Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 5 uppsatser.

Thesis (M.Phil.)--University of Hong Kong, 2003. / Includes bibliographical references (leaves 65-71) Also available in print.

Theses (Ph. D.)--Marshall University, 2004. / Title from document title page. Includes abstract. Document formatted into pages: contains xiv, 192 p. including illustrations. Bibliography: p. 157-192.

Universiẗat, Diss., 2000--Dortmund. / Dateiformat: PDF.

Universiẗat, Diss., 2004--Bielefeld. / Erscheinungsjahr an der Haupttitelstelle: 2003.

Made available in DSpace on 2014-08-20T13:32:48Z (GMT). No. of bitstreams: 1 tese_ciane_xavier_goncalves.pdf: 2548463 bytes, checksum: 75f8193b6bd48d05e54efb24e14099b9 (MD5) Previous issue date: 2013-03-08 / Ethylene is the inductor and acceleratorhormone of the maturation and senescence of climacteric fruits, as is the case of Cantaloupe melons (Cucumismelo var. Cantalupensis, Naud cv. Vedrantais) and tomatoes (LicopersicumesculentumL. cv. Micro-Tom). It is known that the reduction of production and/or the action of this hormone prolongs the shelf life of these fruits. Thus, in order to reduce the production of such hydrocarbon, were genetically transformed melon trees with clones of the ACC oxidase 'antisense', of melon (pMEL1AS, Ayubet al., 1996) and of apple (pAP4AS, Silva et al., 2004). As expected, in both cases, the ethylene production was reduced, prolonging shelf life of fruits in 7 days. However, the production of volatile compounds was also affected. The transgenic melons produced, on average, 70% less esters than the WT, independently of having been transformed with pMEL1AS or pAP4AS. As the intervention made acted in the reduction of the production of ethylene. It was emitted the hypothesis that, with the supplementation of the hormone, the synthesis of flavors would be restored. Such fact was proven in pMEL1AS melons, but not in pAP4AS. The exact causes of this difference were not yet made clear. It is believed that because of the greater reduction of the ethylene production in the fruit pAP4AS, besides affecting genes directly related to the ripening classical metabolic pathways, genes related to other pathways of synthesis of hormones have also been affected, as is the case of cytokininsor polyamines. By observation of the phenotype of pAP4AS plants we realized that these had more vegetative growth and lateral sproutings. Considering such observations, it was launched the hypothesis that with the increase of the levels of cytokinins one could interfere in the responses to ethylene, delaying the normal evolution of the fruits. In pAP4AS melons it was proved that there are higher levels of cytokinins, both in the roots and fruits.However, to confirm that these changes are effectively consequence of higher levels of cytokinins, exogenous application of that growth regulators was made. Nevertheless, were not observed any expected responses. So we decided to test the application of cytokinin in another plant model, the tomato tree cv. Micro- Tom, who is also a producer of climacteric fruits and that provides greater ease of cultivation and obtainment of greater quantity of fruit in protected environments and in areas with limited space. As molecular variables for assessment of the treatments in melons were determined the accumulation of transcript of genes of HPL, LOX and AAT, besides the ACCO and ACCS. In tomatoes, were performed physicochemical analyzes. For melons, when quantifying the accumulation of transcripts of genes of ACC synthase (ACCS), it was found that genes ACCS1 and ACCS3 showed higher expressions in WT fruits, suggesting that they have a strong relation with the evolution of the climacteric crisis. Moreover, the gene ACCS5 was more expressed in fruits pMEL1AS and pAP4AS, indicating that it is negatively regulated by ethylene. For the other variables evaluated (total soluble solids, color, carotenoids, chlorophylls, pulp firmness) during the ripening on the plant, there were no marking differences between pMEL1AS and pAP4AS. After harvesting and treatment of pMEL1AS fruits with ethylene, there was a degreening of the peel and increase of the production of esters, correlated with higher levels of transcripts of the genes of HPL, LOX, AAT1, AAT2, AAT3 and AAT4. In pAP4AS fruits, the levels of these transcripts was significantly lower, not having been observed degreeningor yellowing, nor restoration of the synthesis of esters. In these fruits (pAP4AS) it was detected a higher concentration of zeatin and zeatin ribose than in pMEL1AS and WT. For tomato plants, in which was done application of cytokinins, there was prolongation of the vegetative cycle and delay in the maturation. The fruits from the