La voie PI3Kinase/AKT/mTOR, est une cible thérapeutique du temsirolimus, un inhibiteur de mTORC1. Dans le but d'obtenir une inhibition plus importante de cette voie j’utilise dans ce projet deux nouvelles molécules :- le NVP-BEZ 235 (BEZ) qui inhibe à la fois mTORC1 et la PI3kinase- l'AZD8055 (AZD), un inhibiteur des complexes mTORC1 et mTORC2. En utilisant différentes lignées de LCM, j’ai démontré que l'effet de ces nouveaux inhibiteurs sur la survie cellulaire est plus important que celui du temsirolimus. Cela est probablement dû à l'inhibition de la phosphorylation de l'AKT et la 4EBP. La deuxième partie de ce projet étudie la synergie entre les inhibiteurs de m-TOR kinase et l'aracytine. Un effet additif important a été démontré. J’ai trouvé en western blot que l’aracytine inhibe la phosphorylation des substrats de la voie Akt –mTOR notamment le 4EBP. L’ibrutinib (un inhibiteur de la voie Btk) a un effet modeste mais j’ai pu démontrer qu'il est capable à induire une inhibition plus importante de la survie cellulaire lorsqu'il est associé à l’aracytine. Cependant il s'est révélé antagoniste aux inhibiteurs de la voie PI3K-AKT-mTOR, cela reste difficile à décortiquer. Enfin, j’ai trouvé un effet additif de l’ibrutinib en combinaison avec la doxorubicine. Cependant les inhibiteurs de m-TOR n'ont pas le même effet. Afin d’expliquer ces résultats, j’ai étudié l’effet de ces molécules sur l’expression de GSTPi, enzyme de détoxification connue pour avoir un rôle important dans la résistance de LCM à l’anthracycline. J’ai mis en évidence une diminution de l’expression de cet enzyme par l’Ibrutinib. En revanche, les inhibiteurs de mTOR n’ont pas un effet sur l’expression de GSTPi. L’ibrutinib pourrait donc sensibiliser le LCM à l’anthracycline en diminuant l’expression de GSTPi. / The PI3K / AKT / mTOR pathway is the target of Temsirolimus. However, important resistance is observed. We tried to obtain a more important inhibition of PI3K / AKT pathway using two new molecules :- NVP-BEZ 235 (BEZ) which inhibits both mTORC1 and PI3K- AZD8055 (AZD) an inhibitor of mTORC1 and mTORC2 complexes. Using different cell lines of MCL, we have shown that the effect of these new inhibitors on cell survival was more important than that of Temsirolimus. This is probably because contrary to Temsirolimus, the two new molecules can inhibit AKT and 4EBP phosphorylation. In the second part of this project we studied the synergy between the m-TOR kinase inhibitors and aracytine (conventional treatment of MCL). We revealed a significant additive effect in MCL cell lines. We demonstrated by Western blot analysis that aracytine inhibits S6 and 4EBP phosphorylation. This may explain the results obtained from this drug association. We then showed that Ibrutinib (an inhibitor of Btk pathway) can induce a significant inhibition of cell survival when combined with aracytine. In this study, Ibrutinib proved antagonist effect to PI3K-AKT-mTOR inhibitors. The mechanisms of these results remain unclear. Finally, we demonstrated an additive effect of Ibrutinib in combination with doxorubicin. We did not obtain the same results when we combined m-TOR inhibitors with doxorubicin. To explain these data, we studied the effect of these drugs on the expression of GSTPi by western blot. This enzyme is known to have an important role in MCL resistance to anthracycline. Importantly, Ibrutinib induced a decrease in the expression of GSTPi but AZD8055, Temsirolimus and NVP-BEZ235 had no effect.

