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.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22733号 / 医博第4651号 / 新制||医||1046(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 萩原 正敏, 教授 岩田 想, 教授 渡邊 直樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

As of today, synthetic cannabinoids are one of the biggest groups of new psychoactive substances.These substances can be used as substitutes for the psychoactive drug cannabis, avoiding the legalrestrictions on cannabis. Furthermore, a variety of synthetic cannabinoids are synthesized with eithersignificant or very minor structural differences, making the detection of said novel drugs hard to keepup with and is therefore of great importance to have standards which help in the identification of theintake of the parent synthetic cannabinoid. In this project, several metabolites ofsynthetic cannabinoids with indole/indazole cores with differentside chains was synthesized. The general strategy used in this project was to N-alkylate the desiredcore followed by amide coupling with L-tert-leucine methyl ester or L-Valine methyl esterhydrochloride which resulted in 8 potential synthetic cannabinoid metabolites.

Les alcaloïdes indolo-monoterpéniques (AIMs) constituent une classe de molécules naturelles très étudiée en raison d’un fort potentiel pharmacologique et thérapeutique et d’une grande diversité structurale. D’autre part, les techniques de déréplication par chromatographie liquide couplée à la spectrométrie de masse tandem ont évolué récemment, avec l’émergence de l’approche par réseaux moléculaires (molecular networking). Dans ce contexte, nous avons créé une base de données de spectres de masse tandem moyennés de 172 AIMs témoins, en collaboration avec plusieurs équipes de pharmacognosie dans le monde. Cette base de données, nommée MIADB (Monoterpene Indole Alkaloids DataBase), rendue publique, peut être utilisée comme référence dans des stratégies de déréplication fondées sur l’utilisation des réseaux moléculaires. Nous avons ensuite exploité la MIADB pour l’étude phytochimique de deux Apocynaceae : Geissospermum laeve (Vell.) Miers et Alstonia balansae Guillaumin. Dans un premier temps, l’annotation par la MIADB d’un extrait alcaloïdique des écorces de G. laeve a permis l’isolement et l’élucidation structurale de 3 nouveaux AIMs, dont deux à motif butyrolactone. Par la suite, notre approche a été améliorée par l’emploi du nouvel outil d’annotation in silico MetWork, fondé sur une prédiction métabolique et la modélisation de spectres de masse tandem. C’est ainsi que l’exploration de l’espace chimique d’un extrait alcaloïdique des feuilles d’A. balansae a permis l’identification, puis l’isolement, de 5 nouveaux AIMs du type N-oxyde-sarpagane. La stéréochimie des nouveaux composés a pu être déterminée par l’exploitation de spectres prédits et expérimentaux de dichroïsme circulaire éléctronique.Ce manuscrit décrit, après une introduction consacrée notamment aux emplois du molecular networking pour le ciblage et la découverte de petites molécules naturelles puis aux interconnections biosynthétiques en série AIM, la création puis l’évolution de la MIADB, puis son utilisation dans un workflow de déréplication efficace et de ciblage de nouveaux composés dans des mélanges complexes issus d’Apocynaceae. / Monoterpene indole alkaloids (MIAs) constitute a class of natural products that has been extensively studied due to its important pharmacological and therapeutic potentials, and to its large structural diversity. Dereplication techniques based on liquid chromatography coupled to tandem mass spectrometry have recently evolved, with the implementation of molecular networking-based approaches.In this context, we have created a spectral database that encompasses the averaged tandem mass spectra of 172 reference MIAs, in collaboration with several pharmacognosy research teams around the world. This database, named MIADB (standing for Monoterpene Indole Alkaloids DataBase), was made publicly available and can be used as a reference in the application of molecular networking as a dereplication strategy. Thereafter, we used the MIADB to carry out the phytochemical investigation of two Apocynaceae species: Geissospermum laeve (Vell.) Miers and Alstonia balansae Guillaumin. As a first application, the MIADB-based annotation of an alkaloid extract of the barks of G. laeve led to the isolation and the structural elucidation of three new MIAs, two having a butyrolactone moiety. Afterwards, this approach was improved by the application of a new tool for in silico annotation called MetWork, which is based on metabolic prediction and on the generation of predicted tandem mass spectra. Following this approach, the exploration of the chemical space of an alkaloid extract of the leaves of A. balansae allowed the anticipation and further isolation of five novel MIAs of the N-oxide-sarpagine type. The stereochemistry of all the new molecules could be determined on the basis of experimental and predicted electronic circular dichroism spectra.In the introduction of this manuscript the multiple uses of molecular networking for the identification of small natural molecules are described, as well as the biosynthetic interconnections in the MIAs group. The creation and evolution of the MIADB are then presented, followed by its utilization in efficient dereplication workflows for the targeting of new natural products within complex mixtures from Apocynaceae species.

