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Thèse présentée en cotutelle : Doctorat en Bioingénierie (Politecnico di Milano) et Doctorat en génie des matériaux et de la métallurgie (Université Laval) / Les stratégies de livraison de gènes non viraux ont suscité un intérêt manifeste pour le développement de nouvelles approches thérapeutiques ainsi que pour la recherche fondamentale et appliquée in vitro. Par rapport aux vecteurs viraux populaires, la classe des vecteurs non- viraux, comme les lipides (CLs) et les polymères (CPs) cationiques, capables de réagir spontanément avec des acides nucléiques (NAs) chargés négativement pour former des nanoparticules appelées complexes, connaît actuellement un regain d'intérêt au sein de la communauté scientifique. En effet, en plus d'être relativement sécuritaires, rentables, ils peuvent être facilement produits et fonctionnalisés, même à grande échelle. Cependant, leur efficacité est encore trop faible pour surpasser leurs homologues viraux. L'efficacité des vecteurs non viraux est un équilibre entre leur capacité à introduire des NAs dans les cellules, permettant ou inhibant ainsi leur expression, leur toxicité inhérente et leur capacité à délivrer des gènes aux cellules cibles. Ces dernières années, des efforts considérables en recherche ont donc été déployés pour développer de nouveaux moyens d'améliorer l'efficacité de cette classe de systèmes de livraison. Cette thèse de doctorat avait ainsi pour but de développer des stratégies innovantes pour améliorer l'efficacité des vecteurs non viraux. Dans ce contexte, nous avons considéré la problématique de la livraison sous deux perspectives différentes : d'une part, la modulation de la chimie du vecteur a été évaluée comme moyen de développer des transporteurs multifonctionnels avec une meilleure efficacité ; d'autre part, une stratégie de stimuler les interactions cellules-nanoparticules par modulation mécanique du comportement cellulaire sur la capacité de livraison de vecteurs non viraux a été étudiée. La première partie de cette thèse visait donc à mettre en évidence l'importance de la chimie de ces vecteurs, plus particulièrement ceux à base de lipides, sur la relation structure-fonction de ces matériaux et l'impact de la chimie du vecteur sur leur capacité ultime à livrer des gènes. Cette thèse a ensuite exploré de nouvelles façons d'améliorer les performances de la polyéthylèneimine (PEI), à savoir le vecteur polymère de référence, en configuration linéaire (lPEI) et ramifiée (bPEI). Tout d'abord, une étude approfondie de toutes les variables expérimentales qui peuvent influencer les performances des polyplexes à base de PEI, a été réalisée afin de déterminer les meilleures conditions de fonctionnement des vecteurs à base de PEI et d'améliorer la standardisation des protocoles de criblage in vitro. Ensuite, le focus a été fait sur le développement d'une approche vectorielle pour fonctionnaliser le bPEI avec des entités fonctionnelles ciblées afin d'améliorer la sélectivité du vecteur envers un type de cellule spécifique. Ainsi, il a été synthétisé une gamme de bPEI conjugués incorporant des peptides de ciblage pour délivrer sélectivement des gènes aux cellules musculaires lisses vasculaires (vSMCs). De plus, ces vecteurs de ciblage spécifique ont été incorporés dans une matrice afin de permettre leur libération locale et contrôlée pour des approches liées au domaine cardiovasculaire. Grâce à la conjugaison d'une séquence peptidique dérivée de l'élastine à la structure de la bPEI, l'efficacité du polymère sur les vSMCs a été améliorée tout en laissant les cellules non ciblées intactes. Ceci est particulièrement important pour la translation des approches non virales de livraison de gènes vers le in vivo. D'autre part, une nouvelle approche basée sur la régulation de la réponse cellulaire à l'administration de la nanoparticule a été élaborée. En effet, les cellules in vivo sont constamment exposées à différents facteurs environnementaux qui gouvernent certaines fonctions cellulaires essentielles. L'application d'un stimulus mécanique exogène en termes de sollicitation vibratoire sur des cellules en cours de transfection (processus de livraison de NAs par vecteurs non-viraux) a donc été évaluée avec des polyplexes à base de lPEI et bPEI. Étonnamment, l'efficacité des polyplexes à base d'lPEI a été grandement améliorée grâce à l'activation de certaines réponses cellulaires clés. Plus précisément, les stimulations mécaniques appliquées aux cellules ont amélioré l'internalisation des polyplexes en déclenchant l'activation de l'endocytose médiée par la clathrine (CME). Cette stratégie a mis en évidence l'importance des réponses cellulaires aux signaux exogènes sur l'internalisation et l'expression finales d'un gène d'intérêt et a ouvert la voie à une nouvelle façon de traiter la question de la livraison non virale. En conclusion, ce projet de doctorat a clairement mis en évidence la pertinence de ces approches chimiques et cellulaires comme moyens efficaces d'améliorer l'efficacité des vecteurs non viraux. En combinant les stratégies conçues dans le cadre de ce projet, il serait possible