Ce travail a permis de réaliser une évaluation complète de différentes séquences de siRNA dirigées contre EWS/Fli-1 dans le cadre du traitement des tumeurs de la famille du sarcome d'Ewing Un siRNA a été vectorisé de manière efficace par des nanoparticules polymères ciblées contre un marqueur membranaire spécifique des ESFT. Ces nanoparticules ont été caractérisées et semblent bien tolérées à la fois in vitro et in vivo. Leur évaluation a été réalisée sur des cellules humaines et après prise des tumeurs ce qui représente une avancée intéressante dans la lutte contre les ESFT. La mise au point d'un modèle fluorescent de sarcome d'Ewing permettra de mieux caractériser leur effet sur les métastases, facteur essentiel dans la survie des patients. Enfin, il a été montré que les techniques d'imagerie in vivo permettaient de suivre le devenir in vivo des nanoparticules ce qui permettra de comprendre leur biodistribution et leur mode d'action.

There has been much interest in the use of therapeutics based on ribonucleic acid interference(RNAi) to inhibit synthesis of mutant proteins ever since Elbashir et al. (Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K. and Tuschl, T., 2001. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 411, 494-498.) found that synthetic double stranded small interfering ribonucleic acids (siRNAs) can initiate this evolutionarily conserved process in mammalian cells. Since RNAi is able to target single genes and therefore mitigate the underlying molecular pathology of diseases, RNAi-based therapeutics will most likely benefit monogenic neurodegenerative diseases such as Huntington’s disease. It is however particularly difficult to deliver exogenous materials such as siRNAs into neurons in vivo as the blood-brain barrier (BBB) isolates the brain from the vascular system and prevents permeation of most materials. Neurons also do not take up exogenous materials readily. Therefore, effective delivery of siRNAs into the brain remains one of the biggest challenges impeding their use as a potential neurotherapeutic. Layered double hydroxide (LDH) nanoparticles are a class of anionic clay materials that have demonstrated great potential as a DNA (deoxyribonucleic acid) delivery system for a variety of mammalian cell lines due to their unique physiochemical properties. This thesis examined the feasibility of LDH as a siRNA delivery system for cultured neurons and demonstrated that the delivered siRNAs are able to effectively down-regulate synthesis of a target protein with minimal toxicity. Experiments were conducted using double stranded DNAs (dsDNAs) initially, and siRNAs were then used to verify these results. It was shown that nucleic acids(dsDNAs and siRNAs) could successfully intercalate into pristine LDHs to form nucleic acid-LDH complexes that had properties suitable for use as a delivery system in mammalian cells. These studies established that LDHs and nucleic acid-LDH complexes were biocompatible with neurons isolated from embryonic day 17.5 mouse cerebral cortex, suggesting that LDH can be used for nucleic acid delivery into cultured neurons. LDHs were also shown to successfully deliver nucleic acids into a non-neural mammalian cell line (NIH 3T3 cells). Finally, this thesis demonstrated for the first time that LDHs were able to deliver siRNAs into neurons, providing encouraging preliminary evidence that sequence specific gene silencing of the Mus Musculus Deleted in Colorectal Cancer (DCC) gene had occurred. However, down-regulation of the DCC protein did not occur consistently, suggesting that further optimisation is needed to improve the efficacy of siRNA-LDH complexes to inhibit expression of target protein in neurons. In future, LDHs should be further developed as an efficient siRNA delivery system for therapeutic gene silencing in the central nervous system using a neurodegenerative disease model such as the Huntington’s disease mouse model, which closely phenocopies the human disease. This model will allow the in vivo efficacy of these nanoparticles to be tested and subsequently improved in order to deliver siRNAs locally and systematically into the brain.

