L'augmentation de l'incidence et de la mortalité du cancer colorectal nécessitent la découverte de nouvelles méthodes de prévention et de traitement. Les chalcones ont été identifiées comme des composés intéressants ayant des propriétés chimiopreventives et antitumorales. Dans cette étude, nous avons étudié les effets de la 3-hydroxy-3', 4, 4', 5'-tétra-méthoxy-chalcone (3-HTMC), une chalcone synthétisée par notre laboratoire, sur la prolifération, la distribution du cycle cellulaire, l'apoptose et son mécanisme d'action sur les cellules cancéreuses colorectales humaines HT-29 (COX-2 sauvage) et HCT116 (déficientes en COX-2). Nous avons montré que la 3-HTMC diminue la viabilité cellulaire d'une façon dose dépendante avec un effet antiprolifératif plus puissant sur les cellules HCT116 que les cellules HT-29. L'analyse par cytométrie en flux a révélé une accumulation des cellules en phase G2/M du cycle cellulaire pour les cellules HT-29 et un arrêt significatif en G2/M pour les cellules HCT116 avec une apparition du pic sub-G1 caractéristique de l'apoptose. Nous avons démontré que le traitement des deux lignées cellulaires par la 3-HTMC induit un processus apoptotique associé à la surexpression du récepteur de mort DR5, l'activation des caspase-8 et -3, le clivage de la PARP et la fragmentation de l'ADN. De plus, la 3-HTMC induit l'activation des voies de survie PI3K/Akt et MEK/ERK qui sont impliquées dans la résistance à l'apoptose. De même, la 3-HTMC induit une surexpression de la COX-2 dans les cellules HT-29 qui contribue aussi à la résistance à l'apoptose, ceci expliquant la différence de sensibilité entre les cellules HT-29 et HCT116 traitées par la 3-HTMC. Donc, malgré l'activation des voies de résistance à l'apoptose, la 3-HTMC peut être un agent chimiopréventif ou thérapeutique prometteur pour le traitement du cancer colorectal. / Increasing incidence and mortality of colorectal cancer brings the necessity to uncover new possibilities in its prevention and treatment. Chalcones have been identified as interesting compounds having chemopreventive and antitumor properties. In this study, we investigated the effects of the synthetic chalcone derivative 3-hydroxy-3',4,4',5'-tetra-methoxy-chalcone (3-HTMC) on proliferation, cell cycle distribution, apoptosis and its mechanism of action in human colorectal HT-29 (COX-2 sufficient) and HCT116 (COX-2 deficient) cancer cells. We showed that 3-HTMC decreased cell viability in a dose-dependent manner with a more potent antiproliferative effect on HCT116 than HT-29 cells. Flow cytometric analysis revealed G2/M cell cycle accumulation in HT-29 cells and significant G2/M arrest in HCT-116 cells with a subsequent apoptosis shown by appearance of Sub-G1 peak. We demonstrated that 3-HTMC treatment on both cell lines induced apoptotic process associated with overexpression of death receptor DR5, activation of caspase-8 and -3, PARP cleavage and DNA fragmentation. In addition, 3-HTMC induced activation of PI3K/Akt and MEK/ERK principal survival pathways which delay 3-HTMC-induced apoptosis in both cell lines. Furthermore, COX-2 overexpression in HT-29 cells contributes to apoptosis resistance which explains the difference of sensitivity between HT-29 and HCT116 cells to 3-HTMC treatment. Even if resistance mechanisms to apoptosis reduced chalcone antitumoral potential, our results suggest that 3-HTMC may be considered as an interesting compound for colorectal cancer therapy or chemoprevention.

Les intégrines sont des protéines transmembranaires, formées d’une sous-unité α et d’une sous-unité β, qui sont impliquées dans de nombreuses caractéristiques du cancer. Mes travaux de thèses ont montré que l’axe intégrine α5β1/AKT est impliqué dans la résistance à l’apoptose des cellules de glioblastome et dans leur migration. L’inhibition fonctionnelle de l’intégrine α5β1 associée à la réactivation de p53 sensibilise les cellules de glioblastome à l’apoptose. Mes résultats ont mis en évidence que les protéines anti-apoptotiques PEA-15 et Survivine sont deux intervenants majeurs dans cette résistance à l’apoptose. La migration dépendante de l’intégrine α5β1 implique la β-caténine. Les résultats obtenus par ce travail de thèse, donnent, par conséquent, des arguments supplémentaires en faveur de l’implication de l’intégrine α5β1 dans l’agressivité des glioblastomes. Ces résultats permettent également de proposer de nouvelles cibles thérapeutiques ainsi que des biomarqueurs pertinents pour stratifier les patients potentiellement répondeurs à une stratégie anti-intégrine. / Integrins are αβ membrane localised protein heterodimers involved in numerous hallmarks of cancer. My PhD thesis showed that the integrin α5β1/AKT axis is implicated in glioblastoma cells resistance to apoptosis and migration. The inhibition of α5β1 integrin oncogenic pathway sensitizes glioma cells to p53-reactivation dependent apoptotic cues. My results showed that PEA-15 and Survivin are two anti-apoptotic proteins involved in the resistance to apoptosis. α5β1 integrin dependent migration involves the β-catenin pathway. My results confirm that α5β1 integrin has to be considered as an important player in glioblastoma aggressiveness and resistance to therapy. The results of my thesis also suggest new therapeutic targets and pertinent biomarkers for glioblastoma patient stratification.

