O glioblastoma multiforme (GBM) é um dos tumores mais letais e a radioterapia permanece como um dos principais tratamentos. Novas estratégias são necessárias para coibir a resistência ao tratamento, como o silenciamento de fatores de transcrição (FTs). Nossa hipótese é a de que FTs associados a listas de genes diferencialmente expressos, os quais foram selecionados para linhagens de GBM irradiadas, ou comparando amostras de GBM à amostras de tecido cerebral, possam fornecer alvos moleculares que aumentariam a morte das células tumorais, quando silenciados. Foram analisadas a proliferação, morte e ciclo celular, além da formação e diferenciação de neuroesferas, utilizando, em quase todas as etapas, a citometria de fluxo. Os FTs HEB e E2F1, cujas funções principais estão relacionadas à neurogênese e proliferação celular, foram selecionados a partir das análises in silico de GBM irradiados ou não, ou de GBMs comparados a amostras de cérebro normal, respectivamente. Esses FTs encontram-se expressos em linhagens U87, astrócitos primários e neuroesferas provenientes das mesmas, analisadas por Western blot. O silenciamento de HEB e E2F1 na linhagem U87, de forma geral, reduziu a proliferação, induziu morte celular e diminuiu a porcentagem de células em G0/ G1, em pelo menos um dos tempos analisados (24, 48 e 72h) em relação ao grupo transfectado com a sequência scrambled. O silenciamento de HEB e E2F1 reduziu o número de neuroesferas quando comparadas às células transfectadas com a sequência scrambled. Possivelmente, a capacidade anti-proliferativa do silenciamento dos FTs HEB e E2F1 observada no cultivo em monocamada da U87, possam atuar na capacidade de formação de neuroesferas e, consequentemente, podem ter um papel na manutenção das células tronco do GBM. O silenciamento não alterou a radiorresistência da U87 cultivada em monocamada, com exceção dos efeitos do silenciamento de E2F1 em 24 h, em que houve radioproteção. A irradiação não reduziu o número de neuroesferas silenciadas para HEB em comparação ao grupo não irradiado, mas reduziu o número de células presentes nas neuroesferas, indicando uma possível atuação de HEB na resposta à irradiação em neuroesferas, fato este nunca antes descrito. O silenciamento de E2F1 não interferiu na resposta das neuroesferas à radiação. A expressão de CD133 avaliada oito dias após a dissociação das células silenciadas para E2F1 e HEB, cultivadas em meio de diferenciação, foram superiores ao do grupo scrambled, indicando uma possível diminuição na diferenciação celular. O silenciamento dos dois FTs não atuou na seleção positiva de CD133+ após a irradiação, como observado no grupo das neuroesferas transfectadas com a sequência scrambled e irradiadas, comparado às não irradiadas. Assim, E2F1 e HEB mostraram-se alvos interessantes no sentido de reduzir a proliferação, tanto em células U87 cultivadas em monocamada quanto em neuroesferas. / Glioblastoma multiforme (GBM) is one of the most lethal tumors, and radiation therapy remains one of the main treatments. New strategies are needed to suppress typical GBM treatment resistance and transcription factors (TFs) silencing seems to be a promising strategy. Our hypothesis is that TFs associated with lists of differentially expressed genes which were selected for irradiated compared to shamirradiated GBM cell lines, or GBM samples compared to brain tissue samples, could provide molecular targets that are supposed to increase tumor cell death when they are silenced. We analyzed proliferation, cell death and cell cycle progression, besides the formation and differentiation of neurospheres, using several analyses by flow cytometry. The TFs HEB and E2F1, whose primary functions are related to neurogenesis and cell proliferation, were selected from in silico analysis of GBM irradiated or sham-irradiated GBMs and GBM samples compared with normal brain samples, respectively. These TFs were found expressed in U87 GBM cell line, and primary astrocytes, as well as in neurospheres derivated from both, as analyzed by Western blot. Silencing of E2F1 and HEB in U87 cells, reduced proliferation, induced cell death and decreased the percentage of cells at G0/G1 (24, 48 or 72h) compared to the scrambled sequence transfected group. HEB and E2F1 silencing reduced the number of neurospheres when compared to cells transfected with scrambled sequence. Possibly, the anti-proliferative ability of silencing of HEB and E2F1 TFs observed in monolayer culture of U87, may act in neurospheres forming capacity and therefore may play a role in the maintenance of GBM stem cells. In our experiments, gene silencing did not alter the radio-resistance of U87 grown in monolayer. Irradiation did not reduce the number of neurospheres silenced for HEB compared to non-irradiated group, but reduced the number of cells present in neurospheres, indicating a possible role of HEB in response to ionizing irradiation in neurospheres, a fact that was not described yet. The silencing of E2F1 in neurospheres did not affect the response to irradiation. The expression of CD133, as assessed at eight days after the dissociation of cells silenced for E2F1 and HEB (cultured in differentiation culture media), was superior compared with the scrambled group, indicating a possible decrease in cell differentiation. The silencing of both TFs did not influence the positive selection of CD133 after irradiation, as observed in the group of neurospheres transfected with scrambled sequence, and irradiated compared to nonirradiated. Thus, E2F1 and HEB proved to be interesting targets for decreasing proliferation in both U87 cells grown as monolayer or neurospheres.

