La télomérase est une ribonucléoprotéine, constituée d'un composant ARN (hTR) qui sert dematrice à l'addition des séquences télomériques aux extrémités des chromosomes et d'un composantprotéique catalytique à activité de transcriptase inverse (hTERT). La réactivation de la télomérasedans 90% des cancers compense le raccourcissement des télomères, permettant ainsil'immortalisation et la survie des cellules tumorales. Ce rôle canonique de la télomérase estaujourd'hui bien documenté. Cependant des travaux récents suggèrent que la télomérase pourraitavoir d'autres fonctions indépendantes de son rôle sur le maintien de la longueur des télomères dansla tumorigénèse et/ou la progression tumorale.Dans les neuroblastomes (NB), l'augmentation du niveau d'activité télomérase (AT) estassociée à un stade avancé de la maladie et à un mauvais pronostic. En effet, plusieurs études ontmontré que les neuroblastomes agressifs ont un niveau élevé d'AT alors que les tumeurs de bonpronostic, ont peu ou pas d'AT. En effet, les NB de stade 4S ayant la capacité de régresserspontanément ont un très faible niveau d'AT. Ces observations suggèrent que la télomérase peutjouer un rôle crucial dans le développement des NB.Afin de mieux comprendre l'implication de la télomérase dans le phénotype agressif desneuroblastes malins et dans la chimiorésistance, nous avons caractérisé les modificationsphénotypiques et génotypiques induites par l'inhibition de la télomérase via l'expression ectopiqued'un mutant dominant négatif catalytiquement inactif (DN-hTERT) dans la lignée IGR-N-91 induit unedifférenciation cellulaire de type stromale et une sensibilisation à l'apoptose en réponse à trois agentscytotoxiques (cisplatine, staurosporine, TRAIL). Cette chimiosensibilisation n'est pas la conséquenced'un raccourcissement des télomères mais probablement celui d'une modulation de l'expression decertains gènes impliqués dans la réponse apoptotique (ré-expression de la caspase 8 et de p53sauvage), suggérant une fonction non canonique de la télomérase. De plus, nous avons montréqu'hTERT régule activement l'expression de N-Myc. En effet, l'expression ectopique du mutanthTERT-DN entraîne une perte des copies surnuméraires de N-Myc conduisant à l'extinction del'expression de la protéine alors que la surexpression d'hTERT sauvage augmente au contraire lenombre de copies du gène. Cette élimination de la protéine N-Myc pourrait être le signe d'une pertedu caractère agressif des cellules tumorales comme en témoigne la diminution de l'expression de laNSE (marqueur de mauvais pronostique des NB) et l'induction du CD44 dans les cellules hTERT-DN.L'ensemble de nos résultats démontre donc un nouveau rôle majeur de la télomérase,indépendant de sa fonction canonique d'élongation des télomères, dans l'acquisition du phénotypemalin et dans la chimiorésistance des NB. Ces résultats sont importants en termes de connaissancede la biologie du NB et des possibilités thérapeutiques. En effet, ces résultats suggèrent quel'inhibition de la télomérase comme stratégie anti-cancéreuse est une approche qui présente un intérêttout particulier dans les cas de NB de stade 4 dans lesquels le taux de survie des patients reste trèsinsuffisant malgré les thérapeutiques les plus intensives.

