MicroRNAs (miRNAs) are a class of short (21-25 nt) non-coding endogenous RNAs that mediate the expression of their direct target genes post-transcriptionally. The goal of this thesis is to identify the target genes of miRNAs using computational methods. The most popular computational target prediction methods rely on sequence based determinants to predict targets. However, these determinants are neither sufficient nor necessary to identify functional target sites, and commonly ignore the cellular conditions in which miRNAs interact with their targets \emph{in vivo}. Since miRNAs activity reduces the steady-state abundance of mRNA targets, the main goal of this thesis is to augment large scale gene expression profiles as a supplement to sequence-based computational miRNA target prediction techniques. We develop two computational miRNA target prediction methods: InMiR and BayMiR; in addition, we study the interaction between miRNAs and lncRNAs using long RNA expression data. InMiR is a computational method that predicts the targets of intronic miRNAs based on the expression profiles of their host genes across a large number of datasets. InMiR can also predict which host genes have expression profiles that are good surrogates for those of their intronic miRNAs. Host genes that InMiR predicts are bad surrogates contain significantly more miRNA target sites in their 3 UTRs and are significantly more likely to have predicted Pol II-III promoters in their introns. We also develop BayMiR that scores miRNA-mRNA pairs based on the endogenous footprint of miRNAs on gene expression in a genome-wide scale. BayMiR provides an ``endogenous target repression" index, that identifies the contribution of each miRNA in repressing a target gene in presence of other targeting miRNAs. This thesis also addresses the interactions between miRNAs and lncRNAs. Our analysis on expression abundance of long RNA transcripts (mRNA and lncRNA) shows that the lncRNA target set of some miRNAs have relatively low abundance in the tissues that these miRNAs are highly active. We also found lncRNAs and mRNAs that shared many targeting miRNAs are significantly positively correlated, indicating that these set of highly expressed lncRNAs may act as miRNA sponges to promote mRNA regulation.

MicroRNAs (miRNAs) are a class of short (21-25 nt) non-coding endogenous RNAs that mediate the expression of their direct target genes post-transcriptionally. The goal of this thesis is to identify the target genes of miRNAs using computational methods. The most popular computational target prediction methods rely on sequence based determinants to predict targets. However, these determinants are neither sufficient nor necessary to identify functional target sites, and commonly ignore the cellular conditions in which miRNAs interact with their targets \emph{in vivo}. Since miRNAs activity reduces the steady-state abundance of mRNA targets, the main goal of this thesis is to augment large scale gene expression profiles as a supplement to sequence-based computational miRNA target prediction techniques. We develop two computational miRNA target prediction methods: InMiR and BayMiR; in addition, we study the interaction between miRNAs and lncRNAs using long RNA expression data. InMiR is a computational method that predicts the targets of intronic miRNAs based on the expression profiles of their host genes across a large number of datasets. InMiR can also predict which host genes have expression profiles that are good surrogates for those of their intronic miRNAs. Host genes that InMiR predicts are bad surrogates contain significantly more miRNA target sites in their 3 UTRs and are significantly more likely to have predicted Pol II-III promoters in their introns. We also develop BayMiR that scores miRNA-mRNA pairs based on the endogenous footprint of miRNAs on gene expression in a genome-wide scale. BayMiR provides an ``endogenous target repression" index, that identifies the contribution of each miRNA in repressing a target gene in presence of other targeting miRNAs. This thesis also addresses the interactions between miRNAs and lncRNAs. Our analysis on expression abundance of long RNA transcripts (mRNA and lncRNA) shows that the lncRNA target set of some miRNAs have relatively low abundance in the tissues that these miRNAs are highly active. We also found lncRNAs and mRNAs that shared many targeting miRNAs are significantly positively correlated, indicating that these set of highly expressed lncRNAs may act as miRNA sponges to promote mRNA regulation.

