No description available.

No description available.

No description available.

Populações nativas da América do Sul apresentam diversidade genética reduzida em relação às demais populações do mundo e alta diferenciação interpopulacional dentro do continente. As pressões seletivas sobre a região cis-reguladora do CCR5 geram uma assinatura de diversidade oposta àquela esperada com base na história demográfica, reduzindo a variação interpopulacional e contribuindo para uma elevação das taxas de polimorfismo. No presente estudo investigamos a interação entre esses processos micro-evolutivos, analisando o polimorfismo da região cis-reguladora de ameríndios da América do Sul e comparando os resultados com aqueles obtidos em outras regiões do mundo. Sequenciamos 927 pares de base (pb) da região controladora do CCR5 em 7 tribos indígenas da região amazônica. Em ameríndios, nenhum haplótipo exclusivo foi encontrado em ameríndios e os dois haplogrupos mais comuns foram de diferentes clusters filogenéticos. A diversidade nucleotídica (&#960;) para a amostra total, foi a mais alta dentre todas as regiões do mundo já estudadas (&#960;=0,0027). A estimativa da diversidade genética populacional para ameríndios, baseada no número de sítios polimórficos (&#952;s), foi similar aos valores encontrados para as populações não-africanas, sendo que em africanos o valor foi praticamente o dobro. Conseqüentemente, foi observado um valor de D de Tajima positivo e estatisticamente significativo (D=2,82, p<0,01), maior do que aquele visto para outras regiões do mundo. Através de comparações empíricas e de simulações coalescentes verificamos que o alto valor de D de Tajima encontrado para ameríndios não é explicável por um recente modelo demográfico de evolução humana. O teste Ewens-Watterson indicou para a amostra ameríndia uma taxa de heterozigose maior do que esperada para um população neutra. O valor de Fst observado para comparação envolvendo asiáticos e ameríndios foi mais baixo que 1000 valores de Fst calculados a partir de 783 microssatélites genotipados em amostras de mesmo tamanho de regiões geográficas similares. Essas caraterísticas corroboram a hipótese de seleção balanceadora na região cis-reguladora do CCR5 de ameríndios da região amazônica brasileira. No presente estudo demonstramos também a existência de uma associação estatisticamente significativa (p<0,01) entre os alelos mantidos em freqüências intermediárias na população humana e regiões ativadoras de splicing localizadas em exons (ESEs). Finalmente, nós acreditamos que um modelo demográfico de evolução humana, complementar ao utilizado neste trabalho, deve ser testado para se refutar a hipótese de que a forte assinatura de seleção balanceadora observada em ameríndios não foi causada por seleção natural que atuou em população ancestral à de indígenas americanos / Native american populations show lower genetic diversity and higher interpopulational genetic differences than populations from other continents. Within South America groups from the non-andean geographic region shows extremely high genetic differentiation. The strong signature of balancing selection observed for 5\' cis-regulatory region of the CCR5 gene at the worldwide scale represents an opposite pattern of genetic variation to that expected by demographic process which acted in Amerindians. To evaluate the impact of complex demographic history on the 5\' cis-regulatory region of CCR5 we resequenced 927 bp of this locus in 62 individuals from different native groups located in the Brazilian Amazonian region. No new haplotype was detected and the two most common haplogroups observed were from different phylogenetic clusters, according with the pattern observed for other human populations. The level of heterozigosity of the total sample, measured by nucleotide diversity (&#960;) was the highest yet described for human populations (&#960;=0,0027) while values based on polymorphic sites (&#952;s) were similar among Amerindians and non-Africans populations. Consequently we observed a positive and significant Tajima\'s D value (D=2,82, p <0,01). This value was higher than all D values observed for the other human populations. Observed summary statistics (&#960; and D) were significantly higher than same statistics estimated from 2000 simulated samples assuming a recently proposed demographic model of human evolution. We also found significant deviations in Ewens-Watterson homozygosity test toward an excess of heterozygosity for Amerindian sample. Another striking feature of CCR5 cis-regulatory region was the low Fst value among populations. The Fst among Asians and Amerindians was an outlier among 1000 Fst values estimated by 783 microssatelites of samples from similar geographic regions and with the same sample sizes as those of the CCR5 5\' cis-regulatory data. These features corroborates the strong signature of balancing selection described for CCR5 5\'cis regulatory region. We also contributed to discussion about functional aspects of CCR5 cis-regulatory region demonstrating a significant association between intermediate frequency SNPs and exonic splicing enhancers (ESEs) regions (p<0,01). Finally, we believe that improved models of demographic human evolution must be tested to refute the hypothesis that the strong signature of balancing selection observed in Amerindians was not caused by a selection pressure which occurred in an ancestral population.