Large-scale expansion of lineage-committed stem cells can provide an excellent ex vivo model for studying complex molecular pathways governing cell fate choices. Also, such cells could be useful for implementing cell therapeutic approaches for treatment of specific disorders involving extensive cellular damage within that lineage. Using growth factors, pluri- and multipotent stem cells have been successfully isolated and cultured from pre- and peri-implantation stage embryos, including trophectoderm, primitive ectoderm, epiblast and primitive endoderm. However, ex vivo expansion of lineage restricted cells from later embryonic lineages and adult tissues have been a challenge. N-myc is a well-characterized member of myc gene family that is known to be essential for the proliferation of numerous progenitor cell types during normal embryonic development of diverse organs including lungs, liver, heart, kidneys and brain. Considering this important role of N-myc, we hypothesized that its regulated activation in these progenitors might allow their expansion in culture. To test this hypothesis, we generated a novel doxycycline-inducible transgenic mouse line that expresses N-myc uniformly across all tissues. Using cortical precursors derived from mid-gestation embryos of these mice, we show that upon doxycycline induced N-myc expression, we can achieve at least a million-fold expansion of multipotent neural precursors within a short span of time in culture. When doxycycline is withdrawn, N-myc expression is turned off and the cells differentiate into neurons and glia. An extensive characterization of the expanded cells revealed that the cells retained their differentiation potential, genomic stability and commitment specific to their origin. The tetracycline-inducible N-myc expressing mouse line might also serve as a source for establishing other than neural lineage committed progenitor cell lines where N-myc has a known role in regulating cell proliferation and differentiation decisions.

Large-scale expansion of lineage-committed stem cells can provide an excellent ex vivo model for studying complex molecular pathways governing cell fate choices. Also, such cells could be useful for implementing cell therapeutic approaches for treatment of specific disorders involving extensive cellular damage within that lineage. Using growth factors, pluri- and multipotent stem cells have been successfully isolated and cultured from pre- and peri-implantation stage embryos, including trophectoderm, primitive ectoderm, epiblast and primitive endoderm. However, ex vivo expansion of lineage restricted cells from later embryonic lineages and adult tissues have been a challenge. N-myc is a well-characterized member of myc gene family that is known to be essential for the proliferation of numerous progenitor cell types during normal embryonic development of diverse organs including lungs, liver, heart, kidneys and brain. Considering this important role of N-myc, we hypothesized that its regulated activation in these progenitors might allow their expansion in culture. To test this hypothesis, we generated a novel doxycycline-inducible transgenic mouse line that expresses N-myc uniformly across all tissues. Using cortical precursors derived from mid-gestation embryos of these mice, we show that upon doxycycline induced N-myc expression, we can achieve at least a million-fold expansion of multipotent neural precursors within a short span of time in culture. When doxycycline is withdrawn, N-myc expression is turned off and the cells differentiate into neurons and glia. An extensive characterization of the expanded cells revealed that the cells retained their differentiation potential, genomic stability and commitment specific to their origin. The tetracycline-inducible N-myc expressing mouse line might also serve as a source for establishing other than neural lineage committed progenitor cell lines where N-myc has a known role in regulating cell proliferation and differentiation decisions.

5 Abstract Neuroblastoma is one of the most common cancer diseases diagnosed in children. This rapidly growing solid tumor is usually formed by hypoxic areas which arise as a consequence of inefficient and disorganized neovascularization. The cells stressed by hypoxia triggers transcription of many genes necessary for their survival, and conversely stop the production of proteins which are not necessarily needed for the survival in these severe conditions. The adaptation of cells to hypoxic conditions may appear due to the epigenetic regulation of metabolism associated with chromatin remodeling which involves the DNA methylation and also the posttranslational modifications of histones. Among the most important of these, there is the acetylation of lysine residues of histones associated with the DNA strands loosening, facilitated binding of transcription factors and the activation of gene expression. Thus, the first part of this study is concerned with changes in the acetylation of histones H3 and H4 of human neuroblastoma cell lines UKF-NB-3, UKF-NB-4, SH-SY5Y and SK-N-AS, cultured in parallel under standard culture conditions and in the absence of oxygen (hypoxia, 1% O2) for 24 hours, which are studied by Western blot analysis. Thereupon, the activity of histone deacetylases and histonacetyltransferases,...