Activée par la chimiothérapie, la sénescence est un mécanisme suppresseur de tumeur qui empêche la progression tumorale. Cependant, certaines cellules cancéreuses sont capables d’émerger et de provoquer une rechute clinique. Les mécanismes mis en place par les cellules pour échapper à la sénescence ne sont pas encore clairement connus. Nous avons récemment décrit que les cellules qui échappent à la sénescence résistent à l’anoïkis et sont dépendantes de Mcl-1. Dans cette étude, nous caractérisons plus en détail cette émergence en réponse à l'irinotécan, un traitement de première ligne utilisé dans le cancer colorectal. Nos résultats indiquent que la kinase Aktest activée comme une voie de survie lors de l'étape précoce de la sénescence et qu’elle est également réactivée lors de l’émergence. L'inhibition de la kinase avec le GSK690693 empêche l’émergence et améliore l’efficacité du traitement, à la fois in vitro et in vivo. Cette amélioration a été corrélée à l'inhibition de la sénescence et l’activation concomitante de l'apoptose via l’inactivation de Mcl-1 par Noxa. De plus, en utilisant des cellules p21Waf1 déficientes, nous avons confirmé que la voie p53-p21, principal régulateur de la sénescence, favorise l’émergence. A l’inverse, son inhibition améliore l'efficacité du traitement par induction de l'apoptose. Ces résultats mettent en évidence que l'inhibition d’Akt améliore l’efficacité de l’irinotécan et empêche l'émergence de cellules persistantes en diminuant la sénescence au profit de la réponse apoptotique. Par conséquent, nous proposons que l’utilisation d’inhibiteurs d’Akt en thérapies séquentielles devrait être considérée dans le futur pour améliorer le traitement des cancers colorectaux réfractaires à l’irinotécan. / Activated by chemotherapy, senescence is a tumor suppressive mechanism that prevents tumor progression. However, some cancer cells can emerge to induce clinical relapse. The mechanisms set up by cells to escape senescence are not yet clearly known. We have recently described that cells that escape senescence are more transformed than non treated parental cells ; they resist anoikis and depend on Mcl-1. In this study, we further characterize this emergence in response to irinotecan, a first line treatment used in colorectal cancer. Our results indicate that the Akt kinase was activated as a feedback pathway during the early step of senescence and also during emergence. Inhibition of the kinase with GSK690693 prevented cell emergence and improved treatment efficacy, both in vitro and in vivo. This improvement was correlated with senescence inhibition, p21waf1 down regulation and a concomitant activation of apoptosis due to Noxa upregulation and Mcl-1 inactivation. Indeed, Noxa inactivation prevented apoptosis and increased the number of emergent cells. Moreover, using p21waf1-deficient cells, we further confirmed that an intact p53-p21-senescence pathway favored cell emergence and that its down regulation improved treatment efficacy through apoptosis induction. These results highlight that Akt inhibition improves irinotecan treatment and prevent cell emergence by switching the senescence response to apoptosis. Therefore, we propose that use of Akt inhibitors in sequential therapies should be considered in future to improve the treatment of irinotecan-refractory colorectal cancers.

No description available.