Populações nativas da América do Sul apresentam diversidade genética reduzida em relação às demais populações do mundo e alta diferenciação interpopulacional dentro do continente. As pressões seletivas sobre a região cis-reguladora do CCR5 geram uma assinatura de diversidade oposta àquela esperada com base na história demográfica, reduzindo a variação interpopulacional e contribuindo para uma elevação das taxas de polimorfismo. No presente estudo investigamos a interação entre esses processos micro-evolutivos, analisando o polimorfismo da região cis-reguladora de ameríndios da América do Sul e comparando os resultados com aqueles obtidos em outras regiões do mundo. Sequenciamos 927 pares de base (pb) da região controladora do CCR5 em 7 tribos indígenas da região amazônica. Em ameríndios, nenhum haplótipo exclusivo foi encontrado em ameríndios e os dois haplogrupos mais comuns foram de diferentes clusters filogenéticos. A diversidade nucleotídica (&#960;) para a amostra total, foi a mais alta dentre todas as regiões do mundo já estudadas (&#960;=0,0027). A estimativa da diversidade genética populacional para ameríndios, baseada no número de sítios polimórficos (&#952;s), foi similar aos valores encontrados para as populações não-africanas, sendo que em africanos o valor foi praticamente o dobro. Conseqüentemente, foi observado um valor de D de Tajima positivo e estatisticamente significativo (D=2,82, p<0,01), maior do que aquele visto para outras regiões do mundo. Através de comparações empíricas e de simulações coalescentes verificamos que o alto valor de D de Tajima encontrado para ameríndios não é explicável por um recente modelo demográfico de evolução humana. O teste Ewens-Watterson indicou para a amostra ameríndia uma taxa de heterozigose maior do que esperada para um população neutra. O valor de Fst observado para comparação envolvendo asiáticos e ameríndios foi mais baixo que 1000 valores de Fst calculados a partir de 783 microssatélites genotipados em amostras de mesmo tamanho de regiões geográficas similares. Essas caraterísticas corroboram a hipótese de seleção balanceadora na região cis-reguladora do CCR5 de ameríndios da região amazônica brasileira. No presente estudo demonstramos também a existência de uma associação estatisticamente significativa (p<0,01) entre os alelos mantidos em freqüências intermediárias na população humana e regiões ativadoras de splicing localizadas em exons (ESEs). Finalmente, nós acreditamos que um modelo demográfico de evolução humana, complementar ao utilizado neste trabalho, deve ser testado para se refutar a hipótese de que a forte assinatura de seleção balanceadora observada em ameríndios não foi causada por seleção natural que atuou em população ancestral à de indígenas americanos / Native american populations show lower genetic diversity and higher interpopulational genetic differences than populations from other continents. Within South America groups from the non-andean geographic region shows extremely high genetic differentiation. The strong signature of balancing selection observed for 5\' cis-regulatory region of the CCR5 gene at the worldwide scale represents an opposite pattern of genetic variation to that expected by demographic process which acted in Amerindians. To evaluate the impact of complex demographic history on the 5\' cis-regulatory region of CCR5 we resequenced 927 bp of this locus in 62 individuals from different native groups located in the Brazilian Amazonian region. No new haplotype was detected and the two most common haplogroups observed were from different phylogenetic clusters, according with the pattern observed for other human populations. The level of heterozigosity of the total sample, measured by nucleotide diversity (&#960;) was the highest yet described for human populations (&#960;=0,0027) while values based on polymorphic sites (&#952;s) were similar among Amerindians and non-Africans populations. Consequently we observed a positive and significant Tajima\'s D value (D=2,82, p <0,01). This value was higher than all D values observed for the other human populations. Observed summary statistics (&#960; and D) were significantly higher than same statistics estimated from 2000 simulated samples assuming a recently proposed demographic model of human evolution. We also found significant deviations in Ewens-Watterson homozygosity test toward an excess of heterozygosity for Amerindian sample. Another striking feature of CCR5 cis-regulatory region was the low Fst value among populations. The Fst among Asians and Amerindians was an outlier among 1000 Fst values estimated by 783 microssatelites of samples from similar geographic regions and with the same sample sizes as those of the CCR5 5\' cis-regulatory data. These features corroborates the strong signature of balancing selection described for CCR5 5\'cis regulatory region. We also contributed to discussion about functional aspects of CCR5 cis-regulatory region demonstrating a significant association between intermediate frequency SNPs and exonic splicing enhancers (ESEs) regions (p<0,01). Finally, we believe that improved models of demographic human evolution must be tested to refute the hypothesis that the strong signature of balancing selection observed in Amerindians was not caused by a selection pressure which occurred in an ancestral population.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Immune response enables the body to resist foreign invasions. The Inflammatory response is an important aspect in the immune response which is articulated by elements such as cytokines, APC, T-cell and B-cell, effector cell or natural killer. Of these elements, T-cells especially T-helper cells; a sub class of T-cells plays a pivotal role in stimulating the immune response by participating in various biological reactions such as, the transcription regulatory network. Transcriptional regulatory mechanisms are mediated by a set of transcription factors (TFs), that bind to a specific region (motifs or transcription factor binding sites, TFBS), on the target gene(s) controlling the expression of genes that are involved in T-helper cell mediated immune response. Eukaryotic regulatory motifs, referred to as cis regulatory modules (CRMs) or cistrome, co-occur with the regulated gene’s transcription start site (TSS) thus, providing all the essential components for building the transcriptional regulatory networks that depends on the relevant TF-TFBS interactions. Here, we study IL-12 stimulated transcriptional regulators in STAT4 mediated T helper 1 (Th1) cell development by focusing on the identification of TFBS and CRMs using a set of Stat4 ChIP-on-chip target genes. A region containing 2000 bases of Mus musculus sequences with the Stat4 binding site, derived from the ChIP-on-chip data, has been characterized for enrichment of other motifs and, thus CRMs. Our experiments identify some potential motifs, (such as NF-κB and PPARγ/RXR) being enriched in the Stat4 binding sequences compared to neighboring background sequences. Furthermore, these predicted CRMs were observed to be associated with biologically relevant target genes in the ChIP-on-chip data set by meaningful gene ontology annotations. These analyses will enable us to comprehend the complicated transcription regulatory network and at the same time categorically analyze the IL-12 stimulated Stat4 mediated Th1 cell differentiation.

