A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of requirements for the degree of Master of Science. Johannesburg 2015. / Ovarian cancer is the most lethal gynaecological cancer. About 90% of ovarian cancers are epithelial (ovarian carcinomas), thought to arise from the ovarian surface epithelium. Diagnosed usually at clinically advanced stages, many patients show poor response to chemotherapy, with resistance and recurrent disease being prevalent. siRNA technology is currently being explored in clinical trials as a form of targeted therapeutic strategy in the disease. RBBP6 is a 250kD protein that enhances MDM2-mediated ubiquitination of p53 and also plays a role in cell cycle regulation and cell differentiation. It is upregulated in numerous cancers such as lung, oesophageal, colorectal and cervical cancer. RBBP6 suppresses p53 binding to DNA thereby inhibiting p53-dependent gene transcription. RBBP6 was knocked down using 30 nM siRNA in RMG-1 cells for 48 hours, after which the cells were treated with 50 nM paclitaxel and 0.5μM camptothecin for 24 hours. xCELLigence real time cell analysis was used to evaluate cell proliferation. qPCR and western blot were used to evaluate both gene expression and protein expressions respectively, of Bax, Bcl-2, MDM2, p53 and p21. Flow cytometry was used to determine the mode of cell death elicited apoptosis and also analyse changes in cell cycle progression. qPCR and Western blot analyses showed that RBBP6 expression reduced by approximately 57%. There was a significant upregulation of p53 and a significant downregulation of Bcl-2 in siRBBP6 transfected cells (p<0.05). Knockdown of RBBP6 resulted in a 37±5.8% cell death. There was a significant increase in cell death in paclitaxel and siRBBP6 co-treated cells (81.6±0.79%) as compared to cells treated with paclitaxel only (76.±1.14%). siRNA-mediated knock down of RBBP6 induces cell death in RMG-1 ovarian carcinoma cells. In addition, paclitaxel-induced cell death in RMG-1 cells is potentiated by RBBP6 siRNA transfection. A combination of chemotherapy with paclitaxel or camptothecin and RBBP6 siRNA could be a possible therapeutic strategy in combatting ovarian carcinomas.

Des progrès récents dans l’utilisation préclinique et clinique des petits ARN interférents (siRNA) ont montré leur potentiel en tant qu’inhibiteur de la synthèse protéique dans de nombreuses pathologies comme le cancer. L’administration des siRNA rencontre un certain nombre de problèmes liés à leur dégradation rapide dans les milieux biologiques, ainsi qu’à leur difficulté à pénétrer au sein des cellules cibles en raison de leur hydrophilie et de leur charge négative. Une des clés de l’amélioration de l’efficacité thérapeutique de ces molécules repose sur l’emploi de vecteurs. Au cours de cette thèse, des lipoplexes capables de protéger les siRNA contre la dégradation et de favoriser leur transport jusqu’aux cellules cibles ont été développés et optimisés. Pour ce faire, des lipoplexes recouverts d’HA ont été formulés pour la vectorisation active de siRNA vers des cellules tumorales surexprimant le récepteur CD44. Dans la première partie de cette thèse, la formation des lipoplexes a été étudiée, ainsi que les paramètres influençant leur organisation supramoléculaire. L'insertion de l'HA dans la structure du liposome au moment de la formation des vésicules a entraîné une augmentation de la taille des liposomes en fonction de la concentration d’HA. Leur complexation avec les siRNA a encore augmenté la taille des particules obtenues. L’ajout des acides nucléiques lors de la formation des lipoplexes a provoqué un déplacement d'une partie du conjugué HA-DOPE de la structure des lipoplexes, comme montré par électrophorèse capillaire. La titration calorimétrique isotherme et les études de diffraction des rayons X ont démontré que, sous l’effet des interactions électrostatiques avec les siRNA, un réarrangement des bicouches lipidiques a lieu, conduisant à la formation de vésicules oligolamellaires, confirmé visuellement par cryo-microscopie. Enfin, le positionnement convenable de l’HA sur la surface des lipoplexes et sa capacité de se lier spécifiquement aux récepteurs de CD44 ont été démontrés par la technique de résonance plasmonique de surface. Dans la deuxième partie de cette thèse, la capture cellulaire et localisation intracellulaire des lipoplexes-HA