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A queilite actínica (QA) é uma lesão potencialmente maligna importante para identificar indícios precoces de transformação maligna para o carcinoma epidermoide de lábio (CEL), possibilitando a implementação de um tratamento eficiente e menos invasivo, que promova um melhor prognóstico para os pacientes. Pesquisas recentes indicam que os métodos histopatológicos geralmente são falhos em traçar o risco de malignização de casos de QA, pois além de não demonstrar as alterações genéticas presentes nos queratinócitos, não foram realizados estudos de acompanhamento clínico para avaliar se o grau de displaia epitelial da QA está relacionado ao risco de malignização para CE. Assim, a presente pesquisa teve como objetivo caracterizar, a partir dos casos atendidos no Serviço de Patologia Cirúrgica da FOUSP, qual a diferença de perfil clínico-patológico de pacientes de QA com evolução para CEL, pacientes de QA sem informações e sinais presentes de malignização e pacientes apenas diagnosticados com CEL, e também visou analisar a expressão de Ki67 e pRb nesses três grupos. Para isso, os dados dos pacientes como idade, sexo, cor da pele, aspecto clínico da lesão fundamental, coloração, tamanho e tempo de duração das lesões foram resgatados de 998 casos e distribuídos nessas três categorias. Os resultados da análise clínico-epidemiológica revelaram que o único aspecto clínico estatisticamente significante para diferenciar pacientes apenas diagnosticados com CEL dos demais grupos foi o tempo de duração das lesões. A análise do grau de displasia epitelial nos casos de QA na amostra presente revelou que todos os pacientes de QA posteriormente diagnosticados com CEL foram classificados como lesões de alto risco, e ainda exibiram em maior frequência as atipias: aumento do número de figuras de mitose, variação anormal do tamanho do núcleo, variação anormal do tamanho da célula e alteração da relação núcleo/citoplasma, figuras de mitose anormais e aumento do número e tamanho de nucléolos. Tanto a expressão da proteína Ki-67 como da proteína pRb não demonstraram significância estatística na comparação entre os grupos do estudo. Assim, a avaliação de uma ampla série de casos revelou diferença significante no tempo de duração do CEL com relação à QA. Além disso, algumas alterações morfológicas foram observadas com maior frequência em casos de QA com evolução para CEL. No entanto, outros marcadores biológicos devem ser testados em conjunto, para tentar diagnosticar alterações precoces que levem ao desenvolvimento de CEL. / Actinic cheilitis (AC) is a potentially malignant lesion important to identify early signs of malignant transformation into lip squamous cell carcinoma (LSCC), enabling the implementation of an efficient and less invasive treatment to patients. Recent researches pointed that histopatological methods often fail to trace malignization risk in AC cases, because they are unable to identify genetic damage in keratinocytes and do not exist a clinical follow-up studie to assess if the grading of epithelial dysplasia in AC is related with the malignancy risk to LSCC development. Thus, this research aims to characterize, from cases of Surgical Pathology Service of Universidade de São Paulo, the differences in clinical and pathological profile among AC patients which had evolution to LSCC, AC patients without signs and information about malignization and patients diagnosed only with LSCC. This study also analyzed the expression of Ki-67 and pRb proteins in these three groups. To conduct this study, data as age, gender, race, fundamental lesion aspect, color, size and duration time of the lesion were collected from 998 patients. The clinical-epidemiological analysis revealed that duration time of the lesion was the statistically significant clinical feature to differentiate patients diagnosed only with LSCC from other groups. The grading of epithelial dysplasia analysis showed that all AC patients with a posterior diagnosis of LSCC were classified as high risk lesions and these cases also exhibited most frequently atypia figures as: increased number of mitotic features, abnormal variation in nuclear size, abnormal variation in cellular size, increased nuclear/cytoplasmic ratio, abnormal mitotic features and increased number and size of nucleoli. The immunohistochemical expression of both Ki-67 and pRb protein demonstrated lack of significant statistical difference among the groups. We concluded that the evaluation of a large serie of cases revealed differences in duration time of lesion in patiens only diagnoses with LSCC and some morphological criteria were most frequent in AC cases with a posterior diagnosis of LSCC. However, other biological markers must be tested together, to try to identify early steps of LSCC development.

