L'expression intracellulaire des anticorps (intracorps) est une approche qui permet l'étude et le ciblage des antigènes dans les compartiments intracellulaires. Néanmoins, l'expression d'anticorps entiers fonctionnels dans les cellules reste une tâche difficile en raison de leur grande taille et de leur structure, l'environnement réducteur du milieu intracellulaire étant défavorable à la formation des ponts disulfure. Notre groupe a une forte expertise dans le domaine de l'immunisation intracellulaire et son application pour l'identification de nouvelles cibles thérapeutiques. Pour cela, notre équipe a élaboré des banques de fragments d'anticorps scFv optimisés pour une meilleure expression intracellulaire. Nos travaux antérieurs ont démontré que ces intracorps peuvent cibler spécifiquement des domaines ou des modifications post-traductionnelles de protéines dans des cellules vivantes. Ceci est particulièrement important car il démontre l'un des avantages principaux des intracorps par rapport à l'approche basée sur l'ARNi. Cet avantage a été démontré par un criblage phénotypique dans un modèle d'allergie. En appliquant cette approche à l'étude de l'activation des mastocytes, nous avons pu identifier un nouvel acteur moléculaire impliqué dans la voie de signalisation mise en jeu. Ce travail a été protégé par un brevet européen en 2013 et est publié récemment. Dans le cadre de mon projet de thèse, j'ai construit une nouvelle banque synthétique (HUSCIv) optimisée pour la stabilité, la diversité et l'affinité des scFvs. Pour cela, le scFv 13R4 isolé dans notre équipe a servi de charpente pour le greffage des différentes boucles hypervariables, tout en respectant la diversité des régions CDR observée dans les anticorps naturels humains. Nous avons utilisé la protéine GFP en tant que rapporteur pour étudier le repliement et la solubilité des intracorps. Nos résultats ont clairement démontré que la plupart des intracorps issus de la banque HUSCIv sont soluble dans le cytoplasme des cellules mammifères. Mon projet de thèse décrit ici rapporte l'utilisation de la banque HUSCIv pour un criblage phénotypique dans des cellules de cancer colorectal portant une mutation du gène K-RAS et résistantes au traitement par l'anticorps chimérique Cetuximab. Le projet cherche à sélectionner des scFv capables de restaurer la sensibilité au Cetuximab, avec comme objectif l'identification des cibles intracellulaires impliquées.Pour ce criblage fonctionnel, la banque HUSCIv a été exprimée dans les cellules HCT116 par l'intermédiaire d'un système d'expression rétroviral. Le processus de sélection est basé sur la sélection directe de la prolifération des cellules en utilisant un colorant fluorescent (CMRA). Les cellules dont la prolifération est bloquée sont isolées et un séquençage à haut débit permet de suivre l'évolution des populations de scFv tout au long de l'expérience. Ainsi, ce projet a nécessité un séquençage profond d'un grand nombre de scFv afin de réaliser une analyse statistique. Nous avons réalisé à ce jour deux tours de sélection. Les tests de cytotoxicité réalisés sur les populations sélectionnées ont montré une inhibition significative de la prolifération en présence du Cetuximab d'environ 10%. Ces résultats indiquent l'évolution du phénotype qui tend vers une sélection de scFv inhibiteurs et suggèrent que nous devons réaliser au moins un ou plusieurs tours plus sélectifs avant de formuler des conclusions.L'approche introduite ici est différente de toutes les études existantes en ce qu'elle utilise des banques « naïves », et permet non seulement de répondre à la diversité du protéome, mais aussi d'étudier les messagers secondaires et le métabolisme des cellules. En tant que tel, et par rapport à d'autres approches à grande échelle, celle-ci représente une voie simple pour la découverte de molécules thérapeutiques potentielles. / Intracellular expression of antibodies (intrabodies) permitted the study and targeting of antigens in cellular compartments. However, the expression of functional intrabodies remains a difficult task due to their large size, structure, and the reducing intracellular environment. Our group has a strong expertise in the field of intracellular immunization and the identification of new therapeutic targets. For this purpose, we have developed an scFv library optimized for intracellular expression of scFv antibody fragments. Our previous works have shown the successful use of intrabodies for targeting specific domains or post-translational modifications in living cells. This is particularly important because it demonstrates one of the main advantages of intrabodies compared to the approaches using RNAi. This benefit was demonstrated by a phenotypic screen in a model of allergy. Applying this approach to the study of mast cell activation, we identified a new molecular player involved in the signaling pathway implemented. This work was protected by a European patent in 2013 and was recently published. As part of my thesis project, I designed a new synthetic library (HUSCIv) optimized for scFv stability, diversity and affinity. For this, a highly soluble and hyper-stable framework, scFv13R4 isolated in our group, was used as a scaffold for grafting different hypervariable loops, while respecting the diversity of CDRs observed in human natural antibodies. We used protein GFP as a reporter to study the folding and solubility of intrabodies. Our findings clearly demonstrated that most of the intrabodies from HUSCIv library are soluble in the cytoplasm of mammalian cells. My thesis project described here reports the use of HUSCIv in a phenotypic screen of colorectal cancer cells carrying a mutation in the K-RAS gene and resistant to the treatment with the chimeric antibody Cetuximab. The project seeks to select scFv fragments able to restore the sensitivity to Cetuximab, with the objective to identify the intracellular targets involved. For this functional screen, the HUSCIv library was expressed in HCT116 cells via a retroviral expression system. The selection process is based on the direct selection of cell proliferation using a fluorescent dye (CMRA). The cells whose proliferation is blocked are isolated and the evolution of scFv populations throughout the experiment are tracked via high-throughput sequencing. This sequencing requires a large number of scFvs to perform a statistical analysis. So far, we have achieved two rounds of selection. The cytotoxicity tests carried out on the selected populations showed a significant inhibition of proliferation (10%) in the presence of Cetuximab. These results indicate that the evolving phenotypes are tending towards a selection of scFv inhibitors and suggest that we need to perform at least one or more selective rounds before making conclusions. The approach introduced here is different from all existing studies in that it uses "naive" libraries not only to respond to the diversity of the proteome, but also to study secondary messengers and metabolism in cells. As such, and in comparison to other large-scale approaches, it is a simple way for the discovery of potential therapeutic molecules.

