Does the protection of vulnerable children require a system of mandatory reporting of abuse and neglect? [An issues paper for the New Zealand Government Green Paper for vulnerable children]

In this Issues Paper, I raise some key points relevant for any government which is considering its child protection and family welfare policy. In particular, I will raise questions about whether a form of legislative reporting duty is required, and if so, what consequences this has for child protection. The context of child maltreatment - and each form of maltreatment: physical abuse, sexual abuse, psychological or emotional abuse, and neglect - is extremely complex, and the overarching question of how to deal with these phenomena involve challenging normative, economic and practical questions. There are no easy or perfect solutions. Nor, often, is there the amount and quality of evidence available on which public policy approaches should be devised. However, from the best evidence about the history of this context, from research conducted in this field, and from the best evidence available about the nature, incidence and effects of different subtypes of maltreatment, some observations can be made which may help to inform deliberations. I outline 10 key issues related to mandatory reporting legislation while being mindful of the New Zealand context. My view, based on both research evidence and a concern to protect and promote children’s interests, and society’s interests, is that reporting laws in some form are necessary and can contribute substantially to child protection and enhancing family and community health and wellbeing. However, they are only one necessary part of a sound child protection system, being a method of tertiary and secondary prevention, and primary prevention efforts must also be prioritised. Moreover, it is essential that if a legislative reporting duty is enacted, it must be designed carefully and implemented soundly, and it must be integrated within a properly resourced child protection and family welfare system.

Assessment of flood-related recharge to alluvial aquifers of an irrigated catchment following extended drought conditions using 3D visualisation and environmental tracers, Lockyer Valley, southeast Queensland, Australia.

The Lockyer Valley in southeast Queensland, Australia, hosts an economically significant alluvial aquifer system which has been impacted by prolonged drought conditions (~1997 to ~ 2009). Throughout this time, the system was under continued groundwater extraction, resulting in severe aquifer depletion. By 2008, much of the aquifer was at <30% of storage but some relief occurred with rains in early 2009. However, between December 2010 and January 2011, most of southeast Queensland experienced unprecedented flooding, which generated significant aquifer recharge. In order to understand the spatial and temporal controls of groundwater recharge in the alluvium, a detailed 3D lithological property model of gravels, sands and clays was developed using GOCAD software. The spatial distribution of recharge throughout the catchment was assessed using hydrograph data from about 400 groundwater observation wells screened at the base of the alluvium. Water levels from these bores were integrated into a catchment-wide 3D geological model using the 3D geological modelling software GOCAD; the model highlights the complexity of recharge mechanisms. To support this analysis, groundwater tracers (e.g. major and minor ions, stable isotopes, 3H and 14C) were used as independent verification. The use of these complementary methods has allowed the identification of zones where alluvial recharge primarily occurs from stream water during episodic flood events. However, the study also demonstrates that in some sections of the alluvium, rainfall recharge and discharge from the underlying basement into the alluvium are the primary recharge mechanisms of the alluvium. This is indicated by the absence of any response to the flood, as well as the observed old radiocarbon ages and distinct basement water chemistry signatures at these locations. Within the 3D geological model, integration of water chemistry and time-series displays of water level surfaces before and after the flood suggests that the spatial variations of the flood response in the alluvium are primarily controlled by the valley morphology and lithological variations within the alluvium. The integration of time-series of groundwater level surfaces in the 3D geological model also enables the quantification of the volumetric change of groundwater stored in the unconfined sections of this alluvial aquifer during drought and following flood events. The 3D representation and analysis of hydraulic and recharge information has considerable advantages over the traditional 2D approach. For example, while many studies focus on singular aspects of catchment dynamics and groundwater-surface water interactions, the 3D approach is capable of integrating multiple types of information (topography, geological, hydraulic, water chemistry and spatial) into a single representation which provides valuable insights into the major factors controlling aquifer processes.

