Le locus H19/IGF2 est soumis à l’empreinte génomique. A partir de l’allèle maternel, deux ARN non codants sont exprimés : H19 et 91H. L’ARN H19 est le précurseur de microARN (miR-675) dont aucune cible n’a été décrite dans la glande mammaire. Nous avons donc recherché et validé plusieurs cibles de ses microARN permettant d’expliquer les propriétés oncogéniques d’H19. En effet, le miR-675 régule l’expression des gènes c-Cbl, Cbl-b et FADD dans les cellules cancéreuses mammaires. Ces nouvelles régulations permettent l’augmentation de la prolifération cellulaire, la migration/invasion mais aussi la résistance aux apoptogènes. Dans cette étude, de manière inattendue et surprenante, nous avons également découvert un nouveau mécanisme de recrutement des microARN. Nous montrons que les protéines associées aux ARNm peuvent favoriser le recrutement du complexe RISC, mais surtout moduler l’action de celui-ci. De plus, dans certains cancers, l’ARN H19 peut s’associer aux protéines, notamment la protéine P53 dans les cancers gastriques, pour moduler leur fonction. Dans le cancer du sein, cette interaction empêche l’action de P53 et favorise sa dégradation. Cette nouvelle régulation de P53 peut être responsable de certaines résistances tumorales. Par ailleurs, l’implication d’H19 dans la formation et le maintien des cellules souches a été mise en évidence. Cette nouvelle fonction peut permettre à l’ARN H19 de promouvoir l’initiation ou la récidive tumorale. Les fonctions du long ARN non codant 91H dans les cellules cancéreuses mammaires ont également été étudiées. Cet ARN possède des propriétés oncogéniques et permet l’expression des gènes H19 et IGF2 en modulant la conformation de la chromatine au locus. Pour conclure, j’ai identifié plusieurs modes d’action de l’ARN H19 lui permettant de promouvoir l’apparition de la tumeur, sa progression et sa résistance aux thérapies. J’ai également défini le rôle de l’ARN 91H dans la tumorigenèse et au locus H19/IGF2. / The H19/IGF2 locus is submitted to the genomic imprinting. From the maternal allele, two long non-coding RNA are transcribed: H19 and 91H. The H19 RNA is a precursor of microRNA (miR-675). Few targets have been identified but neither in breast cancer cells lines. I have identified and checked three targets of miR-675 involved in H19 RNA oncogenic properties. Indeed, the miR-675 regulate expression of c-Cbl, Cbl-b and FADD mRNA. These new regulation promotes cell proliferation, cell migration/invasion and resistance of cell apoptosis. Moreover, surprisingly, in these studies, I have highlight new mechanism of microRNA recruitment and function. Indeed, I have established that proteins associated to mRNA regulate microRNA recruitment and function.In cancers, the H19 could be associated with protein, for example the P53 protein in gastric cancer, in order to regulate their function. In breast cancer cells, the H19/P53 association prevents the P53 function and promotes its degradation. This new P53 regulation could be involved in drug resistance in cancer. Otherwise, I have shown that H19 is involved in stem cell formation and maintenance. This new H19 function could be involved in tumor initiation or in tumor recurrence. To finish, I have also determined the function of the H19 antisense long non-coding RNA, 91H. In breast cancer cells, I have demonstrated that 91H acts as an oncogene and promotes H19 and IGF2 expression by modulating chromatin conformation at the locus. In conclusion, I have identified several H19 mechanism involved in tumor formation, progression or resistance to treatment. I have also decipher 91H function in tumorigenesis and at the H19/IGF2 locus.

