Introduction Le cancer bronchique est un problème de santé publique en étant la première cause de décès par cancer dans le monde. Il reste de mauvais pronostic avec une résistance au Cisplatine qui est inéluctable dans l’histoire naturelle de la maladie. Nous nous sommes intéressés à l’association du CDDP aux inhibiteurs de la Poly(ADP-ribose) polymérase. Les inhibiteurs pharmacologiques de PARP sont source d’optimisme en oncologie clinique en monothérapie pour des tumeurs déficientes pour une voie de réparation de l’ADN et en association aux cytotoxiques classiques.Matériel et méthodes Nous avons généré 9 clones résistants au CDDP après culture de la lignée A549 dans des faibles doses de CDDP. Deux inhibiteurs pharmacologiques de PARP, CEP8983 (CEP) et PJ34 (PJ), ainsi que des siRNA spécifiques de PARP1 sont utilisés pour l’inhibition de PARP. L’apoptose est mesurée en cytométrie de flux par l’intermédiaire du potentiel membranaire de la mitochondrie DiOC6(3) et la perméabilisation de la membrane plasmique est évaluée par l’iodide de propidium. Le test de clonogénicité permet d’évaluer la capacité des cellules à échapper à la mort et à former une colonie. L’activité métabolique des cellules est mesurée par la mesure de clivage du sel de tetrazolium WST-1. L’immunofluorescence sur cellules fixées a permis d’étudier les dommages de l’ADN (γH2AX), la voie intrinsèque de l’apoptose (l’activation de la caspase 3 et la libération du cytochrome c) et la recombinaison homologue (BRCA1, RAD51). En Western Blot nous avons mesuré l’expression et l’activité de PARP (PAR) ainsi que l’expression d’acteurs de la réparation par excision de base (BER) (XRCC1 and polymérase β). Nous avons développé une méthode de détection de PAR en immunohistochimie sur des tissus inclus en paraffine. Résultats Nous avons trouvé un effet synergique pour l’association du CDDP aux inhibiteurs de PARP in vitro. De façon inattendue nous avons observé que les clones résistants au CDDP développent une addiction à PARP et sont spécifiquement tués par l’inhibition de PARP contrairement à la lignée parentale. Ces clones exhibent une hyperexpression et une hyperactivité de PARP. La réponse aux inhibiteurs de PARP corrèle plus précisément avec l’activation plutôt qu’avec l’expression de PARP, pointant que PAR est un biomarqueur plus précis que PARP. Nous avons observé que l’hyperactivation de PARP accompagne une résistance induite au CDDP et prédispose à une sensibilité aux inhibiteurs de PARP dans d’autres lignées de cancer bronchique (H460 et H1650), de mésothéliome (P31), de cancer de l’ovaire (TOV112D) et de col (HeLa). Dans des expériences in vivo nous avons noté que dans les xénogreffes obtenues à partir de clones résistants au CDDP, l’expression de PAR est stablement retrouvée en immunohistochimie. Ces tumeurs répondaient à l’inhibition de PARP par le PJ en diminuant l’expression de PAR. Les clones résistants au CDDP sensibilisés aux I PARP ont une recombinaison homologue conservée, cependant ont un déficit dans les étapes terminales du BER.Conclusion Nous avons identifié un effet synergique pour l’association des inhibiteurs de PARP au CDDP de des lignées de cancer bronchique. Nous avons observé une dépendance à PARP dans des lignées de cancer bronchique résistantes au CDDP et déficientes pour l’élongation du BER. Nous postulant que PAR est un biomarqueur spécifique de la réponse aux inhibiteurs de PARP. / Introduction Driven by the facts that non small cell lung cancer (NSCLC) is the leading cause of cancer-related morbidity and mortality worldwide and that NSCLC