L’Splicing de les molècules d’ARN és el procés pel qual les seqüències interposades (“introns”) s’eliminen, i les seqüències restants es concatenen per a formar l’ARN madur. La investigació recent mostra que gairebé tots els gens amb splicing es veuen afectats per splicing alternatiu. Aquí, en primer lloc definim la longitud mínima d’un oligomer d’ARN per a funcionar com a lloc d’unió d’un factor d’splicing. A continuació, explorem la capacitat d’aquests oligomers per a predir estructures completes exó-intró. Destaquem els oligomers que són més informatius per a això, i demostrem que la mateixa precisió com en enfocaments anteriors es pot aconseguir amb menys oligomers. L’observació de que aquest enfocament és lluny de predir amb exactitud tota l’estructura exó-intró ens va portar a investigar els factors que juguen un paper en l’splicing co-transcripcional. Demostrem que els nucleosomes es col.loquen preferentment en els exons i plantegem la hipòtesi que juguen un paper en les decisions de l’splicing. A continuació, introduïm el “completed splicing index” i concluem que l’splicing co-transcripcional és molt generalitzat. A més, l’splicing co-transcripcional mostra vincles amb l’organització de la cromatina. A la llum d’aquests resultats, es van supervisar els canvis de la cromatina en exons diferencialment inclosos en dos teixits. Hem descobert una varietat de marques de les histones, però no totes, mostrant un comportament significativament diferent en els exons més inclosos i més exclosos. Las marques més destacades que apareixen són H3K9ac i dos estats de metilació de lisina 4. / Splicing of RNA molecules is the process, by which intervening sequences (“introns”) in the primary transcript are excised, and the remaining sequences (“exons”) are concatenated to form the mature RNA. Recent evidence shows that almost all spliced genes are affected by alternative splicing. Here, we define the minimal length of RNA oligomers that can sensibly be called splicing factor binding sites. Then, we explore the capacity of these oligomers to predict complete exon-intron structures. We highlight those oligomers that are most informative for this and show, that equal accuracy as in previous approaches can be achieved with less RNA oligomers. The observation, that this approach falls short of accurately predicting the entire exon-intron structure, led us to investigate determinants linked to co-transcriptional splicing. We show that nucleosomes are preferentially positioned on exons and hypothesize that they play a role in splicing decisions. We then introduce the “completed splicing index” and conclude that co-transcriptional splicing is very wide-spread in humans. Furthermore co-transcriptional splicing exhibits links to chromatin organization. In the light of these results, we go on to monitor chromatin changes on differentially included exons in pair-wise tissue comparisons. We find a variety of histone marks, but not all, showing significantly different behavior on up- and downregulated exons. The most prominently appearing marks are H3K9ac and two lysine 4 methylation states.

Lymphomas are neoplasms of the human immune system and can be divided into two categories, Hodgkin’s lymphoma (HL) and non-Hodgkin lymphoma (NHL). Anaplastic large cell lymphoma (ALCL) is a form of NHL that shares a common distinctive feature with HL, the overexpression CD30. The expression of cytokine receptor CD30 is restricted to proliferating B and T lymphocytes in healthy individuals while its overexpression is associated with several lymphoproliferative diseases such as ALCL and HL. The activation of CD30 via ligand or antibodies triggers various cellular responses ranging from apoptosis to cell proliferation and it is thought that the variable cellular response to CD30 activation may be due to cell surface levels of CD30. The human CD30 gene is regulated at the transcriptional level and previous studies characterising its promoter have identified several factors that regulate the expression this gene. However none of these identified factors explain for the high levels of CD30 observed in HL and ALCL. Therefore this study focused on the identification and functional analysis of transcriptionally active regions located up or downstream of the CD30 promoter region. The first aim for this study was to identify and characterise regions within the human CD30 gene that are involved in its transcriptional regulation. Phylogenetic footprinting identified several regions downstream of the CD30 promoter that displayed high levels of sequence homology indicating potential functional significance. Validation of these regions through two in vivo approaches, DNase 1 hypersensitivity assay and chromatin accessibility studies localised potential transcriptionally active regions to intron 1 of the CD30 gene.

