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.

A 3D collision avoidance strategy for UAVs in a non-cooperative environment

This paper presents a feasible 3D collision avoidance approach for fixed-wing unmanned aerial vehicles (UAVs). The proposed strategy aims to achieve the desired relative bearing in the horizontal plane and relative elevation in the vertical plane so that the host aircraft is able to avoid collision with the intruder aircraft in 3D. The host aircraft will follow a desired trajectory in the collision avoidance course and resume the pre-arranged trajectory after collision is avoided. The approaching stopping condition is determined for the host aircraft to trigger an evasion maneuver to avoid collision in terms of measured heading. A switching controller is designed to achieve the spatial collision avoidance strategy. Simulation results demonstrate that the proposed approach can effectively avoid spatial collision, making it suitable for integration into flight control systems of UAVs.

A comparison of security of tenure in Queensland and in Western Europe

Queensland residential tenancies are usually granted for up to 12 months with no guarantee of renewal. On expiration of the term, the landlord, without need to provide an explanation, can require the tenant to leave. Europeans find this unusual. As Hammar observes, to ‘never be sure whether ... you will be allowed to stay for another year ... is ok for a student, or for someone working ... but not for households’. This article informs Queensland policy makers and industry about European practices and concludes by proposing legislative amendments to realise the tenant’s security of tenure.

Normative influences across cultures : conceptual differences and potential confounders among drivers in Australia and China

Normative influences on road user behaviour have been well documented and include such things as personal, group, subjective and moral norms. Commonly, normative factors are examined within one cultural context, although a few examples of exploring the issue across cultures exist. Such examples add to our understanding of differences in perceptions of the normative factors that may exert influence on road users and can assist in determining whether successful road safety interventions in one location may be successful in another. Notably, the literature is relatively silent on such influences in countries experiencing rapidly escalating rates of motorization. China is one such country where new drivers are taking to the roads in unprecedented numbers and authorities are grappling with the associated challenges. This paper presents results from qualitative and quantitative research on self-reported driving speeds of car drivers and related issues in Australia and China. Focus group interviews and questionnaires conducted in each country examined normative factors that might influence driving in each cultural context. Qualitative findings indicated perceptions of community acceptance of speeding were present in both countries but appeared more widespread in China, yet quantitative results did not support this difference. Similarly, with regard to negative social feedback from speeding, qualitative findings suggested no embarrassment associated with speeding among Chinese participants and mixed results among Australian participants, yet quantitative results indicated greater embarrassment for Chinese drivers. This issue was also examined from the perspective of self-identity and findings were generally similar across both samples and appear related to whether it is important to be perceived as a skilled/safe driver by others. An interesting and important finding emerged with regard to how Chinese drivers may respond to questions about road safety issues if the answers might influence foreigners’ perceptions of China. In attempting to assess community norms associated with speeding, participants were asked to describe what they would tell a foreign visitor about the prevalence of speeding in China. Responses indicated that if asked by a foreigner, people may answer in a manner that portrayed China as a safe country (e.g., that drivers do not speed), irrespective of the actual situation. This ‘faking good for foreigners’ phenomenon highlights the importance of considering ‘face’ when conducting research in China – a concept absent from the road safety literature. An additional noteworthy finding that has been briefly described in the road safety literature is the importance and strength of the normative influence of social networks (guanxi) in China. The use of personal networks to assist in avoiding penalties for traffic violations was described by Chinese participants and is an area that could be addressed to strengthen the deterrent effect of traffic law enforcement. Overall, the findings suggest important considerations for developing and implementing road safety countermeasures in different cultural contexts.

