The interplay between chondrocyte redifferentiation pellet size and oxygen concentration

Chondrocytes dedifferentiate during ex vivo expansion on 2-dimensional surfaces. Aggregation of the expanded cells into 3-dimensional pellets, in the presence of induction factors, facilitates their redifferentiation and restoration of the chondrogenic phenotype. Typically 1×105–5×105 chondrocytes are aggregated, resulting in “macro” pellets having diameters ranging from 1–2 mm. These macropellets are commonly used to study redifferentiation, and recently macropellets of autologous chondrocytes have been implanted directly into articular cartilage defects to facilitate their repair. However, diffusion of metabolites over the 1–2 mm pellet length-scales is inefficient, resulting in radial tissue heterogeneity. Herein we demonstrate that the aggregation of 2×105 human chondrocytes into micropellets of 166 cells each, rather than into larger single macropellets, enhances chondrogenic redifferentiation. In this study, we describe the development of a cost effective fabrication strategy to manufacture a microwell surface for the large-scale production of micropellets. The thousands of micropellets were manufactured using the microwell platform, which is an array of 360×360 µm microwells cast into polydimethylsiloxane (PDMS), that has been surface modified with an electrostatic multilayer of hyaluronic acid and chitosan to enhance micropellet formation. Such surface modification was essential to prevent chondrocyte spreading on the PDMS. Sulfated glycosaminoglycan (sGAG) production and collagen II gene expression in chondrocyte micropellets increased significantly relative to macropellet controls, and redifferentiation was enhanced in both macro and micropellets with the provision of a hypoxic atmosphere (2% O2). Once micropellet formation had been optimized, we demonstrated that micropellets could be assembled into larger cartilage tissues. Our results indicate that micropellet amalgamation efficiency is inversely related to the time cultured as discreet microtissues. In summary, we describe a micropellet production platform that represents an efficient tool for studying chondrocyte redifferentiation and demonstrate that the micropellets could be assembled into larger tissues, potentially useful in cartilage defect repair.

Spatio-temporal modelling of ultrafine particle number concentration

This thesis developed semi-parametric regression models for estimating the spatio-temporal distribution of outdoor airborne ultrafine particle number concentration (PNC). The models developed incorporate multivariate penalised splines and random walks and autoregressive errors in order to estimate non-linear functions of space, time and other covariates. The models were applied to data from the "Ultrafine Particles from Traffic Emissions and Child" project in Brisbane, Australia, and to longitudinal measurements of air quality in Helsinki, Finland. The spline and random walk aspects of the models reveal how the daily trend in PNC changes over the year in Helsinki and the similarities and differences in the daily and weekly trends across multiple primary schools in Brisbane. Midday peaks in PNC in Brisbane locations are attributed to new particle formation events at the Port of Brisbane and Brisbane Airport.

Characteristics of spatial variation, size distribution, formation and growth of particles in urban environments

This thesis is the first comprehensive study of important parameters relating to aerosols' impact on climate and human health, namely spatial variation, particle size distribution and new particle formation. We determined the importance of spatial variation of particle number concentration in microscale environments, developed a method for particle size parameterisation and provided knowledge about the chemistry of new particle formation. This is a significant contribution to our understanding of processes behind the transformation and dynamics of urban aerosols. This PhD project included extensive measurements of air quality parameters using state of the art instrumentation at each of the 25 sites within the Brisbane metropolitan area and advanced statistical analysis.

GABAB receptors expressed in human aortic endothelial cells mediate intracellular calcium concentration regulation and endothelial nitric oxide synthase translocation