Les accidents vasculaires cérébraux (AVC) sont considérés comme la pathologie neurologique la plus sévère en termes de mortalité et d’infirmité. Ils touchent plus de 140 000 personnes chaque année. L’AVC ischémique, qui représente 80% des AVC, est causé par l’occlusion localisée d’un vaisseau conduisant à un arrêt de l’apport en oxygène et en glucose au cerveau. Il est ainsi responsable de déficits moteurs, sensitifs et cognitifs qui peuvent gravement compromettre l’autonomie et la qualité de vie des patients. Les patients qui ont subi un AVC ischémique développent notamment une atrophie musculaire qui se produit principalement dans le membre parétique, mais aussi dans une moindre mesure dans le membre non parétique. Toutefois, les mécanismes moléculaires à l’origine de cette atrophie musculaire sont méconnus. Dans une première étude, l’objectif a été d’identifier les déterminants moléculaires mis en jeu dans l’atrophie musculaire induite par une ischémie cérébrale. Pour répondre à cet objectif, les travaux ont été menés sur un modèle d'ischémie cérébrale chez la souris qui consiste en l’occlusion de l'artère cérébrale moyenne par un monofilament en nylon. Nous avons montré que l’ischémie cérébrale entraînait, 3 jours après son induction, une atrophie musculaire des muscles quadriceps, soleus et tibialis anterior du côté parétique. Cette atrophie musculaire était associée à des déficits moteurs touchant l’équilibre, la coordination, la force musculaire, la posture ou la marche. Au niveau moléculaire, nous avons reporté un déséquilibre de la balance entre la synthèse et la dégradation des protéines musculaires en faveur d’une augmentation de la dégradation dans les muscles parétique et non parétique des souris ischémiées. Nous avons notamment montré que l’expression de la myostatine, un régulateur négatif majeur de la masse musculaire, était significativement augmentée. Dans une seconde étude, l’objectif a été d’identifier une cible d’intervention thérapeutique pour préserver la masse musculaire suite à une ischémie cérébrale. Au vu des résultats obtenus dans la première étude, nous avons ciblé la myostatine. Nous avons montré que l’inhibition de la myostatine entraînait, une meilleure récupération du poids de corps et du poids de divers muscles, 15 jours après une ischémie cérébrale. De plus, l’inhibition de la myostatine tendait à améliorer le comportement moteur des souris ischémiées (équilibre, coordination, force musculaire). En revanche, nous n’avons reporté aucune variation majeure des niveaux en ARNm ou protéines d’acteurs impliqués dans les voies de signalisation Akt/mTOR, Smad2/3, ubiquitine-protéasome et autophagie-lysosome, 15 jours après une ischémie cérébrale. Ces données préliminaires suggèrent que l’inhibition pharmacologique de la myostatine pourrait représenter une stratégie thérapeutique efficace pour limiter la perte de masse musculaire suite à une ischémie cérébrale / Strokes are considered as the most severe neurological disease in terms of mortality and disability. The incidence of stroke in France is estimated at 140 000. Ischemic stroke, which represents about 80% of strokes occur as a result of an obstruction of a blood vessel supplying blood to the brain. Motor, cognitive and sensory deficits are common impacts of stroke and can seriously compromise the autonomy and patient quality of life. Ischemic stroke leads to muscle atrophy, wich occurs primarily in the paretic limb, but also to a lesser extent in the nonparetic limb. However, the molecular mechanisms of muscle atrophy is unknown. In a first study, the purpose was to identify the molecular determinants involved in skeletal muscle atrophy following cerebral ischemia. To meet this objective, the work was carried out on a mouse model of cerebral ischemia, which involves the occlusion of the middle cerebral artery (MCAO) with a nylon monofilament. We have shown that cerebral ischemia leads to skeletal muscle atrophy of quadriceps, soleus and tibialis anterior muscles of the paretic side, 3 days after MCAO. This muscular atrophy was associated with motor deficits in the balance, coordination, muscle strength, posture and walking. From a molecular point of view, we reported an imbalance between the rates of synthesis and degradation of muscle protein, in favour of protein degradation in both paretic and nonparetic muscles. In particular, we showed that the expression of myostatin, a master negative regulator of skeletal muscle mass was significantly increased. In a second study, the purpose was to identify a target for therapeutic intervention in order to maintain muscle mass following cerebral ischemia. In view of the results obtained in the first study, we targeted the myostatin. Our results show that myostatin inhibition increases body weight and muscle mass recovery, 15 days after cerebral ischemia. In addition, myostatin inhibition tends to improve motor behavior (balance, coordination, strength). From a molecular point of view, we reported no major change in mRNA or protein level of actors involved in Akt/mTOR, Smad2/3, autophagy-lysosome and ubiquitin-proteasome pathways, involved in the control of muscle mass, 15 days after cerebral ischemia. These preliminary results strongly suggest that pharmacological inhibitors of myostatin may provide significant therapeutic benefit for muscle atrophy following cerebral ischemia

