Genomic imprinting provides an exception to the Mendelian rule of inheritance, as imprinted genes are preferentially expressed in a parent-of-origin specific manner. They play important roles in the development of embryonic and extra-embryonic lineages and postnatally in the maintenance of correct metabolic homeostasis as well as regulation of adult behaviour. The parental conflict theory predicts that maternally expressed genes act as growth suppressors, limiting the usage of maternal resources, and that paternally expressed genes function in an opposite manner to promote growth at the expense of maternal resources. Growth factor bound protein 10 (Grb10) is an imprinted gene encoding an intracellular adaptor protein that can interact with several receptor tyrosine kinases and downstream signalling molecules. Recently, our lab has identified Grb10 as a unique imprinted gene capable of influencing fetal growth, postnatal energy metabolism and adult behaviour depending on functions of each of the parental alleles in distinct tissues. Grb10 predominantly expressed from the maternal allele during embryogenesis affects fetal and placental growth along with postnatal glucose homeostasis, whereas paternal Grb10 expression within the CNS influences social behaviour. Delta-like homologue 1 is (Dlk1) a paternally expressed imprinted gene coding for a protein belonging to the Notch/Delta family that acts as a membrane-associated or a soluble protein known to regulate differentiation of various cell types, notably adipocytes. In vivo Dlk1 has been associated with perinatal survival, regulation of normal growth and development and maintenance of the correct course of adipogenesis. Here a hypothesis is proposed that Grb10, as a predominantly maternally expressed growth inhibitor and Dlk1, a paternally expressed growth promoter, act antagonistically in a common genetic pathway. To test this hypothesis, we have generated Grb10m/+/Dlk1+/p double knockout mice and performed a phenotypic characterisation in comparison with wild type as well as the respective single knockout animals. Results obtained from allometric and metabolic analyses, together with histological studies, reveal strong similarities between the phenotypes of Grb10m/+and Grb10m/+/Dlk1+/p knockout mice. We found that overgrowth of Grb10m/+/Dlk1+/p embryos and placentae resemble the phenotype seen in Grb10m/+ mutants and that tissue overgrowth most likely results from higher proliferation rates of Grb10m/+and Grb10m/+/Dlk1+/p cells. Furthermore, Grb10m/+and Grb10m/+/Dlk1+/p knockout mice each exhibit improved glucose clearance and share an unusual characteristic accumulation of lipid in neonatal liver. These results are consistent with the proposed hypothesis and indicate that the Dlk1 and Grb10 genes might be involved in the same genetic pathway. Moreover, the data suggest Dlk1 is an inhibitor of Grb10 which is in turn acting as a growth suppressor.

Delta-like homologue 1 (Dlk1) and Dlk2 encode vertebrate-specific transmembrane proteins belonging to the Jagged/Delta/Serrate family of Notch ligands. Murine Dlk1 is widely expressed during embryonic development and targeted deletion results in defects in numerous developmental processes, such as adipogenesis, haematopoiesis, neurogenesis and skeletal muscle formation. However, the mechanisms by which DLK1 regulates these processes remains unclear. The purpose of this project is to examine the function of these genes using zebrafish as an in vivo model, allowing insight to the ancestral functions of these genes. We have strong evolutionary evidence that dlk2 is the ancestral version of the gene from which dlk1 is derived; therefore, the thesis focuses primarily on the role of dlk2 in the zebrafish system. I initially examine the expression of zebrafish dlk1 and dlk2 during embryonic development and in the adult brain, determining similarities and differences between mouse and zebrafish. In particular, dlk1 and dlk2 in the fish exhibit a pattern that is more reminiscent of Dlk2 in the mouse. This developmental expression pattern is essential for the interpretation of the modulation of Dlk2 in later chapters, and is aided by the generation of a mammalian Dlk2 antibody that cross-reacts with zebrafish. We obtained a dlk2 mutant and used this line to examine the role of the DLK2 protein in development and in the adult brain. I demonstrate that, in the absence of DLK2, a population of neural precursor cells appear to over-proliferate early in zebrafish development. Later, by larval stages, these cells are absent, suggesting a premature activation and subsequent depletion of the progenitor cell pool in the mutant, reminiscent of the Dlk1 mutant in mouse. Associated with this phenotype are larval behavioral defects in motor response. In this thesis, it will be shown that in the adult dlk2 mutant zebrafish, the radial glial cell population in the telencephalon is completely depleted. These radial glial cells are thought to be responsible for adult neural regeneration in zebrafish, and our characterization of a mutant completely lacking this cell population provides a rich model to further examine and understand the functions of this well-studied but poorly understood cell population. These findings have both functional and evolutionary implications for the relative roles of these two vertebrate specific atypical Notch ligands.

