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.

Base teórica: A artrite reumatoide (AR) é uma doença autoimune sistêmica onde a inflamação crônica da sinóvia articular e a subsequente erosão óssea e da cartilagem resultam em destruição articular, dor e incapacidade funcional. Apesar dos recentes progressos no tratamento da AR, estes ainda apresentam limitações e significativos efeitos adversos, salientando a necessidade de novas estratégias terapêuticas. Plantas da família das Amarilidáceas apresentam em seus bulbos um conjunto de alcaloides muito característicos e exclusivos com atividades farmacológicas, tais como atividade antiviral, anti-inflamatória e atividade anticolinérgica. A montanina é um alcaloide isolado da Rhodophiala bífida, uma planta da família das amarilidáceas utilizada na medicina popular, mas nunca antes testada como terapia para doenças inflamatórias. Objetivo: Avaliar o efeito da montanina como uma terapia anti-inflamatória in vivo em dois modelos de artrite e in vitro sobre a proliferação de linfócitos e sobre a invasão de fibroblastos sinoviais (FLS). Métodos: Artrite induzida por antígeno (AIA) foi realizada em camundongos Balb/C com albumina bovina sérica metilada e a nocicepção e a migração de leucócitos para a articulação do joelho foram os parâmetros avaliados. Artrite induzida por colágeno (CIA) foi realizada em camundongos DBA/1J e o desenvolvimento e severidade da artrite foi avaliado através de escore clínico, nocicepção articular e escore histológico. Montanina foi administrada via intraperitoneal, duas vezes ao dia. A proliferação de linfócitos estimulados por concanavalina A (conA) foi realizada pelo método de MTT e invasão de FLS em 24 horas foi avaliada em um sistema de insertos de Matrigel. Resutados: A administração de montanina diminuiu a migração articular de leucócitos (p0,001) e a nocicepção (p0,01) em camundongos com AIA. Em camundongos com CIA, o tratamento com montanina reduziu a severidade da artrite e o dano articular avaliado pelos escores clínico (p0,01) e histológico (p0,05) e melhorou a nocicepção articular (p0,05), sem causar nenhum dano hepático. Além disso, montanina inibiu in vitro a proliferação de linfócitos estimulados com conA (p0,01) e diminuiu a invasão de FLS (p0,05) em 54%, com uma ação independente de citotoxicidade. Conclusão: Esses resultados indicam que a montanina pode ser explorada para se tornar um possível fármaco para o tratamento de doenças inflamatórias e autoimunes, como a AR. / Background: Rheumatoid arthritis (RA) is an autoimmune and systemic disease where the chronic inflammation of articular synovia and the subsequent bone and cartilage erosion results in joint destruction, pain and functional disability. Despite recent progress in RA treatments, its still have limitations and significant side effects, emphasizing the need of new therapeutic strategies. Amaryllidaceae plants presenting at its bulbs a set of very characteristics and exclusives alkaloids with pharmacological activities such as antiviral, anti-inflammatory and anticholinergic activity. Montanine is an alkaloid isolated from the Rhodophiala bifida, an Amaryllidaceae plant used in alternative medicine but never before tested as a therapy for inflammatory diseases. Objective: To evaluate the effect of montanine as an in vivo anti-inflammatory therapy in two arthritis models and in vitro on lymphocytes proliferation and fibroblast-like synoviocytes (FLS) invasion. Methods: Antigen-induced arthritis (AIA) was performed in Balb/C mice with methylated bovine serum albumin and nociception and leukocytes migration into the knee joint were evaluated. Collagen-induced arthritis (CIA) was performed in DBA/1J mice and arthritis development and severity were assessed by clinical scoring, articular nociception and histological scoring. Montanine was administered intraperitoneally twice a day. Lymphocyte proliferation stimulated by concanavalin A in 48 hours was performed with MTT assay and FLS invasion in 24 hours was assayed in a Matrigel-coated transwell system. Results: Administration of montanine decreased leukocyte articular migration (p0.001) and nociception (p0.01) in mice with AIA. In mice with CIA, treatment with montanine reduced severity of arthritis and joint damage assessed by clinical (p0.01) and histological score (p0.05) and ameliorates articular nociception (p0.05), without causing any hepatic damage. Moreover, montanine inhibited in vitro lymphocyte proliferation stimulated with ConA (p0.01) and decreased FLS invasion by 54% (p0.05), with an action independent of cytotoxicity. Conclusion: These findings suggest that montanine can be explored to become a possible medicament to treat inflammatory and autoimmune diseases such as arthritis.