Inclusive housing in Australia – a voluntary response

The lack of inclusive housing in Australia contributes to the marginalization and exclusion of people with disability and older people from family and community life. The Australian government has handed over the responsibility of increasing the supply of inclusive housing to the housing industry through an agreed national access standard and a voluntary strategy. Voluntary strategies have not been successful in other constituencies and little is known about what would work in Australia today. Findings from a research project into the voluntariness of the housing industry indicate that a reliable and consistent supply is unlikely without an equivalent increase in demand. The strategy has, however, an important role to play in the task of changing housing industry practices towards building more inclusive communities.

The roles of the preproghrelin-derived peptides - ghrelin, desacyl ghrelin and obestatin - in prostate cancer

Prostate cancer is the second most common cause of cancer related deaths in Western men. Despite the significant improvements in current treatment techniques, there is no cure for advanced metastatic, castrate-resistant disease. Early detection and prevention of progression to a castrate-resistant state may provide new strategies to improve survival. A number of growth factors have been shown to act in an autocrine/paracrine manner to modulate prostate cancer tumour growth. Our laboratory has previously shown that ghrelin and its receptors (the functional GHS-R1a and the non-functional GHS-R1b) are expressed in prostate cancer specimens and cell lines. We have shown that ghrelin increases cell proliferation in the PC3 and LNCaP prostate cancer cell lines through activation of ERK1/2, suggesting that ghrelin could regulate prostate cancer cell growth and play a role in the progression of the disease. Ghrelin is a 28 amino-acid peptide hormone, identified to be the natural ligand of the growth hormone secretagogue receptor (GHS-R1a). It is well characterised as a growth hormone releasing and as an orexigenic peptide that stimulates appetite and feeding and regulates energy expenditure and bodyweight. In addition to its orexigenic properties, ghrelin has been shown to play a regulatory role in a number of systems, including the reproductive, immune and cardiovascular systems and may play a role in a number of pathological conditions such as chronic heart failure, anorexia, cachexia, obesity, diabetes and cancer. In cancer, ghrelin and its receptor are expressed in a range of tumours and cancer cell lines and ghrelin has been demonstrated to modulate cell proliferation, apoptosis, migration and invasion in some cell types. The ghrelin gene (GHRL) encodes preproghrelin peptide, which is processed to produce three currently known functional peptides - ghrelin, desacyl ghrelin and obestatin. Prohormone convertases (PCs) have been shown to cleave the preproghrelin peptide into two primary products - the 28 amino acid peptide, ghrelin, and the remaining 117 amino acid C-terminal peptide, C-ghrelin. C-ghrelin can then be further processed to produce the 23 amino acid peptide, obestatin. Ghrelin circulates in two different forms - an octanoylated form (known as ghrelin) and a non-octanoylated form, desacyl ghrelin. The unique post-translational addition of octanoic acid to the serine 3 residue of the propeptide chain to form acylated ghrelin is catalysed by ghrelin O-acyltransferase (GOAT). This modification is necessary for binding of ghrelin to its only known functional receptor, the GHS-R1a. As desacyl ghrelin cannot bind and activate the GHS-R1a, it was initially thought to be an inactive peptide, despite the fact that it circulates at much higher levels than ghrelin. Further research has demonstrated that desacyl ghrelin is biologically active and shares some of the actions of ghrelin, as well as having some opposing and distinct roles. Interestingly, both ghrelin and desacyl ghrelin have been shown to modulate apoptosis, cell differentiation and proliferation in some cell types, and to stimulate cell proliferation through activation of ERK1/2 and PI3K/Akt pathways. The third known peptide product of the ghrelin preprohormone, obestatin, was initially thought to oppose the actions of ghrelin in appetite regulation and food intake and to mediate its effects through the G protein-coupled receptor 39 (GPR39). Subsequent research failed to reproduce the initial findings, however, and the possible anorexigenic effects of obestatin, as well as the identity of its receptor, remain unclear. Obestatin plays some important physiological roles, including roles in improving memory, the inhibition of thirst and anxiety, increased secretion of pancreatic juice, and regulation of cell proliferation, survival, apoptosis and differentiation. Preliminary studies have also shown that obestatin stimulates cell proliferation in some cell types through activation of ERK1/2, Akt and PKC pathways. Overall, however, at the commencement of this PhD project, relatively little was known regarding the functions and mechanisms of action of the preproghrelin-derived functional peptides in modulating prostate cancer cell proliferation. The roles of obestatin, and desacyl ghrelin as potential growth factors had not previously been investigated, and the potential expression and regulation of the preproghrelin processing enzymes, GOAT and prohormone convertases was unknown in prostate cancer cell lines. Therefore, the overall objectives of this study were to: 1. investigate the effects of obestatin on cell proliferation and signaling in prostate cancer cell lines 2. compare the effects of desacyl ghrelin and ghrelin on cell proliferation and signaling in prostate cancer cell lines 3. investigate whether prostate cancer cell lines possess the necessary enzymatic machinery to produce ghrelin and desacyl ghrelin and if these peptides can regulate GOAT expression Our laboratory has previously shown that ghrelin stimulates cell proliferation in the PC3 and LNCaP prostate cancer cell line through activation of