No description available.

Cis-regulatory elements are the short regions of DNA to which specific regulatory proteins bind and these interactions subsequently influence the level of transcription for associated genes, by inhibiting or enhancing the transcription process. It is known that much of the genetic change underlying morphological evolution takes place in these regions, rather than in the coding regions of genes. Identifying these sites in a genome is a non-trivial problem. Experimental (wet-lab) methods for finding binding sites exist, but all have some limitations regarding their applicability, accuracy, availability or cost. On the other hand computational methods for predicting the position of binding sites are less expensive and faster. Unfortunately, however, these algorithms perform rather poorly, some missing most binding sites and others over-predicting their presence. The aim of this thesis is to develop and improve computational approaches for the prediction of transcription factor binding sites (TFBSs) by integrating the results of computational algorithms and other sources of complementary biological evidence. Previous related work involved the use of machine learning algorithms for integrating predictions of TFBSs, with particular emphasis on the use of the Support Vector Machine (SVM). This thesis has built upon, extended and considerably improved this earlier work. Data from two organisms was used here. Firstly the relatively simple genome of yeast was used. In yeast, the binding sites are fairly well characterised and they are normally located near the genes that they regulate. The techniques used on the yeast genome were also tested on the more complex genome of the mouse. It is known that the regulatory mechanisms of the eukaryotic species, mouse, is considerably more complex and it was therefore interesting to investigate the techniques described here on such an organism. The initial results were however not particularly encouraging: although a small improvement on the base algorithms could be obtained, the predictions were still of low quality. This was the case for both the yeast and mouse genomes. However, when the negatively labeled vectors in the training set were changed, a substantial improvement in performance was observed. The first change was to choose regions in the mouse genome a long way (distal) from a gene over 4000 base pairs away - as regions not containing binding sites. This produced a major improvement in performance. The second change was simply to use randomised training vectors, which contained no meaningful biological information, as the negative class. This gave some improvement over the yeast genome, but had a very substantial benefit for the mouse data, considerably improving on the aforementioned distal negative training data. In fact the resulting classifier was finding over 80% of the binding sites in the test set and moreover 80% of the predictions were correct. The final experiment used an updated version of the yeast dataset, using more state of the art algorithms and more recent TFBSs annotation data. Here it was found that using randomised or distal negative examples once again gave very good results, comparable to the results obtained on the mouse genome. Another source of negative data was tried for this yeast data, namely using vectors taken from intronic regions. Interestingly this gave the best results.

