Personnaliser les traitements anticancéreux sur base moléculaire consiste à optimiser la thérapie en fonction des profils d'expression de gènes des cellules saines et tumorales du patient. Les différences au niveau moléculaire entre tissus normaux et cancéreux sont exploitées afin de maximiser l'efficacité du traitement et minimiser sa toxicité. Cette thèse propose une approche pluridisciplinaire expérimentale et mathématique ayant pur but la détermination de stratégies anticancéreuses optimales sur base moléculaire. Cette approche est, tout d'abord, mise en œuvre pour la personnalisation de la chronothérapeutique des cancers, puis pour l'optimisation de la thérapie anticancéreuse dans le cas d'une mutation de l'oncogène SRC. La plupart des fonctions physiologiques chez les mammifères présentent une rythmicité circadienne, c'est-à-dire de période environ égale à 24 h. C'est par exemple le cas de l'alternance activité-repos, de la température corporelle, et de la concentration intracellulaire d'enzymes du métabolisme. Ces rythmes ont pour conséquence une variation de la toxicité et de l'efficacité d'un grand nombre de médicaments anticancéreux selon leur heure circadienne d'administration. De récentes études soulignent la nécessité d'adapter les schémas d'injection chronomodulés au profil moléculaire du patient. La première partie de cette thèse propose une approche combinée expérimentale et modélisatrice pour personnaliser sur base moléculaire la chronothérapie. La première étape a consisté en une preuve de concept impliquant des expérimentations in vitro sur cultures de cellules humaines et des travaux de modélisation in silico. Nous nous sommes focalisés sur l'étude de l'irinotecan (CPT11), un médicament anticancéreux actuellement utilisé en clinique dans le traitement des cancers colorectaux, et présentant des rythmes de chronotoxicité et de chronoefficacité chez la souris et chez l'homme. Sa pharmacocinétique (PK) et pharmacodynamie (PD) moléculaires ont été étudiées dans la lignée de cellules d'adénocarcinome colorectal humain Caco-2. Un modèle mathématique de la PK-PD moléculaire du CPT11, à base d'équations différentielles ordinaires, a été conçu. Il a guidé l'expérimentation qui a été réalisée dans le but de d'évaluer les paramètres du modèle. L'utilisation de procédures d'optimisation, appliquées au modèle calibré aux données biologiques, a permis la conception de schéma d'exposition au CPT11 théoriquement optimaux dans le cas particulier des cellules Caco-2. Le CPT11 s'est accumulé dans les cellules Caco-2 où il a été biotransformé en son métabolite actif le SN38, sous l'action des carboxylestérases (CES). La pré-incubation des cellules avec du verapamil, un inhibiteur non spécifique des transporteurs ABC (ATP-Binding Cassette) a permis la mise en évidence du rôle de ces pompes d'efflux ABC dans le transport du CPT11. Après synchronisation des cellules par choc sérique, qui définit le temps circadien (CT) 0, des rythmes circadiens d'une période de 26 h 50 (SD 63 min) ont été mis en évidence pour l'expression de trois gènes de l'horloge circadienne: REV-ERBα, PER2, et BMAL1; et six gènes de la pharmacologie du CPT11: la cible du médicament la topoisomerase 1 (TOP1), l'enzyme d'activation CES2, l'enzyme de désactivation UGT1A1, et les quatre transporteurs ABCB1, ABCC1, ABCC2, ABCG2. Au contraire, l'expression protéique et l'activité de la TOP1 sont restées constantes. Enfin, la quantité de TOP1 liée à l'ADN en présence de CPT11, un marqueur de la PD du médicament, a été plus importante pour une exposition à CT14 (47±5.2% de la quantité totale de TOP1), que pour une exposition à CT28 (35.5±1.8%). Les paramètres du modèle mathématique de la PK-PD du CPT11 ont ensuite été estimés par une approche de bootstrap, en utilisant des résultats expérimentaux obtenus sur les cellules Caco-2, combinés aux données de la littérature. Ensuite, des algorithmes d'optimisation ont été utilisés pour concevoir les schémas d'exposition théoriquement optimaux des cellules Caco-2 à l'irinotecan. Les cellules synchronisées par un choc sérique ont été considérées comme les cellules saines et les cellules non-synchronisées ont joué le rôle de cellules cancéreuses puisque l'organisation circadienne est souvent perturbée dans les tissus tumoraux. La stratégie thérapeutique optimale a été définie comme celle qui maximise l'efficacité sur les cellules cancéreuses, sous une contrainte de toxicité maximale sur les cellules saines. Les schémas d'administration considérés ont pris la forme d'une exposition à une concentration donnée de CPT11, débutant à un CT particulier, sur une durée comprise entre 0 et 27 h. Les simulations numériques prédisent que toute dose de CPT11 devrait être optimalement administrée sur une durée de 3h40 à 7h10, débutant entre CT2h10 et CT2h30, un intervalle de temps correspondant à 1h30 à 1h50 avant le minimum des rythmes de bioactivation du CPT11 par les CES. Une interprétation clinique peut être établie en ramenant à 24 h ces résultats pour les cellules Caco-2 qui présentent une période de 27 h. Ainsi, une administration optimale du CPT11 chez le patient cancéreux résulterait en une présence du médicament dans le sang pendant 3h30 à 6h30, débutant de 1h30 à 1h40 avant le minimum des rythmes d'activité des CES chez le patient. La deuxième étape de nos travaux a consisté à adapter l'approche mise en œuvre pour optimiser l'exposition des cellules Caco-2 au CPT11, pour l'optimisation de l'administration du médicament chez la souris. Des études récentes mettent en évidence trois classes de chronotoxicité à l'irinotecan chez la souris. La classe 1, les souris de la lignée B6D2F1 femelles, présentent la pire tolérabilité au CPT11 après une administration à ZT3, et la meilleure pour une administration à ZT15, où ZT est le temps de Zeitgeber, ZT0 définissant le début de la phase de lumière. La classe 2, les souris B6D2F1 mâles, montrent une pire heure d'administration à ZT23 et une meilleure à ZT11. Enfin, la classe 3, les B6CBAF1 femelles présentent la pire tolérabilité pour une injection à ZT7, et la meilleure pour ZT15. Nous avons entrepris une approche pluridisciplinaire in vivo et in silico dont le but est la caractérisation moléculaire des trois classes de chronotoxicité, ainsi que la conception de schémas optimaux d'administration pour chacune d'elles. Le modèle mathématique mis au point pour une population de cellules en culture a été adapté pour la construction d'un modèle "corps entier" à base physiologique de la PK-PD de l'irinotecan. Un ensemble de paramètres a été estimé pour la classe 2, en utilisant deux sortes de résultats expérimentaux: d'une part, les concentrations sanguines et tissulaires (foie, colon, moelle osseuse, tumeur) du CPT11 administré aux pire et meilleure heures circadiennes de tolérabilité, et d'autre part, les variations circadiennes des protéines de la pharmacologie du médicament dans le foie et l'intestin. Le