Les pDC représentent la première ligne de défense de l’organisme contre les pathogènes et établissent le lien essentiel entre l’immunité innée et adaptative. Les pDC endocytent et détruisent les particules virales et ainsi détectent leur matériel génétique grâce à des senseurs antiviraux de la famille des Toll-Like Receptors (TLR). L’activation des TLR7/9 induit la production massive d’interféron de type I (IFN-I), un antiviral puissant indispensable au contrôle de la propagation virale lors des phases aigues de l’infection. Cependant, l’IFN-I peut s’avérer avoir des effets délétères dans un grand nombre d’infections chroniques et de maladies auto-immunes. Ainsi, il semble indispensable de découvrir les mécanismes régulateurs des pDC ainsi que des modulateurs de l’activation des pDC. Nous avons ainsi montré que les monoamines (histamine, dopamine, sérotonine) et les polyamines (spermine et spermidine) inhibent l’activation complète des pDC stimulées par divers virus. Par la suite, nous avons identifié CXCR4 comme étant le récepteur des amines sur les pDC. Ainsi nous avons pu montrer que les amines pouvaient réguler les pDC en passant par CXCR4 et que ce récepteur était un interrupteur d’activation potentiel des pDC lors des infections virales. Afin de comprendre le mécanisme des amines, nous avons développé une nouvelle technologie : la transfection de siRNA dans les pDC primaires humaines. D’autre part, nous avons détecté des cellules géantes multinucléées en forme de roue de bicyclette lorsque les pDC sont cultivées in vitro avec de grandes quantités de virus VIH. Ainsi, comme les monocytes et les macrophages, les pDC peuvent former in vitro des cellules géantes multinucléées exprimant de hauts niveaux de protéines virales p24 de VIH-1. Cependant, les pDC ne sont que très peu infectées (moins de 5%). Nous nous sommes alors demandé si le corécepteur CXCR4 du virus VIH était aussi important que le récepteur CD4 pour la reconnaissance de ce dernier lors de l’activation des pDC. / PDC are the first line of defense of our organism against pathogens and establish the essential link between the innate and adaptive immunity. pDC endocyte and destroy the viral particles and thus, detect the genetic material with their antiviral sensors from the Toll-Like Family (TLR). The activation of TLR7/9 induces massive production of type I interferon (IFN-I), a powerful antiviral molecule, essential to control viral propagation during the acute phases of the infection. However, type I IFN can have deleterious effects in a large number of chronic infections and autoimmune diseases. Thus, it seems essential to discover the regulatory mechanism of pDC as well as pDC activation modulators. We showed that monoamines (histamine, dopamine and serotonin) and polyamines (spermine and spermidine) inhibit completely the activation of virus-stimulated pDC. Thus, we showed that amines regulated pDC activation through CXCR4 engagement and that this receptor was a potential switch "on-off" for pDC during viral infections. To better understand the mechanism of action by which amines inhibit pDC activation, we developed a new technology: siRNA transfection in human primary pDC. Furthermore, we detected multinuclear giant cells bearing the shape of a bicycle wheel when pDC are cultured in vitro with high quantities of HIV virus. Thus, on top of monocytes and macrophages, pDC can form in vitro multinuclear giant cells with high levels of p24 viral protein of HIV-1. However, pDC barely get infected (less than 5%). We then wondered if the receptors and co-receptors of the virus were important for the viral recognition during HIV-activation of pDC.

O mecanismo de interferência por RNA (RNAi) é um evento de silenciamento gênico através da degradação do RNA mensageiro. Desta forma, a administração de siRNA (molécula efetora do RNAi) é uma terapia promissora para o tratamento de diversas doenças como o câncer. Porém, para a sua efetiva aplicação terapêutica é necessário o desenvolvimento de sistemas de liberação capazes de liberar o siRNA nas células alvo, promover a sua internalização celular e evitar a sua degradação. Dentre os sistemas de liberação, os de liberação localizada, como os sistemas de geleificação in situ, apresentam vantagens sobre administração sistêmica. Formulações fluidas precursoras (FFP) que formam sistemas líquido cristalinos viscosos in situ podem ser obtidas a partir de lipídeos anfifílicos que absorvem água do meio e se rearranjam. Neste contexto, a presente pesquisa teve como objetivo avaliar o gel formado in situ a partir da FFP (G-FFP), composta por monoglicerídeos (MO), polietilenoimina (PEI), propilenoglicol (PG) e tampão tris, como sistema de liberação localizada de siRNA na terapia do câncer de pele. Os resultados mostraram que o G-FFP é uma mistura de fase cúbica e fase hexagonal. O G-FFP liberou o siRNA de maneira sustentada e complexado ao PEI. A FFP pode ser esterilizada por filtração em membrana e foi capaz de complexar altas concentrações de siRNA (15 mM) e de proteger o siRNA da degradação. A citotoxicidade foi dependente da concentração de FFP e esta quando complexada com siRNA teve a toxicidade diminuída. O siRNA liberado do G-FFP foi internalizado pelas células A431, FaDu, HeLa, A549, WM35/DLC2-GFP e MCF-7/DLC2-GFP. Além disto, siRNAs específicos liberados pelo G-FFP foram capazes de reduzir a expressão da proteína Firefly luciferase em células HeLa e FaDu, pórem não foram capazes de reduzir a expressão do receptor do fator de crescimento epidérmico (EGFR) nas células A431, HeLa, A549 e FaDu. A redução da expressão de EGFR em células A549 foi observado quando a terapia com siRNA foi combinada com internalização fotoquímica. Destes resultados, pode-se inferir que a transfecção celular do siRNA e o silenciamento gênico promovido por ele foi dependente tanto do tipo de linhagem celular como do alvo desejado. Os estudos in vivo mostraram que ocorre a formação de gel intratumoral e após 3 dias da administração intratumoral da FFP contendo siRNA específico para EGFR houve redução de 30% no tamanho dos tumores comparados aos tumores tratados com FFP sem siRNA. Diante destes resultados, pode-se concluir que o sistema desenvolvido tem potencial como sistema de liberação localizada de siRNA quando aplicado subcutaneamente ou intratumoral, uma vez que complexa o siRNA, promove a sua internalização celular e o siRNA liberado no citoplasma das células pode reduzir a expressão de proteínas de interesse. / RNA interference (RNAi) is a mechanism in which small interfering RNA molecules (siRNA) inhibit gene expression, by causing the messenger RNA degradation. Thus, siRNA is a promising therapy for the treatment of several diseases such as cancer. However, the development of delivery systems able to protect the siRNA from degradation and promote its cell uptake is essential for therapeutic use of siRNA. Among the delivery systems, the