No description available.

Le cœur des vertébrés se développe à partir du tube cardiaque et de la participation des cellules progénitrices mésodermiques du second champ cardiaque (SHF). Une perturbation de l’addition des cellules du SHF conduit à des malformations cardiaques congénitales (MCC). Chez l’embryon, l’outflow tract (OFT) dérivé du seul SHF est formé par deux domaines complémentaires qui formeront le myocarde sous-aortique et sous-pulmonaire. Ce travail analyse les cellules progénitrices du SHF qui contribuent aux deux domaines de l’OFT pour former la base de l’aorte et du tronc pulmonaire, l’identité transcriptionnelle des domaines et leur régulation. Nous avons mis en évidence une sous-population de cellules progénitrices Notch-dépendantes, situées en région antérieure du mésoderme pharyngé, qui contribue au myocarde sous-aortique. Nous avons démontré que des cascades de régulation croisées impliquant Notch/Hes1 et Tbx1/Pparg sont importantes pour former les deux domaines fonctionnels régionalisés de l’OFT. Des expériences de culture d’explants et d’embryons ont démontré que Pparg est nécessaire au déploiement des cellules du SHF et pour la régulation transcriptionnelle du futur myocarde sous-pulmonaire. Dans le domaine complémentaire, futur myocarde sous-aortique, nous avons observé l’expression de Dlk1, un régulateur négatif de Pparg. Dlk1 est en amont de la voie de régulation Notch et participe probablement à l’identité régionale de l’OFT. Dans son ensemble, ce travail identifie de nouvelles voies de signalisation et gènes qui régulent l'identité régionale du mésoderme cardio-pharyngé et de nouvelles cibles pour l’étude clinique des MCC. / The vertebrate heart develops from the heart tube and the contribution of mesodermal progenitors termed second heart field (SHF). Perturbation in SHF addition leads to congenital heart defects (CHD). The outflow tract (OFT) myocardium is entirely derived from the SHF. Distinct regions of the embryonic OFT have been shown to give rise to subaortic and subpulmonary myocardium of the heart. The work described here focuses on SHF progenitor subpopulations in mouse giving rise to distinct OFT domains and characterizes the regional transcriptional identity and regulation of future subaortic and subpulmonary myocardium. We identified Notch-dependent subaortic myocardial SHF progenitors in anterior pharyngeal mesoderm. We demonstrated that Notch/Hes1 and Tbx1/Pparg cross regulatory cascades are important to establish functionally important OFT regional domains. Explant and embryo culture experiments revealed that Pparg is required for both the deployment of SHF cells and transcriptional regulation of the future subpulmonary myocardial domain. We also found that Dlk1, a negative regulator of Pparg, is expressed in the complementary subaortic domain upstream of Notch receptor activation and potentially participates in the establishment of OFT regional identity. We also report an overlapping transcriptional profile between future subaortic myocardium and subpopulation of epicardial cells at fetal stages. Finally, we provide evidence for the existence of conserved bipotential myogenic progenitors in cardiopharyngeal mesoderm coexpressing Nkx2-5 and Tbx1. Overall this work identifies novel pathways and genes in cardiopharyngeal mesoderm that may contribute to clinically relevant CHD.

