Evaluation of an inactivated Ross River virus vaccine in active and passive mouse immunization models and establishment of a correlate of protection

Ross River Virus has caused reported outbreaks of epidemic polyarthritis, a chronic debilitating disease associated with significant long-term morbidity in Australia and the Pacific region since the 1920s. To address this public health concern, a formalin- and UV-inactivated whole virus vaccine grown in animal protein-free cell culture was developed and tested in preclinical studies to evaluate immunogenicity and efficacy in animal models. After active immunizations, the vaccine dose-dependently induced antibodies and protected adult mice from viremia and interferon α/β receptor knock-out (IFN-α/βR(-/-)) mice from death and disease. In passive transfer studies, administration of human vaccinee sera followed by RRV challenge protected adult mice from viremia and young mice from development of arthritic signs similar to human RRV-induced disease. Based on the good correlation between antibody titers in human sera and protection of animals, a correlate of protection was defined. This is of particular importance for the evaluation of the vaccine because of the comparatively low annual incidence of RRV disease, which renders a classical efficacy trial impractical. Antibody-dependent enhancement of infection, did not occur in mice even at low to undetectable concentrations of vaccine-induced antibodies. Also, RRV vaccine-induced antibodies were partially cross-protective against infection with a related alphavirus, Chikungunya virus, and did not enhance infection. Based on these findings, the inactivated RRV vaccine is expected to be efficacious and protect humans from RRV disease

Australian higher education evaluation through assurance of learning

A collaborative research project conducted by five Australian universities inquired into the philosophy and motivation for Assurance of Learning (AoL) as a process of education evaluation. Associate Deans Teaching and Learning representing Business schools from twenty-five universities across Australia participated in telephone interviews. Data was analysed using NVIVO9. Results indicated that articulated rationale for AoL was both ensuring that students had acquired the attributes and skills the universities claimed they had, and the philosophy of continuous improvement. AoL was motivated both by ritualistic objectives to satisfy accreditation requirements and virtuous agendas for quality improvement. Closing-the-loop was emphasised, but was mostly wishful thinking for next steps beyond data collection and reporting. AoL was conceptualised as one element within the larger context of quality review, but there was no evidence of comprehensive frameworks or strategic plans.