Le cancer colorectal (CCR) reste encore aujourd'hui une cause majeure de mortalité dans le monde, avec une incidence annuelle d'environ 1 million de cas et une mortalité d'environ 700 000 personnes. La stratégie de dépistage du CCR se base sur la détection de sang occulte dans les selles par le test iFOBT. Malgré une spécificité acceptable, la sensibilité de détection reste toujours insuffisante dans une population asymptomatique (27% de sensibilité pour un adénome, 65% pour un CCR). Aujourd'hui, il existe un réel besoin de trouver de nouveaux biomarqueurs spécifiques du CCR ainsi que de développer des tests de diagnostic faciles à mettre en oeuvre dans les laboratoires de biologie médicale. Les progrès récents de l'analyse protéomique à haut débit permettent aujourd'hui d'identifier et de quantifier un nombre toujours plus grand de protéines. C'est pourquoi, nous avons développé des stratégies en protéomique quantitative pour la recherche et la validation de biomarqueurs du cancer colorectal. L'approche iTRAQ et Label-free ont permis l'identification d'environ 800 protéines et la quantification de 214 protéines. Nous avons entrepris avec succès, la validation de 4 protéines par des tests ELISA et de nouvelles technologie en protéomique quantitative ciblée (LC-SRM). Au Final, nous proposons un test multiplex de 3 protéines qui permettra d'affirmer ou d'infirmer les résultats obtenus par le test iFOBT. L'ajout du test pourrait réduire de manière significative le nombre de coloscopies inutiles, potentiellement dangereuses pour les patients et onéreuses pour les autorités de santé. / Colorectal cancer (CRC) remains a major cause of cancer mortality throughout the world with an annual incidence of approximately 1 million cases and an annual mortality around 700 000. CRC outcome is highly dependent upon the stage of detection. The 5-year survival rate of patients with metastases is less than 20% while patients with localized adenomatous polyps have an excellent outcome with 90% survival rate. Current screening methods of CRC include the iFOBT and colonoscopy. The iFOBT has an acceptable 95% specificity but always an unsatisfactory sensitivity of detection in the asymptomatic population (27% sensitivity for adenoma, 65% for carcinoma). That is why, there is a real need to find new biomarkers and develop new diagnostic test, specific and easy to implement in clinical research. Recent advances in the analysis of biological fluids by high throughput mass spectrometry allow us now to identify and quantify a large number of proteins. Due to these improvements of proteomic strategies, it seems to be a promising way to find new potential proteins markers in the CRC disease. The quantitative iTRAQ & Label-free approaches allowed the identification of 800 proteins and the quantification of 200 proteins. Therefore, we have successfully validated four proteins by targeted proteomics using mass spectrometry (LC-SRM) and ELISA assays. Finally, we suggest a multiplex test of 3 proteins which confirm or contradict the FOBT outcome. The addition of the test could significantly reduce the number of unnecessary colonoscopies which are potentially dangerous for patients and expensive for public health authorities.

BACKGROUND: Colorectal cancer incidence and mortality rates are rising and are still one of the most common cancers worldwide. Its risks are associated with the Western Lifestyle, important facets of this being increase physical inactivity and excess body weight. In sync with colorectal cancer’s increasing rates are obesity rates that are rising in both developed and developing countries. Obesity has been linked to several causes of death that includes cardiovascular disease and certain cancers. Studies have shown the benefits of exercise overtly in alleviating cardiovascular diseases but now have increasing evidence of its benefits in helping with cancer. Some studies have examined the effects of physical activity in colon cancer and have found a link between increase minutes of exercise weekly can slow cancer cell proliferation in colon crypts cells. Myokines are a newly discovered class of proteins that are produced by muscles and have multiple functions. One of those function is its anti-neoplastic activity against certain cancer cell types. Certain myokines evoke an anti-neoplastic effect on cancer, but there has not been an overwhelming agreement within the scientific community. OBJECTIVE: To investigate the anti-proliferative effects of exercise myokines produced by C2C12 cells in colorectal cancer cell lines: HCT116 and HT29, by looking at changes in protein expression through western blot analysis and protein arrays. METHODS: C2C12 cells were grown in cell culture plates and differentiated into myotubules. Myotubules were subjected to Electronic Pulse Stimulation using an apparatus called C-Pace by Ion Optix to generate myokines. Treat HCT116 and HT29 cell lines with exercise myokines and a control treatment and ultimately harvest their protein lysate for western blot analysis and protein array analysis. RESULTS: HCT116 treated with exercise myokines with different dilutions (1:25, 1:50, 1:100) a demonstrated significantly decreased proliferation (p<0.0001). HT29 treated with exercise myokines (1:25 dilution) also demonstrated a significant decrease in proliferation (p<0.0001). Western blot analysis of HCT116 treated with exercise myokines showed significantly lower expression in pRb (p<0.05), PCNA (p<0.05), and cyclin D1 (p<0.05). Western blot analysis of HT29 treated with exercise myokines revealed significant expression downregulation in exercise myokine treatment in PCNA (p<0.05), p42/p44 (p<0.05), pRb (p<0.0001) and Cyclin D1 (p<0.05). Human Phospho-Kinase Array demonstrated downregulation of the following proteins in HT29 cells treated with exercise myokines: GSK-3α/β, STAT3, EGFR, p53, PDGF Rβ, Src, PRAS40, WNK1, and JNK 1/2/3. Lastly, HT29 treated with exercise myokines showed downregulation of the p-EGFR and pHGFR as a result of the Human Phospho-RKT Array. CONCLUSION: Our data demonstrate that exercise myokines produced by Electric Pulse Stimulation of C2C12 cells have an anti-proliferative effect on colorectal cancer cell lines HCT116 and HT29. Further studies should be pursued to identifying and linking anti-proliferative properties to a specific myokine protein and confirming a pathway that this specific myokine exerts its effects.