L'empreinte génomique, une régulation épigénétique unique entraînant une expression génique spécifique aux parents d'origine, est essentielle au développement et à la croissance des mammifères. H19 est un ARN long non codant exprimé en milieu maternel qui est un régulateur central du réseau de gènes à empreinte contrôlant le développement. H19 est exprimé pendant le développement embryonnaire dans de nombreux tissus, y compris toutes les cellules hématopoïétiques. Le rôle de H19 au cours du développement embryonnaire n'a été documenté que pour le placenta où il contrôle la croissance. Le rôle de H19 dans la lymphopoïèse n'a pas été étudié. Notre laboratoire a précédemment trouvé H19 comme principal transcrit exprimé sélectivement par les proB du foie foetal (FF), et exprimé de façon différentielle par des immigrants thymiques précoces et tardives. Cependant, le rôle du gèneH19 dans celui du développement des cellules B, ou même dans le système immunitaire, reste méconnu. Ici, nous avons réalisé une caractérisation complète des perturbations du développement et de la fonction du système immunitaire des souris pour lesquelles un grand segment du locus H19 a été supprimé. Dans cette étude, nous avons constaté que le mutant H19 avait un impact spécifique sur le développement des cellules du FF induisant une augmentation sélective du nombre des cellules proB BP1+ présentant des perturbations importantes du réarrangement du locus IgH.On observe également chez les animaux H19-/- adultes une expansion anormale du compartiment B mature. Bien que H19 ne soit plus exprimé après la naissance, les lymphocytes B des souris adultes mutantes présentent un phénotype altéré. On observe en effet des perturbations importantes du profil d'expression du marqueur B220. Les souris H19-/-présentent un défaut d'expansion des lymphocytes B du centre germinatif, ainsi qu'une chute de la production des IgM spécifiques dans le sérum après immunisation. Indiquant une réponse défectueuse des cellules B. De manière cohérente, nous avons trouvé une réactivité réduite au BCR des cellules B naïves H19-/-, associée les expériences de reconstitution compétitive ont mis en évidence un altération cellule-intrinsèque de la réponse humorale chez les animaux mutants. Un défaut d'induction de l'expression des molécules du CMHII, CD40,et CD86, qui pourrait être à l'origine des perturbations de la réponse humorale observée chez les souris H19-/-. Les analyse de transcriptome réalisées sur les lymphocytes B du centre germinal des animaux mutants ont mis en évidence une expression différentielle des gènes impliqués dans la régulation de l'intensité du signal émanant du récepteur B à l'antigène. Au total ce travail nous a permis de démontrer l'activité régulatrice exercée par l'ARN non codant H19 sur le développement et la fonction du système immunitaire. / Genomic imprinting, a unique epigenetic regulation resulting in a parent-of-origin specificgene expression, is essential for normal mammalian development and growth. H19 is amaternally expressed long non-coding RNA that is a central regulator of the imprinting gene network controlling development and growth. H19 is expressed throughout embryonic development in multiple tissues including all hematopoietic cells. The role of H19 during embryonic development has only been documented for the placenta where it controls growthand the role of H19 in lymphopoiesis has not been investigated. The laboratory has previouslyfound H19 as the major differentially expressed transcript in two microarrays comparing fetalliver (FL) and bone marrow (BM) derived pro-B cells, as well as between early and latethymic settling progenitors. However, a role for imprinting gene H19 in B cell development,or even in immune system remains elusive. Here we sought to analyze mice where a large segment of the H19 locus has been deleted. In our work, we found that loss of H19 have specific impact on the FL B cell development byproducing increased numbers of BP1+ proB cell. Although BP1+ proB cells from H19-/- FLshowed impaired Ig heavy chain V-D-J rearrangement, that increase resulted in a net enlarged B cell compartment in the adult periphery of H19 mutant. In adult mice, although H19 is notexpressed in B lymphocytes after birth, B cells from H19-/- mice exhibited altered B cellsurface phenotype, represented by an upregulated B220 expression on all B cell subsets. After immunization with different T cell dependent antigens, H19-/- exhibits reduced GC B cells, and impaired specific IgM titer in the serum, indicating a defected B cell response in H19-/-mice. Competitive reconstitution analysis showed a B cell autonomous impairment in the Bcell response. Consistently we found a reduced BCR responsiveness of H19-/- naïve B cells that together with less efficient upregulation of MHCII and CD40 expression after immunization might be responsible for the impaired immune response in H19-/- mice. Genome-wide transcription analysis revealed differential expression of genes involved inregulating the intensity of B cell receptor signaling. This work brings new insights on the regulation role of long non-coding RNA H19 in the early B cell development and immune system