localized delivery system such as in situ gelling delivery system, have advantages over systemic administration. Precursor fluid formulations (FFP), which forms in situ viscous liquid crystalline systems, can be obtained from amphiphilic lipids that absorb water from the environment and self-assembling. In this context, the present study aimed to evaluate the in situ gel formed from the FFP (G-FFP), composed of monoglycerides (MO), polyethyleneimine (PEI), propylene glycol (PG) and Tris buffer, as localized delivery system for siRNA in skin cancer therapy. The results showed that the G-FFP is a mixture of cubic and hexagonal phase. The G-FFP sustained release of siRNA and the siRNA is released complexed with PEI. The FFP can be sterilized by membrane filtration at 0.22 ?m. FFP was able to complex high siRNA concentration (15 mM) and protect the siRNA from degradation. The cytotoxicity was dependent on the FFP concentration, when FFP was complexed with siRNA it was observed a decreased in toxicity. The siRNA released from G-FFP was uptake by A431, FaDu, HeLa, A549, WM35/DLC2-GFP and MCF-7/DLC2-GFP cells. In addition, specific siRNAs released from G-FFP were able to reduce the expression of Firefly luciferase in HeLa and FaDu cells, but they were unable to reduce the expression of the epidermal growth factor receptor (EGFR) in A431, HeLa, A549 and FaDu cells. The reduction of expression of EGFR in A549 cells was observed when siRNA therapy was combined with photochemical internalization. From these results, can be inferred that the efficiency of siRNA transfection and knockdown was dependent on both the type of cell line and the desired target protein. In vivo studies showed that the gel was formed in situ after intratumoral injection. 3 days after intratumoral administration of FFP containing EGFR specific siRNA, 30% of reduction in the tumors size compared to tumors treated with FFP without siRNA was observed. Based on these results, could be concluded that the developed system is a potential siRNA delivery system when administered subcutaneously and intratumoral, because it was able to complex siRNA, promoted its cell uptake and the siRNA released into the cytoplasm of the cell may reduce the expression of target proteins

L'interférence ARN est un ensemble de mécanismes de régulation essentiel pour denombreux processus au cours du développement. Ces mécanismes sont caractérisés par lʼinhibition séquence-spécifique de lʼexpression de gènes via lʼaction de petites molécules dʼARN. Parmi les différentes voies de l'interférence ARN, la voie des trans-acting siRNAs dérivés du précurseurTAS3,qui combine des caractéristiques des voies des miRNAs et siRNAs, est spécifique des plantes et en contrôle divers aspects essentiels du développement. Dans cette voie, AGO7, un membre de la famille des protéines ARGONAUTE, interagit spécifiquement avec miR390 pour cibler et cliver le transcrit TAS3 amorçant ainsi la production de tasiARFs par lʼaction de SGS3, RDR6 et DCL4. Cette voie est conservée chez toutes les plantes terrestres. Par leur activité de répression sur des membres des facteurs de réponse à l'auxine ARF2, ARF3 et ARF4, les tasiARFS contrôlent la transition de phase et la régionalisation des feuilles. Notre laboratoire et dʼautres ont montré que TAS3 était également exprimé dans la racine dʼArabidopsis, posant la question du rôle joué par la voie TAS3dans le développement des racines. Nous avons établi que la voie TAS3, par lʼaction des tasiARFs, joue un rôle essentiel dans le contrôle de la croissance des racines latérales. Nous avons démontré que la voie TAS3 agit en aval de lʼauxine pour maintenir la correcte zone et abondance des facteurs de réponse à l'auxine, ARF2, ARF3 et ARF4. De plus nous avons élucidé un ensemble complexe de rétroactions de ces ARFs sur lʼexpression de miR390. Bien que les mécanismes de la voie TAS3 ont été identifié par divers screen génétiques, notre connaissance de lʼorganisation subcellulaire de cette voie reste essentiellement méconnue. Pour cette raison, nous avons choisi dʼétudier la localisation subcellulaire de la voie TAS3, en nous focalisant sur AGO7 qui en constitue un composant spécifique. Nous avons montré que AGO7, RDR6 et SGS3 sʼaccumulent dans des focicytoplasmiques spécifiques, les siRNA bodies. Nous avons observé une colocalisation entre cessiRNA bodies et des marqueurs des stress granules ainsi qu'une protéine virale associée aux membranes. Finalement nous avons démontré lʼimportance de la localisation cytoplasmique dʼAGO7pour la biogenèse des tasiARFs. Notre travail a permis de mieux comprendre les mécanismes de lʼaction de la voie TAS3 au cours du développement dʼArabidopsis. / RNA silencing is a regulatory mechanism essential for many processes during development. This mechanism is characterized by the sequence-specific inhibition of gene expression by small RNA molecules. Among the several pathways of RNA silencing, the TAS3 trans-acting small interfering RNA (ta-siRNA) pathway, which combines features of both micro (mi)RNA and siRNA pathways, is unique to plants and controls several key aspects of plant development. In this pathwayAGO7, a member of the ARGONAUTE family of RNAse, interacts specifically with miR390 to target and cut the TAS3 transcript, priming it for production of tasiARFs by SGS3, RDR6 and DCL4 action. This pathway is conserved across all land plants. By their repressing activity on Auxin Response Factors members, ARF2, ARF3 and ARF4, the tasiARFs control phase change and leaf patterning. Our lab and others have shown that TAS3 is also expressed in the root of Arabidopsis, raising the question of the role played by the TAS3 pathway in root development. We have shown that theTAS3 pathway, through the tasiARFs action, plays an essential role in the control of lateral rootgrowth. We have demonstrated that the TAS3 pathway acts downstream of auxin, to maintain the proper zonation and abundance of the Auxin Response Factors ARF2, ARF3 and ARF4. In addition,we unravelled a complex set of feedbacks of these ARFs on miR390 expression. Although the mechanisms of TAS3 processing have been identified through various genetic screens our knowledge of the subcellular organization of this pathway remains essentially unknown. For this reason we have chosen to study the subcellular localization of the TAS3 pathway, and focused on AGO7 which represents a specific element of this pathway. We have shown that AGO7, RDR6 and SGS3 accumulate in cytoplasmic foci, dubbed siRNA bodies. We have observed colocalization between these siRNA bodies and markers of the stress granules and a membrane-associated viral protein. Finally we have demonstrated the functional relevance of the cytoplasmic localization of AGO7 for the biogenesis of tasiARFs. Our work has led to a better understanding of the mechanisms underlying the action the TAS3 pathway during the development of Arabidopsis.