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.

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.

Base teórica: A artrite reumatoide (AR) é uma doença autoimune sistêmica onde a inflamação crônica da sinóvia articular e a subsequente erosão óssea e da cartilagem resultam em destruição articular, dor e incapacidade funcional. Apesar dos recentes progressos no tratamento da AR, estes ainda apresentam limitações e significativos efeitos adversos, salientando a necessidade de novas estratégias terapêuticas. Plantas da família das Amarilidáceas apresentam em seus bulbos um conjunto de alcaloides muito característicos e exclusivos com atividades farmacológicas, tais como atividade antiviral, anti-inflamatória e atividade anticolinérgica. A montanina é um alcaloide isolado da Rhodophiala bífida, uma planta da família das amarilidáceas utilizada na medicina popular, mas nunca antes testada como terapia para doenças inflamatórias. Objetivo: Avaliar o efeito da montanina como uma terapia anti-inflamatória in vivo em dois modelos de artrite e in vitro sobre a proliferação de linfócitos e sobre a invasão de fibroblastos sinoviais (FLS). Métodos: Artrite induzida por antígeno (AIA) foi realizada em camundongos Balb/C com albumina bovina sérica metilada e a nocicepção e a migração de leucócitos para a articulação do joelho foram os parâmetros avaliados. Artrite induzida por colágeno (CIA) foi realizada em camundongos DBA/1J e o desenvolvimento e severidade da artrite foi avaliado através de escore clínico, nocicepção articular e escore histológico. Montanina foi administrada via intraperitoneal, duas vezes ao dia. A proliferação de linfócitos estimulados por concanavalina A (conA) foi realizada pelo método de MTT e invasão de FLS em 24 horas foi avaliada em um sistema de insertos de Matrigel. Resutados: A administração de montanina diminuiu a migração articular de leucócitos (p0,001) e a nocicepção (p0,01) em camundongos com AIA. Em camundongos com CIA, o tratamento com montanina reduziu a severidade da artrite e o dano articular avaliado pelos escores clínico (p0,01) e histológico (p0,05) e melhorou a nocicepção articular (p0,05), sem causar nenhum dano hepático. Além disso, montanina inibiu in vitro a proliferação de linfócitos estimulados com conA (p0,01) e diminuiu a invasão de FLS (p0,05) em 54%, com uma ação independente de citotoxicidade. Conclusão: Esses resultados indicam que a montanina pode ser explorada para se tornar um possível fármaco para o tratamento de doenças inflamatórias e autoimunes, como a AR. / Background: Rheumatoid arthritis (RA) is an autoimmune and systemic disease where the chronic inflammation of articular synovia and the subsequent bone and cartilage erosion results in joint destruction, pain and functional disability. Despite recent progress in RA treatments, its still have limitations and significant side effects, emphasizing the need of new therapeutic strategies. Amaryllidaceae plants presenting at its bulbs a set of very characteristics and exclusives alkaloids with pharmacological activities such as antiviral, anti-inflammatory and anticholinergic activity. Montanine is an alkaloid isolated from the Rhodophiala bifida, an Amaryllidaceae plant used in alternative medicine but never before tested as a therapy for inflammatory diseases. Objective: To evaluate the effect of montanine as an in vivo anti-inflammatory therapy in two arthritis models and in vitro on lymphocytes proliferation and fibroblast-like synoviocytes (FLS) invasion. Methods: Antigen-induced arthritis (AIA) was performed in Balb/C mice with methylated bovine serum albumin and nociception and leukocytes migration into the knee joint were evaluated. Collagen-induced arthritis (CIA) was performed in DBA/1J mice and arthritis development and severity were assessed by clinical scoring, articular nociception and histological scoring. Montanine was administered intraperitoneally twice a day. Lymphocyte proliferation stimulated by concanavalin A in 48 hours was performed with MTT assay and FLS invasion in 24 hours was assayed in a Matrigel-coated transwell system. Results: Administration of montanine decreased leukocyte articular migration (p0.001) and nociception (p0.01) in mice with AIA. In mice with CIA, treatment with montanine reduced severity of arthritis and joint damage assessed by clinical (p0.01) and histological score (p0.05) and ameliorates articular nociception (p0.05), without causing any hepatic damage. Moreover, montanine inhibited in vitro lymphocyte proliferation stimulated with ConA (p0.01) and decreased FLS invasion by 54% (p0.05), with an action independent of cytotoxicity. Conclusion: These findings suggest that montanine can be explored to become a possible medicament to treat inflammatory and autoimmune diseases such as arthritis.