ont été évalués par cytométrie en flux et microscopie de fluorescence, et ont montré que les lipoplexes modifies par l’HA sont internalisés plus rapidement que les lipoplexes non modifiés, et une fois dans des cellules, ils sont localisés principalement à l’intérieur des endosomes. La capacité des lipoplexes à transporter des molécules de siRNA intactes au cytoplasme a été confirmée par 81 % d'inhibition d'expression de luciferase in vitro sur la lignée cellulaire de cancer du poumon A549-luc. In vivo, le traitement avec les lipoplexes-HA transportant un siRNA anti-luciferase a mené à une diminution statistiquement significative de l’expression de luciferase, ce qui a été confirmé par la réduction de l'expression d’ARNm de la luciferase dans le poumon des animaux traités avec les lipoplexes-HA. L'analyse de la distribution des lipoplexes dans les poumons a montré que les lipoplexes modifiés par l’HA sont distribués de façon plus importante et plus homogène dans le tissu pulmonaire que les lipoplexes non modifiés. Dans la troisième partie de cette thèse, la diffusion des lipoplexes-HA de siRNA dans le mucus a été étudié, afin d’évaluer la faisabilité de l’administration pulmonaire de ces particules. Les études utilisant la technique de « multiple particle tracking (MPT) » ont montré que la présence d’HA combinée à l'ajout de siRNA ont permis l’obtention de deux formulations de lipoplexes présentant une pénétration efficace dans le mucus, les lipoplexes-HA and les lipoplexes-PEG/HA. En conclusion, un système efficace de lipoplexes utilisant siRNA pour l'inhibition de l'expression génique ciblant les récepteurs CD44 a été développé. Les résultats obtenus confirment que les HA-lipoplexes sont capables de libérer efficacement le siRNA dans le cytoplasme des cellules in vitro et in vivo. / Recent progresses in the preclinical and clinical use of small interfering RNA (siRNA) have shown their potential as an inhibitor of protein synthesis in many diseases such as cancer. The administration of siRNA encounters a number of problems related to their rapid degradation in biological media, and their difficulty in penetrating targeted cells due to their hydrophilicity and negative charge. A key to improving the therapeutic efficacy of these molecules is based on the use of vectors. In this thesis, lipoplexes that can protect siRNA against degradation and facilitate their transport into target cells were developed and optimized. To do this, lipoplexes covered with HA were formulated for active vectorization of siRNA to tumor cells overexpressing the receptor CD44.In the first part of this thesis, the formation of lipoplexes was studied, and the parameters influencing their supramolecular organization. Insertion of HA within the liposome structure during vesicle formation resulted in the increase in liposome size as a function of HA concentration. Their complexation with siRNA has further increased the size of the particles obtained. The addition of siRNAs when forming lipoplexes caused a displacement of a portion of the HA-DOPE conjugate from the lipoplexes structure, as shown by capillary electrophoresis. The isothermal titration calorimetry and X-ray diffraction studies showed that a rearrangement of the lipid bilayers occur under the effect of electrostatic interactions with siRNAs, leading to the formation of oligolamellar vesicles, which was visually confirmed by cryo-microscopy. Finally, the proper positioning of the HA on the surface of the lipoplexes and its ability to specifically bind to the CD44 receptors has been demonstrated by the surface plasmon resonance technique.In the second part of this thesis, cellular uptake and intracellular localization of HA-lipoplexes were assessed by flow cytometry and fluorescence microscopy, and showed that lipoplexes modified by HA are internalized more rapidly than unmodified lipoplexes, and once in the cells, they are mainly localized within the endosomes. The ability of lipoplexes to transport intact siRNA molecules to the cytoplasm was confirmed by 81% of luciferase in vitro expression inhibition on the lung cancer cell line A549-luc. In vivo, treatment with HA-lipoplexes carrying anti-luciferase siRNA led to a statistically significant decrease in expression of luciferase, which was confirmed by reducing the mRNA expression of luciferase in lungs of animals treated with