Contexte. Le cancer bronchopulmonaire (CBP) demeure la première cause de mortalité par cancer dans le monde. Malgré l’espoir suscité par le développement des thérapies ciblées, son pronostic demeure sombre, particulièrement dans les cas de CBP à petites cellules (CBP-PC) et de CBP non à petites cellules (CBP-NPC) présentant une activation de l’oncogène KRAS. Matériel et Méthodes. Nous avons mené 3 études successives, visant à (i) radiosensibiliser des modèles de CBP-PC par l’ajout d’un inhibiteur de BCL2, (ii) cibler des modèles de CBP-NPC mutés KRAS par l’association d’un inhibiteur de mTOR et d’un inhibiteur de RAF, et (iii) créer un modèle préclinique orthotopique murin de CBP reproduisant la progression tumorale observée en clinique. Résultats. Dans la première étude, l’inhibiteur de BCL2 oblimersen a présenté un effet radiosensibilisant sur des modèles de CBP-PC, in vitro et in vivo. Dans la seconde étude, l’association de l’inhibiteur de mTOR everolimus et de l’inhibiteur de RAF/VEGFR RAF265 a présenté un effet synergique sur des lignées cellulaires de cancers présentant la double mutation de KRAS et de PIK3CA, in vitro et in vivo. Dans la troisième étude, l’injection orthotopique d’une lignée bioluminescente de CBP-NPC chez des souris nude a permis d’établir des tumeurs intra pulmonaires évoluant vers une extension métastatique ganglionnaire et hématogène, et de détecter la présence de cellules tumorales circulantes. Conclusion. L’association d’un inhibiteur de BCL2 à la radiothérapie est une stratégie intéressante dans le CBP-PC, l’association d’un inhibiteur de mTOR et d’un inhibiteur de RAF/VEGFR est une stratégie intéressante dans le CBP-NPC présentant une double mutation KRAS-PIK3CA, mais ces données doivent être confirmées sur des modèles orthotopiques afin de gagner en pertinence avant d’envisager un transfert en clinique. / Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality worldwide. Activation of phosphatidylinositol-3-kinase (PI3K)-AKT and Kirsten rat sarcoma viral oncogene homologue (KRAS) can induce cellular immortalization, proliferation, and resistance to anticancer therapeutics such as epidermal growth factor receptor inhibitors or chemotherapy. This study assessed the conséquences of inhibiting these two pathways in tumor cells with activation of KRAS, PI3K-AKT, or both. We investigated whether the combination of a novel RAF/vascular endothelial growth factor receptor inhibitor, RAF265, with a mammalian target of rapamycin (mTOR) inhibitor, RAD001 (everolimus), could lead to enhanced antitumoral effects in vitro and in vivo. To address this question, we used cell lines with different status regarding KRAS, PIK3CA, and BRAF mutations, using immunoblotting to evaluate the inhibitors, and MTT and clonogenic assays for effects on cell viability and proliferation. Subcutaneous xenografts were used to assess the activity of the combination in vivo. RAD001 inhibited mTOR downstream signaling in all cell lines, whereas RAF265 inhibited RAF downstream signaling only in BRAF mutant cells. In vitro, addition of RAF265 to RAD001 led to decreased AKT, S6, and Eukaryotic translation initiation factor 4E binding protein 1 phosphorylation in HCT116 cells. In vitro and in vivo, RAD001 addition enhanced the antitumoral effect of RAF265 in HCT116 and H460 cells (both KRAS mut, PIK3CA mut); in contrast, the combination of RAF265 and RAD001 yielded no additional activity in A549 and MDAMB231 cells. The combination of RAF and mTOR inhibitors is effective for enhancing antitumoral effects in cells with deregulation of both RAS-RAF and PI3K, possibly through the cross-inhibition of 4E binding protein 1 and S6 protein. We then focus on animal models. Preclinical models of NSCLC require better clinical relevance to study disease mechanisms and innovative therapeutics. We sought to compare and refine bioluminescent orthotopic mouse models of human localized NSCLC. Athymic nude mice underwent subcutaneous injection (group 1-SC, n = 15, control), percutaneous orthotopic injection (group 2-POI, n = 30), surgical orthotopic implantation of subcutaneously grown tumours (group 3-SOI, n = 25), or transpleural orthotopic injection (group 4-TOI, n = 30) of A549-luciferase cells. Bioluminescent in vivo imaging was then performed weekly. Circulating tumour cells (CTCs) were searched using Cellsearch® system in SC and TOI models Group 2-POI was associated with unexpected direct pleural spreading of the cellular solution in 53% of the cases, forbidding further evaluation of any localized lung tumour. Group 3-SOI was characterized by high perioperative mortality, initially localized lung tumours, and local evolution. Group 4-TOI was associated with low perioperative mortality, initially localized lung tumours, loco regional extension, and distant metastasis. CTCs were detected in 83% of nude mice bearing subcutaneous or orthotopic NSCLC tumours. Transpleural orthotopic injection of A549-luc cells in nude mouse lung induces localized tumour, followed by lymphatic extension and specific mortality, and allowed the first time identification of CTCs in a NSCLC mice model.

