The experiences of learner drivers, provisional drivers and supervisors with graduated driver licensing in two Australian jurisdictions

Newly licensed drivers on a provisional or intermediate licence have the highest crash risk when compared with any other group of drivers. In comparison, learner drivers have the lowest crash risk. Graduated driver licensing is one countermeasure that has been demonstrated to effectively reduce the crashes of novice drivers. This thesis examined the graduated driver licensing systems in two Australian states in order to better understand the behaviour of learner drivers, provisional drivers and the supervisors of learner drivers. By doing this, the thesis investigated the personal, social and environmental influences on novice driver behaviour as well as providing effective baseline data against which to measure subsequent changes to the licensing systems. In the first study, conducted prior to the changes to the graduated driver licensing system introduced in mid-2007, drivers who had recently obtained their provisional licence in Queensland and New South Wales were interviewed by telephone regarding their experiences while driving on their learner licence. Of the 687 eligible people approached to participate at driver licensing centres, 392 completed the study representing a response rate of 57.1 per cent. At the time the data was collected, New South Wales represented a more extensive graduated driver licensing system when compared with Queensland. The results suggested that requiring learners to complete a mandated number of hours of supervised practice impacts on the amount of hours that learners report completing. While most learners from New South Wales reported meeting the requirement to complete 50 hours of practice, it appears that many stopped practising soon after this goal was achieved. In contrast, learners from Queensland, who were not required to complete a specific number of hours at the time of the survey, tended to fall into three groups. The first group appeared to complete the minimum number of hours required to pass the test (less than 26 hours), the second group completed 26 to 50 hours of supervised practice while the third group completed significantly more practice than the first two groups (over 100 hours of supervised practice). Learner drivers in both states reported generally complying with the road laws and were unlikely to report that they had been caught breaking the road rules. They also indicated that they planned to obey the road laws once they obtained their provisional licence. However, they were less likely to intend to comply with recommended actions to reduce crash risk such as limiting their driving at night. This study also identified that there were relatively low levels of unaccompanied driving (approximately 15 per cent of the sample), very few driving offences committed (five per cent of the sample) and that learner drivers tended to use a mix of private and professional supervisors (although the majority of practice is undertaken with private supervisors). Consistent with the international literature, this study identified that very few learner drivers had experienced a crash (six per cent) while on their learner licence. The second study was also conducted prior to changes to the graduated driver licensing system and involved follow up interviews with the participants of the first study after they had approximately 21 months driving experience on their provisional licence. Of the 392 participants that completed the first study, 233 participants completed the second interview (representing a response rate of 59.4 per cent). As with the first study, at the time the data was collected, New South Wales had a more extensive graduated driver licensing system than Queensland. For instance, novice drivers from New South Wales were required to progress through two provisional licence phases (P1 and P2) while there was only one provisional licence phase in Queensland. Among the participants in this second study, almost all provisional drivers (97.9 per cent) owned or had access to a vehicle for regular driving. They reported that they were unlikely to break road rules, such as driving after a couple of drinks, but were also unlikely to comply with recommended actions, such as limiting their driving at night. When their provisional driving behaviour was compared to the stated intentions from the first study, the results suggested that their intentions were not a strong predictor of their subsequent behaviour. Their perception of risk associated with driving declined from when they first obtained their learner licence to when they had acquired provisional driving experience. Just over 25 per cent of participants in study two reported that they had been caught committing driving offences while on their provisional licence. Nearly one-third of participants had crashed while driving on a provisional licence, although few of these crashes resulted in injuries or hospitalisations. To complement the first two studies, the third study examined the experiences of supervisors of learner drivers, as well as their perceptions of their learner’s experiences. This study was undertaken after the introduction of the new graduated driver licensing systems in Queensland and New South Wales in mid- 2007, providing insights into the impacts of these changes from the perspective of