Ce travail de thèse avait pour objectif d’étudier la régulation de l’expression des gènes du métabolisme intermédiaire par les acides aminés alimentaires dans le foie de truite arc-en-ciel. Ces études ont permis de caractériser, pour la première fois dans le foie de truite, les principaux acteurs de la voie de signalisation Akt/TOR et leurs régulations. Nos résultats in vitro montrent qu’un mélange d’acides aminés, seul ou avec l’insuline, est capable de réguler l’expression de nombreux gènes impliqués dans la lipogenèse, la néoglucogenèse et la glycolyse. Les régulations observées en présence conjointe d’un mélange d’acides aminés et d’insuline semblent être, pour la plupart, dépendantes de la voie TOR. Par la suite, nous avons étudié l’effet de certains acides aminés comme la leucine (connue pour son effet « signal ») ainsi que la lysine et la méthionine (souvent ajoutées dans les aliments piscicoles riches en matières premières végétales afin d’atteindre l’équilibre en acides aminés). En présence d’insuline, la leucine, contrairement à la lysine et la méthionine, active la voie de signalisation TOR et régule l’expression de certains gènes (néoglucogenèse et lipogenèse) de façon similaire à un mélange d’acides aminés. Parallèlement, in vivo, nous avons étudié la régulation de l’expression des gènes du métabolisme intermédiaire lors d’un remplacement partiel ou total des huiles et farines de poisson par des produits végétaux dans l’aliment piscicole. Cette expérimentation a montré que, ni les voies de signalisation Akt/TOR, ni l’expression des gènes cibles ne sont affectés par ces nouveaux aliments. En conclusion, ces travaux ont montré que les acides aminés semblent jouer un rôle important dans la régulation de l’expression des gènes hépatiques du métabolisme intermédiaire chez la truite arc-en-ciel. / The objective of my PhD was to characterize the regulation of the intermediary metabolism related gene expression by dietary amino acids in the liver of rainbow trout. This work allowed us to characterize, for the first time in the liver of trout, the main proteins of the Akt/TOR signalling pathway and their regulations. In vitro results showed that a mixture of amino acids, in the presence or absence of insulin, is able to regulate the expression of numerous genes involved in lipolysis, gluconeogenesis and glycolysis. Such regulations induced by an amino acid mix together with insulin appear to be, at least partly, TOR-dependent. Afterwards, I studied the effect of specific amino acids known to be a signalling molecule (leucine) or having potential application as supplements to reach essential amino acid balance in plant ingredients-rich diet (lysine and methionine). In the presence of insulin, leucine, in contrast to lysine and methionine, is able to activate the TOR signalling pathway and regulate the expression of several genes involved in gluconeogenesis and lipogenesis in the same way as a mixture of amino acids. Furthermore, we studied in vivo, the effect of partial or total replacement of fish oil and fish meal by plant products in fish feed on .the regulation of intermediary metabolism related gene expression. This study showed that neither Akt/TOR signalling pathway nor the expression of the target genes were affected by such diets. In conclusion, these studies showed that amino acids seem to play an important role in the hepatic regulation of intermediary metabolism gene expression in the rainbow trout.

1. Adénomes hypophysaires somatotropes : Elaboration d'un modèle in vitro et in vivo d'études du récepteur SST2 et des voies de signalisation somatostatinergiques.Afin de préciser l'étude du récepteur SST2, nous avons mis en place un modèle d'étude in vitro et in vivo, en établissant une lignée polyclonale exprimant le récepteur SST2 humain à de hauts niveaux. Ce modèle nous permettra de tester de nouveaux agonistes somatostatinergiques comme le pasiréotide et d'étudier de façon approfondie les voies de signalisation.2. Méningiomes : Mise en place d'un modèle de culture primaire de méningiome in vitro et étude de l'effet de l'octréotide, du pasiréotide et de l'everolimus sur la prolifération cellulaire et les voies de signalisation intracellulaire.Etant donné la forte expression du récepteur SST2, nous avons démontré in vitro un effet anti-prolifératif de l'octréotide sur les cellules de méningiomes via une inhibition de la voie Pi3kinase-Akt-mTOR. L'octréotide a significativement amélioré l'inhibition par l'everolimus de la prolifération cellulaire.Un effet additif des 2 drogues a été observé. Ces résultats ont permis la mise en place d'une étude clinique . Par ailleurs, on observe un meilleur effet du pasiréotide comparé à celui de l'octréotide.3. Chordomes : Mise au point d'un modèle de culture primaire de cellules de chordomes in vitro et études préliminaires.Les premiers résultats avec octréotide, pasiréotide et everolimus sont concluants, mettant en évidence une diminution de la prolifération cellulaire, et nous incitent à poursuivre les investigations. / 1.Somatotroph adenomas : Development of an in vitro and in vivo model to study SST2 receptor and somatostatin pathway.To precise SST2 role in tumorigenesis, we developed an in vitro and in vivo model, using a polyclonal cell line with high and stable SST2 expression level. This model will provide to test new somatostatin agonists as pasireotide and further studies on intracellular pathway in ''somatotroph'' context.2. Meningiomas : Development of a model of meningioma primary culture in vitro with study of octreotide, pasireoide and everolimus effects on cell proliferation and intracellular pathways. Given the strong SST2 expression, we demonstrated an in vitro antiproliferative effect of octreotide on meningioma cells via an inhibition of Pi3kinase-Akt-mTOR pathway.In vitro, we observed that octreotide significantly improved everolimus induced cell proliferation inhibition. An additive effect of the 2 drugs was observed in each tested tumor. These results supported the development of a clinical trial. Pasireotide provided a better effect than octreotide, alone or in combination with everolimus on cell proliferation and intracellular pathways.3. Chordomas : Development of an in vitro model of chordoma cell primary culture with preliminary studies.We developed a model of in vitro chordoma cell culture. This model is reliable and stable, providing study of different drugs. SST2 receptor expression was lower than in meningiomas but SST2 expression remained significant in the majority of the tumors. First results with octreotide, pasireotide and everolimus are relevant, with a decrease in cell proliferation leading to further studies.