the ERK1/2 pathway. In this study it has been demonstrated that treatments with either ghrelin, desacyl ghrelin or obestatin over 72 hours significantly increased cell proliferation in the PC3 prostate cancer cell line but had no significant effect in the RWPE-1 transformed normal prostate cell line. Ghrelin (1000nM) stimulated cell proliferation in the PC3 prostate cancer cell line by 31.66 6.68% (p<0.01) with the WST-1 method, and 13.55 5.68% (p<0.05) with the CyQUANT assay. Desacyl ghrelin (1000nM) increased cell proliferation in PC3 cells by 21.73 2.62% (p<0.01) (WST-1), and 15.46 7.05% (p<0.05) (CyQUANT) above untreated control. Obestatin (1000nM) induced a 28.37 7.47% (p<0.01) (WST-1) and 12.14 7.47% (p<0.05) (CyQUANT) significant increase in cell proliferation in the PC3 prostate cancer cell line. Ghrelin and desacyl ghrelin treatments stimulated Akt and ERK phosphorylation across a range of concentrations (p<0.01). Obestatin treatment significantly stimulated Akt, ERK and PKC phosphorylation (p<0.05). Through the use of specific inhibitors, the MAPK inhibitor U0126 and the Akt1/2 kinase inhibitor, it was demonstrated that ghrelin- and obestatin-induced cell proliferation in the PC3 prostate cancer cell line is mediated through activation of ERK1/2 and Akt pathways. Although desacyl ghrelin significantly stimulated Akt and ERK phosphorylation, U0126 failed to prevent desacyl ghrelin-induced cell proliferation suggesting ghrelin and desacyl ghrelin might act through different mechanisms to increase cell proliferation. Ghrelin and desacyl ghrelin have shown a proliferative effect in osteoblasts, pancreatic -cells and cardiomyocytes through activation of ERK1/2 and PI3K/Akt pathways. Here it has been shown that ghrelin and its non-acylated form exert the same function and stimulate cell proliferation in the PC3 prostate cancer cell line through activation of the Akt pathway. Ghrelin-induced proliferation was also mediated through activation of the ERK1/2 pathway, however, desacyl ghrelin seems to stimulate cell proliferation in an ERK1/2-independent manner. As desacyl ghrelin does not bind and activate GHSR1a, the only known functional ghrelin receptor, the finding that both ghrelin and desacyl ghrelin stimulate cell proliferation in the PC3 cell line suggests that these peptides could be acting through the yet unidentified alternative ghrelin receptor in this cell type. Obestatin treatment also stimulated PKC phosphorylation, however, a direct role for this pathway in stimulating cell proliferation could not be proven using available PKC pathway inhibitors, as they caused significant cell death over the extended timeframe of the cell proliferation assays. Obestatin has been shown to stimulate cell proliferation through activation of PKC isoforms in human retinal epithelial cells and in the human gastric cancer cell line KATO-III. We have demonstrated that all of the prostate-derived cell lines examined (PC3, LNCaP, DU145, 22Rv1, RWPE-1 and RWPE-2) expressed GOAT and at least one of the prohormone convertases, which are known to cleave the proghrelin peptide, PC1/3, PC2 and furin, at the mRNA level. These cells, therefore, are likely to possess the necessary machinery to cleave the preproghrelin protein and to produce the mature ghrelin and desacyl ghrelin peptides. In addition to prohormone convertases, the presence of octanoic acid is essential for acylated ghrelin production. In this study octanoic acid supplementation significantly increased cell proliferation in the PC3 prostate cancer cell line by over 20% compared to untreated controls (p<0.01), but surprisingly, not in the DU145, LNCaP or 22Rv1 prostate cancer cell lines or in the RWPE-1 and RWPE-2 prostate-derived cell lines. In addition, we demonstrated that exogenous ghrelin induced a statistically significant two-fold decrease in GOAT mRNA expression in the PC3 cell line (p<0.05), suggesting that ghrelin could pontentially downregulate its own acylation and, therefore, regulate the balance between ghrelin and desacyl ghrelin. This was not observed, however, in the DU145 and LNCaP prostate cancer cell lines. The GOAT-ghrelin system represents a direct link between ingested nutrients and regulation of ghrelin production and the ghrelin/desacyl ghrelin ratio. Regulation of ghrelin acylation is a potentially attractive and desirable tool for the development of better therapies for a number of pathological conditions where ghrelin has been shown to play a key role. The finding that desacyl ghrelin stimulates cell proliferation in the PC3 prostate cancer cell line, and responds to ghrelin in the same way, suggests that this cell line expresses an alternative ghrelin receptor. Although all the cell lines examined expressed both GHS-R1a and GHS-R1b mRNA, it remains uncertain whether these cell lines express the unidentified alternative ghrelin receptor. It is possible that the varied responses seen could be due to the expression of different ghrelin receptors in different cell lines. In addition to GOAT, prohormone convertases and octanoic acid availability may regulate the production of different peptides from the ghrelin preprohormone. The studies presented in this thesis provide significant new information regarding the roles and mechanisms of action of the preproghrelin-derived peptides, ghrelin, desacyl ghrelin and obestatin, in modulating prostate cancer cell line proliferation. A number of key questions remain to be resolved, however, including the identification of the alternative ghrelin/desacyl ghrelin receptor, the identification of the obestatin receptor, a clarification of the signaling mechanisms which mediate cell proliferation in response to obestatin treatment and a better understanding of the regulation at both the gene and post-translational levels of functional peptide generation. Further studies investigating the role of the ghrelin axis using in vivo prostate cancer models may be warranted. Until these issues are determined, the potential for the ghrelin axis, to be recognised as a novel useful target for therapy for cancer or other pathologies will be uncertain.