supervisors. The third study involved an internet survey of 552 supervisors of learner drivers. Within the sample, approximately 50 per cent of participants supervised their own child. Other supervisors of the learner drivers included other parents or stepparents, professional driving instructors and siblings. For two-thirds of the sample, this was the first learner driver that they had supervised. Participants had provided an average of 54.82 hours (sd = 67.19) of supervision. Seventy-three per cent of participants indicated that their learners’ logbooks were accurate or very accurate in most cases, although parents were more likely than non-parents to report that their learners’ logbook was accurate (F (1,546) = 7.74, p = .006). There was no difference between parents and non-parents regarding whether they believed the log book system was effective (F (1,546) = .01, p = .913). The majority of the sample reported that their learner driver had had some professional driving lessons. Notwithstanding this, a significant proportion (72.5 per cent) believed that parents should be either very involved or involved in teaching their child to drive, with parents being more likely than non-parents to hold this belief. In the post mid-2007 graduated driver licensing system, Queensland learner drivers are able to record three hours of supervised practice in their log book for every hour that is completed with a professional driving instructor, up to a total of ten hours. Despite this, there was no difference identified between Queensland and New South Wales participants regarding the amount of time that they reported their learners spent with professional driving instructors (X2(1) = 2.56, p = .110). Supervisors from New South Wales were more likely to ensure that their learner driver complied with the road laws. Additionally, with the exception of drug driving laws, New South Wales supervisors believed it was more important to teach safety-related behaviours such as remaining within the speed limit, car control and hazard perception than those from Queensland. This may be indicative of more intensive road safety educational efforts in New South Wales or the longer time that graduated driver licensing has operated in that jurisdiction. However, other factors may have contributed to these findings and further research is required to explore the issue. In addition, supervisors reported that their learner driver was involved in very few crashes (3.4 per cent) and offences (2.7 per cent). This relatively low reported crash rate is similar to that identified in the first study. Most of the graduated driver licensing research to date has been applied in nature and lacked a strong theoretical foundation. These studies used Akers’ social learning theory to explore the self-reported behaviour of novice drivers and their supervisors. This theory was selected as it has previously been found to provide a relatively comprehensive framework for explaining a range of driver behaviours including novice driver behaviour. Sensation seeking was also used in the first two studies to complement the non-social rewards component of Akers’ social learning theory. This program of research identified that both Akers’ social learning theory and sensation seeking were useful in predicting the behaviour of learner and provisional drivers over and above socio-demographic factors. Within the first study, Akers’ social learning theory accounted for an additional 22 per cent of the variance in learner driver compliance with the law, over and above a range of socio-demographic factors such as age, gender and income. The two constructs within Akers’ theory which were significant predictors of learner driver compliance were the behavioural dimension of differential association relating to friends, and anticipated rewards. Sensation seeking predicted an additional six per cent of the variance in learner driver compliance with the law. When considering a learner driver’s intention to comply with the law while driving on a provisional licence, Akers’ social learning theory accounted for an additional 10 per cent of the variance above socio-demographic factors with anticipated rewards being a significant predictor. Sensation seeking predicted an additional four per cent of the variance. The results suggest that the more rewards individuals anticipate for complying with the law, the more likely they are to obey the road rules. Further research is needed to identify which specific rewards are most likely to encourage novice drivers’ compliance with the law. In the second study, Akers’ social learning theory predicted an additional 40 per cent of the variance in self-reported compliance with road rules over and above socio-demographic factors while sensation seeking accounted for an additional five per cent of the variance. A number of Aker’s social learning theory constructs significantly predicted provisional driver compliance with the law, including the behavioural dimension of differential association for friends, the normative dimension of differential association, personal attitudes and anticipated punishments. The consistent prediction of additional variance by sensation seeking over and above the variables within Akers’ social learning theory in both studies one and two suggests that sensation seeking is not fully captured within the non social rewards dimension of Akers’ social