Cancer is essentially a genetic disease of uncontrolled cell survival and proliferation. Medical therapy has traditionally involved chemotherapy and radiation which inhibit the replication of actively dividing cells in a fairly non-selective manner. Research into the molecular pathogenesis of cancer has enabled the recent development of therapy targeted to receptors or signaling pathways crucial to the development and progression of specific cancers and revealed molecular markers that can be used to predict prognosis and treatment response. The work presented in this thesis examines the expression of two potential molecular markers of malignancy, the R1alpha regulatory subunit of cyclic AMP-dependent protein kinase A (PRKAR1A) and insulin-like growth factor 2 (IGF2) retrospectively in a population of canine osteosarcoma patients. Furthermore, it describes the derivation and preliminary characterization of canine osteosarcoma primary cell cultures and the use of these cells to assess the effects of PRKAR1A expression on sensitivity to chemotherapeutic drug treatment in vitro. Post-chemotherapy overall survival was significantly longer in canine osteosarcoma patients with tumours expressing low levels of PRKAR1A, and PRKAR1A expression did not correlate with mitotic index. IGF2 expression was generally high in the small numbers of cases examined, did not differ between axial and appendicular sites and did not correlate with either mitotic index or survival. PRKAR1A expression varied between cell cultures, but did not appear to be related to expression levels of phosphorylated cAMP response element-binding (phospho- CREB), a downstream target of cyclic AMP (cAMP)-dependent protein kinase A (PKA). In vitro chemosensitivity did not correlate with PRKAR1A expression, but faster growing cells with shorter doubling times and higher rates of BrdU incorporation tended to be more chemosensitive. In summary, this work identifies an association between low tumour PRKAR1A expression and prolonged post-chemotherapy survival in dogs with osteosarcoma and provides preliminary evidence suggesting that this survival advantage may not be associated with downstream phosphorylation of CREB or sensitivity of the tumour cells to chemotherapeutic agents.

Indiana University-Purdue University Indianapolis (IUPUI) / The maternal liver exhibits robust adaptations to pregnancy to accommodate the metabolic needs of developing and growing placenta and fetus by largely unknown mechanisms. We found that achaete-scute homolog 1 (Ascl1), a basic helix-loop-helix transcription factor essential for neuronal development, is highly activated in maternal hepatocytes during the second half of gestation in mice. Our aim is to investigate whether and how Ascl1 plays a pregnancy-dependent role. We deleted the Ascl1 gene in the maternal liver using three independent mouse models from mid-gestation until term and identified multiple Ascl1-dependent phenotypes. When Ascl1 was deficient in maternal hepatocytes, maternal livers exhibited aberrant hepatocyte histology, fat accumulation, increased hepatocyte cell cycle, and enlarged size, accompanied by reduced albumin production and elevated levels of free fatty acids, ALT, and AST in the maternal blood, indicating maternal liver dysfunction. In the same situation, maternal spleen and pancreas displayed marked enlargement without an overt structural change; the placenta exhibited striking overgrowth with increased ALP production; and the cecal microbiome showed alterations in the relative abundance of several bacterial subpopulations. Moreover, litters born from maternal hepatic Ascl1 null mutated dam experienced abnormal postnatal growth after weaning. RNA-seq analysis revealed Ascl1-regulated genes in the maternal liver associated with Ascl1-dependent phenotypes. Of particular interest, we found that, in maternal hepatocytes, Ascl1 loss-of-function caused the activation of paternally imprinted gene insulin-like growth factor 2 (Igf2) encoding a major placental and fetal growth factor. IGF2 is also a known mitogen for hepatocytes and several hematopoietic lineages. Thus, IGF2 is a potential inducer of Ascl1-dependent phenotypes including placental overgrowth and maternal organ enlargement. Our studies revealed Ascl1 as a novel regulator of maternal liver physiology during pregnancy. Ascl1 activation in maternal hepatocytes is essential for normal placental growth and appropriate maternal organ adaptations, ensuring the health of both the mother and the fetus.

No description available.

