La tyrosine kinase cytoplasmique Src est un régulateur essentiel de la croissance et de l’adhésion cellulaires induites par de nombreux stimuli extracellulaires, dont les facteurs de croissance et certains composants de la matrice extracellulaire. La dérégulation de son activité catalytique lui confère des propriétés oncogéniques importantes conduisant à la formation de tumeurs agressives chez l’Homme. La plupart des connaissances actuelles sur sa régulation catalytique repose sur des données structurales de cristallographie qui ont révélé l’importance des interactions intramoléculaires SH2 et SH3-dépendantes dans le contrôle des conformations ouvertes et actives de Src. Les domaines SH3, SH2 et kinase de Src sont associés au domaine SH4 N-terminal d'ancrage à la membrane plasmique via un domaine unique (DU) flexible. Le rôle du DU dans la régulation de Src reste obscur car il est intrinsèquement désordonné et par conséquent, cette région n'a pas été incluse dans les analyses structurales originelles. L'analyse par RMN de l'extrémité N-terminale de Src a révélé une conformation partiellement structurée du DU par le contact de séquences peptidiques spécifiques avec les lipides membranaires et le domaine SH3 (Perez et al, 2013; Maffei et al, 2015). De plus, cette interaction est régulée par phosphorylation des Ser69 et Ser75 localisées à proximité de ce domaine. L’objectif de ma thèse était d’adresser la relevance biologique de ces données structurales en me focalisant sur le rôle d’une partie du DU identifiée comme importante par RMN. J’ai montré que l’inactivation de cette région par des mutations perte de fonction, diminue fortement la signalisation de Src dans les cellules non transformées humaines ainsi que ses propriétés tumorales dans les cellules cancéreuses colorectales métastatiques révélant la pertinence biomédicale du système. Des analyses de protéomique m’ont permis de révéler un mécanisme original par lequel cette région contrôle la capacité de Src à phosphoryler ses substrats. En conclusion, ces données confirment un rôle important du DU sur l'activité et la signalisation de Src et révèlent un rôle critique des domaines désordonnés dans les tumeurs chez l’Homme. L’inhibition de cette région unique par de petites molécules devrait conduire à de nouvelles stratégies thérapeutiques dans le cancer. / The membrane-anchored non-receptor tyrosine kinase Src is involved in numerous signal transduction pathways and hyperactive Src is a potent oncogene and driver of human metastasis. Most of our knowledge on Src kinase regulation relies on structural data, which revealed SH2 and SH3-dependent intramolecular interactions to control active conformations of the protein. The kinase, SH3 and SH2 domains of Src are attached to the membrane-anchored SH4 domain through the flexible unique domain (UD). The role of this UD remains obscure due to its intrinsically disordered properties for which reason it was not included in original structural analyses. Interestingly, membrane-associated intrinsic disordered domains are more prevalent among signaling and cancer-related proteins and they are thought to play critical roles in human disease. NMR analysis of Src N-terminus in the presence of lipids revealed a partially structured conformation of the UD through the contact of a small peptide sequence with membrane lipids and the SH3 domain of the protein (Perez et al, 2013; Maffei et al, 2015). This interaction was regulated by phosphorylation of Ser69 and Ser75 surrounded this central region. The aim of my thesis project was to assess the biological relevance of this structural data. Interestingly, I showed that expression of Src mutants with UD loss of function drastically affected Src activity and signaling in human non-transformed cells as well as Src oncogenic properties in metastatic colorectal cancer cells. This highlights the biomedical relevance of the system. Further proteomic analysis revealed an unsuspected mechanism by which this region controls the Src capacity to phosphorylate specific substrates involved in cancer cell activity. These data support a previously unrecognized important role of the UD on Src activity and signaling and reveals a critical role of intrinsic disordered domains in the regulation of kinase signaling in human disease. Targeting this unique region by small molecules may be of therapeutic value in human cancer.