O objetivo geral deste trabalho foi explorar a versatilidade de filmes multicamadas de polieletrólitos (PEM) e suas aplicações em sistemas de entrega de drogas e como filmes funcionais para aplicações biomédicas. Filmes PEM montados pela técnica de camada por camada (layer­-by­-layer, LbL), foram explorados em três aplicações principais. Na primeira, foi explorado o desenvolvimento de um protocolo de funcionalização em filmes de poli(alilamina)/poli (estireno sulfonato), PAH/SPS. Os parâmetros de construção do filme para biotinilação dos grupamentos amina do PAH foram otimizados e aplicados na captura e detecção do antígeno específico da próstata (PSA), na concentração de 100 a 0,1 ng/mL, usando pontos quânticos (Qdots). Em comparação com outros trabalhos, este sistema apresentou uma boa sensibilidade na detecção de PSA, dentro do limite de detecção clínica de 0,4 a 0,1 ng/mL. A segunda aplicação envolveu o desenvolvimento de filmes de sacrifício baseados nas interações naturais da mucina submandibular bovina e da lectina, jacalina (BSM/JAC). Filmes de BSM/JAC apresentaram estabilidade quando submetidos a uma ampla faixa de pH (pH 3-­-9) e em solução de alta força iônica (5 M NaCl). A dissolução dos filmes BSM/JAC pôde ser seletivamente desencadeada mediante à incubação em solução de melibiose, 37 °C, pH 7,4, sem apresentar citotoxicidade às células. Na última parte deste trabalho, a incorporação de lipossomos ecogênicos (ELIP) em mochilas celulares foi investigada. Mochilas celulares são \"patches\" de 7­-10 µm de diâmetro que podem ser fabricados por meio de deposição alternada de polímeros utilizando-­-se a técnica de LbL, sobre uma matriz pré­-moldada obtida por fotolitografia, a fim de criar um sistema composto por três multicamadas estratificadas: uma região de liberação, para promover o destacamento do substrato, uma região de carga de droga, e uma região adesiva às células. O uso de ELIP permitiu incorporação de até 9x mais doxorrubicina (DOX) se comparado com o fármaco livre em solução absorvido pelos dos filmes. A liberação de DOX pelos filmes foi monitorado por 25 dias. Mochilas contendo ELIP-­DOX foram então aderidos a monócitos, e sua viabilidade monitorados por 72h. Mochilas vazias mostraram diminuir a proliferação de monócitos ao longo das 72 horas, enquanto mochilas carregadas com ELIP-­DOX mostraram uma diminuição dramática na população celular, apontando uma potencialização dos efeitos da droga pela sua proximidade com as células. / The overall goal of this thesis was to exploit the versatility of polyelectrolite multilayers (PEM) to be applied in drug delivery systems and biofunctionalizable films for biomedical applications. PEM films assembled by the layer-by­-layer technique were explored in three main applications. In the first part of this work, the development of a functionalization protocol of poly(allylamine)/poly(styrene sulfonate), PAH/SPS was explored. The optimal film parameters to the use of biotinylated multilayers were applied for the capture and detection of prostate specific antigen (PSA) protein in the range of 100 to 0.1 ng/mL, by using quantum dots. Compared to previous work, this system presented a good sensitivity for PSA detection that is within the clinical limit range of 0.4 to 0.1 ng/mL. The second application involved the creation of a novel sacrificial multilayer film. Films based in natural interactions of bovine submaxillary mucin and the lectin jacalin, BSM/JAC were assembled. BSM/JAC films showed stability when underwent a wide rage of pH (pH 3 to 9) and high ionic strength (5 M NaCl) solutions. BSM/JAC dissolution could be triggered released by incubation in melibiose at 37 °C in pH 7.4 buffer, without cytotoxicity. In the last part of this work the incorporation of echogenic liposomes (ELIP) into cell backpacks was investigated. Cell backpacks are 7-10 µm diameter patches that can be fabricated through LbL polymer deposition onto a photopatterned array to create a stacked composite of three stratified multilayer systems: a releasable region for easy detachment from the substrate, a drug payload region, and a cell adhesive region. The use of ELIP allowed up to 9x more doxorubicin (DOX) loading when compared to free drug in solution adsorbed through the films. DOX release from films was monitored for over 25 days. ELIP­-DOX backpacks were then attached to mouse monocytes and their viability monitored by 72h. Empty backpacks showed to decrease monocytes proliferation over the course of 72h, while ELIP­-DOX backpacks showed a dramatic decrease in cell population, showing that DOX effects were enhancement in drug potency by its proximity.