There has been much interest in the use of therapeutics based on ribonucleic acid interference(RNAi) to inhibit synthesis of mutant proteins ever since Elbashir et al. (Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K. and Tuschl, T., 2001. Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature. 411, 494-498.) found that synthetic double stranded small interfering ribonucleic acids (siRNAs) can initiate this evolutionarily conserved process in mammalian cells. Since RNAi is able to target single genes and therefore mitigate the underlying molecular pathology of diseases, RNAi-based therapeutics will most likely benefit monogenic neurodegenerative diseases such as Huntington’s disease. It is however particularly difficult to deliver exogenous materials such as siRNAs into neurons in vivo as the blood-brain barrier (BBB) isolates the brain from the vascular system and prevents permeation of most materials. Neurons also do not take up exogenous materials readily. Therefore, effective delivery of siRNAs into the brain remains one of the biggest challenges impeding their use as a potential neurotherapeutic. Layered double hydroxide (LDH) nanoparticles are a class of anionic clay materials that have demonstrated great potential as a DNA (deoxyribonucleic acid) delivery system for a variety of mammalian cell lines due to their unique physiochemical properties. This thesis examined the feasibility of LDH as a siRNA delivery system for cultured neurons and demonstrated that the delivered siRNAs are able to effectively down-regulate synthesis of a target protein with minimal toxicity. Experiments were conducted using double stranded DNAs (dsDNAs) initially, and siRNAs were then used to verify these results. It was shown that nucleic acids(dsDNAs and siRNAs) could successfully intercalate into pristine LDHs to form nucleic acid-LDH complexes that had properties suitable for use as a delivery system in mammalian cells. These studies established that LDHs and nucleic acid-LDH complexes were biocompatible with neurons isolated from embryonic day 17.5 mouse cerebral cortex, suggesting that LDH can be used for nucleic acid delivery into cultured neurons. LDHs were also shown to successfully deliver nucleic acids into a non-neural mammalian cell line (NIH 3T3 cells). Finally, this thesis demonstrated for the first time that LDHs were able to deliver siRNAs into neurons, providing encouraging preliminary evidence that sequence specific gene silencing of the Mus Musculus Deleted in Colorectal Cancer (DCC) gene had occurred. However, down-regulation of the DCC protein did not occur consistently, suggesting that further optimisation is needed to improve the efficacy of siRNA-LDH complexes to inhibit expression of target protein in neurons. In future, LDHs should be further developed as an efficient siRNA delivery system for therapeutic gene silencing in the central nervous system using a neurodegenerative disease model such as the Huntington’s disease mouse model, which closely phenocopies the human disease. This model will allow the in vivo efficacy of these nanoparticles to be tested and subsequently improved in order to deliver siRNAs locally and systematically into the brain.

Orientador: Kleber Gomes Franchini / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-11T20:15:36Z (GMT). No. of bitstreams: 1 Pereira_AnaHelenaMacedo_M.pdf: 6567293 bytes, checksum: b9a8c070b3b65fe7a4fb095ccfc59d42 (MD5) Previous issue date: 2008 / Resumo: Doenças do coração são freqüentemente associadas à hipertrofia miocárdica. Estímulos mecânicos induzem o crescimento hipertrófico e contribuem para a degeneração e morte dos miócitos cardíacos. Dentre os fatores de transcrição envolvidos no processo de hipertrofia miocárdica, estão os da família MEF2 (Myocyte Enhancer Factor-2), que é composto por 4 membros, MEF2A, B, C e D. O MEF2C é descrito como o principal transcrito no miocárdio. Tanto a deleção quanto a hiperexpressão de seu gene causam efeitos deletérios na formação e na função do músculo cardíaco. Estudos anteriores do nosso laboratório demonstraram que o MEF2 é ativado por estiramento de cardiomiócitos e influencia a expressão de genes do programa hipertrófico. O presente estudo tem como objetivo avaliar os efeitos do silenciamento gênico do MEF2C nas alterações estruturais e funcionais do ventrículo esquerdo de camundongos submetidos à sobrecarga pressórica. Para isso, utilizamos a técnica de interferência por RNA para o MEF2C. A padronização constituiu de: 1) avaliação do silenciamento do MEF2C em cultura de células C2C12 e no ventrículo esquerdo de camundongos Swiss; 2) determinação da dose necessária de siRNA para o silenciamento da expressão protéica