A produção in vitro de embriões expõe o concepto a condições diferentes do ambiente intra-tubárico-uterino. A alta tensão de oxigênio durante o cultivo in vitro induz estresse oxidativo mediante aumento dos níveis das espécies reativas de oxigênio. A condição de estresse oxidativo altera a expressão de RNAm e miRNAs, podendo comprometer vias de interação materno-embrionária. Recentemente, os exossomos têm sido descritos como um mecanismo complementar de transporte de RNAm e miRNAs, que beneficiam a comunicação bidirecional materno-embrionária. Portanto, além do estudo dos próprios embriões, a análise dos exossomos isolados do meio de cultivo da PIVE também é relevante. Comumente são utilizados dois modelos de cultivo na PIVE, a alta tensão (20%) e a baixa tensão (5%) de oxigênio. A hipótese deste estudo é que a alta tensão de oxigênio no cultivo embrionário gera uma condição de estresse oxidativo que causa alterações de expressão de RNAm e miRNAs presentes nos embriões e nos exossomos. Além disso, o estresse oxidativo exerceria efeito sobre o padrão de secreção destes exossomos. Para testar essa hipótese este estudo produziu embriões bovinos in vitro cultivados em diferentes tensões de oxigênio. Os embriões foram coletados no dia 7 e os meios de cultivo, para isolamento os exossomos, foram coletados nos dias 3 e 7 do cultivo. Os blastocistos cultivados em alta tensão de oxigênio apresentaram um aumentou dos níveis de EROs através de análise de fluorescência. Este resultado validou o modelo de estresse oxidativo para embriões. Os resultados iniciais indicaram que apesar do miR-199a-5p, descrito como possível regulador dos genes alvos ERBB2 e ERBB3, ter apresentado maior expressão nos embriões cultivados em alta tensão, os transcritos ERBB2 e ERBB3 e a proteína ERBB2 não apresentaram diferença significativa entre os grupos. Uma vez que não se estabeleceu uma relação de regulação entre o miR-199a-5p e o gene alvo ERBB2 para os embriões bovinos, este estudo se voltou para a análise de outros 96 RNAm e 378 miRNAs, destes 40 RNAm e 8 miRNAs apresentaram alterações significativas entre os embriões cultivados em alta e baixa tensão de oxigênio. Entre as principais funções alteradas estão associadas à resposta ao estresse oxidativo, proliferação e diferenciação celular, remodelação epigenética, apoptose, metabolismo e reconhecimento materno-fetal. Por fim, com o objetivo de compreender um panorama maior dos efeitos do estresse oxidativo, este estudo se propôs a analisar o padrão de expressão e os miRNAs do conteúdo dos exossomos do meio de cultivo. O estresse oxidativo alterou tanto a concentração dos exossomos quanto a expressão dos mRNAs, em ambos os dias do desenvolvimento embrionário (D3 e D7). Dentre os miRNAs, destaca-se o miR-210 que foi considerado por este trabalho como biomarcador não invasivo da condição de normoxia no cultivo in vitro de embriões bovinos. Em conclusão, esse estudo não elucidou como o estresse oxidativo altera a interação materno-embrionária em embriões produzidos in vitro em diferentes condições de tensão de oxigênio, mas gerou conhecimentos adicionais sobre do desenvolvimento embrionário bovino e os efeitos da alta tensão de oxigênio. / The in vitro embryo production exposes the concept to conditions different from the intra-tubal-uterine environment. The high oxygen tension during in vitro production induces the oxidative stress by increasing the concentration of reactive oxygen species. The oxidative stress condition changes the mRNA and miRNAs expression, it can compromise pathways of maternal-embryonic interaction. Recently, the exosomes have been described as a complementary mechanism of mRNA and miRNAs transport, which improve the bidirectional maternal-embryonic communication. Therefore, the studies of the embryos and exosomes isolated from the IVP culture medium are relevant. Two cultivation models are usually used in IVP, the high tension (20%) and the low tension (5%) of oxygen. The hypothesis of this study is that the high oxygen tension in the embryonic culture generates an oxidative stress condition that causes changes in the mRNA and miRNAs expression. In addition, this oxidative stress is able to modulate the secretion pattern of the exosomes isolated from IVP embryos. To test this hypothesis, this study produced in vitro bovine embryos developed at different oxygen tensions. The embryos were sampled on day 7 and the culture media, for exosomes isolation, were collected on days 3 and 7 of the embryo development. Blastocysts cultured in high oxygen tension showed increased levels of ROS through fluorescence analysis. This result validated the oxidative stress model for embryos. In order to understand the effect of oxidative stress on the pathways of maternal-embryonic interaction, this study aimed to analyze the ERBB signaling pathway. Despite the fact that miR-199a-5p, described as a possible regulator of the ERBB2 and ERBB3 target genes, showed higher expression in embryos cultured under high tension, the ERBB2 and ERBB3 transcripts and the ERBB2 protein showed no significant difference between high and low oxygen tension groups. Since a regulatory relationship between miR-199a-5p and the ERBB2 target gene was not established for bovine embryos, this study turned to the analysis of other 96 mRNAs and 378 miRNAs, out of 40 mRNAs and 8 miRNAs showed significant changes among the groups. The main altered functions are the response to oxidative stress, cell proliferation and differentiation, epigenetic remodeling, apoptosis, metabolism and maternal-fetal recognition. Finally, in order to understand the bigger picture of oxidative stress effect we analyze the secretion pattern and the miRNA content of the exosomes from culture medium of IVP embryos. Oxidative stress change both, the exosome concentration and the miRNA expression (at D3 and D7). Among the miRNAs, we highlight the miR-210 that was considered by this work as a non-invasive biomarker of the normoxia condition in the in vitro culture of bovine embryos. In conclusion, this study did not elucidate how oxidative stress changes the maternal-embryonic interaction in IVP embryos, but it generated additional knowledge about bovine embryonic development and the high oxygen tension effects.