Existe um relevante interesse em pesquisar a ocorrência de polimorfismos no genoma bovino por diferentes motivos, e mais recentemente, com a finalidade de agregar mais informações ao estudo de características quantitativas visando selecionar animais geneticamente superiores com considerável valor comercial. Os polimorfismos de base única (SNPs) neste estudo foram identificados como RFLP/MboII e RFLP/HpaII sendo que o polimorfismo RFLP/MboII está situado no exon 6 do gene IGF2 (insulin-like growth factor 2), localizado no cromossomo 29 em bovinos, e desempenha um papel importante na proliferação e diferenciação celular para o crescimento e desenvolvimento dos mamíferos. O polimorfismo RFLP/HpaII encontra-se no elemento Bov-A2 (considerado um elemento SINE - Short Interspersed Nucleotide Element) presente na região promotora do gene CYP21 (Steroid 21-hydroxylase gene) no cromossomo 23 em bovinos. Para avaliar a ocorrência dos SNPs utilizou-se a técnica de PCR-RFLP em amostras de DNA a partir de sangue/sêmen de cerca de 300 animais bovinos das raças zebuínas Gir, Guzerá e Nelore. As frequências alélicas mostraram maior incidência do alelo T quando comparado ao C enquanto que as frequências genotípicas apresentaram alta ocorrência do heterozigoto TC em comparação aos homozigotos CC e TT para o polimorfismo IGF2 - RFLP/MboII. Com relação ao polimorfismo CYP21 RFLP/HpaII, a frequência alélica revelou alto valor do alelo T. A população encontrou-se em equilíbrio de Hardy-Weinberg para os SNPs estudados. Ferramentas de bioinformática foram utilizadas para investigações in silico revelando que os sítios polimórficos estão em regiões com potencial regulatório. A associação desses polimorfismos com DEPs das características reprodutivas e produtivas foram investigadas, entretanto mostrou-se significativas apenas para DP550 (IGF2 - RFLP/MboII) e DP450 (CYP21 - RFLP/HpaII). Os resultados obtidos sugerem que protocolos de Biologia Molecular in vitro podem ser usados para identificar novos marcadores moleculares, como SNPs funcionais adicionando informações que certamente contribuirão para estratégias de melhoramento dessas raças bovinas de grande importância para a produção de carne e leite em nosso país. Este foi o primeiro estudo sobre a ocorrência desses polimorfismos em raças zebuínas criadas no Brasil. / There is a considerable interest in researching the occurrence of polymorphisms in the bovine genome for different reasons, and more recently, in order to add more information to the study of quantitative traits to select genetically superior animals with considerable commercial value. The single nucleotide polymorphisms (SNPs) in this study were identified as RFLP/MboII and RFLP/HpaII polymorphisms being the RFLP/MboII is situated in exon 6 of the IGF2 gene (insulin-like growth factor 2), located on chromosome 29 in cattle, perform an important role in cell proliferation, differentiation for growth and in the development of mammals. Polymorphism RFLP/HpaII is the element Bov-A2 (considered an element SINE - Short Interspersed Nucleotide Element) present in the promoter region of CYP21 gene (Steroid 21-hydroxylase gene) on chromosome 23 in cattle. To evaluate the occurrence of SNPs, we used the PCR-RFLP method on DNA samples from blood/semen of about 300 cattle breeds from Zebu Gyr, Guzerat and Nellore. The allele frequencies showed a higher incidence of T allele compared to C while the genotype frequencies showed high incidence of heterozygous CT compared to CC and TT homozygous for the IGF2 polymorphism - RFLP/MboII. On the subject of the CYP21 polymorphism - RFLP/HpaII, the allele frequency showed high value T. The population was found in Hardy-Weinberg equilibrium for the SNPs studied. Bioinformatics tools, used for in silico investigations, revealed that the polymorphic sites are in regions with regulatory potential. The association of these polymorphisms with EPDs of reproductive and productive traits were investigated, but proved to be significant only for DP550 (IGF2 - RFLP/MboII) and DP450 (CYP21 - RFLP/HpaII). The results suggest that protocols of molecular biology in vitro can be used to identify new molecular markers, such as functional SNPs adding information that certainly will contribute to the improvement strategies of these breeds of great importance for the production of meat and milk in our country. It has been the first study on the occurrence of these polymorphisms in Zebu breeds raised in Brazil.