Evaluation of fibroin-based scaffolds for ocular tissue reconstruction

The epithelium of the corneolimbus contains stem cells for regenerating the corneal epithelium. Diseases and injuries affecting the limbus can lead to a condition known as limbal stem cell deficiency (LSCD), which results in loss of the corneal epithelium, and subsequent chronic inflammation and scarring of the ocular surface. Advances in the treatment of LSCD have been achieved through use of cultured human limbal epithelial (HLE) grafts to restore epithelial stem cells of the ocular surface. These epithelial grafts are usually produced by the ex vivo expansion of HLE cells on human donor amniotic membrane (AM), but this is not without limitations. Although AM is the most widely accepted substratum for HLE transplantation, donor variation, risk of disease transfer, and rising costs have led to the search for alternative biomaterials to improve the surgical outcome of LSCD. Recent studies have demonstrated that Bombyx mori silk fibroin (hereafter referred to as fibroin) membranes support the growth of primary HLE cells, and thus this thesis aims to explore the possibility of using fibroin as a biomaterial for ocular surface reconstruction. Optimistically, the grafted sheets of cultured epithelium would provide a replenishing source of epithelial progenitor cells for maintaining the corneal epithelium, however, the HLE cells lose their progenitor cell characteristics once removed from their niche. More severe ocular surface injuries, which result in stromal scarring, damage the epithelial stem cell niche, which subsequently leads to poor corneal re-epithelialisation post-grafting. An ideal solution to repairing the corneal limbus would therefore be to grow and transplant HLE cells on a biomaterial that also provides a means for replacing underlying stromal cells required to better simulate the normal stem cell niche. The recent discovery of limbal mesenchymal stromal cells (L-MSC) provides a possibility for stromal repair and regeneration, and therefore, this thesis presents the use of fibroin as a possible biomaterial to support a three dimensional tissue engineered corneolimbus with both an HLE and underlying L-MSC layer. Investigation into optimal scaffold design is necessary, including adequate separation of epithelial and stromal layers, as well as direct cell-cell contact. Firstly, the attachment, morphology and phenotype of HLE cells grown on fibroin were directly compared to that observed on donor AM, the current clinical standard substrate for HLE transplantation. The production, transparency, and permeability of fibroin membranes were also evaluated in this part of the study. Results revealed that fibroin membranes could be routinely produced using a custom-made film casting table and were found to be transparent and permeable. Attachment of HLE cells to fibroin after 4 hours in serum-free medium was similar to that supported by tissue culture plastic but approximately 6-fold less than that observed on AM. While HLE cultured on AM displayed superior stratification, epithelia constructed from HLE on fibroin maintained evidence of corneal phenotype (cytokeratin pair 3/12 expression; CK3/12) and displayed a comparable number and distribution of ÄNp63+ progenitor cells to that seen in cultures grown on AM. These results confirm the suitability of membranes constructed from silk fibroin as a possible substrate for HLE cultivation. One of the most important aspects in corneolimbal tissue engineering is to consider the reconstruction of the limbal stem cell niche to help form the natural limbus in situ. MSC with similar properties to bone marrow derived-MSC (BM-MSC) have recently been grown from the limbus of the human cornea. This thesis evaluated methods for culturing L-MSC and limbal keratocytes using various serum-free media. The phenotype of resulting cultures was examined using photography, flow cytometry for CD34 (keratocyte marker), CD45 (bone marrow-derived cell marker), CD73, CD90, CD105 (collectively MSC markers), CD141 (epithelial/vascular endothelial marker), and CD271 (neuronal marker), immunocytochemistry (alpha-smooth muscle actin; á-sma), differentiation assays (osteogenesis, adipogenesis and chrondrogenesis), and co-culture experiments with HLE cells. While all techniques supported to varying degrees establishment of keratocyte and L-MSC cultures, sustained growth and serial propagation was only achieved in serum-supplemented medium or the MesenCult-XF„¥ culture system (Stem Cell Technologies). Cultures established in MesenCult-XF„¥ grew faster than those grown in serum-supplemented medium and retained a more optimal MSC phenotype. L-MSC cultivated in MesenCult-XFR were also positive for CD141, rarely expressed £\-sma, and displayed multi-potency. L-MSC supported growth of HLE cells, with the largest epithelial islands being observed in the presence of L-MSC established in MesenCult-XF„¥ medium. All HLE cultures supported by L-MSC widely expressed the progenitor cell marker £GNp63, along with the corneal differentiation marker CK3/12. Our findings conclude that MesenCult-XFR is a superior culture system for L-MSC, but further studies are required to explore the significance of CD141 expression in these cells. Following on from the findings of the previous two parts, silk fibroin was tested as a novel dual-layer construct containing both an epithelium and underlying stroma for corneolimbal reconstruction. In this section, the growth and phenotype of HLE cells on non-porous versus porous fibroin membranes was compared. Furthermore, the growth of L-MSC in either serum-supplemented medium or the MesenCult-XFR culture system within fibroin fibrous mats was investigated. Lastly, the co-culture of HLE and L-MSC in serum-supplemented medium on and within fibroin dual-layer constructs was also examined. HLE on porous membranes displayed a flattened and squamous monolayer; in contrast, HLE on non-porous fibroin appeared cuboidal and stratified closer in appearance to a normal corneal epithelium. Both constructs maintained CK3/12 expression and distribution of £GNp63+ progenitor cells. Dual-layer fibroin scaffolds consisting of HLE cells and L-MSC maintained a similar phenotype as on the single layers alone. Overall, the present study proposed to create a three dimensional limbal tissue substitute of HLE cells and L-MSC together, ultimately for safe and beneficial transplantation back into the human eye. The results show that HLE and L-MSC can be cultivated separately and together whilst maintaining a clinically feasible phenotype containing a majority of progenitor cells. In addition, L-MSC were able to be cultivated routinely in the MesenCult-XF® culture system while maintaining a high purity for the MSC characteristic phenotype. However, as a serum-free culture medium was not found to sustain growth of both HLE and L-MSC, the combination scaffold was created in serum-supplemented medium, indicating that further refinement of this cultured limbal scaffold is required. This thesis has also demonstrated a potential novel marker for L-MSC, and has generated knowledge which may impact on the understanding of stromal-epithelial interactions. These results support the feasibility of a dual-layer tissue engineered corneolimbus constructed from silk fibroin, and warrant further studies into the potential benefits it offers to corneolimbal tissue regeneration. Further refinement of this technology should explore the potential benefits of using epithelial-stromal co-cultures with MesenCult-XF® derived L-MSC. Subsequent investigations into the effects of long-term culture on the phenotype and behaviour of the cells in the dual-layer scaffolds are also required. While this project demonstrated the feasibility in vitro for the production of a dual-layer tissue engineered corneolimbus, further studies are required to test the efficacy of the limbal scaffold in vivo. Future in vivo studies are essential to fully understand the integration and degradation of silk fibroin biomaterials in the cornea over time. Subsequent experiments should also investigate the use of both AM and silk fibroin with epithelial and stromal cell co-cultures in an animal model of LSCD. The outcomes of this project have provided a foundation for research into corneolimbal reconstruction using biomaterials and offer a stepping stone for future studies into corneolimbal tissue engineering.