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Au sein d’une tumeur des régions hypoxiques se forment ce qui conduit à l’activation du facteur de transcription HIF1. HIF1, composé des deux protéines HIF1a et HIF1b, permet l’adaptation des cellules à des faibles concentrations d’oxygène en activant la transcription de gène cible. Un de ces gènes est celui codant pour la lysyl oxydase (LOX). Cette enzyme structure la matrice extracellulaire et est impliquée dans la tumorigénèse. Pour comprendre les liens entre LOX et HIF1a, nous avons modulé leurs expressions dans des lignées humaines de carcinome du côlon. En condition hypoxique, HIF1a contrôle l’expression de LOX et réciproquement, LOX régule la synthèse protéique d’HIF1a via l’activation de la voie de signalisation PI3K/AKT. Nous avons donc mis en évidence l’existence d’une boucle de régulation positive entre LOX et HIF1 en conditions hypoxique. Sachant que ces deux protéines sont des acteurs majeurs de la progression tumorale, nous avons cherché à comprendre le rôle de cette régulation mutuelle dans ce processus. Nos résultats démontrent que l’activité enzymatique de LOX promeut la croissance tumorale in vitro et in vivo et que son action est potentialisée par la présence de son partenaire HIF1a. De plus, LOX et HIF1a agissent en synergie afin d’augmenter la potentiel métastatiques des cellules tumorale de côlon in vitro. Ainsi, ce travail de thèse a permis de mettre en évidence l’existence d’une boucle de régulation entre HIF1a et LOX qui est critique dans la progression tumorale et semble également être impliquée dans le processus métastatiques / The microenvironment of solid tumors is exposed to hypoxic conditions which lead to the activation of Hypoxia‐Inducible Factor 1 (HIF1). HIF1, composed by a heterodimer of HIF1a and HIF1b protein, is a key transcription factor involved in cellular adaptation to changes in oxygen level, inducing the expression of several transcriptional targets such as Lysyl Oxidase (LOX). LOX is an amine oxidase that catalyzes crosslinking of fibrillar collagens and elastin in the extracellular matrix. Furthermore, LOX is implied in tumor progression. To clarify, the link between LOX and HIF1a, their expression were modulated in human colorectal carcinoma cell lines. We pointed out that besides HIF1‐dependant regulation of LOX, LOX can also act on the HIF1 pathway under hypoxic conditions. Indeed, LOX enzymatic activity upregulates HIF1a protein synthesis, and this action is mediated by the PI3K/AKT pathway. Thus, these results emphasize the existence of a regulation loop between HIF‐1a and LOX, which represent two main actors of tumoral progression. Thus, we wanted to determine the implication of this amplification loop in tumor progression. Our results show that LOX enzymatic activity increase tumor growth in vitro and in vivo, and this role is partially dependant of its partner HIF1a. Furthermore, we established that that LOX and HIF1a act in synergy to foster metastatic potential in colorectal carcinoma cell lines. Taken together, our results demonstrate a regulation loop between LOX and HIF1a with is critical for tumor progression and metastasis formation

Le comportement et le devenir des lymphocytes T (LT) est conditionné par l’intégration de nombreux signaux solubles et cellulaires. Lorsque les LT ne sont pas stimulés, les facteurs de transcription FoxO orchestrent un réseau moléculaire important participant au maintien de la quiescence et à la capacité migratoire des LT. Longtemps considéré comme un état par défaut, le maintien des LT dans cet état quiescent est hautement régulé par un ensemble de signaux parmi lesquels la signalisation via le récepteur à l’interleukine 7 (IL7) et le récepteur à l’antigène (TCR) activé par des molécules du complexe majeur d’histocompatibilité (CMH) chargées avec des peptides du soi. Etonnamment, ces mêmes signaux sont nécessaires pour induire l’entrée des cellules dans le cycle cellulaire. L’inhibition de la prolifération des LT est donc un mécanisme actif qui peut être levé par des signaux externes. Le mécanisme moléculaire permettant le maintien de cet état quiescent reste très peu décrit. Mon projet de thèse a consisté à étudier les conséquences fonctionnelles de l’expression de Fam65b, une nouvelle cible transcriptionnelle de FoxO, sur la prolifération. Au cours de mon travail, j’ai montré que dans des cellules transformées, ayant donc perdu la capacité de réguler leur prolifération, l’expression forcée de Fam65b perturbe la