INTRODUÇÃO: Acredita-se que os fatores de crescimento insulina símile 1 (IGF1) e IGF2 tenham um papel chave na progressão de tumores, resistência à apoptose e terapias. A resistência à insulina tem sido associada ao aumento do volume tireoidiano e aumento do risco de desenvolver nódulos e câncer de tireoide; no entanto, há poucos estudos que avaliaram o papel dos IGFs e seus receptores no carcinoma papilífero de tireoide (CPT) e, até o momento, nenhum estudo foi conclusivo sobre a relação entre a via insulina/IGF e o comportamento do CPT. OBJETIVOS: 1) Estudar a expressão do IGF1, IGF2 e o IGF1R no CPT, incluindo o microcarcinoma papilífero; 2) Correlacionar essa expressão com as características clínicas, variante histopatológica, estadiamento e estratificação de risco. MÉTODOS: Foram selecionados, retrospectivamente, 110 pacientes operados por CPT e atendidos no Ambulatório do Serviço de Endocrinologia do Hospital das Clínicas da FMUSP e separados em dois grupos: 62 microcarcinomas papilíferos (MCP) e 48 CPT > 1,0 cm. A presença e expressão do IGF1, IGF2 e IGF1R foi avaliada, através de exame imunohistoquímico, em 110 tecidos tumorais e 98 tecidos não tumorais (controle). Os casos positivos foram classificados, de acordo com a quantidade de células coradas, em: + (menos de 10% das células); ++ (em 10-50% das células) e +++ (mais de 50% das células). O grau da expressão foi classificada em leve, moderada e forte. A presença e intensidade desses marcadores foram correlacionados com as características clínicas, variante histopatológico, TNM e estratificação de risco. RESULTADOS: O IGF1 e o IGF1R estiveram presentes em 100% e 99% dos CPTs e mostraram-se significativamente hiperexpressos, tanto nos MCPs quanto nos CPTs > 1,0 cm, quando comparados ao tecido adjacente não tumoral (p < 0,001). O IGF2 esteve expresso em 46,7% dos CPTs e mostrou fraca expressão em apenas um tecido não tumoral (p < 0,001). O IGF1 apresentou expressão significativamente maior nos microcarcinomas nos estágios III e IVA quando comparados aos estágios I e II (p=0,022). Não houve diferença significativa na quantidade e intensidade de expressão do IGF1 e IGF1R nos MCT quando comparados aos CPTs > 1,0 cm. O IGF2 apresentou expressão significativamente maior nos MCTs e, nesse grupo, apresentou maior expressão nos tumores multicêntricos (p=0,017) e nos estágios III e IVA, quando comparados aos estagios I e II (p=0,041). CONCLUSÕES: No presente estudo, observamos que o IGF1 e IGF2 foram significativamente mais expressos nos microcarcinomas em estágios mais avançados. O IGF2 apresentou maior expressão nos microcarcinomas papilíferos e esta expressão foi significativamente maior nos tumores multicêntricos / INTRODUCTION: Insulin-like growth factor-1 and 2 (IGF-1 and IGF-2) are believed to play a key role in the progression of tumors, resistance to apoptosis and therapies. The insulin resistence has been associated with increased thyroid volume and increased risk in developing thyroid nodules and thyroid cancer. However, few studies have evaluated the role of IGFs and their receptors on papillary thyroid carcinomas (PTC), and there is no conclusive studies about the relationship between IGF axis and PTC behavior. OBJECTIVES: The aim of this study was to investigate the expression of IGF-1, IGF-2, and IGF-1R in PTC, including papillary microcarcinoma (PTMC), and correlate the expression data with clinical, histologic variants, TNM staging, and risk of recurrence. METHODS: We retrospectively selected 110 paraffin-embedded tumoral tissues from patients with PTC who underwent thyroidectomy at Hospital das Clínicas of FMUSP. These patients were divided into two groups: 62 microcarcinomas (PTMC) and 48 PTC > 1.0 cm. The presence and intensity of expression of IGF-1, IGF-2, and IGF-1R were evaluated through immunohistochemical staining in 110 tumoral tissues, and in 98 non-tumoral tissues (control group). Positive cases were classified according to the numbers of staining cells in: + less than 10% of staining cells; ++ in 10-50% of the staining cells, and +++ in more than 50% of staining cells. The degree of expression was classified as mild, moderate and strong. The presence and degree of IGF1, IGF2, and IGF1R staining were correlated with clinical features, histologic type, TNM