patients often develop resistance against Cisplatin (CDDP)-based therapies, we addressed the question of the combination therapy of CDDP with poly(ADP-ribose) polymerases (PARP) inhibitors. Inhibitors of PARP have raised great expectations for the treatment of a variety of cancers, either as monotherapeutic agent against DNA repair-deficient tumours or combined to DNA-damaging compounds.Material and methods We generated nine CDDP-resistant clones by prolonged exposure to low dose CDDP of the A549 NSCLC parental cell line. Two distinct PARP inhibitors, CEP8983 (CEP) and PJ34 (PJ) as well as PARP1 knockdown with small interfering RNAs (siRNAs) were used for PARP inhibition. Apoptosis was measured by the simultaneous assessment for the loss of the mitochondrial transmembrane potential (m) and the breakdown of the plasma membrane using the m-sensitive fluorochrome DiOC6(3) and the vital dye propidium iodide, respectively. Moreover clonogenic survival was assessed. In vitro assessments of the enzymatic activity of cells were based on the reduction of the colorless tetrazolium salt. Immunofluorescence microscopy determinations were performed with antibodies specific for DNA damage (γH2AX), intrinsic apoptosis (cleaved Caspase-3 and cytochrome c), and homologous recombination (RAD51 and BRCA1). Immunoblotting was assed for PARP1 expression and activity (PAR) and base excision repair (BER) effectors (XRCC1 and polymerase β). We developed an immunohistochemical staining method that specifically detects PAR on paraffin-embedded cell pellets and tissue sections.Results We found that PARP inhibitors and PARP1 siRNAs synergized with CDDP in the killing of NSCLC cells in vitro. Unexpectedly, CDDP-resistant NSCLC cell clones developed addiction to PARP hyperactivation, thereby becoming susceptible to apoptosis induction by PARP inhibition. We showed that these cisplatin-resistant clones, exhibited high PARP protein levels and increased PARP activity, leading to an increased poly-ADP ribosylation of cellular proteins, as compared to their parental, cisplatin-sensitive counterparts. These cisplatin-resistant cells become susceptible to cell death as induced by PARP inhibition, correlating with the hyperactivity of PARP (elevated PAR levels) more accuratly than with the overexpression of PARP. Suggesting that PAR levels may constitute a more accurate biomarker than PARP to predict the sensitivity of cells to PARP inhibition. We expanded the observation that cisplatin resistance causes PARP upregulation and hyperactivation and subsequent sensitization to PARP inhibition to additional five human cancer cell lines including two NSCLC (H1650 and H460), one mesothelioma (P31), one ovarian (TOV112D) and one cervical cancer (HeLa) cell line. To get further insight into this issue, we generated in vivo experiments. Tumors derived from CDDP-resistant cells were characterized by elevated levels of PAR suggesting that PAR levels are preserved during tumor formation. Those PAR-overexpressing tumors responded to the administration of PJ in vivo with a consistent reduction in PAR immunoreactivity. CDDP resistant clones that are specifically killed by PARP inhibitors assessed efficient homologous recombination repair however deficient BER elongation.Conclusion We showed a beneficial effect for the association therapy of PARP inhibitors with CDDP in several NSCLC cell lines. We have identified an addiction to PARP in CDDP resistant cell lines with deficient BER elongation. We postulate that PAR is a specific predictive biomarker for the response to PARP inhibitors.