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V. Hojgrová: Alkaloids of Vinca minor L. and their biological activity VIII. Diploma thesis, Charles University, Faculty of Pharmacy in Hradec Králové, Department of Pharmaceutical Botany. Number of pages 69. This diploma thesis deals with the isolation of alkaloids from Vinca minor L. from the family Apocynaceae. Separation of alkaloids from the selected fraction (VM 215-258) or from their subfractions (VM 34-41, VM 86, VM 87-113) was performed by preparative TLC. Two pure alkaloids were isolated from the subfraction (VH 34-41). The first VH-1 alkaloid not yet isolated and the second VH 2 alkaloid: (-)-raucubainin. Alkaloids were identified by EI-MS, LC-MS, NMR and optical rotation and were compared with data in the literature. Isolated alkaloids were tested for acetylcholinesterase (AChE), butyrylcholinesterase (BuChE) and prolyloligopeptidase (POP) inhibitory activity and for cytotoxicity. Both substances did not show significant cholinesterase inhibitory activity IC50 against AChE after measurement, only (-)-raucubainin showed a slight activity against BuChE (IC50 = 94 ± 7 μM), VH-1 was found to be inactive (IC50 > 100 μM). POP inhibitory activity has so far only been tested for (-)-raucubainin; was found to be inactive (IC50 > 1000 µM). The results of the cytotoxic activity of the alkaloids...

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Catharanthus roseus produces a large array of terpenoid indole alkaloids (TIAs) that are an important source of natural or semi-synthetic anticancer drugs. Biosynthesis of TIAs is tissue-specific and induced by certain phytohormones and fungal elicitors, indicating the involvement of a complex transcriptional control network. However, the transcriptional regulation of the TIA pathway is poorly understood. This study reports the isolation and characterization of the G10H promoter and two WRKY transcription factors regulating TIA biosynthesis. Geraniol 10-hydroxylase (G10H) controls the first committed step in the biosynthesis of terpenoid indole alkaloids (TIA). The C. roseus G10H promoter sequence was isolated by a PCR-based genome walking method. Sequence analysis revealed that the G10H promoter contains several potential eukaryotic regulatory elements involved in regulation of gene expression. For functional characterization, fusion constructs of G10H promoter fragments with the GUS reporter gene were generated and expression was analyzed in a tobacco protoplast transient expression assay. Gain-of-function experiments revealed the presence of three potential transcriptional enhancers located in regions between -191 and -147, -266 and -188, and -318 and -266, respectively. The G10H promoter was capable of conferring stable GUS expression in transgenic tobacco plants and C. roseus hairy roots. In transgenic tobacco seedlings, GUS expression was tissue-specific, restricted to the leaf and actively growing cells around the root tip. GUS expression was not detected in the hypocotyls, root cap and older developing areas of the root. The GUS expression in both transgenic C. roseus hairy roots and tobacco seedlings were responsive to fungal elicitors and methyljasmonate. Compared to other known promoters of TIA pathway genes, the G10H promoter contains unique binding sites for several transcription factors, suggesting that the G10H promoter may be regulated by a different transcriptional cascade. The majority of TIA pathway gene promoters contain typical W-box elements, which are frequently found to be the binding sites of WRKY transcription factors. CrWRKY1 and CrWRKY2 transcription factors were isolated using a degenerate PCR method. The C. roseus WRKY transcription factor, CrWRKY1 is preferentially expressed in roots and induced by phytohormones, jasmonate, gibberellic acid and ethylene. Overexpression of CrWRKY1 in C. roseus hairy roots up-regulated several key TIA pathway genes, especially tryptophan decarboxylase (TDC), as well as transcriptional repressors ZCT1, ZCT2 and ZCT3. In