Purinergic angiogenesis : an investigation of the role of extracellular nucleotide mediated signaling in angiogenesis

Includes bibliographical references.Angiogenesis refers to the growth and maturation of new vessels from pre-existing differentiated blood vessels. Co-ordination of angiogenic responses is crucial for a wide range of physiological and pathological processes. In particular, angiogenesis is hypothesized to facilitate tumour growth and promote metastasis, prompting research into anti-angiogenesis based cancer therapies. CD39/NTPDasel is an ectoenzyme expressed by vascular endothelium that hydrolyses extracellular nucleoside di- and triphosphates to the monophosphate derivatives. As such, this enzyme has a role in modulating endothelial responses to mucleotides that bind specific receptors termed Purinergic-type 2(P2) receptors. Since a number proliferation and migration in vitro, we propose that nucleotide signalling influences the process of angiogenesis in vivo

The reduction of acetylpyridine adenine dinucleotide by NADH: is it a significant reaction of proton-translocating transhydrogenase, or an artefact?

AbstractTranshydrogenase is a proton pump. It has separate binding sites for NAD+/NADH (on domain I of the protein) and for NADP+/NADPH (on domain III). Purified, detergent-dispersed transhydrogenase from Escherichia coli catalyses the reduction of the NAD+ analogue, acetylpyridine adenine dinucleotide (AcPdAD+), by NADH at a slow rate in the absence of added NADP+ or NADPH. Although it is slow, this reaction is surprizing, since transhydrogenase is generally thought to catalyse hydride transfer between NAD(H) – or its analogues and NADP(H) – or its analogues, by a ternary complex mechanism. It is shown that hydride transfer occurs between the 4A position on the nicotinamide ring of NADH and the 4A position of AcPdAD+. On the basis of the known stereospecificity of the enzyme, this eliminates the possibilities of transhydrogenation (a) from NADH in domain I to AcPdAD+ wrongly located in domain III; and (b) from NADH wrongly located in domain III to AcPdAD+ in domain I. In the presence of low concentrations of added NADP+ or NADPH, detergent-dispersed E. coli transhydrogenase catalyses the very rapid reduction of AcPdAD+ by NADH. This reaction is cyclic; it takes place via the alternate oxidation of NADPH by AcPdAD+ and the reduction of NADP+ by NADH, while the NADPH and NADP+ remain tightly bound to the enzyme. In the present work, it is shown that the rate of the cyclic reaction and the rate of reduction of AcPdAD+ by NADH in the absence of added NADP+/NADPH, have similar dependences on pH and on MgSO4 concentration and that they have a similar kinetic character. It is therefore suggested that the reduction of AcPdAD+ by NADH is actually a cyclic reaction operating, either with tightly bound NADP+/NADPH on a small fraction (<5%) of the enzyme, or with NAD+/NADH (or AcPdAD+/AcPdADH) unnaturally occluded within the domain III site. Transhydrogenase associated with membrane vesicles (chromatophores) of Rhodospirillum rubrum also catalyses the reduction of AcPdAD+ by NADH in the absence of added NADP+/NADPH. When the chromatophores were stripped of transhydrogenase domain I, that reaction was lost in parallel with `normal reverse' transhydrogenation (e.g., the reduction of AcPdAD+ by NADPH). The two reactions were fully recovered upon reconstitution with recombinant domain I protein. However, after repeated washing of the domain I-depleted chromatophores, reverse transhydrogenation activity (when assayed in the presence of domain I) was retained, whereas the reduction of AcPdAD+ by NADH declined in activity. Addition of low concentrations of NADP+ or NADPH always supported the same high rate of the NADH→AcPdAD+ reaction independently of how often the membranes were washed. It is concluded that, as with the purified E. coli enzyme, the reduction of AcPdAD+ by NADH in chromatophores is a cyclic reaction involving nucleotides that are tightly bound in the domain III site of transhydrogenase. However, in the case of R. rubrum membranes it can be shown with some certainty that the bound nucleotides are NADP+ or NADPH. The data are thus adequately explained without recourse to suggestions of multiple nucleotide-binding sites on transhydrogenase

