An analysis of the role of glutathione and p53 in the response to oxidative injury

The response to oxidative stress, a process that can lead to genotoxic injury, is thought to involve the abundant cellular antioxidant, glutathione, and the stress response transcription factor, p53. Glutathione (GSH) biosynthesis occurs through a two -step pathway, the first reaction of which is rate limiting and is catalysed by the enzyme gamma glutamylcysteine synthetase (yGCS). yGCS is a heterodimer, composed of a heavy (yGCSh) and a light ( yGCS1) subunit. The heavy subunit contains the active site, whereas the light performs a regulatory function on the heavy by means of a redox -sensitive inter- subunit disulphide bridge.The hypothesis that GSH mediates protection against oxidative stress was investigated by gene targeting of yGCSh in murine embryonic stem (ES) cells. Mouse yGCSh cDNA sequence was isolated by RT -PCR, cloned, characterised and used to screen a mouse genomic ? library. Characterisation of the resultant clone confirmed that it contained yGCSh gene sequences. This information was used to design and construct a replacement targeting vector which was subsequently electroporated into ES cells to delete a segment of the endogenous locus. A total of 285 clones were isolated and analysed for a correct gene targeting event. Unfortunately, no positive clones were identified.The role of GSH and p53 in the response of ES cells to oxidative stress was also examined via a series of in vitro assay strategies measuring cellular viability, apoptosis and intracellular GSH levels. ES cells were shown to express yGCSh. Agents known to induce oxidative stress or lower GSH levels in other cell lines were then tested for toxicity and their potential to modulate GSH levels in ES cells. On the basis of these experiments, the quinone menadione (MQ) and the yGCS inhibitor, buthionine sulphoximine (BSO), were investigated further. Treatment with MQ was associated with a transient elevation of GSH, a strong apoptotic response and reduced clonogenic survival. Addition of BSO depleted GSH levels and prevented the MQ- induced increase in GSH, sensitising cells to oxidative insult. In order to address the role of p53 in the response to oxidative stress, karyotypically normal p53 -/- ES cells were compared to wild -type cells. This showed that both maintenance of basal GSH levels and MQ- induction of GSH were independent of p53 status. However, a role for p53 in this response was demonstrated as the kinetics of MQ- induced apoptosis were delayed in the absence of p53. Taken together, these findings suggest that the pathways involving p53 and GSH act independently to protect against the deleterious effects of oxidative damage.Consistent with studies using a wide spectrum of other DNA damage inducing agents, loss of p53 conferred an immediate survival advantage post oxidative stress. However, the long -term clonogenic survival of p53 -/- ES cells was found to be lower than cells with an intact p53 pathway. This suggests that compensatory mechanisms exist to ensure that, in the absence of functional p53, cells bearing genetic lesions are less likely to be propagated, and furthermore that the ability to engage apoptosis does not necessarily predict long term clonogenic survival.In summary, an attempt was made to address the in vivo significance of GSH by creating a ,GCSh null strain of mice. To this end a targeting vector was generated and used in ES cells, but unfortunately this failed to produce a mutant ),GCSh allele. This thesis has also explored the relationship between oxidative damage and the cellular responses of GSH and p53 in vitro. Evidence is presented to demonstrate that, within embryonic tissues, multiple pathways operate in response to oxidative stress, and that in the absence of p53 cells are prevented from propagating

Satire and sympathy : some consequences of intrusive narration in Tom Jones and other comic novels

This thesis aims to reinterpret Tom Jones by putting it into some previously untried comparative contexts. As well as using the traditional points of reference such as Lucian, Swift and Sterne, I compare Fielding's satire with Flaubert's; his narrative poetics with Dickens's and Beckett's; his strategy of intrusion with George Eliot's; and his literary politics with Brecht's. I start by assuming the ambivalence of Tom Jones, but rather than seeing this as a conscious ironic duality, I argue that it derives from literary, moral and political uncertainty. The intrusive narrator is seen as an index of vacillation between first- and third-person narration, while conservative satiric influences are shown to complicate rather than strengthen the book's moral decisiveness. Its form, moreover, is shown to be dialogic, and unable to keep at bay either the reader's subjectivity or the flux of historical reality. But Fielding's achievement, I finally suggest, is to have put these factors into the service of his awareness of the always judgmental nature of literature. The thesis therefore takes on several previously uncovered areas: it is very specific about the nature and extent of the narrator's presence in Tom Jones; it draws new analogies between social and literary forms (in the sections on conversation) and political and literary structures (in the section on Fielding's plays). It thereby reveals new areas of Fielding's writings which can be treated as literary theory; finds detailed affinities between Fielding and writers not normally associated with him; and eventually constitutes a reading of Tom Jones as an inconclusive and open-ended text which implies not a denial but a redefinition of its historical importance