Experimental evaluation of MCDTK, the Monte Carlo DICOM Tool-Kit

The Monte Carlo DICOM Tool-Kit (MCDTK) is a software suite designed for treatment plan dose verification, using the BEAMnrc and DOSXYZnrc Monte Carlo codes. MCDTK converts DICOM-format treatment plan information into Monte Carlo input files and compares the results of Monte Carlo treatment simulations with conventional treatment planning dose calculations. In this study, a treatment is planned using a commercial treatment planning system, delivered to a pelvis phantom containing ten thermoluminescent dosimeters and simulated using BEAMnrc and DOSXYZnrc using inputs derived from MCDTK. The dosimetric accuracy of the Monte Carlo data is then evaluated via comparisons with the dose distribution obtained from the treatment planning system as well as the in-phantom point dose measurements. The simulated beam arrangement produced by MCDTK is found to be in geometric agreement with the planned treatment. An isodose display generated from the Monte Carlo data by MCDTK shows general agreement with the isodose display obtained from the treatment planning system, except for small regions around density heterogeneities in the phantom, where the pencil-beam dose calculation performed by the treatment planning systemis likely to be less accurate. All point dose measurements agree with the Monte Carlo data obtained using MCDTK, within confidence limits, and all except one of these point dose measurements show closer agreement with theMonte Carlo data than with the doses calculated by the treatment planning system. This study provides a simple demonstration of the geometric and dosimetric accuracy ofMonte Carlo simulations based on information from MCDTK.

Monte Carlo evaluation of collapsed-cone convolution calculations in head and neck radiotherapy treatment plans

The presence of air and bone interfaces makes the dose distribution for head and neck cancer treatments difficult to accurately predict. This study compared planning system dose calculations using the collapsed-cone convolution algorithm with EGSnrcMonte Carlo simulation results obtained using the Monte Carlo DICOMToolKit software, for one oropharynx, two paranasal sinus and three nodal treatment plans. The difference between median doses obtained from the treatment planning and Monte Carlo calculations was found to be greatest in two bilateral treatments: 4.8%for a retropharyngeal node irradiation and 6.7% for an ethmoid paranasal sinus treatment. These deviations in median dose were smaller for two unilateral treatments: 0.8% for an infraclavicular node irradiation and 2.8% for a cervical node treatment. Examination of isodose distributions indicated that the largest deviations between Monte Carlo simulation and collapsed-cone convolution calculations were seen in the bilateral treatments, where the increase in calculated dose beyond air cavities was most significant.