Delta-like 1 Homologue (DLK1), a transmembrane protein of 383 amino acids, belongs to a family of epidermal growth factor (EGF)-like repeat-containing proteins that include Notch/Delta/Serrate, which are involved in cell fate determination. DLK1 is also known as preadipocyte factor-1, pG2, and FA-1, which are identical or polymorphic products of a single gene. Structural analysis revealed that DLK1 consists of an extracellular domain with six EGF-like repeats, a transmembrane domain, and an intracellular domain. The extracellular EGF-like region of DLK1 (DLK1-EC) can be released to the medium by the action of tumor necrosis factor alpha converting enzyme (TACE). DLK1 participates in various differentiation processes including adipogenesis, hematopoiesis, and adrenal gland differentiation. Besides, DLK1 overexpression was observed in patients with biliary atresia and in glioblastoma. Recently, the extracellular domain of thrombomodulin, which also contains six EGF¡Vlike structures, has been delineated to stimulate angiogenesis in vitro and in vivo. This prompted us to investigate whether DLK1-EC played a role in angiogenesis. To test such hypothesis, recombinant DLK1-EC was expressed and purified in E. coli. Adding DLK1-EC recombinant protein inhibited the adipogenesis of adipocytes-derived stem cells in a dose-dependent manner. Despite marginal effect on matrix-metalloproteinase secretion, exogenous DLK1-EC significantly stimulated the proliferation, motility and tube-forming capability of cultured endothelial cells. Above all, implantation of DLK1-EC-containing hydron pellets induced cornea neovascularization in a dose-dependent manner. Western blot analysis revealed that exogenous DLK1-EC induced angiogenesis through Notch1 activating downstream gene Hes1 and subsequently signaling such as Akt/eNOS, p38 MAPK, and ERK pathway to perform its function. Indeed, blockade of Notch1 signaling using £^-secretase inhibitor leads to decreased angiogenesis and inhibits DLK1 EC-induced endothelial cell tubular formation in vitro and in vivo. These findings indicate that DLK1-EC induced Notch1 activation mediated by £^-secretase and tansactivation Akt/eNOS pathway and that Notch1 is critical for DLK1 EC-induced angiogenesis. These results may bring further insights into the physiological and pathological functions of DLK1

La proteïna desacoblant mitocondrial UCP3, expressada en teixits metabòlicament molt importants (muscular i adipós marró) i homòloga a la proteïna termogènica UCP1, ha estat motiu de nombrosos estudis en els darrers anys dirigits a determinar la seva funció. Són moltes les dades bioquímiques i cel·lulars obtingudes, però la realitat és que la seva funció fisiològica bàsica en l'organisme no es coneix encara amb claretat. Amb aquest objetiu i mitjançant l'estudi d'expressió gènica en múscul esquelètic i teixits adiposos de ratolí knockout per UCP3 hem observat que: 1. L'absència d'UCP3 per disrupció dirigida del seu gen en aquests ratolins no causa alteracions significatives d'expressió de gens implicats en estrés oxidatiu en múscul esquelètic de ratolins nounats, tot i que en aquest període l'expressió d'UCP3 es veu fortament induïda en el ratolí wild-type. 2. En ratolins adultsknockout per a UCP3 en procés d'envelliment no es produeixen alteracions significatives dels paràmetres metabòlics circulants i l'expressió de gens implicats en estrés oxidatiu i metabolisme energètic en múscul esquelètic, teixit adipós marró, teixit adipós blanc i fetge resulta mínimament afectada. L'anàlisi massiu d'expressió gènica en múscul mitjançant microarray de genoma complet, tampoc evidencia alteracions substancials. 3. L'exposició perllongada a una dieta hiperlipídica sense carbohidrats en ratolins en envelliment dóna lloc a un increment de pes corporal sense alteracions destacables a nivell de metabolits circulants. Aquesta dieta provoca un fort increment d'expressió de la proteïna d'UCP3 a múscul esquelètic. 4. La dieta hiperlipídica té efectes diferencials en els ratolins knockout per UCP3, que es manifiesten en un increment específic de còssos cetònics circulants i en alteracions de l'expressió de gens implicats en estrés oxidatiu i metabolisme energètic. Això es produeix especialment en teixit adipós marró i fetge, veient-se en general, incrementada l'expressió de gens del catabolisme lipídic. 