INTRODUÇÃO: A obesidade é uma doença multifatorial associada com alto risco de desenvolver resistência à insulina (RI), diabetes melito tipo 2 (DT2), síndrome metabólica (SM) e doenças cardiovasculares (DCV). Os mecanismos moleculares da fisiopatologia das doenças metabólicas não são totalmente compreendidos, emergindo a necessidade de novas estratégias. As modificações epigenéticas, especialmente os miRNAs, podem representar uma importante ferramenta, uma vez que são relacionados com o desenvolvimento de diversas doenças complexas, como as metabólicas. OBJETIVO: Avaliar o perfil de expressão de miRNAs séricos de indivíduos com obesidade, síndrome metabólica e resistência à insulina e sua relação com os mecanismos moleculares envolvidos nas suas fisiopatologias. CASUÍSTICA E MÉTODOS: O estudo foi realizado no Instituto Dante Pazzanese de Cardiologia (IDPC) e no Hospital Universitário da Universidade de São Paulo (HU/USP). Um total de 233 indivíduos foram distribuídos em três grupos: peso normal (n=47), sobrepeso (84) e obesos (102), de acordo com seu índice de massa corpórea (IMC). Os mesmos pacientes também foram re-classificados em com SM (n=89) e sem SM (n=144) de acordo com recomendações da Federação internacional de Diabetes e em com RI (n=123) e sem RI (n=110). As informações biodemográficas e amostras de sangue foram coletadas para testes laboratoriais clínicos e análises de perfis de expressão de miRNA pela PCR array (372 alvos). Analises in silico com a ferramenta Ingenuity Pathway Analysis (IPA) foi realizada para integração do perfil de miRNAs e seus genes alvos com doenças metabólicas. RESULTADOS: Os pacientes obesos e com RI, apresentaram aumento similar das concentrações de marcadores dos perfis lipídico, glicídico e inflamatório quando comparado com indivíduos com peso normal e sem RI. O perfil de miRNAs dos grupos obesos e com RI mostrou que o miR-346 (p=0,010; p=0,019), 483-5p (p=0,034; p=0,020), 296-3p (p=0,010; p=0,008), 1224-3p (p=0,044; p=0,012) e 95-3p (p=0,003; p=0,019) foram expressos similarmente nestes pacientes quando comparados a seus respectivos controles. Estes miRNAs, por predição in silico, desempenham um importante papel na regulação de vias metabólicas associadas a doenças hepáticas gordurosas, inflamação, DCV e doenças metabólicas. Na SM, o grupo portador da doença apresentou altas concentrações de marcadores do perfil lipídico, glicídico e inflamatório. O perfil de expressão de miRNA indicou uma expressão reduzida do miR-183-5p (p=0,008) e miR-301a-3p (p=0,007) e aumentada do miR-542-5p (p=0,003), miR-424-3p (p=0,040), miR-326 (p=0,037), miR-421 (p=0,023) e miR-574-3p (p=0,001) no grupo SM comparado ao sem SM. A análise de regressão linear múltipla stepwise indicou que a expressão aumentada do miR-421 explica em 22,5% o aumento da associação de Hb1Ac, LDL-C, PAI-1, HOMA-IR, glicose e insulina (p <0,001, p=0,014, p=0,009, p <0,001, p=0,001 e p=0,007, respectivamente). Além disso o miR-421 foi predito in silico estar associado com a regulação de 11 mRNA alvos envolvidos em vias regulatórias de doenças como infarto do miocárdio, obesidade, diabetes melito, esteatose hepática, tolerância a glicose, distúrbio do metabolismo da glicose, hiperglicemia e resistência à insulina, bem como atua em vias regulatórias da resposta inflamatória, sendo associado com a variabilidade na quantidade de citocinas, massa do tecido adiposo e quantidade de leptina no sangue. Mediante a relação do miR-421 com a homeostase da glicose, sua expressão foi testada em pacientes com RI e interessantemente se manteve aumentada nesses pacientes em comparação com os sem RI (p=0.014). CONCLUSÕES: Os miR-346, miR-483-5p, miR-296-3p, miR-1224-3p e miR-95-3p podem ser considerados potenciais biomarcadores como preditores de DCV em pacientes obesos e resistência à insulina e o