Le cancer de la vessie est le deuxième cancer urologique après le cancer de la prostate. Le développement de nouveaux agents anticancéreux est nécessaire pour le traitement de cette maladie complexe, car, en dépit d'un large éventail de traitements, aucune augmentation de la survie des patients n'a été observée. - Les effets du GW501516, un agoniste de PPARbeta/delta, n'ont jamais été explorés dans des cellules cancéreuses de la vessie. Nous montrons qu'un traitement par 25 µM de GW501516, pendant 24 h, induit la mort cellulaire par apoptose de cellules dérivées d'un carcinome urothélial invasif mais pas de cellules dérivées d'un cancer urothélial "de bon pronostic". Une stimulation de courte durée par 25 µM de GW501516 induit une augmentation du taux d'espèces réactives de l'oxygène (ROS) qui est associée à une phosphorylation de la protéine de survie Akt. L'apoptose induite par le GW501516 est concomitante à une diminution de l'expression d'une molécule d'adhérence, la cadhérine N qui est une protéine transmembranaire impliquée dans la progression tumorale. Pour la première fois dans un modèle de carcinome, nous montrons que la cadhérine N est la cible d'un clivage protéolytique, libérant des fragments solubles qui peuvent être biologiquement actifs. Le GW501516 module ce clivage à des temps qui précèdent la mort cellulaire. En effet, le GW501516 régule l'expression de protéases telles que ADAMIO à l'origine du clivage de la cadhérine N. - Les fragments de la cadhérine N pourraient intervenir dans les processus de survie ou de mort cellulaires. Par conséquent, cette étude novatrice ouvre le débat quant à la portée thérapeutique du GW501516 dans le traitement du cancer. / Bladder cancer is the most common cancer of the genitourinary tract worldwide and accounts for 5% of ail cancer deaths in human. Despite of a wide range of treatments, no substantial improvement in survival of patients with advanced-stage or metastatic disease has been achieved. The development of nove! effective chemotherapeutic agents is required for the treatment ofthis aggressive disease. Anticancer action of GW501516, an agonist of PPARbeta/delta, has never been investigated in bladder tumor cells. Our results indicated that a 24h treatment by 25 µM of GW501516 induced cell death through both extrinsic and intrinsic apoptotic pathways in T24 cells, derived from an undifferentiated high grade carcinoma but not in RT4 cells, derived from a low grade papillary tumor. GW501516 induced also an early up-regulation of ROS production which was associated with an increase ofphosphorylated Akt level, implicated in cell survival. Apoptosis induced by GW501516 is concomitant with a decrease of N-cadherin expression which is a cell adhesion molecule implicated in tumor progression. For the first time in carcinoma mode!, we showed that N-cadherin was cleaved by proteases, leading to the release of soluble fragments which could be biologically active. Our results indicated that GW501516 modulates N-cadherin cleavage before cell death through the regulation of protease expression such as ADAM l O. N-cadherin fragments could be implicated in cell survival or cell death. By inducing apoptosis of urothelial cancer ce lis and by modulating N-cadherin shedding, GW501516 could be a potential efficient chemotherapeutic drug for bladder cancer through endovesical instillations.