5. Malgrat la baixada d'expressió de Pref-1 en teixits adults, aquest gen es mostra sensiblement regulat, induint-se en teixit adipós marró degut a la manca d'UCP3 i en teixit adipós blanc en resposta a la dieta hiperlipídica.El teixit adipós marró i el teixit adipós blanc tenen un paper oposat al balanç energètic de l'individu. Davant l'evidència de Pref-1 com a controlador negatiu de la diferenciació de l'adipòcit blanc, ens plantejàrem l'estudi de la seva funció biològica en teixit adipós marró així com la seva regulació mitjançant models cel·lulars d'adipòcits marrons en cultiu i models d'animals modificats genèticament. Els nostres resultats indiquen que: 1. L'absència de Pref-1 per disrupció dirigida del gen (ratolins knockout per Pref-1) causa alteracions morfològiques i d'expressió gènica en el teixit adipós marró durant el desenvolupament, consistents en una sobreactivació termogènica del teixit. Això suggereix un potencial papel repressor de Pref-1 en aspectes específics (programa termogènic) de la diferenciació del teixit adipós marró respecte el blanc. No obstant, aquesta alteració sembla no mantenir-se en ratolins adults sota un estímul termogènic. 2. A diferència del teixit adipós blanc, el desenvolupament del teixit adipós marró es dóna en el període fetal, en un context d'elevada expressió del gen Pref-1. 3. En adipòcits marrons, el gen Pref-1 té regulació positiva per C/EBP-beta i sobretot, per C/EBP-delta que actua com a factor de transcripció regulador del promotor gènic de Pref-1. Aquest fet explicaria la potent sobreinducció de Pref-1 en teixit adipós marró de ratolins knockout per C/EBP-alfa, on s'observa sobreexpressió de C/EBP-beta i C/EBP-delta. Aquests resultats indicarien un paper diferencial de C/EBP-delta sobre la diferenciació i expressió gènica de l'adipòcit marró respecte l'adipòcit blanc. / Study of UCP3 and Pref-1 gene regulation in muscular and adipose tissuesAbstract: Mitochondrial uncoupling protein 3 (UCP3) homologous to termogenic UCP1, is mainly expressed in muscular and brown adipose tissues but its physiological function remains unknown. We have carried out gene expression studies in skeletal muscle and adipose tissues of UCP3 knockout mice and we have observed: 1. UCP3 gene deletion in those mice does not impairs oxidative stress related gene expression in skeletal muscle from newborn mice, althought UCP3 gene expression is dramatically induced in wild-type mice during this period. 2. Ageing does not impairs blood parameters neither expression of genes related to oxidative stress and oxidative metabolism in skeletal muscle, brown (BAT) and white (WAT) adipose tissues and liver from knockout mice. Whole genome microarray did not show clear changes in gene expresion. 3. Long term high-fat carbohidrate free diet feeding during agening lead to body weight increase without any change in blood parameters, and a significant increase in UCP3 protein content in skeletal muscle. 4. This diet caused diferential effects on knockout mice: blood ketone bodies increment and impaired expression of genes related to oxidative stress and energetic metabolism. 5. Pref-1 gene is sensitively regulated in adult brown and white fat.BAT and WAT have and oposite role in energy balance. Since Pref-1 has been described as a negative regulator of white adipocyte differenciation, we have studied its biological function and regulation on brown adipocytes and on BAT. 1. Pref-1 gene disruption (Pref-1 knockout mice) impairs BAT morfology and gene expression during its development, consisting on a thermogenic overactivation. This suggest a specific potential repressor rol for Pref-1 compared to WAT differentiation. Nevertheless, these changes do not persist in adult mice under thermogenic stimulus. 2. Unlike WAT, BAT development take place in foetal period, in presence of a high Pref-1 gene expression. 3. In brown adipocytes, Pref-1 gene is positively regulated by C/EBP-beta and specially by C/EBP-delta transcription factors. This fact can explain Pref-1 strong overinduction in BAT from C/EBP-alpha knockout mice which also displays C/EBP-beta and C/EBP-delta overexpression. These results suggest differential rol for C/EBP-delta in brown adipocyte differentiation and gene expression compared to white adipocyte.

