Genome-wide and subcellular nonsense mediated decay

Nonsense mediated decay (NMD) is a translation-dependent RNA quality control pathway that selectively degrades aberrant transcripts before they lead to truncated proteins. NMD causes rapid degradation of target RNAs by assembling multiple factors to drive poly-deadenylation and decapping of the mRNA. NBAS (neuroblastoma amplied sequence) protein was identied to be required for NMD in an RNAi screen in Caenorhabditis elegans. Subsequent evidence has suggested it is a conserved NMD factor in zebrash and human cells. NBAS is localised to the endoplasmic reticulum (ER) suggesting it could be involved in a novel branch of the NMD pathway specic to ER. The key NMD factor UPF1 has been shown to partially localise to the ER and interact with NBAS. NMD targets harbour premature termination codons (PTC)s, upstream open reading frames or long 3' UTRs. However not all transcripts with these features activate NMD, and many NMD targets appear not to contain any of these features. Therefore, the previously dened rules regarding initiation of the NMD pathway do not account for all variation seen in NMD eciency. This thesis presents the creation and investigation of a CRISPR cell line containing an endogenously tagged NBAS protein. Immunoprecipitation mass spectrometry on these cells revealed that NBAS interacts with the ER translocon, where the ribosome docks for translation at the ER membrane, suggesting the presence of an NBAS-UPF1-translocon complex. This interaction was conrmed by proximity ligation assay, a microscopy-based approach. In order to identify the eect of NBAS on transcript expression and stability, RNA-sequencing was performed on total, nascent, cytoplasmic, and membraneassociated fractions, upon siRNA depletion of either NBAS or UPF1 mRNA. Differential expression analysis revealed a correlation in dierentially expressed transcripts when NBAS or UPF1 were depleted with an enrichment in ER-associated RNAs specically in NBAS targets. UPF1 was found to aect expression of a similar number of transcripts in the cytoplasmic and membrane-associated fractions conrming its role in NMD at the ER. Whereas depletion of NBAS led to a 5-fold higher proportion of membrane-associated genes being increased in expression than of non-membrane genes, showing its subcellular specicity. Using both nascent and total RNA-sequencing datasets, estimates of dierential transcript stability upon depletion of either factor were made. Approximately 20% of transcripts increased in expression upon depletion of UPF1 were also found to increase in stability, whereas the remaining 80% of expression changes could be explained by changes in transcription, suggesting these were due to secondary eects. Of those transcripts that changed in expression when NBAS was depleted, none were identied as changed in stability in this study, suggesting NBAS has subtle or negligible eect on transcript stability and is therefore likely to be a peripheral factor in the NMD pathway. Finally, whole genome sequencing was used in conjunction with total and nascent expression datasets to identify correlation between genome variants and NMD eciency. I show that it is possible to identify heterozygous variants with allelespeci c processing and measure the eect of NMD to identify direct NMD activity. This approach can be used in the future to systematically explore the rules of NMD targeting in human cells. In sum, this thesis reveals that NBAS interacts with the ER translocon and aects the expression of hundreds of ER-associated transcripts. However, NBAS does not appear to regulate transcript stability suggesting its role at the translocon is distinct from NMD. My ndings add to the understanding of NMD quality control at the ER and the genome-wide rules governing NMD eciency in human cells

Population dynamics of rhesus macaques and associated foamy virus in Bangladesh.