Orientador: Morgana Rodrigues Guimarães Stabili / Resumo: Células dendríticas têm papel crucial na patogênese das doenças periodontais por orquestrarem a resposta imune adaptativa e por seu papel como precursoras de osteoclastos. A sinalização via Akt tem importante papel em processos como metabolismo, proliferação, apoptose e também na resposta imune. Evidências indicam que Akt1 tem papel de regulador endógeno negativo da resposta inflamatória; porém pode tanto estimular quanto inibir a osteoclastogênese. Considerando que as células dendríticas participam tanto da inflamação/resposta imune quanto do turnover do tecido ósseo como células precursoras de osteoclastos, propusemos avaliar através de um estudo in vivo o papel da atividade de Akt1 na inflamação associada a interações microbiota-hospedeiro, bem como investigar in vitro os efeitos desta via de sinalização sobre os diferentes eventos biológicos das células dendríticas. Para o estudo in vivo foi utilizado um modelo de doença periodontal experimental induzida por P. gingivalis e Fusobacterium nucleatum, em um modelo de animais transgênicos com deleção gênica condicional. Os desfechos avaliados foram: inflamação (morfometria), reabsorção óssea (μCT), osteoclastogênese (IHC), anticorpos específicos para P.gingivalis e Fusobacterium nucleatum (ELISA). No estudo in vitro foi avaliado o papel da via de sinalização Akt1 sobre as seguintes atividades das células dendríticas: proliferação, apoptose, atividade fagocitária, migração, apresentação de antígeno e osteoclastogênese. Resulta... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Dendritic cells play a crucial role in the pathogenesis of periodontal diseases by orchestrating the adaptive immune response and their role as osteoclasts precursors. Akt signaling plays an important role in processes such as metabolism, proliferation, apoptosis and also in the immune response. Evidence indicates that Akt1 plays a role of negative endogenous regulator of the inflammatory response; but can both stimulate and inhibit osteoclastogenesis. Considering that dendritic cells participate in both inflammation/immune response and turnover of bone tissue as osteoclast precursor cells, we have proposed to evaluate in a vivo study the role of Akt1 activity in inflammation associated with microbiota-host interactions, as well as to evaluate in vitro the role of the Akt1 signaling pathway in dendritic cell biology. In vivo study was employed a model of experimental periodontal disease induced by P. gingivalis and Fusobacterium nucleatum in transgenic animals model with conditional gene deletion. The outcomes evaluated were: inflammation (morphometry), bone resorption (μCT), osteoclastogenesis (IHC), antibodies specific for P.gingivalis and Fusobacterium nucleatum (ELISA). In vitro study was evaluated the role of Akt1 signaling pathway on the following outcomes related to dendritic cell biology: proliferation, apoptosis, phagocytic activity, migration, antigen presentation and osteoclastogenesis The results showed that signaling via Akt1 in dendritic cells seems to play an i... (Complete abstract click electronic access below) / Doutor

Orientador: Luis Otavio Zanatta Sarian / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-28T02:45:23Z (GMT). No. of bitstreams: 1 Baptista_MauricioZuccolotto_D.pdf: 4237946 bytes, checksum: b677fee05d5d510e473e6b4c4c856a53 (MD5) Previous issue date: 2015 / Resumo: Introdução: A via de sinalização intracelular PTEN, mTOR, PI3K, IGF-1R e EGFR exerce papel prognóstico importante no câncer de mama. Embora muitos estudos tenham analisado a correlação entre as alterações gênicas de PTEN, mTOR, PI3K, IGF-1R e EGFR e o mau prognóstico em câncer de mama, há uma lacuna na literatura com relação ao valor prognóstico dessas proteínas na adjuvância do câncer de mama. Apesar de existirem fatores prognósticos e preditivos conhecidos, como os receptores hormonais e o HER-2, no campo imunológico, o câncer de mama é um dos tumores menos imunogênicos. As proteínas PD-L1 e PD-L2 fazem parte de uma importante resposta imune antitumoral. Em câncer de mama, o valor prognóstico de PD-L1 e PD-L2 ainda não está definido. Objetivos: Investigar a expressão das proteínas PTEN, mTOR, PI3K, IGF-1R e EGFR e das proteínas PD-L1 e PD-L2, e a correlação com as características clínicas e patológicas do câncer de mama, sobrevida livre de doença e sobrevida global. Métodos: Foi realizada uma coorte que avaliou 192 casos de câncer de mama, estadios I, II e III, tratadas entre 1994 e 2014 no Hospital da Mulher (CAISM) da UNICAMP. Os dados clínicos e de sobrevida foram retirados de prontuários. Blocos de parafina foram utilizados para construção do microarranjo de tecidos (TMA). No TMA foi utilizada a técnica de imunohistoquímica (IHQ) para estudo da expressão dessas proteínas. A terapia adjuvante foi administrada de acordo com o protocolo de tratamento institucional. Resultados: A expressão de PTEN foi encontrada em 40.6% (77/190); mTOR em 47.4% (90/190); PI3K em 29.8% (57/191); IGF-1R em 35.8% (68/190) e EGFR em 25.7% (49/191). Nas