Objectifs: Malgré leur grande hétérogénéité histologique et moléculaire, la prise en charge clinique des carcinomes ovariens de haut-grade (COHG) reste peu variable. Le pronostic sombre de cette pathologie implique un réel besoin des nouvelles thérapies. Au-delà des marqueurs pronostiques histologiques classiques et des enquêtes oncogénétiques, l’objectif de cette étude a consisté à rechercher des cibles moléculaires pharmacologiquement recrutables afin de pouvoir proposer aux patientes un accès à la thérapie innovante et personnalisée. Méthodes: Cette étude a été réalisée chez 53 patientes (pts) (âge moyen 58,9 ans, intervalle 25-87) de COHG histologiquement prouvés dont 45 pts de sous-type séreux. 19 pts ont fait l’objet d’une consultation et d’un test oncogénétique sur la base d’antécédents familiaux / personnel de cancer de sein/ovaire. chez. L’expression de P53 et de PTEN a été évaluée sur des tissus fixés au formol et inclus en paraffine par immunohistochimie. Les mutations somatiques de KRAS, NRAS, BRAF, PIK3CA et MET ont été recherchées par PCR-HRM (Polymerase Chain Reaction High Resolution Melting) puis vérifiées par NGS (Next Generation Sequencing) sur des extraits d'ADN préparés à partir d'échantillons de tumeurs congelés, prélevés au moment du diagnostic. Résultats: Des mutations germinales de BRCA1 / 2 ont été identifiées chez 7 pts, toutes atteintes des carcinomes séreux. Une mutation du gène KRAS (exon 2), 2 mutations du gène NRAS (exon 3), 6 mutations du gène PIK3CA (exon 5, 10 et 21) et 5 mutations du gène MET (exon 14 et 18) ont été identifiées chez les 53 tumeurs par NGS, dont deux mutations du gène NRAS et 2 mutations du gène PIK3CA détectées précédemment par PCR-HRM. Aucun profil mutationnel multiple n’a été retrouvé. La surexpression de P53 et la perte d’expression de PTEN ont été constatées chez 32 sur 53 (60%) et 19 sur 46 (41%) des tumeurs. L’analyse statistique n’a été réalisée que chez le sous-groupe de pts atteintes des carcinomes séreux à cause de l’effectif de l’étude. Avec un suivi médian de 38 mois (intervalle de 6-93), 35 pts ont eu une rechute de la maladie et 25 pts sont décédées. La survie sans progression à 2 ans est 28%, et la survie globale à 5 ans est 37%. La surexpression de P53 a été trouvée associée à une meilleure chimiosensibilité, une meilleure survie sans progression et une meilleure survie globale. Conclusion: Pour des COHG, au-delà des altérations de P53 et PTEN, des anomalies génétiques somatiques concernant les voies de signalisation PI3K et MAPK ne sont pas rares et peuvent être détectées par NGS. L’identification de ces anomalies somatiques pourrait offrir une possibilité des thérapies ciblées innovantes pour les patientes sur la base d’éléments diagnostics moléculaires. / Objectives: Despite the great histological and molecular heterogeneity, the clinical management of high-grade ovarian carcinoma remains univo-cal. As a major subgroup of ovarian carcinoma, high-grade ovarian carci-nomas (HGOC) need novel therapy. Additionally to conventional histolog-ical prognostic markers and oncogenetic investigations, molecular diag-nostic was performed using PCR-HRM (Polymerase Chain Reaction High Resolution Melting) and NGS (Next Generation Sequencing) to identify "druggable" targets that could provide access to innovative personalized therapy. Methods: This study was performed in 53 patients (pts) (mean age 58.9 years, range 25-87) with histologically proven HGOC of which 45 pts with serous carcinoma. BRCA1/2 germline mutations had been screened in 19 pts with familial/personal history of breast/ovarian cancer justifying on-cogenetic investigations. P53 and PTEN expression was assessed on for-malin fixed paraffin-embedded tissues using immunohistochemistry. So-matic mutations of KRAS, NRAS, BRAF, PIK3CA and MET were screened using PCR-HRM and then confirmed using NGS on DNA extracts from frozen tumor specimens taken at diagnosis. Results: Seven pts had BRCA1 / 2 germline mutations, all had serous carcinomas. One mutation of KRAS (exon 2), 2 mutations of NRAS (exon 3), 6 mutations of PIK3CA (exon 5, 10 and 21) and 5 mutations of MET (exon 14 and 18) were identified using NGS, of which 2 mutations of NRAS and 2 mutations de PIK3CA detected previously by PCR-HRM, no multiple mutation was detected. P53 overexpression and PTEN loss of expression was detected respectively in 32 of 53 (60%) and 19 of 46 (41%) of all the tumors. Because of the efffective of the study, statistical analyses were restricted to pts with serous carcinoma. With a median follow-up of 38 months (range 6-93), 35 pts had disease progression and 25 pts died during the follow-up. The 2-year progression-free survival (PFS) rate was 28% and 5-year overall survival (OS) rate was 37%. Overexpression of mutant P53 was found to be associated with chemosensitivity and longer PFS and OS. Conclusion: In HGOC, beside P53 and PTEN alterations, somatic genetic abnormalities of PI3K and MAPK signaling pathways can be detected us-ing NGS and provide molecular rationale for targeted therapies, potential-ly offering new therapeutic opportunities to the patients.