Pólipos adenomatosos colorretais são conhecidos como lesões pré-malignas. Mutações germinativas nas enzimas de reparo (MMR) hMLH1, hMSH2 e hMSH6 são causas reconhecidas de câncer colorretal hereditário não polipóide, induzindo um fenótipo mutante caracterizado por instabilidade de microssatélite (MSI). A MSI também é detectada em cânceres colorretais esporádicos. O Cox-2 é uma enzima induzível que regula a síntese de prostaglandina, sendo fortemente manifestada em locais de inflamação, pólipos adenomatosos colorretais e câncer. Objetivos: Avaliar a imunoexpressão do hMLH1, hMSH2 e Cox-2 em pólipos ressecados através da colonoscopia e associar às características clínico-patológicas (idade, sexo, localização, tamanho, histologia e grau de displasia). Métodos: 138 pacientes com pólipos colônicos, 6 com mucosa colônica normal e 23 pacientes com adenocarcinoma colorretal foram incluídos no estudo. Nenhum paciente apresentava histórico familiar de câncer colorretal. Cento e sessenta e sete amostras foram coletadas e imunocoradas para hMLH1, hMSH2 e Cox-2, usando a técnica ABC-imunohistoquímica e amplificação por tiramida biotinilada. Resultados: A média de idade foi 60,2 + 13,8 (variando de 21 a 90 anos de idade) sendo 77 homens (55,8%) e 61 mulheres (44,2%). Adenomas tubulares estavam presentes em 81,4%, tubulo-viloso em 15,9%, serrilhados em 1,8% e vilosos em 0,9%. A maioria dos adenomas localizava-se na região retossigmoideana (63,5%), seguido pelo ascendente em 14,2%, descendente em 8,2%, ceco em 7,5% e o transverso em 6,7%. Displasia de alto grau foi detectada em 46 (40,4%) e de baixo grau em 68 (59,6%) dos adenomas. Perda de imunoexpressão hMLH1 e hMLH2 foi observada em 20% e 15,5% dos adenomas, respectivamente. O Cox-2 foi positivo em 9% dos adenomas e em 40% dos adenocarcinomas. Além disso, a imunoexpressão Cox-2 foi associada à multiplicidade de adenomas no mesmo paciente (p=0,001). Não houve associação entre a imunoexpressão de marcadores e o sexo, idade, localização, tamanho, histologia ou grau de displasia. Conclusões: 1. A perda de imunoexpressão hMLH1 e hMLH2 em adenomas é relativamente freqüente em pacientes sem histórico familiar de câncer colorretal; 2. O Cox-2 é fortemente manifestado em pólipos adenomatosos e adenocarcinomas colorrretais sendo que sua positividade em adenomas pode indicar um risco maior de lesões múltiplas. / Colorectal adenomatous polyps are known as premalignant lesions. Mutations in the mismatch repair (MMR) enzymes hMLH1, hMSH2 and hMSH6 are recognized causes of hereditary nonpolyposis colorectal cancer and act by inducing a mutator phenotype characterized by microsatellite instability (MSI). MSI is also detected in sporadic colorectal cancers. Cox-2 is an inducible enzyme that regulates prostaglandin synthesis and is overexpressed at sites of inflammation, in colorectal adenomatous polyps and cancer. Aims: To evaluate the immunoexpression of hMLH1, hMSH2 and Cox-2 in resected polyps through colonoscopy, and to associate to the clinicopathologic characteristics (age, gender, location, size, histology and grade of dysplasia). Methods: 138 patients with colonic polyps, 6 with normal colonic mucosa and 23 patients with colorectal adenocarcinoma were enrolled in this study. All patients had no familial history of colorectal cancer. The 167 samples were collected and immunostained for hMLH1, hMSH2 and Cox-2 using the ABC-immunohistochemistry technique and amplification by biotinylated tyramide. Results: The mean age was 60.2 + 13.8 (varying from 21 to 90 years-old) where 77 (55.8%) were men and 61 women (44.2%). Tubular adenomas were present in 81.4%, tubulous-villous in 15.9%, serrated in 1.8%, and villous in 0.9%. The majority of the adenomas were located in the retossigmoid region (63.5%), followed by ascendent in 14.2%, descendent in 8.2%, cecum in 7.5% and transverse in 6.7%. High-grade dysplasia was detected in 46 (40.4%) and low-grade dysplasia in 68 (59.6%) of the adenomas. Loss of hMLH1 and hMLH2 immunoexpression was observed in 20% and 15.5% of the adenomas, respectively. Cox-2 was positive in 9% of the adenomas, and in 40% of the adenocarcinomas. Moreover, Cox-2 immunoexpression was associated to the multiplicity of adenomas in the same patient, p=0.001. There was no association between marker immunoexpression and gender, age, location, size, histology or grade of dysplasia. Conclusions: 1. Loss of hMLH1 and hMLH2 immunoexpression in adenomas is relatively frequent in patients without colorectal cancer familial history; 2. Cox-2 is overexpressed in colorectal adenomatous polyps and adenocarcinomas, and its positivity in adenomas may indicate higher risk for multiple lesions.