contrast, CrWRKY1 overexpression repressed the transcriptional activators ORCA2, ORCA3 and CrMYC2. Overexpression of a dominant-repressive form of CrWRKY1, created by fusing the SRDX-repressor domain to CrWRKY1, resulted in down-regulation of TDC and ZCTs but up-regulation of ORCA3 and CrMYC2. CrWRKY1 bound to the W-box elements of the TDC promoter in electrophoretic mobility shift, yeast one-hybrid and C. roseus protoplast assays. In CrWRKY1 hairy roots, up-regulation of TDC increased TDC activity, tryptamine concentration and resistance to 4-methyl tryptophan inhibition. Compared to control roots, CrWRKY1 hairy roots accumulated up to 3-fold higher levels of serpentine. The preferential expression of CrWRKY1 in roots and its interaction with transcription factors, including ORCA3, CrMYC2 and ZCTs, may play a key role in determining the root-specific accumulation of serpentine in C. roseus plants. CrWRKY2 is induced by methyljasmonate induction. In plant, CrWRKY2 expression is mainly found in young leaves and the stem. The stable transformation of CrWRKY2 in C. roseus hairy roots up-regulated many pathway genes, especially the genes in vindoline biosynthesis. The accumulation of vindoline was observed in CrWRKY2 hairy roots.

Estudos têm mostrado a incidência de mastite bovina associada à alga Prototheca zopfii. O objetivo deste trabalho foi estudar a interação da P. zopfii com neutrófilos recuperados de leite bovino e avaliar o efeito do sistema ácido indol-3-acético/peroxidase de raiz forte (AIA/HRP) sobre a viabilidade deste microrganismo em experimentos in vitro. A P. zopfii foi recuperada de vacas com mastite clínica e, no laboratório, foram realizadas a caracterização molecular, morfológica e crescimento exponencial do microrganismo. Em seguida, neutrófilos recuperados de leite bovino foram incubados na ausência e na presença de P. zopfii opsonizada e foram avaliadas a produção de peróxido de hidrogênio, enzimas antioxidantes dos neutrófilos e microrganismo, e a capacidade fagocitária. Em outro estudo, a P. zopfii foi incubada com o sistema AIA/HRP e foram avaliadas a viabilidade por unidades formadoras de colônias (UFC), atividade de enzimas antioxidantes, integridade de membrana por exclusão com azul de Trypan e integridade do DNA. Os resultados foram analisados pela análise de variância com significância de 5% usando o teste Tukey. Foram observados diversos tamanhos celulares da P. zopfii, presença de autofluorescência, crescimento exponencial ao longo do tempo de incubação em que não foi possível determinar o início da fase de morte. Ainda, foram encontrados os genótipos 1, 2 e 3 nos isolados em estudo. A produção de peróxido de hidrogênio pelos neutrófilos na presença da alga foi estimulada 5 vezes em relação ao controle, estimulou a atividade das enzimas catalase (CAT) em 21% e glutationa redutase (GR) em 27% e não houve diferença significativa quanto à atividade de CAT, GR e superóxido dismutase (SOD) produzido pela P. zopfii. Também foi verificado que a P. zopfii não foi englobada pelo neutrófilo. O sistema AIA/HRP inibiu o crescimento do microrganismo em 45, 82 e 88% nos tempos de 4, 6 e 9 horas de incubação; a atividade da SOD, CAT, Glutationa Peroxidase (GPx) e GR aumentou respectivamente em 90, 120, 150% e 3,4 vezes; houve redução da viabilidade da P. zopfii em 10, 15, 20, 25 e 32% após os tempos de 4, 6, 8, 10 e 12 horas de incubação; e não afetou a integridade do DNA após 6 horas de incubação. Conclui-se que a P. zopfii é altamente resistente frente aos neutrófilos e demonstrou ser susceptível quanto ao efeito microbicida do sistema AIA/HRP. / Studies have shown the incidence of bovine mastitis associated with the algae Prototheca zopfii. The objective of this work was to study the interaction of P. zopfii with neutrophils recovered from bovine milk and to evaluate the effect of system indole-3-acetic acid/horseradish peroxidase (IAA/HRP) on the viability of this microorganism in vitro experiment. P. zopfii was recovered from cows with clinical mastitis and both the molecular