modèle ainsi calibré reproduit de façon satisfaisante les données biologiques. Cette étude est en cours pour les classes 1 et 3. Une fois les ensembles de paramètres validés pour chacune des trois classes, ils seront comparés entre eux pour mettre en évidence de possibles différences moléculaires. L'étape suivante consiste en l'application d'algorithmes d'optimisation sur le modèle corps entier pour définir des schémas d'administration chronomodulés optimaux pour chaque classe. La deuxième partie de cette thèse s'intéresse à l'étude de la tyrosine kinase SRC, dont l'expression est dérégulée dans de nombreux cancers. Des études récentes montrent un contrôle de SRC sur la voie mitochondriale de l'apoptose dans des fibroblastes de souris NIH-3T3 transfectés avec l'oncogène v-src, cette régulation étant inexistante dans les cellules parentales. L'oncogène SRC active la voie RAS / RAK / MEK1/2 / ERK1/2 qui augmente la vitesse de phosphorylation de la protéine pro-apoptotique BIK, menant ainsi à sa dégradation par le protéasome. La faible expression de BIK résultant de ce mécanisme rendrait ainsi les cellules v-src résistantes à la plupart des stress apoptotiques. Notre étude a consisté à déterminer, par une approche combinée mathématique et expérimentale, les stratégies thérapeutiques optimales lorsque les cellules NIH-3T3 parentales jouent le rôle de cellules saines, et les fibroblastes transformés celui de cellules cancéreuses. Pour cela, nous avons, tout d'abord, construit un modèle mathématique de la cinétique de BIK en conditions non-apoptotiques. L'estimation des paramètres de ce modèle, en utilisant des données expérimentales existantes, confirme que la phosphorylation de BIK sous le contrôle de SRC est inactive dans les cellules normales. L'étude exprimentale de l'évolution de BIK après le signal apoptotique que constitue une exposition à la staurosporine, démontre une relocalisation de BIK aux mitochondries, la concentration totale de la protéine restant constante durant le stress. Nous avons ensuite conçu un modèle mathématique de la voie mitochondriale de l'apoptose mettant en jeu les protéines anti-apoptotiques de type Bcl2, les protéines effectrices de type BAX, les protéines BH3-only activatrices et les BH3-only sensibilisatrices. Un ensemble de paramètres a été déterminé pour les cellules NIH-3T3 parentales, et celles transformées v-src, en comparant le modèle aux données expérimentales. Le modèle reproduit le fait expérimentalement démontré que la préincubation des cellules v-src avec un inhibiteur de la tyrosine kinase SRC, avant l'exposition à la staurosporine, annihile la résistance des fibroblastes transformés au stress apoptotique. Le modèle prédit que l'administration de l'ABT-737, un inhibiteur de protéines anti-apoptotiques, avant l'exposition à la staurosporine, ne devrait pas être entreprise dans notre systéme biologique, ce qui a été expérimentalement validé. Enfin, le modèle a été utilisé dans des procédures d'optimisation pour déterminer la stratégie thérapeutique théoriquement optimale, lorsque les cellules normales et transformées sont exposées aux mêmes médicaments. Les combinaisons médicamenteuses considérées consiste en une exposition à la staurosporine, précédée d'une exposition à des répresseurs ou activateurs d'expression des protéines de la famille Bcl2. La stratégie optimale est définie comme celle qui maximise le pourcentage de cellules apoptotiques dans les fibroblastes v-src, sous la contrainte que celui dans les cellules normales reste au-dessous d'un seuil de tolérabilité. Les simulations numériques nous permettent de conclure à une combinaison médicamenteuse optimale constituée d'une exposition à la staurosporine, précédée d'une exposition à un répresseur de l'expression de BAX (de manière à diminuer sa concentration en-dessous du seuil apoptotique dans les cellules normales, mais pas dans les cellules cancéreuses), combinée à un répresseur de BCL2 ou un inhibiteur de tyrosines kinases SRC. Cette stratégie optimale aboutit à moins d'1% de cellules apoptotiques dans les cellules saines et plus de 98% dans les cellules cancéreuses. / Personalizing anticancer treatment on molecular basis consists in optimizing the therapy according to the gene expression profiles of healthy and cancer cells of the patient. The molecular differences between normal and tumor tissues are exploited in order to maximize the treatment efficacy and minimize its toxicities. This PhD thesis presents a combined mathematical and experimental approach to optimize anticancer therapeutic strategies on molecular basis. This approach has been undertaken at first for the personalization of cancer chronotherapeutics, and secondly for the optimization of anticancer therapy in the case of mutated SRC oncogene. Most of the physiological functions in mammals display rhythms of period around 24, also called circadian rhythms. For instance, rest-activity rhythm, core temperature, or intracellular concentrations of metabolic enzymes show variations over the 24 h span. Those rhythms result in variations in the toxicity and efficacy of many anticancer drug with respect to their circadian time of administration. Recent studies highlight the need for chronomodulated injection scheme which would be tailored to the patient's molecular profile. Thus the first sections of this thesis propose a combined experimental and mathematical approach for personalizing cancer chronotherapeutics on molecular basis. The first step consisted in a proof of concept which involved in vitro experiments on human cell culture and in silico mathematical modeling. We focused on the anticancer drug irinotecan (CPT11), which is currently used in the treatment of colorectal cancer and displays chronotoxicity and chronoefficacy rhythms both in mice and in cancer patients. Its molecular pharmacokinetics (PK) and pharmacodynamics (PD) were studied in human colorectal adenocarcinoma Caco-2 cells. An ODE-based mathematical model of its PK-PD was built. It guided the design of experiments which were performed in order to estimate parameter values of the model. Optimization procedures were then applied to this data-calibrated model in order to compute theoretically optimal exposure schemes for the Caco-2 cell line. CPT11 accumulated in Caco-2 cells where it was bioactivated into SN38 under the catalytic activity of carboxylesterases (CES). The pre-incubation of cells with verapamil, a non-specific inhibitor of ATP-Binding Cassette (ABC) transporters, increased CPT11 intracellular accumulation, thus demonstrating the involvement of those efflux pumps in CPT11 transport. After cell synchronization by a seric shock which defines the circadian time (CT) 0, circadian rhythm of period 26 h 50 (SD 63 min) were observed for the expression of the three clock genes REV-ERBα, PER2, and BMAL1; and of six metabolic genes: the drug target topoisomerase 1 (TOP1), the activation enzyme