Trata-se de uma revisão integrativa da literatura, que teve como objetivos buscar e sintetizar as evidências disponíveis na literatura científica relacionado aos fatores de risco alimentares para o câncer colorretal; categorizar os aspectos relacionados aos fatores de risco alimentares levantados e; selecionar as recomendações em educação em saúde especifica para promoção e prevenção para o câncer colorretal, aliada a uma alimentação saudável. Para a seleção dos estudos, foram consultadas as bases de dados LILACS, PUBMED, CINAHL e COCHRANE Library e a amostra constituiu-se de 26 estudos. Foram identificados 21 estudos (80,8%) indexados na base de dados PUBMED e 5 estudos (19,2%) na COCHRANE Library. Houve uma prevalência de 16 estudos (61,5%) com nível de evidência 2 e 10 estudos (38,5%) com nível de evidência 1. Após a análise dos aspectos abordados nestes artigos, reuniu-se em 4 categorias temáticas com a abordagem dos seguintes aspectos: 1- o consumo de carnes e os fatores de risco para câncer colorretal, composta por 6 estudos (23,1%); 2- ácido fólico, fibras, ?-tocoferol e ?-caroteno e os fatores de risco para câncer colorretal, com 6 estudos (23,1%); 3- os fatores de risco associados a suplementação pelo cálcio e vitamina D, com 8 estudos (30,7%); 4- baixo índice glicêmico e de gordura, aumento do consumo de frutas, vegetais e simbióticos como redutores do risco de câncer colorretal, com 6 estudos (23,1%). Na categoria 1 as metanálises encontraram associações de que a ingestão de carne vermelha está relacionada com o aumento do risco para câncer colorretal em 28% a 35%, enquanto que a carne processada está associada com o risco elevado de 20% a 49%. Na categoria 2, as evidências encontradas sobre a suplementação por ácido fólico, antioxidantes e fibras não demonstram segurança em seus dados, uma vez que sugerem a necessidade de mais estudos aprofundados sobre a temática em questão. Quanto a categoria 3, conclui de forma convincente que o alto consumo de cálcio reduz o risco para o câncer colorretal em 45% e quando associado a vitamina D oferece um efeito quimioprotetor ao intervir na carcinogênese colorretal. A categoria 4, encontrou-se uma associação entre o índice e a taxa glicêmicos com o risco para o câncer colorretal, houve uma moderada redução no risco de câncer colorretal associado ao alto consumo de vegetais e frutas, a redução do consumo de gorduras e aumento consumo de frutas e vegetais não reduziu o risco para câncer colorretal invasivo durante o período de seguimento, a intervenção simbiótica não pode afirmar sobre o seu efeito protetor para o câncer colorretal. As recomendações presentes nesta revisão integrativa almejam a promoção e prevenção aos fatores de risco para o câncer colorretal, uma vez que a enfermagem destaca-se como forte agente capaz de estimular a adoção de hábitos alimentares saudáveis, além de oferecer embasamento nutricional juntamente com os demais profissionais da equipe de saúde. / It is an integrative literature review, which aimed to search and synthesize the available evidence in the scientific literature related to dietary risk factors for colorectal cancer; to characterize the the aspects related to risk factors and food surveyed; select the recommendations in health education specific to promotion and prevention of colorectal cancer, combined with a healthy diet. For the studies\' selection were consulted the databases LILACS, PubMed, CINAHL and Cochrane Library, and the sample consisted of 26 studies. It was identified 21 studies (80.8%) indexed in the databases PUBMED and five studies (19.2%) in the Cochrane Library. There was a prevalence of 16 studies (61.5%) with level of evidence 2 and 10 studies (38.5%) with a level of evidence 1. After analyzing the issues addressed in these articles, and were grouped in four themes with the approach of the following: 1- the consumption of meat and the risk factors for colorectal cancer, consisting of six studies (23.1%); 2- folic acid, fiber, ?-tocopherol and ?-carotene and the risk factors for colorectal cancer, 6 studies (23.1%); 3- the risk factors associated with supplementation with calcium and vitamin D, with 8 studies (30.7%); 4- low glycemic index and fat, increased consumption of fruits, vegetables and symbiotic as reducing the risk of colorectal cancer, 6 studies (23.1%). In the category 1, the meta-analysis found that the associations of intake of red meat is related with increased risk for colorectal cancer by 28% to 35%, while the processed meat is associated with increased risk of 20% to 49%. In the category 2, the evidence found on the supplementation of folic acid, antioxidants and fiber do not demonstrate safety of your data, since they suggest the need for more detailed studies on the topic in question. In the category 3, the conclusion was convincingly that high calcium intake reduces the risk for colorectal cancer by 45% and when combined with vitamin D offers a chemoprotective effect by intervening in colorectal carcinogenesis. The fourth category, it was found an association between glycemic index and rate the risk for colorectal cancer, there was a moderate reduction in risk of colorectal cancer associated with high intake of fruits and vegetables, reducing fat intake and increased consumption fruits and vegetables did not reduce the risk to invasive colorectal cancer during the following period, the symbiotic action can not be guaranteed about the protective effect for colorectal cancer. The recommendations in this integrative review aimed the promotion and prevention of the risk factors\' colorectal cancer, since that nursing stands out as a strong agent able to stimulate the adoption of healthy eating habits, and in addition provides nutrition knowledge with other professionals of the health team.