do MEF2C; 3) determinação do curso temporal do silenciamento; 4) avaliação dos efeitos do tratamento com molécula irrelevante de siRNA direcionada para a proteína exógena GFP; 5) avaliação da especificidade do silenciamento (off-targets) pela análise do RNAm para o MEF2A e das proteínas FAK, GAPDH, JNK1/2 e SHP2; 6) avaliação do silenciamento em outros órgãos, como pulmão e rim; 7) avaliação da efetividade do silenciamento de MEF2C em miócitos cardíacos isolados do ventrículo esquerdo de camundongos. O tratamento com siRNA diminuiu a expressão protéica do MEF2 em 70% das células C2C12. Também verificamos que o tratamento com siRNA silenciou 85% da expressão protéica e do RNAm do MEF2C no ventrículo esquerdo de camundongos em até 4 dias de seguimento. Não foi verificada alteração na expressão de RNAm para o MEF2A e das proteínas FAK, GAPDH, JNK1/2 e SHP2. O silenciamento foi efetivo no pulmão e nos cardiomiócitos isolados do ventrículo esquerdo de camundongos tratado com siRNAMEF2C. Após a padronização do silenciamento, procedeu-se à determinação dos efeitos do silenciamento na estrutura e na função do ventrículo esquerdo de camundongos submetidos à sobrecarga pressórica crônica. Para isso, realizaram-se as análises ecocardiográfica, hemodinâmica, gravimétrica e morfométrica do ventrículo esquerdo de camundongos submetidos à coarctação da aorta com seguimento de 15 dias. Demonstramos que o tratamento com siRNAMEF2C atenuou a hipertrofia cardíaca nos animais coarctados. Esta conclusão foi baseada em dados de ecocardiografia que revelaram menor espessura da parede posterior (30% menor) e por gravimetria que revelou atenuação de aproximadamente 45% da massa do ventrículo esquerdo. Apesar de ter havido aumento do gradiente sistólico nos animais coarctados, a pressão arterial sistêmica não apresentou diferença estatisticamente significativa com o tratamento do siRNAMEF2C. Morfologicamente, o siRNA atenuou a fração de colágeno no ventrículo esquerdo de camundongos coarctado com 15 dias de seguimento. Entretanto, o diâmetro dos miócitos e o infiltrado de células inflamatórias foram comparáveis dentre os grupos. Somente os animais coarctados por 24 horas tiveram maior expressão de ß- MHC, e quando tratados com siRNAMEF2C apresentaram menor razão ATP/ADP. Dessa forma, esses dados sugerem que o MEF2C regula múltiplos aspectos da hipertrofia cardíaca induzida por sobrecarga pressórica tais como a expressão de genes sarcoméricos e genes envolvidos na adaptação metabólica do músculo cardíaco. / Abstract: Heart diseases are frequently associated with myocardial hypertrophy. Mechanical stimuli can trigger hypertrophic growth as well as degeneration and death of the cardiac myocytes. The MEF2C family of transcription factors plays a role in the process of myocardial hypertrophy. It is composed by 4 members, MEF2A, B, C and D, and the MEF2C is the main transcript in the heart. Both the deletion and overexpression of mef2c induce deleterious effects in the formation and function of the heart. Previous studies of the our laboratory has shown that the transcription factor MEF2C is activated by mechanical stretch in cardiomyocytes and regulates the expression of genes related to cardiac hypertrophy. This study was performed to address the effects of MEF2C gene silencing in the structural and functional changes of the left ventricle (LV) induced by pressure overload in mice. To silence MEF2C, it was employed the RNA interference technique, specific siRNA target to MEF2C was administered through the mice jugular vein. To optimize the MEF2C knockdown, it was necessary to 1) analyze the MEF2C silencing in C2C12 cells, 2) determine the dose required to induce significant MEF2C silencing in LV of mice, 3) determine the time course of gene silencing, 4) assess the effects of the treatment with irrelevant siRNA target to the protein GFP, 5) evaluate the specificity of gene silencing by siRNAMEF2C through the expression analysis of the transcription factor MEF2A and other non-related proteins, 6) analyze of the MEF2C knockdown in other organs, 7) determine the effectiveness of the MEF2C silencing in cardiac myocytes harverst from the LV of mice treated systemically with siRNAMEF2C. Treatment with 100ng/mL of siRNAMEF2C induced MEF2C silencing (~70%) in C2C12 cells. Intrajugular delivery of 30µg of siRNAMEF2C in mice induced the reduction in the mRNA and protein levels (~85%) until 4 days after the injection. The treatment with siRNAMEF2C did not affect the expression of MEF2A and other non-related proteins. The MEF2C silencing was effective in lung and in cardiac myocytes harverst from LV of mice treated with siRNAMEF2C. After knockdown