learning theory, at least for novice drivers. It appears that novice drivers are strongly influenced by the desire to engage in new and intense experiences. While socio-demographic factors and the perception of risk associated with driving had an important role in predicting the behaviour of the supervisors of learner drivers, Akers’ social learning theory provided further levels of prediction over and above these factors. The Akers’ social learning theory variables predicted an additional 14 per cent of the variance in the extent to which supervisors ensured that their learners complied with the law and an additional eight per cent of the variance in the supervisors’ provision of a range of practice experiences. The normative dimension of differential association, personal attitudes towards the use of professional driving instructors and anticipated rewards were significant predictors for supervisors ensuring that their learner complied with the road laws, while the normative dimension was important for range of practice. This suggests that supervisors who engage with other supervisors who ensure their learner complies with the road laws and provide a range of practice to their own learners are more likely to also engage in these behaviours. Within this program of research, there were several limitations including the method of recruitment of participants within the first study, the lower participation rate in the second study, an inability to calculate a response rate for study three and the use of self-report data for all three studies. Within the first study, participants were only recruited from larger driver licensing centres to ensure that there was a sufficient throughput of drivers to approach. This may have biased the results due to the possible differences in learners that obtain their licences in locations with smaller licensing centres. Only 59.4 per cent of the sample in the first study completed the second study. This may be a limitation if there was a common reason why those not participating were unable to complete the interview leading to a systematic impact on the results. The third study used a combination of a convenience and snowball sampling which meant that it was not possible to calculate a response rate. All three studies used self-report data which, in many cases, is considered a limitation. However, self-report data may be the only method that can be used to obtain some information. This program of research has a number of implications for countermeasures in both the learner licence phase and the provisional licence phase. During the learner phase, licensing authorities need to carefully consider the number of hours that they mandate learner drivers must complete before they obtain their provisional driving licence. If they mandate an insufficient number of hours, there may be inadvertent negative effects as a result of setting too low a limit. This research suggests that logbooks may be a useful tool for learners and their supervisors in recording and structuring their supervised practice. However, it would appear that the usage rates for logbooks will remain low if they remain voluntary. One strategy for achieving larger amounts of supervised practice is for learner drivers and their supervisors to make supervised practice part of their everyday activities. As well as assisting the learner driver to accumulate the required number of hours of supervised practice, it would ensure that they gain experience in the types of environments that they will probably encounter when driving unaccompanied in the future, such as to and from education or work commitments. There is also a need for policy processes to ensure that parents and professional driving instructors communicate effectively regarding the learner driver’s progress. This is required as most learners spend at least some time with a professional instructor despite receiving significant amounts of practice with a private supervisor. However, many supervisors did not discuss their learner’s progress with the driving instructor. During the provisional phase, there is a need to strengthen countermeasures to address the high crash risk of these drivers. Although many of these crashes are minor, most involve at least one other vehicle. Therefore, there are social and economic benefits to reducing these crashes. If the new, post-2007 graduated driver licensing systems do not significantly reduce crash risk, there may be a need to introduce further provisional licence restrictions such as separate night driving and peer passenger restrictions (as opposed to the hybrid version of these two restrictions operating in both Queensland and New South Wales). Provisional drivers appear to be more likely to obey some provisional licence laws, such as lower blood alcohol content limits, than others such as speed limits. Therefore, there may be a need to introduce countermeasures to encourage provisional drivers to comply with specific restrictions. When combined, these studies provided significant information regarding graduated driver licensing programs. This program of research has investigated graduated driver licensing utilising a cross-sectional and longitudinal design in order to develop our understanding of the experiences of novice drivers that progress through the system in order to help reduce crash risk once novice drivers commence driving by themselves.

