Assessment and reduction of the impacts of large freight vehicles on urban traffic corridor performance

Increasing demand for road freight has lead to a widespread adoption of more-productive large freight vehicles (LFVs), such as B-Doubles, by Australia's road freight industry. Individual LFVs have a greater potential to impact traffic efficiency through their greater length and poorer longitudinal performance. However, this is offset to an extent as fewer vehicles are required to perform a given freight task on a tonne-km basis. This research has developed a means of characterising the effects that large freight vehicles have on the performance of an urban arterial corridor managed by signalised intersections. A corridor-level microsimulation model was developed from first principles, which modelled the longitudinal performance of individual vehicles to a greater accuracy than most existing traffic simulation software does. The model was calibrated from traffic counts and GPS-equipped chase car surveys conducted on an urban arterial corridor in Brisbane's southern suburbs. The model was applied to various freight policy and traffic management scenarios, including freight vehicle mode choice, lane utilisation and traffic signal settings; as well as the effectiveness of green time extension for approaching heavy vehicles. Benefits were able to be quantified in terms of reduced travel times and stop rates for both heavy and light vehicles in urban arterial corridors

Predicting sleep disordered breathing in outpatients with suspected OSA

Objective To validate the utilities of Berlin, STOP and STOP-BANG Questionnaires, other patient characteristics, comorbidities, Epworth Sleepiness Scale (ESS), fractional exhaled nitric oxide (FENO) and blood markers for the prediction of sleep disordered breathing (SDB) on limited polygraphy. Setting North Glasgow Sleep Service (a tertiary referral centre). Participants 129 consecutive patients, aged ≥16 years, referred to the sleep clinic for assessment of possible obstructive sleep apnoea. Interventions We selected cut-points of apnoea hypopnoea index (AHI) of ≥5 and ≥15/h from their home polygraphy and determined associations of these with individual symptoms, questionnaire scores and other results. Receiver operating characteristic analysis and univariate and multivariate logistic regression were used to explore these. Primary and secondary outcomes measures Primary: The utility of STOP, STOP-BANG and Berlin Questionnaires for prediction of SDB. Secondary: The utility of other measures for prediction of SDB. Results AHI was ≥5 in 97 patients and ≥15 in 56 patients. STOP and STOP-BANG scores were associated with both AHI cut-points but results with ESS and Berlin Questionnaire scores were negative. STOP-BANG had a negative predictive value 1.00 (0.77–1.00) for an AHI ≥15 with a score ≥3 predicting AHI ≥5 with sensitivity 0.93 (95% CI 0.84 to 0.98) and accuracy 79%, while a score ≥6 predicted AHI ≥15 with specificity 0.78 (0.65 to 0.88) and accuracy 72%. Neck circumference ≥17 inch and presence of witnessed apnoeas were independent predictors of SDB. Conclusions STOP and STOP-BANG Questionnaires have utility for the prediction of SDB in the sleep clinic population. Modification of the STOP-BANG Questionnaire merits further study in this and other patient groups.</p

Resonance energy transfer based detection of G-protein coupled receptor dimerization