miR-421 um potencial biomarcador precoce de predição de risco de DCV em pacientes com síndrome metabólica. / BACKGROUND: Obesity is a multifactorial disease associated with high risk of developing insulin resistance (IR), Diabetes mellitus type 2 (DT2), metabolic syndrome (MetS) and cardiovascular disease (CVD). Molecular mechanisms of metabolic diseases\' pathophysiology are not fully understood, emerging the need for new strategies. Epigenetic modifications, especially miRNAs, may represent an important tool, since they are related to the development of several complex diseases, such as those metabolic. OBJECTIVE: Evaluate miRNA-derived serum expression profile in individuals with obesity, MetS and IR and their relationship with molecular mechanisms involved in the metabolic disease pathophysiology. MATERIAL AND METHODS: The study was conducted at the Dante Pazzanese Institute of Cardiology (IDPC) and at University Hospital of the University of São Paulo (HU/USP). Two hundred thirty-three subjects were distributed into three groups: normal weight (n = 47), overweight (n=84) and obese (n=102), according to their body mass index (BMI). The same patients were also re-classified as MetS (n=89) and Non-MetS (n=144) according to recommendations of the International Diabetes Federation and in IR (n=123) and Non-IR. Biodemographic data were registered and blood samples were collected for clinical laboratory tests and miRNA expression profiles by PCR array (372 targets). In silico analysis using Ingenuity Pathway Analysis (IPA) tool was performed to integrate the miRNA profile and their target genes with metabolic diseases. RESULTS: Obese and IR patients had similar increases in the concentrations of lipid, glucose and inflammatory markers when compared to individuals with normal weight and without IR, respectively. miRNA profile of obese and IR groups showed that miR-346 (p = 0.010, p = 0.019), 483-5p (p = 0.034, p = 0.020), 296-3p (p = 0.010, p = 0.008), 1224-3p (p = 0.044, p = 0.012) and 95-3p (p = 0.003, p = 0.019) were similarly expressed in these patients in comparison to their respective controls. These miRNAs, by in silico prediction, play an important role in the regulation of metabolic pathways associated with fatty liver diseases, inflammation, CVD and metabolic diseases. In MetS, patients diagnosed with the disease showed high concentrations of lipid, glycogen and inflammatory markers. The miRNA expression profile indicated a reduced expression of miR-183-5p (p=0.008) and miR-301a-3p (p=0.007) and increased miR-542-5p (p=0.003), miR- 3 (p=0.040), miR-326 (p=0.037), miR-421 (p=0.023) and miR-574-3p (p=0.001) in the MetS group compared to Non-MetS. Stepwise multiple linear regression analysis indicated that increased expression of miR-421 explain in 22.5% increased of Hb1Ac, LDL-C, PAI-1, HOMA-IR, glucose and insulin (p<0.001, p=0.014, p=0.009, p<0.001, p=0.001 and p=0.007, respectively). In addition the miR-421 was predicted in silico to associated with regulation of eleven mRNA of gene targets involved in regulatory pathways of diseases such as myocardial infarction, obesity, diabetes mellitus, hepatic steatosis, glucose tolerance, glucose metabolism disorder, hyperglycemia and insulin resistance, as well inflammatory response and is also associated with variability in the amount of cytokines, adipose tissue mass and amount of leptin in the blood. Due the relationship of miR-421 with glucose homeostasis, the miRNA expression was tested in patients with IR. Interestingly, miR-421 remained increased in these patients compared to those without IR (p = 0.014). CONCLUSIONS: The miR-346, miR-483-5p, miR-296-3p, miR-1224-3p and miR-95-3p may be potential biomarkers to predict CVD in obese and IR patients, while the miR-421 is a potential biomarker to predict CVD in MetS patients.