GABAB receptors regulate the intracellular Ca2+ concentration ([Ca2+]i) in a number of cells (e.g., retina, airway epithelium and smooth muscle), but whether they are expressed in vascular endothelial cells and similarly regulate the [Ca2+]i is not known. The purpose of this study was to investigate the expression of GABAB receptors, a subclass of receptors to the inhibitory neurotransmitter γ-aminobutyric acid (GABA), in cultured human aortic endothelial cells (HAECs), and to explore if altering receptor activation modified [Ca2+]i and endothelial nitric oxide synthase (eNOS) translocation. Real-time PCR, western blots and immunofluorescence were used to determine the expression of GABAB1 and GABAB2 in cultured HAECs. The effects of GABAB receptors on [Ca2+]i in cultured HAECs were demonstrated using fluo-3. The influence of GABAB receptors on eNOS translocation was assessed by immunocytochemistry. Both GABAB1 and GABAB2 mRNA and protein were expressed in cultured HAECs, and the GABAB1 and GABAB2 proteins were colocated in the cell membrane and cytoplasm. One hundred μM baclofen caused a transient increase of [Ca2+]i and eNOS translocation in cultured HAECs, and the effects were attenuated by pretreatment with the selective GABAB receptor antagonists CGP46381 and CGP55845. GABAB receptors are expressed in HAECs and regulate the [Ca2+]i and eNOS translocation. Cultures of HAECs may be a useful in vitro model for the study of GABAB receptors and vascular biology.

The unequal distribution of road trauma in China : understanding risk factors to improve public health

The World Health Organization identifies road trauma as a major public health issue in every country; most notably among low-to-middle income countries. More than 90% of all road fatalities occur in these countries, although they have only 48% of all registered vehicles [1]. Unprecedented focus has been placed on reducing the global road trauma burden through the United Nations Decade of Action for Road Safety (2011-2020). China is rapidly transitioning from a nation of bicycle riders and pedestrians to one where car ownership and use is increasing. This transition presents important public health, mobility, and safety challenges. Rapid motorisation has resulted in an increased road trauma burden, shouldered disproportionately among the population. Vulnerable road users (bicyclists, pedestrians, and motorcyclists) are of particular concern, representing 70% of all road-related fatalities [1]. Furthermore, those at greatest risk of sustaining a crash-related disability are: male, older, less educated, and earning a lower income [2] and residing in urban areas [3], with higher fatality rates in north-western poorer provinces [3]. Speeding is a key factor in road crashes in China [1, 4] and is one of two risk factors targeted in the Bloomberg Philanthropies-funded Global Road Safety Program operating in two Chinese cities over five year [5] to which the first author has provided expert advice. However, little evidence exists to help understand the factors underpinning speeding behaviour. Previous research conducted by the authors in Beijing and Hangzhou explored personal, social, and legal factors relating to speeding to assist in better understanding the motivations for non-compliance with speed limits. Qualitative and quantitative research findings indicated that speeding is relatively common, including self-reported travel speeds of greater than 30 km/hour above posted speed limits [6], and that the road safety laws and enforcement practices may, in some circumstances, contribute to this [7]. Normative factors were also evident; the role of friends, family members and driving instructors were influential. Additionally, using social networks to attempt to avoid detection and penalty was reported, thereby potentially reinforcing community perceptions that speeding is acceptable [8, 9]. The authors established strong collaborative links with the Chinese Academy of Sciences and Zhejiang Police College to conduct this research. The first author has worked in both institutions for extended time periods and recognises that research must include an understanding of culturally-relevant issues if road safety is to improve in China. Future collaborations to assist in enhancing our understanding of such issues are welcomed. References [1] World Health Organization. (2009). Global status report on road safety: Time for action; Geneva. [2] Chen, H., Du, W., & Li, N. (2013). The socioeconomic inequality in traffic-related disability among Chinese adults: the application of concentration index. Accident Analysis & Prevention, 55(101-106). [3] Wang, S. Y., Li, Y. H., Chi, G. B., Xiao, S. Y., Ozanne-Smith, J., Stevenson, M., & Phillips, M. (2008). Injury-related fatalities in China: an under-recognised public-health problem. The Lancet (British edition), 372(9651), 1765-1773. [4] He, J., King, M. J., Watson, B., Rakotonirainy, A., & Fleiter, J. J. (2013). Speed enforcement in China: National, provincial and city initiatives and their success. Accident Analysis & Prevention, 50, 282-288. [5] Bhalla, K., Li, Q., Duan, L., Wang, Y., Bishai, D., & Hyder, A. A. (2013). The prevalence of speeding and drink driving in two cities in China: a mid project evaluation of ongoing road safety interventions. Injury, 44, 49-56. doi:10.1016/S0020-1383(13)70213-4. [6] Fleiter, J. J., Watson, B., & Lennon, A. (2013). Awareness of risky behaviour among Chinese drivers. Peer-reviewed paper presented at 23rd Canadian Multidisciplinary Road Safety Conference, Montréal, Québec. [7] Fleiter, J. J., Watson, B., Lennon, A., King, M. J., & Shi, K. (2009). Speeding in Australia and China: A comparison of the influence of legal sanctions and enforcement practices on car drivers. Peer-reviewd paper presented at Australasian Road Safety Research Policing Education Conference, Sydney. [8] Fleiter, J. J., Watson, B., Lennon, A., King, M. J., & Shi, K. (2011). Social influences on drivers in China. Journal of the Australasian College of Road Safety, 22(2), 29-36. [9] Fleiter, J. J., Watson, B., Guan, M. Q., Ding, J. Y., & Xu, C. (2013). Characteristics of Chinese Drivers Attending a Mandatory Training Course Following Licence Suspension. Peer-reviewed paper presented at Road Safety on Four Continents, Beijing, China.

