No description available.

No description available.

Cell phenotypic transitions, or cell fate decision making processes, are regulated by complex regulatory networks composed of genes, RNAs, proteins and metabolites. The regulation can take place at the epigenetic, transcriptional, translational, and post-translational levels to name a few. Epigenetic histone modification plays an important role in cell phenotype maintenance and transitions. However, the underlying mechanism relating dynamical histone modifications to stable epigenetic cell memory remains elusive. Incorporating key pieces of molecular level experimental information, we built a statistical mechanics model for the inheritance of epigenetic histone modifications. The model reveals that enzyme selectivity of different histone substrates and cooperativity between neighboring nucleosomes are essential to generate bistability of the epigenetic memory. We then applied the epigenetic modeling framework to the differentiation process of olfactory sensory neurons (OSNs), where the observed 'one-neuron-one-allele' phenomenon has remained as a long-standing puzzle. Our model successfully explains this singular behavior in terms of epigenetic competition and enhancer cooperativity during the differentiation process. Epigenetic level events and transcriptional level events cooperate synergistically in the OSN differentiation process. The model also makes a list of testable experimental predictions. In general, the epigenetic modeling framework can be used to study phenotypic transitions when histone modification is a major regulatory element in the system. Post-transcriptional level regulation plays important roles in cell phenotype maintenance. Our integrated experimental and computational studies revealed such a motif regulating the differentiation of definitive endoderm. We identified two RNA binding proteins, hnRNPA1 and KSRP, which repress each other through microRNAs miR-375 and miR-135a. The motif can generate switch behavior and serve as a noise filter in the stem cell differentiation process. Manipulating the motif could enhance the differentiation efficiency toward a specific lineage one desires. Last we performed mathematical modeling on an epithelial-to-mesenchymal transition (EMT) process, which could be used by tumor cells for their migration. Our model predicts that the IL-6 induced EMT is a stepwise process with multiple intermediate states. In summary, our theoretical and computational analyses about cell phenotypic transitions provide novel insights on the underlying mechanism of cell fate decision. The modeling studies revealed general physical principles underlying complex regulatory networks. / Ph. D.

Osteoarthritis (OA) is a common, multifactorial disease that is characterized by focal degeneration of the smooth articular cartilage in any of the synovial joints. Although the underlying molecular mechanisms for OA are still not fully understood, epidemiological studies have evidenced a significant genetic component to OA susceptibility. Genome-wide linkage scans and large-scale association studies have had success in unraveling the genetic architecture underlying OA with the identification of a number of susceptibility genes. In this work, functional analyses are reported of OA associated polymorphisms within two susceptibility genes: BMP5 and GDF5, both members of the TGF-β superfamily of secreted proteins. The extent of differential allelic expression (DAE) of BMP5 in human mesenchymal tissues was first examined with significant differences in BMP5 allelic output observed (allelic ratios exceeding 4:1 in the tissues of some donors). Significant variability in allelic expression within the different tissues of donors was also observed, suggesting that polymorphism in cis-regulation of BMP5 expression is common and that there is a considerable effect of tissue specific elements on BMP5 expression. DAE was then used as a phenotype to map tissue-specific cis-regulatory polymorphisms with the identification of a single nucleotide polymorphism (SNP) located downstream of BMP5 that was significantly associated with DAE as well as OA, suggesting that variability in cis-regulation of BMP5 is important in OA susceptibility. Moreover, the functional effect of a previously identified OA associated microsatellite within intron 1 of BMP5 was investigated using luciferase reporter assays and electrophoretic mobility shift assays (EMSAs) with significant differences observed both in the ability of various microsatellite alleles to modulate BMP5 promoter activity and to bind GATA-3 nuclear proteins, further suggesting a role for variability in BMP5 expression in OA susceptibility that may in part be due to altered GATA-3 binding. Finally, functional characterization of a previously reported OA associated SNP in the 5′ UTR of GDF5 is presented in which EMSAs show differential binding of nuclear factors between the two SNP alleles, strengthening the possible functional contribution of this SNP to OA susceptibility. Overall, this work demonstrates that polymorphism in cis-regulation is likely to play a role in OA susceptibility.