INTRODUÇÃO: O câncer de mama é uma das mais importantes causas de mortalidade feminina no mundo. Acredita-se que as lesões proliferativas do parênquima mamário sejam marcadoras de risco para câncer ou precursoras do carcinoma mamário. Apesar da intensa pesquisa na área do câncer de mama, os eventos moleculares precoces associados à evolução e progressão do câncer mamário ainda são pouco conhecidos. OBJETIVOS: Com a expectativa de melhor compreender os eventos iniciais da carcinogênese mamária examinamos um conjunto de oitenta e quarto lesões proliferativas mamárias quanto à expressão dos genes N-myc down regulated gene (NDRG1), Prostate Apoptosis Response-4 (PAR-4), Osteonectina e Pontina. A expressão destes genes foi documentada, por trabalhos prévios em nosso laboratório ou por estudos de outros autores, como tendo impacto na evolução do câncer de mama. MÉTODOS: Construímos um TMA com lesões proliferativas da mama e testamos este TMA por método imunohistoquímico para Ndrg1, Par-4, Osteonectina e Pontina, bem como para o receptor de estrógeno e citoqueratinas de alto e baixo peso molecular, com o objetivo de caracterizar as lesões presentes no TMA. A avaliação imunohistoquímica foi feita de forma quantitativa, com o aplicativo e sistema de análise quantitativa para TMA ACIS III, da Dako. RESULTADOS: Após excluirmos amostras não informativas, contamos com 68 amostras de lesões proliferativas mamárias para análise. Nestas, observamos uma notável positividade de NDRG1, em lesões com morfologia apócrina. Observamos ainda alta expressão de NDRG1 em lesões proliferativas mamárias, quando comparadas aos outros tipos de lesões presentes no TMA. Pontina exibiu os mais altos valores de expressão nos casos de lesões proliferativas mamárias, com valor de p estatisticamente significativo. A expressão de PAR-4 foi predominantemente nuclear nas lesões mamárias analisadas no TMA. Osteonectina teve expressão diferenciada no epitélio de lesões hiperplásicas da mama, papilomas e papilomas de pequenos ductos, quando comparada às outras lesões presentes no TMA. CONCLUSÕES: Nossos resultados sugerem uma possível associação entre a expressão de NDRG1 e diferenciação apócrina no parênquima mamário. Esta observação está de acordo com os dados publicados a respeito da superexpressão de NDRG1 e a assinatura apócrina molecular de alguns carcinomas de mama. Também demonstramos superexpressão de NDRG1 em condições hiperproliferativas do parênquima mamário, não associadas à diferenciação apócrina. Até o momento não há informações na literatura que possam explicar a translocação nuclear de Par-4 em lesões benignas da mama observada em nosso estudo. Nossos achados fornecem pela primeira vez evidências de que PAR-4 é ativado em lesões proliferativas da mama, indicando a necessidade de estudos futuros dirigidos à investigação das funções de PAR-4 em tecido mamário não neoplásico. O presente trabalho também indicou uma possível ação coordenada entre a expressão de Osteonectina e NDRG1, pelo menos em lesões apócrinas mamárias. A expressão estromal de Osteonectina pareceu ser menos frequente em lesões mamárias com arquitetura papilífera, quando comparadas a lesões mamárias com tendência a recapitular a arquitetura lobular / INTRODUCTION: Breast cancer is a leading cause of death among women all over the Word. Proliferative lesions of the breast are believed to be precursors of or markers of increased risk for breast carcinoma. Although the active research in the field of breast cancer, the early molecular events associated with cancer evolution and progression are still poorly understood. OBJECTIVES: In order to better understand the early events in the breast carcinogenesis, we examined a set of eighty-four proliferative lesions of the breast for the expression of N-myc down regulated gene (NDRG1), Prostate Apoptosis Response-4 (PAR-4), Osteonectin and Pontin, which expression was previously shown by our laboratory to have impact in breast cancer prognosis. METHODS: A tissue microarray were constructed and immunohistochemically tested for Ndrg1, Par-4, Osteonectin and Pontin together with estrogen receptor, low and high weight cytokeratins, aiming to properly characterize the lesions