and morphological characterization were performed besides the evaluation of exponential growth of the microorganism in the laboratory. Next, neutrophils recovered from bovine milk were incubated in the absence and presence of opsonized P. zopfii and were evaluated the production of hydrogen peroxide, antioxidant enzymes on neutrophils and microorganism, and phagocytic capacity. In another study, P. zopfii was incubated with the system IAA/HRP and the viability assessed by colony forming units (CFU), antioxidant enzymes activity, membrane integrity by exclusion with Trypan blue and DNA integrity. The results were analyzed by analysis of variance with a 5% significance using the Tukey test. Results from P. zopfii characterization showed various cellular sizes, presence of autofluorescence, exponential microorganism growth throughout the incubation time and was not possible to determine the beginning of the death. Moreover it was found genotypes 1, 2 and 3 in the isolates in study. The production of hydrogen peroxide by neutrophils in the presence of algae was stimulated 5 times compared to the control, increase the activity of catalase (CAT) in 21% and glutathione reductase (GR) in 27% was seen in neutrophils; and there was no significant difference in CAT, GR and superoxide dismutase (SOD) activity produced by P. zopfii. P. zopfii was not engulfment by neutrophils. The system IAA/HRP inhibited the growth of the microorganism in 45, 82 and 88% in the times of 4, 6 and 9 hours of incubation, the activity of SOD, CAT, glutathione peroxidase (GPx) and GR increased respectively by 90, 120, 150%, and 3.4 times, decreased the viability of P. zopfii 10, 15, 20, 25 and 32% after the times of 4, 6, 8, 10 and 12 hours of incubation, and did not affect the integrity of DNA after 6 hours of incubation. As a conclusion, P. zopfii is highly resistant to the neutrophils and demonstrated to be susceptible to the effect microbicidal of system IAA/HRP.

A Brachiaria brizantha é considerada uma das forrageiras preferidas entre os agropecuaristas por possuir elevada produção de forragem, tolerância ao calor e ao déficit hídrico, alta resposta à aplicação de fertilizantes, produção em grande massa de raízes e sementes, resistência à cigarrinha das pastagens (exceto as pertencentes ao gênero Mahanarva) e boa competição com plantas invasoras. É considerada a principal fonte de alimento para bovinos, sendo utilizada tanto na cria, recria, como na engorda dos animais. As bactérias fixadoras de nitrogênio ou diazotróficas são procariotos capazes de reduzir o N2 a NH3, forma assimilável pelos organismos, e também podem produzir hormônios vegetais, como ácido-indol-acético, que estimulam o crescimento radicular da planta. Estes micro-organismos apresentam grande importância para a manutenção dos ecossistemas. Sua associação com as raízes de plantas e seu efeito promotor quando associados à Brachiaria brizantha possibilitaria a recuperação de áreas de pastagens que apresentam deficiência de nitrogênio, o que é um mecanismo ainda pouco explorado. Com o objetivo de estudar esta possibilidade, foram escolhidas três áreas (Nova Odessa-SP, São Carlos- SP e Campo Verde-MT), preferencialmente onde o nitrogênio era limitante, constituídas por pastagem de Brachiaria brizantha para a amostragem de solo e raiz. Os três locais demonstraram a ocorrência de diazotróficos, após o isolamento e cultivo das bactérias em meio de cultivo semisólido sem adição de nitrogênio na forma combinada (JNFb). Foram obtidas 110 estirpes bacterianas e, após sorteio aleatório, 72 isolados foram mantidos para realização de testes a fim de se avaliar o potencial biotecnológico das bactérias. Destes, 10 demonstraram atividade da nitrogenase quando submetidos ao método de aumento na concentração de nitrogênio total (Ntotal) em meio de cultura. 