Notre équipe a entrepris des travaux qui ont permis d’établir un lien causal entre la production autocrine de hGH et la stimulation des propriétés prolifératives, anti-apoptotiques, migratoires et invasives des cellules carcinomateuses mammaires humaines. Ces travaux permettent aujourd’hui de considérer l’action de la hGH autocrine comme celle d’un véritable oncogène. Mon travail de thèse s’est intéressé à comprendre les effets de la hGH autocrine dans la progression tumorale mammaire selon deux axes de recherche. Le premier étant de définir le rôle de la GH autocrine dans l’angiogenèse tumorale et le second de déterminer les interactions entre la voie de signalisation de la GH autocrine et la voie de signalisation Wnt. Nous avons démontré dans notre première étude que la production autocrine de hGH par les cellules carcinomateuses mammaires MCF7 favorise la prolifération, la survie, la migration et l’invasion des cellules micro-vasculaires endothéliales HMEC1, ainsi que leurs capacités à former des tubes in vitro. Ces effets cellulaires observés sont médiés par le GHR à exprimé sur les cellules endothéliales et sont la conséquence d’une augmentation d’expression du facteur pro-angiogénique VEGF-A. L’utilisation de l’antagoniste du VEGF-A, le bevacizumab, inhibe les effets vasculaires induits par la hGH autocrine. Ces effets retrouvés in vivo soulignent la pertinence de la production de GH autocrine par les cellules cancéreuses mammaires. Grâce à un modèle de xénogreffe de cellules carcinomateuses mammaires humaines nous avons montré que le développement soutenu de l’angiogenèse et de la lymphogenèse tumorales sont activés par la GH autocrine. La production de hGH autocrine par les cellules cancéreuses mammaires est donc un acteur de l’expansion du réseau vasculaire des cancers du sein. La voie de signalisation Wingless (Wnt) est un déterminant critique du contrôle cellulaire durant le développement mais également chez l’adulte. Nous avons démontré que la hGH autocrine stimule l’expression de différents acteurs de la voie Wnt et notamment la protéine sécrétée WNT4. Une surexpression de WNT4 est identifiée sur des biopsies de cancer du sein par rapport au sein normal. La production de WNT4 exerce un effet synergique avec la GH autocrine sur la stimulation de la prolifération des cellules carcinomateuses mammaires par un mécanisme dépendant de JAK2. Surexprimé dans des cellules épithéliales mammaires humaines, WNT4 stimule leurs propriétés prolifératives, anti-apoptotiques et migratoires et induit la formation de colonies en agar. Ces effets sont médiés par l’activation transcriptionnelle et traductionnelle de protéines clés du cycle et de la survie cellulaires. Des changements phénotypiques sont induits par la surexpression de WNT4 dans les cellules épithéliales mammaires et associés à l’augmentation d’expression de marqueurs mésenchymateux, d’acteurs du remodelage du cytosquelette et d’activateurs de la migration cellulaire. La progression tumorale mammaire humaine est donc induite par les effets autocrines de la hGH sur la production de WNT4 et la surexpression épithéliale mammaire de WNT4 permet l’établissement d’un phénotype pré-oncogénique / Studies of our group demonstrated that autocrine human growth hormone promotes proliferation, apoptotic resistance, invasion and migration of human mammary carcinoma cells. The objective of my Ph.D was to understand the role of autocrine hGH in mammary tumour progression following two aspects. On one hand, characterising its impact on tumour angiogenesis and then analysing the crosstalk between GH and Wnt signalling pathways. In the first part, we demonstrated that autocrine hGH from human mammary carcinoma cell line MCF- 7, stimulates proliferation, survival, migration and invasion of human microvascular endothelial cell line and enhances in vitro tubulogenesis. These effects are mediated through the hGHR and stimulates the expression of the proangiogenic factor VEGF-A. Antagonizing VEGF-A action with bevacizumab inhibited the proangiogenic actions of autocrine hGH in vitro. Using a xenograft nude mice model we have shown that sustained angiogenesis and lymphangiogenesis are activated by autocrine hGH production by mammary carcinoma cells. Thus, autocrine hGH participates in the expansion of the vascular network during breast cancer formation. The Wingless (Wnt) signalling pathway is an important actor during embryonic development and in the adult. Here, we showed that autocrine hGH stimulates the expression of several actors of the Wnt pathway, including WNT4. Human breast cancer biopsies overexpressed WNT4 as compared to normal breast. WNT4 production is JAK2 kinase-dependent and synergizes with autocrine hGH on mammary carcinoma cell proliferation. Forced expression of WNT4 in normal human mammary epithelial cell lines stimulates their proliferative, survival and migratory capacities and the formation of colonies in soft agar. These effects are mediated through the transcriptional and translational activation of key regulators of cell cycle and survival. Phenotypic changes are induced by forced expression of WNT4 in mammary epithelial cells and associated with increased expression of mesenchymal markers, cytoskeletal remodelling factors and cell migration activators. Human breast cancer progression is thus activated by autocrine effects of hGH on WNT4 expression and deregulated expression of WNT4 in mammary epithelial cells induces the establishment of a pre-tumorigenic phenotype

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No description available.