HA-lipoplexes. The analysis of the distribution of lipoplexes in the lungs showed that lipoplexes modified with HA are distributed more evenly in the lung tissue than unmodified lipoplexes.In the third part of this thesis, the movement of siRNA HA-lipoplexes in the mucus was studied to assess the feasibility of administering these particles directly to the lungs. Studies using the technique of "multiple particle tracking (MPT)" showed that the presence of HA combined with the addition of siRNA allowed the preparation of two lipoplexes formulations with efficient mucus-penetration, HA-lipoplexes and PEG/HA-lipoplexes.In conclusion, an efficient siRNA lipoplex system for inhibiting gene expression targeted TO the CD44 receptorS has been developed. The results confirm that the HA-lipoplexes are able to effectively release in vitro and in vivo the siRNA molecules in the cytoplasm of cells.

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. / This work has enabled a comprehensive evaluation of different sequences of siRNAs directed against EWS/Fli-1 in the treatment of tumors of the Ewing sarcoma family of tumors (ESFT). A siRNA was efficiently vectorized by polymeric nanoparticles targeted against a specific membrane marker of ESFT. These nanoparticles were characterized and appear to be well tolerated both in vitro and in vivo. Their evaluation was conducted on human cells and tumors which represents an interesting step forward in the fight against ESFT. The development of a fluorescent model of Ewing's sarcoma will better characterize their effect on metastasis, a key factor in patient survival. Finally, it was shown that in vivo imaging techniques allow to follow the fate of nanoparticles in vivo. That will allow understanding their biodistribution and their mode of action.

La voie de l’ARN interférence (ARNi), en particulier celle des siRNA, constitue la défense antivirale majeure chez les plantes,les nématodes et les insectes. Le génome de l’organisme modèle Drosophila melanogaster code pour trois protéines, Dcr-­‐2, AGO2 et R2D2, indispensables à cette voie. Les mouches mutantes pour une de ces trois protéines sont plus susceptibles et succombent plus rapidement aux infections virales comparées aux mouches sauvages. Beaucoup d’études biochimiques ont permis d’obtenir une image assez précise de la fonction moléculaire de ces trois protéines in vitro. Cependant, plusieurs études in vivo ont révélé une réalité plus complexe, probablement liée à l’association de ces molécules avec des cofacteurs. Ce manuscrit décrit les approches adoptées afin d’identifier les partenaires protéiques de la voie des siRNA et d’étudier leurs rôles, notamment dans un contexte infectieux. Dcr-­‐2, AGO2 et R2D2 ont été étiquetés par génie génétique avec un tag de 16 acides aminés, reconnu par la biotin-­‐ligase BirA, qui permet leur biotinylation après leurs transfections dans les cellules S2. Les cellules transfectées ont été ensuite soumises à différentes infections virales,notamment avec le virus C de la Drosophile (DCV) (Dicistroviridae), le virus de la stomatite vésiculaire (VSV) (Rhabdoviridae) ou le Flock House virus (FHV) (Nodaviridae). Les cellules ont été ensuite lysées au pic de l’infection et les complexes protéiques purifiés et analysés par spectrométrie de masse.[...] / Fighting viral infections is hampered by the scarcity of viral targets and their variability resulting in development of resistance. Viruses depend on cellular molecules for their life cycle, which are attractive alternative targets, provided that they are dispensable for normal cell fonctions. Using the mode! organism Drosophila melanogaster, we identify the ribosomal protein RACK1 as a cellular factor required for infection by the internai ribosome entry site (IRES) containing virus Drosophila C virus (DCV). We further demonstrate that inhibition of RACK1 in human liver cells impairs hepatitis C virus (HCV) IRES-mediated translation and infection. Inhibition of RACK1 in Drosophila and hurnan cells does not affect cell viability and proliferation, and RACK1-silenced adult flies are viable, indicating that this protein is not essential for general translation. Our findings demonstrate a specific function for ribosomal protein RACK 1 in selective mRNA translation and uncover a promising targe! for the development of broad antiviral intervention.