treated plants also prolonged the maturation cycle, but did not decrease sensitivity to ethylene action. It is believed that cytokinins may be the responsible ones for lower sensitivity to ethylene in melons, but this hypothesis was not confirmed and must be tested with greater profundity. The attempt to prove it using tomato plants as model was not efficient. / O etileno é o hormônio indutor e acelerador da maturação e senescência de frutos climatéricos, como é o caso de melões Cantaloupe (Cucumis melo var. Cantalupensis, Naud cv. Vedrantais) e de tomates (Licopersicum esculentum L. cv. Micro-Tom). Sabe-se que a redução de produção e/ou a ação desse hormônio prolonga o período de conservação desses frutos. Assim, com o objetivo de reduzir a produção desse hidrocarboneto, foram transformados geneticamente meloeiros com clones da ACC oxidase antisense , de melão (pMEL1AS, Ayub et al., 1996) e de maçã (pAP4AS, Silva et al., 2004). Como era esperado, em ambos os casos, a produção de etileno foi reduzida, prolongando a vida de prateleira dos frutos em 7 dias. Entretanto, a produção de compostos voláteis também foi afetada. Os melões transgênicos produziram, em média, 70% menos ésteres do que os não transformados (NT), independentemente de terem sido transformados com o pMEL1AS ou pAP4AS. Como a intervenção feita agiu na redução da produção do etileno, emitiu-se a hipótese de que com a suplementação do hormônio, a síntese de aromas seria restaurada. Tal hipótese foi comprovada em melões pMEL1AS, mas não nos pAP4AS. As causas exatas dessa diferença ainda não foram esclerecidas. Acredita-se que pela maior redução da produção de etileno nos frutos pAP4AS, além de ter-se afetado genes diretamente relacionados com as vias metabólicas clássicas do amadurecimento, genes relacionados a outras vias de síntese de hormônios também tenham sido afetados, como é o caso de citocininas ou poliaminas. Pela observação do fenótipo de plantas pAP4AS percebeu-se que essas tiveram maior crescimento vegetativo e brotações laterais. Considerando-se tais observações, lançou-se a hipótese de que com o aumento dos níveis de citocininas poderia interferir nas respostas ao etileno, retardando a evolução normal dos frutos. Em melões pAP4AS comprovou-se haver maiores teores de citocininas, tanto nas raízes quanto nos frutos. Porém, para confirmar que essas alterações são efetivamente consequência de maiores teores de citocininas, fez-se aplicação exógena desse regulador de crescimento. No entanto, não se observaram as respostas esperadas. Então optou-se por testar a aplicação de citocininas noutro modelo vegetal, o tomateiro cv. Micro-Tom, que também é produtor de frutos climatéricos e que propicia maior facilidade de cultivo e obtenção de maior quantidade de frutos em ambientes protegidos e em áreas com limitação de espaço. Como variáveis moleculares para avaliação dos tratamentos em melões determinou-se o acúmulo de transcritos de genes de HPL, LOX e AAT, além da ACCO e ACCS. Em tomates, foram realizadas análises físico-quimicas. Para melões, ao se quantificar o acúmulo de transcritos de genes da ACC sintase (ACCS), verificou-se que os genes ACCS1 e ACCS3 apresentaram maiores expressões nos frutos NT, sugerindo que esses têm forte relação com a evolução da crise climatérica. Por outro lado, o gene ACCS5 foi mais expresso nos frutos pMEL1AS e pAP4AS, indicando que é regulado negativamente pelo etileno. Para as demais variáveis avaliadas (sólidos solúveis totais, coloração, carotenóides, clorofilas, firmeza de polpa) durante o amadurecimento na planta, não houve diferenças marcantes entre pMEL1AS e pAP4AS. Após a colheita e tratamento dos frutos pMEL1AS com etileno, houve desverdeamento da casca, e aumento da produção de ésteres, correlacionado com maiores níveis de transcritos dos genes da HPL, LOX, AAT1, AAT2, AAT3 e AAT4. Nos frutos pAP4AS, os níveis desses transcritos foi significativamente inferior, não tendo-se observado desverdeamento, nem amarelamento, tampouco restauro da síntese de ésteres. Nesses frutos (pAP4AS) foi detectada uma maior concentração de zeatina e zeatina ribose do que nos pMEL1AS e nos NT. Para tomateiros, nos quais fez-se a aplicação de citocininas, houve prolongamento do ciclo vegetativo e retardamento da maturação. Nos frutos provenientes de plantas tratadas verificou-se que a ação do etileno foi regulada, em particular em relação à variação dos principais pigmentos desse fruto (β-caroteno e licopeno). Desse modo, acredita-se que as citocininas possam ser as responsáveis pela menor sensibilidade ao etileno em melões, mas essa hipótese precisa ser testada com maior profundidade. A tentativa de prová-la usando tomateiros como modelo não foi totalmente eficiente.