Metrological assessment of a portable analyzer for monitoring the particle size distribution of ultrafine particles

Adverse health effects caused by worker exposure to ultrafine particles have been detected in recent years. The scientific community focuses on the assessment of ultrafine aerosols in different microenvironments in order to determine the related worker exposure/dose levels. To this end, particle size distribution measurements have to be taken along with total particle number concentrations. The latter are obtainable through hand-held monitors. A portable particle size distribution analyzer (Nanoscan SMPS 3910, TSI Inc.) was recently commercialized, but so far no metrological assessment has been performed to characterize its performance with respect to well-established laboratory- based instruments such as the scanning mobility particle sizer (SMPS) spectrometer. The present paper compares the aerosol monitoring capability of the Nanoscan SMPS to the laboratory SMPS in order to evaluate whether the Nanoscan SMPS is suitable for field experiments designed to characterize particle exposure in different microenvironments. Tests were performed both in a Marple calm air chamber, where fresh diesel particulate matter and atomized dioctyl phthalate particles were monitored, and in microenvironments, where outdoor, urban, indoor aged, and indoor fresh aerosols were measured. Results show that the Nanoscan SMPS is able to properly measure the particle size distribution for each type of aerosol investigated, but it overestimates the total particle number concentration in the case of fresh aerosols. In particular, the test performed in the Marple chamber showed total concentrations up to twice those measured by the laboratory SMPS—likely because of the inability of the Nanoscan SMPS unipolar charger to properly charge aerosols made up of aggregated particles. Based on these findings, when field test exposure studies are conducted, the Nanoscan SMPS should be used in tandem

Spatial distribution of selected persistent organic pollutants (POPs) in Australia's atmosphere

A nation-wide passive air sampling campaign recorded concentrations of persistent organic pollutants in Australia's atmosphere in 2012. XAD-based passive air samplers were deployed for one year at 15 sampling sites located in remote/background, agricultural and semi-urban and urban areas across the continent. Concentrations of 47 polychlorinated biphenyls ranged from 0.73 to 72 pg m-3 (median of 8.9 pg m-3) and were consistently higher at urban sites. The toxic equivalent concentration for the sum of 12 dioxin-like PCBs was low, ranging from below detection limits to 0.24 fg m-3 (median of 0.0086 fg m-3). Overall, the levels of polychlorinated biphenyls in Australia were among the lowest reported globally to date. Among the organochlorine pesticides, hexachlorobenzene had the highest (median of 41 pg m-3) and most uniform concentration (with a ratio between highest and lowest value [similar]5). Bushfires may be responsible for atmospheric hexachlorobenzene levels in Australia that exceeded Southern Hemispheric baseline levels by a factor of [similar]4. Organochlorine pesticide concentrations generally increased from remote/background and agricultural sites to urban sites, except for high concentrations of [small alpha]-endosulfan and DDTs at specific agricultural sites. Concentrations of heptachlor (0.47-210 pg m-3), dieldrin (ND-160 pg m-3) and trans- and cis-chlordanes (0.83-180 pg m-3, sum of) in Australian air were among the highest reported globally to date, whereas those of DDT and its metabolites (ND-160 pg m-3, sum of), [small alpha]-, [small beta]-, [gamma]- and [small delta]-hexachlorocyclohexane (ND-6.7 pg m-3, sum of) and [small alpha]-endosulfan (ND-27 pg m-3) were among the lowest.