células do câncer de mama, a expressão de PTEN ocorreu no citoplasma e na região nuclear; a expressão de mTOR foi constatada de modo intenso na região nuclear e também no citoplasma. A expressão de PI3K foi mais intensa na membrana celular e menos intensa no citoplasma. A expressão de IGF-1R foi detectada na membrana celular das células tumorais. A expressão de todas essas proteínas foi significativamente associada à presença de linfonodos positivos. A idade mais jovem ao diagnóstico foi associada à expressão de PTEN e PI3K. A presença de tumores maiores foi associada à expressão de PTEN. Os receptores de progesterona negativos foram associados à expressão de PI3K. Receptores de estrógeno negativos e recorrência à distância foram ambos associados à expressão de EGFR. As expressões de PTEN, PI3K e EGFR foram fortemente associadas a características clínicas e patológicas de pior prognóstico. A expressão de PI3K foi significativamente associada à pior sobrevida livre de progressão (p=0.04) e pior sobrevida global (p=0.04). A expressão de EGFR foi também significativamente associada à pior sobrevida livre de progressão (p=0.03) e pior sobrevida global (p=0.04). A expressão de PTEN não foi associada à sobrevida. A expressão de PD-L1 foi identificada em 56.7% (107/189) e a de PD-L2 em 50.8% (97/192). Enquanto a expressão de PD-L1 foi detectada na membrana celular e no citoplasma das células do câncer de mama, a expressão de PD-L2 ocorreu no citoplasma e na região nuclear. Idade mais jovem ao diagnóstico, linfonodos positivos, receptor de estrógeno negativo e recorrência à distância foram associados tanto à expressão de PD-L1 quanto à de PD-L2. A presença de tumores maiores e de alto grau histológico foi associada à expressão de PD-L1. A expressão de PD-L1 foi significativamente associada à melhor sobrevida global (p=0.04). Conclusão: A expressão de PTEN, PI3K e EGFR pode representar um tipo de câncer de mama mais agressivo. A expressão de PI3K e EGFR pode ser considerada um marcador de mau prognóstico em câncer de mama. A expressão de PD-L1, apesar de ser associada a características clínicas e patológicas de pior evolução, pode ser considerada um marcador de bom prognóstico em câncer de mama / Abstract: Introduction: The PTEN, mTOR, PI3K, IGF-1R and EGFR signaling pathway plays an important role in prognosis of breast cancer. Although many studies have analyzed the correlation between PTEN, mTOR, PI3K, IGF-1R, and EGFR genes alterations with poor prognosis in breast cancer, there is still a gap in the literature concerning the prognostic value of these proteins in the adjuvant setting. Despite there were some well-known predictive and prognostic factors, such as hormone receptors and HER-2, in the immune field, breast cancer is one of the less immunogenic tumors. PD-L1 and PD-L2 constitute an important antitumor immune response. In breast cancer, the prognostic value of PD-L1 and PD-L2 is still to be defined. Objectives: This study investigates PTEN, mTOR, PI3K, IGF-1R and EGFR proteins expression and PD-L1 and PD-L2 proteins expression, and their correlation with clinicopathological features, disease-free survival and overall survival. Methods: In order to assess these proteins expression, we conducted an immunohistochemistry study using a tissue microarray encompassing 192 breast cancer cases, stage I, II and III, treated between 1994 and 2014 at the Women¿s Hospital (CAISM) from UNICAMP. All clinical and outcome data were retrieved from medical charts. Adjuvant therapy was administered according to the institution¿s treatment protocol. Results: PTEN expression was found in 40.6% (77/190); mTOR expression in 47.4% (90/190); PI3K expression in 29.8% (57/191); IGF-1R expression in 35.8% (68/190); and EGFR expression in 25.7% (49/191). In breast cancer cells PTEN expression showed cytoplasmic and nuclear immunoreactivity, and mTOR expression revealed strong nuclear and cytoplasmic staining. Tumors harboring PI3K expression presented strong immunoreactivity at cell membrane and weak cytoplasmic staining. IGF-1R expression was detected in breast cancer cell membrane. All proteins expression was significantly associated with lymph node positivity. Younger age at diagnosis was related to PTEN and PI3K expression. The presence of larger tumors was associated with PTEN expression. Negative progesterone receptor was correlated to PI3K expression. Estrogen receptor negativity and recurrence at distant sites were associated with EGFR expression. The expression of PTEN, PI3K, and EGFR were strongly associated with clinical and pathological features of poor prognosis. In our cohort, PI3K expression was associated with significantly worse disease-free survival (p=0.04) and overall survival (p=0.04), and EGFR expression was also significantly associated with worse disease-free survival (p=0.03) and overall survival (p=0.04). PD-L1 expression was present in 56.7% (107/189), and PD-L2 expression was identified in 50.8% (97/192). In breast cancer cells PD-L1 expression revealed strong immunoreactivity at cell membrane and cytoplasmic staining, and