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Submitted by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2018-03-20T13:40:44Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Reprodução Não Autorizada.pdf: 47716 bytes, checksum: 0353d988c60b584cfc9978721c498a11 (MD5) / Approved for entry into archive by Divisão de Documentação/BC Biblioteca Central (ddbc@ufam.edu.br) on 2018-03-20T13:51:01Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Reprodução Não Autorizada.pdf: 47716 bytes, checksum: 0353d988c60b584cfc9978721c498a11 (MD5) / Made available in DSpace on 2018-03-20T13:51:02Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Reprodução Não Autorizada.pdf: 47716 bytes, checksum: 0353d988c60b584cfc9978721c498a11 (MD5) Previous issue date: 2017-11-24 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Indisponível. / Introdução: O bócio continua sendo a principal indicação de tireoidectomias em regiões endêmicas. Trata-se de uma doença heterogênea e pouco compreendida molecularmente. Objetivo: Caracterizar a expressão diferencial gênica em tecidos tireoidianos oriundos de peças cirúrgicas diagnosticadas com bócio, comparando-os com tecido tireoidiano com ausência de doença de pacientes submetidos a tireoidectomias na cidade de Manaus-AM utilizando a tecnologia de RNA-seq. Metodologia: Sequenciamento do transcriptoma de fragmentos de tecido tireoideano com bócio e sem doença utilizando a plataforma de sequenciamento de nova geração (NGS) em Illumina HiSeq 2000 com protocolo de RNA-seq e análise de expressão diferencial. Resultados: Os resultados mostraram que há diferença entre os perfis de expressão gênica dos tecidos com bócio em comparação com um controle com ausência de doença, em que 70 sequencias gênicas estiveram diferencialmente expressas. Dentre as sequencias de genes up-regulated no bócio, a proteína nucleolar HOTS, um dos produtos da leitura do locus H19, esteve presente com cópias unicamente no tecido com bócio (p<0,05). Conclusão: Estes resultados demonstraram que o perfil de expressão gênica do bócio é de uma doença pró-tumoral em que os produtos do locus H19 merecem ser investigados quanto ao seu papel na tumorigenese.

Le gène H19 est soumis à l’empreinte génomique parentale et ne code aucune protéine. Le produit de ce gène, le long ARN non codant (lncRNA) H19, agit en tant qu’ARN et est impliqué dans le développement embryonnaire ainsi que dans la tumorigenèse. Le lncRNA H19 est le précurseur du miR-675. Mes travaux de thèse ont permis d’identifier de nouveaux mécanismes impliqués dans la régulation de la tumorigenèse mammaire par le gène H19. Nous avons mis en évidence que le lncRNA H19 régule négativement la protéine p53 dans les cellules cancéreuses mammaires. Mes travaux ont démontré que le lncRNA H19 interagit physiquement avec la protéine p53 et MDM2 afin d’induire sa dégradation et empêcher sa translocation dans le noyau. Ce nouveau mode d’action d’H19 dans les cancers du sein pourrait expliquer le manque de pertinence clinique de l’étude du statut mutationnel de p53 par immunohistochimie dans ce cancer. J’ai également mis en évidence que non seulement le lncRNA H19 est impliqué dans la régulation des cellules souches cancéreuses, mais également le miR-675-5p. En effet, les tumeurs exprimant des signatures géniques associées aux marqueurs de cellules souches cancéreuses sont des tumeurs qui surexpriment le gène H19. De plus, la modulation de l’expression du lncRNA H19 et de son microARN régule les capacités fonctionnelles associées aux cellules souches cancéreuses mammaires. Pour finir, j’ai initié un projet permettant l’identification, sans a priori, des gènes cibles du lncRNA H19 et de son microARN dans les cellules cancéreuses mammaires. Pour conclure, j’ai mis en évidence l’implication du lncRNA H19 et du miR-675-5p dans différents processus impliqués dans la tumorigenèse mammaire. / The H19 gene is subject to genomic imprinting and does not encode protein. The product of this gene, the long non coding RNA (lncRNA) H19, act as an RNA and is involved in development and the tumorigenesis. The H19 RNA is the precursor of miR-675. My thesis work identified new mechanism involved in the regulation of breast tumorigenesis by H19. We have demonstrated that the lncRNA H19 negatively regulates the p53 protein in breast cancer cell lines. My work revealed that H19 interacts with p53 and MDM2 to induce the degradation of p53 and impedes its nuclear localization. This new mechanism of H19 in breast cancer could explain the lack of clinical relevance of the p53 mutational state measured by immunohistochemistry in breast cancer. My work also revealed that not only the lncRNA H19 is involved in the regulation of breast cancer stem cells but also the miR-675-5p. Indeed, we have shown a correlation between overexpression of H19 and expression of a cancer stem cell phenotype in patient tumors. Furthermore, the modulation of H19 or miR-675 expression regulates the functional capacities associated with breast cancer stem cells. I also initiated a project that will allow the identification of H19 and miR-675 target genes in breast cancer cell lines. To conclude, I highlighted the implication of the lncRNA H19 and miR-675 in different process involved in breast cancer tumorigenesis.