Understanding the Role of Science-Specific Literacy Strategies in Supporting Science Teaching and Student Learning: A Case Study of Preservice Elementary Teachers in a Science Methods Course That Integrated a Disciplinary Literacy Framework

The shift to student engagement in scientific and engineering practices to learn science provides opportunities for science learning and language learning to occur in tandem. These opportunities also pose new challenges for elementary pre-service teachers (PSTs) since literacy methods courses have been presented separately from science methods courses. We integrated a disciplinary literacy framework in a science methods course to help elementary PSTs understand the synergistic connections between literacy and science teaching. The purpose of this study was to examine elementary PSTs’ understanding of the use of science-specific literacy strategies to support science teaching and learning through three points of observation. Findings from three data sources indicated that PSTs showed a developing understanding of the role of disciplinary literacy in supporting student engagement in science practices and learning disciplinary core ideas. Implications for future uses of a disciplinary literacy framework for teaching and learning science and elementary PSTs’ science preparation are presented

A Sampling Strategy for High-Dimensional Spaces Applied to Free-Form Gravitational Lensing

We present a novel proposal strategy for the Metropolis-Hastings algorithm designed to efficiently sample general convex polytopes in 100 or more dimensions. This improves upon previous sampling strategies used for free-form reconstruction of gravitational lenses, but is general enough to be applied to other fields. We have written a parallel implementation within the lens modeling framework GLASS. Testing shows that we are able to produce uniform uncorrelated random samples which are necessary for exploring the degeneracies inherent in lens reconstruction.Comment: 10 pages, 9 figures. Accepted for publication in MNRA

Airborne Prokaryotic, Fungal and Eukaryotic Communities of an Urban Environment in the UK

Bioaerosols often contain human pathogens and allergens affecting public health. However, relatively little attention has been given to bioaerosols compared with non-biological aerosols. In this study, we aimed to identify bioaerosol compositions in Manchester, UK by applying high throughput sequencing methods and to find potential sources. Samples were collected at Manchester Air Quality Super Site at the Firs Environmental Research Station in November 2019 and in February 2020. Total DNA has been extracted and sequenced targeting the 16S rRNA gene of prokaryotes, ITS region of fungal DNA and 18S rRNA gene of eukaryotes. We found marine environment-associated bacteria and archaea were relatively more abundant in the February 2020 samples compared with the November 2019 samples, consistent with the North West marine origin based on wind back-trajectory analysis. In contrast, an OTU belonging to Methylobacterium, which includes many species resistant to heavy metals, was relatively more abundant in November 2019 when there were higher metal concentrations. Fungal taxa that fruit all year were relatively more abundant in the February 2020 samples while autumn fruiting species generally had higher relative abundance in the November 2019 samples. There were higher relative abundances of land plants and algae in the February 2020 samples based on 18S rRNA gene sequencing. One of the OTUs belonging to the coniferous yew genus Taxus was more abundant in the February 2020 samples agreeing with the usual pollen season of yews in the UK which is from mid-January until late April. The result from this study suggests a potential application of bioaerosol profiling for tracing the source of atmospheric particles

Diet quality in late midlife is associated with faster walking speed in later life in women, but not men

Healthy diet has been linked to better age-related physical functioning, but evidence on the relationship of overall diet quality in late midlife and clinically relevant measures of physical functioning in later life is limited. Research on potential sex differences in this relationship is scarce. The aim was to investigate the prospective association between overall diet quality, as assessed by the Healthy Eating Index-2015 at age 60-64y and measures of walking speed seven years later, among men and women from the Insight46, a neuroscience sub-study of the Medical Research Council National Survey of Health and Development. Diet was assessed at age 60-64y using five-day food diaries, from which total HEI-2015 was calculated. At age 69-71y, walking speed was estimated during four 10-meter walks at self-selected pace, using inertial measurement units. Multivariable linear regression models with sex as modifier, controlling for age, follow-up, lifestyle, health, social variables and physical performance were used. The final sample was 164 women and 167 men (n=331). Women had higher HEI-2015 scores and slower walking speed than men. A 10 point increase in HEI-2015 was associated with faster walking speed seven years later among women (B: 0.024, 95% CI: 0.006, 0.043), but not men. The association remained significant in the multivariable model (B: 0.021, 95% CI: 0.003, 0.040). In women in late midlife higher diet quality is associated with faster walking speed. A healthy diet in late midlife is likely to contribute towards better age-related physical capability and sex differences are likely to affect this relationship