No presente trabalho, cultivou-se o microrganismo Escherichia coli RR1, contendo o vetor que carrega o gene estrutural para a síntese do hormônio de crescimento humano (hGH) (baseado no promotor pL e pR do fago l sob controle do repressor termosensível cI857) em processos descontínuo e descontínuo-alimentado realizados em biorreatores com capacidade útil de 2 e 4 L. Tal cepa é auxotrófica com relação aos aminoácidos l-leucina e l-prolina e à tiamina (vitamina B1). Nos cultivos descontínuos com concentrações menores de extrato de levedura e bactotriptona em relação ao meio denominado basal, a concentração celular foi baixa, atingindo 2,4 g.L-1, com fator de conversão glicose à células de 0,25 g.g-1. Em cultivos descontínuos com aumento (em relação ao meio basal) da concentração de extrato de levedura e de bactotriptona e com adição de l-prolina, a concentração celular alcançou valores da ordem de 5,9 g.L-1 e fator de conversão glicose à células de 0,48 g.g-1, simultaneamente à maior formação de acetato (2,5 g.L-1), este último prejudicial ao processo. Contudo, este resultado de crescimento celular não se repetiu devido a mudança do lote de células utilizado entre o primeiro e o segundo conjunto de ensaios. Os cultivos descontínuos-alimentados foram realizados com diferentes formas de alimentação bem como diferentes composições de solução de alimentação. Uma alimentação contínua com velocidade exponencial e composição semelhante à do meio, pareceu ser a mais favorável, levando à concentração celular final de 9,2 g.L-1 e fator de conversão glicose a células, na fase descontínua-alimentada, de 0,36 g.g-1. Os ensaios com indução térmica não foram eficientes provavelmente devido à problemas na detecção das concentração de glicose existente no instante inicial da ativação da síntese do hGH. Esta glicose presente pode ter prejudicado a formação do hGH por conseqüência do processo fermentativo causado pelo aumento da temperatura e pela presença de elevada concentração de nutrientes complexos. O meio de cultivo utilizado possivelmente não supriu as necessidades metabólicas da célula para a síntese do hormônio de crescimento humano e em nenhum dos cultivos com indução térmica houve a produção de hGH. / In the present work, the host Escherichia coli RR1, having the vector with the structural gene for human growth hormone (hGH) synthesis, based on pL or pR promoters from bacteriophage l under the control of the thermosensitive repressor cI857, was cultivated in batch and fed-batch cultures in bioreactors with working volumes of 2 and 4 L. This host has amino acids (l-leucine and l-proline) and thiamine (Vitamin B1) auxotrophy. Batch cultures under low yeast extract and bacto-tryptone concentrations (relative to the basal medium) resulted in a low biomass yield (2.4 g.L-1) and cell yield on glucose (0.25 g.g-1). Increasing these concentrations and adding l-proline to the medium led to higher biomass formation (5.9 g.L-1), cell yield on glucose (0.48 g.g-1) and acetate high levels (2.5 g.L-1), which were harmful to the process. However, these results of cellular growth were not reproducible due to different cell stocks applied. The fed-batch cultures were performed under different feeding strategies and different nutrients concentrations of the feeding solution. A continuous exponential feeding rate with growth medium-like composition seemed to be the most favorable, reaching final cellular concentration of 9.2 g.L-1 and yield on glucose on fed-batch mode of 0.36 g.g-1. The heat-shock runs were not efficient probably due to problems in detection of glucose concentration existing on initial instant of hGH activation synthesis. Glucose interferes with the hGH synthesis because the fermentation caused by temperature shift and presence of high complex nutrients concentration. The culture medium used, probably was not able to supply cell metabolic needs for the human growth hormone synthesis and in no other temperature-induced experiment the hGH production was observed.