Determination of coastal ground and surface water processes and character by use of hydrochemistry and stable isotopes, Fraser Coast, Queensland

This study was part of an integrated project developed in response to concerns regarding current and future land practices affecting water quality within coastal catchments and adjacent marine environments. Two forested coastal catchments on the Fraser Coast, Australia, were chosen as examples of low-modification areas with similar geomorphological and land-use characteristics to many other coastal zones in southeast Queensland. For this component of the overall project, organic , physico-chemical (Eh, pH and DO), ionic (Fe2+, Fe3+), and isotopic (ä13CDIC, ä15NDIN ä34SSO4) data were used to characterise waters and identify sources and processes contributing to concentrations and form of dissolved Fe, C, N and S within the ground and surface waters of these coastal catchments. Three sites with elevated Fe concentrations are discussed in detail. These included a shallow pool with intermittent interaction with the surface water drainage system, a monitoring well within a semi-confined alluvial aquifer, and a monitoring well within the fresh/saline water mixing zone adjacent to an estuary. Conceptual models of processes occurring in these environments are presented. The primary factors influencing Fe transport were; microbial reduction of Fe3+ oxyhydroxides in groundwaters and in the hyporheic zone of surface drainage systems, organic input available for microbial reduction and Fe3+ complexation, bacterial activity for reduction and oxidation, iron curtain effects where saline/fresh water mixing occurs, and variation in redox conditions with depth in ground and surface water columns. Data indicated that groundwater seepage appears a more likely source of Fe to coastal waters (during periods of low rainfall) via tidal flux. The drainage system is ephemeral and contributes little discharge to marine waters. However, data collected during a high rainfall event indicated considerable Fe loads can be transported to the estuary mouth from the catchment.