本学位論文は以下の学術論文の内容に基づいて書かれたものである。“A G-quadruplex structure at the 5' end of the H19 coding region regulates H19 transcription” Scientific Reports 8:45815, 2017. DOI: 10.1038/srep45815. / 京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第20593号 / 生博第381号 / 新制||生||50(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 豊島 文子, 教授 朝長 啓造, 教授 藤田 尚志 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Gliomas are aggressive glial cell tumors that are nearly impossible to treat successfully, yielding strikingly low survival rates for patients. Glioblastomas, the most severe type of gliomas, have even poorer prognoses. In the past decade, new literature has shown that H19, a long non-coding RNA (lncRNA), is not only highly expressed in human gliomas, but that it plays several important roles in glioma progression and can even impede certain treatment measures. H19 directly and indirectly promotes several features of glioma cells including their survival, growth, migration, invasion, metastasis – essentially every stage of glioma development – and even stemness. Simply knocking down H19, in vitro, hampered every single one of these features to some degree. High H19 levels have also been linked to a lack of response to temozolomide and radiation treatments, two of the main therapeutic methods currently used to target gliomas. In vivo observations also followed this pattern of high H19 levels correlating with glioma tumorigenicity. So far, due to the accumulation of such findings, H19 has already become valued as both a prognostic and theragnostic marker. However, having seen how damaging H19 knockdown is to gliomas, there is no reason the role of H19 should be limited to that of an indicator; rather, the proto-oncogenic lncRNA should be viewed as a potential therapeutic target. Moreover, given that high H19 expression is an attribute unique to the human embryo stage, any instances of upregulation are typically oncogenic in nature, making H19 an ideal target for cancer therapy. Thus, targeting H19 in glioma patients should be integrated into existing treatment plans as this will obstruct glioma tumorigenesis, improve responsiveness to other therapies, and is not likely to impede normal biological functions.

Biotecnologias reprodutivas como a produção in vitro de embriões e a transferência de núcleo apresentam grande potencial de aplicação na medicina veterinária seja para a correção de infertilidades, para o aumento na eficiência da produção animal ou mesmo para um melhor entendimento sobre os mecanismos envolvidos no desenvolvimento embrionário inicial. Porém, manipulações in vitro de gametas ou embriões levam a alterações na regulação epigenética, podendo causar altas taxas de anormalidades no desenvolvimento e no nascimento de indivíduos derivados. A geração de um modelo de indução da pluripotência in vitro, ou seja, a geração de células iPS (do inglês induced pluripotent stem cells) possibilitou estudar o processo de reprogramação in vitro de maneira robusta e precisa. Os genes OCT4 e SOX2 são fundamentais no processo de aquisição e manutenção da pluripotência celular, e recentemente foi reportado que a ação destes dois fatores exerce grande influência sobre a regulação de alguns genes imprinted, em especial, no locus H19/IGF2, sabidamente importantes para o desenvolvimento normal do embrião e de sua placenta. Este estudo propõe a geração de um modelo experimental in vitro onde os fatores em questão sejam estudados, juntos ou em combinação, quanto à sua influência na regulação do imprinting genômico. Para tal, três linhagens de fibroblastos fetais bovinos (bFF1, bFF2 e bFF3) foram transduzidas com vetores lentivirais contendo cDNAs de OCT4 ou SOX2 humanos. Os fibroblastos foram analisados através de citometria e as células positivas foram separadas e recuperadas (sorted). Os fibroblastos expressando OCT4, SOX2, ambos (OCT4 + SOX2), nenhum (controle) juntamente com um controle recuperado (não sorted) não transgênico (total de cinco tratamentos) foram investigados quanto à expressão de genes relacionados à pluripotência e expressão de genes imprinted, bem como a manutenção dos padrões de metilação do DNA no locus H19/IGF2. Além disso, estas células foram submetidas à