staging, and risk stratification. RESULTS: IGF-1 and IGF-1R were expressed in 100% and 99% of PTC, and were significantly overexpressed in both PTMC and PTC > 1.0 cm, in comparison with non-tumoral tissues (control group) (p < 0.001). IGF-2 was expressed in 46.7% of PTC and had mild positivity expression in only one non-tumoral tissue (p < 0.001). IGF1 was significantly overexpressed in PTMC on stage III and IVa and was less expressed in stage I and II. There was no significant difference on IGF1 and IGF1R expression between PTMC and PTC >1.0 cm. IGF-2 presented greater expression in multicentric PTMC (p=0.017), specially in stage III and IVa. CONCLUSIONS: In our study, both IGF-1 and IGF2 were significantly overexpressed in PTMC group in advanced stages. IGF-2 was also significantly overexpressed in PTMC multicentric tumors

INTRODUÇÃO: Acredita-se que os fatores de crescimento insulina símile 1 (IGF1) e IGF2 tenham um papel chave na progressão de tumores, resistência à apoptose e terapias. A resistência à insulina tem sido associada ao aumento do volume tireoidiano e aumento do risco de desenvolver nódulos e câncer de tireoide; no entanto, há poucos estudos que avaliaram o papel dos IGFs e seus receptores no carcinoma papilífero de tireoide (CPT) e, até o momento, nenhum estudo foi conclusivo sobre a relação entre a via insulina/IGF e o comportamento do CPT. OBJETIVOS: 1) Estudar a expressão do IGF1, IGF2 e o IGF1R no CPT, incluindo o microcarcinoma papilífero; 2) Correlacionar essa expressão com as características clínicas, variante histopatológica, estadiamento e estratificação de risco. MÉTODOS: Foram selecionados, retrospectivamente, 110 pacientes operados por CPT e atendidos no Ambulatório do Serviço de Endocrinologia do Hospital das Clínicas da FMUSP e separados em dois grupos: 62 microcarcinomas papilíferos (MCP) e 48 CPT > 1,0 cm. A presença e expressão do IGF1, IGF2 e IGF1R foi avaliada, através de exame imunohistoquímico, em 110 tecidos tumorais e 98 tecidos não tumorais (controle). Os casos positivos foram classificados, de acordo com a quantidade de células coradas, em: + (menos de 10% das células); ++ (em 10-50% das células) e +++ (mais de 50% das células). O grau da expressão foi classificada em leve, moderada e forte. A presença e intensidade desses marcadores foram correlacionados com as características clínicas, variante histopatológico, TNM e estratificação de risco. RESULTADOS: O IGF1 e o IGF1R estiveram presentes em 100% e 99% dos CPTs e mostraram-se significativamente hiperexpressos, tanto nos MCPs quanto nos CPTs > 1,0 cm, quando comparados ao tecido adjacente não tumoral (p < 0,001). O IGF2 esteve expresso em 46,7% dos CPTs e mostrou fraca expressão em apenas um tecido não tumoral (p < 0,001). O IGF1 apresentou expressão significativamente maior nos microcarcinomas nos estágios III e IVA quando comparados aos estágios I e II (p=0,022). Não houve diferença significativa na quantidade e intensidade de expressão do IGF1 e IGF1R nos MCT quando comparados aos CPTs > 1,0 cm. O IGF2 apresentou expressão significativamente maior nos MCTs e, nesse grupo, apresentou maior expressão nos tumores multicêntricos (p=0,017) e nos estágios III e IVA, quando comparados aos estagios I e II (p=0,041). CONCLUSÕES: No presente estudo, observamos que o IGF1 e IGF2 foram significativamente mais expressos nos microcarcinomas em estágios mais avançados. O IGF2 apresentou maior expressão nos microcarcinomas papilíferos e esta expressão foi significativamente maior nos tumores multicêntricos / INTRODUCTION: Insulin-like growth factor-1 and 2 (IGF-1 and IGF-2) are believed to play a key role in the progression of tumors, resistance to apoptosis and therapies. The insulin resistence has been associated with increased thyroid volume and increased risk in developing thyroid nodules and thyroid cancer. However, few studies have evaluated the role of IGFs and their receptors on papillary thyroid carcinomas (PTC), and there is no conclusive studies about the relationship between IGF axis and PTC behavior. OBJECTIVES: The aim of this study was to investigate the expression of IGF-1, IGF-2, and IGF-1R in PTC, including papillary microcarcinoma (PTMC), and correlate the expression data with clinical, histologic variants, TNM staging, and risk of