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.

L’efficacité de la chimiothérapie contre la croissance tumourale repose sur l'induction d'une mort des cellules tumourales dite immunogène. Les cellules tumourales mourantes agissent alors comme un vaccin thérapeutique en stimulant une réponse immunitaire anti-tumourale capable de contrôler, voire d'éliminer, les cellules cancéreuses résiduel. Les trois marqueurs principales de la mort cellulaire immunogène (MCI), sont (i) l'exposition pré-apoptotique de la calréticuline (CRT) à la surface des cellules, (ii) la sécrétion de l'ATP pendant l'apoptose qui dépend du processus d'autophagie, et (iii) le relargage post-apoptotique de la protéine non-histone de fixation à la chromatine HMGB1 (High Mobility Group B1). CRT, ATP et HMGB1 se lient respectivement à CD91, au récepteur purinergique P2RX7 (Purinergic Receptor P2X, ligand-gated ion channel 7) et au récepteur TLR4 (Toll-like Receptor 4) situés à la surface des cellules dendritiques. En retour, ces interactions initient respectivement la phagocytose des cellules mourantes, la production de l'interleukine-1β et la cross-présentation des antigènes tumouraux aux lymphocytes T.Notre laboratoire a précédemment démontré que la chimiothérapie adjuvante présente une efficacité réduite chez des patients atteints de cancers colorectaux et du sein portant un polymorphisme d'un seul nucléotide ou SNP (Single-Nucleotide Polymorphism) compromettent la fonction des gènes P2RX7 et TLR4, notamment rs3751143 (496Glu>Ala) pour le gène P2RX7 et rs4986790 (299Asp>Gly) pour le gène TLR4.Compte tenu des ces résultats que mettent en évidence la relation étroite entre l’efficacité de la chimiothérapie anticancéreuse et un système immunitaire opérationnel, nous avons décidé d'étudier l'effet de ces SNPs, soit sur la survie globale soit sur la survie sans événement, chez des patients atteints de cancer pulmonaire non à petites cellules ou NSCLC (Non-Small Cell Lung Carcinoma) et de cancers de la tête et du cou ou HNSCC (Head and Neck Squamous Cell Carcinoma). De plus, nous avons porté notre attention sur un autre SNP affectant le gène ATG16L1 (Autophagy-related 16-Like 1), notamment rs2241880 (300Thr>Ala) qui compromet l’activité d’un gène fondamental dans le processus d’autophagie.Dans les cancers NSCLC, les allèles mutants "perte de fonction" des gènes ATG16L1, P2RX7 et TLR4 n'affectent pas la survie globale, indépendamment du type de chimiothérapie administrée. Au contraire, les patientes atteintes de HNSCC, portant au moins un allèle "perte de fonction" d’ATG16L1 et TLR4, présentent un taux de survie sans récidive inferieure par rapport aux patients qui présentent un genotype sauvage. Ce travail décrit pour la première fois un biomarqueur prognostique pour ce type de cancer.De plus, nous avons pu définir, par génotypage à haut débit, une signature de SNPs prédictive de la réponse à la chimiothérapie neoadjuvantes à base d'anthracyclines et des taxanes chez les patients atteints de cancer du sein. En particulier, la combinaison des deux paramètres clinicopathologiques classiques (âge lors du diagnostic et récepteur aux œstrogènes) avec les génotypes rs1076669 du gène ECE1 (Endothelin Converting Enzyme 1; 341Thr>Ile) et rs2277413 du gène PZP (Pregnancy-Zone Protein; 813Val>Ala) a permis de définir trois grandes catégories des patients et leur probabilité respective d'atteindre la réponse complète pathologique après traitement.L'identification de nouveaux biomarqueurs associés à une absence/diminution de réponse à la chimiothérapie apparaît critique pour choisir des alternatives thérapeutiques appropriées et éviter les effets secondaires indésirables chez les non-répondeurs. / Successful chemotherapeutics can induce a type of tumour cell death that is immunogenic, implying that patient’s dying cancer cells function as a therapeutic vaccine and elicit an anti-tumour immune response that controls the residual disease. The three main hallmarks of immunogenic cell death (ICD) are the pre-apoptotic exposure of calreticulin (CRT) on the cell surface, the autophagy-dependent secretion of ATP during the blebbing phase of apoptosis and the post-apoptotic release of the chromatin-binding non-histone protein high mobility group B1 (HMGB1). CRT, ATP and HMGB1 interact with CD91, purinergic receptor P2X, ligand-gated ion channel, 7 (P2RX7) and Toll-like receptor 4 (TLR4), respectively, on the surface of dendritic cells (DCs), thus promoting the engulfment of dying cells, the production of IL-1β and the cross-presentation of tumour-associated antigens to T cells, respectively.Our laboratory has demonstrated that adjuvant chemotherapy exhibits a reduced efficacy in breast and colorectal cancer patients bearing single-nucleotide polymorphisms (SNPs) that compromise the function of P2RX7 or TLR4, such as rs3751143 in P2RX7 (Glu496Ala) and rs4986790 in TLR4 (Asp299Gly).Driven by these results, underpinning the intimate relationship between the success of anti-cancer chemotherapy and an operational immune system, we decided to investigate the effect of these SNPs on disease outcome among non-small cell lung carcinoma (NSCLC) and head and neck squamous cell carcinoma (HNSCC) patients. Additionally, we focused our attention on a SNP in autophagy related 16-like 1 (ATG16L1), namely rs2241880 (Thr300Ala), which compromises the activity of one pivotal autophagic gene. In NSCLC patients, loss-of-function ATG16L1, P2RX7 and TLR4 alleles do not affect overall survival, irrespective of the administration and type of chemotherapy. Conversely, HNSCC patients bearing at least one loss-of-function ATG16L1 or TLR4 allele exhibit a reduced disease-free survival when compared to their wild-type counterparts. This is the first report highlighting a putative prognostic biomarker for this malignancy. Furthermore, taking advantage of a high throughput genotyping study, we delineated a SNP-based signature that predicts the response of breast cancer patients to anthracycline- and taxane-based neoadjuvant chemotherapy. Particularly, the combination of two classical clinicophatological parameters (age at diagnosis and estrogen receptor) and genotype at the ECE1 (rs1076669 Thr341Ile) and PZP (rs2277413 Val813Ala) loci allowed us to define three broad categories with a correspondent probability of achieving pathological complete response. The identification of new biomarkers associated with a reduced/absent response to chemotherapy appears critical for selecting appropriate therapeutic alternatives, and avoiding undesired side effects among non-responders.