Management characteristics and the decision to internationalize : exploration of exporters vs. non-exporters within the Chilean wine industry

The objective of this study was to identify key factors differentiating between exporters and non-exporters in the Chilean wine industry. Based on survey data collected from 61 wineries, the findings show that the main barriers for non-exporters are the lack of financial resources, limited quantities of stock for market expansion, management’s lack of knowledge and experience, and the high cost of travelling and participating in trade shows. The results also show that managers have educational levels and international experience exceeding those of other comparable New World wineries. Finally, in developing their main international markets, Chilean wineries did not target psychically close markets as identified in previous wine industry studies

When non-significance may be significant : lessons learned from a study of the development, implementation and evaluation of a fleet risk assessment tool

This paper reports on the development and implementation of a self-report risk assessment tool that was developed in an attempt to increase the efficacy of crash prediction within Australian fleet settings. This study forms a part of a broader program of research into work related road safety and identification of driving risk. The first phase of the study involved a series of focus groups being conducted with 217 professional drivers which revealed that the following factors were proposed to influence driving performance: Fatigue, Knowledge of risk, Mood, Impatience and frustration, Speed limits, Experience, Other road users, Passengers, Health, and Culture. The second phase of the study involved piloting the newly developed 38 item Driving Risk Assessment Scale - Work Version (DRAS-WV) with 546 professional drivers. Factor analytic techniques identified a 9 factor solution that was comprised of speeding, aggression, time pressure, distraction, casualness, awareness, maintenance, fatigue and minor damage. Speeding and aggressive driving manoeuvres were identified to be the most frequent aberrant driving behaviours engaged in by the sample. However, a series of logistic regression analyses undertaken to determine the DRAS-WV scale’s ability to predict self-reported crashes revealed limited predictive efficacy e.g., 10% of crashes. This paper outlines proposed reasons for this limited predictive ability of the DRAS-WV as well as provides suggestions regarding the future of research that aims to develop methods to identify “at risk” drivers.

A Systems-level approach to building sustainable assessment cultures : moderation, quality task design and dependability of judgement

This paper considers the conditions that are necessary at system and local levels for teacher assessment to be valid, reliable and rigorous. With sustainable assessment cultures as a goal, the paper examines how education systems can support local level efforts for quality learning and dependable teacher assessment. This is achieved through discussion of relevant research and consideration of a case study involving an evaluation of a cross-sectoral approach to promoting confidence in school-based assessment in Queensland, Australia. Building on the reported case study, essential characteristics for developing sustainable assessment cultures are presented, including: leadership in learning; alignment of curriculum, pedagogy and assessment; the design of quality assessment tasks and accompanying standards, and evidence-based judgement and moderation. Taken together, these elements constitute a new framework for building assessment capabilities and promoting quality assurance.

Provincialising Perth? : Satellite television and the Chinese in Perth

Relocated people habitually seek the familiar within their new social milieus as a means of easing their transition into ways strange and alien. However, from networks and food to everyday customs, efforts to hold on to such home comforts are often viewed with a measure of hostile disdain and regarded as preludes to the formation of ethnic enclaves. Through a pilot study on the access of satellite television by mainland Chinese migrants in Perth, Western Australia, this chapter seeks to shed some light on how migrants understand and employ these comforting strategies and how Chinese-Australians are using technology to achieve a sense of sanctuary from the views and habits of Australians while (re)constructing and articulating multiple belongings in their new homes.

Probability analysis of machine angle stability with non-gaussian wind power input

The security of power transfer across a given transmission link is typically a steady state assessment. This paper develops tools to assess machine angle stability as affected by a combination of faults and uncertainty of wind power using probability analysis. The paper elaborates on the development of the theoretical assessment tool and demonstrates its efficacy using single machine infinite bus system.

A new approach for trip generation estimation for TIA

Transport Impact Assessment (TIA) -Generally a short range transport planning activity -Assess transport impacts of new developments or expansions -Present solutions to mitigate impacts Problems with TIA Process -Private vehicles focus (i.e. Veh Trip Ends) -Proxy variables (e.g. 100sqm GFA) -Trip generation rates (e.g. VTE/proxy) -Little info/guidance on trip chaining effects -Little info/guidance on non-PV modes Requires significant professional judgment

Pleading for privilege : Denials and non-admissions under the UCPR

The decision in ASIC v Managed Investments Ltd No 3 [2012] QSC 74 provides practitioners with useful guidance on the relationship between the privileges against self-incrimination and exposure to a penalty, and the UCPR requirements for denials and non-admissions.