A comprovação de que siRNA pode ser usado para supressão de genes em diferentes células de mamíferos atraiu grande atenção como nova possibilidade de tratamento para diversas doenças. No entanto, para aplicação terapêutica de siRNA é necessário o desenvolvimento de um sistema de liberação efetivo e não tóxico, que permita a captação celular do siRNA e também que evite a sua degradação por enzimas. A capacidade de supressão de genes promovido pelo siRNA depende tanto do número de moléculas de siRNA transfectadas quanto da taxa de duplicação da célula. Uma das formas farmacêuticas que vem sendo amplamente utilizadas na literatura com o objetivo de prolongar e proteger a liberação de fármacos são as formulações com capacidade de formação de gel in situ. Desta forma, a presente pesquisa teve por objetivo o desenvolvimento farmacotécnico de formulações líquido cristalinas com formação de gel in situ após administração por via subcutânea para veiculação sustentada de siRNA. Misturas adequadas de monoleína, propilenoglicol, tampão Tris e polietilenoimina ou oleilamina (polímero e lipídeo catiônico, respectivamente) formaram sistemas precursores capazes de se geleificar in situ com excesso de água e, como demonstrado pelo estudo de absorção de água, a formação do gel é um processo rápido. As formulações desenvolvidas também foram eficientes para complexar o siRNA comprovando a importância da incorporação dos aditivos catiônicos aos sistemas. A liberação in vitro dos sistemas líquido cristalinos mostraram que a liberação é dependente da taxa de absorção de água e os estudos in vivo em modelo animal para avaliação da formação do gel in situ e toxicidade demonstraram que o gel se forma in vivo com a absorção de água dos fluidos corporais, sendo biodegradável e biocompatível. Os sistemas desenvolvidos mostraram-se promissores para o tratamento de doenças onde a administração localizada e sustentada de siRNA é necessária. / The evidence that siRNA can be used for suppression of genes in different mammalian cells attracted wide attention as a new possibility of treatment for various diseases. However, for siRNA therapeutic application is necessary to develop an effective non-toxic delivery system, which facilitate the siRNA cell uptake and avoid its degradation by enzymes. The genes suppression promoted by siRNA depends on the number of siRNA molecules transfected as the cell replication rate. One of the dosage forms that have been widely used in literature in order to prolong and protect the drug release is the in situ gelling formulations. Thus, the present study aimed the development and characterization of the in situ gelling liquid crystal-based systems for subcutaneous application of siRNA in gene therapy. Appropriate mixtures of monoolein, propylene glycol, Tris buffer and polyethyleneimine or oleylamine (cationic polymer and lipid, respectively) was able to form precursor formulation that gelling in water excess and, as demonstrated by the swelling studies, the gel formation is a fast process. The developed formulations were also effective for complexing the siRNA, indicating the importance of the incorporation of cationic additives in the systems. The in vitro release study showed that the release is dependent on the water absorption rate. In vivo studies in animal models have shown the gel is formed in vivo after water uptake of body fluids, and it is biodegradable and biocompatible. The systems developed are promising for the treatment of diseases where the local and sustained administration of siRNA is necessary.