Dans un contexte d’amélioration des dispositifs pour la réduction de bruit, l’étude sur le transfert d’énergie irréversible en utilisant des résonateurs purement acoustiques à comportement non linéaire a été réalisée. Les résonateurs acoustiques classiques en régime linéaire agissent comme un Amortisseur de Masse Accordée (TMD, en anglais) et ils sont efficaces pour une gamme de fréquence très étroite. Cependant, lorsqu’ils sont soumis à des excitations très fortes (régime non-linéaire) ils peuvent devenir efficaces pour une plus large gamme de fréquences si des termes non linéaires peuvent être activés. Dans un premier temps, une étude sur ce comportement non-linéaire d’un résonateur d’Helmholtz modifié a été réalisée expérimentalement. Ensuite, l’équation dynamique gouvernante de tels résonateurs ont été développées en prenant en compte les non-linéarités de la force de rappel et d’amortissement. Une approximation de la solution analytique de l’équation gouvernante du résonateur acoustique a été déterminée en utilisant les méthodes des échelles multiples du temps et de transformation du temps non régulière. Dans un deuxième temps, une étude du couplage entre un mode acoustique en basses fréquences et un résonateur (celui étudié précédemment) à comportement non-linéaire a été réalisée. Pour ce faire, des mesures expérimentales avec un montage du système couplé ont permis de vérifier l’atténuation acoustique produite par le résonateur en régime forcé et libre. Une modélisation analytique du couplage a permis d’identifier l’expression de la variété invariante lente, ce qui a permis d’étudier les possibles points d’équilibre et points singuliers du système. Les modèles analytiques développés ont également été vérifiés par des simulations numériques. / Nowadays, there is a need of new types of technologies for sound reduction because of the growing of different industries. In this context, we have studied the targeted energy transfer using a purely acoustic resonator. These acoustic resonators act, in the linear regime, as a Tuned Masse Damper (TMD) and they are efficient for a narrow frequency band. But, when they are excited with high forces, in the nonlinear regime, they are efficient for a wider frequency band if the nonlinear terms are activated. First, an experimental study about the nonlinear behavior of a modified Helmholtz Resonator was done. Then, the governing equation of such resonators were developed considering the nonlinearities in the restitution force and damping. An approximation of the analytical solution of the governing equation of the acoustical resonator is derived using the multiples scales of time method and the non-smooth time transformation method. In a second part, a study about the coupling between an acoustic mode in low frequencies and a resonator (the one studied in the previous part) with a nonlinear behavior is done. In order to do this, experimental measurements of the coupled system to confirm acoustic attenuation by the resonator in forced and free regime were done. Then, an analytical modelling of the coupled system allowed to derive the expression of the Slow Invariant Manifold (SIM), in order to identify the possible equilibrium points and singular points of the system. Derived analytical models were verified by numerical simulations.

Depuis le début des années 2000, les traitements anticancéreux oraux (TAO) sont en développement croissant, notamment dans le cancer du sein. Ils permettent aux patients la prise de comprimés par voie orale à domicile améliorant la qualité de vie. On estime que 10 à 34% des tumeurs du sein sur-expriment la protéine HER2 (HER2+) qui augmente le risque de métastases. Deux thérapies ciblées anti-HER2 sont actuellement disponibles : le trastuzumab, anticorps monoclonal administré par voie intraveineuse et le lapatinib, inhibiteur de tyrosine kinase administré par voie orale. D’après les recommandations de l’Agence Nationale d’Accréditation et d’Evaluation en Santé (ANAES, 2003), les TAO devraient être privilégiées par rapport aux formes intraveineuses prises à l’hôpital lorsque leur efficacité est équivalente. Dans la pratique courante des oncologues, les TAO semblent néanmoins sous-utilisées dans certains cas. En plus des freins médicaux connus (adhésion thérapeutique, gestion des effets indésirables), les TAO induisent une consommation de ressources hopitalières supplémentaires qui n’est pas valorisée dans le modèle de tarification à l’activité (T2A) des établissements hospitaliers, lequel repose sur la nature et la quantité d’activité médicale réalisée. Nous supposons que le modèle de T2A représente un frein économique à l’utilisation des TAO entrainant une disparité d’accès entre les traitements anticancéreux oraux et intraveineux. L’objectif de ce travail de thèse était donc de déterminer le rôle de la T2A dans l’accès des patients aux TAO au moyen d’une évaluation médico- économique de l’impact économique et organisationnel des TAO sur le système de soins. Une revue de littérature a permis de mettre en évidence le rôle du mode de financement des soins sur l’accès aux TAO en France et aux Etats-Unis. L’analyse des bases de données nationales hospitalières du Programme Médicalisé des Systèmes d’Information (PMSI) a permis de quantifier l’enjeu économique de la chimiothérapie pour les établissements de soins et qui représente la deuxième activité des hôpitaux en volume après l’hémodialyse. Le bénéfice lié aux séances