A queilite actínica (QA) é uma lesão potencialmente maligna importante para identificar indícios precoces de transformação maligna para o carcinoma epidermoide de lábio (CEL), possibilitando a implementação de um tratamento eficiente e menos invasivo, que promova um melhor prognóstico para os pacientes. Pesquisas recentes indicam que os métodos histopatológicos geralmente são falhos em traçar o risco de malignização de casos de QA, pois além de não demonstrar as alterações genéticas presentes nos queratinócitos, não foram realizados estudos de acompanhamento clínico para avaliar se o grau de displaia epitelial da QA está relacionado ao risco de malignização para CE. Assim, a presente pesquisa teve como objetivo caracterizar, a partir dos casos atendidos no Serviço de Patologia Cirúrgica da FOUSP, qual a diferença de perfil clínico-patológico de pacientes de QA com evolução para CEL, pacientes de QA sem informações e sinais presentes de malignização e pacientes apenas diagnosticados com CEL, e também visou analisar a expressão de Ki67 e pRb nesses três grupos. Para isso, os dados dos pacientes como idade, sexo, cor da pele, aspecto clínico da lesão fundamental, coloração, tamanho e tempo de duração das lesões foram resgatados de 998 casos e distribuídos nessas três categorias. Os resultados da análise clínico-epidemiológica revelaram que o único aspecto clínico estatisticamente significante para diferenciar pacientes apenas diagnosticados com CEL dos demais grupos foi o tempo de duração das lesões. A análise do grau de displasia epitelial nos casos de QA na amostra presente revelou que todos os pacientes de QA posteriormente diagnosticados com CEL foram classificados como lesões de alto risco, e ainda exibiram em maior frequência as atipias: aumento do número de figuras de mitose, variação anormal do tamanho do núcleo, variação anormal do tamanho da célula e alteração da relação núcleo/citoplasma, figuras de mitose anormais e aumento do número e tamanho de nucléolos. Tanto a expressão da proteína Ki-67 como da proteína pRb não demonstraram significância estatística na comparação entre os grupos do estudo. Assim, a avaliação de uma ampla série de casos revelou diferença significante no tempo de duração do CEL com relação à QA. Além disso, algumas alterações morfológicas foram observadas com maior frequência em casos de QA com evolução para CEL. No entanto, outros marcadores biológicos devem ser testados em conjunto, para tentar diagnosticar alterações precoces que levem ao desenvolvimento de CEL. / Actinic cheilitis (AC) is a potentially malignant lesion important to identify early signs of malignant transformation into lip squamous cell carcinoma (LSCC), enabling the implementation of an efficient and less invasive treatment to patients. Recent researches pointed that histopatological methods often fail to trace malignization risk in AC cases, because they are unable to identify genetic damage in keratinocytes and do not exist a clinical follow-up studie to assess if the grading of epithelial dysplasia in AC is related with the malignancy risk to LSCC development. Thus, this research aims to characterize, from cases of Surgical Pathology Service of Universidade de São Paulo, the differences in clinical and pathological profile among AC patients which had evolution to LSCC, AC patients without signs and information about malignization and patients diagnosed only with LSCC. This study also analyzed the expression of Ki-67 and pRb proteins in these three groups. To conduct this study, data as age, gender, race, fundamental lesion aspect, color, size and duration time of the lesion were collected from 998 patients. The clinical-epidemiological analysis revealed that duration time of the lesion was the statistically significant clinical feature to differentiate patients diagnosed only with LSCC from other groups. The grading of epithelial dysplasia analysis showed that all AC patients with a posterior diagnosis of LSCC were classified as high risk lesions and these cases also exhibited most frequently atypia figures as: increased number of mitotic features, abnormal variation in nuclear size, abnormal variation in cellular size, increased nuclear/cytoplasmic ratio, abnormal mitotic features and increased number and size of nucleoli. The immunohistochemical expression of both Ki-67 and pRb protein demonstrated lack of significant statistical difference among the groups. We concluded that the evaluation of a large serie of cases revealed differences in duration time of lesion in patiens only diagnoses with LSCC and some morphological criteria were most frequent in AC cases with a posterior diagnosis of LSCC. However, other biological markers must be tested together, to try to identify early steps of LSCC development.

