Catharanthus roseus est une plante médicinale produisant divers types d’alcaloïdes indoliques monoterpéniques (AIM) d’intérêt en santé humaine. Ainsi, les AIM dimères comme la vinblastine et la vincristine sont utilisés en chimiothérapie anticancéreuse et les alcaloïdes monomères de type hétéroyohimbine présentent diverses activités pharmacologiques. La fabrication de ces molécules dans la plante est fort complexe. Elle requiert un haut niveau de compartimentation tissulaire et subcellulaire et met en jeu plus d’une trentaine d’étapes enzymatiques, dont certaines sont encore très mal connues. Dans ce contexte, l’objectif de la thèse a consisté à élucider plusieurs étapes enzymatiques de la voie de biosynthèse des AIM. Nos travaux ont permis de caractériser de nouvelles isoformes enzymatiques de la famille des cytochromes P450 ainsi que les réductases qui leur sont associées. Ils ont abouti à l’identification de nouvelles déshydrogénases et mis en évidence, in planta, leurs interactions avec la strictosidine synthase suggérant une biosynthèse orientée vers les divers alcaloïdes de type hétéroyohimbine. Enfin, en ayant recours à l’ingénierie métabolique, un segment de la voie de biosynthèse a été transféré dans la levure Saccharomyces cerevisiae, lui conférant la capacité de bio-transformer la tabersonine en vindoline, l’un des deux précurseurs finaux des alcaloïdes dimères. / Catharanthus roseus is a medicinal plant producing various types of monoterpene indole alkaloids (MIA) with a great interest in human health. Dimeric alkaloids such as vinblastine and vincristine are used in cancer chemotherapy and monomeric heteroyohimbine alkaloids exhibit various pharmacological activities. The production of these molecules in the plant is very complex. It requires a high level of tissular and subcellular compartmentalization and involves more than thirty enzymatic steps, some of which are largely unknown. In this context, the aim of this thesis was to elucidate several enzymatic steps of the MIA biosynthetic pathway. Our work allowed us to characterize new enzyme isoforms of cytochrome P450 and their associated reductases. They also resulted in the identification of new dehydrogenases and highlighted their interactions with the strictosidine synthase suggesting a directed biosynthesis towards various heteroyohimbine type of alkaloids. Finally, engineered yeast containing a segment of the MIA biosynthetic pathway was able to convert tabersonine into vindoline, one of the two final precursors of the dimeric alkaloids.

O estresse de calor prejudica o crescimento e reprodução dos organismos vegetais, ao alterar a permeabilidade de membranas biológicas e desnaturar proteínas, limitando o metabolismo primário. Dentre as respostas ao estresse abiótico, está a síntese de metabólitos secundários. Braquicerina é um alcaloide monoterpeno indólico com ação antioxidante, protetora contra UV e antimutagênica sintetizado por partes aéreas de Psychotria brachyceras. O objetivo deste trabalho é investigar o metabolismo de braquicerina sob estresse de calor. Assim, espera-se contribuir para o conhecimento acerca do metabolismo secundário nas respostas ao estresse de calor, descrever a função in planta do composto, e fornecer ferramentas para obtenção do alcaloide para fins farmacêuticos. O acúmulo de braquicerina foi induzido em discos foliares mantidos a 40ºC por três dias, tanto em regime de elevação abrupta como gradual da temperatura. Baixa temperatura (10ºC) não afetou o acúmulo do alcaloide. Discos foliares de Psychotria carthagenensis, uma espécie que não sintetiza alcaloides monoterpeno indólicos, foram também desafiados por estresse de calor. Clorofila total, teor de peróxido de hidrogênio e peroxidação lipídica foram quantificados em ambas as espécies. P. carthagenensis foi relativamente tolerante ao calor, o que pode estar relacionado à sua elevada concentração de antocianinas, fortemente induzidas por choque térmico de 50ºC por 6h. Peroxidação lipídica foi reduzida nas amostras de P. brachyceras sob estresse de calor agudo ou gradual em comparação à condição controle, sendo este parâmetro inalterado nas duas condições em P. carthagenensis. O teor de peróxido de hidrogênio foi menor em P. brachyceras submetida a choque de térmico em relação ao controle, enquanto o mesmo parâmetro não foi alterado em P. carthagenensis. Discos foliares das espécies sensíveis ao calor Brugmansia suaveolens e Brassica oleracea, pré-tratadas com braquicerina em concentrações similares às encontradas em P. brachyceras, adquiriram fenótipo tolerante ao choque térmico. A expressão do gene que codifica a