de franchir une autre étape en ce qui concerne la recherche sur l'administration de gènes non viraux. En effet, le développement d'approches multidisciplinaires tenant en compte à la fois du vecteur d'administration, de l'environnement dans lequel l'administration des gènes a lieu et de la réponse cellulaire, pourrait ouvrir la voie à des stratégies encore plus efficaces et accélérer le passage du laboratoire au patient pour cette catégorie de matériaux. / Non-viral gene delivery strategies have attracted significant interest in the development of novel therapeutic approaches as well as for basic and applied research in vitro. Compared to popular viral vectors, the class of non-viral carriers, namely cationic lipids (CLs) and polymers (CPs) able to spontaneously interact with negatively charged nucleic acids (NAs) to give nanoparticles called complexes, is now witnessing a surge of interest within the scientific community because they are relatively safe, cost-effective, and they can be easily produced and functionalized even at large scale. However, their efficiency in achieving the delivery tasks is still too low to outperform their viral counterparts. The efficacy of non-viral vectors is a tradeoff between their ability to drive NAs into cells, thus allowing/inhibiting their expression, their inherent toxicity, and their ability to deliver genes to target cells. Extensive research effort has thus been put into developing novel ways to improve the efficiency of such a class of delivery systems. In this context, my Ph.D. aimed at developing innovative strategies to improve non-viral vector effectiveness. To this purpose, we dealt with the delivery issue from two different perspectives: on one hand, the modulation of the vector chemistry was disclosed as a way to develop multifunctional carriers with improved effectiveness; on the other hand, we sought to improve cell-(nano)particles' interactions through the mechanical modulation of the cell behavior in response to the delivery of non-viral vectors. The first part of this thesis was thus aimed at highlighting the importance of vector chemistry on the structure-function relationship of such kinds of materials, with a focus on lipid-based carriers. Furthermore, I dealt with the characterization of a novel class of lipid-based vectors to investigate the interconnection between their structure and ultimate gene transfer ability. This thesis next explored novel ways to improve the performances of polyethyleneimine (PEI), namely the gold standard polymer vector, both in linear (lPEI) and branched (bPEI) topography. First, a thorough investigation of all the experimental variables affecting the performances of PEI-based polyplexes was carried out to disclose the best working conditions of PEI-based carriers and improve the standardization of in vitro screening protocols. Next, I focused on the development of a vector-based approach to functionalize bPEI with targeting moieties to improve the vector's selectivity towards a specific cell type. We thus synthesized a series of bPEI conjugates incorporating targeting peptides to selectively deliver genes to vascular smooth muscle cells (vSMCs). Moreover, the targeting vectors were incorporated into a polyplex releasing matrix to enable their local and controlled release for cardiovascular-related approaches. Through the conjugation of an elastin-derived peptide sequence to the bPEI structure, we were able to improve the polymer's effectiveness on target vSMCs while leaving off-target cells unaffected, a fact that is especially relevant for the translation of non-viral gene delivery approaches in vivo. On the other side, a novel strategy based on the regulation of cell response to the delivery of nanoparticles was devised. Indeed, cells in vivo are constantly subjected to different environmental cues that govern some key cell functions. We thus investigated the application of an exogenous mechanical stimulus in terms of vibrational loading to cells undergoing transfection (i.e., the delivery of NAs utilizing non-viral vectors) using lPEI and bPEI-based polyplexes. Interestingly, mechanical stimuli applied to cells improved polyplex internalization by triggering the activation of clathrin-mediated endocytosis (CME), thus leading to greater transfection outcomes. This strategy outlined the importance of cell responses to exogenous cues on the ultimate internalization and expression of a gene of interest and set the stage for a novel way to deal with the non-viral delivery issue. Overall, the big picture drawn by this Ph.D. project highlighted the suitability of chemical-based approaches and cell-based approaches as promising ways to improve non-viral vector effectiveness. Further improvement in non-viral gene delivery research might be achieved by combining the strategies devised in this project. The development of multidisciplinary approaches taking into account both the delivery vector, the environment in which the delivery of genes takes place, and the cell response may thus pave the way to ever more effective strategies, and expedite the translation from the bench to the bedside of these materials.