mise en place du fuseau mitotique, induisant un arrêt en phase G 2 /M et la mort des cellules. Au cours de ce processus, Fam65b agit avec deux partenaires connus pour leur implication dans le cycle cellulaire, l’histone déacétylase 6 (HDAC6) et la protéine d’échafaudage 14-3-3. J’ai également pu établir que, dans les LT primaires humains, Fam65b est un facteur de quiescence. En effet, l’engagement du TCR induit une diminution d’expression de Fam65b et le maintien de son expression bloque la prolifération des LT, suggérant que l’inhibition de son expression est un pré-requis à la prolifération. Inversement, l’inhibition de l’expression de Fam65b dans des LT naïfs diminue leur seuil d’activation. L’ensemble de ces résultats désigne donc Fam65b comme une nouvelle cible pour le contrôle de la prolifération des cellules primaires et transformées. Nous avons également développé au laboratoire un modèle murin invalidé pour Fam65b dans le lignage T afin d’étudier son rôle dans un modèle plus intégré. J’ai pu initier l’analyse du phénotype de ces souris en l’absence de toute stimulation. L’ensemble de ces travaux, en complément de ceux précédemment obtenus au laboratoire, laissent apparaître Fam65b comme un nouvel effecteur de FoxO capable d’interagir avec divers partenaires afin de contrôler conjointement des fonctions cellulaires majeures. / T cell fate is conditioned by the integration of many soluble and cellular signals. When T cells are not stimulated, FoxO transcription factors orchestrate an important molecular network involved in maintaining the quiescent state and migratory ability of the cells. Considered as a "default" state, it is now known that maintenance of T cell quiescence is a process highly regulated by a set of signals including IL7 signaling and sustained contact with MHC molecules presenting self-peptides. Surprisingly, these same signals are required to induce entry of cells into the cell cycle. Inhibition of T cell proliferation is an active mechanism that can be lifted by external signals. The molecular mechanism maintaining this quiescent state is poorly described. My thesis project was studying the functional consequences of Fam65b expression, a new transcriptional target of FoxO, on proliferation. I showed that, in transformed cells, that have lost the ability to regulate their proliferation, forced expression of Fam65b disrupts the establishment of the mitotic spindle, inducing an arrest in G 2 /M phase and cell death. During this process, Fam65b acts with two partners, known to be involved in the cell cycle process, the histone deacetylase HDAC6 and the 14-3-3 protein scaffold. I have also been able to establish that in human primary T cell, Fam65b is a quiescence factor. Indeed, the TCR stimulation induces a reduction of Fam65b expression and maintaining its expression blocks the proliferation of T cells, suggesting that inhibition of Fam65b expression is a prerequisite for proliferation. Conversely, inhibition of Fam65b expression in naive T cells reduces their activation threshold. Altogether these results show that Fam65b is a new target for the control of the proliferation of primary and transformed cells. We have also developed, in the laboratory, a mouse model invalidated for Fam65b in T cell lineage. I initiated the phenotype analysis of these mice in the absence of any stimulation. This work, in addition to the previous results obtained in the laboratory, reveal that Fam65b is a new effector of FoxO factors, able to interact with various partners to jointly control major cellular functions.

As respostas termorregulatórias ao lipopolissacarídeo (LPS) são influenciadas por moduladores que aumentam (febrigênicos) ou diminuem (criogênicos) a temperatura corporal (Tb). Entre eles, o neurotransmissor gasoso sulfeto de hidrogênio (H2S) modula a inflamação sistêmica induzida por endotoxina em ratos, agindo como uma molécula anti-inflamatória e criogênica, embora os mecanismos subjacentes ainda sejam pouco compreendidos. Considerando que a endotoxina é um ligando para o Toll-like receptor 4 (TLR4) e que evidências recentes revelam um cross-talk entre a via de sinalização TLR e fosfo-Akt (p-Akt), o objetivo do presente estudo foi investigar se o H2S atua como um mediador antiinflamatório e antipirético durante as fases termorregulatórias que ocorrem no choque endotoxêmico (hipotermia e febre) induzido por lipopolissacarídeo bacteriano (LPS, 2,5 mg / kg intraperitoneal (ip)) através da modulação sobre a produção de prostaglandina D2 (PGD2) e a ativação de Akt na área pré-óptica ântero-ventral do hipotálamo (AVPO). A Tb profunda de ratos mantidos a uma temperatura ambiente de 25 °C foi registrada antes e depois da inibição farmacológica da enzima cistationina ?