Live attenuated Salmonella vectors provide a unique alternative in terms of antigen presentation by acting as a vector for heterologous antigens. The efficiency of any live bacterial vector rests with its ability to present sufficient foreign antigen to the human or animal immune system to initiate the desirable protective immune response. Salmonella vectors encoding heterologous protective antigens can elicit the relevant immune responses, be it humoral, mucosal or cell-mediated. STM-1 is a Salmonella mutant developed by RMIT, harbours a mutation in the aroA gene that renders it attenuated, and is a well characterised vaccine strain currently in use to protect livestock against Salmonella infection. In previous work in this laboratory, STM1 was shown to be capable of eliciting immune responses in mice to plasmid-borne antigens. In this study STM-1 was analysed for its ability to vector the model antigen chicken ovalbumin and test antigen C. jejuni major outer membrane protein using in vivo inducible promoters such as pagC and nirB from the plasmid location. The determination of the architecture around the lesion in STM-1 also allowed the development of constructs expressing heterologous antigen from the chromosome. The induction of immune responses, both humoral and cell mediated, was analysed. Another issue addressed in this study was effect of pre-existing immune responses in the animal host against the vector or related strains and the effects on generation of immune responses against the subsequently vectored antigen. Humoral and cellular immune responses to vectored ovalbumin and C. jejuni Momp antigens were observed following vaccination with STM-1, when antigens were expressed from either the plasmid or chromosomal location. Up-regulation of immune responses, both humoral and cell mediated, was observed against the vectored antigens in animals which were pre-exposed to either the bacterial vector or related strains. These results indicate that STM-1 has the potential to be used as a vector to deliver heterologous vaccine antigens from a single copy gene in the field. Lastly, the results from this study indicate that pre-existing immune responses against the bacterial vector or a related strain do in fact enhance both humoral and T cell responses against the heterologous antigen.

Aedes aegypti is an important vector of the viruses that cause dengue fever, dengue hemorrhagic fever, and yellow fever. In depth genetic studies of vector species have been made possible due to the availability of genome sequences and techniques for producing stably transformed mosquitoes. These resources have also contributed to the establishment of new genetics-based approaches to the control of vector borne disease. Genetic studies of Ae. aegypti have benefited from the ability to drive targeted transgene expression, however a ubiquitous inducible promoter has not been identified in this mosquito. The Drosophila melanogaster heat shock 70 promoter has been shown to drive inducible expression in heterologous systems; however, DmHsp70 possesses significant basal activity in Aedes aegypti. This study characterized the sequence and expression of the heat shock 70 genes of Aedes aegypti. AaHsp70 genes were found to be organized in two clusters, each comprised of three divergent pairs. AaHsp70 genes exhibited robust expression upon heat shock in larvae, pupae, and adults as well as in heads, salivary glands, midguts and ovaries. Genomic regions upstream of AaHsp70 genes were found to drive heat-inducible expression of a reporter in both cell and embryo assays. Deletion analysis of AaHsp70-derived promoters yielded two ~1.5 kb genomic fragments that maintained robust heat inducibility in these systems. Aedes aegypti were transformed with AaHsp70-luciferase gene cassettes using the transposable element Mos1. AaHsp70-luciferase transcripts accumulated specifically after heat shock, and displayed a pattern of rapid induction and decay similar to endogenous AaHsp70 genes. Heat-induced expression of luciferase was observed in transgenic larvae, pupae and adults as well as heads, midguts and ovaries but not salivary glands, with levels varying between transgenic strains. The effect of heat shock on the endogenous RNAi pathway as well as the effect of blood feeding on the expression of AaHsp70 genes was investigated, though reproducible results could not be obtained using the assays employed. In conclusion, the heat shock 70 gene family of Aedes aegypti was identified and characterized. The AaHsp70 promoters described could be valuable for gene function studies as well as for the precise timing of the expression of anti-pathogen molecules. / Ph. D.

Researchers of <i>Clostridium perfringens</i>, a Gram-positive anaerobic pathogen, were lacking a tightlyregulated, inducible promoter system in their genetic toolbox. We constructed a lactose-inducible plasmid-based system utilizing the transcriptional regulator, BgaR. Using the <i>E. coli</i> reporter GusA, we characterized its induction in three different strains of <i>C. perfringens</i>. We then used a newly-developed mutation system to create in-frame deletion mutants in three genes with homology to Type IV pilins, and we used the promoter system described above to complement the mutants. We analyzed each pilin for localization and expression, as well as tested each of the mutants for various phenotypes frequently associated with type IV pili (TFP) and type II secretion systems. PilA2, PilA3, and PilA4 localized to the poles of the cells. PilA2 was expressed in the wildtype when <i>C. perfringens</i> was grown on agar plates, and the PilA3 mutant lacked a von Willebrand factor A domain-containing protein in its secretome. We used our promoter system to express GFP-tagged versions of the TFP ATPase homologues and view them in cells growing on surfaces. We saw that PilB1 and PilB2 co-localized nearly all of the time, while a portion of PilT was independent of the PilB proteins. PilT appeared necessary for the localization of PilB, and it localized independently of TFP proteins in <i>Bacillus subtilis</i>. PilT's typical localization in <i>Bacillus subtilis</i> was disrupted when the GTPase and polymerization activity of cell division protein FtsZ was blocked, suggesting that PilT associates with cell division proteins. / Master of Science

