Cancer immunotherapy has proven to be challenging as it depends on overcoming multiple mechanisms that mediate immune tolerance to self-antigens. In situ immunization is based on the concept that it is possible to break immune tolerance by inducing tumor cell death in situ in a manner that provides antigen presenting cells such as dendritic cells (DCs) with a wide selection of tumor antigens that can then be presented to the immune system and result in a therapeutic anticancer immune response. Based on recent advances in the understanding of antitumor immunity, we designed a three-step approach to in situ immunization to lymphoma: (1) Inducing immunogenic tumor cell death with the chemotherapeutic drug Doxorubicin (Dox). Dox enhances the expression of "eat-me" signals by dying tumor cells, facilitating their phagocytosis by dendritic cells (DCs). Due to the vesicant activity of Dox, microparticles (MPs) made of PLGA (a biodegradable polymer) can safely deliver Dox intratumorally and are effective vaccine adjuvants; (2) Enhancing antigen presentation and T cell activation using anti-OX40; (3) Sustaining T cell responses by checkpoint blockade using anti-CTLA-4. In vitro, Dox MPs were less cytotoxic to DCs than to B lymphoma cells, did not require internalization by the lymphoma cells, and significantly enhanced phagocytosis of tumor cells by DCs as compared to soluble Dox. In mice, this three-step therapy induced CD4- and CD8-dependent systemic immune responses that enhanced T cell infiltration into distant lymphoma tumors leading to their eradication and significantly improving survival. Our findings demonstrate that systemic antitumor immune responses can be generated locally by three-step therapy and merit further investigation of three-step therapy for immunotherapy of lymphoma patients. Furthermore, we designed another in situ immunization approach using PLGA MPs loaded with both Dox and CpG oligodeoxynucleotides (CpG). The addition of CpG was to further enhance the Dox MP design by including an agent that addresses Step Two in situ, by enhancing tumor antigen presentation by DCs. In vitro, we show that Dox/CpG MPs can kill B and T lymphoma cells and are less toxic to DCs than soluble Dox. In vivo, Dox/CpG MPs combined with anti-CTLA-4 and anti-OX40 generated systemic immune responses that suppressed injected and distant tumors in a murine B lymphoma model, leading to tumor-free mice. The combination regimen was also effective at reducing T cell lymphoma and melanoma tumor burdens. In conclusion, Dox/CpG MPs represent a versatile, efficient and safe tool for in situ immunization that could provide a promising component of immunotherapy for patients with a variety of types of cancer.

Master of Engineering / This thesis presents an innovative human promoter recognition model HPR-PCA. Principal component analysis (PCA) is applied on context feature selection DNA sequences and the prediction network is built with the artificial neural network (ANN). A thorough literature review of all the relevant topics in the promoter prediction field is also provided. As the main technique of HPR-PCA, the application of PCA on feature selection is firstly developed. In order to find informative and discriminative features for effective classification, PCA is applied on the different n-mer promoter and exon combined frequency matrices, and principal components (PCs) of each matrix are generated to construct the new feature space. ANN built classifiers are used to test the discriminability of each feature space. Finally, the 3 and 5-mer feature matrix is selected as the context feature in this model. Two proposed schemes of HPR-PCA model are discussed and the implementations of sub-modules in each scheme are introduced. The context features selected by PCA are III used to build three promoter and non-promoter classifiers. CpG-island modules are embedded into models in different ways. In the comparison, Scheme I obtains better prediction results on two test sets so it is adopted as the model for HPR-PCA for further evaluation. Three existing promoter prediction systems are used to compare to HPR-PCA on three test sets including the chromosome 22 sequence. The performance of HPR-PCA is outstanding compared to the other four systems.

L'environnement et la santé des populations sont devenus des préoccupations majeures aussi bien au niveau national qu'au niveau européen. REACH (Registration, Evaluation and Authorization of CHemicals) est un système adopté par l'Union Européenne qui regroupe plus de quarante directives afin d'éliminer et de remplacer les substances chimiques les plus toxiques. Dans ce contexte, la mise en place d'un tel système requiert des méthodes d'analyse robuste, fiables et reproductibles dans le but d'évaluer et de détecter à l'état d'ultra-traces les molécules prioritaires persistantes dans les eaux et les sols.<br />Après avoir ciblé plusieurs substances listées sur l'annexe XVII de REACH, nous avons complété cette sélection par des polluants prioritaires de l'environnement Rhône Alpin tels les pesticides. Ainsi, la méthode d'analyse proposée permet de détecter et de quantifier par CPG-SM et CPL-SM/SM 33 substances (10 alkylphénols, 5 fongicides triazole, 1 fongicide morpholine, <br />1 acaricide pyridazinone, 2 phtalates, bisphénol A, 2 insecticides carbamates, un résidu des pesticides dithiocarbamates, 2 pesticides organophosphorés et 8 organochlorés) dans des matrices environnementales aussi complexes que des effluents et des boues de station épuration. <br />L'extraction des analytes est menée par Extraction sur Phase Solide (SPE) pour les échantillons aqueux et par Extraction par Solvant Accélérée (ASE) qui a été entièrement optimisée par étude statistique des paramètres. Les extraits sont ensuite purifiés de la même manière par SPE. La préparation à la validation de ma méthode nous a permis de confirmer des limites de détection basses comprises entre 7,2 ppt et 1,27 ppb dans les eaux et entre 5 ppb et 1,7 ppm dans les boues/sols

