Après intégration dans le génome cellulaire de l’hôte, l’expression des rétrovirus dépend d’éléments agissant en cis localisés dans la longue répétition terminale 5’ (LTR5’) et la région leader, de facteurs de transcription cellulaires et viraux agissant en trans ainsi que de l’organisation chromatinienne du provirus intégré. Notre laboratoire a précédemment identifié dans le génome du rétrovirus HIV-1 (Human Immunodeficiency Virus type 1) une région intragénique importante (nt 4079-6026, où nt +1 est le début de U3 dans le LTR5’) composée du fragment 5103, du site hypersensible aux nucléases SH7 et du fragment 5105. Lors de ce travail, nous avons caractérisé physiquement et fonctionnellement différents sites de liaison pour des facteurs de transcription cellulaires localisés dans la région intragénique du virus HIV-1, dont trois sites de liaison pour le facteur inductible AP-1, dans des expériences de retard de migration sur gel et de transfection transitoire. Nous avons montré l’importance de ces trois sites AP-1 pour la réplication virale au niveau transcriptionnel dans des expériences d’infection et d’immunoprécipitation de la chromatine. De plus, nous avons caractérisé l’activité transcriptionnelle associée à la région intragénique du virus HIV-1. D’autre part, la structure nucléosomale du provirus intégré et les modifications épigénétiques associées jouent un rôle crucial pour l’expression des rétrovirus. La répression transcriptionnelle du rétrovirus oncogène BLV (Bovine Leukemia Virus) lui permet d’échapper au système immunitaire de son hôte bovin et favorise ainsi l’apparition de tumeurs. Dans ce contexte, nous avons montré que la méthylation de l’ADN au niveau du promoteur viral permet le maintient de la latence transcriptionnelle. En effet, la méthylation des dinucleotides CpGs localisés dans le LTR5‘ empêche le recrutement in vivo des facteurs de transcription activateurs CREB/CREM/ATF. Nous avons également montré que l’activation transcriptionnelle de l’expression du BLV par la combinaison PMA/ionomycine s’accompagne d’un remodelage chromatinien rapide mais transitoire au niveau du promoteur viral par des expériences de marquage indirect des extrémités et d’immunoprécipitation de chromatine. Nous avons ensuite démontré l’importance du site de liaison pour le facteur de transcription PU.1 et de la E-box 4 qui lie USF-1/-2, tous deux localisés dans la région dont l’accessibilité aux nucléases s’accroît après traitement des cellules, pour l’activation transcriptionnelle de l’expression virale par cette combinaison d’inducteurs. En conclusion, notre travail devrait permettre une meilleure compréhension des mécanismes transcriptionnels et épigénétiques régulant l’expression des rétrovirus HIV-1 et BLV.

Yin Yang 1 (YY1) is a multifunctional transcription factor that is known mainly for its ability to activate or initiate transcription of a wide assortment of genes involved in cellular growth and differentiation. <i>Xenopus laevis </i>oocytes and embryos were used as a model to identify and characterize a potential developmental role for YY1. Northern and Western blots of oocyte and embryonic extracts showed YY1 mRNA and protein is expressed from the earliest stages of oocyte development through to tadpole stages. Examination of the transcriptional activity of YY1 in both oocytes and embryos using reporter gene constructs containing YY1-binding elements demonstrated that YY1 does not act as a repressor or activator of transcription either in oocytes or in embryos. Sub-cellular fractionation of oocytes and Western blot analysis showed YY1 is localized almost exclusively to the cytoplasm of oocytes and in cells of early embryos. Sequence analysis of YY1 revealed that it contains an established RNA binding motif located within the zinc fingers. A series of biochemical assays were performed to address the possibility that YY1 functions as a component of mRNPs in the oocyte cytoplasm. RNA gel mobility shift analyses using in vitro synthesized histone H2A transcripts and supershifts using YY1-specific antibodies suggested that YY1 or YY1-containing complexes in cytoplasmic extracts were able to bind RNA. Chromatographic analysis of oocyte lysates showed YY1 was specifically retained on oligo (dT) cellulose columns. Treatment of the same lysates with RNase abolished binding to oligo (dT), indicating that retention is dependent on the presence of intact polyadenylated RNAs. This suggested that YY1 may be a component of messenger ribonucleoprotein particles (mRNP). Separation of oocyte lysates by size exclusion chromatography (SEC) revealed that YY1 was present in large complexes with an approximate molecular mass of 480 kDa. RNase or phosphatase treatment of oocyte extracts released YY1 from high mass complexes. Analysis of phosphatase or RNase-treated extracts for DNA binding activity showed that monomeric YY1 was able to bind DNA with high affinity. Immunoprecipitation of YY1 complexes followed by cDNA synthesis and sequencing revealed that YY1 is associated with both ribosomal and messenger RNAs in the cytoplasm of the oocyte. These results indicate a novel function for YY1 as a component of messenger ribonucleoprotein particles.