sorted in the tissue microarray. Immunohistochemistry assessment was made quantitative, with the ACIS III Dako quantitative analysis system and TMA application software. RESULTS: After excluding non informative cores, cores with fibroadipose tissue or mammary parenchyma with normal appearing breast epithelium, we ended up with a TMA with 68 breast lesions. Of those, we observed a noticeable positivity of Ndrg1 for lesions with apocrine morphology. Additionally, all cases of florid epithelial hyperplasia showed variable high immunoexpression levels of protein, when compared to the other sorted out lesions in the TMA. Pontin expression level was highest among the breast hyperplasia cases, with statistically significant p value. PAR-4 protein expression was found to be predominantly localized in the nucleous in the non-malignant breast lesions analyzed. Osteonectin exhibited differentiated expression values in the epithelium of hyperplastic breast lesions, papillomas and multiple papillomas when compared to the other types of breast lesions assorted in the TMA. CONCLUSIONS: Our results suggest a possible association of NDRG1 with apocrine differentiation in the breast parenchyma. This observation are consonant with the publish data about the NDRG1 overexpression and the apocrine signature of some mammary cancers. Additionally, we demonstrated that NDRG1 is also overexpressed in some hyperproliferative breast conditions unassociated with apocrine morphology. At present, there is no data available in the in the literature to explain the nuclear translocation of Par-4 in benign breast lesions as observed in our study. However, our findings provide for the first time evidence that PAR-4 is activated in proliferative lesions of the breast indicating that further clinical and experimental studies aiming to investigate PAR-4 function in non neoplastic breast tissue are warranted. Our study also indicated a possible coordinated expression between Osteonectin and NDRG1, at least in apocrine lesions of the breast. Stromal Osteonectin expression seemed to be less intense in breast lesions with papillary architecture, when compared to breast lesions with tendency to recapitulate breast lobular architecture

INTRODUÇÃO: O câncer de mama é uma das mais importantes causas de mortalidade feminina no mundo. Acredita-se que as lesões proliferativas do parênquima mamário sejam marcadoras de risco para câncer ou precursoras do carcinoma mamário. Apesar da intensa pesquisa na área do câncer de mama, os eventos moleculares precoces associados à evolução e progressão do câncer mamário ainda são pouco conhecidos. OBJETIVOS: Com a expectativa de melhor compreender os eventos iniciais da carcinogênese mamária examinamos um conjunto de oitenta e quarto lesões proliferativas mamárias quanto à expressão dos genes N-myc down regulated gene (NDRG1), Prostate Apoptosis Response-4 (PAR-4), Osteonectina e Pontina. A expressão destes genes foi documentada, por trabalhos prévios em nosso laboratório ou por estudos de outros autores, como tendo impacto na evolução do câncer de mama. MÉTODOS: Construímos um TMA com lesões proliferativas da mama e testamos este TMA por método imunohistoquímico para Ndrg1, Par-4, Osteonectina e Pontina, bem como para o receptor de estrógeno e citoqueratinas de alto e baixo peso molecular, com o objetivo de caracterizar as lesões presentes no TMA. A avaliação imunohistoquímica foi feita de forma quantitativa, com o aplicativo e sistema de análise quantitativa para TMA ACIS III, da Dako. RESULTADOS: Após excluirmos amostras não informativas, contamos com 68 amostras de lesões proliferativas mamárias para análise. Nestas, observamos uma notável positividade de NDRG1, em lesões com morfologia apócrina. Observamos ainda alta expressão de NDRG1 em lesões proliferativas mamárias, quando comparadas aos outros tipos de lesões presentes no TMA. Pontina exibiu os mais altos valores de expressão nos casos de lesões proliferativas mamárias, com valor de p estatisticamente significativo. A expressão de PAR-4 foi predominantemente nuclear nas lesões mamárias analisadas no TMA. Osteonectina teve expressão diferenciada no epitélio de lesões hiperplásicas da mama, papilomas e papilomas