INTRODUÇÃO: A obesidade é uma doença multifatorial associada com alto risco de desenvolver resistência à insulina (RI), diabetes melito tipo 2 (DT2), síndrome metabólica (SM) e doenças cardiovasculares (DCV). Os mecanismos moleculares da fisiopatologia das doenças metabólicas não são totalmente compreendidos, emergindo a necessidade de novas estratégias. As modificações epigenéticas, especialmente os miRNAs, podem representar uma importante ferramenta, uma vez que são relacionados com o desenvolvimento de diversas doenças complexas, como as metabólicas. OBJETIVO: Avaliar o perfil de expressão de miRNAs séricos de indivíduos com obesidade, síndrome metabólica e resistência à insulina e sua relação com os mecanismos moleculares envolvidos nas suas fisiopatologias. CASUÍSTICA E MÉTODOS: O estudo foi realizado no Instituto Dante Pazzanese de Cardiologia (IDPC) e no Hospital Universitário da Universidade de São Paulo (HU/USP). Um total de 233 indivíduos foram distribuídos em três grupos: peso normal (n=47), sobrepeso (84) e obesos (102), de acordo com seu índice de massa corpórea (IMC). Os mesmos pacientes também foram re-classificados em com SM (n=89) e sem SM (n=144) de acordo com recomendações da Federação internacional de Diabetes e em com RI (n=123) e sem RI (n=110). As informações biodemográficas e amostras de sangue foram coletadas para testes laboratoriais clínicos e análises de perfis de expressão de miRNA pela PCR array (372 alvos). Analises in silico com a ferramenta Ingenuity Pathway Analysis (IPA) foi realizada para integração do perfil de miRNAs e seus genes alvos com doenças metabólicas. RESULTADOS: Os pacientes obesos e com RI, apresentaram aumento similar das concentrações de marcadores dos perfis lipídico, glicídico e inflamatório quando comparado com indivíduos com peso normal e sem RI. O perfil de miRNAs dos grupos obesos e com RI mostrou que o miR-346 (p=0,010; p=0,019), 483-5p (p=0,034; p=0,020), 296-3p (p=0,010; p=0,008), 1224-3p (p=0,044; p=0,012) e 95-3p (p=0,003; p=0,019) foram expressos similarmente nestes pacientes quando comparados a seus respectivos controles. Estes miRNAs, por predição in silico, desempenham um importante papel na regulação de vias metabólicas associadas a doenças hepáticas gordurosas, inflamação, DCV e doenças metabólicas. Na SM, o grupo portador da doença apresentou altas concentrações de marcadores do perfil lipídico, glicídico e inflamatório. O perfil de expressão de miRNA indicou uma expressão reduzida do miR-183-5p (p=0,008) e miR-301a-3p (p=0,007) e aumentada do miR-542-5p (p=0,003), miR-424-3p (p=0,040), miR-326 (p=0,037), miR-421 (p=0,023) e miR-574-3p (p=0,001) no grupo SM comparado ao sem SM. A análise de regressão linear múltipla stepwise indicou que a expressão aumentada do miR-421 explica em 22,5% o aumento da associação de Hb1Ac, LDL-C, PAI-1, HOMA-IR, glicose e insulina (p <0,001, p=0,014, p=0,009, p <0,001, p=0,001 e p=0,007, respectivamente). Além disso o miR-421 foi predito in silico estar associado com a regulação de 11 mRNA alvos envolvidos em vias regulatórias de doenças como infarto do miocárdio, obesidade, diabetes melito, esteatose hepática, tolerância a glicose, distúrbio do metabolismo da glicose, hiperglicemia e resistência à insulina, bem como atua em vias regulatórias da resposta inflamatória, sendo associado com a variabilidade na quantidade de citocinas, massa do tecido adiposo e quantidade de leptina no sangue. Mediante a relação do miR-421 com a homeostase da glicose, sua expressão foi testada em pacientes com RI e interessantemente se manteve aumentada nesses pacientes em comparação com os sem RI (p=0.014). CONCLUSÕES: Os miR-346, miR-483-5p, miR-296-3p, miR-1224-3p e miR-95-3p podem ser considerados potenciais biomarcadores como preditores de DCV em pacientes obesos e resistência à insulina e o miR-421 um potencial biomarcador precoce de predição de risco de DCV em pacientes com síndrome metabólica. / BACKGROUND: Obesity is a multifactorial disease associated with high risk of developing insulin resistance (IR), Diabetes mellitus type 2 (DT2), metabolic syndrome (MetS) and cardiovascular disease (CVD). Molecular mechanisms of metabolic diseases\' pathophysiology are not fully understood, emerging the need for new strategies. Epigenetic modifications, especially miRNAs, may represent an important tool, since they are related to the development of several complex diseases, such as those metabolic. OBJECTIVE: Evaluate miRNA-derived serum expression profile in individuals with obesity, MetS and IR and their relationship with molecular mechanisms involved in the metabolic disease pathophysiology. MATERIAL AND METHODS: The study was conducted at the Dante Pazzanese Institute of Cardiology (IDPC) and at University Hospital of the University of São Paulo (HU/USP). Two hundred thirty-three subjects were distributed into three groups: normal weight (n = 47), overweight (n=84) and obese (n=102), according to their body mass index (BMI). The same patients were also re-classified as MetS (n=89) and