No presente trabalho, cultivou-se o microrganismo Escherichia coli RR1, contendo o vetor que carrega o gene estrutural para a síntese do hormônio de crescimento humano (hGH) (baseado no promotor pL e pR do fago l sob controle do repressor termosensível cI857) em processos descontínuo e descontínuo-alimentado realizados em biorreatores com capacidade útil de 2 e 4 L. Tal cepa é auxotrófica com relação aos aminoácidos l-leucina e l-prolina e à tiamina (vitamina B1). Nos cultivos descontínuos com concentrações menores de extrato de levedura e bactotriptona em relação ao meio denominado basal, a concentração celular foi baixa, atingindo 2,4 g.L-1, com fator de conversão glicose à células de 0,25 g.g-1. Em cultivos descontínuos com aumento (em relação ao meio basal) da concentração de extrato de levedura e de bactotriptona e com adição de l-prolina, a concentração celular alcançou valores da ordem de 5,9 g.L-1 e fator de conversão glicose à células de 0,48 g.g-1, simultaneamente à maior formação de acetato (2,5 g.L-1), este último prejudicial ao processo. Contudo, este resultado de crescimento celular não se repetiu devido a mudança do lote de células utilizado entre o primeiro e o segundo conjunto de ensaios. Os cultivos descontínuos-alimentados foram realizados com diferentes formas de alimentação bem como diferentes composições de solução de alimentação. Uma alimentação contínua com velocidade exponencial e composição semelhante à do meio, pareceu ser a mais favorável, levando à concentração celular final de 9,2 g.L-1 e fator de conversão glicose a células, na fase descontínua-alimentada, de 0,36 g.g-1. Os ensaios com indução térmica não foram eficientes provavelmente devido à problemas na detecção das concentração de glicose existente no instante inicial da ativação da síntese do hGH. Esta glicose presente pode ter prejudicado a formação do hGH por conseqüência do processo fermentativo causado pelo aumento da temperatura e pela presença de elevada concentração de nutrientes complexos. O meio de cultivo utilizado possivelmente não supriu as necessidades metabólicas da célula para a síntese do hormônio de crescimento humano e em nenhum dos cultivos com indução térmica houve a produção de hGH. / In the present work, the host Escherichia coli RR1, having the vector with the structural gene for human growth hormone (hGH) synthesis, based on pL or pR promoters from bacteriophage l under the control of the thermosensitive repressor cI857, was cultivated in batch and fed-batch cultures in bioreactors with working volumes of 2 and 4 L. This host has amino acids (l-leucine and l-proline) and thiamine (Vitamin B1) auxotrophy. Batch cultures under low yeast extract and bacto-tryptone concentrations (relative to the basal medium) resulted in a low biomass yield (2.4 g.L-1) and cell yield on glucose (0.25 g.g-1). Increasing these concentrations and adding l-proline to the medium led to higher biomass formation (5.9 g.L-1), cell yield on glucose (0.48 g.g-1) and acetate high levels (2.5 g.L-1), which were harmful to the process. However, these results of cellular growth were not reproducible due to different cell stocks applied. The fed-batch cultures were performed under different feeding strategies and different nutrients concentrations of the feeding solution. A continuous exponential feeding rate with growth medium-like composition seemed to be the most favorable, reaching final cellular concentration of 9.2 g.L-1 and yield on glucose on fed-batch mode of 0.36 g.g-1. The heat-shock runs were not efficient probably due to problems in detection of glucose concentration existing on initial instant of hGH activation synthesis. Glucose interferes with the hGH synthesis because the fermentation caused by temperature shift and presence of high complex nutrients concentration. The culture medium used, probably was not able to supply cell metabolic needs for the human growth hormone synthesis and in no other temperature-induced experiment the hGH production was observed.