Factors affecting 39K NMR detectability in rat tissue

In this study we have found that NMR detectability of 39K in rat thigh muscle may be substantially higher (up to 100% oftotal tissue potassium) than values previously reported of around 40%. The signal was found to consist of two superimposed components, one broad and one narrow, of approximately equal area. Investigations involving improvements in spectral parameters such as signal-to-noise ratio and baseline roll, together with computer simulations of spectra, show that the quality of the spectra has a major effect on the amount of signal detected, which is largely due to the loss of detectability of the broad signal component. In particular, lower-field spectrometers using conventional probes and detection methods generally have poorer signal-to-noise and worse baseline roll artifacts, which make detection of a broad component of the muscle signal difficult.

Tracking the magmatic evolution of island arc volcanism : insights from a high-precision Pb isotope record of Montserrat, Lesser Antilles

The volcanic succession on Montserrat provides an opportunity to examine the magmatic evolution of island arc volcanism over a ∼2.5 Ma period, extending from the andesites of the Silver Hills center, to the currently active Soufrière Hills volcano (February 2010). Here we present high-precision double-spike Pb isotope data, combined with trace element and Sr-Nd isotope data throughout this period of Montserrat's volcanic evolution. We demonstrate that each volcanic center; South Soufrière Hills, Soufrière Hills, Centre Hills and Silver Hills, can be clearly discriminated using trace element and isotopic parameters. Variations in these parameters suggest there have been systematic and episodic changes in the subduction input. The SSH center, in particular, has a greater slab fluid signature, as indicated by low Ce/Pb, but less sediment addition than the other volcanic centers, which have higher Th/Ce. Pb isotope data from Montserrat fall along two trends, the Silver Hills, Centre Hills and Soufrière Hills lie on a general trend of the Lesser Antilles volcanics, whereas SSH volcanics define a separate trend. The Soufrière Hills and SSH volcanic centers were erupted at approximately the same time, but retain distinctive isotopic signatures, suggesting that the SSH magmas have a different source to the other volcanic centers. We hypothesize that this rapid magmatic source change is controlled by the regional transtensional regime, which allowed the SSH magma to be extracted from a shallower source. The Pb isotopes indicate an interplay between subduction derived components and a MORB-like mantle wedge influenced by a Galapagos plume-like source.

Submarine pyroclastic deposits formed during the 20th May 2006 dome collapse of the Soufrière Hills Volcano, Montserrat

The 20th May 2006 lava dome collapse of the Soufrière Hills Volcano, Montserrat, had a total non-dense rock equivalent (non-DRE) collapse volume of approximately 115 × 10 6 m 3. The majority of this volume was deposited into the ocean. The collapse was rapid, 85% of the mobilized volume being removed in just 35 min, giving peak pyroclastic flow flux of 66 × 10 3 m 3 s -1. Channel and levee facies on the submarine flanks of the volcano and formation of a thick, steep-sided ridge, suggest that the largest and most dense blocks were transported proximally as a high concentration granular flow. Of the submerged volume, 30% was deposited from the base of this granular flow, forming a linear, high-relief ridge that extends 7 km from shore. The remaining 70% of the submerged volume comprises the finer grain sizes, which were transported at least 40 km by turbidity currents on gradients of <2°. At several localities, the May 2006 distal turbidity currents ran up 200 m of topography and eroded up to 20 cm of underlying substrate. Multiple turbidites are preserved, representing current reflection from the graben margins and deflection around topography. The high energy of the May 2006 collapse resulted in longer submarine run out than the larger (210 × 10 6 m 3) Soufrière Hills dome collapse in July 2003.

Health status and use of health services among recently arrived men with refugee backgrounds: A comparative analysis of urban and regional settlement in South-east Queensland

Approximately one-third of refugee and humanitarian entrants to Australia are adult men. Many of these men and their families settle in regional areas. Little is known about the health status of refugee men and the use of health services, and whether or not there are differences between those living in urban and regional areas. This paper reports on the cross-sectional differences in health status and use of health services among a group of 233 recently arrived refugee men living in urban and regional areas of South-east Queensland. Overall, participants reported good levels of subjective health status, moderate to good levels of well-being, and low prevalence of mental illness. Men living in urban areas were more likely to have a longstanding illness and report poorer health status than those settled in regional areas. In contrast, men living in regional areas reported poorer levels of well-being in the environment domain and were more likely to visit hospital emergency departments. Targeted health promotion programs will ensure that refugee men remain healthy and develop their full potential as members of the Australian community. Programs that facilitate refugees’ access to primary health care in regional areas may promote more appropriate use of hospital emergency departments by these communities.