CES2, the deactivation enzyme UGT1A1, and the four ABC transporters ABCB1, ABCC1, ABCC2, ABCG2. On the contrary, TOP1 proteic level and activity remained constant. The amount of DNA-bound TOP1 in the presence of CPT11 is a PD marker of the drug and displayed circadian rhythms as it was equal to 47±5.2% of the total amount of TOP1 protein after an exposure at CT14, as compared to 35.5±1.8% after an exposure at CT28. Parameters of CPT11 PK-PD model were estimated from experimental data in Caco-2 cells combined to information from literature by a bootstrap approach. Optimization procedures were then applied to the data-calibrated model in order to compute theoretically optimal exposure schemes for Caco-2 cells. Synchronized cells were considered as healthy cells and non-synchronized cells as cancer ones as the circadian organization is often disrupted in tumor tissues. The adopted therapeutics strategy consisted in maximizing DNA damage in cancer cells under the constraint that DNA damage in the healthy population remained under a tolerability threshold. We considered administration schemes in the form of a cell exposure to an initial extracellular concentration of CPT11, over 1 to 27 h, starting at a particular CT. Numerical procedures predicted that, for all considered doses of CPT11, the optimal exposure lasted from 3h40 to 7h10, and started between CT2h10 and CT2h30, a time interval corresponding to 1h30 to 1h50 before the minimum value of CES activity. A clinical interpretation can be obtained by rescaling to 24 h those results for Caco-2 cells which displayed a period of 26 h 50. Thus, an optimal administration of CPT11 to cancer patients should result in the presence of the drug in the blood during 3h30 to 6h30, starting 1h30 to 1h40 before the minimum value of CES activity in the patient. The second section of our research works has consisted in adapting the pluridisciplinary approach we have undertaken for optimizing CPT11 exposure in Caco-2 cells, for the optimization of CPT11 administration in mice. Recent experimental studies demonstrated the existence of three classes of mice regarding CPT11 chronotoxicity. Female mice of the strain B6D2F1 represented the first class and showed worst tolerability after an injection of CPT11 at ZT3 and best tolerability at ZT15, where ZT is Zeitgeber Time, ZT0 defining the beginning of the light phase. Class 2 was constituted by B6D2F1 male mice and displayed worst toxicity at ZT23 and best toxicity at ZT11. Finally, class 3 was B6CBAF1 female mice and showed worst tolerability at ZT7 and best tolerability at ZT15. Our combined in vivo and in silico approach aimed at characterizing the three chronotoxicity classes at the molecular level and at designing optimal administration schemes for each of them. The mathematical model which was built for Caco-2 cell culture was adapted to design a whole body physiologically based model of CPT11 PK-PD. Parameters were estimated for class 2 by fitting both blood and tissular pharmacokinetics data and measurements of circadian rhythms of proteins involved in CPT11 pharmacology. The calibrated model fitted the biological data. Similar parameter estimations are ongoing for classes 1 and 3. Once three parameter sets are estimated, their comparaison will allow the determination of differences in protein activities and therefore the molecular characterization of the three classes. The next step consists in applying optimization procedures to the calibrated whole body model in order to design theoretically optimal administration schemes for each class. The second project presented in this thesis focuses on the tyrosine kinase SRC, which is deregulated in numerous cancer diseases. Recent studies showed that SRC exerted a control on the mitochondrial pathway of apoptosis in NIH-3T3 mice fibroblasts which were transfected with the oncogene v-src, whereas this control was not observed in parental NIH-3T3 cells. SRC activated the RAS / RAK / MEK1/2 / ERK1/2 pathway which enhanced the phosphorylation of the pro-apoptotic enzyme BIK, thus leading to the protein degradation by the proteasome. As a result, BIK protein expression was low in v-src transformed fibroblasts which possibly contributed to the resistance of those cells to most apoptotic stresses. Our work consisted in designing optimal therapeutics strategies in which parental NIH-3T3 fibroblasts stands for healthy cells and v-src transformed ones for cancer cells. Once again, a combined experimental and mathematical approach was undertaken. First, we built a model for BIK kinetics in non-apoptotic conditions and fitted its parameters to existing experimental data. Estimated parameter values confirmed that SRC-dependent phosphorylation of BIK was inactive in normal cells. Then, we biologically and mathematically investigated BIK kinetics in response to a death signal. We showed a relocalization of BIK to the mitochondria in the first hours of staurosporine exposure, whereas BIK protein total amount remained constant during the apoptotic stress. We then conceived a mathematical model of the mitochondrial pathway of apoptosis in NIH-3T3 cells. It modeled molecular interactions between the anti-apoptotic proteins as BCL2, and the pro-apoptotic enzymes which were divided into three subgroups: the BAX-like effector proteins, the BH3-only activator proteins and the BH3-only sensitizer proteins. Parameters were estimated by fitting experimental results in normal and v-src transformed fibroblasts. The model reproduced the experimentally-demonstrated fact that pre-incubating v-src fibroblasts with an inhibitor of SRC before staurosporine exposure annihilated the cell resistance. The model predicted that an administration of ABT-737, an inhibitor of anti-apoptotic proteins, before staurosporine exposure, should not be undertaken in our particular biological systems, which was experimentally validated. Finally, optimization procedures were applied to the model in order to design optimal therapeutics strategies when both normal and cancer cells were exposed to the same drugs. Considered exposure scheme consisted in the administration of staurosporine after an exposure to activator or of proteins of the mitochondrial pathway of apoptosis. Optimal strategies were defined as the ones which maximized the percentage of apoptotic cells in the cancer cell population, under the constraint that that in the healthy population remained below a toxicity threshold. Optimization procedures allowed to conclude that the optimal drug combinaison consisted in exposing cells to staurosporine after an exposure to a chemical able to repress BAX expression such that its concentration in normal cells was below the needed amount to trigger apoptosis, combined either to a repressor of anti-apoptotic proteins, or an inhibitor of SRC. These optimal strategies led to less than 1% of apoptotic cells in healthy cells, and more than 98% in cancer cells.