La protéine Mlh1 eucaryote est un acteur central de la voie de réparation des mésappariements (MMR). Chez la levure, Mlh1 forme un hétérodimère via sa région C-terminale avec les endonucléases Pms1 et Mlh3. La région C-terminale de Mlh1 est également en interactions avec l’exonucléase Exo1 du MMR et deux protéines Ntg2 et Sgs1 qui sont impliquées dans d’autres voies de réparation. Dans un premier temps, nous avons identifié et caractérisé le site d’interaction de Mlh1 avec les protéines Exo1, Ntg2 et Sgs1, qui utilisent un même motif de 5 acides aminés, (R/K)SK(Y/F)F appelé motif MIP pour Mlh1 Interacting Protein. Nous avons montré que ces 3 protéines interagissent en un même site, appelé site S2. Nous avons identifié 10 positions de Mlh1 impliquées dans le site S2 et caractérisé par microcalorimétrie, une affinité micromolaire entre des peptides contenant le motif MIP et la région C-terminale de Mlh1. Nous avons montré que les protéines EXO1 et BLM humaines qui possèdent également un motif MIP, interagissent spécifiquement avec MLH1 humain par ce motif. Dans un second temps, nous avons résolu la structure cristallographique à 2.6Å de la région C-terminale de l’hétérodimère Mlh1*Pms1. Le site d’hétérodimèrisation présente une surface d’interaction supérieure à celle observée dans les homodimères de MutL bactériens. La structure résolue confirme le rôle des 10 acides aminés de Mlh1 identifiés lors de la caractérisation du site S2. La structure du site endonucléase de Pms1 révèle la présence de deux atomes de zinc chelatés par 5 acides aminés de Pms1 et le dernier acide aminé de la protéine Mlh1, la cystéine C769. Cette première structure d’une région C-terminale d’un complexe Mlh1*Pms1 eucaryote permet d’analyser la position des nombreux mutants ponctuels de MLH1 humain associés à des cancers du côlon HNPCC. / Eucaryotic Mlh1 is a core component of mismatch repair pathway (MMR). In yeast organisms, Mlh1 forms heterodimer with its C-terminal region with endonucleases Pms1 and Mlh3. The C-terminal region of Mlh1 is also involved in interactions with MMR exonuclease Exo1 and two proteins, Ntg2 and Sgs1, which are involved in other DNA repair pathways. First, we identified and charaterised the interaction site between Mlh1 and proteins Exo1, Ntg2, and Sgs1, that share the same motif of 5 amino acids, (R/K)SK(Y/F)F, named MIP box for Mlh1 Interacting Protein. We showed that these 3 proteins bind to the same site, named site S2. 10 positions of Mlh1 important for interactions on site S2 were identified and a micromolar affinity was measured by calorimetry between the C-terminal region of Mlh1 and peptides containing a MIP box. We showed that human EXO1 and BLM specifically with human MLH1 through their MIP box. Secondly, we solved the X-ray structure of the C-terminal region of Mlh1*Pms1 heterodimer at 2.6Å. The structure shows that the surface buried upon heterodimerisation is higher in eucaryotes than in MutL homodimers. The structure confirms the overall structure of the site S2 predicted in the first part of this study. The endonuclease site of Pms1 presents in the crystal two zinc atoms that are bound by five Pms1 residues and the last residue of Mlh1 chain, cystein C769. This structure represents the first image of the C-terminal region of an eucaryote Mlh1*Pms1 heterodimer. It allows localizing the positions of human MLH1 mutants associated with colon cancers named HNPCC.