A multilevel investigation of inequalities in clinical and psychosocial outcomes for women after breast cancer

Background In Australia, breast cancer is the most common cancer affecting Australian women. Inequalities in clinical and psychosocial outcomes have existed for some time, affecting particularly women from rural areas and from areas of disadvantage. We have a limited understanding of how individual and area-level factors are related to each other, and their associations with survival and other clinical and psychosocial outcomes. Methods/Design This study will examine associations between breast cancer recurrence, survival and psychosocial outcomes (e.g. distress, unmet supportive care needs, quality of life). The study will use an innovative multilevel approach using area-level factors simultaneously with detailed individual-level factors to assess the relative importance of remoteness, socioeconomic and demographic factors, diagnostic and treatment pathways and processes, and supportive care utilization to clinical and psychosocial outcomes. The study will use telephone and self-administered questionnaires to collect individual-level data from approximately 3, 300 women ascertained from the Queensland Cancer Registry diagnosed with invasive breast cancer residing in 478 Statistical Local Areas Queensland in 2011 and 2012. Area-level data will be sourced from the Australian Bureau of Statistics census data. Geo-coding and spatial technology will be used to calculate road travel distances from patients' residence to diagnostic and treatment centres. Data analysis will include a combination of standard empirical procedures and multilevel modelling. Discussion The study will address the critical question of: what are the individual- or area-level factors associated with inequalities in outcomes from breast cancer? The findings will provide health care providers and policy makers with targeted information to improve the management of women with breast cancer, and inform the development of strategies to improve psychosocial care for women with breast cancer.

Spatial and temporal clusters of Barmah Forest virus disease in Queensland, Australia

Objective To identify the spatial and temporal clusters of Barmah Forest virus (BFV) disease in Queensland in Australia, using geographical information systems (GIS) and spatial scan statistic (SaTScan). Methods We obtained BFV disease cases, population and statistical local areas boundary data from Queensland Health and Australian Bureau of Statistics respectively during 1992-2008 for Queensland. A retrospective Poisson-based analysis using SaTScan software and method was conducted in order to identify both purely spatial and space-time BFV disease high-rate clusters. A spatial cluster size of a proportion of the population and a 200km circle radius and varying time windows from 1 month to 12 months were chosen (for the space-time analysis). Results The spatial scan statistic detected a most likely significant purely spatial cluster (including 23 SLAs) and a most likely significant space-time cluster (including 24 SLAs) in approximately the same location. Significant secondary clusters were also identified from both the analyses in several locations. Conclusions This study provides evidence of the existence of statistically significant BFV disease clusters in Queensland, Australia. The study also demonstrated the relevance and applicability of SaTScan in analysing on-going surveillance data to identify clusters to facilitate the development of effective BFV disease prevention and control strategies in Queensland, Australia.

Prevalence and prognostic significance of secondary lymphedema following breast cancer

Background The adverse consequences of lymphedema following breast cancer in relation to physical function and quality of life are clear; however, its potential relationship with survival has not been investigated. Our purpose was to determine the prevalence of lymphedema and associated upper-body symptoms at 6 years following breast cancer and to examine the prognostic significance of lymphedema with respect to overall 6-year survival (OS). Methods and Results A population-based sample of Australian women (n=287) diagnosed with invasive, unilateral breast cancer was followed for a median of 6.6 years and prospectively assessed for lymphedema (using bioimpedance spectroscopy [BIS], sum of arm circumferences [SOAC], and self-reported arm swelling), a range of upper-body symptoms, and vital status. OS was measured from date of diagnosis to date of death or last follow-up. Kaplan-Meier methods were used to calculate OS and Cox proportional hazards models quantified the risk associated with lymphedema. Approximately 45% of women had reported at least one moderate to extreme symptom at 6.6 years postdiagnosis, while 34% had shown clinical evidence of lymphedema, and 48% reported arm swelling at least once since baseline assessment. A total of 27 (9.4%) women died during the follow-up period, and lymphedema, diagnosed by BIS or SOAC between 6–18 months postdiagnosis, predicted mortality (BIS: HR=2.5; 95% CI: 0.9, 6.8, p=0.08; SOAC: 3.0; 95% CI: 1.1, 8.7, p=0.04). There was no association (HR=1.2; 95% CI: 0.5, 2.6, p=0.68) between self-reported arm swelling and OS. Conclusions These findings suggest that lymphedema may influence survival following breast cancer treatment and warrant further investigation in other cancer cohorts and explication of a potential underlying biology.