Building a Synthetic Pathway For Nylon precursor Biosynthesis

Biorefineries allow for the sustainable production of higher value products from biomass. In addition to bioethanol, they can produce added value chemicals and pharmaceutical intermediates from isolated component compounds such as sugars. Sugar beet pulp (SBP) is a high volume, low value by-product from sugar beet processing with a low lignin and a high carbohydrate content, making it an attractive biomass feedstock for biorefinery processing. The pectin fraction of SBP can be isolated via steam explosion, which, after complete acid hydrolysis, gives a hydrolysate rich in monosaccharides: primarily L-arabinose (Ara) and D-galacturonic acid (GA), with some D-galactose (Gal) and L-rhamnose (Rha). Isolation of these sugars is therefore a critical step in realising an integrated, whole crop biorefinery. Currently, little work has been reported on the separation and utilisation of SBP hydrolysates. The aim of this thesis is to establish novel, scalable separation processes for the isolation of the component monosaccharides from crude hydrolysed sugar beet pulp pectin. Centrifugal partition chromatography (CPC) is a liquid-liquid separation technique with no solid stationary phase and offers an alternative to traditional resin-based chromatographic techniques. As such it can more easily cope with crude feedstreams such as hydrolysates. Hydrophilic ethanol : ammonium sulphate two-phase systems were examined based on monosaccharide partition coefficients and phase settling times. An ethanol : aqueous ammonium sulphate (300 g L-1 ) (0.8:1.8 v:v) system was chosen for CPC separations of the crude SBP hydrolysate and was shown to be capable of removing the coloured contaminants and isolating three sugar fractions in a single step: Rha, Ara and Gal, and GA. The separation was optimised and the throughput was increased by maximising the sample loading. Operation in an elution-extrusion mode allowed for reproducible separations in 100 min without additional column regeneration. The process was scaled up from a 250 to a 950 mL column providing a final throughput of 1.9 gmonosaccharides L -1 column h -1 using the crude SBP. The following purities and recoveries of the three main fractions were achieved: Rha at 92% purity and 93% recovery; Ara at 84% purity and 97% recovery; and GA at 96% purity and 95% recovery. Simulated moving bed (SMB) allows for continuous chromatographic separations using multiple columns, improving separation performance and throughputs. Isolation of Ara from the neutral sugars Gal and Rha was performed with resins and conditions screened on single columns leading to the selection of a Dowex 50W X8 resin in the Ca2+ form. SMB separation using 8 columns was performed in the 4-zone and 3-zone setups and achieved 94% purity with 99% recovery at a throughput of 4.6 gmonosaccharides L -1 column h -1 with a synthetic mixture of the neutral sugars (Ara, Gal and Rha). However, equivalent separations could not be achieved using the crude SBP hydrolysate which needed pretreatment before SMB. Decolourisation with activated carbon was able to remove 97% of the coloured contaminants with sugar losses of 15% (w/w) in a batch process demonstrated to 50 mL scale. Anion exchange chromatography using a Dowex 1x8 resin was then found to be capable of isolating GA from a synthetic crude mixture of GA and neutral sugars with a dynamic binding capacity of 1.31 mmol mL-1 resin. However, further work is needed to enable this anion exchange step to achieve satisfactory separations with the decolourised crude hydrolysate. The isolated neutral sugars, after GA removal, can be processed on the SMB with comparable separation performance and throughput to a mixture of neutral sugars prepared without GA. In summary, this thesis presents two possible process paths each with their own benefits and drawbacks. CPC is capable of processing the crude SBP hydrolysate directly, isolating the sugars and removing the coloured contaminants in a single step. However, Ara co-elutes with Gal providing a stream that is only 84% pure. In SMB, the potential throughputs and separation performance are higher, however, this could only be experimentally demonstrated with synthetic crude mixtures of sugars and not with the crude SBP hydrolysate. Further pretreatment or SMB method development would be required in order to process the crude hydrolysate, and the resulting multistep processes may reduce the overall viability. Overall this thesis demonstrates two feasible approaches to the preparative scale separation of SBP pectin hydrolysates and supports development of an integrated SBP biorefinery

Relaxing the independent censoring assumption in the Cox proportional hazards model using multiple imputation.