Smart grid-demand side response model for optimization air conditioning

The growing demand of air-conditioning is one of the largest contributors to Australia’s overall electricity consumption. This has started to create peak load supply problems for some electricity utilities particularly in Queensland. This research aimed to develop consumer demand side response model to assist electricity consumers to mitigate peak demand on the electrical network. The model developed demand side response model to allow consumers to manage and control air conditioning for every period, it is called intelligent control. This research investigates optimal response of end-user toward electricity price for several cases in the near future, such as: no spike, spike and probability spike price cases. The results indicate the potential of the scheme to achieve energy savings, reducing electricity bills (costs) to the consumer and targeting best economic performance for electrical generation distribution and transmission.

Toward a shopping typology of primary male grocery shoppers

Purpose – The purpose of this paper is to segment primary male grocery shoppers based on store and product attribute evaluations. A rich profile for each segment is developed. These developed contemporary shopper typologies are contrasted against earlier works. Design/methodology/approach – Data of 280 male grocery shoppers was attained by a survey questionnaire. Factor analysis, cluster analysis and ANOVA were employed to develop specific segments of male shoppers. Findings – Four distinct cohorts of male shoppers emerge from the data of eight constructs, measured by 46 items. One new shopper type, not found in earlier typology literature, emerged from this research. This shopper presented as young, well educated, at the commencement of their career and family lifecycle, attracted by a strong value offer and willingness to share the family food shopping responsibilities. Practical implications – Research outcomes encourage supermarket retailers to implement targeted marketing and rationalized operational strategies that deliver on attributes of importance. Originality/value – This research makes a contribution to segmentation literature and grocery retail practice in several ways. It presents the first retail typology of male supermarket shoppers, employing a cluster analysis technique. The research provides insights into the modern family food shopping behaviour of men, a channel in which men are now recognised as equal contributors. The research provides the basis for further gender comparative and cross-contextual studies.

Perceptions of clinical safety climate of the multicultural nursing workforce in Saudi Arabia : a cross-sectional survey

Purpose The purpose of this study is to explore the safety climate perceptions of the multicultural nursing workforce, and to investigate the influence of diversity of the multicultural nursing workforce on clinical safety in a large tertiary hospital in Saudi Arabia. Background Working in a multicultural environment is challenging. Each culture has its own unique characteristics and dimensions that shape the language, lifestyle, beliefs, values, customs, traditions, and patterns of behaviour, which expatriate nurses must come to terms with. However, cultural diversity in the health care environment can potentially affect the quality of care and patient safety. Method A mixed-method case study (survey, interview and document analysis) was employed. A primary study phase entailed the administration of the Safety Climate Survey (SCS). A population sampling strategy was used and 319 nurses participated, yielding a 76.8% response rate. Descriptive and inferential statistics (Kruskal–Wallis test) were used to analyse survey data. Results The data revealed the nurses’ perceptions of the clinical safety climate in this multicultural environment was unsafe, with a mean score of 3.9 out of 5. No significant difference was found between the age groups, years of nursing experience and their perceptions of the safety climate in this context. A significant difference was observed between the national background categories of nurses and perceptions of safety climate. Conclusion Cultural diversity within the nursing workforce could have a significant influence on perceptions of clinical safety. These findings have the potential to inform policy and practice related to cultural diversity in Saudi Arabia.