de pequenos ductos, quando comparada às outras lesões presentes no TMA. CONCLUSÕES: Nossos resultados sugerem uma possível associação entre a expressão de NDRG1 e diferenciação apócrina no parênquima mamário. Esta observação está de acordo com os dados publicados a respeito da superexpressão de NDRG1 e a assinatura apócrina molecular de alguns carcinomas de mama. Também demonstramos superexpressão de NDRG1 em condições hiperproliferativas do parênquima mamário, não associadas à diferenciação apócrina. Até o momento não há informações na literatura que possam explicar a translocação nuclear de Par-4 em lesões benignas da mama observada em nosso estudo. Nossos achados fornecem pela primeira vez evidências de que PAR-4 é ativado em lesões proliferativas da mama, indicando a necessidade de estudos futuros dirigidos à investigação das funções de PAR-4 em tecido mamário não neoplásico. O presente trabalho também indicou uma possível ação coordenada entre a expressão de Osteonectina e NDRG1, pelo menos em lesões apócrinas mamárias. A expressão estromal de Osteonectina pareceu ser menos frequente em lesões mamárias com arquitetura papilífera, quando comparadas a lesões mamárias com tendência a recapitular a arquitetura lobular / INTRODUCTION: Breast cancer is a leading cause of death among women all over the Word. Proliferative lesions of the breast are believed to be precursors of or markers of increased risk for breast carcinoma. Although the active research in the field of breast cancer, the early molecular events associated with cancer evolution and progression are still poorly understood. OBJECTIVES: In order to better understand the early events in the breast carcinogenesis, we examined a set of eighty-four proliferative lesions of the breast for the expression of N-myc down regulated gene (NDRG1), Prostate Apoptosis Response-4 (PAR-4), Osteonectin and Pontin, which expression was previously shown by our laboratory to have impact in breast cancer prognosis. METHODS: A tissue microarray were constructed and immunohistochemically tested for Ndrg1, Par-4, Osteonectin and Pontin together with estrogen receptor, low and high weight cytokeratins, aiming to properly characterize the lesions sorted in the tissue microarray. Immunohistochemistry assessment was made quantitative, with the ACIS III Dako quantitative analysis system and TMA application software. RESULTS: After excluding non informative cores, cores with fibroadipose tissue or mammary parenchyma with normal appearing breast epithelium, we ended up with a TMA with 68 breast lesions. Of those, we observed a noticeable positivity of Ndrg1 for lesions with apocrine morphology. Additionally, all cases of florid epithelial hyperplasia showed variable high immunoexpression levels of protein, when compared to the other sorted out lesions in the TMA. Pontin expression level was highest among the breast hyperplasia cases, with statistically significant p value. PAR-4 protein expression was found to be predominantly localized in the nucleous in the non-malignant breast lesions analyzed. Osteonectin exhibited differentiated expression values in the epithelium of hyperplastic breast lesions, papillomas and multiple papillomas when compared to the other types of breast lesions assorted in the TMA. CONCLUSIONS: Our results suggest a possible association of NDRG1 with apocrine differentiation in the breast parenchyma. This observation are consonant with the publish data about the NDRG1 overexpression and the apocrine signature of some mammary cancers. Additionally, we demonstrated that NDRG1 is also overexpressed in some hyperproliferative breast conditions unassociated with apocrine morphology. At present, there is no data available in the in the literature to explain the nuclear translocation of Par-4 in benign breast lesions as observed in our study. However, our findings provide for the first time evidence that PAR-4 is activated in proliferative lesions of the breast indicating that further clinical and experimental studies aiming to investigate PAR-4 function in non neoplastic breast tissue are warranted. Our study also indicated a possible coordinated expression between Osteonectin and NDRG1, at least in apocrine lesions of the breast. Stromal Osteonectin expression seemed to be less intense in breast lesions with papillary architecture, when compared to breast lesions with tendency to recapitulate breast lobular architecture