Food insecurity among Australian children : potential determinants, health and developmental consequences

Children in food-insecure households may be at risk of poor health, developmental or behavioural problems. This study investigated the associations between food insecurity, potential determinants and health and developmental outcomes among children. Data on household food security, socio-demographic characteristics and children’s weight, health and behaviour were collected from households with children aged 3–17 years in socioeconomically disadvantaged suburbs by mail survey using proxy-parental reports (185 households). Data were analysed using logistic regression. Approximately one-in-three households (34%) were food insecure. Low household income was associated with an increased risk of food insecurity [odds ratio (OR), 16.20; 95% confidence interval (CI), 3.52–74.47]. Children with a parent born outside of Australia were less likely to experience food insecurity (OR, 0.42; 95% CI, 0.19–0.93). Children in food-insecure households were more likely to miss days from school or activities (OR, 3.52; 95% CI, 1.46–8.54) and were more likely to have borderline or atypical emotional symptoms (OR, 2.44; 95% CI, 1.11–5.38) or behavioural difficulties (OR, 2.35; 95% CI, 1.04–5.33). Food insecurity may be prevalent among socioeconomically disadvantaged households with children. The potential developmental consequences of food insecurity during childhood may result in serious adverse health and social implications.

A parent-led family-focused treatment program for overweight children aged 5 to 9 years : The PEACH RCT

OBJECTIVE: To evaluate a healthy lifestyle intervention to reduce adiposity in children aged 5 to 9 years and assess whether adding parenting skills training would enhance this effect. PARTICIPANTS AND METHODS: We conducted a single-blinded randomized controlled trial of prepubertal moderately obese (International Obesity Task Force cut points) children, aged 5 to 9 years. The 6-month program targeted parents as the agents of change for implementing family lifestyle changes. Only parents attended group sessions. We measured BMI and waist z scores and parenting constructs at baseline, 6, 12, 18, 24 months. RESULTS: Participants (n = 169; 56% girls) were randomized to a parenting skills plus healthy lifestyle group (n = 85) or a healthy lifestyle–only group (n = 84). At final 24-month assessment 52 and 54 children remained in the parenting skills plus healthy lifestyle and the healthy lifestyle–only groups respectively. There were reductions (P < .001) in BMI z score (0.26 [95% confidence interval: 0.22–0.30]) and waist z score (0.33 [95% confidence interval: 0.26–0.40]). There was a 10% reduction in z scores from baseline to 6 months that was maintained to 24 months with no additional intervention. Overall, there was no significant group effect. A similar pattern of initial improvement followed by stability was observed for parenting outcomes and no group effect. CONCLUSIONS: Using approaches that specifically target parent behavior, relative weight loss of ∼10% is achievable in moderately obese prepubertal children and can be maintained for 2 years from baseline. These results justify an investment in treatment as an effective secondary obesity-prevention strategy.

Secular changes in BMI and obesity risk in Japanese children: Considerations from a morphologic perspective

The increasing prevalence of childhood obesity is a global health issue. Past studies in Japan have reported an increase in both body mass index (BMI) and risk of obesity among children and adolescents. However, changes in body size and proportion in this population over time have also influenced BMI. To date, no study of secular changes in childhood obesity has considered the impact of changes in morphological factors. The current study explored the secular changes in BMI and childhood obesity risk among Japanese children from 1950 to 2000 with consideration of changes in body size and the proportions using The Statistical Report of the School Health Survey (SHS). The age of peak velocity (PV) occurred approximately two years earlier in both genders across this period. While the increments in height, sitting height and sub-ischial leg length relative to height levelled off by 1980, weight gain continued in boys. Between 1980 and 2000, the rate of the upper body weight gain in boys and girls were 0.7-1.3 kg/decade and 0.2-1.0 kg/decade, respectively. After considering body proportions, increments in body weight were small. It could be suggested that the increments in weight and BMI across the 50-year period may be due to a combination of changes including the tempo of growth and body size due to lifestyle factors.