PD-L2 expression showed cytoplasmic and nuclear immunoreactivity. Younger age at diagnosis, lymph node positivity, estrogen negative receptor, and recurrence at distant sites were all associated with both PD-L1 and PD-L2 expression. The presence of larger tumors and higher histological grade were both associated with PD-L1 expression. PD-L1 expression was significantly associated with better overall survival (p=0.04) in breast cancer patients. Conclusion: The expression of PTEN, PI3K, and EGFR can represent a more aggressive type of breast cancer. PI3K and EGFR expressions emerge as poor prognostic markers in breast cancer. Despite its association with poor clinical and pathological features, PD-L1 expression seems to be a good prognostic marker in breast cancer / Doutorado / Oncologia Ginecológica e Mamária / Doutor em Ciências da Saúde

A insuficiência cardíaca (IC) é a via final comum da maioria das cardiomiopatias e outras doenças do aparelho circulatório. Considerando a prevalência crescente e a morbimortalidade associada representa um importante problema de saúde pública. Em quadros mais avançados, além do comprometimento funcional, portadores de IC apresentam perda de massa muscular excessiva que pode culminar em caquexia cardíaca; condição que contribui para o mau prognóstico e a mortalidade aumentadas. A massa muscular é regulada pelo balanço entre estímulos anabólicos e catabólicos. A quinase Akt vêm sendo considerando uma importante quinase na regulação do crescimento muscular por controlar o anabolismo proteico. Dessa forma, ativadores da Akt (IGF-I e insulina), bem como proteínas alvo da sinalização da Akt (mTOR e GSK3) são importantes mediadores na manutenção da massa muscular e podem ser regulados por estímulos metabólicos, nutricionais e mecânicos. Assim, o objetivo desse estudo foi avaliar a contribuição da via de sinalização IGF-I/Akt/mTOR na atrofia muscular desencadeada pela IC tanto em humanos quanto em modelo experimental, bem como o efeito do treinamento físico aeróbico (TFA). Nossos resultados demonstraram que em biópsias do vasto lateral de pacientes portadores de IC classe II houve redução na expressão de mRNA de todas as isoformas de IGF-I e na expressão das proteínas IGFBP-3, Akt1, GSK3, pGSK3Ser9, mTOR e tendência a redução na pmTORSer2448 (p=0,08) e aumento na pAMPKThr172. Esses resultados foram acompanhados pela redução no VO2 pico desses pacientes. O TFA de 12 semanas levou a um aumento não significativo na expressão de mRNA da isoforma IGF-I Ea (p=0,07) e IGF-I PAM (p=0,06). Também observou-se aumento na pAMPKThr172 e tendência a aumento na Akt1 (p=0,07) e mTOR (p=0,06). Em modelo experimental de IC, no músculo sóleo observou-se redução na expressão das proteínas IGF-I, PI3K, pAktSer473,pGSK3Ser9 e aumento da pAMPKThr172. O TFA de 8 semanas promoveu aumento na expressão do mRNA das isoformas de IGF-I Ea, Eb e IGF-I PAM, bem como na expressão das proteínas IGFI, PI3K, pAktSer473, pmTORSer2448 e redução na pAMPKThr172. O conjunto de alterações promovido pelo TFA foi associado à maior tolerância ao esforço físico e ganho no desempenho motor em Rota Rod, além de prevenir a atrofia muscular. Quando esses animais foram tratados com rapamicina, um inibidor farmacológico da mTOR, o efeito do TFA na prevenção da atrofia muscular foi abolido. Juntos, esses resultados apoiam a hipótese de que a via de sinalização IGF-I/Akt/mTOR está envolvida no reestabelecimento muscular na IC induzida pelo TFA / Heart failure (HF) is a common ultimate consequence for the most cardiomyopathies and other diseases from circulatory system. Due its growing prevalence and morbimortality is an important public-health problem. In more advanced cases, besides functional impairment, HF patients present an excessive skeletal muscle loss which can lead to cardiac cachexia; a condition associated to a poor prognostic and increased mortality. Skeletal muscle mass is regulated by the balance between anabolic and catabolic stimuli. Akt kinase, although involved with the protein synthesis pathway, is able to regulates the skeletal muscle growth via anabolism and catabolism control. An importante role in the skeletal muscle growing has been attributed to the Akt kinase due its ability to control protein synthesis. Thus, activators (IGF-I and insulin) as well as target proteins in the Akt signaling pathway (mTOR and GSK3) are important mediators in the skeletal muscle mass homeostasis being regulated by anabolic, nutritional and mechanic stimuli. Therefore, the aim of the presente study was to evaluate the contribution of IGF-I/Akt/mTOR contribution to the muscle atrophy induced by HF in humans and mice. Additionally, the effect of aerobic exercise training were evaluated. Our data demonstrated that in biopsies obtained in the vastus lateral from class II IC patients occurred a reduction in the expression of all the forms of IGF investigated and in the expression of proteins such as IGFBP-3, Akt1, GSK3, pGSK3Ser9, mTOR, and tendency to reduce pmTORSer2448 (p=0.08) and to increase pAMPKThr172. These results were accompained by the