Over the past decade there has been a growing body of evidence, obtained from studies employing a wide variety of pharmacological, biochemical and biophysical techniques, suggesting that G protein coupled receptors (GPCRs) exist not as monomeric entities but rather as dimers or other higher order oligomeric arrays. To further the accumulation of knowledge pertaining to this research area, the work presented in this thesis has made use of one such particular biophysical technique called bioluminescence resonance energy transfer (BRET). In order to utilise this system GPCRs were modified at their carboxyl terminal tail with either the anthozoan enzyme Renilla luciferase or the fluorescent protein eYPP. Through this expedient, if the differentially tagged GPCRs are in close proximity when co-expressed within mammalian cells, upon addition of the bioluminescent molecule coelenterazine, there is a non-radiative exchange of energy between the Renilla and eYFP, resulting in a fluorescent emission from eYFP. The technique can be used to monitor interactions in real time, in living cells and does not require any biochemical manipulations/treatments such as are associated with more traditional approaches to this line of enquiry (e.g. co-immunoprecipitation). Using this technique, it was demonstrated that the β2-AR was closely associated when expressed within HEK 293T cells, as were the σ-opioid and K-opioid receptors since all gave robust signals in energy transfer experiments. Contrary to some previous reports however, it was not seen to be the case that the presence of ligand was capable of modulating the magnitude of the energy transfer signal. This indicated that for these GPCRs the binding of ligand did not result in any alteration in the dimerization status of the receptor. It was further shown, through monitoring the energy transfer at varying levels of receptor expression, that energy transfer was favourable between homomers of the K-opioid receptor. At similar receptor densities, energy transfer between coexpressed K-opioid receptor and TRHr was seen to be considerably less favourable, requiring far higher receptor expression levels to be achieved before meaningful levels of energy transfer could be detected. These results strongly indicated that closely related GPCR types had a greater propensity for mutual interaction than did more distantly related ones. Many of these results were confirmed using a modified version of BRET, designated BRET2, which conferred an additional sensitivity to detection of protein-protein interactions. Using BRET2 a previously ill-defined result obtained with traditional BRET, that suggested that the β2-AR might interact with the 6-opioid-receptor, was confirmed. An additional purpose of the work described herein was to explore the potential of GPCR dimerization as a means of providing a novel ligand detection assay suitable for application to industrial high-throughput screening programmes. Since the experiments concentrating on GPCR oligomerization failed to provide such an assay, it was decided that the ability of GPCRs to recruit β-arrestin as part of the process of desensitisation should be evaluated as a possible alternative. Using a cell line stably expressing the GPCR CCR2 the ability of this receptor to recruit various fluorescent proteins conjugated to β-arrestin2 (β-arrestin-red NFP and β-arrestin-cyan NFP) from the cytosol in response to receptor activation was demonstrated. The β-arrestin2 was localized into endocytic vesicles and remained tightly associated with the internalised receptors after sequestration had occurred. This behaviour was in accordance with other previous reports for GPCRs that, like CCR2, possessed serine and threonine clusters within their carboxyl terminal tails. If adapted to a FRET based format this particular protein-protein interaction could form the basis of a ligand screening method for agonists that would be equally applicable to most GPCRs. It was further shown that β-arrestin2-red NFP had a higher affinity for CCR2 than did β-arrestin1- GFP through the monitoring of the respective kinetics and extent of translocation when the constructs were co-expressed within the same cells. As an alternative strategy for the detection of ligands, a constitutively active mutant of β 2-AR (CAM β 2-AR) was modified C-terminally with the bioluminescent enzyme Renilla luciferase. This CAM 2-AR was structurally destabilized to a high degree so that only modest expression levels could be obtained upon expression of the Renilla modified receptor construct (CAM β 2-AR-Rluc) in HEK 293T cells. Upon prolonged exposure to various antagonist ligands, a two to three fold upregulation of the receptor construct could be detected via light output from the luciferase. From parallel competition binding experiments it was also demonstrated that, for each of these ligands, the EC50 for upregulation highly correlated with the dissociation equilibrium constant (Ki). This strongly indicated that it was the presence of the ligand within the receptor binding pocket that alone accounted for the observed upregulation effects. In a similar manner, it was demonstrated that agonist compounds were also capable of mediating a similar degree of upregulation. The increase in receptor density of CAM β2-AR in response to the presence of ligand was subsequently shown to be dependent on the constitutively active nature of the receptor. In an additional experiment co-transfection of CAM β 2-AR-Rluc along with a GFP conjugated version of the βib- adrenoceptor into HEK 293T cells and subsequent monitoring of the upregulation of either construct in response to selective ligands confirmed the necessity for pharmacological specificity in mediating the upregulatory effect. Finally, to show that this assay method would be suitable as a means of detecting ligands in a high- throughput screening format, the ability of β2-AR to be upregulated was assessed in the presence of a wide variety of compounds, only a proportion of which possessed pharmacological specificity for the β2-AR. When tested in this manner it was seen that only compounds that were specific for β2-AR were capable of mediating an upregulatory effect

Prognostic Value of Computed Tomography : Measured Parameters of Body Composition in Primary Operable Gastrointestinal Cancers

Professor Graeme Murray, Department of Pathology, University of Aberdeen provided us access to the colorectal cancer pathology databases from which the colorectal component of the research was based. Conflict of interest There are no conflicts of interest.Peer reviewedPublisher PD

Structural Studies on the Herpes Simplex Virus Type 1 Genome During Latency in Tissue Culture Cells

Herpes simplex virus (HSV) has the pathogenic property of establishing latency during primary infection of man. Following the primary infection at a peripheral site, virions travel via the axons to the neuronal cell bodies in sensory or autonomic ganglia where latency is established and maintained. The virus can be reactivated, return down the axon to the periphery, replicate, be shed and transmitted to other individuals. This gives rise to clinically important recurrent disease, namely herpes labialis, keratitis, uveitis and genitalis. Reactivation is of increasing importance in immunocompromised individuals. Encephalitis, presumed to be due to reactivation of latent virus from the trigeminal ganglion, is the most serious manifestation with a mortality rate of about 50% if untreated and considerable morbidity in half of the survivors