The transition to multicellularity has occurred numerous times in all domains of life, yet its initial steps are poorly understood. The volvocine green algae are a tractable system for understanding the genetic basis of multicellularity including the initial formation of cooperative cell groups. Here we report the genome sequence of the undifferentiated colonial alga, Gonium pectorale, where group formation evolved by co-option of the retinoblastoma cell cycle regulatory pathway. Significantly, expression of the Gonium retinoblastoma cell cycle regulator in unicellular Chlamydomonas causes it to become colonial. The presence of these changes in undifferentiated Gonium indicates extensive group-level adaptation during the initial step in the evolution of multicellularity. These results emphasize an early and formative step in the evolution of multicellularity, the evolution of cell cycle regulation, one that may shed light on the evolutionary history of other multicellular innovations and evolutionary transitions.

Le rétinoblastome est une tumeur rare qui touche la rétine du jeune enfant. L’inactivation bi-allélique du gène RB1 est à l’origine du développement tumoral. RB1 est le premier gène suppresseur de tumeur qui ait été identifié et la prédisposition au rétinoblastome constitue un véritable paradigme de la prédisposition aux cancers. Dans les formes non prédisposées génétiquement, les deux mutations apparaissent dans une cellule rétinienne unique ; le rétinoblastome est alors unilatéral. Dans les formes à prédisposition génétique, la première mutation est constitutionnelle et la deuxième est somatique. La mutation constitutionnelle est une néo-mutation pré- ou post- zygotique dans les formes sporadiques, alors qu’elle est héritée dans les formes familiales. Dans les formes avec prédisposition génétique, le diagnostic est plus précoce que dans les formes sans prédisposition et la bilatérisation du rétinoblastome est généralement la règle. Néanmoins, de rares familles présentent une pénétrance réduite et une variabilité phénotypique se traduisant par la coexistence de patients atteints de rétinoblastome bilatéral ou unilatéral, d’apparentés porteurs sains et d’apparentés présentant des rétinomes. Les mécanismes responsables de la variabilité phénotypique intrafamiliale sont inconnus et l’existence de facteurs génétiques modulant le phénotype tumoral est probable.L’origine de la variabilité de l’expression phénotypique du rétinoblastome peut être la résultante (i) de l’existence de mutations en mosaïque, (ii) de mutations de RB1 et (iii) de facteurs modificateurs génétiques indépendants du locus de RB1. Trois axes distincts et originaux basés sur ces origines possibles de variabilité phénotypique ont été développés pour caractériser les relations génotype/phénotype dans le rétinoblastome. Premièrement, les conséquences d’une mosaïque somatique ont été illustrées grâce à l’étude d’une famille ayant bénéficié de cinq diagnostics prénatals. Dans ces familles, certains fœtus porteurs de l’allèle à risque identifié par une approche indirecte basée sur l’étude de microsatellites au locus de RB1, n’étaient pas porteurs de la mutation du parent atteint, lui-même atteint d’un rétinoblastome bilatéral. Ainsi, nous avons démontré la présence d’une mosaïque somatique et gonadique chez ce parent lourdement atteint. La conséquence de l’existence de patients présentant une mosaïque dans le cadre du conseil génétique a été discutée. La suite de nos travaux a pris en compte ces résultats afin de limiter les biais que pourrait induire la présence de mutations en mosaïque dans des études de corrélation génotype/phénotype dans le rétinoblastome. Deuxièmement, l’association de grandes délétions emportant RB1 avec des retards psychomoteurs chez des patients atteints de rétinoblastome a été étudiée. Une approche de CGH hautement résolutive, ciblée sur le locus de RB1, a été mise en place afin de caractériser le rôle des gènes contigus de RB1 dans ce syndrome. Ainsi, cette approche a permis de définir une zone à risque de retard psychomoteur que nous proposons comme seuil d’alerte pour le généticien. Cette zone définit un gène, PCDH8 d’expression cérébrale exclusive, comme un excellent candidat au retard psychomoteur. Enfin, troisièmement, une approche « gène candidat » reposant sur l’étude du SNP309 du promoteur de MDM2, a été mise en œuvre. / Retinoblastoma is the most common intraocular childhood cancer and occurs when both alleles of the RB1 gene are inactivated in the retina. In patients without genetic predisposition, the two mutations occurred in a single unique retinal cell. In subjects with a genetic predisposition to retinoblastoma, the first RB1 mutation is found in the germline and the second appears as a somatic mutation. Germline carriers usually develop bilateral or multifocal tumors and the diagnosis is earlier. However, some rare families exhibit low penetrance and variable expressivity of the disease because bilaterally affected, unilaterally affected, and unaffected mutation carriers are known to coexist. The existence of genetic modifiers in retinoblastoma therefore appears highly probable and must be considered. The lack of penetrance and the variable