de chimiothérapie a ainsi été estimé à 108 millions d’Euros en 2010 pour l’ensemble des établissements publics et privés en France. L’analyse a également permis de simuler le transfert d’allocation de ressources de l’hôpital vers les soins de ville induit par la substitution des séances de chimiothérapie par l’utilisation des TAO. Un modèle comparant le coût du traitement intraveineux (trastuzumab) aux TAO (lapatinib et capécitabine) dans le cancer du sein métastatique HER2+ a confirmé des coûts moindres pour les TAO (17 165€ versus 36 077€ par an et par patient) liés à une économie sur les transports médicalisés mais surtout sur les consommations hospitalières et ce, malgré un coût d’acquisition plus élevé des TAO. Malgré cet impact budgétaire négatif pour les hôpitaux, une étude de préférences (Méthode des Choix Discrets) conduite auprès d’un échantillon de 203 médecins hospitaliers et libéraux a montré que l’efficacité d’un traitement anticancéreux restait le déterminant principal de la décision thérapeutique (β=2,214, p<0,0001). L’étude a toutefois révélé que, aux stades avancés du cancer, la voie d’administration et son coût étaient également associés au choix du traitement (β=0,612, p=0,035 ;β=0,506, p<0,0001). Les résultats montrent donc que le modèle de financement de l’activité hospitalière influence le choix des modalités de traitement même si les critères d’ordre médicaux et environnementaux du patient demeurent des déterminants essentiels dans le choix du recours aux TAO (profil clinique du patient, adhésion thérapeutique, préférences du patient, environnement familial et socio-économique, conditions d’accès à l’offre de soins) / Since the early 2000’s, oral anticancer drugs (OADs) are increasingly available especially for the treatment of breast cancer. This route of chemotherapy administration allows patients to take oral tablets at home improving their quality-of-life. We estimate that 10 to 34% of breast tumors over-express the HER2 protein (HER2+) that increases the risk of developing metastasis. Two anti-HER2 targeted therapies are currently available: trastuzumab, a monoclonal antibody administered intravenously and lapatinib, a tyrosine kinase inhibitor administered orally. According to the recommendations of the National Agency for Accreditation and Evaluation in Health (ANAES, 2003), OADs should be administered when their efficacy is equivalent to the one of intravenous forms taken at hospital. In the current practice of oncologists, OADs seem to be underused in some cases. The medical brakes to the use of OADs (i.e. adherence, management of side effects) are well known. Nonetheless, OADs induce additional hospital healthcare resources which are not taken into account in the hospital payment system that is based on the nature and the quantity of medical activities performed (i.e. per-case payment system (PPS)). We assume that the current model of PPS represents an economic barrier to the use of OADs and which induces a disparity of access between oral and intravenous cancer treatments. The objective of this thesis was to determine the role of the PPS on the patient access to OADs based on a medico-economic evaluation of the economical and organizational impacts of OADs on the health care system. A literature review has highlighted the role of the funding of care on the access to OADs in the French and US healthcare systems. From an analysis of the national hospital database (PMSI database), we have quantified the economic implications of chemotherapy administration that is the second hospital activity in volume after hemodialysis. Earnings associated with chemotherapy sessions have been estimated at 108 million Euros in 2010 for all private and public institutions in France. This analysis also allowed us to simulate the transfer of resources allocation from hospital to community setting induced by the substitution of chemotherapy sessions by the use of OADs. A model comparing the cost of intravenous anticancer drug (trastuzumab) to OADs (lapatinib and capecitabine) in the treatment of HER2+ metastatic breast cancer confirmed the lower costs for OADs (€ 17,165 versus € 36,077 per year per patient). The higher acquisition cost of OADs was offset by the cost savings in terms of medical transportation and hospital resources. Despite this negative budget impact for hospitals, a preference study (Discrete Choice Experiment) conducted among 203 physicians showed that the efficacy of cancer treatment remained the main determinant of the therapeutic decision (β=2.214, p<0.0001). The study has also revealed that, in the advanced stages of cancer, the route of administration and its associated cost was also associated with the treatment choice (β=0.612, p= 0.035; =β0.506, p<0.0001). Overall, the results show that the hospital payment system influences the choice of treatment modalities. Nonetheless, medical criterions related to the patient remain essential in the choice of using OADs (clinical profile of the patient, adherence, patient preferences, familial and socio-economic environment, and conditions of access to health care)