Yin Yang 1 (YY1) is a multifunctional transcription factor that is known mainly for its ability to activate or initiate transcription of a wide assortment of genes involved in cellular growth and differentiation. <i>Xenopus laevis </i>oocytes and embryos were used as a model to identify and characterize a potential developmental role for YY1. Northern and Western blots of oocyte and embryonic extracts showed YY1 mRNA and protein is expressed from the earliest stages of oocyte development through to tadpole stages. Examination of the transcriptional activity of YY1 in both oocytes and embryos using reporter gene constructs containing YY1-binding elements demonstrated that YY1 does not act as a repressor or activator of transcription either in oocytes or in embryos. Sub-cellular fractionation of oocytes and Western blot analysis showed YY1 is localized almost exclusively to the cytoplasm of oocytes and in cells of early embryos. Sequence analysis of YY1 revealed that it contains an established RNA binding motif located within the zinc fingers. A series of biochemical assays were performed to address the possibility that YY1 functions as a component of mRNPs in the oocyte cytoplasm. RNA gel mobility shift analyses using in vitro synthesized histone H2A transcripts and supershifts using YY1-specific antibodies suggested that YY1 or YY1-containing complexes in cytoplasmic extracts were able to bind RNA. Chromatographic analysis of oocyte lysates showed YY1 was specifically retained on oligo (dT) cellulose columns. Treatment of the same lysates with RNase abolished binding to oligo (dT), indicating that retention is dependent on the presence of intact polyadenylated RNAs. This suggested that YY1 may be a component of messenger ribonucleoprotein particles (mRNP). Separation of oocyte lysates by size exclusion chromatography (SEC) revealed that YY1 was present in large complexes with an approximate molecular mass of 480 kDa. RNase or phosphatase treatment of oocyte extracts released YY1 from high mass complexes. Analysis of phosphatase or RNase-treated extracts for DNA binding activity showed that monomeric YY1 was able to bind DNA with high affinity. Immunoprecipitation of YY1 complexes followed by cDNA synthesis and sequencing revealed that YY1 is associated with both ribosomal and messenger RNAs in the cytoplasm of the oocyte. These results indicate a novel function for YY1 as a component of messenger ribonucleoprotein particles.

Eighteenth-century London was a hotbed for instrumental arrangements, and many of these works were derived from the operas of George Frideric Handel (1685-1759). Thirty-one of his operas, in whole or in part, were arranged for recorder or flute, and there were over seventy keyboard transcriptions of the overtures to these operas. While the transcriptions of Handel overtures have been thoroughly examined, opera aria transcriptions have never received an appropriate level of study and analysis. The Ladys Banquet or The Lady’s Entertainment provides an excellent starting point. Not only does it include numerous opera aria arrangements, but its volumes were re-issued several times, suggesting a wide circulation. Its study raises a number of issues, including publication and authorship of Handel transcriptions, gendered music of the eighteenth century and analysis of opera transcriptions. The Ladys Banquet or The Lady’s Entertainment is a collection of six volumes of keyboard music published by John Walsh in the first half of the eighteenth century. The first two books were issued in 1704 and 1706 respectively, and included many undemanding pieces by fashionable composers such as Jeremiah Clarke (c.1674-1707) and Henry Purcell (1659-1695). The Third and Fourth Books followed in circa 1715 and 1716 and contain predominantly dance tunes and popular songs revised for the keyboard. When, in the early 1730s, the Fifth and Sixth Books appeared, the original four volumes were revised, and included wholly different material than the first editions. The publications of John Walsh are notoriously confusing owing to their lack of publication dates, repeated use of title pages, and misleading advertisements. The Ladys Banquet, as a whole, is especially bewildering because of the reissues of the collection and the changing repertoire. My research focuses on the Fifth Book of The Ladys Banquet, first printed around 1734, due to its abundance of opera aria transcriptions and consistency of content within editions. This document compiles relevant background information and offers a lucid guide to The Ladys Banquet. It provides historical context, examination and discussion of the contents of each volume, with specific details about the music in the Fifth Book, as well as analysis of the Handel aria transcriptions.

Eighteenth-century London was a hotbed for instrumental arrangements, and many of these works were derived from the operas of George Frideric Handel (1685-1759). Thirty-one of his operas, in whole or in part, were arranged for recorder or flute, and there were over seventy keyboard transcriptions of the overtures to these operas. While the transcriptions of Handel overtures have been thoroughly examined, opera aria transcriptions have never received an appropriate level of study and analysis. The Ladys Banquet or The Lady’s Entertainment provides an excellent starting point. Not only does it include numerous opera aria arrangements, but its volumes were re-issued several times, suggesting a wide circulation. Its study raises a number of issues, including publication and authorship of Handel transcriptions, gendered music of the eighteenth century and analysis of opera transcriptions. The Ladys Banquet or The Lady’s Entertainment is a collection of six volumes of keyboard music published by John Walsh in the first half of the eighteenth century. The first two books were issued in 1704 and 1706 respectively, and included many undemanding pieces by fashionable composers such as Jeremiah Clarke (c.1674-1707) and Henry Purcell (1659-1695). The Third and Fourth Books followed in circa 1715 and 1716 and contain predominantly dance tunes and popular songs revised for the keyboard. When, in the early 1730s, the Fifth and Sixth Books appeared, the original four volumes were revised, and included wholly different material than the first editions. The publications of John Walsh are notoriously confusing owing to their lack of publication dates, repeated use of title pages, and misleading advertisements. The Ladys Banquet, as a whole, is especially bewildering because of the reissues of the collection and the changing repertoire. My research focuses on the Fifth Book of The Ladys Banquet, first printed around 1734, due to its abundance of opera aria transcriptions and consistency of content within editions. This document compiles relevant background information and offers a lucid guide to The Ladys Banquet. It provides historical context, examination and discussion of the contents of each volume, with specific details about the music in the Fifth Book, as well as analysis of the Handel aria transcriptions.