Nous avons voulu évaluer si les gènes CHE2, ATM et TP53, associés à un risque intermédiaire de cancer du sein, étaient soumis à une différence d'expression allélique (DEA). Nous avons étudié des lignées cellulaires lymphoblastiques (LCLs) dérivées de patientes à haut risque, négatives pour des mutations dans CRCA1 et BRCA2. Nous avons développé une méthode basée sur la "fusion haute-résolution" (High-resolution melting curve analysis, HRM) et l'utilisation d'une sonde d'hybridation fluorescente pour détecter de la DEA chez des individus hétérozygotes pour un SNP marqueur exonique. Cette méthode permet de corréler le signal fluorescent à la quantité relative des transcrits alléliques. Nous avons développé un outil d'analyse adapté aux besoins spécifiques de l'étude de la DEA par HRM. Dans nos échantillons, une DEA statistiquement significative a été identifiée pour le gène CHEK2, chez les porteurs de la mutation tronquante 1100delC. En revanche, en combinant les données du criblage mutationnel des gènes candidats et de l'étude de DEA, nous n'avons pas identifié de variant régulateur localisé en cis qui induirait de la DEA significative dans les gènes étudiés, dans le contexte de régulation transcriptionnelle propre aux LCLs proliférant librement. Nos résultats montrent que le HRM est une méthode sensible et précise pour mesurer de la DAE et qui peut être appliquée à d'autres tissus, mammaires ou sanguins. Ces derniers présentent un grand intérêt pour les études de criblage mutationnel à haut-débit cherchant à identifier des variants dysfonctionnels dans les régions régulatrices de gènes candidats. / We aimed to assess whether the breast-cancer intermediate-risk genes CHEK2, ATM ant TP53 were subject to differential allelic expression (DAE) in lymphoblastoid cell lines (LCLs) from high-risk breast cancer patients for whom no mutation in BRCA1 or BRCA2 had been identified.We implemented an assay based on high-resolution melting curve analysis (HRM) of single labeled fluorescent probes to detect allelic expression imbalance. The method relies on the distinction of the two alleles of an exonic marker SNP in heterozygous individuals with a fluorescent signal correlated to the relative abundance of each transcript. We developed an analysis tool for HRM data processing, specifically dedicated to DAE assessment. In our series, we found evidence for DAE for CHEK2, in carriers of the truncating mutation 1100delC. When combining mutation-screening data and assessment of DAE, we did not identify functional regulatory variant located in cis of the studied genes that would lead to DAE, in the transcriptional regulatory milieu of freely proliferating LCLs. Our results support that HRM is a method with high sensitivity and accuracy that can be used for DAE assessment. This approach can be applied to study breast and blood tissue samples. The latter would be of great interest for high-throughput mutation screening projects aiming to identify dysfunctional regulatory variants in candidate genes.