RNA silencing with direct delivery of siRNA has been used to suppress ldhA gene expression in filamentous fungus Rhizopus oryzae. Here, for the first time we show that, introducing small interfering RNA which consequently forms silencing complexes can alter the gene expression and we report a significant reduction of lactic acid production for isolates containing short (25 nt) synthetic siRNA. In all samples lactic acid production was reduced comparing with wild types. The average concentration of lactic acid production by Rhizopus oryzae during batch fermentation process where glucose has been used as a sole carbon source, diminished from 2.06 g/l in wild types to 0.36 g/l in knockdown samples which signify 5.7 times decrease. Interestingly, the average concentration of ethanol production was increased from 0.38 g/l in wild types to 0.45 g/l in knockdown samples. In some samples we were able to report even a 10 fold decrease in lactic acid production. Since R.oryzae is capable to assimilate a wide range of carbohydrates hydrolysed from lignocellulosic material in order to produce many economically valuable bulk material such as ethanol, these results suggest that RNA silencing is a useful method for industrial biotechnology to be applied in fungus Rhizopus oryzae in order to trigger the metabolism and gene expression toward a desired product.

Orientador: Márcio José Tiera / Banca: Julio Cesar Fernandes / Banca: Luis Alexandre Pedro de Freitas / Banca: Éder Tadeu Gomes Cavalheiro / Banca: Marinônio Lopes Cornélio / Resumo: A quitosana, polímero catiônico de origem natural, biocompatível, biodegradável e de baixa toxicidade vem sendo largamente estudada e utilizada no desenvolvimento de vetores não-virais para uso em terapia gênica. Nesse estudo, derivados de quitosana foram modificados com grupos hidrofílicos dietilaminoetil (DEAE) e diisopropiletilamina (DIPEA) tendo como objetivo melhorar a eficiência de transfecção e a liberação do material genético no ambiente intracelular. Poliplexos dos derivados de quitosana com diferentes massas molares foram investigados em ensaios de transfecção in vitro realizados com células HeLa, utilizando siRNA SSB como agente silenciador. Os resultados obtidos mostraram bons níveis de silenciamento e bloqueio de expressão do RNAm SSB, apresentando, em algumas situações, eficiência superior aos vetores não-virais Lipofetamina®, TransIT-siQUEST® e PEI. Fatores que podem afetar a eficiência de transfecção como pH, massa molar, grau de ionização, tamanho dos poliplexos, potencial zeta e razão N/P foram avaliados. Tanto as quitosanas desacetiladas quanto os seus derivados mostraram níveis de viabilidade celular superiores a 80% em ensaios de citotoxicidade realizados com MTS-PMS. Os resultados sugerem que os derivados de quitosana sintetizados e caracterizados nesse estudo possuem características favoráveis que os habilitam como potenciais vetores não-virais para serem usados em terapia gênica / Abstract: Chitosan, a natural biocompatible, biodegradable and low toxicity cationic polymer has been widely studied and used in the development of non-viral vectors for use in gene therapy. In this study, chitosan derivatives were modified with hydrophilic groups diethylaminoethyl (DEAE) and diisopropylethylamine (DIPEA) aiming to improve the transfection efficiency and the release of the genetic material in the intracellular environment. Poliplexes of chitosan derivatives with different molar masses were investigated in vitro transfection assays performed with HeLa cells using SSB siRNA as the silencing agent. The results showed significant knockdown of SSB mRNA expression for chitosan and its derivatives, presenting, in some situations, superior efficiency to the non-viral vectors Lipofetamina®, TransIT-siQUEST® and PEI. Factors that could affect transfection efficiency such as pH, molar mass, degree of ionization, nanoparticle size, zeta potential and N/P ratio were evaluated. Both deacetylated chitosan and its derivatives showed cell viability above 80% in MTS-PMS cytotoxicity assays. The results suggest that the chitosan derivatives synthesized and characterized in this study have favorable characteristics that enable them as potential non-viral vectors to be used in gene therapy / Doutor