reprogramação in vitro e produção de células iPS. A indução à pluripotência foi realizada através da transdução dos fibroblastos com o vetor policistrônico contendo o cDNAs murino ou humano dos fatores de transcrição OCT4, SOX2, c-MYC e KLF4 (OSMK, vetor STEMCCA). Os resultados da análise de fluorescência por citometria de fluxo foram, em média, de 40,4% para OCT4, 6,1% para SOX2 e 0,63% para OCT4 + SOX2. A bFF1 foi a única linhagem a apresentar uma recuperação pós-sorting, o que possibilitou sua utilização para a indução da pluripotência. De maneira interessante, as células que não passaram pela citometria geraram colónias de células iPS, enquanto que os demais grupos não. A quantificação de transcritos por qRT-PCR mostrou que a expressão de OCT4 e de SOX2 estava aumentada nos respectivos grupos, a expressão do gene H19 mostrou-se aumentada no grupo controle que passou pelo procedimento de sorting e a expressão do gene imprinted IGF2R não variou entre os grupos. Já a análise preliminar da manutenção do padrão de metilação de DNA na DMR do locus H19/IGF2 mostrou que o grupo controle sorted apresentou uma leve diferença no padrão de metilação quando comparada aos outros grupos. Neste estudo, portanto, o procedimento de separação e recuperação celular por citometria de fluxo celular, aliado ao elevado número de repiques celulares durante o cultivo prolongado pode ter levado a um efeito prejudicial sobre a eficiência de reprogramação in vitro / Reproductive biotechniques such as in vitro embryo production and somatic cell nuclear transfer may greatly contribute for fertility improvements, to enhance animal production or else to contribute to a better understanding of the underlying mechanism involved during initial embryonic development. However, in vitro manipulation of gametes or embryos may lead to possible disruptions on epigenetic regulation, causing high developmental abnormalities and decreased healthy calves born at term. The generation of induced pluripotency models (induced pluripotent stem cells, or iPS) made it possible to study the process of in vitro reprogramming in a more solid and precise manner. OCT4 and SOX2 are fundamental genes for the acquisition and maintenance process of cellular pluripotency. Recently, it has been reported that both factors may have a huge influence on the regulation of some imprinted genes, specially at locus H19/IGF2, known to be important for the normal development of embryo and placenta. Therefore, this study aimed to generate an in vitro experimental model where the above transcription factors will be studied together or separately regarding their influence on genomic imprinting regulation. For that, three bovine fetal fibroblasts cell lines (bFF1, bFF2 and bFF3) were transduced with lentiviral vectors containing human OCT4 or SOX2 cDNAs. The fibroblasts were analyzed trough cell cytometry and positive cells were sorted. Fibroblasts expressing OCT4, SOX2, both (OCT4+SOX2), none (control) together with a non-sorted and non-transgenic control (five treatments) were investigated regarding pluripotency and imprinted gene expression, as well maintenance of DNA methylation patterns at H19/IGF2 locus. Further, these cells were also submitted to in vitro induced reprogramming and production of iPS cell colonies. Induction into pluripotency was realized by transducing fibroblasts with polycistronic excisable vector containing the murine or human cDNA of OCT4, SOX2, c-MYC and KLF4 transcription factors (OSMK, STEMCCA vector). The results of fluorescence analysis by flow cytometry were, on average, 40.4% for OCT4, 6.1% for SOX2 and 0,63% for OCT4+SOX2 groups. bFF1 was the only lineage presenting a post-sorting recovery that enabled its use for pluripotency induction. Interestingly, non-sorted cells generated biPS colonies whereas sorted cells (control non transgenic, OCT4, SOX2 and OCT4+SOX2 expressing cells) did not generate biPS cells. The transcript quantification by qRT-PCR showed that OCT4 and SOX2 expression were increased in the respective groups, the expression of H19 gene was increased in the control sorted group and IGF2R expression was not different between groups. Preliminary results of imprinting pattern methylation at H19/IGF2 locus showed that sorted group was slightly different from others. In this study, therefore, analysis and sorting procedure by flow citometry, together with an extended period in culture may have lead to a detrimental effect on in vitro reprogramming efficiency

