Cette thèse est consacrée au traitement d’images de bioluminescence chez le petit animal. Ce type d’imagerie, bien qu'utilisé en routine pour la recherche en cancérologie par exemple, présente néanmoins des problèmes liés aux phénomènes de diffusion et d'absorption par les tissus internes à l'animal. Il s'ajoute à cela le bruit du système d'acquisition ainsi que le bruit lié aux rayonnements cosmiques. Ceci influe sur la qualité des images acquises et rend leur exploitation délicate. Le but de cette thèse est de compenser ces effets perturbateurs. Les travaux menés ont abouti à la proposition d’un modèle de formation des images de bioluminescence ainsi qu’à une chaîne de traitement adaptée composée d’une étape de filtrage suivie d’une étape de déconvolution. Après étude de la nature des différents bruits liés à l'acquisition, nous avons mis au point un nouveau filtre médian pour la suppression du bruit impulsionnel aléatoire présent sur les images acquises ; ce filtre représente le premier bloc de la chaîne proposée. Pour l'étape de déconvolution, nous avons mené une étude comparative de différents algorithmes de déconvolution. Cela a conduit à choisir un algorithme de déconvolution aveugle initialisé avec la réponse impulsionnelle estimée du système d'acquisition. Nous avons validé notre approche globale en comparant les résultats à la réalité terrain. Au travers de différents essais cliniques, nous avons montré que le traitement que nous proposons permet une amélioration significative de la mesure des sources bioluminescentes et une meilleure distinction de sources très proches, ce qui représente un apport non négligeable pour les utilisateurs d'images de bioluminescence. / This thesis is devoted to the analysis of bioluminescence images applied to the small animal. This kind of imaging modality is used in cancerology studies. Nevertheless, some problems are related to the diffusion and the absorption of the tissues of the light of internal bioluminescent sources. In addition, system noise and the cosmic rays noise are present. This influences the quality of the images and makes it difficult to analyze. The purpose of this thesis is to overcome these disturbing effects. We first have proposed an image formation model for the bioluminescence images. The processing chain is constituted by a filtering stage followed by a deconvolution stage. We have proposed a new median filter to suppress the random value impulsive noise which corrupts the acquired images; this filter represents the first block of the proposed chain. For the deconvolution stage, we have performed a comparative study of various deconvolution algorithms. It allowed us to choose a blind deconvolution algorithm initialized with the estimated point spread function of the acquisition system. At first, we have validated our global approach by comparing our obtained results with the ground truth. Through various clinical tests, we have shown that the processing chain allows a significant improvement of the spatial resolution and a better distinction of very close tumor sources, what represents considerable contribution for the users of bioluminescence images.

L-arginine is one of the most versatile amino acids due to the fact that it serves as a precursor for many molecules which have important roles in bodily functions including mammalian reproduction. The current studies sought to further examine the role that L-arginine has in mammalian reproduction utilizing both in vivo and in vitro approaches. In the first study, a novel bioluminescent murine pregnancy model was developed to monitor VEGFR2 transcription activity non-invasively in the fetoplacental unit. Secondly, the effect that dietary L-arginine supplementation has during mouse gestation was examined. L-arginine supplementation increased weight gain during the latter third of gestation, total litter size, number of implantation sites, and litter birth weight. Additionally, L-arginine supplementation increased VEGFR2 transcription activity in the fetoplacental unit which may create a more favorable environment for fetal survival. Moreover, the increased number of implantation sites observed suggests an effect of L-arginine at the level of the endometrium. To this end, the effect that L-arginine has on apoptosis and cell proliferation in an established endometrial cell line was examined. The addition of L-arginine at physiological (200 micromolar) and supra-physiological (800 micromolar) concentrations increased cell proliferation , and this effect was achieved through biosynthesis of polyamines and nitric oxide. L-arginine also decreased the proportion of cells that were experiencing mitochondrial mediated apoptosis, and it was observed that this decrease in mitochondrial mediated apoptosis was concurrent with increased phosphorylation of BAD protein, which induces apoptosis when not phosphorylated. The final study examined the ability of porcine uterine epithelial (PUE) cells to synthesize L-arginine from L-citrulline. L-citrulline was able to support PUE cell proliferation in the absence of L-arginine. Additionally, ASS-1 and ASL, L-arginine synthesizing enzymes, were expressed in PUE cells and were regulated by the presence of L-arginine and L-citrulline, respectively. This data would support the hypothesis that PUE cells may be able to convert L-citrulline to L-arginine. Together, the current findings along with the plethora of relevant literature provide further evidence for the role of L-arginine in mammalian reproduction and allow for new questions to be investigated regarding this particular amino acid’s role in mammalian reproduction.