Dans les régions de fortes endémicités, 70-80% des carcinomes hépatocellulaires sont induits par le VHB. Bien qu’un vaccin prophylactique très efficace existe, il n’est d’aucune utilité pour les 250 millions de personnes chroniquement infectées. Les traitements actuels pour contrôler l'infection chronique par le VHB montrent des limites et le besoin de nouvelles thérapies se fait ressentir. La Polo-like kinase-1 (PLK1), qui joue un rôle essentiel dans la mitose et est surexprimée dans de nombreux cancers, représente une cible prometteuse. Outre son rôle lors de la division cellulaire, PLK1 est impliquée dans la régulation de l'expression des gènes en interphase. Il a été montré que la protéine X du VHB (HBx) active PLK1 dans des modèles de cellules murines. Cependant, il restait à déterminer si PLK1 jouait un rôle au niveau de la réplication du VHB dans des hépatocytes quiescents. Des études récentes ont mis en évidence un lien positif entre l'activation de PLK1 et la réplication du VHB. Le but de ce projet de thèse a été d'étudier le(s) mécanisme(s) par le(s)quel(s) PLK1 jouait un rôle positif sur la réplication virale, avec pour objectif futur d'explorer l’inhibition de PLK1 comme cible antivirale. L'interaction entre PLK1 et la réplication du VHB a d'abord été décrite à l'aide du modèle HepAD38. Dans ce contexte, l'ADN viral est intégré dans le génome hôte, sous le contrôle d'un système d'expression Tet-off. La transcription de l'ARN prégénomique (pgRNA), à la base de la réplication virale, est initiée par la suppression de tétracycline. Dans ce contexte, l'augmentation de l'expression de PLK1 est corrélée avec la régulation négative de deux protéines; SUZ12 et ZNF198, faisant partie de complexes de remodelage de la chromatine. L'inhibition de PLK1 bloque la réplication du VHB, en agissant au niveau de la transcription virale. D'autre part, dans les modèles de réplication du VHB qui miment au mieux une infection, comprenant les hépatocytes primaires humains (PHH) et les cellules non transformées/différenciées HepaRG (dHepaRG), où le VHB se réplique dans des cellules quiescentes, nous avons mis en évidence que: 1) L'inhibition pharmacologique de PLK1 bloque la réplication virale, semblablement en perturbant l’encapsidation du pgRNA via une interaction avec la protéine core du VHB (HBc). 2) Un knocking-down de PLK1 en utilisant des ARN interférents délivrés par nanoparticules lipidiques résulte en une forte baisse de la production de pgRNA et dans la sécrétion des antigènes HBeAg/HBsAg, sans impact sur la viabilité cellulaire. Ce projet a donc permis la preuve de concept que PLK1 pouvait être une cible thérapeutique afin de controler la réplication du VHB. De plus, grâce à la technologie de délivrance par nanoparticules lipidiques d’ARN interférents, nous avons pu cibler spécifiquement les hépatocytes, augmentant de ce fait la spécificité et l’efficacité de nos traitements. Un travail sur la compréhension précise des méchanismes cellulaires impliqués permettra de mieux cerner cette interaction hôte/virus afin de poursuivre le développement de stratégies antivirales innovantes portant sur l’inhibition de PLK1. De manière significative, l'inhibition de PLK1 est non toxique pour les cellules quiescentes par rapport à des cellules cancéreuses à fort taux réplicatif, ce qui fait de PLK1 une cible thérapeutique attrayante. Des inhibiteurs spécifiques sont déjà en essais cliniques pour certains cancers (e.g., Volasertib pour le traitement de la leucémie myéloïde aiguë) et pourraient servir de thérapie bimodale dans le cadre de patients infectés par le VHB, en inhibant la réplication virale, ainsi qu’en prévenant l'émergence de cellules néoplasiques. L'inhibition de la PLK1 est une approche