ARAUJO, F. M. Ocorrência Familial e Associação de Polimorfismos dos Genes H19 e IGF2 com as Síndromes Hipertensivas Gestacionais. 2007. 118f. Disertação (Mestrado) Faculdade de Medicina, Universidade de São Paulo, Ribeirão Preto, 2007. As síndromes hipertensivas gestacionais [Pré-eclâmpsia/eclâmpsia (PE/E), hipertensão gestacional (HG) e hipertensão arterial crônica (HAC)] estão entre as maiores causas de morte materna e fetal. A PE é a mais prevalente dessas síndromes e o papel dos fatores genéticos na sua etiologia é bem aceito, embora o padrão de herança seja ainda assunto para debate. Os genes H19 e IGF2 sofrem imprinting (marcação) genômico e estão envolvidos na formação placentária e no desenvolvimento fetal. O objetivo do presente trabalho foi a pesquisa de ocorrência familial e da associação com os polimorfismos H19/RsaI e do IGF2/ApaI das síndromes hipertensivas gestacionais e do peso do recém-nascido. Todas as pacientes do estudo foram atendidas no Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo e o projeto foi aprovado pelo Comitê de Ética deste hospital e pela Comissão Nacional de Ética em Pesquisa. Para a condução do estudo familial foram selecionadas 226 mulheres (75 apresentavam PE, 49 com HG e 102 do grupo controle). Os dados foram analisados pelos Testes Exato de Fisher e do Qui-quadrado, resultando em uma maior freqüência estatisticamente significativa (p <0,05) de parentes de primeiro-grau com PE/E entre o grupo de PE/E comparado aos outros grupos. Não foi observada influência da cor da pele na distribuição entre os grupos de pacientes. Para a pesquisa de polimorfismos de comprimento de fragmento de restrição H19/RsaI (alelos A e B) e IGF2/ApaI (alelos A e G) através da reação em cadeia da polimerase , foi extraído DNA de sangue periférico de 236 pacientes (55 apresentavam PE, 40 com HG, 34 com HAC e 107 do grupo controle). Os resultados, analisados através dos Testes do Qui-quadrado e G, não mostraram associação estatisticamente significativa entre os polimorfismos e as síndromes hipertensivas gestacionais ou HAC. Houve uma maior freqüência do alelo G na população estudada. Foi observado que em torno de 80% das pacientes dos quatro grupos estudados apresentou pelo menos uma cópia do alelo B e uma do alelo G, concomitantemente. A associação do peso do recém-nascido com os polimorfismos foi analisada utilizando-se os Testes Kolmogorov-Smirnov (a P<0,05) e os Não-paramétricos de Kruskal-Wallis (a P<0,05), não tendo sido evidenciadas diferenças estatisticamente significativas. No grupo da PE houve uma diminuição estatisticamente significativa do peso dos recém-nascidos quando não havia correção para a idade gestacional. Embora não tenha sido evidenciada correlação entre os polimorfismos e os fenótipos estudados, trabalhos futuros com um número amostral maior serão importantes para auxiliar no entendimento do envolvimento de fatores epigenéticos nas síndromes hipertensivas gestacionais e fornecer indícios para a prevenção, o tratamento e o aconselhamento genético. / ARAUJO, F. M. Familial Occurrence and H19 and IGF2 Polymorphism Association with Gestational Hypertensive Disorders. 2007. 118p. Dissertation (Master\'s degree) - University of Medicine, University of São Paulo, Ribeirão Preto, 2007. Gestational hypertensive disorders [preeclampsia/eclampsia (PE/E), gestational hypertension (GH) and chronic hypertension (CH)] are among the largest causes of maternal and fetal death. PE is the more prevalent of those syndromes and the role of the genetic factors in its etiology is well accepted, although the pattern of inheritance is still subject for debate. The imprinted genes H19 and IGF2 are involved in the placental formation and in the fetal development. The objective of the present study was to verify the familial occurrence of these disorders and the H19/RsaI and IGF2/ApaI polymorphism association with gestational hypertensive disorders and the weight of the newborn. All patients