O dimorfismo sexual relacionado à hipertensão arterial surge na adolescência e persiste por toda vida adulta. Homens apresentam maior incidência de doenças cardiovasculares quando comparados a mulheres de mesma faixa etária. O mesmo perfil é observado em modelos animais de hipertensão, nos quais machos apresentam maiores níveis pressóricos quando comparados a fêmeas. Dessa forma, a testosterona é frequentemente relacionada à hipertensão arterial. Entretanto, os mecanismos pelos quais a testosterona exerce efeitos vasculares ainda não estão esclarecidos. O objetivo deste trabalho foi investigar os efeitos da testosterona sobre a geração de espécies reativas de oxigênio (EROs), importantes mediadores do processo hipertensivo, em células da musculatura lisa vascular (CMLV) de ratos normotensos e espontaneamente hipertensos (SHR). Os receptores para andrógenos, as fontes de EROs (papel da NADPH oxidase), bem como os efeitos funcionais celulares (migração celular) relacionados aos efeitos da testosterona também foram analisados. Para tanto, CMLV do leito mesentérico de ratos Wistar (W), Wistar-Kyoto (WKY) e SHR foram isoladas, cultivadas e estimuladas com testosterona 10-7mol/L em diferentes tempos, de acordo com cada protocolo. Sempre que necessário, as células foram pré-incubadas por 30 minutos com inibidores específicos para o estudo dos mecanismos envolvidos, tais como: flutamida (inibidor do receptor clássico para andrógenos), apocinina (inibidor da NADPH oxidase), PP2 (inibidor da c-Src), actinomicina D (inibidor da transcrição gênica) e cicloheximida (inibidor da síntese protéica). Nossos resultados indicam que a testosterona induz a geração de EROs por mecanismos dependentes do tempo e da linhagem de ratos, de modo que células isoladas de animais SHR são mais sensíveis a testosterona. Esta geração ocorre por dois mecanismos principais: um mediado pelo receptor clássico para andrógenos (AR) e outro mediado pelo receptor de membrana para andrógenos (ARm), resultando em efeitos genômicos e não-genômicos, respectivamente. Enquanto os efeitos genômicos são comuns, isto é, são observados em células de animais normotensos e hipertensos, os efeitos não-genômicos são específicos, e ocorrem exclusivamente em células de animais hipertensos. A geração genômica de EROs, mediada pelo AR, depende da modulação da expressão de subunidades da NADPH oxidase. Por outro lado, a geração não-genômica, é mediada pelo ARm, independe de síntese protéica, e ocorre devido à ativação de vias de sinalização específicas, reguladoras do complexo enzimático NADPH oxidase. As EROs formadas a partir do estímulo com a testosterona tanto por mecanismos genômicos ou não-genômicos levam a migração celular por mecanismos mediados pelo RA. Nossos resultados sugerem que a testosterona tem papel importante na função de células da musculatura lisa vascular, o que pode contribuir para algumas alterações vasculares características do processo hipertensivo. Portanto, nosso trabalho é o primeiro a demonstrar que a testosterona regula vias de sinalização redox em CMLV levando a efeitos funcionais importantes, relacionados ao remodelamento vascular, os quais podem contribuir para o desenvolvimento e manutenção da hipertensão arterial. / Sexual dimorphism related to hypertension begins at childhood and persists through adulthood. The incidence of cardiovascular diseases is higher in men when compared to age-matched women. Although testosterone has been associated to the sexual dimorphism in hypertension, the mechanisms whereby testosterone acts in the vasculature remain unclear. The main objective of this study was to determine whether testosterone induces reactive oxygen species (ROS) generation, key players on hypertension, in vascular smooth muscle cells (VSMC) isolated from normotensive and hypertensive rats. The signaling pathways and the androgen receptors activated by testosterone, the role of NADPH oxidase in ROS generation and the cellular outcomes (cell migration) were also determined. Accordingly, VSMC isolated from the mesenteric bed of Wistar (W), Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats were stimulated with testosterone 10-7mol/L for different periods of time, according to each protocol. Whenever appropriate, cells were pre-incubated with specific inhibitors, such as flutamide 10-5mol/L (nuclear androgen receptor antagonist), apocynin 3x10-5mol/L (NADPH oxidase inhibitor), PP2 10-5mol/L (c-Src inhibitor), actinomycin D 10-5mol/L (inhibitor of gene transcription), and cycloheximide 10-5mol/L (protein synthesis inhibitor). Our findings demonstrate that testosterone induces ROS formation in a time and strain-dependent manner. Augmentation of ROS formation is higher in SHR-VSCMC, indicating an increased sensitivity of SHR-VSMC to testosterone stimuli. Testosterone-induced ROS production occurs by two main mechanisms: the first mediated through the classical androgen receptor (AR) and the second mediated through membrane-associated androgen receptor (ARm), leading to genomic and non-genomic effects, respectively. Whereas the genomic effects occur in VSMC from both strains, non-genomic effects are only observed in SHR-VSMC. The genomic ROS production is mediated through AR and depends on modulation of NADPH oxidase subunits. On the other hand, non-genomic ROS formation is mediated through RAm, does not rely on protein synthesis and occurs via specific signaling pathways that regulate NADPH oxidase. Genomic and non-genomic ROS production by testosterone leads to a common final effect: VSMC migration, indicating that testosterone plays a key role in VSMC function. These results indicate that testosterone signals through redox-sensitive pathways, important in c-Src-mediated migration of VSMCs in SHR. Such processes may contribute to vascular remodeling in hypertension.

The altered protein expression and activity of receptor tyrosine kinases (TK) are implicated in the progression of various types of cancers. One such dysfunction is the overexpression of the epidermal growth factor receptor (EGFR) that correlates with aggressive tumor progression and poor prognosis. On the other hand, c-Src non-receptor tyrosine kinase is overexpressed and activated in a large number of human malignancies and has been strongly linked to progression to distant metastases. c-Src-induced phosphorylation of EGFR is required for EGF-mediated mitogenesis, tumorigenesis and tumour invasiveness. Thus we surmised that molecules termed "combi-molecules" designed to block both EGFR and c-Src should not only possess significant growth inhibitory potency but also strong anti-invasive properties. In this thesis, we utilized molecular modeling to design molecules containing two moieties: one that straddles the structure of the known Src inhibitor PP2 and the other that mimics the backbone of Iressa, a potent EGFR inhibitor. Of all the molecules synthesized, only SB163 containing the longest spacer between the two moieties was capable of inducing a dose dependent inhibition of both Src and EGFR. More importantly, SB163 blocked cell motility in the wound healing assay and showed significantly greater anti-invasive activity than a PP2+Iressa combination. The observation that SB163 was a less potent EGFR or Src inhibitor than Iressa and PP2 suggests that its superior potency when compared with the PP2+ Iressa combination may be at least partially attributed to mechanisms other than EGFR or Src blockade. This was also corroborated by the fact that SB163, despite its significant bulkiness (>700) could induce dose dependent inhibition of other kinase such PDFGR and Abl. The results in toto suggest that conferring multiple kinase targeting properties to single molecules can lead to highly anti-proliferative and anti-invasive agents. Traditionally, multi-kinase targeted molecules were discovered serendipitously through multi-kinase testing. Here we initiated a more rational approach to the design of single multi-targeted molecules. Cancer being a complex disease driven by tumours characterized by multiple disordered signaling pathways, this approach may well represent a novel avenue in the therapy of refractory malignancies.