antivirale innovante, qui, en combinaison avec les thérapies actuelles de type IFN-α ou analogues nucléotidiques offre de grandes promesses pour endiguer l'infection chronique par le VHB mais également prévenir les événements carcinogéniques / In highly HBV endemic regions, 70-80% of hepatocellular carcinoma cases are attributable to this virus. Despite the existence of an HBV vaccine, the World Health Organization estimates 240 million individuals are chronically infected with HBV worldwide. Current antiviral treatments to control chronic HBV infections, and consequently reduce the incidence of liver cancer, are ineffective. New and effective therapies are needed not only for fighting the virus but also to prevent HCC emergence or progression. The polo-like-kinase 1 (PLK1), which plays pivotal roles in mitosis and is over-expressed in many human cancers, represents a promising druggable target in oncology. Beside its role during cell division, PLK1 is also thought to be involved in gene expression regulation during interphase. It was shown that the X protein (HBx) could activate PLK1 in murine cell transformation models. Yet it remained to be determined whether PLK1 could also play a role for HBV replication in non-dividing hepatocytes. Our, and collaborators, recent studies have identified a positive link between PLK1 activation and HBV replication. The goal of this thesis project was to investigate the mechanism(s) by which PLK1 exerts a positive effect on HBV replication, with the future goal of exploring PLK1 as an antiviral target. The interplay between PLK1 and HBV replication was firstly described using the HepAD38 cellular model of HBV replication. In this context, the HBV DNA is stably integrated into the host genome, under control of a Tet-off expression system. Transcription of HBV pregenomic RNA (pgRNA), the template of viral replication, is initiated by tetracycline removal. It has been shown that in HBV-replicating HepAD38 cells, increased PLK1 expression correlates with down-regulation of two proteins that are components of chromatin modifying complexes; SUZ12 protein of the PRC2 complex, and ZNF198 of the LSD1-CoREST-HDAC1 complex. PLK1 inhibition was described to inhibit HBV replication by reducing viral transcription. How PLK1 regulates HBV transcription remains unknown. On the other hand, in HBV replication models that resemble physiologic HBV infection, comprised of Primary Human Hepatocytes (PHH) and non-transformed/differentiated HepaRG cells (dHepaRG), where HBV replicates in non-transformed and non-dividing cells, thus enabling the study of the inter-phasic role of PLK1, irrespective of its well-established cell division implication, we have demonstrated that: 1) A pharmacological inhibition of PLK1 suppressed HBV replication by a different mechanism, likely targeting the packaging of pgRNA by the HBV core antigen (HBc). 2) Knocking-down PLK1 using siRNA delivered by lipid nanoparticles (LNP siPLK1) results in a strong drop of HBV DNAs, RNAs and HBe/HBsAg secretion without affecting the cell viability. This thesis project brought the proof of concept that PLK1 could be a drug target in HBV infection. Furthermore, the use of LNP allowed us to improve the delivery of siPLK1 to hepatocytes. Significantly, PLK1 inhibition is not toxic to quiescent cells in comparison to fast growing cancer cells, rendering PLK1 an attractive therapy target. High level of viremia in chronic HBV patients is a risk factor for progression to liver cancer. PLK1 specific inhibitors are already in clinical trials for other types of cancer (e.g., acute myeloid leukaemia) and could serve as bimodal therapy in HBV infected patients, by inhibiting virus replication as well as preventing emergence and spreading of neoplastic cells. This project was part of a full-working group of experts and thus, has beneficiated of a strong support. The proximity of the oncology-specialized hospital, the Centre Léon Bérard provided us with fresh hepatic biopsy [etc...]