HEF1 (Human Enhancer of Filamentation 1) est une protéine d’ancrage multi-domaine de la famille CAS. Ces protéines sont impliquées dans des modifications du cytosquelette et sont des médiateurs de l’activation par les intégrines des cascades de signalisation au niveau des adhérences focales. Des données récentes ont mis en évidence une expression élevée de HEF1 associée à une capacité de migration et d’invasion cellulaire accrue dans plusieurs types de cancer. Ainsi, un rôle de la protéine dans la progression tumorale et le développement métastatique a été suggéré. HEF1 est une phosphoprotéine. Outre sa phosphorylation sur résidus tyrosine par les kinases FAK et Src bien décrite, elle est également phosphorylée sur des sérines: la sérine 296 et la sérine 369 identifiée par notre laboratoire comme étant un régulateur de la dégradation protéosomale de la protéine. A ce jour, peu d’informations sont disponibles sur le rôle de HEF1 dans la progression tumorale colorectale, ou sur l’implication de sa phosphorylation sur sérine dans ses fonctions biologiques.Mon travail de thèse a permis de montrer une régulation positive de l’expression de HEF1 dans des échantillons du cancer de côlon, associée à une augmentation de la migration cellulaire dans des lignées tumorale colorectales. L’induction de la migration de la lignée HCT116 lors de la surexpression de HEF1 est corrélée à une augmentation de la phosphorylation sur tyrosine de FAK de manière dépendante de la kinase Src. Dans un deuxième temps, nous nous sommes intéressé à la phosphorylation sur sérine de HEF1, et avons montré que, comparativement à la protéine sauvage, la mutation sur la Sér-369 amplifie son effet pro-migratoire ainsi que la phosphorylation de FAK régulée par HEF1 consécutivement à une stabilisation de la protéine. Nous avons également caractérisé, pour la première fois, une mutation fonctionnelle de HEF1 sur l’Arg-367 qui mime l'effet de la mutation sur la Sér-369. Enfin, nous avons identifié des protéines adaptatrices de la famille BCAR3 comme partenaires de HEF1 et démontré que l’effet pro-migratoire de HEF1 est requiert son interaction avec BCAR3. En conclusion, ce travail nous a permis d’identifier une mutation de HEF1 (R367Q) qui stabilise la protéine, accroissant ainsi son effet pro-migratoire. Nous avons également associé la migration induite par HEF1 à son interaction avec BCAR3 et à une augmentation de la phosphorylation sur tyrosine de FAK. / Human Enhancer of Filamentation 1 (HEF1) is a member of the p130Cas family of docking proteins. HEF1 is present at focal adhesions and is involved in integrin-mediated cytoskeleton reorganization associated with cell migration. Elevated expression of HEF1 promotes invasion and metastasis in multiple cancer cell types. To date, little is known on the role of HEF1 in CRC tumor progression. HEF1 is phosphorylated on several Ser/Thr residues but the effects of these post-translational modifications on the functions of HEF1 are poorly understood. In this manuscript, we investigated the role of HEF1 in colorectal adeno-carcinoma migration capacities. First, we found that HEF1 expression was augmented in high stages CRC samples and then showed that overexpression of HEF1 in colo-carcinoma cell line HCT116 increases cell migration. Moreover, in these cells, HEF1 increases Src-mediated phosphorylation of FAK. We then showed that HEF1 mutation on Ser-369 enhances HEF1-induced migration and FAK phosphorylation as a result of protein stabilization. We also, for the first time characterized a functional mutation of HEF1 on Arg-367 which mimics the effect of Ser-369 to Ala mutation. Finally through mass spectrometry experiments, we identified BCAR3 as an essential interactor and mediator of HEF1-induced migration. We demonstrated that single amino acid mutations that prevent formation of the HEF1-BCAR3 complex impair HEF1-mediated migration. Therefore, amino-acid substitutions that prevent Ser-369 phosphorylation stabilize HEF1 which increases the migration of CRC cells and this requires the interaction of HEF1 with the NSP family adaptor protein BCAR3. Collectively, these data reveal the importance of HEF1 expression level in cancer cell motility and then support the utilization of HEF1 as a biomarker of tumor progression.