Cellular senescence is a permanent cell cycle arrest that is induced as a response to cellular stress. Replicative senescence is a well-described mechanism that limits the replicative capacity of cells and must be overcome by cancer cells. Oncogene-induced senescence (OIS) is a form of premature senescence and a potent tumor suppressor mechanism. OIS is induced in normal cells as a result of deregulated oncogene or tumor suppressor gene expression. An exciting area of research is the identification of novel targets that induce senescence in cancer cells as a therapeutic approach. In this study, a novel mechanism is described where the inhibition of Hsp90 in small cell lung cancer (SCLC) cells induced premature senescence rather than cell death. The senescence induced following Hsp90 inhibition was p21-dependent and the loss of p21 allowed SCLC cells to bypass the induction of senescence. Additionally, we identified a novel mechanism where the depletion of PKCι induced senescence in glioblastoma multiforme (GBM) cells. PKCι depletion-induced senescence did not activate the DNA-damage response pathway and was p21-dependent. Further perturbations of mitosis, using an aurora kinase inhibitor, increased the number of senescent cells when combined with PKCι depletion. This suggests that PKCι depletion-induced senescence involves defects in mitotic progression. Senescent glioblastoma cells at a basal level of senescence in culture, induced by p21 overexpression, and induced after PKCι depletion had aberrant centrosomes. Mitotic slippage is an early exit from mitosis without cell division that occurs when the spindle assembly checkpoint (SAC) is not satisfied. Senescent glioblastoma cells had multiple markers of mitotic slippage. Therefore, PKCι depletion-induced senescence involves mitotic slippage and results in aberrant centrosomes. A U87MG cell line with a doxycycline-inducible shRNA targeting PKCι was developed to deplete PKCι in established xenografts. PKCι was depleted in established glioblastoma xenografts in mice and resulted in decreased cell proliferation, delayed tumor growth and improved survival. This study has demonstrated that both Hsp90 and PKCι are novel targets to induce senescence in cancer cells as a potential therapeutic approach.

No description available.

Cellular senescence is a permanent cell cycle arrest that is induced as a response to cellular stress. Replicative senescence is a well-described mechanism that limits the replicative capacity of cells and must be overcome by cancer cells. Oncogene-induced senescence (OIS) is a form of premature senescence and a potent tumor suppressor mechanism. OIS is induced in normal cells as a result of deregulated oncogene or tumor suppressor gene expression. An exciting area of research is the identification of novel targets that induce senescence in cancer cells as a therapeutic approach. In this study, a novel mechanism is described where the inhibition of Hsp90 in small cell lung cancer (SCLC) cells induced premature senescence rather than cell death. The senescence induced following Hsp90 inhibition was p21-dependent and the loss of p21 allowed SCLC cells to bypass the induction of senescence. Additionally, we identified a novel mechanism where the depletion of PKCι induced senescence in glioblastoma multiforme (GBM) cells. PKCι depletion-induced senescence did not activate the DNA-damage response pathway and was p21-dependent. Further perturbations of mitosis, using an aurora kinase inhibitor, increased the number of senescent cells when combined with PKCι depletion. This suggests that PKCι depletion-induced senescence involves defects in mitotic progression. Senescent glioblastoma cells at a basal level of senescence in culture, induced by p21 overexpression, and induced after PKCι depletion had aberrant centrosomes. Mitotic slippage is an early exit from mitosis without cell division that occurs when the spindle assembly checkpoint (SAC) is not satisfied. Senescent glioblastoma cells had multiple markers of mitotic slippage. Therefore, PKCι depletion-induced senescence involves mitotic slippage and results in aberrant centrosomes. A U87MG cell line with a doxycycline-inducible shRNA targeting PKCι was developed to deplete PKCι in established xenografts. PKCι was depleted in established glioblastoma xenografts in mice and resulted in decreased cell proliferation, delayed tumor growth and improved survival. This study has demonstrated that both Hsp90 and PKCι are novel targets to induce senescence in cancer cells as a potential therapeutic approach.

