Orientadores: Sérgio Roberto Peres Line, Marcelo Rocha Marques / Texto em português e inglês / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-24T17:45:16Z (GMT). No. of bitstreams: 1 Ribeiro_MarianaMartins_D.pdf: 2903322 bytes, checksum: 9e0e5e91a22262495ca9bf8ae1d84cec (MD5) Previous issue date: 2014 / Resumo: G-Quadruplexes são estruturas secundárias presentes nas moléculas de DNA e RNA, os quais são formados pelo empilhamento de G-quartetos (interação de quatro guaninas (G-tratos) delimitadas por ligações de hidrogênio do tipo Hoogsteen. O intron 1 do gene PAX9 humano tem um G-quadruplex formado na região localizada perto do exon 1, que é conservada entre os mamíferos placentários. Análises de Dicroísmo Circular (CD), e CD melting mostraram que estas sequências são capazes de formar estruturas quadruplex altamente estáveis. Devido à proximidade da estrutura quadruplex ao limite éxon-íntron foi utilizado um ensaio validado de splicing duplo repórter e PCR em tempo real para analisar o seu papel na eficiência de splicing. O quadruplex humano mostrou ter um papel chave na eficiência de splicing do íntron 1 do gene PAX9, já que uma mutação que aboliu a formação do quadruplex diminuiu drasticamente a eficiência de splicing. O quadruplex de rato, menos estável, mostrou menor eficiência quando comparado com sequências humanas. Além disso, o tratamento com 360A, um forte ligante que estabiliza estruturas quadruplex, aumentou ainda mais a eficiência de splicing do íntron 1 do PAX9 humano. Em conjunto estes resultados fornecem evidências de que as estruturas de G-quadruplex estão envolvidas na eficiência de splicing do intron 1 do gene PAX9 / Abstract: G-Quadruplex are secondary structures present in DNA and RNA molecules, which are formed by stacking of G-quartets (i.e. interaction of four guanines (G-tracts) bounded by Hoogsteen hydrogen bonding). Human PAX9 intron 1 has a putative G-quadruplex- forming region located near exon 1, which is conserved among placental mammals. Using Circular Dichroism (CD) analysis, and CD melting we showed that this region is able to form highly stable quadruplex structures. Due to the proximity of the quadruplex structure to exon-intron boundary we used a validated double reporter splicing assay and real time PCR to analyze its role on splicing efficiency. The human quadruplex was shown to have a key role on splicing efficiency of PAX9 intron 1, as a mutation that abolished quadruplex formation decreased dramatically splicing efficiency. The less stable, rat quadruplex had a less efficient splicing when comparing to human sequences. Additionally, the treatment with 360A, a strong ligand that stabilizes quadruplex structures, further increased splicing efficiency of human PAX9 intron 1. Altogether these results provide evidences that G-quadruplex structures are involved in splicing efficiency of PAX9 intron 1 / Doutorado / Histologia e Embriologia / Doutora em Biologia Buco-Dental

Le développement des nanoparticules fluorescentes, appelées quantum dots (QDs) est devenu l'un des domaines les plus prometteurs de la science des matériaux. Dans cette étude une procédure de synthèse de QDs a été mise au point, comprenant la synthèse de noyaux ultra-minces de CdSe, la purification de noyau haute performance, le revêtement central avec une coquille épitaxiale en ZnS. Cette approche a permis d’obtenir des QDs d’une taille de 3,7 nm possédant un rendement quantique supérieur à 70%. Les QDs développés ont été utilisés pour concevoir des conjugués de QDs compacts avec les nouveaux dérivés d'acridine, ayant une affinité élevée pour le G-quadruplex des télomères, ainsi que leur effet inhibiteur sur la télomérase, une cible importante du traitement du cancer. Les résultats de cette étude ouvrent la voie à l'ingénierie de nanosondes multifonctionnelles possédant une meilleure pénétration intracellulaire, une plus forte brillance et une stabilité colloïdale plus importante. / Development of the fluorescent nanoparticles referred to as quantum dots (QDs) has become one of the most promising areas of materials sciences. In this study, a procedure of synthesis of QDs, which includes the synthesis of ultrasmall CdSe cores, high-performance purification, core coating with an epitaxial ZnS shell has been developed. This approach has allowed obtaining 3.7-nm QDs with a quantum yield exceeding 70%. The QDs have been used: to engineer compact conjugates of QDs with the novel acridine derivatives, which have a high affinity for the telomere G-quadruplex; to demonstrate their inhibitory effect on telomerase, an important target of anticancer therapy; and to accelerate transmembrane penetration of ultrasmall QDs into cancer cells while retaining a high brightness and colloidal stability. The results of this study pave the way to the engineering of multifunctional nanoprobes with improved intracellular penetration, brightness, and colloidal stability.

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