of the study were referred to the Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto, University of São Paulo, and the project was approved by the Hospital Ethic Committee and the National Commission of Ethics in Research. For the familial study, 226 women were selected (75 presented PE/E, 49 with GH and 102 from the control group). The data were analyzed by Exact of Fisher and Qui-square tests, and the frequency of families with at least one female first-degree relative (mothers and/or sisters) with PE/E was higher among the PE/E group compared to the other groups, and it was statistically significant (P<0.05). There was no statistically significant influence of the \"skin color\". Blood samples of 236 pregnant women (55 with PE/E, 40 with GH, 34 with CH and 107 from the control group) were obtained for DNA extraction, and PCR. Genotyping was carried out by enzymatic digestion with ApaI (IGF2) and RsaI (H19). The statistical analyses were performed by Qui-square and G tests. The genotypes were not significantly associated with the different groups. A higher frequency of the G allele (IGF2) was observed. Around 80% of the patients presented at least one copy of the allele B (H19) and G (IGF2), concomitantly. The association of the weight of the newborn with the polymorphisms was analyzed using the Kolmogorov-Smirnov (P <0.05) and the No-parametric Test of Kruskal-Wallis (P <0.05) tests, and statistically significant differences were not evidenced. In the group of the PE/E there was a statistically significant decrease of the weight of the newborn when the correction for the gestational age was not carried out. Although correlation has not been evidenced between the polymorphisms and the phenotypes, future studies with a higher number of patients and other imprinted genes will be important to elucidate the involvement of epigenetic factors for the prevention, treatment and genetic counseling of the gestational hypertensive disorders

Biotecnologias reprodutivas como a produção in vitro de embriões e a transferência de núcleo apresentam grande potencial de aplicação na medicina veterinária seja para a correção de infertilidades, para o aumento na eficiência da produção animal ou mesmo para um melhor entendimento sobre os mecanismos envolvidos no desenvolvimento embrionário inicial. Porém, manipulações in vitro de gametas ou embriões levam a alterações na regulação epigenética, podendo causar altas taxas de anormalidades no desenvolvimento e no nascimento de indivíduos derivados. A geração de um modelo de indução da pluripotência in vitro, ou seja, a geração de células iPS (do inglês induced pluripotent stem cells) possibilitou estudar o processo de reprogramação in vitro de maneira robusta e precisa. Os genes OCT4 e SOX2 são fundamentais no processo de aquisição e manutenção da pluripotência celular, e recentemente foi reportado que a ação destes dois fatores exerce grande influência sobre a regulação de alguns genes imprinted, em especial, no locus H19/IGF2, sabidamente importantes para o desenvolvimento normal do embrião e de sua placenta. Este estudo propõe a geração de um modelo experimental in vitro onde os fatores em questão sejam estudados, juntos ou em combinação, quanto à sua influência na regulação do imprinting genômico. Para tal, três linhagens de fibroblastos fetais bovinos (bFF1, bFF2 e bFF3) foram transduzidas com vetores lentivirais contendo cDNAs de OCT4 ou SOX2 humanos. Os fibroblastos foram analisados através de citometria e as células positivas foram separadas e recuperadas (sorted). Os fibroblastos expressando OCT4, SOX2, ambos (OCT4 + SOX2), nenhum (controle) juntamente com um controle recuperado (não sorted) não transgênico (total de cinco tratamentos) foram investigados quanto à expressão de genes relacionados à pluripotência e expressão de genes imprinted, bem como a manutenção dos padrões de metilação do DNA no locus H19/IGF2. Além disso, estas células foram submetidas à reprogramação in vitro e produção de células iPS. A indução à pluripotência foi realizada através da transdução dos fibroblastos com o vetor policistrônico contendo o cDNAs murino ou humano dos fatores de transcrição OCT4, SOX2, c-MYC e KLF4 (OSMK, vetor STEMCCA). Os