Les micro-ARNs (miRNAs) sont des ARNs de 20-22 nucléotides, transcrits à partir du génome, dont la fonction est de réguler l’expression génique en s’appariant à des ARNm cibles, inhibant ainsi leur traduction et/ou entrainant leur dégradation. Dans les cancers, l’expression des miRNAs est fortement dérégulée. Une majorité de miRNAs est diminuée dans les tissus tumoraux par rapport aux tissus normaux, et un lien causal a été décrit entre inhibition globale des miRNAs et tumorigenèse. Par ailleurs, des miRNAs agissant comme des suppresseurs de tumeurs et d’autres comme des oncogènes ont été décrits. Dans ce contexte impliquant de plus en plus les miRNAs dans les pathologies néoplasiques, l’objectif de ce travail était d’étudier le rôle de la voie miRNA dans la biologie des cellules tumorales. Afin d’identifier des cellules tumorales dépendant de miRNAs oncogènes endogènes pour survivre ou proliférer, nous avons développé une stratégie d’inhibition globale de la biogenèse des miRNAs en ciblant Drosha ou DGCR8, les deux composants du microprocesseur, complexe nucléaire de maturation des miRNAs. Cette stratégie nous a permis d’identifier des lignées cellulaires tumorales dans lesquelles l’inhibition du microprocesseur conduit à un phénotype d’arrêt de prolifération durable. Nous avons mis à profit cette dépendance à la voie miRNA pour réaliser un crible positif de complémentation du défaut de prolifération observé grâce à l’expression de miRNAs individuels. Nous avons ainsi pu mettre en évidence des miRNAs capables de soutenir individuellement la prolifération de ces cellules tumorales. Cette stratégie nous a également permis de montrer des différences fonctionnelles entre miRNAs homologues ou de la même famille. La recherche des cibles régulées par ces miRNAs nous a permis d’élaborer des hypothèses concernant les cibles potentiellement impliquées dans le phénotype observé. Nous avons ainsi démontré la participation du suppresseur de tumeur PTEN à l’arrêt de prolifération induit par l’inhibition du microprocesseur. La stratégie d’inhibition globale de la voie miRNA suivie d’une complémentation phénotypique par des miRNAs individuels permet de s’affranchir de la grande redondance de séquence et de fonction des miRNAs et devrait pouvoir s’appliquer d’une manière plus générale à l’étude d’autres processus régulés par les miRNAs. / MicroRNAs (miRNAs) are 20-22 nucleotides RNAs, transcribed from the genome, which regulate gene expression by base-pairing to target mRNAs, thus inhibiting their translation and/or leading to their degradation. In cancers, miRNAs expression is strongly deregulated. A majority of miRNAs is diminished in tumoral tissues compared to normal tissues, and a causal link has been established between global inhibition of the miRNA pathway and tumorigenesis. In addition, miRNAs acting like tumor suppressors or oncogenes have been described. In this context of growing evidences implicating miRNAs in neoplasic diseases, this work aimed to investigate the role played by miRNA pathway in the biology of tumoral cells. In order to identify tumoral cells depending on endogenous oncogenic miRNAs to proliferate or survive, we developed a strategy of global inhibition of miRNAs biogenesis by targeting Drosha or DGCR8, the two components of the “microprocessor”, the nuclear miRNA maturation complex. This strategy allowed us to identify tumoral cell lines in which microprocessor inhibition led to a sustained growth arrest. We took advantage of this miRNA pathway dependency to screen for individual miRNAs able to complement the observed growth defect. This complementation screen allowed us to identify individual miRNAs able to sustain growth in those tumoral cells. This strategy also highlighted functional differences between homologous miRNAs or between miRNAs from the same family. The search for targets regulated by those miRNAs allowed us to develop hypothesis concerning the potential targets involved in the observed phenotype. By using this approach, we demonstrated that the tumor suppressor PTEN was involved in the growth arrest induced by microprocessor inhibition. The strategy of global miRNA pathway inhibition followed by phenotypic complementation by individual miRNAs allows overcoming the high sequence and function redundancy of miRNAs. We thus think it could be applied more generally to the study of other cellular processes regulated by miRNAs.