enzima triptofano descarboxilase (TDC), envolvida na biossíntese de braquicerina em P. brachyceras, foi fortemente inibida em discos foliares submetidos à 40ºC por 6h, 12h e 24h, sugerindo que o efeito da temperatura na estimulação de acúmulo de alcaloide ocorra em nível pós-transcricional. Em conjunto, os dados indicam que a exposição ao calor é um meio efetivo de aumentar o rendimento de braquicerina, cuja acumulação contribui para proteção contra os danos oxidativos associados. / Heat stress impairs plant growth and reproduction by altering membrane permeability and promoting protein denaturation, which limits primary metabolism. Secondary metabolites often take part in protection against abiotic stress responses. Brachycerine is a monoterpene indole alkaloid with antioxidant, UV protectant, and antimutagenic activity synthesized by Psychotria brachyceras shoots. Brachycerine metabolism was analyzed under heat stress, in order to shed light on brachycerine‘s in planta function and to provide potential tools to improve alkaloid yields for pharmaceutical analysis. Accumulation was induced in leaf disks kept at 40ºC for three days, both by abrupt and stepwise temperature increase. Brachycerine concentration was not affected by low temperature (10ºC) exposure. Leaf disks of Psychotria carthagenensis, a species devoided of alkaloids, were also challenged by heat. Total chlorophyll, hydrogen peroxide and lipid peroxidation concentrations were determined in both species. P. carthagenensis was relatively tolerant to heat treatments which may be explained by its high anthocyanin concentration, strongly induced by heat shock of 50ºC for 6h. Brugmansia suaveolens and Brassica oleracea, pre-treated with brachycerine in concentrations equivalent to those found in P. brachyceras, had a heat shock tolerant phenotype, based on chlorophyll content. Expression of the TRYPTOPHAN DECARBOXYLASE gene, which encodes for an enzyme involved in alkaloid biosynthesis (TDC) was strongly repressed in leaf disks exposed to 40ºC for 6h, 12h e 24h, suggesting that temperature effect may occur at post-transcriptional level. Taken together, data indicate that heat exposure is an effective means to increase yields of brachycerine, whose accumulation contributes to protect against associated oxidative damage.

Submitted by Marcondes Lopes Leite (marcondesleite@ifma.edu.br) on 2018-07-17T03:02:40Z No. of bitstreams: 1 DISSERTAÇÃO_Marcondes (1).pdf: 855505 bytes, checksum: 87f199a0b3c25e7ac6c9ca3496aaff6b (MD5) / Approved for entry into archive by Karina Gimenes Fernandes null (karinagi@fcav.unesp.br) on 2018-07-17T11:51:19Z (GMT) No. of bitstreams: 1 leite_ml_me.pdf: 825599 bytes, checksum: 98eaa2d4a84e378b555cde009287328e (MD5) / Made available in DSpace on 2018-07-17T11:51:19Z (GMT). No. of bitstreams: 1 leite_ml_me.pdf: 825599 bytes, checksum: 98eaa2d4a84e378b555cde009287328e (MD5) Previous issue date: 2018-05-30 / Outra / PROPAGAÇÃO ASSEXUADA DE FRUTEIRAS DA FAMILIA CLUSIACEA: BACURIZEIRO (Platonia insignis Mart.) E BACUPARIZEIRO (Garcinia gardneriana (Planch & Triana) Zappi). RESUMO As fruteiras bacuri (Platonia insignis Mart.) e bacupari (Garcinia gardneriana (Planch & Triana) Zappi).pertencem à família Clusiaceae e predominam no Norte do Brasil. Estas espécies não são domesticadas e a obtenção de frutos é feita principalmente por meios extrativos sendo raros pomares comerciais. Para à propagação por estaquia do bacurizeiro, ramos jovens foram retirados de uma planta matriz e as estacas foram obtidas como: i. estacas lenhosas, ii. estacas herbáceas com duas folhas e duas gemas na base e iii. estacas herbáceas com duas folhas, sem gemas na base. As estacas foram tratadas com ácido indol butírico (AIB) nas seguintes concentrações: 0.0 (controle), 1.000, 2.000, 3.000 e 4.000 mg.L-1. As estacas lenhosas apresentaram a maior taxa de sobrevivência (95,83%) em relação as estacas herbáceas (70 e 67,5%), sendo que em apenas uma dessas foi observado o desenvolvimento radicular (0,28%). Em relação ao bacuparizeiro, como o estiolamento tem sido utilizado para melhorar o enraizamento em espécies de difícil propagação e como não foram encontrados relatos sobre o uso de estacas estioladas para propagar esta espécie, o objetivo deste estudo foi avaliar a viabilidade dessa técnica de propagação e estudar o efeito de diferentes doses de AIB. As estacas estioladas continham um par de folhas (~12 cm de comprimento) e foram tratadas com AIB por 10 segundos (0 – controle, 500, 1.000 e 2.000 mg.L-1). O estiolamento resultou em 2,5% de estacas enraizadas quando tratadas com 500 e 1.000 mg.L-1 de AIB. O tratamento