Baseball's Steroid Era put many different high-profile athletes under pressure to explain steroid allegations that were made against them. This thesis used textual analysis of news reports and media portrayals of the athletes, along with analysis of their image repair strategies to combat those allegations, to determine how successful the athletes were in changing public opinion as evidenced through the media. The contexts, media reports, and strategies of Jason Giambi, Mark McGwire, Andy Pettitte, and Roger Clemens were analyzed and revealed important implications involving effective use of image repair strategies. They provided a deeper framework for the success of mortification strategies. An authentic, sincere mortification strategy has more power to change the media's reporting and portrayal of the athlete, while stunted or incentivized mortification strategies provide diminishing results. The four different situations of the players and the different combinations of strategies used provide insight into how much a public persona matters in confronting allegations. They show how ineffective the strategy of minimization is against allegations that involve on-field performance. The situations reveal how the promise of future on-field actions, along with actual on-field success can help repair an athlete's image without a solid rhetorical strategy. They show the amount of information offered, along with the strategies used, influences the amount of persuasion that occurs. The different situations also showed how a complete image repair strategy is successful in ending news coverage of the allegations and not just changing the media portrayal.

Nous avons pour objectif de jeter les bases d’une technologie en totale rupture avec celles existantes pour la fabrication d’une nouvelle génération de composants électroniques à base du Carbure de Silicium pour les applications à très hautes températures (jusqu’à 600°C). Cette nouvelle technologie est basée sur l'emploi d'une nouvelle génération de matériaux pour les contacts ohmiques haute température. Nous avons ciblé la phase Ti3SiC2, qui est une phase céramique/métallique, pour former un bon contact ohmique stable et fiable à haute et très haute température. A savoir que l’aspect céramique est nécessaire pour assurer une bonne stabilité thermique à haute température, et l’aspect métallique est nécessaire pour obtenir des bonnes propriétés électriques (bonne conductivité électrique, faible résistance électrique…). Dans le but d’élaborer le Ti3SiC2 sur SiC, un film mince de 200 nm d’un alliage TixAl1-x a été déposé sur SiC-4H suivit d’un recuit sous Ar. Dans cette étude, on a fait varier la concentration du Ti et d’Al dans le dépôt métallique (Ti20Al80, Ti30Al70, Ti50Al50 et Ti), et on a aussi varié la température de recuit de 900°C à 1200°C. Des analyses structurales comme le DRX, MET, MEB et XPS ont été effectuées après recuit. Pour caractériser électriquement la couche Ti3SiC2 synthétisée sur SiC, des motifs TLM ont été réalisés. Des caractérisations électriques à température ambiante et à très haute température (jusqu’à 600°C) ont été mis en œuvre pour chaque type de dépôt et par conséquence la hauteur de barrière de potentielle a été également déterminée. Enfin, pour étudier la stabilité thermique du Ti3SiC2 sur SiC, des tests de vieillissement ont été réalisé à 600°C sous Ar. / The growth of Ti3SiC2thin films was studied onto 4H-SiC (0 0 0 1) 8◦and 4◦-off substrates by thermalannealing of TixAl1−x(0.5 ≤ x ≤ 1) layers. The annealing time was fixed at 10 min under Argon atmosphere.The synthesis conditions were also investigated according to the annealing temperature (900–1200◦C)after deposition. X-Ray Diffraction (XRD) and Transmission Electron Microscope (TEM) show that thelayer of Ti3SiC2is epitaxially grown on the 4H-SiC substrate. In addition the interface looks sharp andsmooth with evidence of interfacial ordering. Moreover, during the annealing procedure, the formationof unwanted aluminum oxide was detected by using X-Ray Photoelectron Spectroscopy (XPS); this layercan be removed by using a specific annealing procedure. Using TLM structures, the Specific Contact Resistance (SCR) at room temperature of all contacts was measured. The temperature dependence up to 600°C of the SCR of the best contacts was studied to understand the current mechanisms at the Ti3SiC2/SiC interface. Experimental results are in agreement with the thermionic field emission (TFE) theory. With this model, the barrier height of the contact varies between 0.71 to 0.85 eV.