Abstract The epithelial cells form barriers that compartmentalize the organs. Carcinomas are cancers stemming from epithelial cells and are the most common cancer type. The aim of this study was to understand the differentiation and malignant transformation of epithelial Madin-Darby canine kidney (MDCK) cells and to analyse the electrophysiological parameters which regulate their transport capacity. Emphasis was placed on comparing different culture environments, both in 2D and 3D. First, the effects of drugs or basal extracellular fluid composition on MDCK cell, cyst and lumen volumes were analysed using time-lapse microscopy. The results showed that MDCK cells were capable of both water secretion and reabsorption. The cells were able to perform these functions in a hyperpolarizing or depolarizing environment; change in osmolality of basal fluid was not required. Taken together, these results validate MDCK cells as a good basic model for studying kidney function. Next, the aim was to analyse the effect of 2D and 3D culture environments on the gene expression of untransformed MDCK and temperature sensitive ts-Src -transformed MDCK cells and the changes a single oncogene can induce. Microarray analysis revealed a decrease in the expression of survivin, an inhibitor of apoptosis protein, when switching the untransformed cells from 2D environment to 3D. This downregulation of survivin occurs in adult tissues as well, indicating that the cells grown in 3D are closer to the in vivo state than 2D cells. Src oncogene induced disintegration of cell junctions, but did not downregulate E-cadherin expression. The last part was to study further the factors controlling survivin expression and its significance to cell survival. MDCK cells grown in 3D did not suffer apoptosis if the cells remained in contact with the extracellular matrix. If MDCK cells were denied of ECM contacts they were more susceptible to apoptosis than survivin-expressing ts-Src MDCK cells. Finally, if cells were denied of cell-cell junctions, cells lacking survivin suffered apoptosis even though they had proper cell-matrix contacts. Taken together, these results highlighted the importance of cellular contacts to the cells: MDCK cells needed ECM contacts to differentiate and cell-cell contacts to avoid apoptosis. / Tiivistelmä Epiteelisolut ovat erikoistuneet toimimaan rajapintana elimen ja ympäristön välillä. Ihmisten yleisin syöpä on epiteelisoluista alkunsa saanut karsinooma. Tämän tutkimuksen tarkoituksena oli ymmärtää Madin-Darby-koiran munuaisen solujen (MDCK) erilaistumista ja pahanlaatuistumista sekä analysoida sähköfysiologisia tekijöitä, jotka säätelevät näiden solujen kuljetustoimintaa. Erityisenä kiinnostuksen kohteena oli erilaisten kasvuympäristöjen vertailu. Farmakologisten aineiden tai basaalisen, solunulkopuolisen nesteen koostumuksen vaikutusta MDCK-solujen, -kystan sekä luumenin kokoon tutkittiin valomikroskooppisten aikasarjojen avulla. Tulokset osoittivat MDCK-solujen olevan kykeneviä sekä veden eritykseen että absorptioon, niin hyperpolarisoivassa kuin depolarisoivassakin ympäristössä. Basaalisen nesteen osmolaliteetin muutosta ei tarvittu. Nämä tulokset osoittavat MDCK-solujen olevan hyvä munuaisen tutkimuksen perusmalli. Seuraavaksi analysoitiin kaksi- ja kolmiulotteisten (2D ja 3D) viljely-ympäristöjen vaikutusta ei-transformoitujen MDCK-solujen ja lämpötilaherkkien ts-Src-transformoitujen MDCK-solujen geenien ilmentymiseen sekä yhden onkogeenin aktivoimisen aikaansaamia muutoksia. Microarray-analyysi osoitti apoptoosin estäjän, surviviinin, ilmentymisen vähenemisen, kun kasvuympäristö vaihdettiin 2D-ympäristöstä 3D-ympäristöön. Koska surviviinin väheneminen on normaali tapahtuma aikuisissa kudoksissa, voitiin todeta, että 3D-ympäristössä kasvatetut solut ovat lähempänä luonnonmukaista olotilaa kuin 2D-ympäristössä kasvaneet. Src-onkogeeni sai aikaan soluliitosten hajoamisen, mutta ei vähentänyt E-kadheriinin ilmentymistä. Tutkimuksen viimeinen osa keskittyi surviviinin ilmentymistä säätelevien tekijöiden analysoimiseen ja surviviinin merkitykseen solujen eloonjäämiselle. 3D-ympäristössä kasvaneet MDCK-solut eivät kärsineet apoptoosista edellyttäen, että solut pysyivät kosketuksissa soluväliaineeseen. Jos solut irtautuivat soluväliaineesta, ne päätyivät herkemmin apoptoosiin kuin surviviinia ilmentävät ts-Src MDCK-solut. Mikäli solujen väliset liitokset pakotettiin avautumaan, solut joutuivat apoptoosiin, vaikka ne olivat kosketuksissa soluväliaineeseen. Yhteenvetona nämä tulokset korostavat solujen kontaktien merkitystä: MDCK-solut tarvitsevat soluväliainekontakteja erilaistumiseen ja solujen välisiä kontakteja välttyäkseen apoptoosilta.

O dimorfismo sexual relacionado à hipertensão arterial surge na adolescência e persiste por toda vida adulta. Homens apresentam maior incidência de doenças cardiovasculares quando comparados a mulheres de mesma faixa etária. O mesmo perfil é observado em modelos animais de hipertensão, nos quais machos apresentam maiores níveis pressóricos quando comparados a fêmeas. Dessa forma, a testosterona é frequentemente relacionada à hipertensão arterial. Entretanto, os mecanismos pelos quais a testosterona exerce efeitos vasculares ainda não estão esclarecidos. O objetivo deste trabalho foi investigar os efeitos da testosterona sobre a geração de espécies reativas de oxigênio (EROs), importantes mediadores do processo hipertensivo, em células da musculatura lisa vascular (CMLV) de ratos normotensos e espontaneamente hipertensos (SHR). Os receptores para andrógenos, as fontes de EROs (papel da NADPH oxidase), bem como os efeitos funcionais celulares (migração celular) relacionados aos efeitos da testosterona também foram analisados. Para tanto, CMLV do leito mesentérico de ratos Wistar (W), Wistar-Kyoto (WKY) e SHR foram isoladas, cultivadas e estimuladas com testosterona 10-7mol/L em diferentes tempos, de acordo com cada protocolo. Sempre que necessário, as células foram pré-incubadas por 30 minutos com inibidores específicos para o estudo dos mecanismos envolvidos, tais como: flutamida (inibidor do receptor clássico para andrógenos), apocinina (inibidor da NADPH oxidase), PP2 (inibidor da c-Src), actinomicina D (inibidor da transcrição gênica) e cicloheximida (inibidor da síntese protéica). Nossos resultados indicam que a testosterona induz a geração de EROs por mecanismos dependentes do tempo e da linhagem de ratos, de modo que células isoladas de animais SHR são mais sensíveis a testosterona. Esta geração ocorre por dois mecanismos principais: um mediado pelo receptor clássico para andrógenos (AR) e outro mediado pelo receptor de membrana para andrógenos (ARm), resultando em efeitos genômicos e não-genômicos, respectivamente. Enquanto os efeitos genômicos são comuns, isto é, são observados em células de animais normotensos e hipertensos, os efeitos não-genômicos são específicos, e ocorrem exclusivamente em células de animais hipertensos. A geração genômica de EROs, mediada pelo AR, depende da modulação da expressão de subunidades da NADPH oxidase. Por outro lado, a geração não-genômica, é mediada pelo ARm, independe de síntese protéica, e ocorre devido à ativação de vias de sinalização específicas, reguladoras do complexo enzimático NADPH oxidase. As EROs formadas a partir do estímulo com a testosterona tanto por mecanismos genômicos ou não-genômicos levam a migração celular por mecanismos mediados pelo RA. Nossos resultados sugerem que a testosterona tem papel importante na função de células da musculatura lisa vascular, o que pode contribuir para algumas alterações vasculares