com IBA afetou a sobrevivência das estacas na fase de propagação, possivelmente ao reduzir a retenção foliar quando estas foram tratadas com concentrações de IBA superiores a 1.000 mg.L-1. Os experimentos foram realizados no outono-inverno, estes devem ser repetidos na primavera-verão, pois espécies neotropicais requerem clima quente e úmido para seu desenvolvimento. / Bacuri (Platonia insignis Mart.) and bacupari (Garcinia gardneriana (Planch & Triana) Zappi.) fruit trees belong to the Clusiaceae family and they predominate in the North of Brazil in the Amazonia basin. These species are not domesticated and fruit are obtained mainly by extractive means due to the inexistence of commercial orchards. To propagate bacuri by cutting, young branches were taken from an matrix bacuri tree and cuttings were obtained as: i. hardwood cuttings, ii. herbaceous stem cuttings with two leaves and two buds on the base, and iii. herbaceous stem cuttings with two leaves without buds on the base. Cuttings were treated with indolbutyric acid (IBA) in the following concentrations: 0,0 mg.L-1 (control), 1,000 mg.L-1 , 2,000 mg.L-1 , 3,000 mg.L-1 , and 4,000 mg.L-1 . The hardwood cuttings presented the biggest survival rate (95.83%) in relation to the herbaceous stem cuttings (70%, 67.50%), and in only one of this cutting was observed root development (0.28%). Regarding bacupari, as etiolation has been used to improve rooting in species which are difficult to propagate and as there is no reports regarding the use of etiolate cuttings to propagate this species, the objective of this study was to evaluate the feasibility of this technique to propagate bacupari and to study the effect of the different doses of IBA. Etiolate cuttings contained on pair of leaves (~12 cm long), and were treated with IBA for 10 seconds (0,0-control, 500, 1,000, and 2,000 mg.L-1 ). Etiolation resulted in 2.5% rooted cuttings when treated with 500 and 1,000 mg.L-1 IBA. The IBA treatment affected the cuttings surviving the propagate phase possibly by reducing the foliar retention when treated with IBA concentrations superior than 1,000 mg.L-1 . The experiments were set during the autumn-winter season, they should be repeated in the spring-summer season as these species require hot and humid climate to develop.

O estresse de calor prejudica o crescimento e reprodução dos organismos vegetais, ao alterar a permeabilidade de membranas biológicas e desnaturar proteínas, limitando o metabolismo primário. Dentre as respostas ao estresse abiótico, está a síntese de metabólitos secundários. Braquicerina é um alcaloide monoterpeno indólico com ação antioxidante, protetora contra UV e antimutagênica sintetizado por partes aéreas de Psychotria brachyceras. O objetivo deste trabalho é investigar o metabolismo de braquicerina sob estresse de calor. Assim, espera-se contribuir para o conhecimento acerca do metabolismo secundário nas respostas ao estresse de calor, descrever a função in planta do composto, e fornecer ferramentas para obtenção do alcaloide para fins farmacêuticos. O acúmulo de braquicerina foi induzido em discos foliares mantidos a 40ºC por três dias, tanto em regime de elevação abrupta como gradual da temperatura. Baixa temperatura (10ºC) não afetou o acúmulo do alcaloide. Discos foliares de Psychotria carthagenensis, uma espécie que não sintetiza alcaloides monoterpeno indólicos, foram também desafiados por estresse de calor. Clorofila total, teor de peróxido de hidrogênio e peroxidação lipídica foram quantificados em ambas as espécies. P. carthagenensis foi relativamente tolerante ao calor, o que pode estar relacionado à sua elevada concentração de antocianinas, fortemente induzidas por choque térmico de 50ºC por 6h. Peroxidação lipídica foi reduzida nas amostras de P. brachyceras sob estresse de calor agudo ou gradual em comparação à condição controle, sendo este parâmetro inalterado nas duas condições em P. carthagenensis. O teor de peróxido de hidrogênio foi menor em P. brachyceras submetida a choque de térmico em relação ao controle, enquanto o mesmo parâmetro não foi alterado em P. carthagenensis. Discos foliares das espécies sensíveis ao calor Brugmansia suaveolens e Brassica oleracea, pré-tratadas com braquicerina em concentrações similares às encontradas em P. brachyceras, adquiriram fenótipo tolerante ao choque térmico. A expressão do gene que codifica a enzima triptofano descarboxilase (TDC), envolvida na biossíntese de braquicerina em P. brachyceras, foi fortemente inibida em discos foliares submetidos à 40ºC por 6h, 12h e 24h, sugerindo que o efeito da temperatura na estimulação de acúmulo de