-sintase (CBS - responsável pela produção endógena de H2S no cérebro) usando aminooxiacetato (AOA, 100 pmol, intracerebroventricular (icv)), combinado ou não com administração de LPS. Para esclarecer os mecanismos responsáveis por esses ajustes da resposta imune, foram determinados na AVPO os níveis de H2S, a produção de PGD2 e o perfil de expressão das proteínas CBS, p-Akt e p-CREB. Além disso, foi analisada a concentração de citocinas plasmáticas (IL-1?, IL-6, IL-10, TNF?, IFN-? , E IL-4). A injeção ip de LPS causou hipotermia típica seguida de febre. Os níveis de AVPO H2S aumentaram significativamente durante a hipotermia quando comparado com ratos eutérmicos e febris. A microinjeção icv de AOA não causou nenhuma alteração na Tb nem na produção basal de PGD2 durante a eutermia. Em ratos tratados com LPS, o AOA causou uma atenuação na queda da Tb durante a fase de hipotermia e uma febre exacerbada, simultaneamente com o aumento na produção de PGD2 e abolição do aumento induzido pela endotoxina na atividade de Akt. Durante a fase de febre, a expressão relativa de CBS esteve significativamente diminuída enquanto a expressão relativa de p-Akt esteve aumentada, quando comparado com ratos eutérmicos e hipotérmicos. As citocinas plasmáticas aumentaram durante a inflamação sistêmica, mas apenas a IL-4 mostrou um padrão semelhante em relação à Akt. Estes dados são consistentes com a noção de que o neurotransmissor gasoso H2S modula as fases de hipotermia e febre durante o choque endotoxêmico, atuando como uma molécula criogênica. Este papel anti-inflamatório durante a inflamação sistémica envolve uma regulação positiva da PGD2, de Akt e da IL-4 plasmática. / Thermoregulatory responses to lipopolysaccharide (LPS) are affected by modulators that increase (pro-pyretic) or decrease (cryogenic) body temperature (Tb). Among them, the gaseous messenger hydrogen sulfide (H2S) modulates endotoxin-induced systemic inflammation being an anti-inflammatory and cryogenic molecule, although the underlying mechanisms are still poorly understood. Since endotoxin is a Toll-like receptor 4 (TLR4) ligand and recent evidence indicates that there is a possible a cross-talk between the TLR and phospho-Akt (p-Akt) signaling pathway, the current study aimed to investigate whether H2S acts as an anti-inflammatory and anti-pyretic mediator during thermoregulatory phases of endotoxic shock (hypothermia and fever) induced by bacterial lipopolysaccharide (LPS, 2.5 mg/kg intraperitoneal (ip)) through the modulation of prostaglandin D2 (PGD2) production and activation of Akt in the anteroventral preoptic region of the hypothalamus (AVPO). Deep Tb in rats kept at an ambient temperature of 25 °C, was recorded before and after pharmacological inhibition of the enzyme cystathionine ?-synthase (CBS - responsible for H 2S endogenous production in the brain) using aminooxyacetate (AOA; 100 pmol/1 ?l intracerebroventricular (icv)) combined or not with endotoxin administration. To clarify the mechanisms responsible for these adjustments on immune response were verified in the AVPO H 2S levels, PGD2 production and expression profiles of CBS, p-Akt and p-CREB. In addition, plasma cytokines concentration (IL-1?, IL-6, IL-10, TNF?, IFN-?, and IL-4) was analyzed. Intraperitoneal injection of LPS caused typical hypothermia followed by fever. Intracerebroventricular microinjection of AOA neither affected Tb nor basal PGD2 production during euthermia. Levels of AVPO H2S were significantly increased during hypothermia when compared to both euthermic and febrile rats. In LPS-treated rats, AOA increased Tb values during hypothermia and fever, along with enhanced PGD2 production and abolition of endotoxin-induced increase in Akt activity. During fever, CBS relative expression was significantly decreased whereas p-Akt was significantly increased when compared to both euthermic and hypothermic rats. Plasma cytokines were increased during systemic inflammation, but only IL-4 showed a similar pattern in relation to Akt. These data are consistent with the notion that the gaseous messenger H2S modulates hypothermia and fever during endotoxic shock, acting as a cryogenic molecule. This anti-inflammatory role during systemic inflammation involves a H2S-induced up-modulation of PGD2, Akt and plasma IL-4.