Estudos recentes demonstraram que a variação de expressão alelo-específica é mais comum do que se imaginou, podendo chegar, em humanos, a 50% dos genes. Identificar os genes submetidos ao controle de expressão alelo-específica é muito importante para o entendimento de várias doenças, incluindo o câncer. A identificação dos alvos desse tipo de regulação diferencial é difícil, principalmente devido à dificuldade de se avaliar a expressão de cada alelo individualmente. Neste trabalho, abordamos este problema com uma estratégia de análise in-silico, fundamentada na integração de dados públicos do genoma humano, dados de expressão (como cDNAs, SAGE e MPSS) e dados sobre polimorfismos (SNPs). Desenvolvemos um banco de dados de polimorfismos de base única (Single-Nucleotide Polymorphism - SNPs) associados a etiquetas alternativas de SAGE (Serial Analysis of Gene Expression) e MPSS (massively parallel signature sequencing). SAGE e MPSS são técnicas desenvolvidas para análise da expressão de genes em larga escala. Ambas as técnicas têm como princípio a produção de pequenas seqüências marcadoras (etiquetas), adjacentes aos sítios de enzimas de restrição que estiverem mais próximo da cauda poli-A do RNA mensageiro. Tais etiquetas são seqüenciadas em grande escala e a quantidade de etiquetas é usada para medir a abundância relativa dos RNAs mensageiros correspondentes. A presença de SNPs nos sítios de restrição ou nas seqüências das etiquetas pode gerar etiquetas distintas para alelos do mesmo gene, que denominamos etiquetas alternativas. Neste trabalho, empregamos o banco de dados de etiquetas alternativas associadas a SNPs para identificar genes com expressão alélica diferencial. Usando esta estratégia, identificamos 812 genes com expressão monoalélica, Estudos anteriores comprovaram que, dentre os 812 genes identificados, cinco estão sujeitos ao fenômeno de imprinting genômico. Durante o decorrer deste estudo, trabalhos realizados por outros grupos apontaram outros 73 genes do nosso repertório como genes que apresentam variação no nível de expressão dos alelos em heterozigotos. Com objetivo de confirmar a expressão alélica diferencial dos nossos candidatos, selecionamos 29 genes para validação experimental. Para 12 destes genes não achamos indivíduos heterozigotos, impossibilitando a análise da expressão dos alelos. Dentre os outros 17 genes, três apresentaram expressão bialélica e 14 apresentaram expressão alélica diferencial nos indivíduos heterozigotos, sendo que 3 deles apresentaram expressão monoalélica. Estes resultados sugerem que nossa estratégia pode contribuir significativamente na identificação de genes com expressão alélica diferencial. / Recent studies have shown that variation of allelic-specific gene expression is more common than previously thought, reaching up to 50% of human genes. To identify genes displaying differential expression among alleles it is important for the understanding of several diseases, including the cancer. Identification of genes submitted to allelic-specific differential expression is hard, mostly due to the difficulty in evaluating the expression levels of each allele independently. In this work, we developed an in-silico approach, based on the integration of public data about the human genome, gene expression data (such as cDNAs, SNPs, SAGE and MPSS) and data on polymorphisms (SNPs). We developed a database of Single Nucleotide Polymorphisms (SNPs) associated to alternative SAGE (Serial Analysis of Gene Expression) and MPSS (Massively Parallel Signature Sequencing) tags. SAGE and MPSS are genome-wide techniques developed for analysis of gene expression. Both techniques rely on the production of short marker sequences (known as tags), adjacent to restriction sites closer to the poly-A tail of messenger RNAs. Such tags are sequenced in a large scale and tag counts are used to measure the relative abundance of their corresponding transcripts. The presence of SNPs in the restriction sites or in the tag sequences might generate allelic-specific tags for the same gene, which we call alternative tags. In this work, we used the database of SNPs and associated alternative tags to identify genes submitted to allelic-specific differential gene expression. Using this approach, we identified 812 genes showing allelic-specific differential gene expression. Previous studies have shown that, among the 812 candidates, five genes are targets for genomic imprinting. While this study was being performed, work done by other groups suggested other 73 genes in our candidates list to have different expression levels for alleles in heterozygous. Aiming to verify whether variations in the expression levels of alleles existed among our candidate genes, we submitted 29 genes for experimental validation. For 12 genes, we couldnt find heterozygous individuals, thus rendering it impossible to ascertain whether the supposed expression variation was true. Among the other 17 genes analyzed, three genes presented bi-allelic expression and 14 genes have shown clear differential expression among alleles, three of the last ones displaying strict mono-allelic expression. These results suggest that our approach may contribute significantly to the identification of genes with allelic-specific differential expression.

O desenvolvimento de uma vacina contra malária causada por P. falciparum é prejudicado pelo alto nível de polimorfismo apresentado pelos antígenos desse parasito. O dimorfismo alélico é um padrão no qual os alelos observados de um gene se encontram divididos em duas famílias. A proteína dimórfica MSP-6 se associa à proteína MSP-1 (também dimórfica) na superfície do merozoíto. Genes de msp-6 de 21 isolados obtidos de pacientes do Brasil, mais 2 isolados da Tanzânia, África, foram seqüenciados para estudo da diversidade nucleotídica e distribuição geográfica dos alelos. As duas famílias possuem distribuição global. Não foi verificada associação entre o dimorfismo de MSP-1 e MSP-6. O gene ortólogo de msp-6 em P. reichenowi, grupo irmão de P. falciparum, foi seqüenciado para estudos evolutivos. Os alelos dimórficos de MSP-6 aparentam ter surgido de uma população ancestral polimórfica, tendo sido mantidos no presente por seleção balanceada. O alto grau de conservação encontrado dentro de cada família alélica torna MSP-6 um potencial alvo de uma vacina contra a malária. / The development of a vaccine against malaria caused by Plasmodium falciparum has been hampered by the high level of antigen polymorphism exhibited by this parasite. Allelic dimorphism is a pattern in which every observed allele of a gene is clearly grouped into one of two families. The dimorphic protein MSP-6 forms a complex with MSP-1 (also dimorphic) on merozoite surface. The msp-6 genes were sequenced in isolates obtained from 21 patients from Brazil, plus 2 isolates from Tanzania, Africa, to study nucleotide diversity and geographic distribution of alleles. Both families are globally distributed. Moreover, no association was observed between the MSP-1 and MSP-6 allelic types. Orthologous gene of msp-6 in P. reichenowi, chimpanzee parasite and sister group of P. falciparum, was sequenced for evolutionary studies. Dimorphic alleles of MSP-6 seem to have originated from an ancestral polymorphic population and are maintained by balancing selection. The high degree of conservation observed within each allelic family makes MSP-6 an promising target for vaccine development.