Waving or drowning? Managing resource constraints in entrepreneurial firm with bricolage as a response to the global financial crisis

These are challenging times for new entrepreneurial firms. The development of the Global Financial Crisis shook the very foundations of global markets and institutions that most firms relied on to do business (Claessens, et al., forthcoming). In the midst of institutional flux and resource constraints, entrepreneurial firms, which have been shown to make a range of contributions to the economy (van Praag & Versloot 2007) faced increasing constraints. The Australian Federal Government quickly implemented the Green Loan program in response to the financial crisis. Unfortunately, the green loans program was flawed with obsolete processes and information (Faulkner, 2011), further constraining new firms. Prior research provides few clues regarding how resource-constrained entrepreneurial firms deal with these institutional flaws within institutional change and how they might overcome these challenges and prosper. One promising theory that evaluates behavioural responses to constraints and institutional flaws is bricolage (Levi Strauss, 1967). Bricolage aligns with notions of resourcefulness: defined here as “making do by applying combinations of the resources at hand to new problems and opportunities” (Baker and Nelson 2005: 333). Using three case studies, we consider how institutional flaws impact firm behaviours and illustrate the use of bricolage in attempts to reinforce, shape and change the GL program further extending bricolage domains of Baker and Nelson (2005).

Measurement of in-service insulated rail joint tie plate response

This paper presents the results of a recent investigation into Insulated Rail Joint Tie Plate fatigue failures. In particular it focuses on the results of data obtained through field strain gauge and accelerometer measurements of in-service Insulated Rail Joint Tie Plates. These measurements have identified a significant variability in the strains present in similar joints operating under identical load conditions. This variability in stress invariably has a significant influence on the life of the joints. The results of rainflow counting and a fatigue analysis are also presented.

Literacy in Early Childhood and Primary Education : Issues, Challenges and Solutions

Literacy in Early Childhood and Primary Education provides a comprehensive introduction to literacy teaching and learning. The book explores the continuum of literacy learning and children’s transitions from early childhood settings to junior primary classrooms, and then to senior primary and beyond. Reader-friendly and accessible, this book equips pre-service teachers with the theoretical underpinnings and practical strategies and skills needed to teach literacy. It places the ‘reading wars’ firmly in the past as it examines contemporary research and practices. The book covers important topics such as literacy acquisition, family literacies and multiliteracies, foundation skills for literacy learning, reading difficulties, assessment, and supporting diverse literacy learners in early childhood and primary classrooms. It also addresses some of the challenges that teachers may face in the classroom and provides solutions to these. Each chapter includes learning objectives, reflective questions and definitions to key terms to engage and assist readers. Further resources are also available at www.cambridge.edu.au/academic/literacy. Written by an expert author team and featuring real-world examples from literacy teachers and learners. Literacy in Early Childhood and Primary Education will help pre-service teachers feel confident teaching literacy to diverse age groups and abilities.

The intraocular pressure response to dehydration : a pilot study

This study was designed to determine the Intraocular Pressure (IOP) response to differing levels of dehydration. Seven males participated in a 90 minute treadmill walk (5 km/h and 1 % grade) in both a cool (22 °C) and hot (43 °C) climate. At Baseline and at 30 minute intervals measurements of IOP, by tonometery, and indicators of hydration status (nude weight and plasma osmolality (Posm)) were taken. Body temperature and heart rate were also measured at these time points. Statistically significant interactions (time point (4) by trial (2)) were observed for IOP (F = 10.747, p = 0.009) and body weight loss (F = 50.083, p < 0.001) to decrease, and Posm (F = 34.867, p < 0.001) to increase, by a significantly greater amount during the hot trial compared to the cool. A univariate general linear model showed a significant relationship between IOP and body weight loss (F = 37.63, p < 0.001) and Posm (F = 38.53, p < 0.001). A significant interaction was observed for body temperature (F = 20.908, p < 0.001) and heart rate (F = 25.487, p < 0.001) between the trials and time points, but there was negligible association between these variables and IOP (Pearson correlation coefficient < ±0.5). The present study provides evidence to suggest that IOP is influenced by hydration status.