The porcine antral follicle, which consists of an oocyte and surrounding follicular components, including theca, granulosa, and cumulus cells and follicular fluid, is an essential microenvironment for oocyte development and maturation. Investigating cellular and molecular events in the context of the whole follicle will aid in our understanding of interactions between the oocyte and the follicular components. The objective of this dissertation was to develop a novel bioluminescent imaging model to visualize and measure cellular and molecular events in living intact ovarian follicles in vitro. Bioluminescence imaging was employed to facilitate noninvasive, dynamic, and real-time transgene analysis in living intact follicles. The time courses of luciferase-luciferin reactions, effective plasmid DNA and D-luciferin doses and their combinations were determined as the first step toward developing a new real-time bioluminescence imaging model. In addition, the efficient nonviral gene delivery methods: cationic lipid mediated gene transfer (chemical) and electroporation (physical) for the living intact follicles were determined. For the cationic lipid mediated gene transfer method, the 1:3 DNA lipid ratio was optimal. It was also found that the optimal condition of electroporation (4 electric pulses with 100 ms duration at field strength of 100 V/cm) resulted in 15 times higher luciferase activity and increased granulosa cell viability over the cationic lipid mediated gene transfer method. Moreover, increased granulosa cell viability, increased follicular fluid progesterone content, and oocytes with expanded cumulus cells were observed in intact follicles transfected by electroporation at a field strength of 100 V/cm. Finally, bioluminescence imaging was applied to quantify functional and ligand-activated estrogen receptor (ER) activity within living intact follicles. The functional ERs were differentially activated during the different stages of the estrous cycle in the mature sow; the levels of functional ER activity in cultured granulosa cells and intact follicles in vitro were increased from late luteal phase to early follicular phase and then significantly decreased at late follicular phase. The methodology developed herein can be applicable to further our understanding of oocyte and follicle development and oocyte maturation.

