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

Measuring Gravitational Lensing Flexions in Abell 1689 Using an Analytic Image Model

Measuring dark matter substructure within galaxy cluster haloes is a fundamental probe of the Lambda-CDM model of structure formation. Gravitational lensing is a technique for measuring the total mass distribution which is independent of the nature of the gravitating matter, making it a vital tool for studying these dark-matter dominated objects. We present a new method for measuring weak gravitational lensing flexions, the gradients of the lensing shear field, to measure mass distributions on small angular scales. While previously published methods for measuring flexions focus on measuring derived properties of the lensed images, such as shapelet coefficients or surface brightness moments, our method instead fits a mass-sheet-transformation-invariant Analytic Image Model (AIM) to the each galaxy image. This simple parametric model traces the distortion of lensed image isophotes and constrains the flexion fields. We test the AIM method using simulated data images with realistic noise and a variety of unlensed image properties, and show that it successfully reproduces the input flexion fields. We also apply the AIM method for flexion measurement to Hubble Space Telescope observations of Abell 1689, and detect mass structure in the cluster using flexions measured with the AIM method.Comment: 44 pages, 4 figures, 3 tables. Accepted to ApJ. V2 (published version) has minor changes from V1; ApJ 736 (2011

RXTE-PCA observations of 1A 1118--61: timing and spectral studies during an outburst

We report detailed timing and spectral analysis of RXTE-PCA data obtained from observations during the outburst of a transient X-ray pulsar 1A 1118--61 in January 2009. The pulse profile showed significant evolution during the outburst and also significant energy dependence - a double peaked profile upto 10 keV and a single peak at higher energy. We have also detected quasi-periodic oscillations (QPO) at 0.07--0.09 Hz. The rms value of the QPO is 5.2% and it shows a significant energy dependence with highest rms of 7% at 9 keV. The QPO frequency changed from 0.09 Hz to 0.07 Hz within 10 days. The magnetic field strength calculated using the QPO frequency and the X-ray luminosity is in agreement with the magnetic field strength measured from the energy of the cyclotron absorption feature detected in this source. The 3-30 keV energy spectrum over the 2009 outburst of 1A 1118--61 can be well fitted with a partial covering power-law model with a high energy cutoff and an iron fluorescence line emission. The pulse phase resolved spectral analysis shows that the partial covering and high energy cutoff model parameters have significant changes with the pulse phase.Comment: Accepted for publication in MNRA

Five-Year Cost of Dementia: Medicare

About 5.5 million older adults are living with dementia, a chronic, progressive disease characterized by severe cognitive decline. This number will likely grow significantly as the U.S. population ages, which has cost implications for the Medicare program. A full accounting of these additional expenses will help policymakers plan for them in their Medicare budgets. In this study, Norma Coe and colleagues examined survival and Medicare expenditures in older adults with and without dementia to estimate dementia’s incremental costs to Medicare in the five years after diagnosis

Suzaku observation of the transient X-ray pulsar GRO J1008-57

We report the timing and broad-band spectral properties of the Be transient high mass X-ray binary pulsar GRO J1008-57 using a Suzaku observation in the declining phase of its 2007 November-December outburst. Pulsations with a period of 93.737 s were clearly detected in the light curves of the pulsar up to the 80-100 keV energy band. The pulse profile was found to be strongly energy dependent, a double peaked profile at soft X-ray energy bands (< 8 keV) and a single peaked smooth profile at hard X-rays. The broad-band energy spectrum of the pulsar, reported for the first instance in this paper, is well described with three different continuum models viz. (i) a high energy cut-off power-law, (ii) a Negative and Positive power-law with EXponential cut-off (NPEX), and (iii) a partial covering power-law with high energy cut-off. Inspite of large value of absorption column density in the direction of the pulsar, a blackbody component of temperature ~0.17 keV for the soft excess was required for the first two continuum models. A narrow iron K_\alpha emission line was detected in the pulsar spectrum. The partial covering model, however, is found to explain the phase averaged and phase resolved spectra well. The dip like feature in the pulse profile can be explained by the presence of an additional absorption component with high column density and covering fraction at the same pulse phase. The details of the results are described in the paper.Comment: 9 pages, 7 figures, Accepted for publication in MNRA

Black carbon physical and optical properties across northern India during pre-monsoon and monsoon seasons

Black carbon (BC) is known to have major impacts on both climate and human health and is therefore of global importance, particularly in regions close to large populations that have strong sources. The size-resolved mixing state of BC-containing particles was characterised using a single-particle soot photometer (SP2). The study focusses on the Indo-Gangetic Plain (IGP) during the pre-monsoon and monsoon seasons. Data presented are from the UK Facility for Airborne Atmospheric Measurements BAe-146 research aircraft that performed flights during the pre-monsoon (11 and 12 June) and monsoon (30 June to 11 July) seasons of 2016. Over the IGP, BC mass concentrations were greater (1.95 µg m−3) compared to north-west India (1.50 µg m−3) and north-east India (0.70 µg m−3) during the pre-monsoon season. Across northern India, two distinct BC modes were recorded; a mode of small BC particles (core diameter <0.16 µm and coating thickness <50 nm) and a mode of moderately coated BC (core diameter <0.22 µm and coating thickness of 50–200 nm). The IGP and north-east India locations exhibited moderately coated black carbon particles with enhanced coating thicknesses, core sizes, mass absorption cross sections, and scattering enhancement values compared to much lower values present in the north-west. The coating thickness and mass absorption cross section increased with altitude (13 %) compared to those in the boundary layer. As the monsoon arrived across the region, mass concentration of BC decreased over the central IGP and north-east locations (38 % and 28 % respectively), whereas for the north-west location BC properties remained relatively consistent. Post-monsoon onset, the coating thickness, core size, mass absorption cross section, and scattering enhancement values were all greatest over the central IGP much like the pre-monsoon season but were considerably reduced over both north-east and north-west India. Increases in mass absorption cross section through the atmospheric column were still present during the monsoon for the north-west and central IGP locations, but less so over the north-east due to lack of long-range transport aerosol aloft. Across the Indo-Gangetic Plain and north-east India during the pre-monsoon and monsoon seasons, solid-fuel (wood burning) emissions form the greatest proportion of BC with moderately coated particles. However, as the monsoon develops in the north-east there was a switch to small uncoated BC particles indicative of traffic emissions, but the solid-fuel emissions remained in the IGP into the monsoon. For both seasons in the north-west, traffic emissions form the greatest proportion of BC particles. Our findings will prove important for greater understanding of the BC physical and optical properties, with important consequences for the atmospheric radiative forcing of BC-containing particles. The findings will also help constrain the regional aerosol models for a variety of applications such as space-based remote sensing, chemistry transport modelling, air quality, and BC source and emission inventories