La télomérase, enzyme qui permet le maintien de la longueur des télomères est activée dans la majorité des cellules cancéreuses. Du fait de son rôle dans l’immortalité cellulaire, elle a été proposée comme cible de nouvelles stratégies anticancéreuses; il est donc fondamental d’identifier les mécanismes de sa régulation. Des travaux récents du laboratoire ont démontré, en utilisant une lignée de leucémie aiguë promyélocytaire résistante à la différenciation par les rétinoïdes (NB4-LR1), que l’acide rétinoïque tout trans (ATRA) induit une répression transcriptionnelle de hTERT, sous-unité catalytique de la télomérase, indépendamment de la différenciation. Cette répression est également associée à la mort des clones résistants. Il a également été montré lors du traitement par l’ATRA de cellules résistantes à cette répression, NB4-LR1SFD, qu’il existait une hyperméthylation de la région distale du promoteur de hTERT associée à la persistance de sa transcription et à la prolifération cellulaire. Ceci nous a conduit à proposer un modèle de régulation de hTERT dans lequel l'induction de modifications épigénétiques de la région distale de son promoteur empêcherait la fixation d'éventuels répresseurs. Des résultats récents suggèrent que WT1, facteur connu pour contribuer à la répression de hTERT, pourrait être un de ces facteurs. WT1 code pour un facteur de transcription à doigt de zinc et a été décrit pour la première fois comme délété dans les tumeurs de Wilms (tumeurs rénales de l’enfant). Cependant le rôle de WT1 dans développement tumoral varie en fonction des modèles cellulaires ; il peut avoir un rôle d’oncogène ou au contraire agir comme un gène suppresseur de tumeur. Dans le but d’élucider les mécanismes de régulation de la télomérase, nous avons donc voulu étudier le rôle de WT1 dans la répression de hTERT. Pour ce travail, nous avons utilisé deux modèles cellulaires :• La Leucémie aigue promyélocytaire (LAP) : leucémie aigue myéloblastique caractérisée par un transcrit de fusion impliquant le récepteur aux rétinoïdes.• Le neuroblastome : tumeur solide extra crânienne la plus fréquente chez l’enfant. Cette maladie est très hétérogène aussi bien au niveau biologique que clinique ; en effet certaines formes peuvent régresser spontanément alors que d’autres, très agressives, sont résistantes à toutes les thérapeutiques actuelles. Cette hétérogénéité clinique est notamment liée à la différenciation de la tumeur et également à la présence ou non de l’amplification de l’oncogène N-Myc qui a un rôle pronostic majeur dans cette maladie. Les voies de signalisations impliquées dans le fort pouvoir tumorigène des neuroblastomes ne sont pas encore clairement identifiées.Nous avons pu montrer dans ces modèles que WT1 réprime hTERT et que cette répression semble fonction de l'état de méthylation de la région distale du promoteur de hTERTLa 2ème partie de mon travail a été d’étudier le rôle de WT1 dans les neuroblastomes. Les résultats de nos travaux montrent que WT1 est plus exprimé dans les tumeurs sans amplification de N-Myc et dont la différenciation est stromale. Cependant la surexpression de WT1 dans des tumeurs sans amplification de N-Myc semble associée à un moins bon pronostic. L’étude de l’expression de WT1 pourrait constituer un outil pronostic intéressant dans ces tumeurs. / Telomerase is expressed and active in most immortalized cells. Whereas telomerase becomes activated during neoplastic transformation, its activity decreases during differentiation of various immortal cells in response to pharmacological agents, including retinoids. We showed using both an Acute Promyelocytic Leukemia (APL) cellular model (NB4 cell model) and Neuroblastoma cells that all-trans-retinoic acid (ATRA) induced a transcriptional repression of the catalytic subunit of telomerase, hTERT, associated with differentiation. This repression also occurred independently of differentiation, as demonstrated during long term treatment of ATRA-induced maturation