Efficient targeting of genes to either inhibit or increase their expression in specific tissues in vivo remains a challenge. Adeno-Associated Virus (AAV) has emerged as an efficacious delivery method in both humans and murine model systems. AAV is a non-enveloped, single-stranded DNA virus that is non-integrating with long-term expression. Due to its low immunogenicity and various serotypes with specific tissue tropisms, AAV is a preferred choice for organ specific-gene delivery in many experimental settings. This project focused on protocol optimization for high-volume production of AAV plasmids, improved transfection efficiency, and increased viral yield and purity to specifically target noncoding RNAs (ncRNAs) expressed from the imprinted Dlk1-Dio3 locus. Five AAV9 viruses were produced, each containing one of the following transgenes: 1) human Meg3 cDNA for overexpression of this long noncoding RNA, 2) Meg3-specific short hairpin RNA for knockdown analysis, 3) eGFP cDNA to demonstrate AAV9 tissue tropism, 4) Cas9 cDNA, and 5) gene-specific guide RNAs to target the Meg3 proximal promoter. The AAV9 virus production protocol optimized in this project expands the tools available for in vivo study of the Dlk1-Dio3 ncRNA locus.

Avanços recentes na etiologia da puberdade precoce foram obtidos a partir da análise do genoma por sequenciamento global. Mutações inativadoras do gene MKRN3 representam uma causa importante de puberdade precoce central (PPC) familial (33-46% dos casos). O objetivo do estudo foi a análise do DNA genômico de pacientes com PPC de origem familial ou esporádica sem mutações deletérias no gene MKRN3. Foram selecionados 68 indivíduos com PPC (37 com a forma familial e 31, aparentemente, esporádicos). O DNA genômico foi extraído do sangue periférico ou da saliva dos pacientes com PPC. A técnica de sequenciamento genômico em larga escala (ILLUMNA -Clonal Single Molecule Array Technology - CSMA) foi usada na busca de novos genes implicados com o desenvolvimento puberal prematuro em seis indivíduos, sendo três afetados e três não afetados, pertencentes a uma grande família brasileira com PPC (Família 1). Mutações em um gene candidato foram pesquisadas em 64 pacientes por sequenciamento automático direto (método de Sanger). Em um subgrupo de pacientes, foi realizada a técnica de MLPA com sondas customizadas na busca de deleções. Por sequenciamento genômico global, foi identificado um novo complexo rearranjo no gene DLK1, caracterizado por uma deleção de, aproximadamente, 14.000 pb na região 5\' não traduzida (5\'UTR), englobando o início do exon 1, associada a uma duplicação de uma região do intron 3 de 269 pb. O gene DLK1 está localizado no braço longo do cromossomo 14 (14q32.2) e sofre imprinting materno. Este lócus está associado à síndrome de Temple, uma doença complexa com múltiplas manifestações, incluindo puberdade precoce central em até 90% dos casos. Para investigar o efeito dessa deleção genômica, as concentrações séricas da proteína DLK1 pelo método ELISA foram medidas nas pacientes afetadas da Família 1. Valores indetectáveis de DLK1 foram encontrados nestas pacientes. O fenótipo das pacientes afetadas da Família 1 caracterizou-se por uma PPC típica, sem sinais sindrômicos (excluída a síndrome de Temple). Posteriormente, por meio do sequenciamento direto, duas novas mutações inativadoras no gene DLK1 foram identificadas (p.Val272Cysfs*14 e p.Pro160Leufs*50) em duas famílias (Famílias 2 e 3) com PPC ou história de menarca precoce. O estudo de segregação nas Famílias 1 e 2 confirmou o padrão de herança autossômico dominante com penetrância completa e transmissão exclusiva pelo alelo paterno. A média de idade de início da puberdade nas pacientes afetadas do sexo feminino foi de 5,4 anos. A técnica