La conception de biomatériaux actifs est actuellement encouragée par le manque de thérapies régénératives efficaces pour des tissus endommagés présentant une faible capacité d’autoréparation. Les progrès récents concernant les techniques de préparation de matériaux structurés (électrospinning, microfluidique) ainsi que la découverte du fort potentiel régénératif des cellules souches ont suscité un regain d’intérêt pour des projets collaboratifs à l’interface entre biologie et sciences des matériaux. Une approche prometteuse de régénération tissulaire repose donc sur la combinaison de cellules souches et de biomatériaux implantables. Des biomatériaux innovants, injectables et servants à la fois de support aux cellules et de réservoir de molécules actives telles que des protéines ou des agents de thérapie génique (Matrice Génétiquement Activée) ont été développés. Se plaçant plus particulièrement dans le contexte de l’ingénierie du cartilage, ce travail a pour objectif de développer une stratégie complémentaire concernant l’orientation de la différenciation de cellules souches mésenchymateuses (CSM) grâce au mécanisme d’interférence ARN.La principale difficulté rencontrée lors de l’utilisation d’acides nucléiques pour induire la différenciation des CSM reste leur faible capacité à traverser les membranes cellulaires, due à leur nature hydrophile et leur charge négative. De plus, les acides nucléiques sont dégradés très facilement par les nucléases extracellulaires, ce qui rend nécessaire l’utilisation d’un vecteur. Les vecteurs non-viraux sont d’excellents candidats pour des applications in vivo en raison de leur faible coût de production et leur faible immunogénicité. Toutefois, la plupart des systèmes de vectorisation trouvés dans la littérature présentent un manque de reproductibilité associé à une cytotoxicité vis-à-vis des cellules primaires. Nous souhaitions donc développer un système de transfection synthétique à la fois efficace et biocompatible. Pour cela, nous nous sommes basés sur les résultats encourageants concernant l’utilisation des micelles de complexes polyioniques (PIC) pour la transfection des cellules dendritiques. Ces micelles sont formées par complexation de deux polyélectrolytes : un copolymère à blocs double-hydrophiles (CBDH) avec un bloc anionique et un homopolymère cationique. Dans ce travail, nous avons évalué le polyoxyde d’éthylène – b – polyacide méthacrylique en tant que CBDH et la poly-L-lysine ou le polyéthylènimine en tant que polycation. L’influence des caractéristiques des composantes (asymétrie du CBDH, nature du polycation, taille des blocs, ratio de charges…) sur les propriétés physico-chimiques des micelles formées (taille, charge de surface) a d’abord été étudiée. Puis, la possibilité de complexation d’un siRNA au sein des micelles ainsi que leur stabilité en conditions physiologiques ont été évaluées. La formulation des micelles a été conçue pour permettre une dissociation des objets à un pH comparable à celui des endosomes ; ceci a été vérifié par diffusion dynamique de la lumière. Une analyse par cytométrie en flux avec un siRNA marqué TAMRA ont démontré l’internalisation effective des micelles dans les CSM. Plus important encore, l’inhibition spécifique d’un gène cible, Runx2, a été démontrée à un niveau comparable à celui d’un vecteur commercial standard, la Lipofectamine2000®. La seconde partie de la thèse a consisté en l’élaboration de microparticules. A cet effet, nous avons préparé des microsphères de collagène par un dispositif de microfluidique, et ce à partir de diverses sources de collagène (murin, porcin, bovin). Des expériences préliminaires démontrent qu’il est possible d’imprégner les micelles dans les microsphères. De même, de premiers résultats encourageants ont été obtenus quant à la capacité du système globale à assurer l’adhésion cellulaire et permettre une transfection efficace des CSM dans un environnement 3D par les micelles PIC vectorisant un siRNA anti-Runx2. / The relative lack of efficient regenerative therapies for damaged tissues with low capacity for self-repair is one major motivation for the design of new active biomaterials. Recent progress in hierarchical materials processing techniques (electrospinning, microfluidics…) and the demonstration of the strong regenerative potential of stem cells have prompted renewed interest for collaborative projects at the biology / materials science interface. The combination of stem cells and active implantable materials has emerged as a high potential approach for the regeneration of damaged tissues. In particular, injectable cell carriers also acting as a reservoir for active molecules like proteins or gene therapy agents (Gene Activated Matrices) bring about innovative solutions to current issues in the field of tissue engineering. In the context of cartilage regeneration, the main objective of this work was to investigate a complementary strategy to orient mesenchymal stem cell (MSC) fate by the use of RNA interference. One major difficulty to reach high transfection levels and efficiently direct MSC differentiation comes from the low ability of nucleic acids (NA) to cross cellular membranes, largely due to their hydrophilicity and negative charge. This, along with a strong susceptibility to extracellular nucleases, calls for efficient gene delivery vectors. Their low production cost and low immunogenic potential make non viral vectors good candidates for in vivo applications. Besides, most systems reported in the literature show reproducibility and cytotoxicity issues with primary cells that we intended to address to achieve a safe and efficient synthetic vector for MSC. Based on previous encouraging results on the transfection of dendritic cells, we chose to investigate tripartite polyionic complex (PIC) micelles. Their formation is based on the polyelectrolyte complexation of a polyanionic double-hydrophilic block copolymer (DHBC) with a cationic homopolymer. In this work, we investigated polyethylene oxide – b – polymethacrylic acid as the DHBC and Poly-L-Lysine or Polyethyleneimine as the polycation. One major part of the work was to study the influence of micelles components characteristics (block size, DHBC asymmetry, polycation nature and molecular weight, polyelectrolyte charge ratios, etc.) on the physical characteristics (dimensions, surface charge) of the obtained nanoparticles. We then studied the ability of micelles to stably complex siRNA at high loading levels, and their stability in physiological conditions. Importantly, the PIC micelles’ formulation was designed to allow for pH-triggered disassembly in acidic conditions similar to those found in endosomes, as assessed by light scattering measurements. These nanoparticles were shown to be efficiently internalized inside MSC by flow cytometry using a fluorescently labeled SiRNA-TAMRA. Most importantly, they were shown to efficiently down-regulate Runx2 mRNA in MSC, at levels similar to those reached with the gold standard Lipofectamine2000®. The second major step for the development of a GAM suited for cartilage regeneration was to elaborate injectable microparticles. To this purpose, we prepared collagen microspheres through a microfluidic-based process and with different collagen sources (murine, bovine, and porcine). Preliminary experiments show that micelles can be efficiently loaded into the microspheres. First encouraging results were also obtained regarding the ability of the created GAM to support cell adhesion, and to allow for the efficient transfection of MSC in this 3D environment, thanks to an anti-runX2 siRNA vectorized with PIC micelles. This proof-of-concept study has demonstrated that the main elements of the nano-in-micro system are ready and mostly meet the assigned requirements. This opens the way for further work to assess the ability of this GAM to effectively improve MSC chondrogenesis and ultimately cartilage repair.