Foamy viruses are complex retroviruses that have been shown to be transmitted from nonhuman primates to humans. In Bangladesh, infection with simian foamy virus (SFV) is ubiquitous among rhesus macaques, which come into contact with humans in diverse locations and contexts throughout the country. We analyzed microsatellite DNA from 126 macaques at six sites in Bangladesh in order to characterize geographic patterns of macaque population structure. We also included in this study 38 macaques owned by nomadic people who train them to perform for audiences. PCR was used to analyze a portion of the proviral gag gene from all SFV-positive macaques, and multiple clones were sequenced. Phylogenetic analysis was used to infer long-term patterns of viral transmission. Analyses of SFV gag gene sequences indicated that macaque populations from different areas harbor genetically distinct strains of SFV, suggesting that geographic features such as forest cover play a role in determining the dispersal of macaques and SFV. We also found evidence suggesting that humans traveling the region with performing macaques likely play a role in the translocation of macaques and SFV. Our studies found that individual animals can harbor more than one strain of SFV and that presence of more than one SFV strain is more common among older animals. Some macaques are infected with SFV that appears to be recombinant. These findings paint a more detailed picture of how geographic and sociocultural factors influence the spectrum of simian-borne retroviruses

Infrared Dark Clouds in the Small Magellanic Cloud?

We have applied the unsharp-masking technique to the 24 $\mu$m image of the Small Magellanic Cloud (SMC), obtained with the Spitzer Space Telescope, to search for high-extinction regions. This technique has been used to locate very dense and cold interstellar clouds in the Galaxy, particularly infrared dark clouds (IRDCs). Fifty five candidate regions of high-extinction, namely high-contrast regions (HCRs), have been identified from the generated decremental contrast image of the SMC. Most HCRs are located in the southern bar region and mainly distributed in the outskirts of CO clouds, but most likely contain a significant amount of H2. HCRs have a peak-contrast at 24 $\mu$m of 2 - 2.5 % and a size of 8 - 14 pc. This corresponds to the size of typical and large Galactic IRDCs, but Galactic IRDCs are 2 - 3 times darker at 24 $\mu$m than our HCRs. To constrain the physical properties of the HCRs, we have performed NH3, N2H+, HNC, HCO+, and HCN observations toward one of the HCRs, HCR LIRS36-EAST, using the Australia Telescope Compact Array and the Mopra single-dish radio telescope. We did not detect any molecular line emission, however, our upper limits to the column densities of molecular species suggest that HCRs are most likely moderately dense with n ~ 10^{3} cm-3. This volume density is in agreement with predictions for the cool atomic phase in low metallicity environments. We suggest that HCRs may be tracing clouds at the transition from atomic to molecule-dominated medium, and could be a powerful way to study early stages of gas condensation in low metallicity galaxies. Alternatively, if made up of dense molecular clumps < 0.5 pc in size, HCRs could be counterparts of Galactic IRDCs, and/or regions with highly unusual abundance of very small dust grains.Comment: accepted for publication in the Astronomical Journa

Acute Responses in Agonists of uEGF to Moderate-Intensity and High-Intensity Interval Exercise in Mid-Spectrum CKD

Urine epidermal growth factor (uEGF) is a novel biomarker utilized in assessing renal health in various renal diseases, specifically chronic kidney disease (CKD). uEGF promotes multiple intracellular pathways, stimulating renal cell growth, survival, and replication. uEGF production is activated by multiple agonists that bind to the uEGF receptor. Aerobic exercise initiates the upregulation of several of these agonists to increase the production of uEGF. Depending on the mode and intensity of aerobic exercise, uEGF agonists may activate differently in CKD populations. PURPOSE: To determine the influence of an acute bout of steady-state exercise (SSE) and high-intensity interval exercise (HIIE) on concentrations of uEGF agonists (serum insulin-like growth factor 1 (IGF-1), angiotensin II receptor type 1 (AGTR-1), and transforming growth factor beta 1 (TGF-β1)) in mid-spectrum CKD. METHODS: Twenty participants (n = 6 men; n = 14 women; age 62.0 + 9.9 yr; weight 80.9 + 16.2 kg; body fat 37.3 + 8.5% of weight; VO2max 19.4 + 4.7 ml/kg/min) completed 30 min of SSE at 65% VO2reserve or HIIE by treadmill walking (90% and 20% of VO2reserve in 3:2 min ratio) in a randomized crossover design. Both exercise conditions averaged ~ 65% VO2reserve. Blood and urine samples were obtained under standardized conditions just before, 1hr, and 24hrs after exercise. uEGF (ng/mL), serum IGF-1 (ng/mL), AGTR-1 (ng/mL), and TGF-β1 (pg/mL) responses were analyzed using 2 (condition) by 3 (sample point) repeated measures ANOVAs and Pearson Correlations. RESULTS: Serum IGF-1 and AGTR-1 increased 1hr and 24hr post-exercise in both exercise conditions; however, statistical significance was not achieved (p = 0.28 and p = 0.09). Similarly, serum TGF-β1 decreased at 24hrs in both exercise conditions but statistically remained unaltered (p = 0.42). IGF-1 was significantly correlated to uEGF in both conditions at all three-time points (p = 0.03), while AGTR-1 was significantly correlated to uEGF at 1hr in HIIE. uEGF findings were previously reported in ACSM abstract (DOI: 10.1249/01.mss.0000560710.72569.11). CONCLUSION: Agonists of uEGF remained unaltered following an acute bout of SSE and HIIE in mid-spectrum CKD. Further research is needed to understand better uEGF response activation to aerobic exercise in mid-spectrum CKD