The delivery of biomolecules into living cells is an important methodology in cell and molecular biology. Optical methods using lasers are attractive tools for such application. However, the interaction of the laser with the cell depends on the laser type and the parameters used. Hence, in this thesis, optical transfection and injection of both mammalian and embryonic cells is demonstrated using a variety of laser sources. Furthermore, some key issues are addressed by demonstrating alternative configurations of optoinjection and transfection systems to develop a robust, user-friendly device with potential for commercialisation. Most optical methods for the delivery of molecules rely on complex and expensive laser systems that occupy a large footprint. In order for the system to be accessible to end-users, transient transfection of plasmid DNA into mammalian cells using an inexpensive continuous wave 405 nm diode laser is demonstrated. In this work, the laser parameters are varied in order to optimise the transfection efficiency. By calculating the temperature change upon irradiation of the focused violet light, the mechanism of violet diode laser transfection is elucidated. Furthermore, the system is used to deliver small interfering RNA molecules to specifically knock down a particular protein within the cell. This work is a major step towards an inexpensive and portable optical transfection system. The critical issue of accurate targeting of the cell membrane is also addressed in conventional near-infrared femtosecond optical transfection systems. A near-infrared femtosecond holographic system is built utilising a spatial light modulator in order to provide fast three dimensional beam translation. Computer control of dosage and targeting allows us to explore the potential of different targeting modalities. An enhanced optoinjection and transfection on mammalian cells is demonstrated. Furthermore, the system is applied to optically manipulate a developing Pomatoceros lamarckii embryo. The holographic system can be employed to optoinject a variety of macromolecules into the embryo, as well as orient and position the embryo by switching to the continuous wave mode of the laser. Such development of optical techniques to deliver biomolecules and orient embryos will benefit the field of developmental biology. Lastly, to achieve controlled cavitation, limiting the mechanical effects of a nanosecond laser source, an optically trapped microsphere undergoes laser induced breakdown in the presence of a cell monolayer. Laser induced breakdown of a trapped microsphere allows control over several parameters, such as the microsphere material, position of the breakdown from the monolayer and the size of the microsphere. Optimising these parameters provide limited mechanical effects, particularly suited for cell transfection. This technique is an excellent tool for plasmid-DNA transfection of multiples of cells with both reduced energy requirements and cell lysis compared to previously reported approaches. Demonstrating optimised and successful delivery of macromolecules with the variety of laser sources used in this thesis will advance the applicability of optical injection and transfection and allow more potential users to access the technique. This thesis advances optical injection and transfection for optimised delivery of macromolecules to both mammalian cells and a developing embryo.

L’objectif de ce travail était de mettre au point un modèle de culture cellulaire, afin d’étudier in vitro l’incidence sur la physiologie des cellules pulpaires et en particulier de l’odontoblaste, de biomatériaux utilisés pour traiter les effractions de pulpes dentaires. Ce modèle repose sur l’utilisation de culture sphéroïdes dont la conformation spatiale reproduit plus fidèlement l’environnement in vivo que les cultures bidimensionnelles. Après avoir élaboré le modèle sphéroïde à partir de lignées murines, des expérimentations visant à déterminer la cytotoxicité des matériaux ont été effectuées. Leurs capacités à induire la biominéralisation ont également été évaluées. La dernière partie de ce travail avait pour objectif d’immortaliser des primocultures de cellules pulpaires humaines en transfectant les gènes SV40 et hTERT, afin d’établir une lignée cellulaire / The aim of this study was to develop a cell culture model to assess in vitro the effects on the physiology of pulp cells, in particular the odontoblasts, of biomaterials used to treat dental pulp exposures. This model is based on the use of spheroid culture whose spatial configuration reproduces the in vivo environment more faithfully than do two-dimensional cultures. After developing the spheroid model from mouse lines, experiments to determine the cytotoxicity of the materials were conducted. Their ability to induce bio-mineralisation was also assessed. The last part of this work aimed to immortalise primo-cultures of human pulp cells by transfecting hTERT and SV40 genes, in order to establish a new cell line

This thesis investigates the application of a variety of optical techniques for the manipulation of single cells and their local micro-environment. The methodologies developed provide enhanced control over a single cell under study affording exquisite spatial and temporal control over biological processes of interest. The work presented within the thesis can be split into three distinct categories. The first of these provides an investigation in light activated “caged” molecular probes. This work generated several new compounds which were then applied to providing control over processes involved in pain, mitochondrial intracellular signalling and memory processes in the central nervous system. Application of caged neurotransmitters then demonstrates the first in vitro wavelength orthogonal photolysis of biologically relevant substances. Such a technique has great potential in the study of fundamental interactions within the processes underpinning memory and cognitive