Indoor and outdoor particle concentrations and elemental and organic carbon at 17 primary schools

The focus of this paper is on the measured particle number concentrations (PNC) as well as elemental and organic carbon in 17 primary schools. This study is part of the “Ultrafine Particles from Traffic Emissions and Children’s Health (UPTECH)”, which aims to determine the relationship between exposure to traffic related ultrafine (UF) particles and children’s health (http://www.ilaqh.qut.edu.au/Misc/UPTECH%20Home.htm). To achieve this, air quality and health data are being collected at 25 schools within Brisbane Metropolitan Area in Australia over two years. This paper presents the general aspects of UF particles data and preliminary results from the first 17 schools (S01 to S17), tested from Oct 2010 to Dec 2011.

Networked reading : using AustLit to assist reading and understanding of texts from the past

In response to a focus on reading, this paper examines the notion of reading online; as such it uses the term ‘networked reading’ to describe any act of reading in an online or digital environment. In accordance with this notion of ‘networked’ reading, the paper provides a broad introduction to AustLit: the Australian Literature Resource. This is followed by an examination of a suite of services and digital tools (LORE) developed by the Aus-e-Lit project that extends the scope of AustLit records and facilitates links to external resources. The focus of the final section of the paper is on a collection of Full Text resources located within the AustLit subset Children’s Literature Digital Resources (CLDR). It proposes a number of ways in which these texts, and an accompanying anthology of critical articles, can be utilised in classrooms across the Primary, Middle and Senior School spectrum.