Grey matter in shadow banking: international organizations and expert strategies in global financial governance

Who controls global policy debates on shadow banking regulation? We show how experts secured control over how issues in shadow banking regulation are treated by examining the policy recommendations of the Bank of International Settlements, the International Monetary Fund and the Financial Stability Board. The evidence suggests that IO experts embedded a bland reformism opposed to both strong and ‘light touch’ regulation at the core of the emerging regulatory regime. Technocrats reinforced each other's expertise, excluded some potential competitors (legal scholars), co-opted others (select Fed and elite academic economists), and deployed measurement, mandate, and status strategies to assert issue control. In the field of shadow banking regulation, academic economists’ influence came from their credibility as arbitrageurs between several professional fields rather than their intellectual output. The findings have important implications for how we study the relationship between IO technocrats and experts from other professional field

Effects of antiplatelet therapy on stroke risk by brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases: subgroup analyses of the RESTART randomised, open-label trial

Background Findings from the RESTART trial suggest that starting antiplatelet therapy might reduce the risk of recurrent symptomatic intracerebral haemorrhage compared with avoiding antiplatelet therapy. Brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases (such as cerebral microbleeds) are associated with greater risks of recurrent intracerebral haemorrhage. We did subgroup analyses of the RESTART trial to explore whether these brain imaging features modify the effects of antiplatelet therapy

Investigation into the effects of surface stripping ZnO nanosheets

ZnO nanosheets are polycrystalline nanostructures that are used in devices including solar cells and gas sensors. However, for efficient and reproducible device operation and contact behaviour the conductivity characteristics must be controlled and surface contaminants removed. Here we use low doses of argon bombardment to remove surface contamination and make reproducible lower resistance contacts. Higher doses strip the surface of the nanosheets altering the contact type from near-ohmic to rectifying by removing the donor-type defects, which photoluminescence shows to be concentrated in the near-surface. Controlled doses of argon treatments allow nanosheets to be customised for device formation

Evaluating the Effects of SARS-CoV-2 Spike Mutation D614G on Transmissibility and Pathogenicity.

Global dispersal and increasing frequency of the SARS-CoV-2 spike protein variant D614G are suggestive of a selective advantage but may also be due to a random founder effect. We investigate the hypothesis for positive selection of spike D614G in the United Kingdom using more than 25,000 whole genome SARS-CoV-2 sequences. Despite the availability of a large dataset, well represented by both spike 614 variants, not all approaches showed a conclusive signal of positive selection. Population genetic analysis indicates that 614G increases in frequency relative to 614D in a manner consistent with a selective advantage. We do not find any indication that patients infected with the spike 614G variant have higher COVID-19 mortality or clinical severity, but 614G is associated with higher viral load and younger age of patients. Significant differences in growth and size of 614G phylogenetic clusters indicate a need for continued study of this variant