de MLPA com sondas customizadas para o gene DLK1 não encontrou outras deleções no subgrupo estudado. Em conclusão, foram identificadas três mutações inativadoras no gene DLK1 associadas à PPC familial de origem paterna. O DLK1 é o segundo gene imprintado associado a distúrbios puberais em humanos. Este achado sugere um papel dos genes imprintados no controle da puberdade. O mecanismo pelo qual esse gene afeta a puberdade ainda é desconhecido / Recent advances in the etiology of precocious puberty were obtained from the whole-genome sequencing analysis. Inactivating mutations of the MKRN3 gene represent a major cause of familial central precocious puberty (CPP) (33%- 46% of the cases). The objective of the study was to analyze the genomic DNA of patients with familial or sporadic CPP without deleterious mutations in the MKRN3 gene. Sixty-eight individuals with CPP (37 with familial form and 31 apparently sporadic cases) were selected. The genomic DNA was extracted from the peripheral blood or saliva of patients with CPP. We used the whole-genomic sequencing technique (ILLUMNA - Clonal Single Molecule Array Technology - CSMA) searching for a new candidate genes implicated in premature pubertal development in 6 individuals, 3 affected and 3 non-affected, belonging to a large Brazilian family with CPP (Family 1). Mutations in one candidate gene were investigated in 64 patients through automatic sequencing (Sanger\'s method). In a subgroup of patients, MLPA using synthetic MLPA probes was performed to search for deletions. A new complex rearrangement in the DLK1 gene characterized by a deletion of approximately 14.000pb in the 5\' untranslated (5\'UTR), encompassing the start of exon 1, associated with a duplication of a region of intron 3 of 269 bp was identified by whole-genomic sequencing. The DLK1 gene is located on the long arm of chromosome 14 (14q32.2) and it is maternally imprinted gene. This locus is associated with Temple syndrome, a complex disorder with multiple alterations, including central precocious puberty in up to 90% of cases. To investigate the effect of this genomic deletion, a serum measurement of DKL1 protein using ELISA method was performed in the affected patients from Family 1. Undetectable serum DLK1 levels were found in these patients. The phenotype of affected patients from Family 1 was characterized by a typical CPP, without syndromic signs (excluding Temple syndrome). Posteriorly, two new inactivating mutations in the gene DLK1 were identified (p.Val272Cysfs*14 and p.Pro160Leufs*50) through direct sequencing in two families (Families 2 and 3) with CPP or precocious menarche history. The segregation studies in Families 1 and 2 confirmed the pattern of dominant autosomal inheritance with complete penetrance and exclusive transmission by the paternal allele. The average age of puberty onset in the affected female patients was 5.4 years. The MLPA technique with synthetic MLPA probes for the DLK1 gene did not find other deletions in the studied subgroup. In conclusion, we identified 3 paternally inherited inactivating mutations in the DLK1 gene associated with familial CPP. The DLK1 is the second imprinted gene associated with pubertal disorders in humans. This finding suggests a role of the imprinted genes in puberty control. The mechanism through which this gene affects puberty is still unknown