Background Renal cell carcinoma (RCC) is the most common type of kidney cancer. Patients diagnosed with metastatic RCC (mRCC) have shorter overall survival compared to those diagnosed at earlier stages. Several targeted therapies, which cost from $7,000 - $16,000 per month have been approved since 2005 to treat mRCC. In addition, there is a growing interest in the use of cytoreductive nephrectomy (CN) with targeted therapies among mRCC patients. However, little is known regarding the economic burden of RCC and role of CN and prescribing patterns of targeted therapies among older mRCC patients. Objectives 1) To assess the economic burden of RCC among older adults in the targeted therapy era 2) To compare the overall survival (OS) and total healthcare cost (THC) among older mRCC patients receiving CN and targeted therapy versus patients receiving targeted therapy alone 3) To describe prescribing patterns of targeted therapies and associated OS and THC among older mRCC patients. Methods This dissertation was conducted using the Surveillance Epidemiology and End Results (SEER) - Medicare linked data. For the first objective, the study included a prevalent cohort of RCC patients from 2013, diagnosed during 2005 - 2013 and continuously enrolled in Medicare. RCC patients were matched to non-cancer beneficiaries using propensity score matching. Generalized linear models estimated the incremental healthcare costs. Incremental total healthcare cost (THC) was multiplied by the estimated number of RCC patients on Medicare to calculate the total economic burden of RCC. For the second objective, we included patients diagnosed with mRCC between 2007-2014 and compared overall survival (OS), and THC between patients who received CN + targeted therapy and targeted therapy alone. A propensity score based inverse probability of treatment weighting (IPTW) method was used to balance the two treatment groups. A Cox proportional hazard model assessed the risk for death and a GLM compared healthcare costs between the groups. For the third objective, patients with mRCC were defined as patients who were diagnosed at stage-IV or at earlier stages but were currently using targeted therapies. Further, we restricted our sample to patients who initiated targeted therapy. We described the frequencies of the most common first and second line targeted therapies. We also described OS and THC per month for clear-cell and non-clear cell mRCC for each therapy and line of therapy. Results The first study included 10,392 each of RCC and control patients. The average THC associated with RCC was $7,419. The average THC was $4,584 for patients diagnosed at stage-I, $4,727 for stage-II, $9,331 for stage-III, and $31,637 for stage-IV. The annual economic burden of RCC on Medicare was estimated to be $1.5 billion. The second study included 471 mRCC patients that received CN + targeted therapy or targeted therapy alone. The median OS from the adjusted survival curves was significantly higher (p Conclusions The economic burden of RCC varied substantially between early stage and metastatic patients. Among mRCC patients, use of CN among targeted therapy users was associated with a higher median OS and similar monthly THC over a lifetime. Sunitinib and everolimus were the most common first and second line targeted therapies among mRCC patients. The descriptive analysis suggested that OS and THC were similar across types of targeted therapy sequences.

Improvement in the in vitro oxidative folding of disulfide-containing proteins, such as extracellular and pharmaceutically important proteins, is required. Traditional folding methods using small molecule aliphatic thiol and disulfide, such as glutathione (GSH) and glutathione disulfide (GSSG) are slow and low yielding. Small molecule aromatic thiols and disulfides show great potentiality because aromatic thiols have low pKa values, close to the thiol pKa of protein disulfide isomerase (PDI), higher nucleophilicity and good leaving group ability. Our studies showed that thiols with a positively charged group, quaternary ammonium salts (QAS), are better than thiols with negatively charged groups such as phosphonic acid and sulfonic acid for the folding of bovine pancreatic trypsin inhibitor (BPTI). An enhanced folding rate of BPTI was observed when the protein was folded with a redox buffer composed of a QAS thiol and its corresponding disulfide. Quaternary ammonium salt (QAS) thiols and their corresponding disulfides with longer alkyl side chains were synthesized. These QAS thiols and their corresponding disulfides are promising small molecule thiols and disulfides to fold reduced BPTI efficiently because these thiols are more hydrophobic and can enter the core of the protein. Conformational changes of disulfide-containing proteins during oxidative folding influence the folding pathway greatly. We performed the folding of BPTI using targeted molecular dynamics (TMD) simulation and investigated conformational changes along with the folding pathway. Applying a bias force to all atoms versus to only alpha carbons and the sulfur of cysteines showed different folding pathways. The formation of kinetic traps N' and N* was not observed during our simulation applying a bias force to all atoms of the starting structure. The final native conformation was obtained once the correct antiparallel β-sheets and subsequent Cys14-Cys38 distance were decreased to a bond distance level. When bias force was applied to only alpha carbons and the sulfur of cysteines, the distance between Cys14-Cys38 increased and decreased multiple times, a structure similar to the confirmation of N*, NSH were formed and native protein was ultimately obtained. We concluded that there could be multiple pathways of conformational folding which influence oxidative folding.