La croissance post natale est dépendante des effets physiologiques de l'hormone de croissance ou GH («Growth Hormone») qui se traduisent au niveau cellulaire par une augmentation de la prolifération, de la différenciation et du métabolisme. La libération pulsatile de GH hypophysaire induit une réponse cellulaire adaptée et régulée en fonction des différentes situations physiologiques et conditions homéostatiques. La transduction du signal de la GH est médiée par l'activation de la voie canonique de transduction des signaux des cytokines, Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT). La GH est également produite en des sites extrahypophysaires par les cellules endothéliales, fibroblastiques et épithéliales suivant un mode de régulation bien distinct de celui de l'hypophyse. Cette GH ectopique synthétisée en faible quantité par rapport à la production hypophysaire ne contribue pas à l'élévation de la concentration de GH circulante et agirait comme un facteur local de régulation exerçant ses effets de façon autocrine et paracrine. L'objectif principal exposé dans ce mémoire est de comprendre le rôle physiologique et les conséquences pathologiques de la synthèse de GH autocrine. Cette problématique a été abordée depuis la cellule jusqu'à l'animal entier et expérimentalement testée par à un ensemble de techniques de biologie cellulaire, moléculaire et biochimiques. Nous avons dans un premier temps identifié certains sites de production ectopique de GH tels que les organes du système immunitaire et la glande mammaire et démontré que son expression était régulée en fonction de l'ontogenèse ou de l'état physiologique des animaux. Dans la glande mammaire de souris la GH autocrine participe à son développement en activant les mécanismes de prolifération épithéliale et en s'opposant à la différenciation lactogène. Nous avons également mis en évidence l'expression de GH humaine (hGH) dans les cellules épithéliales et fibroblastiques du sein humain et montré qu'elle était augmentée dans les cas de cancers agressifs. L'élaboration d'un modèle cellulaire de production autocrine de hGH nous a permis de caractériser en détail son rôle dans les cellules carcinomateuses mammaires. La GH induit une augmentation significative du nombre de cellules carcinomateuses par stimulation des mécanismes moléculaires de la prolifération cellulaire et par activation des mécanismes anti-apoptotiques. La production autocrine de hGH induit également une remarquable augmentation des capacités migratoires et invasives des cellules associées à un changement morphologique en type fibroblastique conjointement à l'acquisition des marqueurs de la transition épithélio-mésenchymateuse. Nous avons par la suite démontré que les mécanismes moléculaires de la progression tumorale mammaire induite par la GH autocrine résultent de l'activation d'un programme transcriptionnel complexe médié par l'action de protéines spécifiques (CHOP, HOXA1, Cycline D1, c-Myc, Bcl-2, Catalase). Cette plateforme transcriptionnelle permet l'activation soutenue de la voie des MAPK ainsi que la stimulation des fonctions antiapoptotiques, antiradicalaires et de chimiorésistance des cellules carcinomateuses. Par la suite nous avons découvert que l'expression constante et forcée de hGH dans une lignée épithéliale mammaire humaine est suffisante pour induire leur transformation oncogénique in vitro et in vivo, suggérant que la GH pourrait exercer la fonction d'un oncogène impliqué dans les cancers du sein humains. L'ensemble de nos travaux indique que les effets autocrines et paracrines de la GH se traduisent en situation physiologique par le développement prolifératif de l'épithélium mammaire et en conditions pathologiques stimulent la progression tumorale. L'expression constante de hGH par les cellules épithéliales mammaires humaines est potentiellement responsable de leur transformation oncogénique. L'antagonisme de la voie de transduction du signal de la GH représente donc un nouveau niveau d'intervention thérapeutique.