Avanços recentes na etiologia da puberdade precoce foram obtidos a partir da análise do genoma por sequenciamento global. Mutações inativadoras do gene MKRN3 representam uma causa importante de puberdade precoce central (PPC) familial (33-46% dos casos). O objetivo do estudo foi a análise do DNA genômico de pacientes com PPC de origem familial ou esporádica sem mutações deletérias no gene MKRN3. Foram selecionados 68 indivíduos com PPC (37 com a forma familial e 31, aparentemente, esporádicos). O DNA genômico foi extraído do sangue periférico ou da saliva dos pacientes com PPC. A técnica de sequenciamento genômico em larga escala (ILLUMNA -Clonal Single Molecule Array Technology - CSMA) foi usada na busca de novos genes implicados com o desenvolvimento puberal prematuro em seis indivíduos, sendo três afetados e três não afetados, pertencentes a uma grande família brasileira com PPC (Família 1). Mutações em um gene candidato foram pesquisadas em 64 pacientes por sequenciamento automático direto (método de Sanger). Em um subgrupo de pacientes, foi realizada a técnica de MLPA com sondas customizadas na busca de deleções. Por sequenciamento genômico global, foi identificado um novo complexo rearranjo no gene DLK1, caracterizado por uma deleção de, aproximadamente, 14.000 pb na região 5\' não traduzida (5\'UTR), englobando o início do exon 1, associada a uma duplicação de uma região do intron 3 de 269 pb. O gene DLK1 está localizado no braço longo do cromossomo 14 (14q32.2) e sofre imprinting materno. Este lócus está associado à síndrome de Temple, uma doença complexa com múltiplas manifestações, incluindo puberdade precoce central em até 90% dos casos. Para investigar o efeito dessa deleção genômica, as concentrações séricas da proteína DLK1 pelo método ELISA foram medidas nas pacientes afetadas da Família 1. Valores indetectáveis de DLK1 foram encontrados nestas pacientes. O fenótipo das pacientes afetadas da Família 1 caracterizou-se por uma PPC típica, sem sinais sindrômicos (excluída a síndrome de Temple). Posteriormente, por meio do sequenciamento direto, duas novas mutações inativadoras no gene DLK1 foram identificadas (p.Val272Cysfs*14 e p.Pro160Leufs*50) em duas famílias (Famílias 2 e 3) com PPC ou história de menarca precoce. O estudo de segregação nas Famílias 1 e 2 confirmou o padrão de herança autossômico dominante com penetrância completa e transmissão exclusiva pelo alelo paterno. A média de idade de início da puberdade nas pacientes afetadas do sexo feminino foi de 5,4 anos. A técnica de MLPA com sondas customizadas para o gene DLK1 não encontrou outras deleções no subgrupo estudado. Em conclusão, foram identificadas três mutações inativadoras no gene DLK1 associadas à PPC familial de origem paterna. O DLK1 é o segundo gene imprintado associado a distúrbios puberais em humanos. Este achado sugere um papel dos genes imprintados no controle da puberdade. O mecanismo pelo qual esse gene afeta a puberdade ainda é desconhecido / Recent advances in the etiology of precocious puberty were obtained from the whole-genome sequencing analysis. Inactivating mutations of the MKRN3 gene represent a major cause of familial central precocious puberty (CPP) (33%- 46% of the cases). The objective of the study was to analyze the genomic DNA of patients with familial or sporadic CPP without deleterious mutations in the MKRN3 gene. Sixty-eight individuals with CPP (37 with familial form and 31 apparently sporadic cases) were selected. The genomic DNA was extracted from the peripheral blood or saliva of patients with CPP. We used the whole-genomic sequencing technique (ILLUMNA - Clonal Single Molecule Array Technology - CSMA) searching for a new candidate genes implicated in premature pubertal development in 6 individuals, 3 affected and 3 non-affected, belonging to a large Brazilian family with CPP (Family 1). Mutations in one candidate gene were investigated in 64 patients through automatic sequencing (Sanger\'s method). In a subgroup of patients, MLPA using synthetic MLPA probes was performed to search for deletions. A new complex rearrangement in the DLK1 gene characterized by a deletion of approximately 14.000pb in the 5\' untranslated (5\'UTR), encompassing the start of exon 1, associated with a duplication of a region of intron 3 of 269 bp was identified by whole-genomic sequencing. The DLK1 gene is located on the long arm of chromosome 14 (14q32.2) and it is maternally imprinted gene. This locus is associated with Temple syndrome, a complex disorder with multiple alterations, including central precocious puberty in up to 90% of cases. To investigate the effect of this genomic deletion, a serum measurement of DKL1 protein using ELISA method was performed in the affected patients from Family 1. Undetectable serum DLK1 levels were found in these patients. The phenotype of affected patients from Family 1 was characterized by a typical CPP, without syndromic signs (excluding Temple syndrome). Posteriorly, two new inactivating mutations in the gene DLK1 were identified (p.Val272Cysfs*14 and p.Pro160Leufs*50) through direct sequencing in two families (Families 2 and 3) with CPP or precocious menarche history. The segregation studies in Families 1 and 2 confirmed the pattern of dominant autosomal inheritance with complete penetrance and exclusive transmission by the paternal allele. The average age of puberty onset in the affected female patients was 5.4 years. The MLPA technique with synthetic MLPA probes for the DLK1 gene did not find other deletions in the studied subgroup. In conclusion, we identified 3 paternally inherited inactivating mutations in the DLK1 gene associated with familial CPP. The DLK1 is the second imprinted gene associated with pubertal disorders in humans. This finding suggests a role of the imprinted genes in puberty control. The mechanism through which this gene affects puberty is still unknown

<p>Understanding signalling pathways that control pancreatic endocrine tumour (PET) development and proliferation may reveal novel targets for therapeutic intervention. The pathogenesis for sporadic and hereditary PETs, apart from mutations of the <i>MEN1</i> and <i>VHL</i> tumour suppressor genes, is still elusive. The protein product of the <i>MEN1</i> gene, menin, regulates many genes. The aim of this thesis was to identify genes involved in pancreatic endocrine tumourigenesis, with special reference to Notch signalling.</p><p>Messenger RNA and protein expression of NOTCH1, HES1, HEY1, ASCL1, NEUROG3, NEUROD1, DLK1, POU3F4, PDX1, RPL10, DKK1 and TPH1 were studied in human PETs, sporadic and MEN 1, as well as in tumours from heterozygous <i>Men1</i> mice. For comparison, normal and <i>MEN1</i> non-tumourous human and mouse pancreatic specimens were used. Nuclear expression of HES1 was consistently absent in PETs. In mouse tumours this coincided with loss of menin expression, and there was a correlation between <i>Men1</i> expression and several Notch signalling factors. A new phenotype consisting of numerous menin-expressing endocrine cell clusters, smaller than islets, was found in <i>Men1</i> mice. Expression of NEUROG3 and NEUROD1 was predominantly localised to the cytoplasm in PETs and islets from MEN 1 patients and <i>Men1</i> mice, whereas expression was solely nuclear in wt mice. Differences in expression levels of Pou3f4, Rpl10 and Dlk1 between islets of <i>Men1</i> and wt mice were observed.</p><p>In addition, combined RNA interference and microarray expression analysis in the pancreatic endocrine cell line BON1 identified 158 target genes of ASCL1. For two of these, DKK1 (a negative regulator of the WNT/β-catenin signalling pathway) and TPH1, immunohistochemistry was performed on PETs. In concordance with the microarray finding, DKK1 expression showed an inverse relation to ASCL1 expression.</p><p>Altered subcellular localisation of HES1, NEUROD1 and NEUROG3 and down-regulation of DKK1 may contribute to tumourigenesis.</p>