expressivity could be the sum of three independent causes. The presence of a mosaic can affect the phenotype, the nature of the mutation can drive low penetrance and particular phenotype like psychomotor delay in case of large genomic deletions and genetic modifier factors could modulate the phenotype. These three major keys have been studied in order to highlight the relations between the phenotype and the genotype. Firstly, the consequences of mosaicism have been illustrated by a prenatal diagnosis concerning a couple with a bilateral retinoblastoma-affected male patient who requested five successive prenatal diagnoses and in whom RB1 mutation mosaicism had important implications. Implications of mosaicism in genetic counseling have been discussed and taken into consideration in order to limit bias in the two following genotype/phenotype studies. Secondly, the association between whole germline monoallelic deletions of the RB1 gene and psychomotor delay was studied by a high-resolution CGH array focusing on RB1 and its flanking region. Comparative analysis detected a four megabase critical interval including a candidate gene, protocadherin 8 (PCDH8). PCDH8 is thought to function in signaling pathways and cell adhesion in a central nervous system-specific manner, making loss of PCDH8 one of the probable causes of psychomotor delay in RB1-deleted patients. Thirdly, a candidate gene approach based on partners that are necessary for the development of the tumor attempted to find possible genetic modifiers. MDM2, which increases p53 and pRB catabolism, was therefore a prominent candidate. The minor allele of MDM2 that includes a 309T>G transversion (SNP rs2279744) in the MDM2 promoter is known to enhance MDM2 expression. In family-based association analyses performed in 70 retinoblastoma families, the MDM2 309G allele was found to be statistically significantly associated with incidence of bilateral or unilateral retinoblastoma among members of retinoblastoma families under a recessive model (Z = 3.305, two-sided exact P = .001). The strong association of this genotype with retinoblastoma development designates MDM2 as the first modifier gene to be identified among retinoblastoma patients

L'analyse de données d'expression montre qu'il est avantageux d'analyser les processus biologiques en termes de modules plutôt que simplement considérer les gènes un par un. Dans ce projet nous avons conduit une analyse non supervisée des données d'expression de gènes de plusieurs cohortes de tumeurs urothéliales en appliquant la méthode d'Analyse en Composantes Indépendantes. Plusieurs études ont démontré les meilleures performances de l'ACI par rapport à l'ACP et les méthodes de clustering, pour obtenir une décomposition plus réaliste des données d'expression en patterns d'expression pertinents et associés avec le phénotype d'intérêt.Les tumeurs urothéliales apparaissent et évoluent selon deux voies distinctes dont la probabilité de progression en cancer musculo-invasif diffère radicalement. Excepté la mutation de FGFR3 dans le groupe le moins agressif, les processus moléculaires sous-jacents n'ont pas été complètement identifiés. Le principal objectif de cette thèse était dédié aux interprétations biologiques des différentes composantes indépendantes pour aider à confirmer et étendre la liste des processus biologiques connus pour être impliqués dans le cancer de vessie.Chaque composante indépendante est caractérisée par une liste de projections de gènes et de contributions pondérées d'échantillons tumoraux . En combinant expertise biologique et comparaison des listes de gènes à des voies existantes et en étudiant conjointement l'association des composantes aux annotations cliniques et moléculaires, nous avons pu différencier les CIscausées par des facteurs techniques, tels que le prélèvement chirurgical de celles ayant des interprétations biologiques pertinentes. De plus, parmi les signaux pertinents biologiquement, cette analyse nous a permis de différencier les signaux provenant du stroma, comme la réponse immunitaire médiée par les lymphocytesB&T, de ceux produits par les tumeurs elles-mêmes, comme les signaux reliés à la prolifération ou à la différenciation. La classification des tumeurs selon leurs contributions à certaines CIs a pu être étroitement associée à des annotations anatomo-cliniques, et a mis en évidence de nouveaux sous-types de tumeur spotentiels, qui suggèrent l'existence de voies de progression supplémentaires dans le cancer de vessie. De façon similaire, l'étude des contributions de groupes de tumeurs basés sur des annotations cliniques ou moléculaires a montré différents niveaux de contamination par le stroma entre les tumeurs mutées et nonmutées pour FGFR3. La reproductibilité des composantes a été étudiée en utilisant des graphes de corrélation. La majeure partie des CIs interprétées a été validée sur trois jeux de données indépendants, et plusieurs d'entre elles ont aussi détectées dans un jeu de données de lignées cellulaires.Une deuxième étude sur le rétinoblastome a montré que nous pouvions tirer partie de l'ACI dans des contextes variés. Le rétinoblastome est initié par la perte des deux alléles du gène suppresseur de tumeur RB1. D'autres