resistant NB4-LR1, leading to telomere shortening, growth arrest and cell death. Changes in chromatin environment of hTERT promoter and binding of transcriptional factors have been demonstrated in differentiating cells when hTERT is repressed. However, it is not clear whether these changes are directly involved in hTERT repression or only linked to differentiation. A variant cell line was isolated from NB4-LR1 cells, (NB4-LR1SFD), which bypassed the death step because of a re-activated telomerase and resisted to hTERT repression despite the continuous presence of ATRA. Using both NB4-LR1 and NB4-LR1SFD cells, it was shown that the mechanisms of ATRA-induced hTERT repression involved: 1) a switch from c-myc to mad1 binding at the proximal domain of hTERT promoter, 2) epigenetic modifications of the distal domain of hTERT promoter (-600 -250 nucleotides). Indeed, the persistence of hTERT transcription was associated with an hypermethylation of the distal domain in ATRA-treated NB4-LR1SFD. Interestingly, the binding of a known hTERT repressor, WT1, to this distal domain, was defective in NB4-LR1SFD. The first part of my thesis was to investigate the role in hTERT transcriptionnal regulation of both c-myc and WT1, two transcription factors, which bind to the proximal and the distal region of hTERT promoter, respectively. The second part was to determine the role of WT1 in neuroblastoma. Neuroblastoma is the most common extra cranial solid tumor in childhood and the most frequently diagnosed neoplasm during the infancy. A striking feature of this tumor is its clinical heterogeneity. Among tumor progression markers delineated so far, MYCN amplification occurs in about 25% of total neuroblastoma cases and this percentage increases at 30% in advanced stage NEUROBLASTOMA. Despite the fact that MYCN amplification is strongly correlated with neuroblastoma of poor outcome, MYCN status cannot predict all cases of poor survival in neuroblastoma. In addition, neuroblastoma without MYCN amplification (about 70-80% of neuroblastoma) are far to display favorable behavior. WT1 was initially identified as a tumor suppressor gene involved in the development of a pediatric renal tumor (Wilm’s tumor). Here, we describe an inverse correlation between WT1 expression and MYCN amplification and expression. However and most notably, our results show that WT1 gene expression is associated with a poor outcome for patients showing non-MYCN-amplified tumors. Thus WT1 expression may be a prognostic marker for a better risk-stratification and for an optimized therapeutic management of neuroblastoma.

MYC is a family of three regulator genes that codes for transcription factors. Expression of Myc proteins from MYC genes is found to be deregulated in 70 % of all cancer forms. The three human homologs C-Myc, N-Myc and L-Myc are mainly associated with cancer in the lymphatic system, nerve tissues and lung cancer, respectively. Even though N-Myc is associated with Neuroblastoma, the cancer variant that is most common among children, the field is focused towards C-Myc. The activation of C-Myc begins with phosphorylation of Serine 62, followed by trans-to-cis isomerisation of Proline 63. Then Threonine 58 becomes phosphorylated leading to that Serine 62 is dephosphorylated and subsequent cis-to-trans isomerisation of Proline 63, and C-Myc is marked for degradation. Cis-trans isomerisation is necessary for regulation of gene expression, and is therefore important to understand. Since N-Myc and C-Myc have identical sequences between residues 47 to residue 69, the hypothesis is that N-Myc is activated in the same manner, but this has not been confirmed. In this project the first 69 amino acids of N-Myc were analysed with NMR spectroscopy. This resulted in a near complete assignment of the major conformation, and of the alternative minor conformations as well. The traditional assignment experiments HNCACB, HN(CO)CACB, HNCO, HN(CA)CO in combination with CCH-TOCSY and HN(CCO)C revealed that the majority of the minor configurations can be explained by cis/trans isomerisation of prolines. In addition, the protein was analysed with direct carbon detected NMR spectroscopy to be able to detect the prolines.