57 isolados foram capazes de reduzir o gás acetileno a etileno quando submetidos à técnica de redução de acetileno. As estirpes bacterianas C4 (Pseudomonas sp.) e C7 (Azospirillum sp.), isoladas da rizosfera de Brachiaria brizantha da área de Campo Verde-MT, se destacaram das demais por apresentar atividade da nitrogenase muito superior até a de bactérias diazotróficas que foram incluídas na avaliação como testemunhas positivas. Outros 68 isolados produziram o hormônio vegetal ácido-indol-acético quando cultivados em meio de cultivo LB, na presença de triptofano. A produção variou de 0,39µg/mL a 195 µg/mL de AIA. Todos os 72 isolados foram utilizados em experimento em casa de vegetação para avaliar o efeito de inoculação em B. brizantha quando com eles inoculada. Avaliaram-se a matéria seca da parte aérea e raiz e o teor de nitrogênio total da parte aérea através do método micro-Kjeldhal. Nenhum isolado diferiu significativamente do controle sem inoculação bacteriana que continha a mesma dose de nitrogênio fornecido às plantas. O seqüenciamento parcial do gene 16S rRNA dos 72 isolados permitiu a caracterização de sete grupos genotípicos: Stenotrophomonas sp, Pseudomonas sp., Xanthomonas sp., Bacillus sp., Rhizobium sp, Sphingomonas sp. e Azospirillum sp. O gênero Stenotrophomonas sp. predominou (69%) nas três áreas de estudo. / Brachiaria brizantha is considered one of preferred fodders among farmers for having high forage yield, tolerance to heat and drought, high response to fertilizer application, large production of root mass and seeds, resistance to grassland leafhopper (except those belonging to the genus Mahanarva) and good competition with weeds. It is considered the main source of food for cattle, being used in the raising, breeding, and fattening of animals. The nitrogen fixing bacteria or diazotrophs are prokaryotes able to reduce N2 to NH3, which is assimilated by organisms, and may also produce plant hormones such as indole-acetic acid, which stimulates root growth. These micro-organisms have great importance for the maintenance of ecosystems. Their association with plant roots and their promoting effect when combined with Brachiaria brizantha enable recovery of nitrogen-deficient grazing areas, which is a mechanism still little explored. Therefore, three areas were chosen (Nova Odessa-SP, Sao Carlos-SP and Campo Verde-MT), preferably where nitrogen was limiting, consisting of Brachiaria brizantha from which samples of soil and roots were collected. The three sites showed the occurrence of diazotrophs after the isolation and cultivation of bacteria in semi-solid culture medium with no nitrogen added in the combined form (JNFb). It was obtained 110 bacterial strains and, after the raffle random, 72 were kept isolated for testing in order to assess the biotechnological potential of bacteria. From which, 10 showed nitrogenase activity when subjected to the method of total nitrogen concentration increase (N-total) in the culture medium. 57 isolates were able to reduce acetylene to ethylene when subjected to the acetylene reduction technique. The strains C4 (Pseudomonas sp.) and C7 (Azospirillum sp.), isolated from the rhizosphere of Brachiaria brizantha in the area of Campo Verde-MT, stood out from the others by presenting nitrogenase activity far superior to that of diazotrophs recommended as positive controls. Other 68 isolates produced the plant hormone indole-acetic acid when grown in LB culture medium in the presence of tryptophan. Production ranged from 0.39 g/mL to 195 g/mL of IAA. All 72 isolates were used in an experiment in a greenhouse to evaluate the effect of inoculation on B. brizantha. Evaluations were carried out on dry matter of shoot and root and total nitrogen content of the shoot through the micro-Kjeldahl method. None of the isolates differed significantly from the control without bacterial inoculation which contained the same amount of nitrogen supplied to plants. Partial sequencing of the 16S rRNA of the 72 isolates allowed the characterization of seven genotype groups: Stenotrophomonas sp., Pseudomonas sp., Xanthomonas sp., Bacillus sp., Rhizobium, Sphingomonas sp. and Azospirillum sp. The genus Stenotrophomonas sp. predominated (69%) in the three study areas.