O mecanismo de interferência por RNA (RNAi) é um evento de silenciamento gênico através da degradação do RNA mensageiro. Desta forma, a administração de siRNA (molécula efetora do RNAi) é uma terapia promissora para o tratamento de diversas doenças como o câncer. Porém, para a sua efetiva aplicação terapêutica é necessário o desenvolvimento de sistemas de liberação capazes de liberar o siRNA nas células alvo, promover a sua internalização celular e evitar a sua degradação. Dentre os sistemas de liberação, os de liberação localizada, como os sistemas de geleificação in situ, apresentam vantagens sobre administração sistêmica. Formulações fluidas precursoras (FFP) que formam sistemas líquido cristalinos viscosos in situ podem ser obtidas a partir de lipídeos anfifílicos que absorvem água do meio e se rearranjam. Neste contexto, a presente pesquisa teve como objetivo avaliar o gel formado in situ a partir da FFP (G-FFP), composta por monoglicerídeos (MO), polietilenoimina (PEI), propilenoglicol (PG) e tampão tris, como sistema de liberação localizada de siRNA na terapia do câncer de pele. Os resultados mostraram que o G-FFP é uma mistura de fase cúbica e fase hexagonal. O G-FFP liberou o siRNA de maneira sustentada e complexado ao PEI. A FFP pode ser esterilizada por filtração em membrana e foi capaz de complexar altas concentrações de siRNA (15 mM) e de proteger o siRNA da degradação. A citotoxicidade foi dependente da concentração de FFP e esta quando complexada com siRNA teve a toxicidade diminuída. O siRNA liberado do G-FFP foi internalizado pelas células A431, FaDu, HeLa, A549, WM35/DLC2-GFP e MCF-7/DLC2-GFP. Além disto, siRNAs específicos liberados pelo G-FFP foram capazes de reduzir a expressão da proteína Firefly luciferase em células HeLa e FaDu, pórem não foram capazes de reduzir a expressão do receptor do fator de crescimento epidérmico (EGFR) nas células A431, HeLa, A549 e FaDu. A redução da expressão de EGFR em células A549 foi observado quando a terapia com siRNA foi combinada com internalização fotoquímica. Destes resultados, pode-se inferir que a transfecção celular do siRNA e o silenciamento gênico promovido por ele foi dependente tanto do tipo de linhagem celular como do alvo desejado. Os estudos in vivo mostraram que ocorre a formação de gel intratumoral e após 3 dias da administração intratumoral da FFP contendo siRNA específico para EGFR houve redução de 30% no tamanho dos tumores comparados aos tumores tratados com FFP sem siRNA. Diante destes resultados, pode-se concluir que o sistema desenvolvido tem potencial como sistema de liberação localizada de siRNA quando aplicado subcutaneamente ou intratumoral, uma vez que complexa o siRNA, promove a sua internalização celular e o siRNA liberado no citoplasma das células pode reduzir a expressão de proteínas de interesse. / RNA interference (RNAi) is a mechanism in which small interfering RNA molecules (siRNA) inhibit gene expression, by causing the messenger RNA degradation. Thus, siRNA is a promising therapy for the treatment of several diseases such as cancer. However, the development of delivery systems able to protect the siRNA from degradation and promote its cell uptake is essential for therapeutic use of siRNA. Among the delivery systems, the localized delivery system such as in situ gelling delivery system, have advantages over systemic administration. Precursor fluid formulations (FFP), which forms in situ viscous liquid crystalline systems, can be obtained from amphiphilic lipids that absorb water from the environment and self-assembling. In this context, the present study aimed to evaluate the in situ gel formed from the FFP (G-FFP), composed of monoglycerides (MO), polyethyleneimine (PEI), propylene glycol (PG) and Tris buffer, as localized delivery system for siRNA in skin cancer therapy. The results showed that the G-FFP is a mixture of cubic and hexagonal phase. The G-FFP sustained release of siRNA and the siRNA is released complexed with PEI. The FFP can be sterilized by membrane filtration at 0.22 ?m. FFP was able to complex high siRNA concentration (15 mM) and protect the siRNA from degradation. The cytotoxicity was dependent on the FFP concentration, when FFP was complexed with siRNA it was observed a decreased in toxicity. The siRNA released from G-FFP was uptake by A431, FaDu, HeLa, A549, WM35/DLC2-GFP and MCF-7/DLC2-GFP cells. In addition, specific siRNAs released from G-FFP were able to reduce the expression of Firefly luciferase in HeLa and FaDu cells, but they were unable to reduce the expression of the epidermal growth factor receptor (EGFR) in A431, HeLa, A549 and FaDu cells. The reduction of expression of EGFR in A549 cells was observed when siRNA therapy was combined with photochemical internalization. From these results, can be inferred that the efficiency of siRNA transfection and knockdown was dependent on both the type of cell line and the desired target protein. In vivo studies showed that the gel was formed in situ after intratumoral injection. 3 days after intratumoral administration of FFP containing EGFR specific siRNA, 30% of reduction in the tumors size compared to tumors treated with FFP without siRNA was observed. Based on these results, could be concluded that the developed system is a potential siRNA delivery system when administered subcutaneously and intratumoral, because it was able to complex siRNA, promoted its cell uptake and the siRNA released into the cytoplasm of the cell may reduce the expression of target proteins

O glioblastoma multiforme (GBM) é um dos tumores mais letais e a radioterapia permanece como um dos principais tratamentos. Novas estratégias são necessárias para coibir a resistência ao tratamento, como o silenciamento de fatores de transcrição (FTs). Nossa hipótese é a de que FTs associados a listas de genes diferencialmente expressos, os quais foram selecionados para linhagens de GBM irradiadas, ou comparando amostras de GBM à amostras de tecido cerebral, possam fornecer alvos moleculares que aumentariam a morte das células tumorais, quando silenciados. Foram analisadas a proliferação, morte e ciclo celular, além da formação e diferenciação de neuroesferas, utilizando, em quase todas as etapas, a citometria de fluxo. Os FTs HEB e E2F1, cujas funções principais estão relacionadas à neurogênese e proliferação celular, foram selecionados a partir das análises in silico de GBM irradiados ou não, ou de GBMs comparados a amostras de cérebro normal, respectivamente. Esses FTs encontram-se expressos em linhagens U87, astrócitos primários e neuroesferas provenientes das mesmas, analisadas