Orientador: Fernando Antonio Santos Coelho / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-15T03:19:35Z (GMT). No. of bitstreams: 1 Abella_CarlosAlbertoMiranda_D.pdf: 8234084 bytes, checksum: 0320b741bad5e28b8ad206ec73935836 (MD5) Previous issue date: 2009 / Resumo:O capítulo 1 relata os intermediários atingidos visando à síntese do alcalóide (±)-8-a-etoxiprecrivelina. Obteve-se um intermediário com todos os grupos funcionais necessários para atingir o alvo. A adição de metilamina sobre o aduto de Morita-Baylis-Hillman sililado proveniente do bromopiperonal levou ao produto de adição syn com elevada diastereosseletividade que foi racionalizada através de cálculos teóricos. Através de uma mudança na rota sintética inicialmente proposta, foi possível inserir um álcool secundário necessário através de uma reação de ozonólise seguida de redução quimiosseletiva. Dada a ausência na literatura deste tipo de reação sobre olefinas de adutos de Morita-Baylis-Hillman com anéis aromáticos, realizaram-se testes com adutos possuindo diferentes demandas eletrônicas no anel aromático os quais foram obtidos na sua maioria com sucesso. O capítulo 2 descreve a aplicação de uma metodologia desenvolvida no nosso grupo de pesquisa visando a síntese de alcalóides indolizidínicos a partir de lactamas bicíclicas polifuncionalizadas. Estas últimas foram obtidas nas suas formas racêmicas e quiral. O aldeído quiral sintetizado não racemizou nas condições empregadas na síntese do aduto de Morita-Baylis-Hillman, o qual foi obtido de forma majoritária como anti. Os diastereoisômeros foram separados e o majoritário ciclizado para fornecer a lactama bicíclica. A primeira parte do capítulo 3 relata as tentativas de usar bases de Tröger como catalisadores na reação de Morita-Baylis-Hillman, as quais se mostraram infrutíferas. Na segunda parte do mesmo capítulo, o mecanismo de reação de formação de bases de Tröger foi estudado por ESI-MS usando tanto formaldeído como urotropina como fontes de metileno. Os intermediários chaves foram caracterizados via ESI-MS/MS e determinou-se que o mecanismo envolve uma seqüência de reações de substituição eletrofílica aromática / Abstract: Chapter 1 describes the intermediates obtained towards the total synthesis of (±)-8-a-ethoxyprecriwelline. An intermediate with all required functionalities was obtained in order to achieve the target. Methylamine addition on silylated Morita-Baylis-Hillman adduct obtained from bromopiperonal gave mostly the syn diastereoisomer which was rationalized by the use of theoretical calculations. By means of changing the original synthetic proposal, it was possible to introduce a necessary secondary alcohol through an ozonolysis reaction followed by chemoselective reduction. Since there was no evidence of this kind of reaction on olefin of Morita-Baylis-Hillman adducts with aromatic ring in the literature, some test were done with adducts having different electronic requirements on the aromatic ring, and most were successfully obtained. Chapter 2 shows the application of a methodology developed in our research group envisaging the synthesis of indolizidinic alkaloids from polyfunctionalized bicyclic lactams. The latter were obtained in its racemic and chiral form. The chiral aldehyde synthesized didn' t racemize in the conditions used in the synthesis of Morita-Baylis-Hillman adduct, which was obtained as the anti as the major diastereomer. Both diastereomers were separated and the major one was cyclized to produce the bicyclic lactam. The first part of chapter 3 tells the tentative of use of Tröger' s bases as catalyst in the reaction of Morita-Baylis-Hillman, which didn' t prove useful. In the second part of the same chapter, the mechanism of formation of Tröger' s bases was studied by ESI-MS using formaldehyde as well as urotropine as the source of methylene. Key intermediates were characterized by ESI-MS/MS and it was found that the mechanism involves a sequence of electrophilic aromatic substitution reaction / Doutorado / Quimica Organica / Doutor em Ciências