The Cox proportional hazards model is frequently used in medical statistics. The standard methods for fitting this model rely on the assumption of independent censoring. Although this is sometimes plausible, we often wish to explore how robust our inferences are as this untestable assumption is relaxed. We describe how this can be carried out in a way that makes the assumptions accessible to all those involved in a research project. Estimation proceeds via multiple imputation, where censored failure times are imputed under user-specified departures from independent censoring. A novel aspect of our method is the use of bootstrapping to generate proper imputations from the Cox model. We illustrate our approach using data from an HIV-prevention trial and discuss how it can be readily adapted and applied in other settings

Methods to improve recruitment to randomised controlled trials : Cochrane systematic review and meta-analysis

Peer reviewedPublisher PD

Winds and tides of the Extended Unified Model in the mesosphere and lower thermosphere validated with meteor radar observations

The mesosphere and lower thermosphere (MLT) is a critical region that must be accurately reproduced in general circulation models (GCMs) that aim to include the coupling between the lower and middle atmosphere and the thermosphere. An accurate representation of the MLT is thus important for improved climate modelling and the development of a whole atmosphere model. This is because the atmospheric waves at these heights are particularly large, and so the energy and momentum they carry is an important driver of climatological phenomena through the whole atmosphere, affecting terrestrial and space weather. The Extended Unified Model (ExUM) is the recently developed version of the Met Office s Unified Model which has been extended to model the MLT. The capability of the ExUM to model atmospheric winds and tides in the MLT is currently unknown. Here, we present the first study of winds and tides from the ExUM. We make a comparison against meteor radar observations of winds and tides from 2006 between 80 and 100 km over two radar stations - Rothera (68° S, 68° W) and Ascension Island (8° S, 14° W). These locations are chosen to study tides in two very different tidal regimes - the equatorial regime, where the diurnal (24 h) tide dominates, and the polar regime, where the semi-diurnal (12 h) tide dominates. The results of this study illustrate that the ExUM is capable of reproducing atmospheric winds and tides that capture many of the key characteristics seen in meteor radar observations, such as zonal and meridional wind maxima and minima, the increase in tidal amplitude with increasing height, and the decrease in tidal phase with increasing height. In particular, in the equatorial regime some essential characteristics of the background winds, tidal amplitudes and tidal phases are well captured but with significant differences in detail. In the polar regime, the difference is more pronounced. The ExUM zonal background winds in austral winter are primarily westward rather than eastward, and in austral summer they are larger than observed above 90 km. The ExUM tidal amplitudes here are in general consistent with observed values, but they are also larger than observed values above 90 km in austral summer. The tidal phases are generally well replicated in this regime. We propose that the bias in background winds in the polar regime is a consequence of the lack of in situ gravity wave generation to generate eastward fluxes in the MLT. The results of this study indicate that the ExUM has a good natural capability for modelling atmospheric winds and tides in the MLT but that there is room for improvement in the model physics in this region. This highlights the need for modifications to the physical parameterization schemes used in the model in this region - such as the non-orographic spectral gravity wave scheme - to improve aspects such as polar circulation. To this end, we make specific recommendations of changes that can be implemented to improve the accuracy of the ExUM in the MLT. </p

Spatial Fingerprint of Younger Dryas Cooling and Warming in Eastern North America

The Younger Dryas (YD, 12.9–11.7 ka) is the most recent, near‐global interval of abrupt climate change with rates similar to modern global warming. Understanding the causes and biodiversity effects of YD climate changes requires determining the spatial fingerprints of past temperature changes. Here we build pollen‐based and branched glycerol dialkyl glycerol tetraether‐based temperature reconstructions in eastern North America (ENA) to better understand deglacial temperature evolution. YD cooling was pronounced in the northeastern United States and muted in the north central United States. Florida sites warmed during the YD, while other southeastern sites maintained a relatively stable climate. This fingerprint is consistent with an intensified subtropical high during the YD and demonstrates that interhemispheric responses were more complex spatially in ENA than predicted by the bipolar seesaw model. Reduced‐amplitude or antiphased millennial‐scale temperature variability in the southeastern United States may support regional hotspots of biodiversity and endemism

A measurement of the cosmological mass density from clustering in the 2dF Galaxy Redshift Survey

The large-scale structure in the distribution of galaxies is thought to arise from the gravitational instability of small fluctuations in the initial density field of the Universe. A key test of this hypothesis is that forming superclusters of galaxies should generate a systematic infall of other galaxies. This would be evident in the pattern of recessional velocities, causing an anisotropy in the inferred spatial clustering of galaxies. Here we report a precise measurement of this clustering, using the redshifts of more than 141,000 galaxies from the two-degree-field (2dF) galaxy redshift survey. We determine the parameter β = Ω0.6/b = 0.43 +/- 0.07, where Ω is the total mass-density parameter of the Universe and b is a measure of the `bias' of the luminous galaxies in the survey. (Bias is the difference between the clustering of visible galaxies and of the total mass, most of which is dark.) Combined with the anisotropy of the cosmic microwave background, our results favour a low-density Universe with Ω ~ 0.3