Jatropha curcas L., também conhecida no Brasil como pinhão-manso, é uma oleaginosa que atraiu a atenção do mundo para bioenergia dado a qualidade e o alto conteúdo de óleo na semente. Contudo, a espécie necessita de programas de melhoramento genético para fixar características de interesse em bancos de germoplasma. Este trabalho objetivou identificar e analisar a diversidade alélica, metabólica e físico-química relacionada à síntese de óleo e ésteres de forbol em uma amostra de 22 genótipos do BAG do Instituto Agronômico de Campinas para seleção de genótipos elite. Os genes MFP2, KASIII e 3-N-D, envolvidos na biossíntese de óleo e de taxol, foram selecionados a partir de Gomes et al. (2010). Os resultados mostraram que há baixa diversidade alélica para os genes KAS III e MFP2, por outro lado, a expressão desses mesmos genes e a composição de ácidos graxos foi bastante variável entre os genótipos e ao longo do desenvolvimento da semente de J. curcas indicando uma provável regulação diferenciada da via de óleo. O estudo realizado permite combinar a diversidade alélica, expressão dos genes, conteúdo metabólico e poder calorífico para a seleção de genótipos e identificação de parentais direcionando cruzamentos no quadro do programa de melhoramento do IAC. / Jatropha curcas L., also know in Brazil as physic nut, is an oilseed that call world´s attention to bioenergy due to quality and high oilseed content. However, this species needs of breeding programs so that interest traits may to be fixed in germplasm banks. This study aimed to identify and analyze allelic, metabolic and physicochemical diversity related to oil and phorbol esters synthesis in a 22 genotypes sample from Instituto Agronômico de Campinas-BAG for elite genotypes selection. The MFP2, KASIII and 3-ND genes involved in oil and taxol biosynthesis were selected from Gomes et al. (2010). The results showed that there is low allelic diversity for KAS III and MFP2 genes, on the other hand, the expression of these same genes and fatty acid composition was quite variable between genotypes through J. curcas seed development indicating a putative differential regulation in oil pathway. This study allows combining allelic diversity, gene expression, metabolic content and calorific values for parental genotypes identification and selection driving crosses within IAC breeding program.

Plasmodium vivax é a espécie de malária humana de maior distribuição geográfica, com 35 milhões de casos por ano. No Brasil, é a espécie mais prevalente, sendo responsável por cerca de 70% dos casos de malária. Diferentemente do P. falciparum, o P. vivax apresenta hipnozoítas, formas que se mantêm em estágio dormente no fígado e que, após um período de tempo variável, por mecanismos ainda desconhecidos, causam novo ataque malárico denominado recaída. Para contribuir para um melhor conhecimento acerca das recaídas causadas por P. vivax, neste trabalho foram analisadas amostras pareadas referentes ao ataque primário e à recaída de 10 pacientes que se infectaram na Amazônia Brasileira. Através da amplificação de um segmento polimórfico do gene que codifica a Proteína de Superfície do Merozoíta 1 (PvMSP1), foi encontrado um índice de 40% de infecções mistas, presentes inclusive durante a recaída, indicando que a ativação de hipnozoítas não é clonal. Em análise mais detalhada deste segmento polimórfico, utilizando técnicas de clonagem e sequenciamento, foi possível verificar que a população de parasitas obtida durante o ataque primário é idêntica àquela que surge nas recaídas. O estudo da resposta IgG específica naturalmente adquirida contra a região C-terminal da PvMSP1, a mais imunogênica da molécula, demonstrou, durante a recaída, um aumento nos títulos acompanhado por uma maturação na afinidade destes anticorpos além de um predomínio de IgG1. / Plasmodium vivax is the most widely distributed human malarial parasite causing an estimated 35 million cases annually. In some parts of the world, including Brazil where it reaches almost 70% of malaria cases, this is the most prevalent species. Unlike P. falciparum, P. vivax has hypnozoites, hepatocyte dormant stages that cause clinical and parasitological relapses. Unfortunately, the molecular basis of relapses remain poorly understood. This work compared paired primary attack and relapse samples obtained from 10 infected patients from the brazilian Amazon Region using a polymorphic segment of the gene encoding the Merozoite Surface Protein 1 (PvMSP1) as a genetic marker. PCR, Southern blot and DNA sequence analysis demonstrated that the parasite population from the primary attack is identical to the one arising during relapses and that the activation of hypnozoites is not clonal; moreover, a large percentage (40%) of mixed infections, were detected. Studies on the naturally acquired human specific IgG response of these patients against the C-terminal region of the PvMSP1 molecule, the most immunogenic region, demonstrated an increase in the titers, affinity maturation and a predominance of the IgG1 subclass during the relapse.