por Western blot. O silenciamento de HEB e E2F1 na linhagem U87, de forma geral, reduziu a proliferação, induziu morte celular e diminuiu a porcentagem de células em G0/ G1, em pelo menos um dos tempos analisados (24, 48 e 72h) em relação ao grupo transfectado com a sequência scrambled. O silenciamento de HEB e E2F1 reduziu o número de neuroesferas quando comparadas às células transfectadas com a sequência scrambled. Possivelmente, a capacidade anti-proliferativa do silenciamento dos FTs HEB e E2F1 observada no cultivo em monocamada da U87, possam atuar na capacidade de formação de neuroesferas e, consequentemente, podem ter um papel na manutenção das células tronco do GBM. O silenciamento não alterou a radiorresistência da U87 cultivada em monocamada, com exceção dos efeitos do silenciamento de E2F1 em 24 h, em que houve radioproteção. A irradiação não reduziu o número de neuroesferas silenciadas para HEB em comparação ao grupo não irradiado, mas reduziu o número de células presentes nas neuroesferas, indicando uma possível atuação de HEB na resposta à irradiação em neuroesferas, fato este nunca antes descrito. O silenciamento de E2F1 não interferiu na resposta das neuroesferas à radiação. A expressão de CD133 avaliada oito dias após a dissociação das células silenciadas para E2F1 e HEB, cultivadas em meio de diferenciação, foram superiores ao do grupo scrambled, indicando uma possível diminuição na diferenciação celular. O silenciamento dos dois FTs não atuou na seleção positiva de CD133+ após a irradiação, como observado no grupo das neuroesferas transfectadas com a sequência scrambled e irradiadas, comparado às não irradiadas. Assim, E2F1 e HEB mostraram-se alvos interessantes no sentido de reduzir a proliferação, tanto em células U87 cultivadas em monocamada quanto em neuroesferas. / Glioblastoma multiforme (GBM) is one of the most lethal tumors, and radiation therapy remains one of the main treatments. New strategies are needed to suppress typical GBM treatment resistance and transcription factors (TFs) silencing seems to be a promising strategy. Our hypothesis is that TFs associated with lists of differentially expressed genes which were selected for irradiated compared to shamirradiated GBM cell lines, or GBM samples compared to brain tissue samples, could provide molecular targets that are supposed to increase tumor cell death when they are silenced. We analyzed proliferation, cell death and cell cycle progression, besides the formation and differentiation of neurospheres, using several analyses by flow cytometry. The TFs HEB and E2F1, whose primary functions are related to neurogenesis and cell proliferation, were selected from in silico analysis of GBM irradiated or sham-irradiated GBMs and GBM samples compared with normal brain samples, respectively. These TFs were found expressed in U87 GBM cell line, and primary astrocytes, as well as in neurospheres derivated from both, as analyzed by Western blot. Silencing of E2F1 and HEB in U87 cells, reduced proliferation, induced cell death and decreased the percentage of cells at G0/G1 (24, 48 or 72h) compared to the scrambled sequence transfected group. HEB and E2F1 silencing reduced the number of neurospheres when compared to cells transfected with scrambled sequence. Possibly, the anti-proliferative ability of silencing of HEB and E2F1 TFs observed in monolayer culture of U87, may act in neurospheres forming capacity and therefore may play a role in the maintenance of GBM stem cells. In our experiments, gene silencing did not alter the radio-resistance of U87 grown in monolayer. Irradiation did not reduce the number of neurospheres silenced for HEB compared to non-irradiated group, but reduced the number of cells present in neurospheres, indicating a possible role of HEB in response to ionizing irradiation in neurospheres, a fact that was not described yet. The silencing of E2F1 in neurospheres did not affect the response to irradiation. The expression of CD133, as assessed at eight days after the dissociation of cells silenced for E2F1 and HEB (cultured in differentiation culture media), was superior compared with the scrambled group, indicating a possible decrease in cell differentiation. The silencing of both TFs did not influence the positive selection of CD133 after irradiation, as observed in the group of neurospheres transfected with scrambled sequence, and irradiated compared to nonirradiated. Thus, E2F1 and HEB proved to be interesting targets for decreasing proliferation in both U87 cells grown as monolayer or neurospheres.

Ce travail concerne le développement d’un système de vectorisation nanoparticulaire pour l’interférence ARN, constituant une nouvelle proposition thérapeutique applicable à des pathologies virales ou tumorales, et possiblement complémentaire aux traitements existants. La vectorisation de siRNA est ici basée sur l’enrobage multicouche de nanoparticules de phosphate de calcium, la multicouche étant constituée de dépôts alternés de PEI modifié et de siRNA. Ce système permet d’obtenir une efficacité de transfection des cellulaires cibles supérieure à celle des procédés conventionnels et une rémanence fonctionnelle in vitro jusqu’à neuf jours. Les résultats d’interférence ARN obtenus ont permis notamment d’inhiber l’infection par le virus de l’hépatite C jusqu’à 99,95%, l’inhibition de l’expression d’une protéine intrinsèque jusqu’à90,5%, et le ralentissement de la croissance cellulaire dans un modèle 3D mimant une tumeur hépatique jusqu’à 46,5%. Ces nanoparticules pourraient présenter un intérêt majeur, en offrant une action à long terme et en résolvant la plupart des difficultés rencontrées en utilisant des siRNA en thérapie. / This work concerns the development of a nanoparticle vector system for RNA interference,constituting a new therapeutic option applicable to viral and tumor pathologies,and possibly complementary to existing treatments.siRNA vectorisation is here based on multi layer coating of alcium phosphate nanoparticles,the multi layer being constituted of alternate coatings of modified PEI and siRNA.This system triggers a better transfection efficiency of target cells than classic techniques,as well as a functional persistence up to 9 days in vitro.RNA interference results using CPnp allowed inhibition of hepatitis C virus infection up to 99.95%,of intrinsic protein expression up to 90.5%,and of cell growth in a 3 D model mimicking an hepatic tumor up to 46.5%.These nanoparticles could be of major interest,by offering a long term action,and resolving most of the issues found in the use of siRNA in therapy.