recurrence. METHODS: We retrospectively selected 110 paraffin-embedded tumoral tissues from patients with PTC who underwent thyroidectomy at Hospital das Clínicas of FMUSP. These patients were divided into two groups: 62 microcarcinomas (PTMC) and 48 PTC > 1.0 cm. The presence and intensity of expression of IGF-1, IGF-2, and IGF-1R were evaluated through immunohistochemical staining in 110 tumoral tissues, and in 98 non-tumoral tissues (control group). Positive cases were classified according to the numbers of staining cells in: + less than 10% of staining cells; ++ in 10-50% of the staining cells, and +++ in more than 50% of staining cells. The degree of expression was classified as mild, moderate and strong. The presence and degree of IGF1, IGF2, and IGF1R staining were correlated with clinical features, histologic type, TNM staging, and risk stratification. RESULTS: IGF-1 and IGF-1R were expressed in 100% and 99% of PTC, and were significantly overexpressed in both PTMC and PTC > 1.0 cm, in comparison with non-tumoral tissues (control group) (p < 0.001). IGF-2 was expressed in 46.7% of PTC and had mild positivity expression in only one non-tumoral tissue (p < 0.001). IGF1 was significantly overexpressed in PTMC on stage III and IVa and was less expressed in stage I and II. There was no significant difference on IGF1 and IGF1R expression between PTMC and PTC >1.0 cm. IGF-2 presented greater expression in multicentric PTMC (p=0.017), specially in stage III and IVa. CONCLUSIONS: In our study, both IGF-1 and IGF2 were significantly overexpressed in PTMC group in advanced stages. IGF-2 was also significantly overexpressed in PTMC multicentric tumors

Existe um relevante interesse em pesquisar a ocorrência de polimorfismos no genoma bovino por diferentes motivos, e mais recentemente, com a finalidade de agregar mais informações ao estudo de características quantitativas visando selecionar animais geneticamente superiores com considerável valor comercial. Os polimorfismos de base única (SNPs) neste estudo foram identificados como RFLP/MboII e RFLP/HpaII sendo que o polimorfismo RFLP/MboII está situado no exon 6 do gene IGF2 (insulin-like growth factor 2), localizado no cromossomo 29 em bovinos, e desempenha um papel importante na proliferação e diferenciação celular para o crescimento e desenvolvimento dos mamíferos. O polimorfismo RFLP/HpaII encontra-se no elemento Bov-A2 (considerado um elemento SINE - Short Interspersed Nucleotide Element) presente na região promotora do gene CYP21 (Steroid 21-hydroxylase gene) no cromossomo 23 em bovinos. Para avaliar a ocorrência dos SNPs utilizou-se a técnica de PCR-RFLP em amostras de DNA a partir de sangue/sêmen de cerca de 300 animais bovinos das raças zebuínas Gir, Guzerá e Nelore. As frequências alélicas mostraram maior incidência do alelo T quando comparado ao C enquanto que as frequências genotípicas apresentaram alta ocorrência do heterozigoto TC em comparação aos homozigotos CC e TT para o polimorfismo IGF2 - RFLP/MboII. Com relação ao polimorfismo CYP21 RFLP/HpaII, a frequência alélica revelou alto valor do alelo T. A população encontrou-se em equilíbrio de Hardy-Weinberg para os SNPs estudados. Ferramentas de bioinformática foram utilizadas para investigações in silico revelando que os sítios polimórficos estão em regiões com potencial regulatório. A associação desses polimorfismos com DEPs das características reprodutivas e produtivas foram investigadas, entretanto mostrou-se significativas apenas para DP550 (IGF2 - RFLP/MboII) e DP450 (CYP21 - RFLP/HpaII). Os resultados obtidos sugerem que protocolos de Biologia Molecular in vitro podem ser usados para identificar novos marcadores moleculares, como SNPs funcionais adicionando informações que certamente contribuirão para estratégias de melhoramento dessas raças bovinas de grande importância para a produção de carne e leite em nosso país. Este foi o primeiro estudo sobre a ocorrência desses polimorfismos em raças zebuínas criadas no Brasil. / There is a considerable interest in researching the occurrence of polymorphisms in the bovine genome for different reasons, and more recently, in order to add more information to the study of quantitative traits to select genetically superior animals with considerable commercial value. The single nucleotide