In this study, we have explored the combination of a novel Purine Nucleoside Phosphorylase mediated Gene-Directed Enzyme Prodrug Therapy (PNP-GDEPT) with chemotherapeutics, Taxotere and/or Carboplatin to target prostate and ovarian cancer (PC & OC). PNP converts the prodrug (Fludarabine-phosphate) to a toxic purine, 2-fluoroadenine (2FA) that inhibits RNA/DNA synthesis. Taxotere is active against late stage PC whilst carboplatin is first line therapy for OC. Neither modality is adequately effective. We expect that a combination will target heterogeneity via cytotoxicity to diverse cancer cell populations leading to effective synergies, which may improve efficacy and quality of life. For PC, Synergy between Ad-PNP-GDEPT and Taxotere were assessed in vitro and in vivo. Cell killing effects of combination led to significant synergistic killing of human PC-3 & murine RM1 PC cells accompanied by enhanced apoptosis. A lower individual dose (by up to 8 fold) led to enhanced efficacy. In vivo, the combination regimen given at the suboptimal doses led to reduction in local tumour (PC-3 & RM1) growth in nude and in C57BL/6 mice, respectively. A significant reduction in lung RM1 colony numbers indicated enhanced systemic efficacy. Combination treated mice also displayed significantly improved survival (25 days vs 15 days for control mice). Importantly, the condition of combination treated mice (e.g. weight loss) was better than those given individual treatments. The possible involvement of the immune system in this enhanced effect is under investigation. For OC, three-way synergy between Ad-PNP-GDEPT, Taxotere and carboplatin was effectively demonstrated in SKOV-3 and OVCAR-3 cells. This was significantly greater than bimodal or individual treatments. A 10-50 fold dose reduction of individual treatments was effective when combined, accompanied by enhanced apoptosis. Western-blotting analyses revealed a shift in the expression of anti-apoptotic and proapoptotic proteins upon treatment with various combinations. This is the first demonstration of synergy between these modalities.

Published information on the structure and regulation of H. pylori promoters is limited. The work presented in this thesis describes the cloning and characterisation of promoter regions from a clinical isolate of H. pylori, and the development of an alternative, non-radioactive method for verifying the location of transcriptional start sites of bacterial promoters. H. pylori 4187E promoters were randomly cloned into the promoter-trap vector pKK232-8 in Escherichia coli DH5α using two sets of restriction enzymes. Vector pKK232-8 contains a promoterless chloramphenicol acetyltransferase (CAT) gene. Seventy-four promoter-containing clones were isolated from selective media based on their resistance to chloramphenicol. The strength of each promoter was analysed qualitatively, using chloramphenicol minimum inhibitory concentrations, and quantitatively, using CAT assays following exposure of the clones to pH 4 and pH 7. Selected promoter fragments were subcloned into the GFP reporter vector pFPV25, containing a promoterless gfp gene. The subclones were exposed to buffered LB broth at pH 4, 5, 6, 7 and 8, for varying lengths of time, to study acid-induced regulation of gene expression. Subclones were examined qualitatively, using visual examination of GFP fluorescence and fluorescence microscopy, and quantitatively, using flow cytometry following acid shock. DNA sequences were determined for 61 of the 74 H. pylori promoters, and sequence alignments with the published H. pylori strains (26695 and J99) were performed. The transcriptional start site of 27 H. pylori promoter fragments was experimentally mapped using a fluorescence-based primer extension protocol developed by our group. Potential -35 and -10 sequences were identified for each promoter, and a new consensus sequence for H. pylori promoters was proposed based upon these results. This study has considerably expanded knowledge of H. pylori promoter sequences and transcriptional start sites based on those which also function in E. coli. It has also revealed several H. pylori promoters which appear to respond to acid stress

Thesis (Ph.D. in Biochemistry) -- University of Colorado at Denver and Health Sciences Center, 2006. / Typescript. Includes bibliographical references (leaves 135-145). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

Thesis (Ph.D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed on June 24, 2009). Includes bibliographical references.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2008. / Title from PDF title page (viewed on Sept. 17, 2009). Includes bibliographical references.