En France, le cancer colorectal (CCR) est la 2ème cause de décès par cancer. Chez les patients, lorsque la tumeur est localisée, elle est réséquée par chirurgie. Toutefois, 50% des patients sont diagnostiqués à un stade métastatique, ces patients sont alors traités par chimiothérapie (FOLFOX/FOLFIRI), souvent en combinaison avec des thérapies ciblées incluant des anticorps tel que le Cetuximab (anti-EGFR) ou le Bevacizumab (anti-VEGF). Cependant, il reste encore environ 40% des patients qui ne répondent pas au traitement et l’une des causes les mieux établies est l'influence des mutations de la voie RAS en aval du récepteur à l'EGF sur la réponse au Cetuximab. C’est pourquoi l’identification de nouvelles cibles moléculaires accessibles aux anticorps permettrait le développement de nouveaux modèles de thérapie ciblée. Depuis peu, les Claudines ont suscité un intérêt pour le ciblage tumoral comme la claudin-4 dans les cancers de l’endomètre ou de la prostate. Très récemment, les résultats d'une étude clinique ont démontré que la combinaison d'un anticorps anti-Claudin-18.2, l'Acm IMAB362, avec la chimiothérapie prolongeait nettement la survie chez des patients atteints de cancer gastrique avancé.C’est dans cette optique que nous nous sommes focalisés sur les claudines dans le CCR. Tout d’abord en étudiant leur expression dans des échantillons de patients atteints de CCR métastatique. En analysant l’expression des claudines dans les nouveaux sous-types moléculaires, nous avons montré que certaines pouvaient avoir une valeur pronostique. Nous avons également identifié des claudines comme cibles thérapeutiques potentielles dans le CCR, parmi elles la CLDN1. En effet, nous avons montré que la forme membranaire de la CLDN1 était surexprimée dans les tumeurs primaires et métastases du CCR. Nous avons développé un anticorps monoclonal (Acm) ciblant les parties extracellulaires de la CLDN1. Nous avons montré que le ciblage thérapeutique de la CLDN1 par Acm provoquait un ralentissement significatif de la croissance, la migration et l'invasion des cellules tumorales aussi bien in vitro que in vivo. Afin d’améliorer l’efficacité du ciblage de la CLDN1 par Acm, nous avons couplé ce dernier avec une toxine, générant ainsi un Antibody Drug conjugated (ADC). Nous avons montré que le ciblage de la CLDN1 par un ADC diminuait la survie cellulaire in vitro en culture cellulaire 2D, mais également celle des sphéroïdes via un effet cytotoxique. Ce travail a permis d'établir la preuve de concept du ciblage de la CLDN1 aussi bien par Acm que par ADC. Afin de finaliser ce travail, ces résultats doivent être confirmés in vivo. A terme, nous espérons que ce ciblage puisse trouver sa place au sein de l’arsenal thérapeutique du CCR métastatique, en particulier chez les patients résistants aux traitements. / Colorectal cancer (CRC) is one of the major causes of cancer-related deaths in the Western world. When localized, CRC is often curable by surgery. However, 50% of patients are diagnosed at a metastatic stage, these patients are then treated with chemotherapy (FOLFOX / FOLFIRI), often in combination with targeted therapies including antibodies such as Cetuximab (anti-EGFR) or Bevacizumab (anti -VEGF). Despite these treatment, almost 40% of patients develop resistance. One of the most known resistance mechanisms of resistance is due to the RAS pathway downstream of the EGF receptor in response to Cetuximab. Therefore, more therapeutic options are required particularly by identifying new molecular targets that can be reached by antibodies. Recently, Claudines have generated interest as targets in cancer, such as claudin-4 in endometrial or prostate cancer. More recently, the results of a clinical study demonstrated that the combination of an anti-Claudin-18.2 antibody, IMAB362 mAb, with chemotherapy significantly prolonged survival in patients with advanced gastric cancer.Therefore, one of our aims was to focus on claudins in CCR. First, by studying their expression in metastatic CRC patient samples. We demonstrated the prognostic value of some claudins, after analyzing their gene expression in the new molecular subtype of CRC. Beside, we identified some claudins as potential therapeutic targets in CCR, among them claudin-1 (CLDN1). Indeed, we showed that the membrane form of CLDN1 is overexpressed in primary tumors and metastases of CRC. Therefore, we developed a monoclonal antibody (mAb) targeting the extracellular parts of CLDN1. We showed that therapeutic targeting of CLDN1 by mAb significantly decreased tumor cell growth, migration and invasion both in vitro and in vivo. In order to improve the efficiency of targeting CLDN1 by mAb, we conjugated it with a toxin, thus generating an Antibody Drug Conjugate (ADC). We showed that CLDN1 targeting by an ADC decreased cell survival in vitro in 2D cell culture, but also spheroids growth via a cytotoxic effect.This work demonstrated the proof of concept of CLDN1 targeting by both mAb and ADC. In order to achieve this work, the next steps remains on testing ADC affect in vivo models. CLDN1 is a good therapeutic target in the CCR. In the long term, we hope that its targeting can find its place within the therapeutic arsenal of metastatic CRC, particularly in treatment of resistant patients.