Le but de ce travail de thèse a été d'analyser l'implication du tronc dans la locomotion, notamment sa commande rythmique, afin d'en comprendre les mécanismes de contrôle et les différentes activités segmentaires qui amènent ses mouvements. Ces m´ecanismes peuvent alors être modélisés pour reproduire les différentes synchronisations observées au niveau des activités de chaque segment vertébral considéré. Enfin, cette modélisation permet de spécifier les activités du tronc à observer pour suivre en continu le cycle de marche d'un individu se déplacant. Dans un premier temps nous rappellerons les données de la littérature sur la locomotion, et notamment l'activité du tronc, qui nous ont permis de définir les axes principaux dans lesquels nous allons orienter notre travail. Cet état de l'art nous a notamment amenés à étudier les structures de type ”générateurs de rythme centraux” (Central pattern generator, CPG, en anglais). Dans un second temps nous avons réalisé une série de mesures expérimentales pour analyser de facon systématique et précise l'activité musculaire et cinématique du tronc lors de diverses situations locomotrices (marche, course, bond, pédalage). Ces mesures nous ont permis de mieux comprendre la manière dont le tronc se mettait en mouvement et comment ses activités musculaires et cinématiques, notamment les synchronisations intersegmentaires, évoluaient afin de tirer le meilleur parti des mouvements du haut du corps lors de la locomotion proprement dite. Dans un troisième temps, les résultats obtenus par les mesures d'activités du tronc ayant mis en évidence l'utilité du tronc et les mécanismes de contrôle dans une locomotion efficace, nous avons exploré la modélisation de l'activité du tronc au moyen d'un réseau d'oscillateurs mimant un CPG. Le choix d'un modèle de CPG pour représenter l'activité du tronc fait suite à des considérations phylogénétiques qui semblent indiquer qu'une telle structure pourrait exister chez l'homme. Une des propriétés de ce type de réseaux est leur capacité à exprimer différentes synchronisations sans changer de structure, c'est ce que nous avons appliqué aux observations faites lors de la marche, la course... Une autre propriété de ces réseaux est leur aptitude à se synchroniser avec un signal externe, nous avons donc explor´e diff´erents moyens de commander ce réseau d'oscillateur en phase avec la locomotion, encore une fois à partir de l'activité du tronc, de son accélération pour être plus précis.

<p>Bacillus Calmette Guérin (BCG), or attenuated Mycobacterium bovis, is the gold standard of immunotherapy in the clinic to treat superficial bladder cancer. However, setbacks remain due to a high recurrence rate, side effects, and BCG-refractory disease. In this thesis, we explored the use of novel immunotherapeutic agents such as CpG oligodeoxynucleotides (CpG ODNs) or synthetic ODNs containing unmethylated CpG dinucleotides. Since unmethylated CpG motifs are predominant in bacterial but not vertebrate DNA, they function as a “danger signal” leading to a potent immune response.</p><p>To be able to test various immunotherapeutic agents, we optimized subcutaneous (s.c.), metastatic, and orthotopic models using the murine bladder-49 (MB49) cancer cell line. In the orthotopic model, we show that poly-L-lysine promotes MB49 attachment to the bladder leading to 100% tumor take. In addition, Clorpactin (sodium oxychlorosene) potently enhances adenoviral transduction in the bladder.</p><p>Utilizing the MB49 model, we compare CpG ODNs with BCG and demonstrate the increased efficacy of CpG ODNs which could cure both s.c. and aggressive orthotopic bladder cancer. In our model, type B ODNs were most optimal and the antitumor response required T cells in order to induce regression and tumor-specific immunity. We also combined CpG ODNs with adenoviral vectors (Ad) expressing the immunostimulatory molecules CD40L, TRANCE, lymphotactin, IL2 or IL15. However, we show that CpG ODNs are effective as a monotherapy and adenoviral vectors did not enhance the effect.</p><p>AdCD40L was also used to genetically modify human dendritic cells (DCs). AdCD40L-transduced DCs not only had a higher and prolonged expression of the Th1 cytokine IL12 compared to TNFα-matured DCs, but CD40L-activated DCs could also resist the suppressive effects of IL10 and TGFβ. Since TNFα is commonly used in clinical DC vaccination protocols and because tumors often secrete immunosuppressive cytokines, these data have important implications for optimizing cancer immunotherapy.</p>