function. Secondly the application of optical injection techniques for the introduction of membrane impermeable species of interest is presented. An exploration of laser sources and optical systems has yielded two new strategies for optical injection. The targeted introduction of fluorescent stains, nucleic acids and gold nanoparticles to the interior of live mammalian cells demonstrates the power of these techniques. Thirdly, an investigation in optical trapping and optical injection provides simplified micromanipulation techniqes for application to biological studies. The use of capillaries as reservoirs for reagents of interest has realised a procedure for the reduction of large-scale chemical assays to a single cell level in static flow. When this technique is combined with intelligent control over the trapping laser source’s temporal behaviour, the interaction with the sample under study can be tailored for biological amiability or sample ablation. In this way a single laser source can be employed for the optical trapping and nanosurgery of a biological sample. A final study is presented demonstrating initial results for the targeted optical injection of caged compounds into mammalian cells. This methodology draws on the strengths of optical injection and caging technologies and presents a significant step forward in the level of control afforded over a biological system under study by optical techniques. The studies presented highlight the level of control and flexibility afforded by the application of optical manipulation and excitation strategies. Such optical methodologies extend the photonic tools available for enhanced studies in the life sciences.

published_or_final_version / Dentistry / Doctoral / Doctor of Philosophy

Sarcocystis neurona is an apicomplexan parasite that is a major cause of equine protozoal myeloencephalitis (EPM). During intracellular development of S. neurona, many genes are temporally regulated. To better understand gene regulation, it is important to identify and characterize regulatory elements controlling gene expression in S. neurona. To perform this study, it was essential to establish transfection system for this parasite. Hence, the 5 flanking region of the SnSAG1 gene was isolated from a genomic library and used to construct expression plasmids. In transient assays, the reporter molecules -galactosidase (-gal) and yellow fluorescent protein (YFP) were expressed by electroporated S. neurona, thereby confirming the feasibility of performing molecular genetic experiments in this organism. Stable transformation of S. neurona was achieved using a mutant dihydrofolate reductase thymidylate synthase (DHFR-TS) gene of T. gondii that confers resistance to pyrimethamine. This selection system was used to create transgenic S. neurona that stably express -gal and YFP. These transgenic clones were shown to be useful for analyzing growth rate of parasites in-vitro and for assessing drug sensitivities. To uncover possible sequence elements involved in promoter activity, the 5 flanking regions of five S. neurona genes were subjected to comparative analysis. This revealed the presence of a 7-base conserved motif GCGTCTC. Using a dual luciferase assay system, the SnSAG1 promoter was subjected to functional analysis. The motif GAGACGC located between -136 and -129 upstream of the transcription start site was found to be essential for SnSAG1 expression. This motif functions in an orientation dependent manner and was shown to play a role in binding nuclear proteins of S. neurona.

Clavicipitaceous fungal endophytes provide several ecological benefits to their hosts. Besides improving host’s growth characteristics, Neotyphodium coenophialum, the endophyte of tall fescue (Lolium arundinaceum), produces ergot alkaloids that have been proposed to be involved in fescue toxicosis. One approach to address the toxicosis problem is to genetically manipulate and modify N. coenophialum by knocking out a pair of homologous genes, (dmaW1 and dmaW2), encoding dimethylallyltryptophan synthase, the enzyme for the first and determinant step in ergot-alkaloid biosynthesis. In this study, disruption of dmaW2 was attempted using several disruption methods. Out of 1522 transformants screened, three putative knockouts were identified. Southern blot analysis of digested genomic DNA indicated that homologous gene replacement at dmaW2 locus took place while dmaW1 was still present. Chromosome separation followed by Southern-blot hybridization showed that the dmaW genes in N. coenophialum are located on different chromosomes. The aim of this study was to obtain a nontoxic endophyte free of marker genes that could be used to inoculate popular tall fescue cultivars. Therefore the Cre/loxP system developed in this study allows reusing the marker gene for sequential transformations. Protoplasts from Neotyphodium coenophialum, Neotyphodium uncinatum, or Epichloë festucae isolates, containing a floxed hygromycin phosphotransferase (hph) gene (loxP::hph::loxP), were transfected with a Crerecombinase expression plasmid and then cultured without selection. The marker was excised in 0.5-2% of the colonies, leaving a single loxP sequence. This strategy will help to reduce the concerns related to field release or commercialization of economically important grasses associated with manipulated fungal strains. It is expected that the technology will likely be adapted and applied in other fungal species. Manipulation of the ergot alkaloid (EA) gene cluster from C. purpurea and C. fusiformis by introducing and expressing its genes in different fungal-grass symbionts was also investigated. Heterologous expression of the ergot alkaloid cluster could result either in the synthesis of compounds similar to the ones produced by the host or in synthesis of novel compounds with new modes of action. Even though the results indicated that several EA genes were expressed in the new symbiota, none of the ergot alkaloids intermediates were detected.