Serum bilirubin concentration is modified by UGT1A1 Haplotypes and influences risk of type-2 diabetes in the Norfolk Island Genetic Isolate

Background Located in the Pacific Ocean between Australia and New Zealand, the unique population isolate of Norfolk Island has been shown to exhibit increased prevalence of metabolic disorders (type-2 diabetes, cardiovascular disease) compared to mainland Australia. We investigated this well-established genetic isolate, utilising its unique genomic structure to increase the ability to detect related genetic markers. A pedigree-based genome-wide association study of 16 routinely collected blood-based clinical traits in 382 Norfolk Island individuals was performed. Results A striking association peak was located at chromosome 2q37.1 for both total bilirubin and direct bilirubin, with 29 SNPs reaching statistical significance (P < 1.84 × 10−7). Strong linkage disequilibrium was observed across a 200 kb region spanning the UDP-glucuronosyltransferase family, including UGT1A1, an enzyme known to metabolise bilirubin. Given the epidemiological literature suggesting negative association between CVD-risk and serum bilirubin we further explored potential associations using stepwise multivariate regression, revealing significant association between direct bilirubin concentration and type-2 diabetes risk. In the Norfolk Island cohort increased direct bilirubin was associated with a 28 % reduction in type-2 diabetes risk (OR: 0.72, 95 % CI: 0.57-0.91, P = 0.005). When adjusted for genotypic effects the overall model was validated, with the adjusted model predicting a 30 % reduction in type-2 diabetes risk with increasing direct bilirubin concentrations (OR: 0.70, 95 % CI: 0.53-0.89, P = 0.0001). Conclusions In summary, a pedigree-based GWAS of blood-based clinical traits in the Norfolk Island population has identified variants within the UDPGT family directly associated with serum bilirubin levels, which is in turn implicated with reduced risk of developing type-2 diabetes within this population.

Elevated concentration of TNF-a induces trophoblast differentiation in mouse blastocyst outgrowths

Elevated expression of tumour necrosis factora (TNF-a) is associated with adverse pregnancy outcome. This study has examined the expression of TNF-a and its receptors (TNF-Rs) by mouse blastocysts and blastocyst outgrowths from day 4 to 9.5 of pregnancy and investigated the effects of elevated TNF-a on the inner cell mass (ICM) and trophoblast cells of blastocyst outgrowths. RTPCR demonstrated TNF-a mRNA expression from day 7.5 to 9.5, TNF-R1 from day 6.5 to 9.5 and TNF-R2 from day 5.5 to 7.5 of pregnancy, and in situ hybridisation revealed the trophoblast giant cells (TGCs) of the early placenta as the site of TNF-a expression. Day 4 blastocysts were cultured in a physiologically high concentration of TNF-a (100 ng/ml) for 72 h to the outgrowth stage and then compared to blastocysts cultured in media alone. TNF-a-treated blastocyst outgrowths exhibited a significant reduction in ICM cells (mean € SD 23.90€10.42 vs 9.37€7.45, t-test, P<0.0001) with no significant change in the numbers of trophoblast cells (19.97€8.14 vs 21.73€7.79, t-test, P=0.39). Within the trophoblast cell population, the TNF-a-treated outgrowths exhibited a significant increase in multinucleated cells (14.10€5.53 vs 6.37€5.80, t-test, P<0.0001) and a corresponding significant decrease in mononucleated cells (5.87€3.60 vs 15.37€5.87, t-test, P<0.0001). In summary, this study describes the expression of TNF-a and its receptors during the peri-implantation period in the mouse. It also reports that elevated TNF-a restricts ICM proliferation in the blastocyst and changes the ratio of mononucleated to multinucleated trophoblast cells. These findings suggest a mechanism by which increased