<p>The main aim of the thesis was to explore the potential of supercritical fluid (SF) techniques in the field of drug delivery. In particular, the relatively recently developed solution-enhanced dispersion by supercritical fluids (SEDS) technology has been employed in the preparation of particles/powders. </p><p>The manufacturing, stability and bioavailability of a dosage form strongly depend on the physicochemical properties of the formulation particles. For example, dry powder inhalation (DPI) for administering drugs to the respiratory tract require particles in a narrow size range (1-5 μm) to be effective. The identification of polymorphs and control of purity are also important issues since the physicochemical properties and therapeutic effects of the alternative forms of a drug may differ substantially. Solvent-based traditional crystallisation processes provide the product that may require further down-stream processing to obtain particles for advanced drug delivery applications. This can result in unwanted changes in the physicochemical properties of the particles and thus affect the performance of the dosage form. SF processing has addressed many of the challenges in particle formation research. Among several SF technologies developed for particle processing over the last decade, the SEDS process with its specially designed co-axial nozzle with mixing chamber has resulted in improved control over the particle formation process. Carbon dioxide (CO₂) was used as the SF, because it has low critical points and is non-toxic, non-flammable and relatively inexpensive. </p><p>The initial part of the thesis concerns the formation of particles of model drugs such as hydrocortisone, budesonide and flunisolide using SEDS technology and the determination of the influence of processing conditions and solvents on particle characteristics such as size, shape and crystal structure. Particles of model drugs of differing shapes in a size range suitable for inhalation delivery were prepared. In the process, two new polymorphic forms of flunisolide were identified. This was the first report of SEDS technology being shown as a polymorph-screening tool. The remainder of the thesis deals with the development of SEDS technology for precipitating therapeutic proteins such as recombinant human growth hormone (hGH) from aqueous solutions. Powders of hGH were precipitated using SEDS without significant changes in the chemical or physical stability of the protein. The addition of sucrose to hGH in the feed solution promoted precipitation and minimised the detrimental effects of the solvent and/or the process on the physical aggregation of the protein. </p><p>In conclusion, this thesis highlights the applicability of the SEDS process in drug delivery research and advances general understanding of the particle formation phenomenon. The SEDS process may also prove to be a potential alternative technology for the precipitation of stable powders of therapeutic proteins.</p>

The main aim of the thesis was to explore the potential of supercritical fluid (SF) techniques in the field of drug delivery. In particular, the relatively recently developed solution-enhanced dispersion by supercritical fluids (SEDS) technology has been employed in the preparation of particles/powders. The manufacturing, stability and bioavailability of a dosage form strongly depend on the physicochemical properties of the formulation particles. For example, dry powder inhalation (DPI) for administering drugs to the respiratory tract require particles in a narrow size range (1-5 μm) to be effective. The identification of polymorphs and control of purity are also important issues since the physicochemical properties and therapeutic effects of the alternative forms of a drug may differ substantially. Solvent-based traditional crystallisation processes provide the product that may require further down-stream processing to obtain particles for advanced drug delivery applications. This can result in unwanted changes in the physicochemical properties of the particles and thus affect the performance of the dosage form. SF processing has addressed many of the challenges in particle formation research. Among several SF technologies developed for particle processing over the last decade, the SEDS process with its specially designed co-axial nozzle with mixing chamber has resulted in improved control over the particle formation process. Carbon dioxide (CO2) was used as the SF, because it has low critical points and is non-toxic, non-flammable and relatively inexpensive. The initial part of the thesis concerns the formation of particles of model drugs such as hydrocortisone, budesonide and flunisolide using SEDS technology and the determination of the influence of processing conditions and solvents on particle characteristics such as size, shape and crystal structure. Particles of model drugs of differing shapes in a size range suitable for inhalation delivery were prepared. In the process, two new polymorphic forms of flunisolide were identified. This was the first report of SEDS technology being shown as a polymorph-screening tool. The remainder of the thesis deals with the development of SEDS technology for precipitating therapeutic proteins such as recombinant human growth hormone (hGH) from aqueous solutions. Powders of hGH were precipitated using SEDS without significant changes in the chemical or physical stability of the protein. The addition of sucrose to hGH in the feed solution promoted precipitation and minimised the detrimental effects of the solvent and/or the process on the physical aggregation of the protein. In conclusion, this thesis highlights the applicability of the SEDS process in drug delivery research and advances general understanding of the particle formation phenomenon. The SEDS process may also prove to be a potential alternative technology for the precipitation of stable powders of therapeutic proteins.