optimization, echocardiographic, hemodynamic, gravimetric and morphometric analysis was performed to address the effects of MEF2C silencing in the structure and function of the LV from 15 days aorticbanded mice. Myocardial MEF2C silencing attenuated the load-induced hypertrophy in banded mice, indicated by the reductions of the wall thickness and the mass (~45%) of the LV. An increase in transconstriction gradient was observed in banded mice but the systemic blood pressure did not shown a significant statistically difference with the siRNAMEF2C treatment. The siRNAMEF2C injection reduced the collagen fraction in the LV of 15 days banded mice. On the other hand, the myocytes diameter and inflammatory cells level were comparable between the groups. Only the 24 hours banded mice showed an increase in the â-MHC expression and the treatment with siRNAMEF2C reduced ATP/ADP ratio. This study indicate that MEF2C regulates many aspects of the cardiac hypertrophy induced by pressure overload, like the expression of sarcomeric genes and genes involved in metabolic adaptation of the heart muscle. / Mestrado / Medicina Experimental / Mestre em Fisiopatologia Médica

SUMMARYThe diversity of cancer molecular origins associated with the genetic variability of patients has encouraged the development of chemotherapeutic treatments adapted not only to the target tumor, but also to a specific patient. This personalized strategy is based on cancer biomarkers allowing a better identification and characterization of each tumor where predictive biomarkers provide the distinction between various factors indicative of the response to the treatment. In this context, several studies highlighted the role of the solute carrier transporter family 22 (solute carriers 22 or SLC22) in the uptake of platinum anticancer drugs. This mechanism being not well understood, our work intends to establish the potential role of SLC22 member A18 (SLC22A18) as predictive biomarker in the aim to help to a better targeted chemotherapeutic strategy for each patient. We optimized a system overexpressing SLC22A18 stably or transiently in HeLa cancer cell line. SLC22A18 expression was confirmed by qRT-PCR, western blotting, microscopy and flow cytometry. The cell lines were treated with taxane, anthracyclin, vinca alkaloid and nitrosoureas anticancer drug families. We showed that doxorubicin, camptothecin, chloroquine, tetracycline and carmustin had no effect on the cell viability assays suggesting that they are not substrates of SLC22A18. Interestingly, the cell line was sensitized in the presence of antimitotic drug with a sensitivity factor of 2.7 in the presence of paclitaxel, 1.4 with docetaxel, 1.8 with vinblastin and 2.2 in the presence of vincristine. To confirm these results, we elaborated a SLC22A18 knockdown cell line in HS683 cells using siRNA technology. The downexpression of SLC22A18 was correlated to a tendency to resist to the accumulation of paclitaxel thereby confirming the previous results. Simultaneously, a knockout cell line was established using the transcription activator-like effectors nuclease (TALEN) technology in U373 cell line. Our studies constitute a robust base of knowledge for further investigation on SLC22A18 transporter as a predictive biomarker promoting antimitotic treatment in tumors where this transporter is detected. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Therapeutics based on siRNA represent a promising hope for the treatment of many congenital and acquired disorders. This method is based on posttranscriptional silencing of pathological gene or set of genes (RNAi process), which are responsible for the actual cause of the disease. Access is therefore based on the assumption of treatment options for the disease at the point of origin of the defect intervention at the molecular level, which is different from the conventional, so-called symptomatic therapy, which focuses only on the treatment or suppression of symptoms. Despite rapidly increasing understanding of gene function and cause a number of genetic diseases, the expansion of siRNA therapeutics limited the development of efficient and safe transport systems (vectors). In order to ensure efficient transport of siRNA in vivo conditions, the vectors must sufficiently reduce the size of the siRNA, protect it against degradation during transport, and release in the cytoplasm of the target cell. For this purpose they were developed sophisticated transport systems based on viral and non-viral origin. This diploma thesis is focused on the preparation of new transport systems, siRNA-based synthetic hydrophilic polymers, such as non-viral vectors. For in vitro testing the effectiveness during transport of siRNA...