In an Experimental Rat model for endometrial carcinoma (EC) a minimal region of recurrent deletion/allelic loss at the neighborhood of the Tp53 gene has been identified. A similar observation of deletion at the homologous position on human chromosome 17 unassociated with TP53 mutation has been reported in several human cancer types. Thus an important tumor suppressor activity located close to, but distinct of TP53 is suggested. Detailed molecular analysis of this candidate region in a tumor model suggested Myo1c (myosin 1C) and Inpp5k (inositol polyphosphate-5-phosphatase K), also known as Skip (skeletal muscle and kidney enriched inositol polyphosphate phosphatase), as the best candidates. These two genes are suggested to be involved in glucose metabolism through PI3K/AKT signaling and neither of them has earlier been reported as a tumor suppressor gene. The present work aimed to investigate the potential correlation of MYO1C and/or INPP5K proteins with components of PI3K/AKT signaling pathway involved in cell growth and survival. Cells were transfected with increasing amounts of MYO1C- or INPP5K- gene expression constructs and protein extracts of the cells were subjected to Western Blot analysis for 13 important components of the signaling pathway: p110β\α\δ, p85, pAkt308&473, 14-3-3β, PTEN, Akt, pErk, Erk, Ras, p4EBP1 and 4EBP1. The analysis showed dose-dependent changes in the expression levels of several of these proteins, and the observed changes for the most part were directed towards negative regulation of cell proliferation and survival. The presented data further extended the initial hypothesis for potential tumor suppressor activities of MYO1C and INPP5K proteins through PI3K/AKT pathway.

Dans le cadre de ce travail, nous avons synthétisé, isolé et caractérisé de nouvelles molécules amphiphiles dérivées d’acides nucléiques. Les modifications de ces structures ont été réalisées par voie chimique au moyen d’une réaction de chimie clic, la réaction de Huisgen. Les amphiphiles développés peuvent être classés dans deux catégories différentes : - Les amphiphiles de faible masse moléculaire qui dérivent de nucléosides et de glycosylnucléosides. Les propriétés d’auto organisation de ces composés ont été étudiées par différentes techniques, notamment de microscopie électronique et de diffraction des rayons X. La capacité de ces amphiphiles à former des gels a été évaluée dans différents solvants (eau et solvants organiques). Les propriétés de complexation des acides nucléiques de ces molécules ont également été mises en évidence. Les premiers résultats de transfection montrent que les glycosylnucléosides amphiphiles permettent l’internalisation des oligonucléotides à visée thérapeutique dans des cellules humaines de carcinome hépatocellulaire (Huh 7) en présence de sérum. - Les amphiphiles de masse moléculaire élevée qui dérivent d’oligonucléotides. La formation d’agrégats a été mise en évidence par différentes techniques telles que la microscopie électronique et la diffusion de la lumière. Les propriétés de reconnaissance associées à la séquence oligonucléotide ont été étudiées par des expériences de thermodénaturation. L’auto vectorisation de ces composés a pu être observée par microscopie d’épi-fluorescence et confocale. Cette auto vectorisation a également pu être quantifiée par cytométrie en flux sur une gamme variée de types cellulaires humains tels que les cellules épithéliales (Hela T4), les cellules gastriques (NCI) ou encore les cellules de carcinome hépatocellulaire (Huh-7). Ces travaux présentent également pour la première fois l’évaluation in cellulo d’oligonucléotides amphiphiles ciblant le virus de l’hépatite C. / *