Dans la recherche de thérapie antitumorale de plus en plus innovante, nous avons évalué un traitement local basé sur l’utilisation de plasma froid. Le plasma froid (dans ce cas, <40°C) est un gaz ionisé par un apport d’énergie. Il contient des charges (électrons, ions), des radicaux libres et des molécules excitées. Il peut être généré à l’extrémité de cathéter permettant un traitement locorégional comme le traitement de dysplasie ou encore de tumeurs non résécables. Une activité antitumorale importante du plasma a été mise en évidence in vitro sur diverses lignées tumorales (colorectale, pulmonaire, pancréatique et cérébrale). Par ailleurs les cellules tumorales sont plus sensibles au plasma que les cellules normales. Les ROS générés sont à l’origine des principaux mécanismes d’action du plasma. Ils induisent de nombreux dommages à l’ADN, suivi d’un arrêt du cycle cellulaire conduisant à l’apoptose des cellules. Les études de tolérance ont mis en évidence l’innocuité de faibles doses de plasma sur le tissu traité permettant de définir les doses de plasmas utilisable dans le cadre de traitements antitumoraux. En utilisant des tumeurs xénogreffées en sous cutané et l’imagerie de bioluminescence, une activité antitumorale du plasma froid a été mise en évidence pour la première fois in vivo avec une augmentation de la survie des souris traitées d’environ 60%. Le traitement induit un arrêt de la prolifération tumoral avec une induction d’apoptose dans l’ensemble de la tumeur sans augmenter la surface de nécrose. L’effet antitumoral a également été démontré en utilisant le plasma gun sur un modèle de tumeurs colique et pancréatique en situation orthotopique chez la souris avec une augmentation de la survie (115%) accompagné d’une diminution de la métastasie. Ces résultats obtenues dans une démarche de recherche translationnelle montrent l’intérêt potentiel du plasma comme nouvel agent antitumoral. / In the context of new innovated antitumor treatment discovery, we evaluated the efficacy of a new local treatment based on non-thermal plasma (NTP). NTP is a cold (in this case, <40°C) ionized gas (a ir or noble gas) thanks to an electric discharge. It contains free charges (electrons, ions), free radicals and excited molecules. It can be generated at the end of a catheter allowing a local treatment that is compatible with usual endoscopes for dysplasia or non resecable tumors. We showed that NTP has a significant antitumor effect in vitro on various cell lines including colorectal, pancreatic, lung and brain tumor cells. The major action mechanisms of NTP was linked to a high rate generation of ROS in the vicinity of tumors cells and others plasma components have a minor implications. These ROS induce lethal DNA damages leading to a multiphase cell cycle arrest and finally to apoptosis. In vivo, a good tolerance of plasma treatment was highlighted and NTP treatment parameter was defined. Using subcutaneous xenografts and bioluminescence imaging, we showed a major antitumor effect of plasma in vivo with a 60% increase of mice life span. NTP treatments of tumor induce a tumor cell cycle arrest with a significant apoptosis induction in the whole tumor without increase of necrotic area. This in vivo antitumor effect was also observed with an in situ treatment using plasma gun of colorectal and pancreatic orthotopic tumor xenografts. A significant increase of mice lifespan (115%) was obtained together with a metastasis decrease. These results obtained in translational research showed the potential antitumor activity of NTP as a new type of treatment for cancer treatment.

Non-viral gene delivery is plagued by low transfection levels compared to viral delivery. The nuclear envelope presents a significant obstacle for non-viral vectors. A peptide-based nuclear localizing sequence has been incorporated into non-viral vectors to traverse the nuclear envelope. Here, we selected a photo-chemical method for covalently labeling the peptide onto plasmid DNA. The hypothesis of this work was to incorporate a nuclear localizing sequence into a non-viral delivery vector, demonstrate increased nuclear uptake and show a subsequent increase in transgene expression both in vitro and in vivo. We focused on pursuing in vitro and in vivo methods by which to test non-viral vectors for increases in gene expression based on the nuclear localizing sequence. Hydrodynamic dosing and intramuscular dosing (followed by electroporation) are two efficient delivery routes for dosing DNA in vivo. Through preliminary experiments, we became confident that whole animal bioluminescent imaging was a reliable and quantitative method by which to detect luciferase expression by either delivery route. Moving forward, both hydrodynamic and intramuscular dosing would be used to test formulations for nuclear localizing ability in vivo. Nuclear localizing peptides containing a photo-activatable functionality were synthesized and characterized. We quantitatively explored the photo-labeling capabilities on plasmid DNA via a radioactive peptide. In vitro, tissue culture-based experiments were carried out to show increased nuclear uptake by confocal microscopy as well as increased transgene expression. Throughout the literature, achieving an increase in expression by incorporating a nuclear localizing sequence into a non-viral vector has been elusive. The complexity of achieving this goal is increased when considering an in vivo system for improving gene transfer efficiency. Several strategies have been explored to demonstrate an increase in reporter gene expression from this type of non-viral vector, and the methods developed herein can be applied to other nuclear localizing vectors.