Bacillus Calmette Guérin (BCG), or attenuated Mycobacterium bovis, is the gold standard of immunotherapy in the clinic to treat superficial bladder cancer. However, setbacks remain due to a high recurrence rate, side effects, and BCG-refractory disease. In this thesis, we explored the use of novel immunotherapeutic agents such as CpG oligodeoxynucleotides (CpG ODNs) or synthetic ODNs containing unmethylated CpG dinucleotides. Since unmethylated CpG motifs are predominant in bacterial but not vertebrate DNA, they function as a “danger signal” leading to a potent immune response. To be able to test various immunotherapeutic agents, we optimized subcutaneous (s.c.), metastatic, and orthotopic models using the murine bladder-49 (MB49) cancer cell line. In the orthotopic model, we show that poly-L-lysine promotes MB49 attachment to the bladder leading to 100% tumor take. In addition, Clorpactin (sodium oxychlorosene) potently enhances adenoviral transduction in the bladder. Utilizing the MB49 model, we compare CpG ODNs with BCG and demonstrate the increased efficacy of CpG ODNs which could cure both s.c. and aggressive orthotopic bladder cancer. In our model, type B ODNs were most optimal and the antitumor response required T cells in order to induce regression and tumor-specific immunity. We also combined CpG ODNs with adenoviral vectors (Ad) expressing the immunostimulatory molecules CD40L, TRANCE, lymphotactin, IL2 or IL15. However, we show that CpG ODNs are effective as a monotherapy and adenoviral vectors did not enhance the effect. AdCD40L was also used to genetically modify human dendritic cells (DCs). AdCD40L-transduced DCs not only had a higher and prolonged expression of the Th1 cytokine IL12 compared to TNFα-matured DCs, but CD40L-activated DCs could also resist the suppressive effects of IL10 and TGFβ. Since TNFα is commonly used in clinical DC vaccination protocols and because tumors often secrete immunosuppressive cytokines, these data have important implications for optimizing cancer immunotherapy.

Synthetic CpG containing oligodeoxynucleotides (ODNs) are recognized by Toll like Receptor 9 (TLR9) and induce a strong pro-inflamatory immune response. To date, four different CpG ODN classes have been described. K-Class ODNs (also known as B-ODN) are potent B cell activators and stimulate TNF

Methylation of CpG islands located at the promoter regions is a mechanism which controls gene silencing and expression. Hyper or hypo methylation of these sites on promoter sequences have been associated with many diseases, like cancer. Even though promoter CpG islands and their methylation profiles are important regulators of gene expression, the exact mechanism of gene silencing through methylation is not known. Here, we have investigated the status of promoter CpG methylation under various evolutionary pressures by calculating the differences in promoter CpG content and methylation profiles at different pass points. In order to determine the list of genes under each category we have analyzed and compared the orthologs among 58 genomes available through ENSEMBL. The total number of CpG dinucleotides at the promoter regions of all groups of genes have been calculated and compared. Additionally, we have compared the experimentally determined methylation profiles of these CpG&#039 / s between human blood cells and fibroblast cells. While the promoter CpG content changed through common to newer genes, the number of the CpG units methylated found to be consistent. Here, we present the functional level analysis of common gene lists at different pass points and report the differences of the promoter CpG content and the methylation profiles among these groups with distinct evolutionary conservation status. We have also observed the conservation status of individual methylated CpG units on the low and high methylated genes. Our analysis revealed that the surrounding methylation content had a positive effect on the conservation of individual CpG&rsquo / s.