polymorphisms (SNPs) in this study were identified as RFLP/MboII and RFLP/HpaII polymorphisms being the RFLP/MboII is situated in exon 6 of the IGF2 gene (insulin-like growth factor 2), located on chromosome 29 in cattle, perform an important role in cell proliferation, differentiation for growth and in the development of mammals. Polymorphism RFLP/HpaII is the element Bov-A2 (considered an element SINE - Short Interspersed Nucleotide Element) present in the promoter region of CYP21 gene (Steroid 21-hydroxylase gene) on chromosome 23 in cattle. To evaluate the occurrence of SNPs, we used the PCR-RFLP method on DNA samples from blood/semen of about 300 cattle breeds from Zebu Gyr, Guzerat and Nellore. The allele frequencies showed a higher incidence of T allele compared to C while the genotype frequencies showed high incidence of heterozygous CT compared to CC and TT homozygous for the IGF2 polymorphism - RFLP/MboII. On the subject of the CYP21 polymorphism - RFLP/HpaII, the allele frequency showed high value T. The population was found in Hardy-Weinberg equilibrium for the SNPs studied. Bioinformatics tools, used for in silico investigations, revealed that the polymorphic sites are in regions with regulatory potential. The association of these polymorphisms with EPDs of reproductive and productive traits were investigated, but proved to be significant only for DP550 (IGF2 - RFLP/MboII) and DP450 (CYP21 - RFLP/HpaII). The results suggest that protocols of molecular biology in vitro can be used to identify new molecular markers, such as functional SNPs adding information that certainly will contribute to the improvement strategies of these breeds of great importance for the production of meat and milk in our country. It has been the first study on the occurrence of these polymorphisms in Zebu breeds raised in Brazil.

According to the World Health Organization (WHO) depression is the leading cause of disability worldwide with more than 300 million patients affected. Current antidepressants have a delayed onset of action and moreover, only two-thirds of patients suffering from depressive disorder respond to antidepressant drug treatment. The N-methyl-D-aspartate (NMDA) receptor antagonist ketamine offers promising perspectives for the treatment of major depressive disorder. Although ketamine demonstrates rapid and long-lasting effects, even in treatment-resistant patients, to date, the underlying mode of action remains elusive. Thus, the aim of this thesis was to investigate the molecular mechanism of ketamine and its major metabolites at clinically relevant concentrations by establishing an in vitro model based on human induced pluripotent stem cells (iPSCs)-derived neural progenitor cells (NPCs). As the pathophysiology of depression correlates with decreased adult neurogenesis, I aimed to investigate the molecular effects of ketamine on neural progenitor cell proliferation using a human-based iPSC-model. The findings from this thesis substantially contribute to an enhanced understanding of the molecular mode of action of ketamine as a novel signaling pathway involved in ketamine-induced effects was identified. Ketamine induced proliferation of human iPSC-derived NPCs and bioinformatic analysis of RNA-Seq data revealed significant upregulation of insulin-like growth factor2 (IGF2) and p11, a member of the S100 EF-hand protein family, which are both implicated in the pathophysiology of depression, 24 hours after ketamine treatment. In line with this, ketamine dependent proliferation was significantly impaired after IGF2 knockdown. Moreover, ketamine was able to enhance cAMP signaling in NPCs and both, cell proliferation as well as IGF2 expression, were reduced after protein kinase A (PKA)-inhibition. Noteworthy, the Nestin-expressing NPCs do not express functional NMDA receptors, suggesting that the proproliferative effect of ketamine in NPCs is NMDA receptor-independent. Furthermore, 24 hours post administration of ketamine (15 mg/kg) in vivo confirmed phosphorylation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in the subgranular zone (SGZ) of the hippocampus in C57BL/6 mice. In conclusion, ketamine promotes proliferation of NPCs presumably by involving cAMP-IGF2 signaling.

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