Made available in DSpace on 2014-12-17T14:05:22Z (GMT). No. of bitstreams: 1 CristianeFA_TESE.pdf: 2715743 bytes, checksum: 62bfe71508cd021b46627368e67b663d (MD5) Previous issue date: 2010-01-18 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Chitosan is a natural polymer, biodegradable, nontoxic, high molecular weight derived from marine animals, insects and microorganisms. Oligomers of glucosamine (GlcN) and N-acetylglucosamine (GlcNAc) have interesting biological activities, including antitumor effects, antimicrobial activity, antioxidant and others. The alternative proposed by this work was to study the viability of producing chitooligosaccharides using a crude enzymes extract produced by the fungus Metarhizium anisopliae. Hydrolysis of chitosan was carried out at different times, from 10 to 60 minutes to produce chitooligosaccharides with detection and quantification performed by High Performace Liquid Chromatography (HPLC). The evaluation of cytotoxicity of chitosan oligomers was carried out in tumor cells (HepG2 and HeLa) and non-tumor (3T3). The cells were treated for 72 hours with the oligomers and cell viability investigated using the method of MTT. The production of chitosan oligomers was higher for 10 minutes of hydrolysis, with pentamers concentration of 0.15 mg/mL, but the hexamers, the molecules showing greater interest in biological properties, were observed only with 30 minutes of hydrolysis with a concentration of 0.004 mg/mL. A study to evaluate the biological activities of COS including cytotoxicity in tumor and normal cells and various tests in vitro antioxidant activity of pure chitosan oligomers and the mixture of oligomers produced by the crude enzyme was performed. Moreover, the compound with the highest cytotoxicity among the oligomers was pure glucosamine, with IC50 values of 0.30; 0.49; 0.44 mg/mL for HepG2 cells, HeLa and 3T3, respectively. Superoxide anion scavenging was the mainly antioxidant activity showed by the COS and oligomers. This activity was also depending on the oligomer composition in the chitosan hydrolysates. The oligomers produced by hydrolysis for 20 minutes was analyzed for the ability to inhibit tumor cells showing inhibition of proliferation only in HeLa cells, did not show any effect in HepG2 cells and fibroblast cells (3T3) / A quitosana ? um pol?mero natural, biodegrad?vel, n?o t?xico e de alta massa molecular obtido a partir de animais marinhos, insetos e microrganismos. Os olig?meros de glicosamina (GlcN) e N-acetilglicosamina (GlcNAc) t?m atividades biol?gicas interessantes, incluindo efeitos antitumorais, atividade antimicrobiana, antioxidante entre outras. A alternativa proposta por este trabalho foi estudar a viabilidade de produ??o de quitooligossacar?deos utilizando um extrato bruto de enzimas produzidas pelo fungo Metarhizium anisopliae. A hidr?lise da quitosana foi realizada em diferentes tempos, de 10 a 60 minutos para a produ??o de quitooligossacar?deos e a detec??o e quantifica??o foi realizado pela Cromatografia L?quida de Alta Efici?ncia. A avalia??o da citotoxicidade dos olig?meros de quitosana foi realizada em c?lulas tumorais (HepG2 e HeLa) e n?o tumoral (3T3). As c?lulas foram tratadas durante 72 horas com os olig?meros e a viabilidade celular foi feita usando o m?todo do MTT. A produ??o de olig?meros de quitosana teve maiores rendimentos durante 10 minutos de hidr?lise, os pent?meros apresentaram concentra??o de 0,15 mg/mL, por?m os hex?meros, que apresentam maior interesse pelas suas propriedades biol?gicas, s? foram detectados com 30 minutos de hidr?lise apresentando uma concentra??o de 0,004 mg/mL. Um estudo visando avaliar as atividades biol?gicas dos QCOS entre elas a citotoxicidade em c?lulas tumorais e normais e v?rios testes de atividade antioxidante in vitro entre olig?meros puros de quitosana e a mistura dos olig?meros produzidos pelo extrato bruto enzim?tico foi realizado. A glicosamina foi o composto com a maior toxicidade dentre os olig?meros puros, apresentando valores de IC50 0,30; 0,49; 0,44 mg/mL para c?lulas HepG2, HeLa e 3T3, respectivamente. O seq?estro do ?nion super?xido foi a principal atividade antioxidante mostrada pelos QCOS. Sendo que essa atividade tamb?m foi dependende da composi??o dos hidrolisados de quitosana. Os olig?meros produzidos por hidr?lise durante 20 minutos foram analisados quanto ? capacidade de inibir c?lulas tumorais mostrando inibi??o da prolifera??o apenas nas c?lulas HeLa, n?o apresentando nenhum efeito em c?lulas HepG2 e c?lulas de fibroblastos (3T3)