alcaloide ocorra em nível pós-transcricional. Em conjunto, os dados indicam que a exposição ao calor é um meio efetivo de aumentar o rendimento de braquicerina, cuja acumulação contribui para proteção contra os danos oxidativos associados. / Heat stress impairs plant growth and reproduction by altering membrane permeability and promoting protein denaturation, which limits primary metabolism. Secondary metabolites often take part in protection against abiotic stress responses. Brachycerine is a monoterpene indole alkaloid with antioxidant, UV protectant, and antimutagenic activity synthesized by Psychotria brachyceras shoots. Brachycerine metabolism was analyzed under heat stress, in order to shed light on brachycerine‘s in planta function and to provide potential tools to improve alkaloid yields for pharmaceutical analysis. Accumulation was induced in leaf disks kept at 40ºC for three days, both by abrupt and stepwise temperature increase. Brachycerine concentration was not affected by low temperature (10ºC) exposure. Leaf disks of Psychotria carthagenensis, a species devoided of alkaloids, were also challenged by heat. Total chlorophyll, hydrogen peroxide and lipid peroxidation concentrations were determined in both species. P. carthagenensis was relatively tolerant to heat treatments which may be explained by its high anthocyanin concentration, strongly induced by heat shock of 50ºC for 6h. Brugmansia suaveolens and Brassica oleracea, pre-treated with brachycerine in concentrations equivalent to those found in P. brachyceras, had a heat shock tolerant phenotype, based on chlorophyll content. Expression of the TRYPTOPHAN DECARBOXYLASE gene, which encodes for an enzyme involved in alkaloid biosynthesis (TDC) was strongly repressed in leaf disks exposed to 40ºC for 6h, 12h e 24h, suggesting that temperature effect may occur at post-transcriptional level. Taken together, data indicate that heat exposure is an effective means to increase yields of brachycerine, whose accumulation contributes to protect against associated oxidative damage.

A banana é um fruto consumido no mundo todo e, ao contrário do que acontece na maioria dos países nos quais predomina o grupo Cavendish, uma grande variedade de cultivares são consumidas nas diferentes regiões do Brasil. No entanto, as informações bioquímicas e fisiológicas a respeito das cultivares consumidas no país são ainda restritas e, apesar de serem muito diferentes entre si, são tratadas da mesma maneira na pré e pós-colheita, o que muitas vezes compromete a qualidade do fruto. Diante disso, dois pontos são fundamentais para a obtenção de frutos de melhor qualidade: a definição do ponto de colheita e o tratamento dos frutos com etileno após a colheita. Hoje a colheita é feita em função do diâmetro dos frutos e o tratamento com etileno é o mesmo para todas as cultivares. Porém, frutos com o mesmo diâmetro nem sempre estão no mesmo estágio de maturidade fisiológica e, usualmente, o tratamento pós-colheita feito com etileno, visando o amadurecimento mais rápido e uniforme dos frutos, não segue nenhuma orientação técnica. A consequência da falta de critérios definidos para a colheita e para a aplicação de etileno resulta em bananas com baixa qualidade e com vida-de-prateleira curta. A produção de banana é dificultada pelos problemas fitossanitários que ocorrem nas plantações, incluindo doenças como as Sigatokas Negra e Amarela e o Mal-do-Panamá. Tendo em vista a ameaça que as doenças da bananeira representam e os prejuízos que podem causar, a introdução de cultivares resistentes é a melhor forma para reduzir a pressão desses patógenos sobre a cultura. Por ser resistente às Sigatokas e ao Mal de Panamá este trabalho visou conhecer melhor a cultivar Thap Maeo (Musa acuminata AAB cv. Thap Maeo) que tem como defeito principal uma vida-de-prateleira muito curta. Os objetivos deste trabalho foram: (1) estabelecer o ponto de colheita das bananas da cultivar Thap Maeo utilizando a metodologia da soma de temperatura a que a planta está exposta durante o desenvolvimento dos frutos; (2) estabelecer, a partir da caracterização físico-química dos frutos, o teor ideal de etileno exógeno para promover o amadurecimento uniforme dos frutos e, (3) estudar o balanço hormonal no amadurecimento dos frutos. Em uma primeira etapa foi implantado um experimento de campo para determinar a Temperatura Base e a Idade Fisiológica Máxima para esta cultivar. Estes parâmetros são necessários para o cálculo da idade fisiológica e na determinação do ponto de colheita. Com o término desta etapa, foram realizadas colheitas de frutos em diferentes épocas do ano para