The tumour suppressor p53 actually exists as 9 protein isoforms. Among them, D133p53a, b and g result from the use of an alternative promoter and lack the N-terminal transactivation domain. In addition to its multiple functions maintaining cell integrity, p53 is also able to block angiogenesis, a process strongly contributing in tumour development. Here I have examined the role of p5 isoforms in the regulation of angiogenesis and tumor progression. I also focused my work on FGF-2 regulation by p53. In a first part, full length p53 (p53) and/or D133p53 isoforms were selectively knocked-down with siRNAs in human glioblastoma cells U87. Conditioned medium produced by tumour cells knocked- down for D133p53 inhibited endothelial cell - EC - migration and tubulogenesis. Furthermore, in the chicken chorioallantoïd membrane CAM, D133p53 knockdown gave rise to smaller tumours devoid of vessels, whereas, in mice, it strongly inhibited tumour growth. Interestingly, the double knockdown of p53 and D133p53 also slowed town tumour growth in mice. Taqman Low Density Array revealed distinct gene expression profiles of pro and anti-angiogenic factors regulation following D133p53 and/or p53 knockdown. In particular, D133p53 knockdown resulted in specific down-regulation of Angiogenin and hepatocyte growth factor, whereas the main angiogenic factors FGF-2 and VEGF-A were not significantly affected. Secondly we investigated the regulation of FGF-2 by p53 and its isoforms D133p53 in a human osteosarcoma cell line U2OS, at translational, transcriptional and secretion levels. It resulted in a sophisticated mode of regulation mediated by a transient IRES-dependent translation inhibition of FGF-2. Our data reveal D133p53 isoforms as activators of angiogenesis and tumour progression, through a specific modulation of the angiogenic balance. These isoforms exhibit dominant-negative effect towards p53 but also intrinsic activities, while underlining the importance of considering D133p53 expression in cancers, as well as the potential antitumoural interest of drugs targeting this p53 isoform.

The airway epithelium has many roles in innate immunity including detection of pathogens and transmitting danger signals to other cell types. However, its role as a primary defender against infection is not well recognized. We have investigated methods of augmenting antiviral immunity by application of agents that stimulate viral killing, either in the extracellular space or within the cytoplasm. A recently described property of airway epithelial cells is direct oxidative killing of bacteria through the coordination of Duox and lactoperoxidase enzymes. We have exploited this property by supplementing airway cells with the lactoperoxidase substrate iodide to prevent viral infection. A second method for enhancing antiviral defenses is to supply small interfering RNAs (siRNAs) targeting essential viral genes. We have optimized antiviral siRNAs targeting respiratory syncytial virus by designing them to specifically target positive sense viral RNAs. Finally, we have initiated a project to discover host defense genes that are expressed in either the submucosal glands surface epithelium of human airway. This information will enable a better characterization of the roles for these structures in host defense pathways, and may identify other targets for augmentation of antiviral immunity.