Evaluation of the iPhone with an acrylic sleeve versus the Scoliometer for rib hump measurement in scoliosis

Background. Vertebral rotation found in structural scoliosis contributes to trunkal asymmetry which is commonly measured with a simple Scoliometer device on a patient's thorax in the forward flexed position. The new generation of mobile 'smartphones' have an integrated accelerometer, making accurate angle measurement possible, which provides a potentially useful clinical tool for assessing rib hump deformity. This study aimed to compare rib hump angle measurements performed using a Smartphone and traditional Scoliometer on a set of plaster torsos representing the range of torsional deformities seen in clinical practice. Methods. Nine observers measured the rib hump found on eight plaster torsos moulded from scoliosis patients with both a Scoliometer and an Apple iPhone on separate occasions. Each observer repeated the measurements at least a week after the original measurements, and were blinded to previous results. Intra-observer reliability and inter-observer reliability were analysed using the method of Bland and Altman and 95% confidence intervals were calculated. The Intra-Class Correlation Coefficients (ICC) were calculated for repeated measurements of each of the eight plaster torso moulds by the nine observers. Results. Mean absolute difference between pairs of iPhone/Scoliometer measurements was 2.1 degrees, with a small (1 degrees) bias toward higher rib hump angles with the iPhone. 95% confidence intervals for intra-observer variability were +/- 1.8 degrees (Scoliometer) and +/- 3.2 degrees (iPhone). 95% confidence intervals for inter-observer variability were +/- 4.9 degrees (iPhone) and +/- 3.8 degrees (Scoliometer). The measurement errors and confidence intervals found were similar to or better than the range of previously published thoracic rib hump measurement studies. Conclusions. The iPhone is a clinically equivalent rib hump measurement tool to the Scoliometer in spinal deformity patients. The novel use of plaster torsos as rib hump models avoids the variables of patient fatigue and discomfort, inconsistent positioning and deformity progression using human subjects in a single or multiple measurement sessions.

Meta-evaluation to improve learning, evaluation capacity development and sustainability : Findings from a participatory evaluation project in Nepal

Recent studies suggest that meta-evaluation can be valuable in developing new approaches to evaluation, building evaluation capacities, and enhancing organizational learning. These new extensions of the concept of meta-evaluation are significant, given the growing emphasis on improving the quality and effectiveness of evaluation practices in the South Asian region. Following a review of the literature, this paper presents a case study of the use of concurrent meta-evaluation in the four-year project Assessing Communication for Social Change which developed and trialled a participatory impact assessment methodology in collaboration with a development communication Non-government organization (NGO) in Nepal. Key objectives of the meta-evaluation included to: continuously develop, adapt and improve the impact assessment methodology, Monitoring and Evaluation (M&E) systems and process and other project activities; identify impacts of the project; and build capacities in critical reflection and review. Our analysis indicates that this meta-evaluation was essential to understanding various constraints related to the organizational context that affected the success of the project and the development of improved M&E systems and capacities within the NGO. We identified several limitations of our meta-evaluation methods, which were balanced by the strengths of other methods. Our case study suggests that as well as assessing the quality, credibility and value of evaluation practices, meta-evaluations need to focus on important contextual issues that can have significant impacts on the outcomes of participatory evaluation projects. They include hierarchical organizational cultures, communication barriers, power/knowledge relations, and the time and resources available. Meta-evaluations also need to consider wider issues such as the sustainability of evaluation systems and approaches.

Large area noise evaluation

A software tool (DRONE) has been developed to evaluate road traffic noise in a large area with the consideration of network dynamic traffic flow and the buildings. For more precise estimation of noise in urban network where vehicles are mainly in stop and go running conditions, vehicle sound power level (for acceleration/deceleration cruising and ideal vehicle) is incorporated in DRONE. The calculation performance of DRONE is increased by evaluating the noise in two steps of first estimating the unit noise database and then integrating it with traffic simulation. Details of the process from traffic simulation to contour maps are discussed in the paper and the implementation of DRONE on Tsukuba city is presented.