811 research outputs found

    Investigating the cellular localisation of DEAD box helicase DDX3

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    DDX3 is an RNA helicase that has been shown to have a range of nuclear and cytoplasmic functions, including transcription, translation, splicing and mRNA export. DDX3 has also been shown to play a role in innate immune signalling and tumorigenesis. DDX3 is targeted by multiple viruses: HIV, HCV , HBV and Vaccinia Virus have all been shown to interact with DDX3; either using DDX3 to replicate or inhibiting DDX3’s function in antiviral signalling. Human DDX3 was reported to be exported from the nucleus independently of its N-terminal NES and to interact with CRM-1 through DDX3's C-terminal region. It was suggested that DDX3 acts as a CRM-1 cofactor, rather than cargo, and that it mediates Rev-dependent export of HIV RNAs. I confirmed that DDX3 exports in a CRM-1 dependent manner, and DDX3's N-terminus is required for nuclear export. We have also investigated the nuclear import of DDX3. Putative NLS were found using bioinformatic software, and tested for functionality by mutating key basic residues. We also used a range of nuclear import inhibitors to examine how DDX3 is imported into the nucleus. We found that the DDX3's two Rec-A like domains could be imported independently, and also that DDX3 imported independently of Importin- and Calmodulin. We were also interested as to whether DDX3's cellular localisation was regulated during viral infection, cellular stress and during the cell cycle. We investigated the relationship of DDX3 with HIV and HCV viral proteins, and found that both HIV and HCV target the cellular localisation of DDX3. We found that DDX3 directly interacts with HIV-Rev protein, and also that HIV-Rev recruits DDX3 to the nucleolus. We also found that HCV Core protein recruited DDX3 to cytoplasmic speckles, and prevented the nucleocytoplasmic shuttling of DDX3. We found DDX3 to be recruited to Stress Granules after cellular stress. DDX3's cellular localisation and expression levels were also found to change throughout the cell cycle

    Genetic factors modulating mitochondrial DNA copy number

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    PhD ThesisMitochondria are dynamic organelles whose principal role is the generation of cellular energy (ATP) through oxidative phosphorylation (OXPHOS). 13 OXPHOS subunits are encoded by the mitochondrion’s polyploid circular genome (mtDNA), and the nuclear genome (nDNA) encodes the remaining subunits as well as proteins required for mtDNA maintenance. In addition to mitochondrial number, mtDNA copy number (mtDNA CN) varies between cell and tissue type, depending on metabolic demand and baseline mtDNA quality, and ranges from hundreds to thousands of copies per cell. mtDNA CN is often linked to mitochondrial dysfunction and the ubiquity of mitochondria results in a broad spectrum of dysfunction and clinical phenotypes; ranging from primary mitochondrial disorders to complex diseases such as cancer, type 2 diabetes, and Parkinson’s disease. Given the variability in mtDNA between individuals, it is possible that mtDNA CN is influenced by secondary factors. I hypothesise that nDNA diversity is a major component of mtDNA variability between individuals and will test this hypothesis by conducting a genome wide association study (GWAS) in a large, European, asymptomatic cohort (>8000 individuals), comparing nDNA genotype to mtDNA copy-number as a QTL. Peripheral blood mtDNA CN was correlated to array-based and imputed nDNA genotype in a two-stage QTL analysis, utilising three independent replicative cohorts: UKBS, Newcastle, and ALPAC. In addition the effect of potential confounding biological variables such as age, gender, blood count, and potential methodological confounders such as assay variation, technical and biological replicate numbers, and differences in genotype platform were all assessed and used to improve the GWAS analysis. Individual cohort analysis identified nuclear gene UNC13C (Unc-13 Homolog C), two intergenic, and one intronic SNP, which is in close proximity to PSMD3 (Proteasome 26S Subunit, Non-ATPase 3), to be genome wide significant (GWS) (p< 1.00E-07) in individual cohort analysis. However these hits could not be replicated in meta-analysis. mtDNA variant analysis in all three cohorts revealed that mtDNA SNPs G5460A and G5046A, which identify as mitochondrial haplogroup W, were significantly associated to a significant reduction in mtDNA CN. Furthermore, our work identified gender-specific genetic differences, which was supported by a Preliminary iv significant decrease in mtDNA CN in males with age, but not females, and significant changes in mtDNA CN relative to blood cell type and proportions highlighted the importance of regulating for cellular heterogeneity. Additionally, no difference in mtDNA CN was observed between pre- and post-menopausal women. This work indicates that there are likely genetic variants present at the population level modulating mtDNA CN, but that this process is complex and multifaceted.Wellcome Trust and Barbour Foundatio

    An EXAFS study of rare-earth phosphate glasses in the vicinity of the metaphosphate composition

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    A study of rare-earth phosphate glasses, in the vicinity of the metaphosphate composition, has been undertaken at different temperatures using the extended X-ray absorption fine structure (EXAFS) facility at the Synchrotron Radiation Source, Daresbury Laboratory, UK. The metaphosphate-like glasses examined contained the rare-earth elements La, Sm. Eu and Gd as R3+ ions. The experiments were carried out at room temperature, 145 and 79 K. The data show that the first shell surrounding rare-earth ions contains only oxygen atoms at a mean distance of similar to 2.2-2.4 Angstrom. The 'Iantfianide contraction' is clearly observed, i.e., the R-O distance decreases with increasing atomic number. The observed R(-O) coordination numbers are in the range 5-7. A second correlation shell was found associated with phosphorus atoms around the central rare-earth atom (similar to 3.5 Angstrom), and another rare-earth:oxygen correlation was also identified (similar to 4 Angstrom). Static disorder dominates the Debye-Waller term, but thermal disorder is not negligible. Within the accuracy of the EXAFS data, no significant structural variations were observed over the temperature range studied. We conclude that the anomalous bulk properties are associated with either subtle structural features or primarily due to rare-earth ion Interactions

    Reviews

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    The Making of a Mystic: New and Selected Letters of Evelyn Underhill. Ed. Carol Poston. Reviewed by Joe R. Christopher. From Girl to Goddess: The Heroine\u27s Journey through Myth and Legend. Valerie Estelle Frankel. Reviewed by Janet Brennan Croft. The Wizard of Oz and Philosophy: Wicked Wisdom of the West (Popular Culture and Philosophy, Vol. 37). Ed. Randall E. Auxier and Phillip S. Seng. Reviewed by Ruth Berman. C.S. Lewis\u27s Lost Aeneid: Arms and the Exile. Edited with an Introduction by A.T. Reyes. Reviewed by Richard C. West. The Ring and the Cross: Christianity and The Lord of the Rings. Edited by Paul E. Kerry. Reviewed by Harley J. Sims. Fastitocalon: Studies in Fantasticism Ancient to Modern. 1.2 (2010). Ed. Thomas Honegger and Fanfan Chen. Reviewed by Janet Brennan Croft. Journal of Inklings Studies. 1.1 (March 2011). Ed. Judith Wolfe. Reviewed by Janet Brennan Croft. VII: An Anglo-American Literary Review. 27 (2010). Ed. Marjorie Lamp Mead. Reviewed by Janet Brennan Croft. Tolkien Studies: An Annual Scholarly Review. 7 (2010). Ed. Douglas A. Anderson, Michael D.C. Drout, and Verlyn Flieger. Reviewed by Janet Brennan Croft. Tolkien and the Study of His Sources: Critical Essays. Edited by Jason Fisher. Reviewed by Mike Foster
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