L’ARN est sans aucun doute la molécule biologique la plus versatile qui soit. Tout comme l’ADN, il peut contenir et transmettre de l’information génétique. Tout comme les protéines, il peut accomplir une multitude de fonctions biologiques. De plus, son rôle le plus connu demeure celui d’intermédiaire entre l’ADN et les protéines. L’ARN est donc au cœur d’un bon nombre de processus biologiques. Ceci lui confère un immense potentiel thérapeutique qui jusqu’à présent demeure largement inexploité. Pour accomplir ses fonctions, l’ARN doit adopter une structure tridimensionnelle précise qui est dépendante à la fois de sa séquence et de son environnement. Ainsi, en modifiant la structure d’un ARN, il est possible d’en moduler sa fonction. C’est l’objectif global des travaux présentés dans cette thèse. Pour y parvenir, de courts oligonucléotides antisens (OA) ont été utilisés. Cette stratégie revêt plusieurs avantages. Comme les OA s’apparient à leur cible en formant des paires de bases Watson-Crick, ils offrent une grande spécificité et leur design est facile. De plus, en se fiant aux données structurales et aux logiciels de prédictions de structures des ARN, on peut aisément identifier les régions à cibler avec les OA. Enfin, cette technique est versatile puisqu’on peut cibler différents motifs d’ARN. La première cible a été le ribozyme du virus de l’hépatite D. Cet ARN, qui catalyse une réaction d’auto-coupure, a été modifié afin que son activité devienne dépendante à la liaison d’OA. Plusieurs modules ont ainsi été créés et combinés afin d’obtenir des ribozymes qui répondaient à la présence d’un ou plusieurs OA. En insérant ces interrupteurs moléculaires dans les régions non traduites d’un ARNm, nous avons ainsi modulé l’expression de ce gène avec les OA. Cet outil a des applications intéressantes pour la régulation de gènes en biologie synthétique. Un autre motif ciblé a été le G-quadruplex (G4). Cette structure non canonique exerce de nombreuses fonctions biologiques et représente donc une cible thérapeutique intéressante. Lorsque présent dans la région 5’ non traduite d’un ARNm, le G4 mène généralement à une diminution de la traduction. En utilisant des OA qui empêchent la formation du G4, nous avons été en mesure d’augmenter la traduction du gène ciblé. De plus, il a été possible de développer des OA qui favorisent la formation d’un G4 dans le but de diminuer l’expression de la cible. Finalement, dans le dernier chapitre de cette thèse, il est démontré que les G4 présents dans les microARN primaires influencent leur maturation en microARN matures. Des OA ciblant ces G4 ont été utilisés afin de favoriser la maturation de microARN suppresseurs de tumeurs, ce qui présente un potentiel thérapeutique intéressant. En bref, les travaux présentés dans cette thèse démontrent clairement que les OA sont un outil de choix pour cibler et modifier la structure de motifs d’ARN spécifiques. / Abstract : RNA is a versatile biological molecule. Like DNA, it can contain and transmit genetic information. Like proteins, it can accomplish multiple biological functions. Also, its most known role remains that of intermediary between DNA and proteins. RNA is thus a key player in many biological processes. This gives it an immense therapeutic potential which remains largely untapped. To fulfill its functions, RNA must adopt a precise threedimensional structure that is dependent on both its sequence and its environment. Thus, by modifying the structure of an RNA, it is possible to modulate its function. This is the overall objective of the work presented in this thesis. To achieve this, small antisense oligonucleotides (ASO) have been used. This strategy has several advantages. As ASO bind their target with Watson-Crick base pairs, they offer great specificity and their design is easy. Moreover, reliance on structural data and RNA structure prediction softwares makes it easy to identify the regions to be targeted with ASO. Finally, this technique is versatile since it is possible to target different RNA motifs. The first target was the HDV self-cleaving motif. This RNA, which catalyzes a self-cleaving reaction, has been modified so that its activity became dependent on the binding of ASO. Several modules were thus created and combined in order to obtain ribozymes which responded to the presence of one or more ASO. By inserting these molecular switches into an mRNA’s UTR, the expression of this gene was modulated with the ASO. This has interesting applications for the regulation of genes in synthetic biology. Another target motif was the G-quadruplex (G4). This non-canonical structure exerts many biological functions and therefore represents an interesting therapeutic target. When present in the mRNA’s 5’UTR, G4 generally lead to a decrease in translation. Using ASO that prevent G4 formation, we were able to increase the translation of the target gene. In addition, it has been possible to develop ASO which promote the formation of a G4 in order to decrease the expression of the target. Finally, in the last chapter of this thesis, it is demonstrated that the G4 present in the primary microRNAs influence their maturation in mature microRNAs. ASO targeting these G4 have been used in order to promote the maturation of tumor suppressor microRNAs, which has an interesting therapeutic potential. The work presented in this thesis clearly demonstrates that ASO are ideal for targeting and altering the structure of specific RNA motifs.