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

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 est une plante tropicale de la famille des Apocynacées d’intérêt thérapeutique en raison de sa capacité à synthétiser des alcaloïdes indoliques monoterpéniques (AIM) utilisés en chimiothérapie anticancéreuse. La teneur en AIM in planta est très faible notamment en raison d’une haute compartimentalisation cellulaire et subcellulaire de la voie de biosynthèse. Si la compartimentalisation cellulaire était bien caractérisée, très peu de données de localisation subcellulaire in situ étaient disponibles au début de cette thèse. Une connaissance fine de cette compartimentalisation est cependant nécessaire pour identifier les transports inter-compartiment de métabolites intermédiaires, limitant potentiellement le flux métabolique, afin d’améliorer ensuite le rendement de biosynthèse des AIM par ingénierie métabolique. Dans ce contexte nous avons réalisé une étude exhaustive de la localisation subcellulaire des enzymes de cette voie par imagerie GFP dans des cellules de C. roseus transformées par biolistique permettant d’établir un nouveau modèle intégré d’organisation cellulaire et subcellulaire de la biosynthèse des AIM. / Catharanthus roseus is a tropical plant from the Apocynaceae family with a great therapeutic value due to its ability to synthesize monoterpene indole alkaloids (MIA) used in cancer treatment. The yields of these molecules in planta are very low due to a very high level of compartmentation of the biosynthetic pathway at both cellular and subcellular levels. While the cellular compartmentation was widely characterized, very few in situ subcellular localization data were available at the beginning of this PhD. An accurate knowledge of this compartmentation is necessary to identify intermediate metabolites transport events from one compartment to another one, in order to increase the MIA biosynthesis yield by metabolic engineering approaches. In this context we have proceed to the exhaustive study of the subcellular localization of these enzymes by in vivo GFP imaging in C. roseus cells transformed by biolistic. Potential interprotein interactions of these enzymes have also been studied by BiFC. Altogether, our results enabled us to draw an integrated model of the cellular and subcellular organization of MIA biosynthesis in situ.

Les alcaloïdes sont parmi les molécules naturelles les plus intéressantes pour leurs propriétés biologiques et médicinales. Parmi ceux-ci, les amaryllidacés et notamment la clivonine et ses analogues présentent de nombreuses propriétés peu étudiées et la synthèse de molécules de cette famille permettra des avancées dans ces études. Notre équipe s’intéresse particulièrement à cette molécule car sa synthèse pourrait se faire à partir d’une réaction de nitroso Diels-Alder énantiosélective qui est un sujet majeur de recherche dans notre laboratoire. Dans la suite de la synthèse, une deuxième étape clé doit faire intervenir une insertion C-H intramoléculaire. La réaction de nitroso Diels-Alder a été réalisée avec un excès énantiomérique de 96% et la synthèse a été poursuivie pour accéder à l’intermédiaire diazo clé, substrat de départ de l’insertion C-H. Cette dernière réaction n’a pas permis d’obtenir les produits escomptés en utilisant des catalyseurs commerciaux et une collaboration a été entamée avec le Pr Huw Davies pour pouvoir utiliser des catalyseurs plus élaborés. Les résultats de cette collaboration ne sont pas encore définitifs. Parallèlement, trois autres voies de synthèse de la clivonine ont été conçues et expérimentées, des variations de la synthèse originale, mais aucune n’a abouti. En revanche, une de ces synthèses nous a permis de découvrir une réactivité nouvelle des cycloadduits de nitroso Diels-Alder : en présence d’organolithiens, une réaction de β-fragmentation a lieu, suivie d’une addition, qui permet d’obtenir des composés monocycliques très fonctionnalisés avec une très bonne stéréosélectivité. Cette réaction et son étendue doivent encore être étudiées mais pourraient avoir des applications en synthèse totale. / Alkaloids are among the most interesting natural compounds because of their biological and medicinal properties. Among them amaryllidaceae alkaloids and especially clivonine and its analogues bear many under-studied properties and the synthesis of members of this family should allow more advances in this field. Our team is particularly interested in this target since its synthesis could be achieved starting from an enantioselective nitroso Diels-Alder reaction which is a major research topic of our group. Later in the synthesis a second key step would involve an intramolecular C-H insertion. The nitroso Diels-Alder reaction was performed with an enantiomeric excess of 96% and the synthesis was continued to access the key diazo intermediate, starting material for the C-H insertion step. This last reaction did not allow to access the desired products using commercially available catalysts and a collaboration was started with Pr Huw Davies in order to use more advanced catalysts. The final results of this collaboration are not available yet. Meanwhile three other synthetic routes for clivonine were designed and investigated as variants of the original strategy but none of them allowed access to clivonine. However one of those syntheses led to the discovery of a new reactivity of nitroso Diels-Alder cycloadducts: when exposed to organolithium reagents, a β-fragmentation occurs followed by an addition leading to highly fonctionnalized monocyclic products with high diastereoselectivity. This reaction and its scope need to be investigated further but may have applications in total synthesis.