A atividade da serotonina (5-HT) pode estar envolvida no desenvolvimento do câncer colorretal, embora detalhes desse processo permaneçam desconhecidos. Camundongos knockout (KO) para triptofano hidroxilase 1 (Tph1) falham em sintetizar 5-HT e fornecem um sistema modelo apropriado para estudar o papel deste neuro-hormônio na carcinogênese colorretal. Animais Tph1KO apresentaram um desenvolvimento reduzido de tumores alográficos e tumores colorretais induzidos por colite. No entanto, o tratamento carcinogênico colorretal promoveu um aumento do número de tumores induzidos pela exposição à um carcinógeno. Também se observou uma alta intensidade de dano de DNA no nicho de células-tronco do cólon, bem como uma redução na populações de células enteroendócrinas e aumento daquelas unidades caliciformes. Antes da ativação da ataxia telangiectasia relacionada a Rad 3 (ATR) e proteína 53 relacionada à transformação (Trp53), a síntese de 5-HT promoveu a atividade da O-6-metilguanina-DNA metiltransferase (MGMT) em resposta à exposição carcinogênica. O resgata da síntese de 5-HT através do tratamento com 5-hidroxitriptofano reduziu o dano de DNA induzido pela radiação. O papel protetor da 5-HT foi confirmado em camundongos transgênicos com perda específica da expressão de Tph1 nos intestinos. Esta investigação demonstra um papel protetor da síntese de 5-HT contra o desenvolvimento da carcinogênese colorretal. / Serotonin (5-HT) activity may impact on the development of colorectal cancer. However, details of this process remain unknown. Tryptophan hydroxylase 1 knockout (Tph1KO) mice fail to synthesize 5-HT and provide an appropriate model system to study the role of this neurohormone in colorectal carcinogenesis. Tph1KO mice have a decreased development of allograft tumors and colitis-induced colorectal tumors. However, colorectal carcinogen treatment led to increased tumor numbers, with high DNA damage intensity in the colonic stem cell niche. It was related to decreased numbers of enteroendocrine cells but an increase in the goblet cell population. Indeed, 5-HT synthesis promoted O-6-methylguanine-DNA methyltransferase activity in response to azoxymethane before activating ataxia telangiectasia and Rad3 related (ATR) and transformation related protein 53 (Trp53) expression. Radiation-induced DNA damage could be rescued by 5-hydroxytryptophan. The protective role of 5-HT was confirmed in transgenic mice with intestine-specific loss of Tph1 expression. This investigation reveals a protective role of 5-HT synthesis against the development of colorectal carcinogenesis.

Introdução: O Câncer Colorretal (CCR) localmente avançado é caracterizado pela aderência ou invasão do tumor primário a órgãos ou estruturas vizinhas. Nesses pacientes, a realização de cirurgia alargada por meio de ressecção multivisceral em bloco constitui a melhor alternativa para prover perspectivas de cura. Os objetivos desse estudo foram estimar a incidência das lesões localmente avançadas em nosso meio, avaliar os resultados operatórios e investigar os fatores que influenciaram o prognóstico. Pacientes e Métodos: realizou-se estudo coorte retrospectivo incluindo 679 pacientes com CCR que foram submetidos a tratamento cirúrgico entre 1995 a 2007. Foram anotados dados clínicos (idade, sexo e co-morbidades), cirúrgicos e histológicos (localização da neoplasia, órgãos adjacentes comprometidos e disseminação tumoral) dos pacientes portadores de neoplasia colorretal localmente avançada. Os índices de sobrevida foram estimados pela curva de Kaplan Meier considerando apenas os doentes que foram submetidos à operações com intenção curativa. Resultados: 90 pacientes (13,2%) com tumor localmente avançado foram identificados. A idade média foi de 59 anos e houve predomínio no sexo feminino (61%). A distribuição topográfica demonstrou que 66% dos tumores localizavam-se no cólon e 34% no reto. A distribuição quanto ao sexo revelou maior prevalência dos tumores retais entre as mulheres (77%; p=0,02). Complicações no pós-operatório foram registradas em 25,6% dos doentes, representados principalmente por deiscência e/ou infecção da parede abdominal (22%), íleo prolongado (14,8%) e deiscência de anastomose (11,1%). A mortalidade peri-operatória foi de 3,3%. Os órgãos mais freqüentemente envolvidos foram o intestino delgado (19,9%), bexiga (16,4%) e útero (12,9%). Quanto à penetração tumoral, foram detectadas lesões T4 em 58% e lesões T3 em 42%. A sobrevida de 5 anos foi menor entre os tumores T4 em relação às lesões T3 (50% vs. 75%; p=0,01). Em média, 21,6 linfonodos (LN) foram ressecados durante os procedimentos e o envolvimento linfonodal pela neoplasia determinou sobrevida menor (35% LN+ vs. 80%LN -; p=0,004). Observou-se também redução dos índices de sobrevida associados a outros fatores como a presença da invasão vascular, linfática e perineural (35% vs. 80%; p=0,02), e localização retal dos tumores quando comparada às lesões colônicas (45% vs. 65%; p=0,01). Por outro lado, o caráter neoplásico (59%) ou inflamatório (41%) das aderências não influenciou significativamente os índices de sobrevida (55% vs. 65%, p=0,60). Conclusão: 1) durante o período de estudo, detectaram-se lesões localmente avançadas em 13,2% dos pacientes; 2) ocorreram complicações operatórias em 25% dos procedimentos; 3) a sobrevida dos pacientes submetidos a operações com intenção curativa sofreu impacto negativo na presença de maior penetração na parede, invasão vascular, linfática e perineural, nos tumores de localização retal e naqueles com linfonodos comprometidos; 4) outras variáveis como tipo histológico, grau