Les éléments transposables (ET) sont des constituants majeurs des génomes eucaryotes. Leur mobilisation joue un rôle important dans l'évolution et l'adaptation des organismes. Cependant, la transposition des ET peut conduire à des dommages irréversibles du génome et elle doit donc être étroitement contrôlé. Chez Drosophila melanogaster, la transposition des ET est contrôlée par les siRNA (small interfering RNAs) et les piRNA (Piwi-interacting RNAs) qui agissent en réprimant des cibles ARN de séquences complémentaires. Les siRNA et piRNA ont des modes distincts de biogenèse, de reconnaissance de cibles et d'activité repressive. Les piRNAs sont seulement présents en abondance dans les gonades, et transmis maternellement aux embryons. Par une approche de séquençage à haut débit, j'ai pu montrer que bien qu'ils induisent une répression transcriptionnelle des ET à ce stade du développement, ils sont pratiquement absents des têtes de drosophiles adultes. Cet état est cependant hérité et il est suffisant pour limiter l'expression des ET dans l'adulte, même en l'absence de siRNA. A l'inverse, si la répression transcriptionnelle précoce n'est pas établie, les siRNA agissent comme un système de sauvegarde en limitant l'expression des ET. En cas de perte conjointe des piRNA et siRNA, l'expression des ET augmente significativement et la durée de vie des mouches adultes se trouve réduite. Les analyses de sequences à grande échelle m'ont par ailleurs conduit à développer des outils logiciels intégrés dans Galaxy et à m'impliquer significativement dans la communauté qui développe ce système au niveau international. / Transposable elements are major components of eukaryotic genomes and have been proposed as important drivers of gene network evolution, as they can move or “transpose” in their host genome, creating gene duplications, gene inactivations or altogether altering gene function. Nevertheless, uncontrolled high-rate transposition leads to DNA damage and genomic instabilities, and therefore needs to be kept at a low level. In the fruitfly Drosophila melanogaster, transposition is counteracted by multiple mechanisms, amongst which the generation of small interfering RNAs (siRNAs) and Piwi-interacting RNAs (piRNAs). siRNAs and piRNAs belong to the category of small RNAs, and these are involved in negative regulation of complementary target RNAs abundance, but siRNAs and piRNAs have distinct mechanisms of biogenesis, target recognition and mechanisms of target regulation. Notably, piRNAs are only abundant in gonads and are transmitted to the embryo. By sequencing small RNAs and normal transcripts in adult heads, I conclude that, while piRNAs are likely absent in adult heads, they induce a repressive state on TEs. If this repressive state is lost, the siRNA pathway can compensate and limit Transposable element levels. If siRNAs are lost, the repressive state induced by piRNAs suffices to limit Transposable element levels. If both piRNAs and siRNAs are lost, the expression level of Transposable elements increases, and flies have a shorter life span. The requirement to analyse large-scale sequencing data led to the development of multiple tools for the reproducible research platform Galaxy.

Oncolytic measles virus is a promising cancer therapeutic in clinical trials which possesses multiple characteristics that are advantageous over traditional therapies. Currently, clinical oncolytic measles virus vectors are unmodified or express reporter transgenes that benefit its therapeutic efficacy. The next phase in its development will see genetically engineered vectors encoding transgenes that enhance its antineoplastic effects. To this end, preclinical research has focused on studying novel transgenes which favour viral replication, cytotoxicity, and the anti-cancer immune response. We sought to encode artificial micoRNAs targeting RIG-I as a strategy to interfere with innate immunity. Silencing RIG-I with multiple siRNAs yielded one which promotes measles virus syncytia formation through a mechanism that appears to be independent of RIG-I. The mechanism caused by the siRNA leads to enhanced measles virus cell-cell fusion and has peculiar characteristics which are not fully understood.

Type II diabetes mellitus is associated with many detrimental health situations including heart complications. The purpose of this study was to identify a role for PKA-dependent Rap1a signaling in the AGE-RAGE cascade. My hypothesis was Rap1a GTPase increased the downstream effects of AGE-RAGE signaling in diabetes via a PKA-dependent pathway leading to elevated ECM remodeling in the heart. Cardiac fibroblasts were isolated from heterozygous (Het) and diabetic (db/db) mice. To test the hypothesis, gain-ofunction and loss-ofunction treatments were used. PKC-Zeta is known as a major signaling hub that potentially links PKA-dependent and AGE-RAGE signaling cascades so PKC-Zeta inhibition to downregulate PKA-dependent cascade at PKC-Zeta was also used. Results showed a downregulation of signaling markers in the AGE-RAGE cascade when disrupting Rap1a crosstalk at PKC-Zeta. By understanding where the PKA-dependent and AGE-RAGE signaling cascades crosstalk, a new molecular mechanism is understood possibly leading to decreasing remodeling in a diabetic heart.

No description available.