Les carcinomes mammaires invasifs sont des tumeurs solides, au sein desquelles le fort pouvoir de prolifération des cellules cancéreuses entraîne la formation de zones hypoxiques. Les mécanismes cellulaires d’adaptation à la diminution des apports en oxygène sont principalement déterminés par des facteurs de transcription hétérodimériques induits par l’hypoxie (HIF-1 et HIF-2, hypoxia inducible factor). Le monomère alpha de ces complexes est déstabilisé en situation de normoxie. Les récepteurs aux facteurs de croissance épidermique (EGFR) peuvent également augmenter la traduction de l’ARNm codant ces monomères indépendamment de l’oxygène. La première partie de la thèse repose sur le développement de deux clones cellulaires fluorescents stables et inductibles par l’hypoxie, à partir d’une lignée de carcinome mammaire invasif triple négatif surexprimant le récepteur EGFR (MDA- MB-231). Le modèle CA9-GFP permet de restituer l’activité transcriptionnelle des complexes HIFs et le modèle GFP-P564 traduit la stabilité de leur sous-unité alpha. La fluorescence des deux modèles cellulaires est fortement induite en situation d’hypoxie (1% O2) et en présence d’agents mimétiques de l’hypoxie (cobalt, desferrioxamine et diméthyl-oxalyl glycine). Ces modèles ont ensuite été utilisés pour évaluer l’effet de trois thérapies anti-EGFR sur les voies de réponses à l’hypoxie. Le cétuximab et le lapatinib n’ont aucun effet sur la fluorescence des cellules alors que le géfitinib diminue l’intensité du signal en situation d’hypoxie. Ces différences sont associées à un impact similaire sur la migration et la viabilité cellulaire traduisant ainsi une sensibilité différentielle à ces trois composés anti-EGFR. La deuxième partie de la thèse s’est ensuite orientée vers la quantification par RT-qPCR de 45 gènes inductibles par l’hypoxie et impliqués dans le développement du cancer du sein sur une série rétrospective portant sur 32 exérèses de carcinomes mammaires invasifs précoces. Une analyse comparative des données a été effectuée en fonction des critères anatomo-pathologiques (stade, grade, statut en récepteur HER2, rechute). Une surexpression coordonnée de l’ensemble des gènes est observée dans les tumeurs de haut grade, HER2+ et pour les sujets ayant récidivé. La comparaison des groupes « rechute » versus « non rechute » a permis de sélectionner six marqueurs de référence s’exprimant de manière significative. Elle permet en outre la réalisation d’un algorithme basique de classement des individus en fonction du risque de récidive. Celui-ci a été validé par la construction de courbes de survie (méthode de Kaplan-Meier et test du Mantel-Haenszel). L’utilisation combinée des deux modèles cellulaires fluorescents permet l’identification et la caractérisation dynamique de molécules agissant directement ou indirectement sur les réponses cellulaires à l’hypoxie tumorale. L’analyse de l’expression de gènes connus pour leur profil agressif et régulés par le microenvironnement tumoral, constitue un outil clinique d’aide au pronostic des cancers du sein. / The invasive breast cancers are solid tumors, wherein the proliferation of cells causes the formation of hypoxic zones. Cellular adaptive responses to reduced oxygen concentrations are mainly determined by heterodimeric transcription factors induced by hypoxia (HIF-1 & HIF-2, Hypoxia Inducible Factors). The alpha subunits of HIF are rapidly degraded under normoxic conditions. HIF complexes are also regulated by activation of epidermal growth-factor receptor (EGFR) signaling pathways in an oxygen-independent manner. The first part of this work was to develop stable fluorescent clones for examining responses to hypoxia from a metastatic triple-negative breast cancer cell line overexpressing EGFR (MDA-MB-231). The Ca9-GFP and GFP-P564 cell models allow to assess HIF activity and alpha subunits stability, respectively. In both models, fluorescence signals were strongly increased with hypoxia-mimicking reagents (cobalt, desferrioxamine and dimethyl-oxalyl glycine) and under hypoxia (1% O2). The impact of three EGFR inhibitors on HIF activity and stability was assessed. Cetuximab and lapatinib did not affect the signal induced by hypoxia, whereas gefitinib sharply reduced its intensity in both models. The differential effect of these three EGFR-targeted therapies on hypoxia responses was correlated with a similar impact on viability and cell migration. The second part of this work focused on the relative quantification using qPCR analysis of 45 genes involved in hypoxia responses and in the development of breast cancer, from a retrospective series of 32 tumor samples from patients with early- stage invasive breast cancer. A comparative analysis was performed according to anatomo-pathological criteria (stage, grade, HER2 status, and relapse). A coordinated overexpression of all genes was observed in high-grade and HER2+ tumors and in the group of patients that relapsed. The comparison of gene expression between "relapse" and "no relapse" groups was used to identify six reference markers. It also allowed to develop a basic algorithm to classify patients according to the risk of relapse. This algorithm was validated by constructing survival curves (Kaplan-Meier method and Mantel-Haenszel test). Combined use of two fluorescent cell models allows identification and dynamic characterization of compounds able to directly or indirectly inhibit cellular responses to tumor hypoxia. Analysis of gene expression known for their aggressive character and regulated by the tumor microenvironment is a clinical tool for assessing breast cancer prognosis.