Infecção pelo HPV é o principal fator de risco para câncer cervical. Os tumores apresentam microambiente complexo, células tumorais e inflamatórias integram sinais modulando a atividade de vias de sinalização. Caracterizamos o estado de ativação de três vias de sinalização importantes para a progressão tumoral. Com o aumento do grau da lesão, observamos: aumento da expressão de NFκB e Akt fosforiladas no microambiente tumoral; redução de NFκB fosforilada e aumento de STAT3 e Akt fosforiladas em leucócitos circulantes. Em modelos experimentais, a via de NFκB também encontra-se inibida, principalmente em células apresentadoras de antígenos. Para testar se a modulação de vias de sinalização poderiam alterar as respostas a tumores, tratamos linfócitos B de pacientes com agonista de CD40, ativador de NFκB, e observamos aumento do potencial de apresentação antigênica. Nossos resultados mostram o panorama do estado de ativação de importantes vias de sinalização para a progressão tumoral e trazem a possibilidade de uma ferramenta imunomoduladora. / Cervical cancers have HPV infection as a main factor. HPV tumors recruit leucocytes and change their phenotype as an evasion mechanism. We tried to understand how the tumor influences the immune cells behavior, analyzing signaling pathways important to tumor progression in cervical biopsy and PBMC from patients with high-grade lesions and cancer. We find an increase in NFκB activation in immune cells from tumor microenvironment, but a decrease in PBMC. We could not find any difference in STAT3 signaling pathway in the tumor microenvironment, however, there was an activation increase in PBMC. Both tumor microenvironment immune cells and PBMC had an increase in Akt signaling pathway. To identify a possible strategy to reverse the tumor influence, we stimulated B lymphocytes from patients with sCD40L, achieving an increase in the numbers of CD80+CD86+ cells. Our results demonstrate a tumor effect over the immune cells, with an important systemic effect. However, the approach used to stimulate B lymphocytes from patients present us with a possible immunotherapy.

A disfunção cardíaca, decorrente de um prejuízo na contratilidade miocárdica, tem sido reconhecida como um fator importante que contribui para as altas taxas de mortalidade na sepse. Outro fato importante aponta para o envolvimento das calpaínas na inibição da via de sinalização PI3K/mTOR levando a uma diminuição potencial das taxas globais de síntese proteica através da redução da maquinaria de tradução disponível, o que reforça o envolvimento destes elementos na progressão da disfunção cardíaca na sepse. Metodologia: Foram utilizados camundongos da linhagem C57/BL6 para indução de sepse através da técnica de ligadura e perfuração do ceco separados em quatro grupos: controle com e sem tratamento e sepse moderada com e sem tratamento, o tratamento foi realizado 2 horas antes da cirurgia com inibidor da via mTOR, rapamicina. Foi realizada análise histopatológica em metacrilato e coloração picrosirius para colágeno, western blotting para quantificação da expressão proteica e real-time PCR para quantificação da expressão gênica, por fim realizamos análise funcional através da ecocardiografia. Resultados: Foi encontrado aumento das lesões teciduais e depósito de colágeno no grupo séptico tratado com rapamicina. A análise por western blotting e real-time PCR demonstrou redução das proteínas envolvidas na via mTOR nos grupos sépticos com e sem tratamento com ênfase no grupo tratado e por fim a avaliação funcional mostrou redução dos parâmetros débito cardíaco e fração de ejeção nos grupos sépticos com e sem tratamento. Conclusão: Nossos resultados demonstram que a via mTOR é de extrema importância na estrutura e função cardíacas, visto que sua inibição ocasionou o aumento de lesões e deposição de colágeno juntamente com alterações funcionais, podendo se transformar em um possível alvo terapêutico para