Non-MetS (n=144) according to recommendations of the International Diabetes Federation and in IR (n=123) and Non-IR. Biodemographic data were registered and blood samples were collected for clinical laboratory tests and miRNA expression profiles by PCR array (372 targets). In silico analysis using Ingenuity Pathway Analysis (IPA) tool was performed to integrate the miRNA profile and their target genes with metabolic diseases. RESULTS: Obese and IR patients had similar increases in the concentrations of lipid, glucose and inflammatory markers when compared to individuals with normal weight and without IR, respectively. miRNA profile of obese and IR groups showed that miR-346 (p = 0.010, p = 0.019), 483-5p (p = 0.034, p = 0.020), 296-3p (p = 0.010, p = 0.008), 1224-3p (p = 0.044, p = 0.012) and 95-3p (p = 0.003, p = 0.019) were similarly expressed in these patients in comparison to their respective controls. These miRNAs, by in silico prediction, play an important role in the regulation of metabolic pathways associated with fatty liver diseases, inflammation, CVD and metabolic diseases. In MetS, patients diagnosed with the disease showed high concentrations of lipid, glycogen and inflammatory markers. The miRNA expression profile indicated a reduced expression of miR-183-5p (p=0.008) and miR-301a-3p (p=0.007) and increased miR-542-5p (p=0.003), miR- 3 (p=0.040), miR-326 (p=0.037), miR-421 (p=0.023) and miR-574-3p (p=0.001) in the MetS group compared to Non-MetS. Stepwise multiple linear regression analysis indicated that increased expression of miR-421 explain in 22.5% increased of Hb1Ac, LDL-C, PAI-1, HOMA-IR, glucose and insulin (p<0.001, p=0.014, p=0.009, p<0.001, p=0.001 and p=0.007, respectively). In addition the miR-421 was predicted in silico to associated with regulation of eleven mRNA of gene targets involved in regulatory pathways of diseases such as myocardial infarction, obesity, diabetes mellitus, hepatic steatosis, glucose tolerance, glucose metabolism disorder, hyperglycemia and insulin resistance, as well inflammatory response and is also associated with variability in the amount of cytokines, adipose tissue mass and amount of leptin in the blood. Due the relationship of miR-421 with glucose homeostasis, the miRNA expression was tested in patients with IR. Interestingly, miR-421 remained increased in these patients compared to those without IR (p = 0.014). CONCLUSIONS: The miR-346, miR-483-5p, miR-296-3p, miR-1224-3p and miR-95-3p may be potential biomarkers to predict CVD in obese and IR patients, while the miR-421 is a potential biomarker to predict CVD in MetS patients.

A produção in vitro de embriões expõe o concepto a condições diferentes do ambiente intra-tubárico-uterino. A alta tensão de oxigênio durante o cultivo in vitro induz estresse oxidativo mediante aumento dos níveis das espécies reativas de oxigênio. A condição de estresse oxidativo altera a expressão de RNAm e miRNAs, podendo comprometer vias de interação materno-embrionária. Recentemente, os exossomos têm sido descritos como um mecanismo complementar de transporte de RNAm e miRNAs, que beneficiam a comunicação bidirecional materno-embrionária. Portanto, além do estudo dos próprios embriões, a análise dos exossomos isolados do meio de cultivo da PIVE também é relevante. Comumente são utilizados dois modelos de cultivo na PIVE, a alta tensão (20%) e a baixa tensão (5%) de oxigênio. A hipótese deste estudo é que a alta tensão de oxigênio no cultivo embrionário gera uma condição de estresse oxidativo que causa alterações de expressão de RNAm e miRNAs presentes nos embriões e nos exossomos. Além disso, o estresse oxidativo exerceria efeito sobre o padrão de secreção destes exossomos. Para testar essa hipótese este estudo produziu embriões bovinos in vitro cultivados em diferentes tensões de oxigênio. Os embriões foram coletados no dia 7 e os meios de cultivo, para isolamento os exossomos, foram coletados nos dias 3 e 7 do cultivo. Os blastocistos cultivados em alta tensão de oxigênio apresentaram um aumentou dos níveis de EROs através de análise de fluorescência. Este resultado validou o modelo de estresse oxidativo para embriões. Os resultados iniciais indicaram que apesar do miR-199a-5p, descrito como possível regulador dos genes alvos ERBB2 e ERBB3, ter apresentado maior expressão nos embriões cultivados em alta tensão, os transcritos ERBB2 e ERBB3 e a proteína ERBB2 não apresentaram diferença significativa entre os grupos. Uma vez que não se estabeleceu uma relação de regulação entre o miR-199a-5p e o gene alvo ERBB2 para os embriões bovinos, este estudo se voltou para a análise de outros 96 RNAm e 378 miRNAs, destes 40 RNAm e 8 miRNAs apresentaram alterações significativas entre os embriões cultivados em alta e baixa tensão de oxigênio. Entre as principais funções alteradas estão associadas à resposta ao estresse oxidativo, proliferação e diferenciação celular, remodelação epigenética, apoptose, metabolismo e reconhecimento materno-fetal. Por fim, com o objetivo de compreender