Programa de Pós-Graduação em Ciências Farmacêuticas
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Doctoral Thesis Integração de dados moleculares e químicos para análise da biossíntese de metabólitos especializados em Erythrina velutina Willd(Universidade Federal do Rio Grande do Norte, 2024-03-27) Chacon, Daisy Sotero; Giordani, Raquel Brandt; Fett Neto, Arthur Germano; http://lattes.cnpq.br/5032750980466610; https://orcid.org/0000-0003-1932-2301; http://lattes.cnpq.br/6025915331568147; Aragão, Cicero Flávio Soares; http://orcid.org/0000-0002-3434-2602; http://lattes.cnpq.br/9657118649043311; Fett, Janette Palma; Ferreira, Leandro de Santis; Araújo, Wagner LuizThe study of plants is under increasing transformation due to the availability of molecular and bioinformatics tools to understand metabolism and point out potential sources of new bioactive chemical entities with pharmaceutical interest. In this scenario, Erythrina velutina stands out as a medicinal plant found in the northeast region of Brazil, mainly in the Caatinga biome. This plant has a remarkable ability to produce alkaloids and flavonoids, which are the main secondary metabolites of the genus and are often associated with pharmacological activities, such as modulators of the central nervous system (with antidepressant and anxiolytic effects) and anti-inflammatory action. Although some natural products (NP) have been effective as a source of new drugs, the pharmaceutical industry still faces challenges in the identification and optimized production of these compounds and this has led to a reduction in interest in NP-based drugs. In order to minimize these gaps, this study aims to integrate molecular and chemical data to investigate the biosynthesis of the main metabolites present in E. velutina under three aspects: to evaluate the metabolic production in plants collected in natural habitat (Caatinga) and in cultivated plants submitted to different elicitors (ii); identify reference genes that normalize RT-qPCR tests for the species (ii) and evaluate the expression of target genes responsible for the production of alkaloids and flavonoids with bioactive potential (iii). The study focuses on transcriptome, proteome and metabolome data obtained from samples collected in their natural habitat, in addition to seedling cultivation subjected to environmental stresses (temperature, UV, water stress, salt stress and mechanical damage) and phytohormones (methyljasmonate , salicylic acid, nitric oxide and abscisic acid). As a result, the predominant flavonoids observed were flavones, isoflavones, and flavonols, along with several isoquinoline alkaloids from both dienoids and alkenoids. Transcriptome analysis revealed the presence of enzymes involved in the flavonoid and alkaloid biosynthesis pathways, including those differentially expressed, such as EvCHI, EvIF7GT, EvCHS, EvCOR, EvNMT and EvODM. Among the treatments tested, the most significant accumulation of metabolites occurred in response to NO, MeJA, water stress and heat, while ABA caused the least pronounced effect. For normalization of RT-qPCR assays, EvCSTF64, EvRAB7, EvNLE, EvUPF and EvARP4 were identified as the most stable reference genes, while EvNADH and EvPBL27 exhibited lower stability. These genes serve as reliable reference points to ensure accurate normalization of gene expression data in subsequent analyses. The results pointed to NO as crucial in increasing the expression of CHI and the amounts of isoflavones noted. Drought stress negatively affected early precursors of the Erythrina alkaloid pathway, while MeJA had the opposite effect. Exposure to heat increased alkaloid concentrations. This study will contribute to the understanding of the secondary metabolism of plants in the semi-arid/xeric environmental conditions, characteristic of the Caatinga region. In addition, may help to improve the production of bioactive molecules in the species and to value a plant from the Caatinga of Rio Grande do Norte as a source of compounds with pharmaceutical potential. This research represents the first step towards guiding the management for optimal target metabolite production in E. velutina.Master Thesis Avaliação do transcriptoma, proteoma e perfil metabólico na investigação da biossíntese de alcaloides de Erythrina velutina Willd(Universidade Federal do Rio Grande do Norte, 2020-03-20) Chacon, Daisy Sotero; Giordani, Raquel Brandt; http://lattes.cnpq.br/5032750980466610; https://orcid.org/0000-0003-1932-2301; http://lattes.cnpq.br/6025915331568147; Ferreira, Leandro de Santis; https://orcid.org/0000-0002-8408-5886; http://lattes.cnpq.br/6622861873027635; Furlan, MaysaPlants belonging to the genus Erythrina are sources of tetracyclic alkaloids of the benzylisoquinoline type, can be found in northeastern Brazil, in the Caatinga biome and used in folk medicine mainly as a central nervous system depressant (sedative, sedative). One of the limiting factors that many natural products present as medicines is their low yield and difficult to obtain in sufficient quantities to supply the pharmaceutical industry. Thus, according to border studies in the area, it is important to understand aspects of alkaloids biosynthesis through molecular tools. This work aims to contribute to the study in the area of biosynthesis with regard to the alkaloids present in the leaves and seeds of the species Erythrina velutina Willd through “omics” technologies (transcriptome and metabolic profile) in order to understand the dynamics of production of these molecules with bioactive potential. Therefore, for the investigation of the transcriptome we performed the sequencing again for the first time on a species of the genus and on a plant native to the Caatinga biome with this approch. The analysis of the targeted metabolic profile were performed with a Liquid Chromatography strategy coupled to Mass Spectrometry [LC-MS/MS] combined with the Molecular Networking tool for secondary metabolites. As a result, 17 transcripts were identified to form the biosynthetic pathway of isoquinoline alkaloids in seeds and leaves of this species. In addition, 25 alkaloids were noted belonging to the two classes of alkaloids already reported for the genus: dienoid and alkenoid, with 8 alkaloids common to leaves and seeds, 8 found exclusively in seeds and 9 exclusively in leaves. These data contribute to scientific knowledge in the design of biotechnological intervention strategies for future studies, in order to improve the production of these bioactive molecules in the plant, in addition to the enhancement of a plant in the Caatinga of RN as a source of alkaloids with potential pharmaceutical interest.