futuras pesquisas clínicas em animais e humanos. / Cardiac dysfunction, due to impairment in myocardial contractility, has been recognized as an important factor contributing to the high mortality rates in sepsis. Another important fact is the involvement of the calpain in the inhibition of the PI3K / mTOR signaling pathway leading to a potential decrease in the overall rates of protein synthesis through the reduction of available translation machinery, which reinforces the involvement of these elements in the progression of cardiac dysfunction in sepsis. Methods: C57 / BL6 mice were used for induction of sepsis through the technique of ligation and perforation of the cecum separated into four groups: control with and without treatment and moderate sepsis with and without treatment, treatment was performed 2 hours before surgery with mTOR pathway inhibitor, rapamycin. Histopathological analysis was performed on methacrylate and picrosirius staining for collagen, western blotting for quantification of protein expression and real-time PCR for quantification of gene expression. Finally we performed functional analysis through echocardiography. Results: Increased tissue lesions and collagen deposition were found in the septic group treated with rapamycin. Western blotting and real-time PCR analysis showed reduction of the proteins involved in the mTOR pathway in the septic groups with and without treatment with emphasis in the treated group and finally the functional evaluation showed a reduction of the parameters cardiac output and ejection fraction in the septic groups with and without treatment. Conclusion: Our results demonstrate that the mTOR pathway is extremely important in cardiac structure and function, since its inhibition has resulted in increased lesions and collagen deposition along with functional alterations, and may become a possible therapeutic target for future clinical research in animals and humans.

O carcinoma de cabeça e pescoço é uma neoplasia maligna de origem epitelial que resulta em aproximadamente 500.000 novos casos por ano ao redor do mundo. Diversos estudos têm sido conduzidos de maneira a elucidar os mecanismos de proliferação e invasão desta doença, sendo a via de sinalização Akt/mTOR e proteínas relacionadas, apontada como uma das principais vias envolvidas em sua progressão. Sabe-se que células neoplásicas, bem como células de diferentes tecidos, podem ter seu comportamento modificado após terem sido irradiadas com laser em baixa intensidade (LLLT). Porém, os mecanismos de atuação da luz laser de baixa potência sobre estas células permanecem ainda não completamente esclarecidos. Portanto, o objetivo deste estudo foi o de analisar a viabilidade celular e expressão das proteínas Akt, pAkt, Hsp90, S6, pS6 e Ciclina D1 em duas linhagens celulares de carcinoma de boca (SCC9 e SCC25), bem como em uma linhagem de queratinócitos orais humanos com displasia (DOK) após irradiação com laser em baixa intensidade. O laser utilizado foi um diodo semicondutor de arseneto de Gálio e Alumínio (GaAlAs) operando nos comprimentos de onda vermelho (660nm) e infravermelho (780nm), com potência fixa em 40mW e três densidades de energia para cada comprimento de onda disponível: 2.05J/cm², 3.07J/cm² e 6.15J/cm². A análise de apoptose foi realizada por meio do teste de TUNEL e a expressão proteica foi obtida com imunofluorescência e western blotting. Após análise estatística por meio do método ANOVA dois critérios e testes de Tukey ou teste T de estudante, todos com nível de significância de 5%, pôde-se concluir que a LLLT induziu comportamentos distintos em cada uma das linhagens celulares utilizadas. Foi notado aumento, bem como diminuição da viabilidade celular, dependendo do comprimento de onda utilizado e das células irradiadas. A densidade de energia de 2.05J/cm² foi a que produziu efeitos mais significativos em SCC9. Para a linhagem celular SCC25, a dose mais relevante foi a de 