Master of Science in Medicine (by research) / Assisted reproductive technologies (ARTs) are very efficient in producing embryos, however many of these embryos have poor viability. No more than 50% of IVF embryos complete preimplantation development (Hardy et al. 2001). The poor viability is manifested as a reduced rate of cell proliferation and increased rates of apoptosis in the early embryo, resulting in high rates of embryo mortality (Hardy et al. 2001). The reduced viability occurs as a response to a range of cellular stressors that are a consequence of embryo culture (Hardy et al. 2001). The stress of culture disrupts some survival signalling pathways, metabolism of substrates and induces redox stress (Hardy et al. 2001). The cellular stress sensor p53 is expressed in the early embryo but is normally kept at very low levels (Li et al. 2005). This latency may be breached in IVF embryos following culture of zygotes in vitro for 96 hours, resulting in the up-regulation and nuclear accumulation of p53 (Li et al. 2005). Activation of the p53 stress-sensing pathway in the early mouse embryo by culture in vitro causes a marked loss of their developmental competence (Li et al. 2005). This study aimed to establish whether benefits could be obtained by culturing mice IVF embryos in the presence of p53 protein inhibitors. IVF zygotes were cultured individually in 10µl drops of 1.25, 2.5, 5 or 10µM Pifithrin-a (PFTa) in 0.05% DMSO for 96 hours. On day 5 the development stage was assessed. Embryos reaching the blastocyst stage were fixed and stained with Hoechst 33342 for total cell count and the proportion of nuclei with normal and abnormal morphology. There was an increase in the blastocyst rate, total cell count and the proportion of nuclei in a blastocyst with normal nuclei in 10µM-treated embryos. This study also aimed to determine whether benefits could be obtained by incubating mouse IVF sperm with p53 protein inhibitors during IVF. IVF sperm was treated with 1.25, 2.5, 5 or 10µM of PFTa in 0.05% DMSO during incubation with oocytes for 6 hours. Resulting zygotes were cultured for 96 hours individually in 10µl drops of MODHTFM. On day 5 the development stage was assessed. Embryos reaching the blastocyst stage were fixed and stained with Hoechst 33342 for total cell count and the proportion of nuclei with normal and abnormal morphology. There was a reduction in the proportion of fragmented nuclei in blastocysts derived from 1.25 and 10µM-treated sperm. 10µM treated sperm increased the total cell count, the proportion of normal nuclei in a blastocyst and the blastocyst development rate. IVF sperm incubated with 1.25µM PFTa during insemination of oocytes increased the fertilisation rate. Another aim of this study was to establish whether p53 siRNA could inhibit p53 mRNA in mice IVF embryos and if so, whether this would improve embryo viability in culture. IVF zygotes were transfected with 15nM p53 small inhibiting RNA (siRNA) and 0.8% Oligofectamine Reagent immediately, 24 h, 48 h and 72 h after IVF then cultured individually in 10µl drops of MOD-HTFM for a total of 96 hours. On day 5 the blastocyst rate was assessed and immunofluorescence performed probing for p53. There was no significant reduction in p53 expression and no improvement in blastocyst rate at any of the transfection times. However, there was a decrease in the proportion of nuclei which expressed p53 when p53 siRNA was transfected 72 hours after IVF. Also, it was determined that siRNA was efficiently being delivered into the preimplantation embryo with Oligofectamine Reagent. Lastly, this study aimed to determine whether mice sperm with p53 gene deletions have a selective advantage in fertilising the oocyte compared to their wild-type counterparts. p53+/- males were mated with p53+/+ females and the resulting zygotes genotyped after 24 hours of culture. More than 50% of offspring had a p53+/+ genotype. There was no selective advantage for p53 null sperm to fertilise the oocyte, there was actually a disadvantage. The selective disadvantage for p53 null sperm to fertilise the F1 hybrid oocyte in IVF compared to its wild-type counterparts may imply that p53 null sperm are not as viable and may have a survival disadvantage. The reduction in fertility of p53 null sperm in vitro infers that p53 function may be important for the fertility of the mouse sperm in vitro. The results of this thesis could establish means of improving human embryo viability in ART, some examples being P53 protein inhibition in preimplantation embryos during culture prior to transfer to the uterus, or P53 protein inhibition in IVF sperm. The use of the new technology, p53 siRNA was not effective in inhibiting p53 expression, although the build-up experiments determined that siRNA is efficiently delivered into the preimplantation embryo with Oligofectamine Reagent. The demonstration that p53 null sperm has a selective disadvantage in fertilising the oocyte compared to their wild-type counterparts does not indicate a positive selection pressure for naturally occurring mutations to this gene. And so, there is no concern regarding the genetic and epigenetic risks to progeny arising from assisted reproductive technologies with respect to sperm.