Made available in DSpace on 2014-12-17T14:03:39Z (GMT). No. of bitstreams: 1 RaquelHBA_DISSERT.pdf: 1772501 bytes, checksum: 51cea5d11ac1524fb197874e85c15f64 (MD5) Previous issue date: 2012-07-20 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Heparin is a pharmaceutical animal widely used in medicine due to its potent anticoagulant effect. Furthermore, it has the ability to inhibit the proliferation, invasion and adhesion of cancer cells to vascular endothelium. However, its clinical applicability can be compromised by side effects such as bleeding. Thus, the search for natural compounds with low bleeding risk and possible therapeutic applicability has been targeted by several research groups. From this perspective, this study aims to evaluate the hemorrhagic and anticoagulant activities and citotoxic effect for different tumor cell lines (HeLa, B16-F10, HepG2, HS-5,) and fibroblast cells (3T3) of the Heparin-like from the crab Chaceon fenneri (HEP-like). The HEP-like was purified after proteolysis, ion-exchange chromatography, fractionation with acetone and characterized by electrophoresis (agarose gel) and enzymatic degradation. Hep-like showed eletroforetic behavior similar to mammalian heparin, and high trisulfated /Nacetylated disaccharides ratio. In addition, HEP-like presented low in vitro anticoagulant activity using aPTT and a minor hemorrhagic effect when compared to mammalian heparin. Furthermore, the HEP-like showed significant cytotoxic effect (p<0.001) on HeLa, HepG2 and B16-F10 tumor cells with IC50 values of 1000 ug/mL, after incubation for 72 hours. To assess the influence of heparin-like on the cell cycle in HeLa cells, analysis was performed by flow cytometry. The results of this analysis showed that HEP-like influence on the cell cycle increasing S phase and decreasing phase G2. Thus, these properties of HEP-like make these compounds potential therapeutic agents / A heparina ? um agente farmac?utico amplamente utilizado em medicina devido ao seu potente efeito anticoagulante. Al?m disso, tem a capacidade de inibir a prolifera??o, invas?o e ades?o de c?lulas cancerosas ao endot?lio vascular. No entanto, a sua aplicabilidade cl?nica pode ser comprometida por efeitos secund?rios tais como hemorragia. Assim, a busca de compostos naturais com baixo risco hemorr?gico e poss?vel aplicabilidade terap?utica tem sido alvo de v?rios grupos de pesquisa. A partir desta perspectiva, este estudo visa avaliar as atividades hemorr?gica, anticoagulante e efeito citot?xico para as diferentes linhagens de c?lulas tumorais (HeLa, B16-F10, HepG2, HS-5,) e c?lulas de fibroblastos (3T3) proporcionadas pelo composto tipo heparina obtido do caranguejo Chaceon fenneri. Dessa forma, o composto foi purificado ap?s prote?lise, cromatografia de troca i?nica e fracionamento com acetona, e caracterizado por eletroforese em gel de agarose e degrada??o enzim?tica. O composto em estudo mostrou comportamento eletrofor?tico semelhante ? heparina de mam?fero, e alta raz?o de propor??o de dissacar?deos trissulfatado / N-acetilado. Al?m disso, o composto apresentou baixa atividade anticoagulante in vitro usando aPTT e um efeito hemorr?gico menor quando comparado com heparina de mam?fero. O composto tipo heparina obtido do caranguejo Chaceon fenneri mostrou efeito citot?xico significativo (p <0,001) em c?lulas linhagens de c?lulas tumorais HeLa, HepG2 e B16-F10 com valores de IC50 de 1000 ug / mL, ap?s a incuba??o durante 72 horas. Para avaliar a influ?ncia do composto sobre o ciclo celular em c?lulas HeLa, foi realizada uma an?lise por citometria de fluxo. Os resultados desta an?lise mostraram que a influ?ncia do composto sobre o ciclo celular aumenta a fase S e diminui a fase G2. Assim, essas propriedades do composto tipo heparina obtido do caranguejo Chaceon fenneri sugerem este composto como um agente terap?utico em potencial