La maladie de Huntington (MH) est causée par une expansion de répétitions CAG sur l’exon 1 du gène huntingtine (htt), codant pour une protéine mutée. Il a été montré que la diminution d’expression de cette protéine est une piste thérapeutique très prometteuse. Dans ce projet, nous avons étudié et comparé trois approches dites «antisens» : une stratégie allèle non-spécifique, visant à diminuer de manière générale l’expression de htt ; une stratégie allèle spécifique ciblant les répétitions CAG afin d’impacter préférentiellement l’allèle muté ; et enfin une stratégie de saut d’exon permettant d’enlever des sites de clivage à l’origine d’une forme raccourcie et toxique de la protéine htt. Nous avons évalué ces approches grâce à deux outils différents : les tricyclo-DNA (TcDNA), qui sont une nouvelle classe d’oligonucléotides antisens (AON) plus performante que les chimies précédentes, et le système U7snRNA vectorisé, permettant d’induire une expression stable des séquences antisens. Dans un premier temps, ces différentes molécules ont été évaluées in vitro dans des lignées de fibroblastes de patients en quantifiant le niveau d’ARNm et de protéines htt par RTqPCR et Western blot respectivement. Par la suite, les séquences les plus efficaces in vitro ont été sélectionnées et les AON et AAV-U7snRNA correspondants ont été injectés en intracérébroventriculaire (ICV) dans un modèle murin de la MH (souris YAC128). Les résultats les plus encourageants ont été obtenus avec le TcDNA-NS (pour allèle Non Spécifique), permettant une diminution significative de l’expression de htt dans le cortex, l’hippocampe et le striatum 2 et 6 semaines après une injection ICV. Ces résultats prometteurs suggèrent le potentiel des TcDNA comme nouvel outil thérapeutique pour la MH. / Huntington’s disease (HD) is caused by a CAG repeat expansion in the exon 1 of huntingtin gene (htt), encoding for a mutant protein. It has been shown that the silencing/down regulation of huntingtin protein is a promising therapeutic lead. In this project, I have explored and compared three strategies using the antisense approach: a non-allele specific strategy, aiming to silence the global expression of htt; an allele specific strategy targeting CAG repeats to silence preferentially the mutant allele; and an exon-skipping strategy in order to remove cleavage sites which originally cause a shorter and toxic form of the htt protein. These strategies have been evaluated using two different tools: tricyclo-DNA (TcDNA), a new class of antisense oligonucleotides (AON) more efficient than the previous chemistries, and a vectorized approach using U7snRNA system allowing a stable expression of antisense sequences. Firstly, these different molecules have been assessed in vitro in HD fibroblasts quantifying mRNA and htt protein levels with RTqPCR and Western blot respectively. Subsequently, the most efficient sequences have been selected and intracerebroventricular (ICV) injections have been performed with corresponding AON and AAV-U7snRNA in a HD mouse model (YAC128). The most encouraging results have been obtained with the TcDNA-NS (for Non Specific allele), allowing a significant decrease of htt expression in cortex, hippocampus and striatum 2 and 6 weeks after ICV injection. These promising results suggest the potential of TcDNA as a new therapeutic tool for HD.