Understanding the mechanisms that regulate gene expression is a major challenge in biology. One of the most important tasks in this challenge is to identify the transcription factors binding sites (TFBS) in DNA sequences. The common representation of these binding sites is called “motif” and the discovery of TFBS problem is also referred as motif finding problem in computer science. Despite extensive efforts in the past decade, none of the existing algorithms perform very well. This dissertation focuses on this difficult problem and proposes three new methods (MotifEnumerator, PosMotif, and Enrich) with excellent improvements. An improved pattern-driven algorithm, MotifEnumerator, is first proposed to detect the optimal motif with reduced time complexity compared to the traditional exact pattern-driven approaches. This strategy is further extended to allow arbitrary don’t care positions within a motif without much decrease in solvable values of motif length. The performance of this algorithm is comparable to the best existing motif finding algorithms on a large benchmark set of samples. Another algorithm with further post processing, PosMotif, is proposed to use a string representation that allows arbitrary ignored positions within the non-conserved portion of single motifs, and use Markov chains to model the background distributions of motifs of certain length while skipping these positions within each Markov chain. Two post processing steps considering redundancy information are applied in this algorithm. PosMotif demonstrates an improved performance compared to the best five existing motif finding algorithms on several large benchmark sets of samples. The third method, Enrich, is proposed to improve the performance of general motif finding algorithms by adding more sequences to the samples in the existing benchmark datasets. Five famous motif finding algorithms have been chosen to run on the original datasets and the enriched datasets, and the performance comparisons show a general great improvement on the enriched datasets.

Vascular endothelial growth factor receptor-2 (VEGFR2) is a key angiogenic factor, and angiogenesis is an important physiological process associated with neovascularization, growth, and metastasis of many different tumors. The mechanism of VEGFR2 gene expression was investigated in MiaPaCa-2, Panc-1, and AsPC-1 pancreatic cancer cells transfected with a series of VEGFR2 promoter deletion/mutated constructs, and the results indicated that the GC-rich –60 to –37 region of the promoter was essential for VEGFR2 expression in these cell lines. EMSA and ChIP assays showed that Sp proteins are expressed and bind to the proximal GC-rich region of the VEGFR2 promoter. RNA interference studies on Sp proteins demonstrated that Sp1, Sp3, and Sp4 all contributed to VEGFR2 gene/protein expression in pancreatic cancer cells. VEGFR2 gene expression was also investigated in ZR-75 and MCF-7 breast cancer cells. ZR-75 cells treated with 10 nM 17b-estradiol (E2) increased VEGFR2 mRNA levels/protein expression. The VEGFR2 promoter was induced by E2 in ZR-75 cells, and analysis of the VEGFR2 promoter identified the GC rich -60 to -37 region that was required for E2-mediated transactivation. EMSA and ChIP assays confirmed that Sp1, Sp3, and Sp4 proteins are expressed in ZR-75 cells and bind the proximal GC-rich region of the VEGFR2 promoter. RNA interference was used to determine the relative contributions of Sp proteins on hormonal regulation of VEGFR2 through ER/Sp complexes, and interestingly, in ZR-75 cells, hormone-induced activation of VEGFR2 involves ERa/Sp3 and ERa/Sp4 but not ERa/Sp1. In MCF-7 cells treated with 10 nM E2, VEGFR2 mRNA levels were decreased. Analysis of the VEGFR2 promoter revealed that the same GC-rich region important for E2-mediated upregulation in ZR-75 cells was responsible for E2-dependent downregulation of VEGFR2 gene expression in MCF-7 cells. EMSA and ChIP assays confirmed that Sp1, Sp3, and Sp4 proteins are expressed in MCF-7 cells and bind to the proximal GC-rich region of the VEGFR2 promoter. RNA interference studies showed that Sp1, Sp3, and Sp4 are involved in the E2-mediated downregulation of VEGFR2 in MCF-7 cells, and ERa/Sp protein-promoter interactions are accompanied by recruitment of the corepressor SMRT using the ChIP assay.

Thèse de doctorat : Sciences du vivant : Strasbourg 1 : 2007. / Titre provenant de l'écran-titre. Bibliogr. f. 144-155.

Thèse de doctorat : Bioinformatique : Evry-Val d'Essonne : 2008. / Titre provenant de l'écran-titre.

Thèse de doctorat : Informatique : Evry-Val d'Essonne : 2009. / Titre provenant de l'écran-titre.