confirmar a metodologia usada. Foi possível estabelecer uma metodologia para estimar o ponto de colheita dos frutos de acordo com a estação do ano em que estes se desenvolveram. Para estabelecer o teor ideal de etileno exógeno aplicado na pós-colheita, os frutos foram testados com diferentes concentrações de etileno (0 a 1000 ppm) em dose única. Os resultados indicam que mesmo as menores concentrações de etileno aplicadas promovem o amadurecimento uniforme dos frutos, sem diferenças aparentes entre os tratamentos. Além disso, as análises do perfil de compostos voláteis da polpa indicam que a maior diferença entre o aroma dos tratamentos é entre frutos tratados e não tratados. Os frutos que não foram tratados apresentaram maior quantidade de compostos identificados. Em todas as colheitas realizadas, o período entre o início do amadurecimento e o amadurecimento completo dos frutos foi consideravelmente curto se comparado a bananas do subgrupo Nanica. Para melhor compreender estes resultados, além do etileno, foram quantificados os teores livres dos hormônios ácido indol-3-acético (AIA) e ácido abcísico (ABA). Em comparação com estudos anteriores, com cultivares do subgrupo Nanica, a cultivar Thap Maeo produz mais etileno e ABA durante o amadurecimento, além de apresentar menores teores de AIA quando verde. Estas diferenças hormonais podem explicar o curto período de amadurecimento desta cultivar. / Banana is an important staple food. Unlike the world Market, which has only the Cavendish Bananas as export bananas, Brazilian market has many Bananas varieties. However, biochemical and physiological information about these varieties are still scarce and different bananas cultivars are treated the same way in pre or post-harvest, which often affects the quality of the fruit. In this context, two issues are important to reach best fruit quality: definition of harvest time and the exogenous ethylene treatment. Now a day the harvest time is defined by the diameter of the fruits and the ethylene treatment is equal for all cultivars. However, fruits with the same diameter not always are in the same development grade and the post-harvest ethylene treatment, aiming a faster and uniform ripening, does not follow any technical recommendation. The consequences for the lack of criteria in the harvest and for the ethylene treatment might be a low quality and shelf life banana. Banana production is hampered by plant health problems occurring in the production fields, including the diseases such as the Sigatokas and the Panama Wilt. Given the threat of the banana diseases, and the damage they can cause, the introduction of resistant cultivars is the best way to reduce the pressure of these pathogens on this crop. Being resistant to Sigatokas and Panama diseases, this work aimed to know better the cultivar Thap Maeo (Musa acuminata AAB cv. Thap Maeo) whose main defect is a short shelf life. The objectives of this work were: (1) to establish the harvest time of Thap Maeo bananas using the thermal sum techniques, (2) using the physico-chemical characterization of the fruit, establish the ideal concentration of exogenous ethylene to promote uniform ripening of fruits and (3) study the hormonal balance in the fruit ripening. The first step was a field experiment to determine the base temperature and the maximum physiological age. These parameters are used to calculate the thermal sum. The next step was to harvest fruits from different times of the year to confirm the methodology. It was established a methodology to estimate the harvest time according to the season in which the fruit has developed. Five ethylene concentration were tested (0-1000 ppm). Results showed that even the low ethylene concentration applied could promote the uniform ripening of the fruits with no apparent differences between the treatments. Furthermore, the analysis of volatile compounds in the pulp indicate that the major difference between the aroma of treatments is between treated and untreated fruits. The fruits that were not treated showed a higher amount of identified compounds. For all harvested fruits, the period between the ripening start and full ripening of the fruit was short when compared to the Nanica bananas. Levels of the hormones indole-3-acetic acid and abscisic acid, in addition to ethylene, were quantified to better understand these results. Compared to previous studies, with cultivars of Nanica subgroup, the cultivar Thap Maeo produce more ethylene and ABA during ripening, and have lower IAA level in the green stage. These hormonal differences may explain the short maturity period of this cultivar.