Os microRNAs (miRNAs) são pequenos RNAs não codificadores de proteínas presentes na maioria dos eucariotos. Esses RNAs regulam a expressão gênica em nível pós-transcricional através do silenciamento de mRNAs-alvo que possuem sítios complementares às suas sequências, atuando em praticamente todos os processos celulares. Embora a estrutura e função dos miRNAs estejam bem caracterizadas, aspectos relacionados à sua organização genômica, evolução e atuação em doenças são tópicos que apresentam enormes lacunas. Nesta tese, utilizamos abordagens computacionais para investigar estes temas em três trabalhos. No primeiro, processamos e integramos um vasto volume de dados publicamente disponíveis referentes aos miRNAs e genes codificadores de proteínas para cinco espécies de vertebrados. Com isso, construimos uma ferramenta web que permite a fácil inspeção da organização genômica dos miRNAs em regiões inter e intragênicas, o acesso a dados de expressão de miRNAs e de genes codificadores de proteínas (classificados em genes hospedeiros e não hospedeiros de miRNAs), além de outras informações pertinentes. Verificamos que a ferramenta tem sido amplamente utilizada pela comunidade científica e acreditamos que ela possa facilitar a geração de hipóteses associadas à regulação dos miRNAs, principalmente quando estão inseridos em genes hospedeiros. No segundo estudo, buscamos compreender como o contexto genômico e a origem evolutiva dos genes hospedeiros influenciam a expressão e evolução dos miRNAs humanos. Nossos achados mostraram que os miRNAs intragênicos surgem preferencialmente em genes antigos (origem anterior à divergência de vertebrados). Observamos que os miRNAs inseridos em genes antigos têm maior abrangência de expressão do que os inseridos em genes novos. Surpreendentemente, miRNAs jovens localizados em genes antigos são expressos em um maior número de tecidos do que os intergênicos de mesma idade, sugerindo uma vantagem adaptativa inicial que pode estar relacionada com o controle da expressão dos genes hospedeiros, e como consequência, expondo-os a contextos celulares e conjuntos de alvos diversos. Na evolução a longo prazo, vimos que genes antigos conferem maior restrição nos padrões de expressão (menor divergência de expressão) para miRNAs intragênicos, quando comparados aos intergênicos. Também mostramos possíveis associações funcionais relacionadas ao contexto genômico, tais como o enriquecimento da expressão de miRNAs intergênicos em testículo e dos intragênicos em tecidos neurais. Propomos que o contexto genômico e a idade dos genes hospedeiros são fatores-chave para a evolução e expressão dos miRNAs. Por fim, buscamos estabelecer associações entre a expressão diferencial de miRNAs e a quimioresistência em câncer colorretal utilizando linhagens celulares sensíveis e resistentes às drogas 5-Fluoruracil e Oxaliplatina. Dentre os miRNAs identificados, o miR-342 apresentou níveis elevados de expressão nas linhagens sensíveis à Oxaliplatina. Com base na análise dos alvos preditos, detectamos uma significativa associação de miR-342 com a apoptose. A superexpressão de miR-342 na linhagem resistente SW620 evidenciou alterações na expressão de genes da via apoptótica, notavelmente a diminuição da expressão do fator de crescimento PDGFB, um alvo predito possivelmente sujeito à regulação direta pelo miR-342. / MicroRNAs (miRNAs) are short non-coding RNAs found in most eukaryotic species. These RNAs regulate gene expression at post-transcriptional level by silencing target mRNAs through base-pairing of complementary sequences, thus acting on virtually all cellular processes. Although the structure and function of miRNAs are well understood, several aspects related to their genomic organization, evolution and involvement with diseases are largely underexplored. In this thesis, we employed computational methods to investigate such issues in three different studies. In the first one, we have processed and integrated