Submitted by Aline Batista (alinehb.ufpel@gmail.com) on 2017-08-28T21:54:31Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_Patricia_Costa.pdf: 751161 bytes, checksum: e817a14f53a6b9a7c3652132bcc1750f (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-08-28T21:55:22Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_Patricia_Costa.pdf: 751161 bytes, checksum: e817a14f53a6b9a7c3652132bcc1750f (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-08-28T21:55:30Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_Patricia_Costa.pdf: 751161 bytes, checksum: e817a14f53a6b9a7c3652132bcc1750f (MD5) / Made available in DSpace on 2017-08-28T21:55:30Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Dissertacao_Patricia_Costa.pdf: 751161 bytes, checksum: e817a14f53a6b9a7c3652132bcc1750f (MD5) Previous issue date: 2017-03-20 / Sem bolsa / O estado nutricional está diretamente ligado à saúde da criança, influenciando no seu processo de crescimento e desenvolvimento. O estudo teve por objetivo investigar o estado nutricional e identificar a existência da associação com os polimorfismo R577X da ACTN3 e ECA I/D, em escolares do ensino da rede pública e privada. É um estudo com delineamento transversal no período de novembro de 2015 a julho de 2016. Foram incluídos no estudo todos os escolares matriculados e que estavam frequentando regularmente as aulas. Para a coleta dos dados foi realizada avaliação antropométrica por meio das medidas de peso e estatura. Posteriormente, os dados foram analisados através da utilização do programa Stata 13.0. Após a avaliação dos dados pode-se observar os seguintes resultados, em relação ao estado nutricional observa-se que apenas 0,2% (n=1) foram classificados com magreza e 57,8% como eutróficos, enquanto que 16,5% (n=76) e 23,7% (n=109) foram classificados com sobrepeso e obesidade, respectivamente. Em relação ao polimorfismo da ECA, encontrou-se 223 indivíduos DD (52,5%), 131 ID (30,8%) e 71 II (16,70). A prevalência do alelo D foi de 68% e do alelo I foi de 32%. Quanto ao polimorfismo da ACTN3 observou-se 155 indivíduos RR (38,8%), 161 RX (40,25%) e 84 XX (21%). A prevalência do alelo R de 59% e do alelo X foi de 41%. Não foi encontrada associação entre o estado nutricional das crianças e os polimorfismos estudados. Os dois genes apresentaram estarem em desequilíbrio de distribuição quanto a Hardy-Weinberg na amostra, o que deverá ser estudado para verificar se este desequilíbrio também reflete a população adulta de Arroio Grande. / The nutritional status is directed to the health of the child, influencing its process of growth and development. The objective of the study was to investigate the nutritional status and to identify the existence of the association with ACTN3 (R577X) and ECA I / D gene polymorphisms in public and private school students. It is a study with a crosssectional design from November 2015 to July 2016. All the studies enrolled in school are included and are frequently considered as classes. To collect the data on an anthropometric evaluation by means of measures of weight and height. Subsequently, the data were analyzed for the use of the Stata 13.0 program. After an evaluation of the results, the following results can be observed regarding nutritional status, observing that only 0.6% (n = 1) were evaluated with 57.8% as eutrophic, while 16.5% (N = 76) and 23.7% (n = 109) were overweight and obese. Regarding the RCT polymorphism, 223 individuals were found to be DD (52.5%), 131 ID (30.8%) and 71 II (16.70). The prevalence of the D allele was 68% and the I allele was 32%. Regarding the ACTN3 polymorphism, 155 RR (38.8%), 161 RX (40.25%) and 84 XX (21%) individuals were observed. The prevalence of the 59% R allele and the X allele was 41%. No association was found between the nutritional status of the children and the polymorphisms studied. The two genes show a balance of distribution disequilibrium for Hardy-Weinberg in the sample, which should be studied to verify if this imbalance also reflects an adult population of Arroio Grande.