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.

A randomised controlled trial of a theory-based intervention to improve sun protective behaviour in adolescents ('you can still be HOT in the shade') : study protocol

Background: Most skin cancers are preventable by encouraging consistent use of sun protective behaviour. In Australia, adolescents have high levels of knowledge and awareness of the risks of skin cancer but exhibit significantly lower sun protection behaviours than adults. There is limited research aimed at understanding why people do or do not engage in sun protective behaviour, and an associated absence of theory-based interventions to improve sun safe behaviour. This paper presents the study protocol for a school-based intervention which aims to improve the sun safe behaviour of adolescents. Methods/design: Approximately 400 adolescents (aged 12-17 years) will be recruited through Queensland, Australia public and private schools and randomized to the intervention (n = 200) or 'wait-list' control group (n = 200). The intervention focuses on encouraging supportive sun protective attitudes and beliefs, fostering perceptions of normative support for sun protection behaviour, and increasing perceptions of control/self-efficacy over using sun protection. It will be delivered during three × one hour sessions over a three week period from a trained facilitator during class time. Data will be collected one week pre-intervention (Time 1), and at one week (Time 2) and four weeks (Time 3) post-intervention. Primary outcomes are intentions to sun protect and sun protection behaviour. Secondary outcomes include attitudes toward performing sun protective behaviours (i.e., attitudes), perceptions of normative support to sun protect (i.e., subjective norms, group norms, and image norms), and perceived control over performing sun protective behaviours (i.e., perceived behavioural control). Discussion: The study will provide valuable information about the effectiveness of the intervention in improving the sun protective behaviour of adolescents.

3D mesenchymal stem/stromal cell osteogenesis and autocrine signalling

Mesenchymal stem/stromal cells (MSC) are rapidly becoming a leading candidate for use in tissue regeneration, with first generation of therapies being approved for use in orthopaedic repair applications. Capturing the full potential of MSC will likely require the development of novel in vitro culture techniques and devices. Herein we describe the development of a straightforward surface modification of an existing commercial product to enable the efficient study of three dimensional (3D) human bone marrow-derived MSC osteogenic differentiation. Hundreds of 3D microaggregates, of either 42 or 168 cells each, were cultured in osteogenic induction medium and their differentiation was compared with that occurring in traditional two dimensional (2D) monolayer cultures. Osteogenic gene expression and matrix composition was significantly enhanced in the 3D microaggregate cultures. Additionally, BMP-2 gene expression was significantly up-regulated in 3D cultures at day 3 and 7 by approximately 25- and 30-fold, respectively. The difference in BMP-2 gene expression between 2D and 3D cultures was negligible in the more mature day 14 osteogenic cultures. These data support the notion that BMP-2 autocrine signalling is up-regulated in 3D MSC cultures, enhancing osteogenic differentiation. This study provides both mechanistic insight into MSC differentiation, as well as a platform for the efficient generation of microtissue units for further investigation or use in tissue engineering applications.

Properties of the bovine viral diarrhoea virus replicase in extracts of infected MDBK cells

An assay for the bovine viral diarrhoea virus (BVDV) replicase was developed using extracts from BVDV-infected cells. The replicase activity was maximal approximately 8 h post-infection as measured by the generation of a genomic length radiolabelled RNA. Using a semi-denaturing gel system, three virus-specific in vitro radiolabelled nascent RNA species were identified. A fast-migrating RNA was demonstrated to be the double-stranded replicative form (RF). A second form was shown to be a partially single-stranded/partially doublestranded RNA, characteristic of the replicative intermediate (RI). A third form, which was often undetectable, migrated between the RF and RI and was probably genomic viral RNA. The optimal replicase activity was dependent on 5–10mM Mg2+ and although it was also active in 1–2mM Mn2+ it was inhibited at higher concentrations. The optimum KCl concentration for labelling of the RI and RF were different, suggestive of at least two distinct replicase activities. These results are supportive of a semi-conservative model of BVDV RNA replication.