Nicotinic acetylcholine receptors (nAChR) are found widely throughout the body. Like all members of the cys-loop family of receptors, nAChRs are composed of five protein subunits, each with a large extra-cellular domain and four transmembrane domains. Together these subunits form a binding domain, transmembrane pore, and selectivity filter. Neuronal nicotinic acetylcholine receptors, formed exclusively from α2-10 and β2-4 subunits, can form in many arrangements and stoichiometries. Each arrangement can have varying binding affinities and channel kinetics, resulting in great modulatory control. α3 and β2 subunit mRNA is found in CA1 interneurons in the stratum radiatum and stratum oriens of the rat hippocampus, and in surprising expression frequency and ratios. Further study of α3 and β2 subunit mRNA injected into Xenopus laevis oocytes yields interesting results about the potential for two α3β2 subtypes. These results were in intriguing, and prompted further study to better characterize and screen the α3β2 nAChR. In order to do so, a model was needed where the α3β2 nAChR could be studied in a more physiologically relevant mammalian environment, with consistent control over α3 and β2 subunit expression ratios, and sufficient protein expression and functionality. To this end, we created a doxycycline inducible HEK-293 cell line, stably transfected with the genetic sequences for the α3 and β2 subunits and NACHO, a transmembrane protein of the neuronal endoplasmic reticulum, which has been shown to mediate the assembly of α3β2 and other nAChRs. This new model is able to induce expression various ratios between α3 and β2 subunits in a consistent, manner, proving to be valuable tool in the characterization and screening of the α3β2 nAChR.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Ces travaux de thèse d’imagerie moléculaire et translationnelle proposent, sur des modèles murins, deux approches innovantes pour les thérapies géniques. La plupart des cancers sont associés à des dérégulations de l’expression génique et certains gènes sont surexprimés. L’utilisation de microARN (miARN) permet d’envisager une réduction de l’expression d’un gène spécifique mais il est nécessaire de limiter cette inhibition au tissu pathologique. L’utilisation des promoteurs thermo-inductibles couplés à un dépôt local de chaleur autorise un contrôle spatial et temporel de l’expression génique in vivo. Notre projet a été de coupler le contrôle spatio-temporel et l’inhibition d’un gène cible. A cette fin, un miARN synthétique a été placé sous contrôle du promoteur thermo-inductible Hsp70B pour induire l’inhibition d’un gène d’imagerie (luciférase firefly) surexprimé dans une tumeur. L’étude a été menée in vitro sur des lignées cellulaires génétiquement modifiées puis in vivo sur un modèle de xénogreffes chez la souris grâce au suivi en imagerie optique de bioluminescence (BLI). Nos résultats montrent la faisabilité d’induire transitoirement l’inhibition génique au sein d’une tumeur. L’induction est modulable par la température. Cette stratégie peut être couplée à des méthodes couramment utilisées en clinique et ouvre des perspectives thérapeutiques intéressantes. Notre travail de thèse s’intéresse également à l’utilisation d’ARN comme molécule thérapeutique pour la thérapie génique. L’électroporation intra-dermique d’ARN codant pour la luciférase permet de suivre et de quantifier in vivo par BLI l’expression génique. Plusieurs types d’ARN ont été utilisés pour comparer les efficacités respectives des différentes voies traductionnelles. Notre travail démontre que les ARN permettent l’expression transitoire, sans risque d’insertion génomique, d’un gène in vivo. Nous montrons ainsi tout le potentiel de l’utilisation des ARN en thérapie génique. / The present thesis work in molecular and translational imaging establishes two innovative approaches for gene therapy in mouse models. Abnormal regulation of gene expression is the hallmark of cancer, and some of them are overexpressed. MicroRNA (miRNA) can be used as tools to reduce specific gene expression but requires inhibition to be limited to the pathological tissue. Thermo-inducibles promoters associated with local hyperthermia allow for spatial and temporal control of gene expression in vivo. The goal of the present study was to achieve gene inhibition with spatio-temporal control of miRNA expression to inhibit a target gene. In our strategy, a synthetic miRNA was placed under transcriptional control of the heat-inducible promoter Hsp70B to induce inhibition of the imaging reporter gene firefly luciferase overexpressed in a tumor. The study was conducted both in vitro using genetically modified cells lines and in vivo using a xenograft model in mice monitored by optical bioluminescence imaging (BLI). Our data show the feasibility of transient induction and heat-modulation of gene inhibition within a tumor. This strategy can be performed with currently clinically available methods and thus, offers interesting therapeutics prospects. Our work also includes a study on RNA as therapeutic vector for gene therapy. The intradermic electroporation of RNA encoding the imaging reporter gene firefly luciferase allows to monitor and quantify gene expression by BLI in vivo. Several types of RNA have been used to investigate efficiency of the different translational mechanisms. Our data clearly demonstrate that RNA allows for transient gene expression in vivo without any risk of insertion into the target cell’s genome. Altogether, our data highlight the potential use of RNA in gene therapy.