a large amount of public data related to miRNAs and coding genes for five vertebrate species. Then, we developed a webtool to allow the analysis of the miRNA genomic context in inter and intragenic regions, the access of miRNA and gene expression data (classified as host and non-host genes), as well as other relevant information. We noticed that the webtool has been largely used by the scientific community, and we believe that it can facilitate hypothesis generation related to miRNA regulation, especially when they are within host genes. In the following study, we sought to understand how the genomic context and the evolutionary origin of host genes can affect the expression and evolution of human miRNAs. Our findings showed that intragenic miRNAs are preferentially embedded within old host genes (originated before the split of vertebrates). We observed that miRNAs within old genes are more broadly expressed than those within young genes. Surprisingly, young miRNAs within old genes were expressed in more tissues than their intergenic counterparts, suggesting an initial adaptive advantage which might be related to their hosts expression control, and as a consequence, exposing them to a more diverse cellular contexts and target genes. In the long run, we found that old host genes lead to expression constraints (lower expression divergence) between species for intragenic miRNAs, in respect to intergenic ones. We also showed possible functional associations related to miRNA genomic context, such as the enrichment of young intergenic miRNAs in testis, while young intragenic miRNAs were enriched in neural tissues. Thus, we propose that the genomic context and the age of the host genes are key factors in shaping the expression and evolution of miRNAs. Finally, we sought to establish associations between differential expression of miRNAs and chemoresistance in colorectal cancer using resistant and sensitive cell lines to 5-Fluoruracil and Oxaliplatin. Among differentially expressed miRNAs, miR-342 was highly expressed in sensitive cell lines to Oxaliplatin. Based on target prediction analysis, miR-342 is likely associated with apoptosis. The induced overexpression of miR-342 in SW620, a cell line resistant to Oxaliplatin, changed the expression levels of genes linked to the apoptosis pathway, notably the downregulation of PDGFB growth factor, which is a predicted target possibly subjected to direct regulation by miR-342.

Despite therapeutic advances, colorectal cancer still has a 45% mortality rate, and one of the most crucial problems is the development of acquired resistance to treatment with anticancer drugs. Thus the aims of this project are to develop drug-resistant colon cancer cell lines in order to identify mechanisms of resistance for the most commonly drugs used in colorectal cancer: 5-fluorouracil, oxaliplatin, and irinotecan. Following evaluation of drug sensitivity to these agents in an initial panel of eight colorectal cancer cell lines, 3 lines (DLD-1, KM-12 and HT-29) were selected for the development of 5-FU (3 lines), oxaliplatin (2) and irinotecan (1) resistant sublines by continuous drug exposure, with resistance confirmed using the MTT assay. Consistently resistant sublines were subject to a „stable isotope labelling with amino acids in cell culture‟ (SILAC) approach and a MudPIT proteomics strategy, employing 2D LC and Orbitrap Fusion mass spectrometric analysis, to identify novel predictive biomarkers for resistance. An average of 3622 proteins was quantified for each resistant and parent cell line pair, with on average 60-70 proteins up-regulated and 60-70 down-regulated in the drug resistant sublines. The validity of this approach was further confirmed using immunodetection techniques. These studies have provided candidate proteins which can be assessed for their value as predictive biomarkers, or as therapeutic targets for the modulation of acquired drug resistance in colorectal cancer.