de diferenciação tumoral, número de órgãos ressecados, transfusão de sangue e caráter das aderências entre órgãos não influenciaram as chances de sobrevida / Locally advanced colorectal tumors are characterized by adherence or invasion of the primary tumor into surrounding structures and organs. For these patients, an en-bloc multivisceral resection represents the best alternative for cure. The aims of this study were to estimate the incidence of locally advanced lesions in our service, to evaluate operative results and to investigate factors that could influence prognosis. A retrospective cohort study was performed including 679 patients with colorectal cancer (CRC) who underwent surgery from 1995 to 2007. Clinical (age, gender, comorbidities), surgical and histological (tumor location, involved organs and tumor spreading) data were collected from patients with locally advanced colorectal cancer. Survival rates were estimated by the Kaplan-Meier curve considering only patients who underwent curative procedures. Ninety patients (13.2%) with locally advanced tumors were identified. Average age was 59 years and there was a female predominance (61%). Regarding topographic distribution, 66% of the lesions were colonic and 34% were located in the rectum. Gender distribution showed a higher prevalence of rectal tumors among women (77%; p=0.02). Postoperative morbidity occurred in 25.6% of the patients, the most common being abdominal wall infection (22%), prolonged ileus (14.8%) and anastomosis dehiscence (11.1%). Mortality rate was 3.3%. Involvement of adjacent organs was more frequently detected with the small intestine (19.9%), bladder (16.4%) and uterus (12.9%). Concerning tumor penetration, there were detected T4 lesions in 58% and T3 lesions in 42%; Five years survival was smaller in the former lesions (50% vs. 75%; p=0.01). During surgery 21.6 lymph nodes were resected on average, and the presence of positive nodes determined shorter survival (35% vs. 80%, p=0.004). It was also observed shorter survival rates associated with other factors such as the presence of vascular, lymphatic, and perineural invasion (35% vs. 80%, p=0.02) and rectal tumor location compared to colonic ones (45% vs. 65%, p=0.01). On the other hand, the neoplastic (59%) or inflammatory nature (41%) of adhesions did not significantly influence survival rates (55% vs. 65%, p=0.60). During the period of study it was possible to conclude that 1) locally advanced lesions represented 13.2% of the patients; 2) there were operative complications in 25% of the procedures; 3) survival of patients undergoing curative-intended surgery had a negative impact with deeper tumor wall penetration, vascular, lymphatic and/or perineural invasion, rectal location (compared to colonic) and positive lymph nodes. 4) other variables such as histological type, tumoral differentiation and number of resected organs, blood transfusion and character of adhesions between organs did not affect chances of survival

O rearranjo da anatomia gastrointestinal é atualmente o foco de estudos para a remissão e cura do diabetes mellitus tipo 2. Há evidências a favor da melhora desta comorbidade após a gastrectomia em pacientes não obesos, entretanto sem análise de longo prazo. O intestino grosso, como integrante do aparelho digestório e potencialmente produtor de incretinas, ainda não foi estudado em relação à influência de sua retirada (colectomia) no desfecho do diabetes. Nestas circunstâncias, faz-se necessário um estudo em longo prazo destas duas cirurgias na homeostase glicídica. Objetivos: Analisar a resposta em longo prazo do diabetes e pré-diabetes pré-existentes após gastrectomia, bem como, colectomia por câncer. Métodos: Foram analisados pacientes adultos submetidos à gastrectomia subtotal e total (Y de Roux) por câncer gástrico e colectomia (direita ou retossigmoidectomia) por câncer de colo e reto há mais de 3 anos e sem sinais de doença em atividade. Incluíram-se controles nas duas situações de pós-operatório tardio com homeostase glicídica normal, a fim de averiguar também sua evolução em longo prazo. Agregou-se ainda um grupo controle pré-operatório atual constituído de doentes diabéticos com câncer gástrico, candidatos à gastrectomia curativa, pois alguns exames analisados no pós-operatório não haviam sido coletados no período pré-operatório. Os pacientes foram divididos de acordo com a presença e curso clínico do diabetes. O grupo diabético foi subdividido em Refratários (permaneceram diabéticos) e Responsivos (remissão parcial ou completa). O grupo controle foi dividido em Estáveis (permaneceram sem diabetes) e Neodiabetes (ficaram diabéticos ou pré-diabéticos). Também foram avaliados de acordo com o tipo de cirurgia. Foram coletados exames de albumina, transferrina, ferritina, ferro sérico, hemoglobina, leucócitos, colesterol total, LDL, VLDL e HDL, triglicérides, insulina, hemoglobina A1C, Peptídeo C, IGF-1, Leptina, proteína C reativa, fibrinogênio, tempo de protrombina, dímero D, complemento C3 e C4, ácido fólico e vitamina B12. Peso, altura e perímetro abdominal e do quadril também foram analisados. Resultados: Os seguimentos atingiram 86,8 ± 25,1 meses nos gastrectomizados e 79,2 ±27,4 nos colectomizados. Os pacientes gastrectomizados beneficiaram-se com remissão do diabetes em 41,2% dos casos e os colectomizados em 32,4%. No grupo controle de gastrectomizados surgiram em longo prazo 63,2% de neodiabéticos e no de colectomizados 30,8%. Nos pacientes diabéticos responsivos com câncer gástrico houve paralelismo entre queda da glicemia e do IMC, algo que não se sucedeu nos