No capítulo 1 apresentamos uma síntese eficiente de compostos 5-organoteluro-1H- 1,2,3-triazóis realizada via reação de cicloadição [3+2] entre azidas orgânicas e alquinos substituídos com organotelúrio. Além disso, os 5-organoteluro-1H-1,2,3-triazóis foram funcionalizados na posição 5 do anel triazólico por reação de acoplamento cruzado de Sonogashira. A regioquímica dos produtos de cicloadição foram descritas com base em experimentos de RMN, cálculos teóricos e cristalografia de raio-x. Apresentamos uma proposta mecanística para a cicloadição mediada por cobre, baseada em experimentos de espectrometria de massas de alta resolução. No capítulo 2, investigamos a eficiência de reações one-pot com indol, cloreto de oxalila e diferentes nucleófilos para obtermos derivados do indol-3-glioxila em condições adequadas. Do mesmo modo, envolvendo a adição de azidas orgânicas, levando à síntese de indol-3-glioxil-1,2,3-triazóis, os produtos foram obtidos com rendimentos que variaram de 59 a 85%. / In chapter 1 we present an efficient synthesis of 5-organotelluro-1H-1,2,3-triazole compounds that was accomplished via the [3+2]-cycloaddition reaction of organoazides and organotelluro alkynes. Additionally, 5-organotelluro-1H-1,2,3-triazoles were readily functionalized at the 5-position via the Sonogashira cross-coupling reaction, leading to highly functionalised triazoles. The regiochemistry of the products was assessed by bidimensional NMR experiments, theoretical calculations and x-ray crystallography. We presented a mechanistic proposal for the cycloaddition mediated by copper, based on high resolution mass spectrometry experiments. In chapter 2 we investigated a general and efficient reaction of indole with oxalyl chloride and nucleophiles providing indole-3-glyoxyl derivatives which has been developed in mild conditions. In the same fashion, the other reaction involved the addition of organic azides leading to the synthesis of indole-3-glyoxyl-1,2,3-triazoles, which proceeds smoothly generating the products in moderate to high yields.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Considered viral diseases of major re-emerging in the world, the dengue, chikungunya and Zika have as the main vector Aedes aegypti (L.) (Diptera: Culicidae). Larvicidal vector control is an important measure to prevent the transmission of such infections. A major challenge in the control of this arthropod is the low sensitivity of its population through the use of conventional larvicides. Thus, the insect acquires resistance, reducing the effectiveness of these pesticides. Consequently, it will lead to an increase in the risks of toxicity in nontarget organisms and a change in the environment. An alternative to avoid problems caused by the use of these products is the search for new compounds with less environmental impact and better benefits to human health. The indole molecule ring represents one of the subunits of great importance in the discovery of new pesticide products for the pharmaceutical market. By Friedel-Crafts acylation reaction, the C-3 of this ring makes it susceptible to chemically react. Thus, 12 indole analogues were synthesized as potential larvicidal agents against Ae. aegypti in its 3rd larval stage followed by the evaluation of the toxicity in nauplii of Artemia sp. The compounds were identified by analytical thin-layer chromatography, purified on a silica gel 60 chromatographic column (using the Hexane: Ethyl acetate (90:10, v / v) binary system as the mobile phase) and characterized by melting point, 13C and 1H NMR (using a residual solvent peak or TMS as reference for 1 H NMR spectra), mass spectrum and infrared. Bioassays were performed using 20 larvae per test, disposable cups containing 20mL of the test solution in triplicate. Branched aliphatic side chain derivatives were more potent than the others were, and the linear ones exhibited potency oscillation as the addition of the methylene chains. Toxicity tests indicated that (3-chlorophenyl)1-(1H-indol-3-yl)methanone, with moderate larvicidal potency (LC50 = 50.59 ppm), showed the highest selectivity index (SI >19.7), being less toxic to Artemia sp. than Ae. aegypti. The relationships between structural changes in indole derivatives and their LC50 results provide information that may contribute to the understanding of the influence of physicochemical properties on the larvicidal action of this class of compounds, without damage to the ecosystem. / Consideradas doenças virais de grande reemergência no mundo, a dengue, chikungunya e zika têm como principal vetor o Aedes aegypti (L.) (Diptera:Culicidae). O controle vetorial baseado em larvicidas é uma medida importante de prevenir a transmissão de tais infecções. Um grande desafio nas formas de controle desse artrópode está na baixa sensibilidade de sua população através do uso de larvicidas convencionais. Assim, o inseto adquire resistência, reduzindo a eficácia desses pesticidas. Consequentemente, levará a um aumento nos riscos de toxicidade em organismos não-alvo e uma alteração ao meio ambiente. Uma alternativa para evitar problemas ocasionados pela utilização desses produtos é a pesquisa por novos compostos com menos impacto ambiental e melhores benefícios à saúde humana. O anel da molécula do indol representa uma das subunidades com grande importância na descoberta de novos produtos pesticidas para o mercado farmacêutico. Por reação de acilação de Friedel-Crafts, o C-3 deste anel torna suscetível a reagir quimicamente. Assim, foram