3.07J/cm², enquanto que para a linhagem DOK, a dose de 6.15J/cm² causou efeitos mais proeminentes. Estas respectivas doses foram escolhidas para cada uma das linhagens para dar continuidade aos experimentos de Western Blotting e Imunofluorescência. Dentre os resultados mais relevantes obtidos com estas técnicas, pode-se citar a variação dos níveis de pS6 e Ciclina D1 para a linhagem DOK em determinados períodos. Já a linhagem SCC9 apresentou variação dos níveis de pAkt e Ciclina D1 nos períodos estudados. A linhagem SCC25 também teve as expressões de pAkt, pS6 e Ciclina D1 modificadas por LLLT. De maneira interessante, o aparecimento ou manutenção de uma isoforma de Hsp90 foi encontrado em SCC9 e SCC25 após irradiação laser. Por fim, a indução de apoptose foi detectada na linhagem SCC25. Em conclusão, pode-se dizer que a LLLT, como empregada neste estudo, foi capaz de aumentar a expressão de proteínas relacionadas à progressão e invasão em todas as linhagens estudadas. Além disso, a irradiação laser foi única, apesar de ter causado efeitos prolongados, algumas vezes até o último período estudado. / Head and neck squamous cell carcinoma (HNSCC) is an epithelial malignant neoplasm that accounts for approximately 500.000 new cases yearly around the world. Several studies have been conducted to elucidate the mechanisms of proliferation and invasion of this lesion, whereas the Akt/mTOR signaling pathway with its related proteins is being pointed out as one of the main pathways involved in HNSCC`s progression. Neoplastic cells, as well as cells that originate from different tissues may have their behavior modified by low level laser therapy (LLLT); however, the mechanisms through which the low level laser light interacts with these cells remain poorly understood. Thus, this study sought to evaluate the cell viability and the expression levels of Akt, pAkt, Hsp90, S6, pS6 and Cyclin D1 proteins in two oral squamous cell carcinoma cell lineages (SCC9 and SCC25) and in one oral dysplastic human keratinocyte cell line (DOK) after they had been treated with LLLT. The laser device was a semiconductor diode of Gallium and Aluminum Arsenate (GaAlAs), operating with wavelengths of 660nm (red) and 780nm (infrared), with a fixed power of 40mW and giving three different energy densities: 2.05J/cm², 3.07J/cm² and 6.15J/cm². Apoptosis was analyzed through TUNEL test and the protein expression was accessed with Immunofluorescence and Western blotting. After statistical analysis through two-way ANOVA and Tukey or Student`s T test, all of them with a level of significance of 5%, it was concluded that LLLT induced distinct behaviors to each of the studied cell lines. Increases and inhibitions in cell viabilities were detected depending on the wavelength and also on the irradiated cell line. The energy density of 2.05J/cm² produced the most significant findings over SCC9. On the other hand, in SCC25 the most relevant results were detected with 3.07J/cm², while the most prominent findings were seen with 6.15J/cm² when the cell line DOK was evaluated. In that way, these respective doses were chosen for each cell line to continue with Western blotting and Immunofluorescence. Among the most relevant findings, the variation of pS6 and Cyclin D1 levels can be cited for DOK in some evaluated periods. SCC9 presented both pAkt and Cyclin D1 variations in the studied periods. Besides that, SCC25 also had pAkt, pS6 and Cyclin D1 levels modified by LLLT. Interestingly, the appearance and maintenance of an Hsp90 isoform was found in SCC9 and SCC25 after laser irradiation. Moreover, the induction of apoptosis was detected for the SCC25 cell line. Finally, the LLLT employed herein was able to enhance the expression of proteins related to progression and invasion in all of the studied cell lines. In addition, there was a single laser irradiation, although it caused prolonged effects, sometimes through the latest evaluated period.