características do processo hipertensivo. Portanto, nosso trabalho é o primeiro a demonstrar que a testosterona regula vias de sinalização redox em CMLV levando a efeitos funcionais importantes, relacionados ao remodelamento vascular, os quais podem contribuir para o desenvolvimento e manutenção da hipertensão arterial. / Sexual dimorphism related to hypertension begins at childhood and persists through adulthood. The incidence of cardiovascular diseases is higher in men when compared to age-matched women. Although testosterone has been associated to the sexual dimorphism in hypertension, the mechanisms whereby testosterone acts in the vasculature remain unclear. The main objective of this study was to determine whether testosterone induces reactive oxygen species (ROS) generation, key players on hypertension, in vascular smooth muscle cells (VSMC) isolated from normotensive and hypertensive rats. The signaling pathways and the androgen receptors activated by testosterone, the role of NADPH oxidase in ROS generation and the cellular outcomes (cell migration) were also determined. Accordingly, VSMC isolated from the mesenteric bed of Wistar (W), Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats were stimulated with testosterone 10-7mol/L for different periods of time, according to each protocol. Whenever appropriate, cells were pre-incubated with specific inhibitors, such as flutamide 10-5mol/L (nuclear androgen receptor antagonist), apocynin 3x10-5mol/L (NADPH oxidase inhibitor), PP2 10-5mol/L (c-Src inhibitor), actinomycin D 10-5mol/L (inhibitor of gene transcription), and cycloheximide 10-5mol/L (protein synthesis inhibitor). Our findings demonstrate that testosterone induces ROS formation in a time and strain-dependent manner. Augmentation of ROS formation is higher in SHR-VSCMC, indicating an increased sensitivity of SHR-VSMC to testosterone stimuli. Testosterone-induced ROS production occurs by two main mechanisms: the first mediated through the classical androgen receptor (AR) and the second mediated through membrane-associated androgen receptor (ARm), leading to genomic and non-genomic effects, respectively. Whereas the genomic effects occur in VSMC from both strains, non-genomic effects are only observed in SHR-VSMC. The genomic ROS production is mediated through AR and depends on modulation of NADPH oxidase subunits. On the other hand, non-genomic ROS formation is mediated through RAm, does not rely on protein synthesis and occurs via specific signaling pathways that regulate NADPH oxidase. Genomic and non-genomic ROS production by testosterone leads to a common final effect: VSMC migration, indicating that testosterone plays a key role in VSMC function. These results indicate that testosterone signals through redox-sensitive pathways, important in c-Src-mediated migration of VSMCs in SHR. Such processes may contribute to vascular remodeling in hypertension.

L’invasion métastatique des tumeurs humaines est un mécanisme complexe qui repose sur l’acquisition de nouvelles fonctionnalités par les cellules tumorales. Les protéines ADAM et plus particulièrement la protéine ADAM9, grâce à leur domaine extracellulaire se composant d’une activité métalloprotéasique et disintégrine, possèdent des fonctions importantes et nécessaires au processus d’invasion. Cependant, les mécanismes de régulation de la protéine restent globalement méconnus dans la pathologie cancéreuse. L’objectif de ce travail a consisté à évaluer le rôle de l’expression d’ADAM9 dans les mécanismes d’agressivité tumorale tels que l’adhérence cellulaire, la migration cellulaire ou l’angiogénèse ainsi que l’étude des mécanismes de régulation de l’expression de cette protéine. Compte tenu du fait que le peroxyde d’hydrogène est connu pour induire l’expression d’ADAM9, les premiers travaux ont eu pour objectif d’établir le lien entre stress oxydant, ADAM9 et adhérence tumorale. L’exposition des cellules d’adénocarcinome pulmonaire au peroxyde d’hydrogène induit une augmentation dose dépendante de l’expression de la protéine ADAM9 transmembranaire et de sa forme sécrétée. Les études in vitro ont permis d’établir que les capacités d’adhérence et d’invasion tumorale induites par le stress oxydant sont principalement médiées par les deux isoformes de la protéine ADAM9. Par ailleurs, l’expression d’ADAM9 provoque un accroissement de la néo-angiogénèse par l’intermédiaire d’un accroissement non transcriptionel de la biosynthèse d’IL8. Cette cytokine proangiogénique va interagir avec le récepteur CXCR2 et va permettre la mise en place d’une néovascularisation in vitro. Le développement d’un modèle de xénogreffe de cellule d’adénocarcinome pulmonaire a permis de confirmer le rôle majeur d’ADAM9 dans les processus de dissémination métastatique et d’angiogénèse tumoraux. L’étude de la modulation pharmacologique d’ADAM9 a reposé sur deux stratégies pharmacologiques différentes : d’une part l’interaction directe avec les différentes isoformes d’ADAM9 au moyen d’un anticorps neutralisant et d’autre part une action sur les mécanismes de transduction cellulaire tels que la protéine SRC ou la protéine kinase C. Ce travail a permis de mieux comprendre l’implication de la protéine ADAM9 au cours du processus de cancérogenèse de part sa participation aux étapes majeures que sont la dissémination métastatique induite par le stress oxydant et l’angiogenèse / Tumor invasion is a complex mechanism that is based on the acquisition of tumor cells new functions. ADAM proteins, especially protein ADAM9, through their extracellular domain consisting of a disintegrin and metalloprotease activity, have important functions and processes necessary for invasion. However, the mechanisms regulating protein remain largely unknown in cancer pathology. The objective of this work was to evaluate the role of ADAM9 expression in tumor aggressiveness such as cell adhesion, cell migration and angiogenesis and to study the mechanisms regulating this protein expression. Given the fact that hydrogen peroxide is known to induce the expression of ADAM9 protein, the first work aimed to establish the relationship between oxidative stress, adhesion and tumor ADAM9 expression. Hydrogen peroxide induces a dose-dependent increase of both expression and activity of ADAM9 on adenocarcinoma pulmonary cells. Oxidative stress induced ADAM9 expression and activity are mainly supported by the secreted form of ADAM9 protein. In vitro studies have shown that capacity of adhesiveness and invasiveness induced by oxidative stress are mainly mediated by the two forms of ADAM9 protein. In addition, ADAM9 protein expression induces neoangiogenesis through increased production of interleukin 8. This proangiogenic cytokine that interacts with the CXCR2 receptor is able to stimulate neovascularization in vitro studies. Development of a lung adenocarcinoma xenograft model confirmed that ADAM9 protein have an important role on metastasis process and tumor angiogenesis. The study of pharmacological modulation of ADAM9 expression was based on two different pharmacological strategies: the first interacts with different isoforms of ADAM9 using a neutralizing antibody and the second strategy modulate cell transduction mechanism such as SRC protein or protein kinase C (PKC). This work aims to understand the involvement of ADAM9 protein during the process of carcinogenesis, such as tumor invasion induced by oxidative stress and neoangiogenesis