sintetizados 12 análogos do indol como potenciais agentes larvicidas contra o Ae. aegypti no seu 3º estágio larvar, seguido pela avaliação da toxicidade em náuplios de Artemia sp. Os compostos sintetizados foram identificados por cromatografia em camada delgada analítica, purificados em coluna cromatográfica de sílica gel 60 (utilizando o sistema binário Hexano: Acetato de etila (90:10, v/v) como fase móvel), e caracterizados por ponto de fusão, RMN de 13C e 1H (utilizando um pico de solvente residual ou TMS como referência para os espectros de RMN 1H), espectro de massas e infravermelho. Os bioensaios foram realizados utilizando 20 larvas por teste, copos descartáveis contendo 20 mL da solução teste em triplicata. Derivados de cadeias laterais alifáticas ramificadas foram mais potentes que os demais, e os lineares exibiram oscilação de potência conforme acréscimo das cadeias de metileno. Ensaios de toxicidade apontaram que a (3-clorofenila)1-(1H-indol-3- ila)metanona, com potência larvicida moderada (CL50 = 50,59 ppm), exibiu o maior índice de seletividade (IS >19,7), sendo menos tóxico para Artemia sp do que para o Ae. aegypti. As relações entre mudanças estruturais dos derivados do indol e seus resultados de CL50 fornecem informações que podem contribuir para a compreensão da influência de propriedades físico-químicas na ação larvicida desta classe de compostos, sem prejuízos ao ecossistema. / São Cristóvão, SE

Catharanthus roseus (Madagascar periwinkle), is a well-known medicinal plant that produces a vast array of terpenoid indole alkaloids (TIAs), including two anticancer compounds vinblastine and vincristine. Industrial scale production of TIAs is hampered by the difficulties of total chemical synthesis of these compounds and the fragmented knowledge on TIA pathway. Transcriptional regulation of the TIA biosynthetic pathway has not been thoroughly investigated in Catharanthus and only a few structural genes have been identified as the targets of two master regulators: the basic helix-loop-helix (bHLH) transcription factor (TF) CrMYC2 and APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF), ORCA3. Next generation sequencing (NGS) has been used as a tool to isolate novel genes encoding enzymes and regulators of TIA pathway in Catharanthus. In this dissertation, I have performed the transcriptomic analysis of transgenic Catharanthus hairy roots ectopically expressing a dominant repressive form of CrMYC2 or ORCA3 in order to understand their potential impact on the TIA transcriptional regulatory network and to identify and characterize novel target(s) of these two key TFs. MYC2 acts as regulatory hub involved in diverse aspects of plant growth, development, and specialized metabolite biosynthesis by coordinating the crosstalk among different phytohormone signals. CrMYC2 was initially identified in Catharanthus as a regulator of ORCA3. CrMYC2 transactivates ORCA3 by binding to the T/G-box in jasmonate-responsive element (JRE) of ORCA3 promoter. RNA interference (RNAi) mediated knockdown of CrMYC2 strongly reduced TIA accumulation in Catharanthus cell suspension culture. However, the potential influence of CrMYC2 on the expression of other regulatory and structural genes in the TIA pathway remains poorly understood. Transcriptomic analyses revealed that CrMYC2 plays an essential role in JA-induced gene expression and the differentially expressed genes are involved in diverse aspects of growth and development as well as abiotic and biotic stress responses in Catharanthus. Additionally, the expression of genes related to auxin, ethylene, and abscisic acid signaling cascades were affected in hairy roots with modified CrMYC2 expression, suggesting this TF mediates cross-talk between JA and other phytohormones. Surprisingly, overexpression of CrMYC2 resulted in repressed expression of TIA pathway genes in transgenic hairy roots. Expressions of key activators of indole and iridoid pathway were downregulated whereas expression of repressors were upregulated in CrMYC2 hairy roots. Activators (i.e. CrMYC2 and ORCA3) and repressors (i.e. G-box binding factors; GBFs) have been isolated and characterized for their role in regulation of TIA pathway. However, the interconnection between those regulators and the underlying molecular mechanism has not been throughly studied. I identified (i) the interaction of CrMYC2 with CrGBFs and (ii) how this cross-family transcription factor interactions fine-tunes TIA biosynthesis in Catharanthus. The expression profiles of CrMYC2 and CrGBFs were highly correlated in different tissues and in response JA. Moreover, CrMYC2 interacted with CrGBF1 and CrGBF2 in both yeast and plant cells. CrGBF1 and CrGBF2 could form homo- and hetero-dimer which bound T/G-box elements of TIA pathway gene promoters. In plant cells, CrGBF1 antagonizes the activity of CrMYC2 on target promoters in a dosage dependent-manner. Similarly, CrMYC2 can overcome CrGBF1-mediated repression of target promoters in a dosage dependent manner. ORCA3 is another major regulator of TIA biosynthesis in Catharanthus. The transcriptomic analysis of ORCA3 transgenic hairy roots revealed (i) the effect of ORCA3 on newly identified TIA pathway biosynthetic enzymes; (ii) identify the potential effect of ORCA3 on three biological processes: abiotic stress response, plant secondary metabolic process, and response to hormonal stimulus; and (iii) the identification of potential regulator(s) of TIA biosynthesis using ORCA3 based co-expression analysis.