reduction of peak VO2 in these patients. Twelve weeks of aerobic exercise training promoted a non-significant increase in the expression of the IGF-I Ea (p=0.07) and IGF-I PAM (p=0.06) mRNA expression. Moreover, it was observed an increase to pAMPKThr172, and tendency of increase for Akt1 (p=0.07) and mTOR (p=0.06) mRNA expression. In a experimental model of IC, it was observed a reduction in the expression of IGF I, PI3K, pAktSer473, pGSK3Ser9 and increase of pAMPKThr172. Eight weeks of aerobic exercise training increased the mRNA of IGF-I Ea, Eb and IGF-I PAM isoforms, as well as in the expression of IGF-I, PI3K, pAktSer473, pmTORSer2448 and pAMPKThr172 reduction. The changes induced by aerobic exercise training were associated with a higher tolerance to exercise and motor performance in rota rod besides prevent muscular atrophy. When treated with a inhibitor of mTOR, rapamycin, the adaptations induced by aerobic exercise training were blunted, supporting the hypothesis that the IGF-I/Akt/mTOR signaling pathway is involved in the recovering of muscle mass induced by physical training

Bcl-xL et Mcl-1 sont deux protéines anti-apoptotiques de la famille Bcl-2 dont dépendent les cancers de l’ovaire pour leur survie, leur inhibition semble donc être une stratégie pertinente. La molécule BH3-mimétique ABT 737 (ou son analogue oral, l’ABT-263), est un puissant inhibiteur de Bcl-xL mais l’inhibition de Mcl-1 reste problématique. Les voies de signalisation PI3K/Akt/mTOR et MAPK/ERK régulent l’expression et l’activité de cette dernière protéine et de ses partenaires BH3-only (Bim, Puma, Noxa). Nous nous sommes donc intéressés à l’intérêt de leur inhibition pour sensibiliser les cellules cancéreuses ovariennes à l’ABT-737. La première étude menée avec le BEZ235, double inhibiteur PI3K/mTOR développé par le laboratoire Novartis, montre qu’il inhibe l’expression de Mcl-1 et induit celle de Puma, et qu’il sensibilise les cellules cancéreuses ovariennes à l’ABT-737 à condition que l’expression de Bim soit également induite. La deuxième étude a évalué les effets de l’AZD8055, inhibiteur du site actif de mTOR développé par le laboratoire AstraZeneca, et du trametinib, inhibiteur allostérique de MEK développé par le laboratoire GlaxoSmithKline et actuellement en clinique, sur trois lignées cancéreuses ovariennes. L’inhibition de l’expression de Mcl-1 et l’induction de celle de Puma par l’AZD8055 ne permettent pas de diminuer suffisamment le ratio [Mcl-1/protéines BH3-only] pour sensibiliser les cellules à l’ABT-737. En revanche, la forte induction de Bim sous forme active déphosphorylée par le trametinib permet de diminuer suffisamment ce ratio pour sensibiliser deux des trois lignées testées à l’ABT-737. C’est cependant la triple combinaison AZD8055/trametinib/ABT-737 qui est la plus efficace pour induire une apoptose massive dans les trois lignées. Par ailleurs, de façon intéressante, l’association de l’AZD8055 et du trametinib est cytotoxique sans ABT 737 dans une des lignées testées. Ces résultats mettent en évidence l’efficacité de différentes stratégies thérapeutiques multi-cibles et la nécessité de définir des marqueurs prédictifs de la réponse afin d’évoluer vers un traitement personnalisé pour améliorer la prise en charge des cancers de l’ovaire. / Ovarian cancers depend on Bcl-xL and Mcl-1, two anti-apoptotic protein of the Bcl-2 family, for their survival and their inhibition seems to by a relevant strategy. The BH3-mimetic molecule ABT-737 (or its oral form, ABT-263), is a strong Bcl-xL inhibitor, but Mcl-1 inhibition remains problematic. Signaling pathways PI3K/Akt/mTOR and MAPK/ERK regulate expression and activity of Mcl-1 and its BH3-only partners (Bim, Puma, Noxa). We focused on the interest of their inhibition to sensitize ovarian cancer cells to ABT-737. The first study with BEZ235, a PI3K/mTOR dual inhibitor developed by Novartis, inhibits Mcl-1 expression and induces the one of Puma, and sensitizes ovarian cancer cells to ABT-737 provided that Bim expression is induced. The second study evaluated the effects of AZD8055, mTOR active site inhibitor developed by AstraZeneca, and of trametinib, MEK allosteric inhibitor developed by GlaxoSmithKline and currently in clinic, on three ovarian cancer cell lines. Mcl-1 expression inhibition and Puma expression induction by AZD8055 does not sufficiently reduce [Mcl-1/BH3-only proteins] ratio to sensitize cells to ABT-737. On the other hand, strong Bim induction in its active dephosphorylated form by trametinib sufficiently reduce this ratio to sensitize two of the three cell lines tested to ABT-737. Nevertheless, the triple combination AZD8055/trametinib/ABT-737 is the most efficient to induce massive apoptosis in the three cell lines. Besides, interestingly, AZD8055 and trametinib association is cytotoxic without ABT-737 in one of the tested cell lines. These results highlight the efficacy of different multi-targets therapeutic strategies and the need of predictive marker definition of the response to develop personalized treatment and to improve ovarian cancer management.