resultados da análise de fluorescência por citometria de fluxo foram, em média, de 40,4% para OCT4, 6,1% para SOX2 e 0,63% para OCT4 + SOX2. A bFF1 foi a única linhagem a apresentar uma recuperação pós-sorting, o que possibilitou sua utilização para a indução da pluripotência. De maneira interessante, as células que não passaram pela citometria geraram colónias de células iPS, enquanto que os demais grupos não. A quantificação de transcritos por qRT-PCR mostrou que a expressão de OCT4 e de SOX2 estava aumentada nos respectivos grupos, a expressão do gene H19 mostrou-se aumentada no grupo controle que passou pelo procedimento de sorting e a expressão do gene imprinted IGF2R não variou entre os grupos. Já a análise preliminar da manutenção do padrão de metilação de DNA na DMR do locus H19/IGF2 mostrou que o grupo controle sorted apresentou uma leve diferença no padrão de metilação quando comparada aos outros grupos. Neste estudo, portanto, o procedimento de separação e recuperação celular por citometria de fluxo celular, aliado ao elevado número de repiques celulares durante o cultivo prolongado pode ter levado a um efeito prejudicial sobre a eficiência de reprogramação in vitro / Reproductive biotechniques such as in vitro embryo production and somatic cell nuclear transfer may greatly contribute for fertility improvements, to enhance animal production or else to contribute to a better understanding of the underlying mechanism involved during initial embryonic development. However, in vitro manipulation of gametes or embryos may lead to possible disruptions on epigenetic regulation, causing high developmental abnormalities and decreased healthy calves born at term. The generation of induced pluripotency models (induced pluripotent stem cells, or iPS) made it possible to study the process of in vitro reprogramming in a more solid and precise manner. OCT4 and SOX2 are fundamental genes for the acquisition and maintenance process of cellular pluripotency. Recently, it has been reported that both factors may have a huge influence on the regulation of some imprinted genes, specially at locus H19/IGF2, known to be important for the normal development of embryo and placenta. Therefore, this study aimed to generate an in vitro experimental model where the above transcription factors will be studied together or separately regarding their influence on genomic imprinting regulation. For that, three bovine fetal fibroblasts cell lines (bFF1, bFF2 and bFF3) were transduced with lentiviral vectors containing human OCT4 or SOX2 cDNAs. The fibroblasts were analyzed trough cell cytometry and positive cells were sorted. Fibroblasts expressing OCT4, SOX2, both (OCT4+SOX2), none (control) together with a non-sorted and non-transgenic control (five treatments) were investigated regarding pluripotency and imprinted gene expression, as well maintenance of DNA methylation patterns at H19/IGF2 locus. Further, these cells were also submitted to in vitro induced reprogramming and production of iPS cell colonies. Induction into pluripotency was realized by transducing fibroblasts with polycistronic excisable vector containing the murine or human cDNA of OCT4, SOX2, c-MYC and KLF4 transcription factors (OSMK, STEMCCA vector). The results of fluorescence analysis by flow cytometry were, on average, 40.4% for OCT4, 6.1% for SOX2 and 0,63% for OCT4+SOX2 groups. bFF1 was the only lineage presenting a post-sorting recovery that enabled its use for pluripotency induction. Interestingly, non-sorted cells generated biPS colonies whereas sorted cells (control non transgenic, OCT4, SOX2 and OCT4+SOX2 expressing cells) did not generate biPS cells. The transcript quantification by qRT-PCR showed that OCT4 and SOX2 expression were increased in the respective groups, the expression of H19 gene was increased in the control sorted group and IGF2R expression was not different between groups. Preliminary results of imprinting pattern methylation at H19/IGF2 locus showed that sorted group was slightly different from others. In this study, therefore, analysis and sorting procedure by flow citometry, together with an extended period in culture may have lead to a detrimental effect on in vitro reprogramming efficiency