Radiological imaging already has a key role in the detection and management of patients with metastatic colorectal cancer (mCRC). With the evolution of personalised medicine there is a need for non-invasive imaging biomarkers that can detect early tumour response to targeted therapies. Translation from bench to bedside requires a multicentre approach that follows an agreed development roadmap to ensure that the proposed biomarker is precise (reproducible/ repeatable) and accurate in its characterisation of a meaningful physiological, pathological or post treatment response. The following thesis (organized in the alternative format with experimental studies written as individual complete manuscripts) investigates methods to improve precision and accuracy of the Apparent Diffusion Coefficient (ADC), a proposed quantitative imaging biomarker with a potential role in characterisation of post treatment responses in mCRC. The first objective was to establish baseline multicentre reproducibility (n=20) for ADC. A change in ADC greater than 21.1% was required to determine a post treatment response. Using a statistical error model, the dominating factors that influenced reproducibility were motion artefact and tumour volume. In the second study these factors were addressed using a single centre cohort with pre and post treatment data. Correcting for errors due to motion and tumour volume improved sensitivity from 30.3% to 1.7%, so a post treatment response was detected in 6/12 tumours compared to 0/12 using the baseline approach. In the third study, motion correction was implemented and the statistical error model was applied successfully to a multicentre cohort of 15 patients (1.9% sensitivity). The results of this thesis highlights that with careful consideration and correction of factors that negatively influence sensitivity, ADC is a potential imaging biomarker for use in post treatment response for patients with mCRC.

This project set out to examine the possibility that by acquiring Raman spectra and performing multi-photon imaging we can get better diagnosis and understanding of the biochemistry of an individual cancerous tumour and distinguish it from the healthy tissue. Within the frame of this study, colorectal primary and secondary cancer cells are examined with Raman spectroscopy in order to (i) study and distinguish them according to their chemical composition by applying multivariate methods and (ii) determine whether Raman spectroscopy can identify the cells which are the link between primary and secondary colorectal cancer cells, the so-called Cancer Stem Cells. The second part of this thesis is based on tissue studies. Human colorectal tissue sections are examined in a label-free manner with the use of multi-photon imaging modes (i) Two photon excitation fluorescence, (ii) stimulated Raman scattering and (iii) second harmonic generation, in order to determine whether these can provide fast and accurate diagnosis of colorectal cancer. These techniques were able to distinguish between healthy and cancerous tissue regions, based on the chemically-specific images of the tissue microenvironment and architecture. The hypothesis of Cancer stem cell is examined with the use of Raman spectroscopy shown that the CSCs have some small differences according to their tissue origin.

O câncer colorretal (CCR) é importante causa de morte no Brasil, representando a segunda causa de morbimortalidade por câncer nas regiões Sudeste e Sul. Foi uma das primeiras neoplasias malignas a ter modelo de carcinogênese identificado. Os objetivos deste trabalho foram avaliar a expressão do NF-KB e da survivina na progressão do CCR, sua relação com alvos moleculares envolvidos na sobrevivência celular [proibitina, fator de necrose tumoral (TNFR1), p53, B- catenina]; invasão (metaloproteinases 2 e 9 - MMP), angiogênese (fator de crescimento endotelial vascular VEGF) e apoptose (caspase 3 e método do Túnel). Trata-se de estudo retrospectivo baseado na análise histopatológica de dezoito casos de adenomas com displasia de alto (AG) e baixo grau (BG), respectivamente; dez casos de adenocarcinoma bem, moderado e pouco diferenciado, respectivamente, nove casos de lesões serrilhadas e nove biópsias colorretais (controle) selecionados aleatoriamente no Serviço de Patologia do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo, no período de 2000 a 2009. Foram construídos arranjos de matriz tecidual para representar os casos de adenocarcinomas e cinco casos de adenomas >1 cm de diâmetro. A detecção do NF-KB foi realizada pelo método de Southwestern imunoistoquímica. Avaliou-se a expressão imunoistoquímica da survivina, proibitina, TNFR1, p53, B-catenina, MMPs 2 e 9, VEGF, caspase 3 e Túnel. Não observamos expressão imunoistoquímica do