évènements génomiques non identifiés sont nécessaires à la progression de la maladie. Nous avons observé une association entre âge des patients et altérations génomiques. Les patients jeunes présentant moins d'altérations que les patients âgés, ces derniers présentant des gains du 1q et des pertes du 16q. Cette séparation des tumeurs selon l'âge est également observée sur les données d'expression, notamment en appliquant l'ACI dont l'une des composantes discrimine les patients selon leur âge. Ces résultats suggèrent l'existence de deux voies de progression dans le rétinoblastome. L'analyse des données à haut débit fournit de nombreuses listes de gènes. Afin de les interpréter, une possibilité est d'extraire les dernières publications groupées par sujets prédéfinis (fonction, localisation,...). / Practice of gene expression data analysis shows that it is advantageous to analyze biologicalprocesses in terms of modules rather than simply consider gene one by one. In this project, we conducted anunsupervised analysis of the gene expression data of several cohorts of urothelial tumors, applying theIndependent Component Analysis method. Several studies demonstrated the outperformance of ICA overPCA and clustering-based methods in obtaining a more realistic decomposition of the expression data intoconsistent patterns of coexpressed genes associated with the studied phenotypes[1, 2, 3, 4].Urothelial tumors arise and evolve through two distinct pathways which radically differ on their probabilityof progression to muscle invasion. Except the mutation of FGFR3 in the less aggressive group, theunderlying molecular processes have not been completely identified. The first and main objective of the PhDthesis was dedicated to the biological interpretation of the different independent components to help toconfirm and extend the list of biological processes known to be involved in bladder cancer.Each independent component (IC) is characterized by a list of gene projections on the one hand and weightedcontributions of tumor samples on the other hand. By combining biological expertise and comparison of theassociated list of genes to known pathways, and jointly studying the association of the components tomolecular and clinical annotations, we have been able to differentiate components that were caused bytechnical factors, such as surgical sampling, from those having consistent biological interpretationin terms of tumor biology. Moreover, among the biologically meaningful signals, this analysis allowed us todifferentiate the signals from stroma of the tumor, like immune response mediated by B- and T-lymphocytes,from the signals produced by the tumors themselves, like signals related to proliferation, or differentiation.The clustering of the tumor samples according to their contributions on some ICs can be closely associated toanatomo-clinical annotations, and highlighted new potential subtypes of tumors which suggest existence ofadditional pathways of bladder cancer progression. Similarly, the study of the contributions of preestablishedgroups of tumors based on clinical or molecular criteria showed different levels of stromacontamination between FGFR3 non-mutated and mutated tumors. The reproducibility of the components wasinvestigated using correlation graphs. The major part of the interpreted ICs was validated on threeindependent bladder cancer datasets, and several of them were also identified in an urothelial cancer celllines data set.A second study about retinoblastoma gave us the occasion to show that we can take advantage ofICA in various contexts. Retinoblastoma is initiated by the loss of both alleles of the RB1 tumor suppressorgene. Although necessary for initiation, other genomic events, that remain to be identified, are needed for theprogression of the disease [5]. We observed, as it was previously described [6], an association between age ofthe patients and levels of genomic aberrations, the younger patients having fewer alterations than the olderpatients, which generally present gain of 1q and loss of 16q. We showed that this tendency of the tumors tobe clustered into two groups of age can also be observed on the expression data by applying ICA whose oneof the component was highly correlated to the age of the patients. These results suggest the existence of twopathways of progression in retinoblastoma.The analysis of high throughput data provides many lists of genes. To interpret them, a possibility isto study the latest related publications grouped by pre-defined group of topics (function, cellular location...).To that aim, in a third study, we introduced a web-based Java application tool named GeneValorization whichgives a clear and handful overview of the bibliography corresponding to one particular gene list [7].

<p>The aim of this thesis was to investigate the cellular role of the mouse RBBP6 gene using the interference RNA (RNAi) gene targeting technology and also to understand the relevance of two promoters for the RBBP6 gene.</p>

<p>The aim of this thesis was to investigate the cellular role of the mouse RBBP6 gene using the interference RNA (RNAi) gene targeting technology and also to understand the relevance of two promoters for the RBBP6 gene.</p>