damaging thousands of plants, affecting yield and competitiveness of the sector. Currently, Huanglongbing (HLB), associated with phloem bacteria Candidatus Liberibacter spp., is considered one of the most destructive citrus diseases. The lack of sweet orange cultivars resistant to HLB makes genetic transformation an important tool in the disease control. To defend from pest and pathogen attack, plants developed a series of defense mechanisms during the evolutionary process, which may include the production of peptides with antimicrobial activity. Plant defensins are small peptides related to pathogenesis (PR) which have antimicrobial activities, associated with plant defense mechanisms . The objective of this study was to obtain transgenic plants of sweet orange (Citrus sinensis L.) cultivars \'Hamlin\', \'Natal\', \'Valência\' and \'Pera\' with Agrobacterium tumefaciens overexpressing the defensin gene (def), isolated from Citrus sinensis cv. \'Valência\', controlled by the promoter with preferential expression in the phloem AtSUC2, (sucrose transporter, cloned from Arabidopsis thaliana) or by the constitutive promoter CaMV 35S (cloned from the mosaic virus of cauliflower). The explants used in genetic transformation were epicotyl segments obtained from germinated plants in vitro. The identification of transgenic plants was accomplished by PCR analysis using primers for the detection of nptII gene fragment. The PCR+ plants were acclimatized and transferred to greenhouse. The analysis of Southern blot confirmed the transgene integration in 36 plants. Seven transgenic plants were obtained for the cultivar \'Hamlin\', nine for \'Natal\', one for \'Pera\' and nine for \'Valência\' containing the gene construct pC35S/def and three transgenic plants for \'Hamlin\', six for \'Natal\' and one for \'Valência\' containing the gene construct pcAtSUC2/def. The results obtained in this work are important for future evaluation of the plants for resistance to Candidatus Liberibacter spp..

L’immunothérapie a permis des avancées majeures dans la thérapie du cancer. Le traitement par des cellules T modifiées pour exprimer un récepteur chimérique d’antigène (CAR) a changé complètement la vision de la thérapie de la leucémie. L’efficacité de ce traitement sur des cancers résistants, a ouvert la voie à la thérapie cellulaire et génique dans ce contexte. Malgré les premiers résultats très positifs, il s’avère que l’épuisement cellulaire et la perte des cellules T thérapeutiques est un problème majeur pour maintenir l’efficacité de la thérapie CAR et prévenir les rechutes. Les travaux présentés dans cette thèse visent à permettre l’utilisation d’autres types cellulaires pour la thérapie CAR. L’hypothèse de travail est que les cellules NK ou les cellules souches hématopoïétiques (HSC) permettrait de dépasser les limites de la thérapie CAR utilisant les cellules T. Pour permettre l’utilisation des cellules NK, un des problèmes technique est la transduction par les vecteurs viraux. Les travaux présentés ici démontrent que l’utilisation de l’enveloppe BaEV permet une transduction efficace des NK avec un vecteur lentiviral. Par cette méthode nous avons pu générer de grandes quantités de cellules NK transduites avec un CAR, prouvant la possibilité d’utiliser les NK dans la thérapie CAR. L’utilisation des HSC dans la thérapie CAR, permettrait de produire des cellules CAR T en permanence pour renouveler les cellules T épuisées. Cependant, la surexpression d’un récepteur CAR sur toutes les cellules dérivant des HSC pourrait être un problème. Pour permettre l’utilisation des HSC, nous avons développé des promoteurs spécifiques courts restreignant l’expression du transgène à une population précise. Nous avons prouvé la spécificité d’un promoteur T et démontré la possibilité de l’utiliser dans le contexte de la thérapie CAR utilisant les HSC. Ces travaux sont une preuve de concept de l’utilisation d’autres cellules que les cellules T dans la thérapie CAR. / Immunotherapy has allowed major advances in cancer therapy. The treatment using modified T cells with a chimeric antigen receptor (CAR) completely changed the vision of leukemia therapy. The efficiency against resistant cancer paved the way to cellular and gene therapy in this context. Despite very positive results at first, the disappearance and exhaustion of therapeutic cells seems to be a major problem to maintain the efficiency of the CAR treatment and prevent relapses. The work of this thesis is to allow the use cell types other than T cells for CAR therapy. The hypothesis is that NK cells or hematopoietic stem cells (HSC) could overcome the limitation of CAR therapy using T cells. To allow the use of CAR NK cells, a major technical issue is the transduction by viral vectors. The work presented here shows the use of BaEV envelope to pseudotype vectors allows an efficient transduction of NK cells. Using this method, we were able to produce large amounts of CAR NK cells, showing the possibility to use NK cells in CAR therapy. The use of HSC in CAR therapy, could allow the permanent replenishment of the pool of CAR T cells once exhausted. Despite that advantage, the overexpression of a CAR receptor on all hematopoietic cells coming from those HSC could be an issue. To allow the use of HSC, we developed short specific promoters restraining the expression of the transgene to a precise population. We prove the specificity of a T cell promoter and demonstrated the possibility to use it in CAR therapy using HSC. This work is a proof of concept of the use of other population than T cells in CAR therapy.