DNA can be represented abstrzctly as a language with only four nucleotides represented by the letters A, C, G, and T, yet the arrangement of those four letters plays a major role in determining the development of an organism. Understanding the signi cance of certain arrangements of nucleotides can unlock the secrets of how the genome achieves its essential functionality. Regions of DNA particularly enriched with cytosine (C nucleotides) and guanine (G nucleotides), especially the CpG di-nucleotide, are frequently associated with biological function related to gene expression, and concentrations of CpGs referred to as \CpG islands" are known to collocate with regions upstream from gene coding sequences within the promoter region. The pattern of occurrence of these nucleotides, relative to adenine (A nucleotides) and thymine (T nucleotides), lends itself to analysis by machine-learning techniques such as Hidden Markov Models (HMMs) to predict the areas of greater enrichment. HMMs have been applied to CpG island prediction before, but often without an awareness of how the outcomes are a ected by the manner in which the HMM is applied. Two main ndings of this study are: 1. The outcome of a HMM is highly sensitive to the setting of the initial probability estimates. 2. Without the appropriate software techniques, HMMs cannot be applied e ectively to large data such as whole eukaryotic chromosomes. Both of these factors are rarely considered by users of HMMs, but are critical to a successful application of HMMs to large DNA sequences. In fact, these shortcomings were discovered through a close examination of published results of CpG island prediction using HMMs, and without being addressed, can lead to an incorrect implementation and application of HMM theory. A rst-order HMM is developed and its performance compared to two other historical methods, the Takai and Jones method and the UCSC method from the University of California Santa Cruz. The HMM is then extended to a second-order to acknowledge that pairs of nucleotides de ne CpG islands rather than single nucleotides alone, and the second-order HMM is evaluated in comparison to the other methods. The UCSC method is found to be based on properties that are not related to CpG islands, and thus is not a fair comparison to the other methods. Of the other methods, the rst-order HMM method and the Takai and Jones method are comparable in the tests conducted, but the second-order HMM method demonstrates superior predictive capabilities. However, these results are valid only when taking into consideration the highly sensitive outcomes based on initial estimates, and nding a suitable set of estimates that provide the most appropriate results. The rst-order HMM is applied to the problem of producing synthetic data that simulates the characteristics of a DNA sequence, including the speci ed presence of CpG islands, based on the model parameters of a trained HMM. HMM analysis is applied to the synthetic data to explore its delity in generating data with similar characteristics, as well as to validate the predictive ability of an HMM. Although this test fails to i meet expectations, a second test using a second-order HMM to produce simulated DNA data using frequency distributions of CpG island pro les exhibits highly accurate predictions of the pre-speci ed CpG islands, con- rming that when the synthetic data are appropriately structured, an HMM can be an accurate predictive tool. One outcome of this thesis is a set of software components (CpGID 2.0 and TrackMap) capable of ef- cient and accurate application of an HMM to genomic sequences, together with visualization that allows quantitative CpG island results to be viewed in conjunction with other genomic data. CpGID 2.0 is an adaptation of a previously published software component that has been extensively revised, and TrackMap is a companion product that works with the results produced by the CpGID 2.0 program. Executing these components allows one to monitor output aspects of the computational model such as number and size of the predicted CpG islands, including their CG content percentage and level of CpG frequency. These outcomes can then be related to the input values used to parameterize the HMM.

Les protéines à " Methyl-CpG-binding domain " (MBD) jouent un rôle important dans l'interprétationde la méthylation de l'ADN conduisant à la répression transcriptionnelle via le recrutement decomplexes remodelant la chromatine. Dans les cancers, MBD2 jouerait un rôle essentiel dans la perted'expression des gènes hyperméthylés. Ainsi, MBD2 serait une cible potentielle pour rétablir, enpartie au moins, leur expression. Caractériser, à l'échelle du génome, la distribution de MBD2 et sesconséquences sur la répression transcriptionnelle au cours de la cancérogenèse est donc une étapeincontournable. (1) L'impact sur l'expression génique de l'inhibition de MBD2 par interférence àl'ARN, a été étudié en utilisant des puces, dans des cellules normales MRC5. La perte de MBD2n'induit pas de surexpression génique globale et la densité en CpG des promoteurs méthylés sembleêtre une composante importante dans la force de répression par MBD2. (2) Les profils de méthylationde l'ADN, de liaisons de MBD2 et de l'ARN polymérase II dans les cellules HeLa ont été analysés parChIP-on-chip avec des puces promoteurs. Ces mêmes approches couplées à l'analyse de l'acétylationdes histones H3 ont été réalisées dans un modèle cellulaire syngénique de progression tumoralemammaire humain. Dans les modèles étudiés, une forte proportion de gènes silencieux et méthylés estliée par MBD2. Les comparaisons entre cellules immortalisées et transformées ne montrent pas dechangements majeurs de la méthylation de l'ADN ou de la répression transcriptionnelle, par contreune redistribution de MBD2 parmi ces sites est observée, suggérant une redondance entre les protéinesliant l'ADN méthylé.