In vitro and in vivo studies on protease-activated receptor-2

Protease-activated receptor-2 (PAR2) is a G protein coupled receptor (GPCR) that is activated by proteolytic cleavage of its amino terminal domain by trypsin-like serine proteases. Cleavage of this receptor exposes a neoepitope, termed the tethered ligand (TL), which binds intramolecularly within the receptor to stimulate signal transduction via coupled G proteins. PAR2-mediated signal transduction is also experimentally stimulated by hexapeptides (agonist peptides; APs) that are homologous to the TL sequence. Due to the irreversible nature of PAR2 proteolysis, downstream signal transduction is tightly regulated. Following activation, PAR2 is rapidly uncoupled from downstream signalling by the post-translational modifications phosphorylation and ubiquination which facilitate interactions with â- arrestin. This scaffolding protein couples PAR2 to the internalisation machinery initiating its desensitisation and trafficking through the early and late endosomes followed by receptor degradation. PAR2 is widely expressed in mammalian tissues with key roles for this receptor in cardiovascular, respiratory, nervous and musculoskeletal systems. This receptor has also been linked to pathological states with aberrant expression and signalling noted in several cancers. In prostate cancer, PAR2 signalling induces migration and proliferation of tumour derived cell lines, while elevated receptor expression has been noted in malignant tissues. Importantly, a role for this receptor has also been suggested in prostate cancer bone metastasis as coexpression of PAR2 and a proteolytic activator has been demonstrated by immunohistochemical analysis. Based on these data, the primary focus of this project has been on two aspects of PAR2 biology. The first is characterisation of cellular mechanisms that regulate PAR2 signalling and trafficking. The second aspect is the role of this receptor in prostate cancer bone metastasis. In addition, to permit these studies, it was first necessary to evaluate the specificity of the commercially available anti-PAR2 antibodies SAM11, C17, N19 and H99. The evaluation of the four commercially available antibodies was assessed using four techniques: immunoprecipitation; Western blot analysis; immunofluorescence; and flow cytometry. These approaches demonstrated that three of the antibodies efficiently detect ectopically expressed PAR2 by each of these techniques. A significant finding from this study was that N19 was the only antibody able to specifically detect N-glycosylated endogenous PAR2 by Western blot analysis. This analysis was performed on lysates from prostate cancer derived cell lines and tissue derived from wildtype and PAR2 knockout mice. Importantly, further evaluation demonstrated that this antibody also efficiently detects endogenous PAR2 at the cell surface by flow cytometry. The anti-PAR2 antibody N19 was used to explore the in vitro role of palmitoylation, the post-translational addition of palmitate, in PAR2 signalling, trafficking, cell surface expression and desensitization. Significantly, use of the palmitoylation inhibitor 2-bromopalmitate indicated that palmitate addition is important in trafficking of PAR2 endogenously expressed by prostate cancer cell lines. This was supported by palmitate labelling experiments using two approaches which showed that PAR2 stably expressed by CHO cells is palmitoylated and that palmitoylation occurs on cysteine 361. Another key finding from this study is that palmitoylation is required for optimal PAR2 signalling as Ca2+ flux assays indicated that in response to trypsin agonism, palmitoylation deficient PAR2 is ~9 fold less potent than wildtype receptor with a reduction of about 33% in the maximum signal induced via the mutant receptor. Confocal microscopy, flow cytometry and cell surface biotinylation analyses demonstrated that palmitoylation is required for efficient cell surface expression of PAR2. Importantly, this study also identified that palmitoylation of this receptor within the Golgi apparatus is required for efficient agonist-induced rab11amediated trafficking of PAR2 to the cell surface. Interestingly, palmitoylation is also required for receptor desensitization, as agonist-induced â-arrestin recruitment and receptor degradation were markedly reduced in CHO-PAR2-C361A cells compared with CHO-PAR2 cells. Collectively, these data provide new insights on the life cycle of PAR2 and demonstrate that palmitoylation is critical for efficient signalling, trafficking, cell surface localization and degradation of this receptor. This project also evaluated PAR2 residues involved in ligand docking. Although the extracellular loop (ECL)2 of PAR2 is known to be required for agonist-induced signal transduction, the binding pocket for receptor agonists remains to be determined. In silico homology modelling, based on a crystal structure for the prototypical GPCR rhodopsin, and ligand docking were performed to identify PAR2 transmembrane (TM) amino acids potentially involved in agonist binding. These methods identified 12 candidate residues that were mutated to examine the binding site of the PAR2 TL, revealed by trypsin cleavage, as well as of the soluble ligands 2f-LIGRLO-NH2 and GB110, which are both structurally based on the AP SLIGRLNH2. Ligand binding was evaluated from the impact of the mutated residues on PAR2-mediated calcium mobilisation. An important finding from these experiments was that mutation of residues Y156 and Y326 significantly reduced 2f-LIGRLO-NH2 and GB110 agonist activity. L307 was also important for GB110 activity. Intriguingly, mutation of PAR2 residues did not alter trypsin-induced signalling to the same extent as for the soluble agonists. The reason for this difference remains to be further examined by in silico and in vitro experimentation and, potentially, crystal structure studies. However, these findings identified the importance of TM domains in PAR2 ligand docking and will enhance the design of both PAR2 agonists and potentially agents to inhibit signalling (antagonists). The potential importance of PAR2 in prostate cancer bone metastasis was examined using a mouse model. In patients, prostate cancer bone metastases cause bone growth by disrupting bone homeostasis. In an attempt to mimic prostate cancer growth in bone, PAR2 responsive 22Rv1 prostate cancer cells, which form mixed osteoblastic and osteolytic lesions, were injected into the proximal aspect of mouse tibiae. A role for PAR2 was assessed by treating these mice with the recently developed PAR2 antagonist GB88. As controls, animals bearing intra-tibial tumours were also treated with vehicle (olive oil) or the prostate cancer chemotherapeutic docetaxel. The effect of these treatments on bone was examined radiographically and by micro-CT. Consistent with previous studies, 22Rv1 tumours caused osteoblastic periosteal spicule formation and concurrent osteolytic bone loss. Significantly, blockade of PAR2 signalling reduced the osteoblastic and osteolytic phenotype of 22Rv1 tumours in bone. No bone defects were detected in mice treated with docetaxel. These qualitative data will be followed in the future by quantitative micro-CT analysis as well as histology and histomorphometry analysis of already collected tissues. Nonetheless, these preliminary experiments highlight a potential role for PAR2 in prostate cancer growth in bone. In summary, in vitro studies have defined mechanisms regulating PAR2 activation, downstream signalling and trafficking and in vivo studies point to a potential role for this receptor in prostate cancer bone metastasis. The outcomes of this project are that a greater understanding of the biology of PAR2 may lead to the development of strategies to modulate the function of this receptor in disease.