Les « cell penetrating peptides » (CPPs) sont des vecteurs peptidiques capables de délivrer diverses molécules (acides nucléiques, protéines, peptides, petites molécules, etc.) à l'intérieur des cellules de mammifères. Notre laboratoire a élaboré le vecteur amphipatique secondaire CADY capable de transporter, indépendamment de toute voie d'endocytose, des molécules thérapeutiques telles que les petits ARN interférents (siRNA). En effet, leur potentiel thérapeutique réside dans leur capacité à inhiber de manière spécifique l'expression de protéines dérégulées dans un cadre pathologique.Le sujet de ma thèse s'est centré sur la caractérisation et l'optimisation des complexes CADY/siRNA. Au cours de mes travaux, nous avons pu mettre en évidence que CADY adoptait une structure en hélice alpha en présence du siRNA ce qui conduit à la formation des nanoparticules. Notre étude a eu pour but d'optimiser la séquence de CADY et de contrôler sa formulation pour permettre le transfert de notre système de vectorisation d'une application in cellulo à une application in vivo. En premier lieu, nous avons mené une étude de relations structure-activité avec six peptides analogues de CADY, en réalisant des mutations sur les résidus tryptophanes (PSF1, PSF2, PSF3 et PSW) et sur la zone initiatrice de l'hélice alpha (PG9, PG16). L'analyse approfondie de ces analogues a permis de confirmer que la limitation du caractère amphipathique et du polymorphisme structural des vecteurs conduisaient à une réduction de l'efficacité d'internalisation. Parmi les 6 analogues, seules les nanoparticules à base de PG9 et PG16 présentent des résultats in cellulo comparables à ceux obtenus avec les nanoparticules CADY/siRNA. A ce jour, la séquence primaire de CADY étant la plus adaptée pour la transfection de siRNA, nous avons établi une procédure de formulation standardisée permettant un autoassemblage CADY/siRNA reproductible et homogène, dont la taille moyenne est de 106 ± 31 nm et l'indice de polydispersité de 0,357 ± 0,053. De plus, nous avons mis en place une procédure d'extrusion/lyophilisation afin de stocker les nanoparticules sous forme de poudre. Celle-ci peut être resuspendue en milieu aqueux sans modifications des propriétés colloïdales des nanoparticules ni de leur capacité de transfection.Dans le but d'améliorer la spécificité tissulaire et la biodisponibilité des nanoparticules CADY/siRNA pour une application in vivo, nous avons greffé des motifs de ciblage (YIGSR-S) et de furtivité (PEG) sur la séquence de CADY. L'ajout de ces deux entités, de natures très différentes, ne modifie que faiblement les caractéristiques physico-chimiques (ex. taille moyenne des complexes) et biologiques (transfection cellulaire) des nanoparticules. Ces résultats sont très encourageants pour le développement de nanoparticules dites de 3ème génération, sur lesquelles on peut greffer plusieurs sortes de molécules d'intérêts (ciblage, polymère, agent de contraste etc.).L'ensemble des résultats obtenus au cours de ma thèse marque un réel progrès dans l'optimisation de la formulation du vecteur CADY, et nous incitent à exploiter davantage son potentiel pour le transfert de siRNA in vivo. / Cell penetrating peptides (CPPs) are short peptides that can enter many cell types and transduce into cells a wide range of molecular therapeutics (nucleic acid, proteins, peptides, small molecules, etc.). Our laboratory has developed the secondary amphipathic peptide CADY able to promote the transport of small interfering RNA (siRNA) independently of all endocytotic pathways. Indeed, siRNA therapeutic interest lies on its ability to inhibit specifically deregulated proteins in the context of pathology. The subject of my thesis focused on the characterization and optimization of CADY/siRNA complexes. During my work, we have been able to show that CADY adopts a helical structure while interacting with the siRNA leading to the formation of nanoparticles. The goal of my study was to optimize CADY sequence and control its formulation to consider the transferring from an in cellulo to an in vivo application of our vectorization system. First, we conducted a structure-activity study with six analogues by mutating CADY on tryptophane residues (PSF1, 2, 3 and PSW) and in the area initializing helical structure (PG9, PG16). A thorough analysis of these analogues has confirmed that the limitation of the amphipathic character and structural polymorphism is directly related to the reduction of internalization efficiency of our CPPs. Among the six analogues, only PG16/siRNA and PG9/siRNA nanoparticles show in cellulo results equal to those obtained with CADY/siRNA. Based on the fact, that CADY is the most suitable vector for the transfection of therapeutic molecules such as siRNA, we have established a standard formulation procedure to obtain reproducible and homogeneous CADY/siRNA complexes with an average size of 106 ± 31 nm and a polydispersity index of 0.357 ± 0.053. In addition, we have implemented an extrusion/lyophilization step to allow nanoparticle storage as powder, which can be re-suspended in an aqueous solution without losing their colloidal and transfection properties.In order to improve tissue specificity and bioavailability of CADY/siRNA nanoparticles for an in vivo application, we have grafted ether a targeting sequence (YIGSR-S) or a stealth motif (PEG) to the CADY sequence. These two entities of very different nature provoke only few changes in the physicochemical (e.g. average size) and biological (cell transfection) characteristics of the nanoparticles formed with a siRNA. These results are very encouraging for the development of the so-called 3rd nanoparticle generation which includes several kinds of molecules (targeting, polymer, contrast agent etc.).These outcomes mark the real progress in CADY formulation optimization, and encourage us to further exploit its potential for the in vivo transfer of siRNA.