NF-KB em 70% dos casos de adenocarcinomas, em 72% dos adenomas AG e em 89% das lesões serrilhadas; a marcação foi positiva em 66% e 56% dos casos de adenoma do BG e controle, respectivamente. Expressões da survivina, proibitina citoplasmática, TNFR1, p53, MMP 2 e 9 foram crescentes na seqüência adenoma-carcinoma, sem aparente modulação pelo NF-KB. A survivina suprimiu ação da caspase 3, exceto nos adenomas BG e controle, com baixos níveis de apoptose ao túnel. Lesões serrilhadas e controle apresentaram baixa expressão do TNFR1 e da proibitina citoplasmática, ausência de marcação da p53 e MMPs 2 e 9, exceto um caso controle. Identificou-se marcação nuclear da proibitina nos adenocarcinomas pouco diferenciados, adenomas AG e BG. A expressão da p53 relacionou-se ao grau de displasia nos adenomas e à desregulação da survivina. Observou-se expressão citoplasmática e nuclear da B- catenina nos adenocarcinomas, adenomas AG e BG. As lesões serrilhadas exibiram expressão citoplasmática em 44% dos casos. O grupo controle exibiu expressão preservada da B-catenina. Identificou-se expressão do VEGF nos adenocarcinomas, relacionada à perda de diferenciação celular, presença de metástases, não correlacionada ao NF-KB. Com base nos nossos resultados, sugerimos a desregulação da B- catenina como possível responsável pela inibição do NF-KB; além de sua participação na desregulação da survivina juntamente com a p53. As lesões serrilhadas não exibiram indícios sugestivos de inibição do NF- KB pela B-catenina.A survivina, devido propriedades anti-apoptóticas, emerge como potencial alvo terapêutico no tratamento do CCR, confirmando estudos anteriores. / Colorectal cancer (CRC) is an important cause of death in Brazil, representing the second leading cause of cancer mortality in the Southeast and South. It was among the first malignancies that have the carcinogenesis model identified.The aim of this study was to evaluate the expression of NF-KB and survivin in the progression of CRC, its relationship with molecular targets involved in cell survival [prohibitin, tumor necrosis factor (TNFR1), p53,B-catenina], invasion (metalloproteinases 2 and 9 - MMP), angiogenesis (vascular endothelial growth factor VEGF) and apoptosis (caspase 3 and method Tunnel). This is a retrospective study based on the histopathological analyses of eighteen cases of high- (HG) and low- grade dysplastic (LG) adenomas, respectively; ten cases of adenocarcinoma well, moderately and poorly differentiated, respectively, nine cases of serrated lesions e nine biopsies (control); randomly selected in the Pathology Service of the University Hospital of the Faculty of Medicine of Ribeirão Preto, University of São Paulo from 2000 to 2009. Tissue microarray were constructed to represent the cases of adenocarcinomas and five cases of adenomas > 1cm in diameter. Detection of NF-KB was performed by the Southwestern immunohistochemistry method. We evaluated immunohistochemical expression of survivin, prohibitin, TNFR1, p53, B- catenin, MMPs 2 and 9, VEGF, caspase 3 and method Tunnel. We did not observe immunohistochemical expression of NF-KB in 70% of cases of adenocarcinomas, in 72% of HG adenomas and 89% of lesions serrated; staining was positive in 66% and 56% of cases of LG adenoma and control, respectively. Expressions of survivin, cytoplasmic prohibitin, TNFR1, p53, MMPs 2 and 9 were increasing in adenoma-carcinoma sequence, without apparent modulation by NF-KB. Survivin suppressed action of caspase 3, except in BG adenomas and control, with low levels of apoptosis to the tunnel. Serrated lesions and control showed low expression of TNFR1 and cytoplasmic prohibitin, the absence of staining of p53 and MMPs2 and 9, except for one control. We identified nuclear staining of prohibitin in poorly differentiated adenocarcinomas, HG and LG adenomas. Expression of p53 was related to the grade of dysplasia in adenomas and deregulation of survivin.The expression of the cytoplasmic and nuclear B-catenin was observed in adenocarcinoma, HG and LG adenoma. Serrated lesions exhibited cytoplasmic expression in 44% of cases. Control group exhibited preserved expression of B-catenin. It was identified VEGF expression in adenocarcinomas, related to the loss of cellular differentiation, metastasis, not correlated with NF-KB. According to our results, we suggest that deregulation of catenin is possible responsible for the inhibition of NF-KB besides their participation in the deregulation of survivin with p53.Serrated lesions exhibited no evidence suggestive of inhibition of NF-KB by B-catenin. Survivin emerges as a potential therapeutic target in the treatment of CRC due to their anti-apoptotic properties, confirming previous studies.

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.