Plusieurs lymphomes à cellules B présentent des anomalies génétiques qui sont importantes pour déterminer leurs caractéristiques biologiques et peuvent être utiles pour le diagnostic. Les types les plus courants sont le lymphome folliculaire et le lymphome diffus à grandes cellules B (LDGCB), qui représentent à eux deux plus de 60 % de tous les lymphomes. Les LDGCB sont agressifs mais peuvent être traités par chimiothérapie à agents multiples. Cependant, les gènes suppresseurs de tumeur (GST) potentiellement responsables de la lymphomagenèse ne sont pas tous connus. Le rationnel de ce projet reposait sur des données non publiées du projet translationnel GHEDI (déchiffrer l'hétérogénéité génétique du lymphome diffus à grandes cellules B à l'ère du rituximab). Une hybridation génomique comparative (CGH) (puce Agilent 180 K) a été réalisée sur une série de 202 LDGCB de la série GHEDI et 40 % des délétions de la région 6q21 ont été identifiées, dont la région minimale commune délétée qui contient le gène HACE1. Par ailleurs, l'analyse transcriptomique a montré une corrélation significative entre le nombre de copies du gène et le niveau d'expression. Le gène HACE1, situé sur le chromosome 6q, code pour une ubiquitine ligase E3 et est régulé négativement chez l'homme dans les tumeurs, y compris les neuroblastomes et les lymphomes à cellules tueuses naturelles (NK). Il a été montré que le gène HACE1 ubiquityle Rac1, une protéine impliquée dans la prolifération cellulaire et la progression G2/M du cycle cellulaire. La fonction du gène HACE1 et les facteurs impliqués dans sa régulation transcriptionnelle sont en grande partie inconnus dans le contexte des lymphomes à cellules B. Dans cette étude, nous avons examiné si le gène HACE1 était un gène candidat dans la région génomique 6q impliqué dans la lymphomagenèse des LDGCB et plus largement dans les lymphomes B. Nous avons déterminé la fréquence de l'inactivation du gène HACE1 dans le lymphome à cellules B et analysé les mécanismes impliqués dans son extinction. / Several B-cell lymphomas have characteristic genetic abnormalities that are important in determining their biologic features and can be useful in differential diagnosis. Historically, classical Hodgkin lymphomas have been distinguished from non-Hodgkin lymphomas (NHL). The most common types are follicular lymphoma and diffuse large B-cell lymphoma (DLBCL), which together make up more than 60% of all lymphomas. DBCL are aggressive but potentially curable with multi-agent chemotherapy. However the putative tumor suppressor genes (TSG) responsible for lymphomagenesis still remain unknown. The rational of this project was based on unpublished data from the translational project GHEDI (Deciphering the Genetic Heterogeneity of Diffuse large B-cell lymphoma in the rituximab era). Array comparative genomic hybridization (aCGH) (Agilent 180 K) was performed in a series of 202 DLBCL and found 40% of deletions of 6q21 region, whose minimal commune deleted region (MCR) contains HACE1 gene. Furthermore, transcriptomic analysis showed a significant correlation between gene copy number and expression level. HACE1, located on chromosome 6q, encodes an E3 ubiquitin ligase and is downregulated in human tumors such as neuroblastomas and natural killer (NK) lymphomas. HACE1 has been shown to ubiquitylate Rac1, a protein involved in cell proliferation and G2/M cell cycle progression. The function of HACE1 and the factors involved in its transcriptional regulation are largely unknown in the context of B-cell lymphomas. In this study, we investigated whether HACE1 is a candidate gene in the 6q genomic region involved in DLBCL lymphomagenesis. We determined the frequency of HACE1 inactivation in B-cell lymphoma and analyzed the mechanisms involved in its silencing. We show, by RT-qPCR, that HACE1 gene is constitutively expressed in normal lymph nodes and in normal B-cells isolated from peripheral blood, contrasting with a strong downregulation of its expression in more than 70% (77/111) of B-cell lymphoma cases and in four tested B-Lymphoma cell lines. HACE1 gene copy number was assessed by quantitative multiplex PCR of short fluorescent fragments (QMPSF) and array for comparative genomic hybridization (aCGH) in 91 DLBCL cases.

Protein phosphorylation by members of the cyclin-dependent kinase (CDK) family determines the cell cycle and regulates gene transcription. CDK12 and CDK16 are relatively poorly characterised family members containing atypical domain extensions and represent novel targets for structural studies, as well as cancer drug discovery. In this thesis, I developed protocols to express and purify the human CDK12 kinase domain in complex with its obligate partner, CycK. I solved three distinct crystal structures of the complex providing insights into the structural mechanisms determining CycK assembly and kinase activation. These structures revealed a C-terminal kinase extension that folded flexibly across the active site of CDK12 to potentially gate the binding of the substrate ATP. My structures also identified Cys1039 in the C-terminal extension as the binding site for the first selective covalent inhibitor of CDK12, which has enormous potential as a pharmacological probe to investigate the functions of CDK12 in the DNA damage response and cancer. I also identified rebastinib and dabrafenib as potent, clinically-relevant inhibitors of CDK16 and solved a co-crystal structure that defined the extended type II binding mode of rebastinib. Preliminary trials using these relatively non-selective compounds to inhibit CDK16 in melanoma and medulloblastoma cancer cell lines revealed rebastinib as the more efficacious drug causing loss of cell proliferation in the 1-2 micromolar range. Use of the co-crystal structure to design more selective derivatives would be advantageous to further explore the specific role of CDK16. Finally, I identified a D-type viral cyclin from Kaposi's sarcoma-associated herpesvirus that could bind to the CDK16 kinase domain and interfere with its functional complex with human CycY causing loss of CDK16 activity. These studies provide novel insights into the structural and regulatory mechanisms of two underexplored CDK family subgroups and establish new opportunities for cancer drug development.

Busoni's contribution to the art of the piano transcription is formidable. His chorale prelude transcriptions make him responsible for giving over to the piano repertoire a small portion of sacred literature. His special admiration of J. S. Bach, evidenced throughout his life, make Busoni's transcriptional practices all the more significant. Bach himself was a prolific transcriber of his own works and the works of others. This paper presents a brief history of keyboard transcriptional practices, emphasizing Busoni's methods by comparing the original works for organ with the transcriptions for piano. Four chorale preludes form the basis for this study: Ich ruf' zu dir, Herr Jesu Christ (BWV 639), Komm, Gott, Schopfer, Heiliger Geist (BWV 667), Nun komm' der Heiden Heiland (BWV 659), and In dir ist Freude (BWV 615).