In vitro differentiation of stem cells in biocompatible scaffolds in a bioreactor is a promising method for creating functional engineered tissue replacements suitable for implantation. Basic studies have shown that mechanical, chemical, and pharmaceutical stimuli enhance biological functionality of the replacement as often defined by parameters such as cell viability, gene expression, and protein accumulation. Most of the assays to evaluate these parameters require damage or destruction of the cell-scaffold construct. Therefore, these methods are not suitable for monitoring the development of a functional tissue replacement in a spatial and temporal manner prior to implantation. Bioluminescence imaging is a technique that has been utilized to monitor cell viability and gene expression in various in vivo applications. However, it has never been applied in an in vitro setting for the specific purpose of evaluating a cell-scaffold construct. This research describes the design of flow perfusion bioreactor system suitable for bioluminescence imaging. In the first experimental chapter, the system was tested using MC3T3-E1 cells transfected with a constitutive bioluminescent reporter. It was found that bioluminescence imaging was possible with this system. In the second experimental chapter, MC3T3-E1 cells transfected with BMP-2 linked bioluminescence reporter were cultured by flow perfusion for a period of 11 days. Bioluminescence was detectable from the cells starting at day 4, while peaking in intensity between days 7 and 9. Further, it was also found that bioluminescence occurred in distinct regions within the scaffold. These results indicate that these strategies may yield information not available with current assays. / Master of Science

No description available.

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.

Children with high-risk SHH/TP53-mut and Group 3 medulloblastoma (MB) have a 5-year overall survival of only 40%. Innovative approaches to enhance survival while preventing adverse effects are urgently needed. We investigated an innovative therapy approach combining irradia- tion (RT), decitabine (DEC), and abacavir (ABC) in a patient-derived orthotopic SHH/TP53-mut and Group 3 MB mouse model. MB-bearing mice were treated with DEC, ABC and RT. Mouse survival, tumor growth (BLI, MRT) tumor histology (H/E), proliferation (Ki-67), and endothelial (CD31) staining were analyzed. Gene expression was examined by microarray and RT-PCR (Ki-67, VEGF, CD31, CD15, CD133, nestin, CD68, IBA). The RT/DEC/ABC therapy inhibited tumor growth and enhanced mouse survival. Ki-67 decreased in SHH/TP53-mut MBs after RT, DEC, RT/ABC, and RT/DEC/ABC therapy. CD31 was higher in SHH/TP53-mut compared to Group 3 MBs and decreased after RT/DEC/ABC. Microarray analyses showed a therapy-induced downregulation of cell cycle genes. By RT-PCR, no therapy-induced effect on stem cell fraction or immune cell inva- sion/activation could be shown. We showed for the first time that RT/DEC/ABC therapy improves survival of orthotopic SHH/TP53-mut and Group 3 MB-bearing mice without inducing adverse effects suggesting the potential for an adjuvant application of this multimodal therapy approach in the human clinic.

Bioluminescence is the chemical production of light that results when a luciferase enzyme catalyzes the luminogenic oxidation of a small-molecule luciferin substrate. The numerous luciferases and luciferins nature has evolved can be used to illuminate biological processes, from in vitro assays to imaging processes in live animals. However, we can improve the utility of bioluminescence through modification of these enzymes and substrates. My thesis work focuses on developing reporters that expand the bioluminescent toolkit and improving our understanding of how bioluminescence works on a molecular level. The first part of my thesis focuses on characterizing luciferases and luciferins that improve bioluminescence imaging in vivo. Some of our luciferins can outperform the natural D-luciferin substrate in live mouse imaging, while others are selectively utilized by mutant luciferases in live mouse brain. We also engineered luciferins that can selectively report on endogenous enzymatic activity in live mice. The second part of my thesis focuses on determining the molecular details of how enzymes related to firefly luciferase, long-chain fatty acyl-CoA synthetases (ACSLs), can function as latent luciferases. I have determined the structure for one of these enzymes and improved its bioluminescent activity with synthetic luciferins enough to image in live mouse brain. I also characterized the selectivity in chimerized enzymes that combine firefly luciferase and ACSLs. In summary, my work improves the utility of bioluminescence for in vivo use and informs us about how evolutionarily-related enzymes function as luciferases on a molecular level.