La famille des kinases Src, dont Lyn fait partie, joue un rôle clé dans le contrôle de nombreux processus biologiques. Lyn a une fonction bien établie dans les cellules hématopoïétiques et est notamment impliqué dans le maintien de différentes leucémies et son expression protéique est altérée dans les tumeurs solides. Plusieurs études ont mis en évidence qu’elle avait un rôle anti-apoptotique. Lyn peut être clivée par les caspases en donnant une protéine tronquée cytosolique. Nous avons ainsi montré que Lyn cytosolique (cLyn) régulait Bim, un membre pro-apoptotique de la famille Bcl-2, en le phosphorylant sur les tyrosines 92 et 161 en inhibant la fonction pro-apoptotique de Bim, en augmentant son interaction avec Bcl-XL, limitant ainsi la perméabilisation de la membrane externe mitochondriale et l’apoptose. Lyn possède également un rôle pro-inflammatoire. Nous avions préalablement montré que la surexpression de cLyn, chez la souris, conduit à un syndrome inflammatoire de la peau, ressemblant au psoriasis. Sur la base de ce résultat, nous avons voulu savoir si Lyn jouait un rôle dans cette maladie chronique de la peau. L’analyse de l’expression de Lyn chez des patients souffrant de psoriasis a montré que Lyn était surexprimée dans la peau lésionnelle par rapport à la peau non lésionnelle ou saine, résultats confirmés dans deux modèles de psoriasis chez la souris. De façon intéressante, nous avons montré que l’augmentation de l’expression de Lyn se situe à la fois dans le derme et dans l’épiderme chez l’homme et chez la souris, indiquant que le recrutement de cellules immunitaires dans la peau lésionnelle mais aussi la modulation de Lyn dans les kératinocytes sont impliqués. Par ailleurs, une augmentation de l’expression de Lyn a été observée dans les kératinocytes humains stimulés par le TNF-α et l'IL-17A. Afin de déterminer le rôle de Lyn dans cette maladie cutanée nous l’avons induit chez des souris déficientes pour Lyn. Une réduction significative du phénotype cutanée a été observée dans les souris LynKO, identifiant Lyn comme un nouvel acteur dans la pathogénie du psoriasis. De plus, nos résultats ont établi que l’expression de Lyn dans les kératinocytes semblait suffisante pour le maintien du phénotype psoriasique, indiquant un nouveau rôle de Lyn dans les kératinocytes. Au cours de ce travail, nous avions observé que les caspases inflammatoires étaient activées dans la peau lésionnelle de patients atteints du psoriasis. Les caspases inflammatoires, suite à leur activation, vont cliver l’IL-1β et l’IL-18, ce qui conduit à leur maturation. Nous avons alors voulu savoir si les caspases participaient au développement du psoriasis. Nous avons pu montrer que lorsque nous induisions du psoriasis chez des souris, l’invalidation des caspases inflammatoires ou son inhibition pharmacologique réduisait de façon significative le développement de la maladie. Bien que les cellules immunitaires et les kératinocytes soient capables de secréter de l’IL-1β via l’activation de l’inflammasome, nos données ont établi que seule l’activation des caspases inflammatoires dans le système immunitaire semblait nécessaire pour une réponse inflammatoire complète. En résumé, l’ensemble de mon travail de thèse a permis de montrer un mécanisme moléculaire par lequel la kinase Lyn régule négativement la voie apoptotique mitochondriale, ce qui peut contribuer à la transformation et/ou la résistance des cellules cancéreuses. D’autre part, nos résultats montrent que Lyn pourrait être un régulateur important du psoriasis, et notre étude indique que les caspases inflammatoires activées dans les cellules immunitaires sont impliquées dans la pathogenèse du psoriasis. A ce jour, bien que plusieurs traitements aient été développés pour le psoriasis, la maladie reste non résolue, donc le développement de cibles thérapeutiques contre Lyn et les caspases inflammatoires pourraient être intéressant pour le traitement de la maladie. / The Src family kinase, of which Lyn is one member, plays a key role in controlling many biological processes. Lyn has a well-established function in hematopoietic cells, presenting an important role in the regulation of hematopoietic abnormalities. In fact, Lyn plays a key role in maintaining several kind of leukemia, and furthermore it expression is altered in solid tumors. Different studies have shown that Lyn has an anti-apoptotic role. Lyn can be cleaved by caspases, cysteine proteases involved in apoptosis and inflammation, giving a new protein with a cytosolic location, different from the WT and membrane-anchored form. We have shown that the cytosolic form of Lyn (cLyn) regulated Bim, a pro-apoptotic member of the Bcl-2 family, involved in the control of mitochondrial apoptosis. We have identified that Bim is phosphorylated on tyrosine 92 and 161 by Lyn, resulting in an inhibition of its pro-apoptotic function, increasing its interaction with anti-apoptotic members such as Bcl-XL, thus limiting the permeabilization of mitochondrial outer membrane and impairing cell apoptosis. Lyn also has a pro-inflammatory role. We have previously shown that overexpression of the caspase-cleaved form of Lyn, in mice, leads to an inflammatory skin syndrome, resembling human psoriasis. Based on this result, we wanted to know if Lyn played a role in this chronic skin disease. Analysis of Lyn's expression in psoriasis patients showed that Lyn was overexpressed in lesional skin compared to non-lesional or healthy skin, which was subsequently confirmed in two mouse models of psoriasis disease. Interestingly, we have shown that the increase in Lyn expression is in both the dermis and the epidermis in humans and in mice, indicating that recruitment of immune cells into lesional skin but also the modulation of Lyn in keratinocytes are involved. To determine the role of Lyn in this skin disease we induced a psoriasis-like phenotype in Lyn deficient mice. A significant reduction in cutaneous phenotype was observed in LynKO mice compared to WT mice, identifying Lyn as a new player in the pathogenesis of psoriasis. In addition, our results established that Lyn expression in keratinocytes seemed crucial and sufficient for the maintenance of the psoriasis phenotype, indicating a new role of Lyn in the regulation of keratinocytes. During this work, we observed that inflammatory caspases were activated in lesional skin from psoriasis patients. Inflammatory caspases, following their activation within the inflammasome, will cleave IL-1β and IL-18, leading to their maturation. We then wanted to know if inflammatory caspases participated in the development of psoriasis. We were able to show that when we induced a psoriasis-like disease in mice, the invalidation of inflammatory caspases or its pharmacological inhibition significantly reduced the development of the disease compared to WT mice. Although immune cells and keratinocytes were able to secrete IL-1β via activation of the inflammasome, our data established that only activation of inflammatory caspases in the immune system seemed necessary for a complete inflammatory response. In summary, my thesis shows a molecular mechanism by which Lyn tyrosine kinase negatively regulates the mitochondrial apoptotic pathway, which may contribute to the transformation and/or chemotherapeutic resistance of cancer cells. On the other hand, our results show that Lyn could be an important regulator of psoriasis and our study indicates that inflammatory caspases activated in immune cells are involved in the pathogenesis of psoriasis. To date, although several treatments have been developed for psoriasis, the disease remains unresolved, so the development of therapeutic targets against Lyn and inflammatory caspases could be of interest for the treatment of the disease.