Les microfaisceaux d’ions ont, au cours de ces dernières décennies, montré leur efficacité dansl’étude des effets des rayonnements ionisants sur le vivant notamment concernant les effets des faiblesdoses ou l’étude de l’effet de proximité. Le CENBG dispose depuis 2003 d’un dispositif permettant la micro-irradiation ciblée d’échantillons biologiques vivants. Les applications des microfaisceaux dans ce domainese sont récemment diversifiées et des études plus fines sur les mécanismes de réparation desdommages ADN radio-induits aux échelles cellulaire et multicellulaire sont devenues possibles via lesévolutions en imagerie par fluorescence et en biologie cellulaire. Ces approches ont nécessité une évolutionimportante de l'instrumentation de la ligne de micro-irradiation du CENBG qui a été entièrementredessinée et reconstruite dans un souci d’optimisation d’apport de nouvelles fonctionnalités. Les objectifsde mes travaux ont été i) la mise en service du dispositif, ii) la caractérisation des performances dusystème, iii) la mise en place de protocoles pour l’irradiation ciblée à dose contrôlée aux échelles cellulaireet multicellulaire, in vitro et in vivo, et le suivi en ligne des conséquences précoces de cette irradiation,iv) la modélisation des irradiations afin d’interpréter les observables biologiques au regard des donnéesphysiques calculées.Ces travaux ont permis i) de caractériser les performances du dispositif : une taille de faisceau d’environ2 μm sur cible et une précision de tir de ± 2 μm, de développer des systèmes de détection d’ions pour uncontrôle absolu de la dose délivrée, ii) d’induire des dommages ADN fortement localisés in vitro, et devisualiser en ligne le recrutement de protéines impliquées dans la réparation de ces dommages,iii) d’appliquer ces protocoles pour générer des dommages ADN in vivo au sein d’un organisme multicellulaireau stade embryonnaire, Caenorhabditis elegans.Ces résultats ouvrent la voie vers des expériences plus fines sur la ligne de micro-irradiation ciblée duCENBG pour étudier les effets de l’interaction des rayonnements ionisants avec le vivant, aux échellescellulaire et multicellulaire, in vitro et in vivo. / The main goal of radiobiology is to understand the effects of ionizing radiations on the living.These past decades, ion microbeams have shown to be important tools to study for example the effects oflow dose exposure, or the bystander effect. Since 2003, the CENBG has been equipped with a system toperform targeted micro-irradiation of living samples. Recently, microbeams applications on this subjecthave diversified and the study of DNA repair mechanisms at the cellular and multicellular scales, in vitroand in vivo, has become possible thanks to important evolutions of fluorescence imaging techniques andcellular biology. To take into account these new approaches, the CENBG micro-irradiation beamline hasbeen entirely redesigned and rebuilt to implement new features and to improve the existing ones. My PhDobjectives were i) commissioning the facility, ii) characterizing the system on track etch detectors, and onliving samples, iii) implementing protocols to perform targeted irradiations of living samples with a controlleddelivered dose, at the cellular and multicellular scales, and to visualize the early consequencesonline, iv) modelling these irradiations to explain the biological results using the calculated physical data.The work of these past years has allowed us i) to measure the performances of our system: a beam spotsize of about 2 μm and a targeting accuracy of ± 2 μm, and to develop ion detection systems for an absolutedelivered dose control, ii) to create highly localized radiation-induced DNA damages and to see onlinethe recruitment of DNA repair proteins, iii) to apply these protocols to generate radiation-induced DNAdamages in vivo inside a multicellular organism at the embryonic stage: Caenorhabditis elegans.These results have opened up many perspectives on the study of the interaction between ionizing radiationsand the living, at the cellular and multicellular scales, in vitro and in vivo.

Large and ever-increasing numbers of chemicals, including large quantities of a broad spectrum of organic compounds are used in modern society. More than 30 000 of the more than 100 000 chemical substances registered in the EU are estimated to be daily used, of which many will be discharged into the waste-streams handled by municipal sewage treatment plants (STPs). The main objective of the work underlying this thesis was to improve understanding of the relationships between the characteristics of sewage contaminants and their sewage treatment efficiency. Further objectives were to examine the relationships between socio-economic uses of chemicals and sludge quality, and the effects of regulatory actions on sludge quality. The quality of the sewage sludge and the levels and distribution patterns of the sludge contaminants, both within and between the STPs, seem to remain quite constant over time. The overall findings indicate that the levels of contaminants in sewage sludge seem to be largely independent of the location, size and treatment techniques applied at the STPs, and generally, of the types of human activity connected to them. The total and relative concentrations of the sludge contaminants were found to be fairly constant on a dry weight basis, with some exceptions, indicating that the pollutants originate from broad usage and diffuse dispersion rather than (industrial) point sources. The proportion of cyclic methylsiloxanes recovered in sludge seems to strongly depend on their vapour pressure, which decreases with the number of siloxane units. The higher water solubility and biodegradability of organophosphorus flame retardants and plasticizers than polybrominated diphenyl ethers (PBDEs) were also reflected in lower percentages (relative to their national use) found in sludge. Significant time-trends in levels of a-third of the sludge contaminants included in the annual national measurement program were detected over a period of seven years. The levels of compounds displaying significant time-trends generally decreased following declines in the quantities used nationally. However, a quarter of these compounds showed increasing trends, of which the linear methylsiloxanes followed the same trend as used quantities. The decaBDE was also found to be increasing in sludge, probably as a result of the phase-out of pentaBDE and octaBDE. The results indicate that the STP removal efficiency of anthropogenic substances, in Sweden, is generally good and that STPs, at least those in cold climates, do not efficiently remove certain polar contaminants. A non-targeted screening (by use of environmetrics and GCxGC-TOFMS) was performed and found to fulfil the objective to assess the STP removal efficiency, with emphasis to systematically analyse which compound classes that are not efficiently removed using the current STP technology. Many polar aromatic compounds were identified to be poorly removed. The acquired data on levels, profiles and variations in sludge contaminants (sludge quality) extend both the available information and understanding of the degree and nature of sludge contamination, which should help attempts to track changes in its contaminants and revisions, if necessary, of guideline values. This thesis also contributes to improve the knowledge base for the development of future STP technologies, and that archived sewage sludge can be used in retrospective analysis of new and emerging pollutants.