acometidos de câncer colorretal. Em nenhuma das duas populações refratárias pode-se atribuir um papel para a variação do IMC no desfecho do diabetes. Nos pacientes neodiabéticos nenhuma contribuição do IMC foi despistada em quaisquer dos grupos estudados. Os pacientes diabéticos gastrectomizados e colectomizados não revelaram diferenças significativas nos valores de glicemia e prevalência de diabetes quando separados por tipo de cirurgia. Conclusão: Nos pacientes gastrectomizados, comprovou-se remissão do diabetes a longo prazo ainda que em proporções menores que as documentadas usualmente, com obesos tratados por cirurgia bariátrica. A perda de peso demonstrou influência positiva na resposta do diabetes. A exclusão duodenal pode ser outro fator envolvido na melhora do diabetes ao lado deste moderado emagrecimento. Já a remoção do fundo gástrico não demonstrou influência na evolução destes pacientes. Não há indícios de que a gastrectomia protegeu contra o aparecimento de novos casos de diabetes e pré-diabetes, pois estes se manifestaram em elevadas proporções. Os pacientes colectomizados diabéticos apresentaram taxa ligeiramente menor de responsivos quando comparados com o grupo bariátrico, sem diferenças significativas. A variação do peso e o local de ressecção não se relacionaram com o desfecho do diabetes. As conversões de pacientes normoglicêmicos para diabetes, ao final do tempo de seguimento, foram mais baixas que nos gastrectomizados. São necessários mais estudos para elucidar os mecanismos envolvidos na história natural da homeostase glicídica, tanto após gastrectomia quanto cirurgia colorretal / The rearrangement of gastrointestinal anatomy is currently the focus of research for the cure and remission of type 2 diabetes mellitus. There is evidence suggesting that this comorbidity improves after cancer gastrectomy in non-obese patients, however no long-term analysis is available. The large intestine, as part of the digestive system and potentially producing incretins, has not been studied with regard to the influence of surgical removal (colectomy) on the outcome of diabetes. In these circumstances, a study focusing the long-term outcome of glucose homeostasis after these two surgeries was deemed appropriate. Objectives: Analysis of the long-term response of preexisting diabetes and pre-diabetes after gastrectomy and colectomy for cancer. Population: Adult patients who underwent subtotal and total (Roux-en Y) gastrectomy for gastric cancer, as well as colorectal operation (right hemicolectomy or anterior resection) for colon and rectum cancer, with at least 3 years of follow up and no signs of active disease. Controls with normal glucose homeostasis were included in both contexts, in order to investigate long term evolution also in euglycemic subjects. A current preoperative control group was added consisting of diabetic patients with gastric cancer, aiming to provide information not available in the retrospective analysis of the preoperative period. Patients were divided according to the presence and clinical course of diabetes. The diabetes group was divided into Refractory (remained diabetic) and Responsive cases (partial or complete remission). The control group was similarly divided into Stable (remained without diabetes) and New onset diabetes/NOD (became diabetic or pre-diabetic). Surgical modality was also considered in the stratification (subtotal versus total gastrectomy and right colectomy versus anterior resection). Biochemical tests included albumin, transferrin, ferritin, serum iron, hemoglobin, white blood cell count, total cholesterol, LDL, VLDL, and HDL, triglycerides, insulin, hemoglobin A1C, C-peptide, IGF-1, leptin, C-reactive protein, fibrinogen, prothrombin time, D-dimer, complement C3 and C4, folic acid and vitamin B12. Weight, height and waist/hip ratio were also documented. Results: The follow-up reached 86.8 ± 25.1 months in patients submitted to gastrectomy and 79.2 ± 27.4 in colorectal surgery. Gastrectomized patients benefited from diabetes remission in 41.2% of cases and 32.4% after large bowel operation. NOD was detected in 63.2% and 30.8% of nondiabetic subjects, respectively. Among responsive diabetic patients with gastric cancer direct relation between fall in blood glucose and BMI was demonstrated, but not with colorectal cancer. In both refractory populations BMI failed to correlated with outcome of diabetes. In NOD patients no contribution of BMI was shown in any group either. Prevalence of diabetes was not different when stratified according to type of surgery. Conclusion: In gastrectomized patients, long term remission of diabetes was confirmed, even though is smaller proportions than those reported after bariatric surgery. Weight loss showed a positive influence in the responsive diabetes population submitted to gastrectomy. There are reasons to believe that duodenal exclusion was involved as well in diabetes amelioration, besides moderate weight loss. The removal of the gastric fundus was not relevant for the evolution of these patients. No evidence in favor of gastrectomy protection against the onset of new diabetes and pre-diabetes was detected. On the contrary, these were registered in high proportions. Colorectal diabetic patients had slightly lower rate of response when compared to the gastrectomy group, without significance. Weight shift and location of the resection were unrelated to the outcome of diabetes. NOD cases at the end of follow-up period were less frequent than after gastric surgery. Further studies are needed to elucidate the mechanisms involved in the natural history of glucose homeostasis, after both gastric and colorectal surgery