L'épissage des ARN pré-messagers est une étape essentielle pour l'expression des gènes chez les eucaryotes supérieurs. La reconnaissance des exons par la machinerie d'épissage est réalisée grâce à différents éléments cis-régulateurs incluant les séquences consensus d'épissage et les séquences auxiliaires activatrices ou inhibitrices d'épissage. Le pré-ARNm représente une nouvelle cible thérapeutique pour le traitement des maladies génétiques. L'approche du saut d'exon thérapeutique, destinée à restaurer l'expression d'une protéine totalement ou partiellement fonctionnelle en interférant avec le processus d'épissage, suscite un grand intérêt notamment pour la dystrophie musculaire de Duchenne où la modification du transcrit permettrait d'obtenir une forme modérée de la maladie, la Dystrophie musculaire de Becker. Des oligonucléotides antisens sont utilisés pour masquer les signaux d'épissage de reconnaissance d'un exon par le spliceosome, et induire son excision (ou saut) du transcrit mature. La détermination de la meilleure séquence cible des AONs est une difficulté majeure de cette approche. Pour le gène DMD, nous avons pu établir grâce à des analyses bioinformatiques et statistiques combinées avec des tests fonctionnels utilisant des minigènes rapporteurs d'épissage, que le ciblage de motifs exoniques qui fixent le facteur d'épissage SF2/ASF permettait d'obtenir la meilleure efficacité des AONs. Par ailleurs, nous avons exploré la régulation de l'épissage des pseudoexons dans le gène DMD, et notamment les mécanismes conduisant à l'inclusion de ces séquences introniques dans le transcrit mature en condition pathologique. L'étude de deux cas exceptionnels d'activation de pseudoexons associée à des remaniements introniques rares (double délétion, inversion) élargit le spectre des mutations à l'origine de ces défauts d'épissage, et illustre le rôle encore mal connu des remaniements introniques en pathologie humaine. / Splicing of pre-messenger RNAs to mature transcripts is a crucial step in eukaryotic gene expression. The recognition of exon by the splicing machinery involves different cis-regulatory elements, including the splice site motifs and auxiliary sequences, which can act by stimulating or repressing splicing. The pre-mRNA represents a new therapeutic target for the treatment of genetic diseases. Notably, the exon skipping strategy is currently one of the most promising therapeutic approaches for the Duchenne muscular dystrophy. It intends to restore the expression of a partially functional protein by interfering with the splicing process, and converts the severe DMD phenotype into the moderate form of the disease, Becker muscular Dystrophy (BMD). Antisense oligonucleotides are used to mask the splicing signals involved in exon recognition by the spliceosome to induce its skipping from the mature transcript and restore an open reading frame. The determination of the best target sequence of the AONs is one of the major hurdles to overcome. For the DMD gene, a bioinformatic and statistical analysis combined with minigenes studies allowed us to establish that targeting binding sites for the splicing factor SF2/ASF maximizes the AONs efficiency. In a second part of this work, we investigated the splicing regulation of pseudoexons in the DMD gene, in particular the mechanisms leading to the inclusion of these intronic sequences in the mature transcript in pathological conditions. The study of two exceptional cases of pseudoexons activation associated with rare intronic rearrangements (double-deletions, inversion) expands the spectrum of missplicing mutations, and demonstrates the potential role of pure intronic rearrangements in human pathology.