um panorama maior dos efeitos do estresse oxidativo, este estudo se propôs a analisar o padrão de expressão e os miRNAs do conteúdo dos exossomos do meio de cultivo. O estresse oxidativo alterou tanto a concentração dos exossomos quanto a expressão dos mRNAs, em ambos os dias do desenvolvimento embrionário (D3 e D7). Dentre os miRNAs, destaca-se o miR-210 que foi considerado por este trabalho como biomarcador não invasivo da condição de normoxia no cultivo in vitro de embriões bovinos. Em conclusão, esse estudo não elucidou como o estresse oxidativo altera a interação materno-embrionária em embriões produzidos in vitro em diferentes condições de tensão de oxigênio, mas gerou conhecimentos adicionais sobre do desenvolvimento embrionário bovino e os efeitos da alta tensão de oxigênio. / The in vitro embryo production exposes the concept to conditions different from the intra-tubal-uterine environment. The high oxygen tension during in vitro production induces the oxidative stress by increasing the concentration of reactive oxygen species. The oxidative stress condition changes the mRNA and miRNAs expression, it can compromise pathways of maternal-embryonic interaction. Recently, the exosomes have been described as a complementary mechanism of mRNA and miRNAs transport, which improve the bidirectional maternal-embryonic communication. Therefore, the studies of the embryos and exosomes isolated from the IVP culture medium are relevant. Two cultivation models are usually used in IVP, the high tension (20%) and the low tension (5%) of oxygen. The hypothesis of this study is that the high oxygen tension in the embryonic culture generates an oxidative stress condition that causes changes in the mRNA and miRNAs expression. In addition, this oxidative stress is able to modulate the secretion pattern of the exosomes isolated from IVP embryos. To test this hypothesis, this study produced in vitro bovine embryos developed at different oxygen tensions. The embryos were sampled on day 7 and the culture media, for exosomes isolation, were collected on days 3 and 7 of the embryo development. Blastocysts cultured in high oxygen tension showed increased levels of ROS through fluorescence analysis. This result validated the oxidative stress model for embryos. In order to understand the effect of oxidative stress on the pathways of maternal-embryonic interaction, this study aimed to analyze the ERBB signaling pathway. Despite the fact that miR-199a-5p, described as a possible regulator of the ERBB2 and ERBB3 target genes, showed higher expression in embryos cultured under high tension, the ERBB2 and ERBB3 transcripts and the ERBB2 protein showed no significant difference between high and low oxygen tension groups. Since a regulatory relationship between miR-199a-5p and the ERBB2 target gene was not established for bovine embryos, this study turned to the analysis of other 96 mRNAs and 378 miRNAs, out of 40 mRNAs and 8 miRNAs showed significant changes among the groups. The main altered functions are the response to oxidative stress, cell proliferation and differentiation, epigenetic remodeling, apoptosis, metabolism and maternal-fetal recognition. Finally, in order to understand the bigger picture of oxidative stress effect we analyze the secretion pattern and the miRNA content of the exosomes from culture medium of IVP embryos. Oxidative stress change both, the exosome concentration and the miRNA expression (at D3 and D7). Among the miRNAs, we highlight the miR-210 that was considered by this work as a non-invasive biomarker of the normoxia condition in the in vitro culture of bovine embryos. In conclusion, this study did not elucidate how oxidative stress changes the maternal-embryonic interaction in IVP embryos, but it generated additional knowledge about bovine embryonic development and the high oxygen tension effects.

Stem cell research, especially the one dealing with human embryonic stem cells, is a major topic nowadays. In the last few years studies about human embryonic stem cell derived cardiomyocytes highlighted the importance of those, as their characteristics are almost identical as of the cardiomyocytes in the heart (i.e. the contraction of those cells). The studies concentrate on the ability of using cardiomyocytes in the drug development for cardiac diseases or in regenerative medicine and cell replacement therapies. In contrast some researchers concentrate on microRNAs (miRNAs) as regulators in the development of cardiomyocytes. This study combines both research topics as it deals with stem cells and miRNAs (as well as their target mRNAs). A main objective is to find differentially expressed genes by using Significance Analysis of Microarrays (SAM) as method. Furthermore miRNA target prediction is applied and the identified targets are compared with the ones found by SAM. With an intersection approach we derived 41 targets of up-regulated miRNAs and 25 targets of down-regulated miRNAs, which can be the basis for further studies (i.e. knock-out experiments).