Nitric oxide synthase type-II is synthesized by human gingival tissue and cultured human gingival fibroblasts

Nitric oxide is known to be an important inflammatory mediator, and is implicated in the pathophysiology of a range of inflammatory disorders. The aim of this study was to determine the localization and distribution of endothelial NOS (NOS-II) in human gingival tissue, and to ascertain if human gingival fibroblasts express NOS-II when stimulated with interferon gamma (IFN-gamma) and bacterial lipopolysaccharide (LPS). The distribution of NOS-II in inflamed and non-inflamed specimens of human gingivae was studied using a monoclonal antibody against nitric oxide synthase II. Cultures of fibroblasts derived from healthy human gingivae were used for the cell culture experiments. The results from immunohistochemical staining of the tissues indicated an upregulation of NOS-II expression in inflamed compared to non-inflamed gingival tissue. Fibroblasts and inflammatory cells within the inflamed connective tissue were positively stained for NOS-II. In addition, basal keratinocytes also stained strongly for NOS-II, in both healthy and inflamed tissue sections. When cultured human gingival fibroblasts were stimulated by INF-gamma and Porphyromonas gingivalis LPS, NOS-II was more strongly expressed than when the cells were exposed to LPS or IFN-gamma alone. These data suggest that, as for other inflammatory diseases, NO plays a role in the pathophysiology of periodontitis.

The pedagogical, linguistic and content features of popular English language learning websites in China : a framework for analysis and design

As increasing numbers of Chinese language learners choose to learn English online (CNNIC, 2012), there is a need to investigate popular websites and their language learning designs. This paper reports on the first stage of a study that analysed the pedagogical, linguistic and content features of 25 Chinese English Language Learning (ELL) websites ranked according to their value and importance to users. The website ranking was undertaken using a system known as PageRank. The aim of the study was to identify the features characterising popular sites as opposed to those of less popular sites for the purpose of producing a framework for ELL website design in the Chinese context. The study found that a pedagogical focus with developmental instructional materials accommodating diverse proficiency levels was a major contributor to website popularity. Chinese language use for translations and teaching directives and intermediate level English for learning materials were also significant features. Content topics included Anglophone/Western and non-Anglophone/Eastern contexts. Overall, popular websites were distinguished by their mediation of access to and scaffolded support for ELL.

Interpretations of formative assessment in the teaching of English at two Chinese universities : a sociocultural perspective

Formative assessment is increasingly being implemented through policy initiatives in Chinese educational contexts. As an approach to assessment, formative assessment derives many of its key principles from Western contexts, notably through the work of scholars in the UK, the USA and Australia. The question for this paper is the ways that formative assessment has been interpreted in the teaching of College English in Chinese Higher Education. The paper reports on a research study that utilised a sociocultural perspective on learning and assessment to analyse how two Chinese universities – an urban-based Key University and a regional-based Non-Key University – interpreted and enacted a China Ministry of Education policy on formative assessment in College English teaching. Of particular interest for the research were the ways in which the sociocultural conditions of the Chinese context mediated understanding of Western principles and led to their adaptation. The findings from the two universities identified some consistency in localised interpretations of formative assessment which included emphases on process and student participation. The differences related to the specific sociocultural conditions contextualising each university including geographical location, socioeconomic status, and teacher and student roles, expectations and beliefs about English. The findings illustrate the sociocultural tensions in interpreting, adapting and enacting formative assessment in Chinese College English classes and the consequent challenges to and questions about retaining the spirit of formative assessment as it was originally conceptualised.