Cellular senescence is a permanent cell cycle arrest that is induced as a response to cellular stress. Replicative senescence is a well-described mechanism that limits the replicative capacity of cells and must be overcome by cancer cells. Oncogene-induced senescence (OIS) is a form of premature senescence and a potent tumor suppressor mechanism. OIS is induced in normal cells as a result of deregulated oncogene or tumor suppressor gene expression. An exciting area of research is the identification of novel targets that induce senescence in cancer cells as a therapeutic approach. In this study, a novel mechanism is described where the inhibition of Hsp90 in small cell lung cancer (SCLC) cells induced premature senescence rather than cell death. The senescence induced following Hsp90 inhibition was p21-dependent and the loss of p21 allowed SCLC cells to bypass the induction of senescence. Additionally, we identified a novel mechanism where the depletion of PKCι induced senescence in glioblastoma multiforme (GBM) cells. PKCι depletion-induced senescence did not activate the DNA-damage response pathway and was p21-dependent. Further perturbations of mitosis, using an aurora kinase inhibitor, increased the number of senescent cells when combined with PKCι depletion. This suggests that PKCι depletion-induced senescence involves defects in mitotic progression. Senescent glioblastoma cells at a basal level of senescence in culture, induced by p21 overexpression, and induced after PKCι depletion had aberrant centrosomes. Mitotic slippage is an early exit from mitosis without cell division that occurs when the spindle assembly checkpoint (SAC) is not satisfied. Senescent glioblastoma cells had multiple markers of mitotic slippage. Therefore, PKCι depletion-induced senescence involves mitotic slippage and results in aberrant centrosomes. A U87MG cell line with a doxycycline-inducible shRNA targeting PKCι was developed to deplete PKCι in established xenografts. PKCι was depleted in established glioblastoma xenografts in mice and resulted in decreased cell proliferation, delayed tumor growth and improved survival. This study has demonstrated that both Hsp90 and PKCι are novel targets to induce senescence in cancer cells as a potential therapeutic approach.

This PhD research relates to the design, fabrication, characterization, and optimization of on-chip electrochemical microelectrode arrays (MEAs) for measurement of transient concentrations and gradients, focusing on fast transients and physiological applications. In particular, this work presents the determination of kinetic mechanisms taking place at an active interface (either physiological or non-physiological) in contact with a liquid phase using the MEA device to simultaneously estimate the concentration and gradient of the analyte of interest at the surface of the active interface. The design approach of the MEA device and the corresponding measurement methodology to acquire reliable concentration information is discussed. The ability of the MEA device to measure fast (i.e., in sub-second time scale) transient gradients is demonstrated experimentally using a controllable diffusion-reaction system which mimics the consumption of hydrogen peroxide by a 2D cell culture. The proposed MEA device and measurement methodology meet effectively most of the requirements for physiological applications and as a demonstration of this, two physiological applications are presented. In one application, the MEA device was tailored to measure the hydrogen peroxide uptake rate of human astrocytes and glioblastoma multiforme cells in 2D cell culture as a function of hydrogen peroxide concentration at the cell surface; the results allowed to quantitatively determine the uptake kinetics mechanisms which are well-described by linear and Michaelis-Menten expressions, in agreement with the literature. In the other application, further customization of the MEA device was realized to study the glucose uptake kinetics of human bronchial epithelial and small cell lung cancer cells, these latter with and without DDX5 gene knockdown; the results allowed to distinguish mechanistic differences in the glucose uptake kinetics among the three cell lines. These results were complemented with measurements of glycolytic and respiration rates to obtain a bigger picture of the glucose metabolism of the three cell lines. Finally, additional applications, both physiological and non-physiological, are proposed for the developed MEA device.

Deregulation of gene expression caused by genetic or epigenetic changes plays an important role in pathogenesis of cancer. The thesis is a commented collection of ten publications dealing with the molecular biology of tumours. The author has significantly contributed to all of them. All the articles contained in the thesis are linked to the topic of assessment of molecules involved in gene expression regulation (microRNAs) or DNA alterations that affect gene expression (promoter methylation, presence of a fusion gene). MicroRNAs are short single-stranded RNA molecules involved in posttranscriptional regulation of gene expression by triggering mRNA degradation or inhibiting translation. It is a basic mechanism with an impact on all cellular processes including the pathogenesis of various diseases. MicroRNAs can either act as oncogenes by decreasing the expression of tumour-suppressor genes or as tumour-suppressor genes by decreasing the expression of oncogenes. However, the network of microRNA - RNA interactions is much more complex. Our published results that are part of this thesis are focused on colorectal carcinoma (CRC), prostate cancer, head and neck squamous cell carcinoma (HNSCC), gastric cancer and non-small cell lung cancer (NSCLC). In patients with CRC, we demonstrated the prognostic...

No description available.