Lors du développement d’une tumeur au sein d’un tissu, les cellules cancéreuses se retrouvent entourées par les cellules saines. Les interactions entre ces deux types cellulaires, transformé et normal, jouent un rôle important dans le devenir de la tumeur, mais restent à ce jour mal comprises. L’objectif de cette thèse a été de mettre en place des systèmes in vitro qui permettent d’étudier les interactions entre une population de cellules normales et une population de cellules transformées.Nous avons tiré profit d’une lignée de cellules épithéliales sensibles à la lumière, élaborée par Olivier Destaing (IAB, Grenoble). Lorsqu’elles sont exposées à la lumière bleue, ces cellules suractivent la protéine Src, connue pour être surexprimée dans de nombreux cancers. Sinon, elles gardent un phénotype normal. L’utilisation de ces cellules, appelées « OptoSrc », combinée à un dispositif optique, permet de créer des tissus mosaïques dans lesquels le motif des cellules mutées est déterminé par le motif d’illumination bleue. Notre système présente plusieurs avantages : le contrôle dans le temps et dans l’espace du motif de cellules transformées, mais aussi l’activation graduelle et réversible de l’oncoprotéine.Nous avons montré qu’en illuminant dans le bleu un îlot circulaire de cellules au sein d’une monocouche OptoSrc, les cellules activées s’extrudent collectivement, donnant naissance à un agrégat tri-dimensionnel cohésif surplombant la monocouche. Nous pouvons contrôler la taille et le temps d’apparition de ce sphéroïde en ajustant respectivement l’aire éclairée et la fréquence d’illumination. De plus, ce phénomène d’extrusion collective est réversible lorsque le stimulus de lumière bleue s’arrête. Finalement, nous avons montré que la formation de cet agrégat s’accompagne d’une diminution des E-cadhérines à la membrane, et de l’apparition de la vimentine, pour les cellules éclairées. Nos résultats suggèrent qu'un groupe de cellules surexprimant la protéine Src, au sein d’une monocouche de cellules normales, subit une transition epithéliale- mesenchymateuse partielle. / During the development of a tumour in a tissue, the cancer cells are surrounded by healthy cells. The interactions between these two cell types, transformed and normal, play an important role in the tumour stability, but remain to this day poorly understood. The aim of this thesis was to establish in vitro assays to study the interactions between populations of normal and transformed cells.We benefited from a light-sensitive cell line, constructed by Olivier Destaing (IAB, Grenoble). When they are exposed to blue light, these cells overactivate the protein Src, which is known to be overexpressed in many cancers. Otherwise, they keep a normal phenotype. Using these cells, called “OptoSrc”, in combination with an optical setup, we are able to create mosaic tissues in which the pattern of mutated cells is determined by the blue illumination pattern. Our system has several advantages: a selective control in time and space of the group of transformed cells, and a gradual and reversible activation of the oncoprotein.We have shown that when we illuminate a circular islet of cells from a monolayer of OptoSrc cells, the activated cells were collectively extruded, resulting in a cohesive three-dimensional aggregate on top of the monolayer. We can control the size and appearance time of this spheroid by tuning, respectively, the area and frequency of illumination. Besides, this collective extrusion is reversible when the blue light stimulation is stopped. Finally, we have shown that the formation of this three-dimensional aggregate coincides with the loss of E-cadherin at the membrane, as well as the apparition of vimentin, for the illuminated OptoSrc cells. Our results suggest that a group of cells overexpressing the protein Src, in a monolayer of normal cells, undergoes a partial epithelial-to-mesenchymal transition.

The development of autoinflammatory diseases is caused by the dysregulation of innate immune mechanisms. This leads to the development of spontaneous inflammation. Mice lacking adaptor protein PSTPIP2 develop chronic autoinflammatory osteomyelitis due to higher activity of neutrophil granulocytes and their increased production of IL-1β. .β. PSTPIP2 interacts with PEST phosphatases and kinase CSK. These proteins are impor- tant negative regulators of Src family kinases. In this diploma thesis, the role of Src family kinases and the role of their positive regulator phosphatase CD45 in the development of chronic autoinflammatory osteomyelitis was studied. For this purpose, a mouse model of chronic autoinflammatory osteomyelitis (CMO) lacking CD45 was used. These mice deve- lop the disease with delayed kinetics. Bone marrow cells isolated from these mice produce less IL-1β. upon silica activation and have lower phosphorylation of ERK MAP kinase. It isβ. probably caused by higher phosphorylation of the inhibitory tyrosine of Src family kinases resulting in their lower activity. The presence of different immune cell populations in the bone marrow, spleen and blood of these mice was also monitored in these mice. The re- sults of this work contribute to a better understanding of the role of Src family...

8 1 ABSTRACT (EN) This thesis is focused mainly on understanding mechanisms of regulatory roles of C-terminal Src kinase (CSK) and phosphoprotein associated with glycosphingolipid- enriched microdomains (PAG) in the high-affinity IgE receptor (FcɛRI)-mediated signaling of murine mast cells. FcɛRI activation is initiated by aggregation of the receptor by complexes of multivalent antigen with IgE, followed by activation and enhanced activities of protein tyrosine kinases, phosphatases, adaptor proteins and number of other signal transduction molecules. The signaling events result in mast cell degranulation and release of variety of proinflammatory mediators, responsible for initiation of allergy and other inflammatory diseases. Understanding the function of key regulatory molecules controlling FcεRI-mediated mast cell activation, degranulation, and cytokines production could have therapeutic impact. CSK is a major negative regulator of Src family tyrosine kinases (SFKs) that play a critical role in various immunoreceptor signaling events. However, its function in mast cell activation has not been completely understood. Because of its cytoplasmic localization, CSK was assumed to be brought to the vicinity of the plasma membrane- bound SFKs via binding to membrane-bound adaptors and PAG was a major candidate....