No description available.

Philosophiae Doctor - PhD / Ovarian cancer (OC) is the most fatal gynaecologic malignancy that is generally diagnosed in the advanced stages, resulting in a low survival rate of about 40%. This emphasizes the need to identify a biomarker that can allow for accurate diagnosis at stage I. MicroRNAs (miRNAs) are appealing as biomarkers due to their stability, non-invasiveness, and differential expression in tumour tissue compared to healthy tissue. Since they are non-coding, their biological functions can be uncovered by examining their target genes and thus identifying their regulatory pathways and processes. This study aimed to identify miRNAs and genes as candidate biomarkers for early stage OC diagnosis, through two distinct in silico approaches. The first pipeline was based on sequence similarity between miRNAs with a proven mechanism in OC and miRNAs with no known role. This resulted in 9 candidate miRNAs, that have not been previously implicated in OC, that showed 90-99% similarity to a miRNA involved in OC. Following a series of in silico experimentations, it was uncovered that these miRNAs share 12 gene targets that are expressed in the ovary and also have proven implications in the disease. Since the miRNAs target genes contribute to OC onset and progression, it strengthens the notion that the miRNAs may be dysregulated as well. Using TCGA, the second pipeline involved analysing patient clinical data along with implementing statistical measures to isolate miRNAs and genes with high expression in OC. This resulted in 26 miRNAs and 25 genes being shortlisted as the potential candidates for OC management. It was also noted that targeting interactions occur between 15 miRNAs and 16 genes identified through this pipeline. In total, 35 miRNAs and 37 genes were identified from both pipelines.

Cutaneous melanoma is associated with significant morbidity and mortality representing the most significant cutaneous malignancy. As it is known that early diagnosis and treatment are the most efficient approaches to cure cutaneous melanoma, an improved understanding of the molecular pathogenesis of melanoma and exploration of more reliable molecular biomarkers are particularly essential. Two different types of molecular biomarker for melanoma have been investigated in this thesis. microRNAs (miRNAs) are single-stranded RNA molecules of 20-23 nucleotides in length that are found in both animal and plant cells. miRNAs are involved in the RNA interference (RNAi) machinery to regulate gene expression posttranscriptionally. miRNAs have important roles in cancer: by controlling the expression level of their target genes they can affect cell signalling pathways and have been shown to have both prognostic and therapeutic potential. Importantly for melanoma research, reproducible miRNA expression profiles from formalin-fixed paraffin-embedded (FFPE) tissues can be obtained that are comparable to those from fresh-frozen samples. The aims of the miRNA project were: first, to identify a melanoma-specific miRNA expression profile; secondly, to investigate roles of some of the melanoma-specific miRNAs identified in melanomagenesis. Using miRNA microarray on FFPE samples, I obtained a melanoma-specific miRNA expression profile. 9 of these differentially expressed miRNAs between benign naevi and melanomas (7 downregulated, 2 upregulated in malignancies) were verified by qRT-PCR and the functions of four of these miRNAs were studied. Ectopic overexpression of miR- 200c and miR-205 in A375 melanoma cells inhibited colony forming ability in methylcellulose, an in vitro surrogate assay for tumourigenicity. Moreover, elevation of miR-200c resulted in increased expression levels of E-cadherin through negative regulation of the zinc finger E-box-binding homeobox 2 (ZEB2) gene. Ectopic overexpression of miR-211 in A375 melanoma cells repressed both colony formation in methylcellulose and migratory ability in matrigel, an in vitro surrogate assay for invasiveness. These findings indicate that miR-200c, miR-205 and miR-211 act as tumour suppressors in melanomagenesis. The second biomarker investigated, mutated BRAF, has been seen in 50-70% of spontaneous cutaneous melanoma. The commonest mutation in melanoma is a glutamic acid for valine substitution at position 600 (V600E). Oncogenic BRAF controls many aspects of melanoma cell biology. The aim of this part of the work was: firstly, to study BRAF V600E mutation status in our melanoma tissue microarray (TMA) panel; secondly, to correlate this mutation to various clinicopathological features and evaluate its prognostic value through statistical analyses. BRAF V600E mutations were seen in 20% of the primary and 69% of the metastatic melanomas, respectively. More BRAF V600E mutations were seen in males relative to females. The mutation was also related to cell pigmentation, but not to age, ulceration or solar elastosis. Melanoma patients with the BRAF V600E mutation relapse earlier than patients without this mutation. However, no significant association between the BRAF V600E mutation and overall survival and melanoma specific survival was found.