Catharanthus roseus (Madagascar periwinkle) is the exclusive source of an array of terpenoid indole alkaloids (TIAs) that are used in the treatments of hypertension and certain types of cancer. TIA biosynthesis is under stringent spatiotemporal control and is induced by jasmonate (JA) and fungal elicitors. Tryptamine, derived from the indole branch, and secologanin from the iridoid branch are condensed to form the first TIA, strictosidine. Biosynthesis of TIA is regulated at the transcriptional level and several transcription factors (TFs) regulating the expression of genes encoding key enzymes in the pathway have been isolated and characterized. The JA-responsive APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF), ORCA3, and the basic helix-loop-helix (bHLH) factor, CrMYC2, are the key activators of the TIA biosynthesis. Recently, two other TFs, the bHLH IRIDOID SYNTHESIS 1 (BIS1) and BIS2 were also identified as regulators of TIA pathway. Analysis of C. roseus genome sequence has revealed that ORCA3 forms a physical cluster with two uncharacterized AP2/ERFs, ORCA4 and ORCA5. In plants, physically linked clusters of TFs are less characterized. Moreover, the regulation of TF clusters is relatively unexplored. My research uncovered that the ORCA gene cluster is differentially regulated. ORCA4 and ORCA5, while functionally overlapping with ORCA3, regulate an additional set of TIA pathway genes. ORCA4 or ORCA5 overexpression has resulted in significant increase of TIA accumulation in C. roseus hairy roots. In addition, ORCA5 directly regulates the expression of ORCA4 and indirectly regulates ORCA3, likely via unknown factor(s). Interestingly, ORCA5 also activates the expression of ZCT3, a negative regulator of the TIA pathway. In addition CrMYC2 is capable of activating ORCA3 and co-regulating pathway genes concomitantly with ORCA3. Several lines of evidence suggest that, in addition to the transcriptional control, biosynthesis of TIAs is also controlled at the posttranslational level, such as protein phosphorylation. Available literature indicates that a mitogen-activated protein kinase (MAPK) cascade is involved in this process. Analysis of C. roseus MAP kinome, identified two independent MAPK cascades regulating the indole and iridoid branches of the TIA pathway. We showed that the ORCA cluster and CrMYC2 act downstream of a MAP kinase cascade consisting of CrMAPKK1, CrMAPK3 and CrMAPK6. Overexpression of CrMAPKK1 in C. roseus hairy roots upregulates TIA pathway genes expressions and boosts TIA accumulation. The other cascade, consisting of CrMAPKK6 and CrMAPK13, mostly regulates the iridoid branch of the TIA pathway. Overexpression of CrMAPK13 in C. roseus hairy roots significantly upregulates iridoid pathway genes and boosts tabersonine accumulation. Moreover, we recently identified the third MAPK cascade, consisting of CrMAPKK1 and CrMAPK20, that negatively regulates the indole branch of the TIA pathway. Overexpression of CrMAPK20 in C. roseus hairy roots represses the genes regulated by CrMYC2-ORCAs and reduces catharanthine accumulation. These findings significantly advance our understanding of transcriptional and post-translational regulatory mechanisms that govern TIA biosynthesis in C. roseus.

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