O câncer colorretal (CCR) é a segunda de causa morte relacionada ao câncer, com aproximadamente 500.000 mortes por ano em países desenvolvidos. Os pacientes com doença localmente avançada ou metastática possuem um prognóstico ruim e, desta forma, a detecção precoce é necessária para reduzir a alta mortalidade associada à essa doença. Ainda, os métodos de rastreamento utilizados no momento apresentam boa exatidão, porém são de alto custo, consomem muito tempo e são incômodos para os pacientes. A colonoscopia é o método mais comumente utilizado para a detecção de CCR. Entretanto, apresentam duas limitações importantes: i) necessidade da luz intestinal estar limpa, sem resíduos fecais, o que só é conseguido após um preparo rigoroso, com dieta e laxativos, nos dias que antecedem a realização do exame, e, ii) é examinador-dependente, ou seja, dependem da experiência do profissional em localizar as alterações anatômicas, e ainda da habilidade em selecionar o melhor local para a realização da biópsia. Portanto, o desenvolvimento de um método rápido e sensível para o diagnóstico do câncer colorretal é extremamente importante.<br /> A proposta desta dissertação foi desenvolver um método eficaz para o preparo dos tecidos parafinizados e extração de proteínas para experimentos em eletroforese bidimensional (2DE). Tecidos fixados em formaldeído e embebidos em parafina apresentam como vantagens a preservação da qualidade morfológica, abundância e facilidade de armazenamento, o que permite estudos futuros. Entretanto, para análises proteômicas, a recuperação do material biológico é ineficiente. A partir deste estudo, 156 proteínas foram obtidas de tecidos parafinizados, sendo possível identificar por espectrometria de massas, levando a estudos posteriores para identificação de possíveis biomarcadores relacionados ao CCR. / Colorectal cancer (CRC) is the second cause of cancer-related death, with approximately 500,000 deaths per year in developed countries. Patients with locally advanced or metastatic disease have a poor prognosis and, thus, early detection is needed to reduce the high mortality associated with this disease. Yet, the screening methods used at the time have good accuracy, but are expensive, time consuming and are uncomfortable for patients. Colonoscopy is the most commonly used method for the detection of CRC. However, present two major limitations: i) the need to be clean intestinal lumen without fecal waste, which is achieved only after rigorous preparation with laxatives and diet, in the days before the exam, and ii) it is examiner-dependent, i.e., depends on the professional\'s experience in locating anatomical changes, and even the ability to select the best site for biopsy. Therefore, the development of a rapid and sensitive method for the diagnosis of colorectal cancer is extremely important. The purpose of this dissertation was to develop an effective method for the preparation of tissues and protein extraction experiments for two-dimensional electrophoresis (2DE). Tissues fixed in formaldehyde and embedded in paraffin have the advantage of preserving the morphological quality, abundance and storage facility, which allows for further studies. However, for proteomic analysis, the recovery of biological material is inefficient. From this study, 156 proteins were obtained from paraffinized tissues, and can be identified by mass spectrometry, leading to further studies to identify potential biomarkers related to CCR.

The DNA Mismatch repair (MMR) pathway is responsible for the repair of base-base mismatches and insertion/deletion loops, formed during DNA replication. Mutations in MMR genes significantly increase the predisposition to cancer with MMR deficiency estimated to be present in 15-17 % of all colorectal cancers. 5-fluorouracil is the main treatment for advanced colorectal cancer however the majority of studies suggest that MMR deficient tumours are more resistant to 5-fluorouracil than MMR proficient tumours. Therefore, there is a critical clinical need to identify novel therapeutics to treat these tumours. To this end, we have performed a high-throughput compound screen, to identify compounds that cause selective lethality in MMR deficient cell lines. We identified the potassium-sparing diuretic drug, Triamterene, as selectively lethal in vitro and in vivo in MMR deficient cell lines. Our data suggest that this selectivity is through its antifolate activity, leading to the accumulation of reactive oxygen species and DNA double strand breaks in MMR deficient cells. Interestingly, we identified a requirement, for thymidylate synthase expression, the only de novo enzyme for dTTP synthesis for the Triamterene cytotoxicity. NRF2 and NRF2-induced antioxidants were regulated upon Triamterene treatment and thymidylate synthase silencing, therefore suggesting a role for the antioxidant response in Triamterene toxicity. Taken together, our results suggest Triamterene as a promising novel therapeutic for the treatment of MMR deficient cancers. In order to identify novel therapeutics to treat MMR deficient tumours, we have also performed a high-throughput siRNA screen, to identify genes that cause selective lethality in MMR deficient cell lines. We identified AURKA gene as synthetically lethal in MSH6 deficient cell lines which suggests AURKA as a promising novel therapeutic target for the treatment of MMR deficient cancers. Taken together, in this PhD thesis we have identified two novel therapeutic strategies for the treatment of MMR deficient cancers.