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.

Modeling resource management in the building design process by information constraint Petri nets

The effect of resource management on the building design process directly influences the development cycle time and success of construction projects. This paper presents the information constraint net (ICN) to represent the complex information constraint relations among design activities involved in the building design process. An algorithm is developed to transform the information constraints throughout the ICN into a Petri net model. A resource management model is developed using the ICN to simulate and optimize resource allocation in the design process. An example is provided to justify the proposed model through a simulation analysis of the CPN Tools platform in the detailed structural design. The result demonstrates that the proposed approach can obtain the resource management and optimization needed for shortening the development cycle and optimal allocation of resources.

Food for thought : designing for critical reflection on food practices

This workshop is a continuation and extension to the successful past workshops including [4, 5, 6]. The workshop addresses the opportunities and challenges for the design of digital interactive systems that engage individuals in critical reflection on their everyday food practices - including designing for engagement in more environmentally aware, socially inclusive, and healthier behaviour. These three themes represent the focus of much recent HCI work related to food. The workshop aims to further the conversation on these themes through understanding specifically how the process of critical reflection can be encouraged by interactive technology. While the focus will be on food as an application area, the intention is to also explore, more generally, how the process of critical reflection can be facilitated through interactive technology. The workshop provides a unique forum to discuss existing theoretical and pragmatic approaches, and to envision novel ways to design technology that encourages sustained critical reflection.

Daylighting performance of subtropical multi-residential towers : simulations tools for design decisions

During an intensive design-led workshop multidisciplinary design teams examined options for a sustainable multi-residential tower on an inner urban site in Brisbane (Australia). The main aim was to demonstrate the key principles of daylight to every habitable room and cross-ventilation to every apartment in the subtropical climate while responding to acceptable yield and price points. The four conceptual design proposals demonstrated a wide range of outcomes, with buildings ranging from 15 to 30 storeys. Daylight Factor (DF), view to the outside, and the avoidance of direct sunlight were the only quantitative and qualitative performance metrics used to implement daylighting to the proposed buildings during the charrette. This paper further assesses the daylighting performance of the four conceptual designs by utilizing Climate-based daylight modeling (CBDM), specifically Daylight Autonomy (DA) and Useful Daylight Illuminance (UDI). Results show that UDI 100-2000lux calculations provide more useful information on the daylighting design than DF. The percentage of the space with a UDI <100-2000lux larger than 50% ranged from 77% to 86% of the time for active occupant behaviour (occupancy from 6am to 6pm). The paper also highlights the architectural features that mostly affect daylighting design in subtropical climates.

Sustainable urban stormwater management : water sensitive urban design perceptions, drivers and barriers

Stormwater has been recognised as one of the main culprits of aquatic ecosystem pollution and as a significant threat to the goal of ecological sustainable development. Water sensitive urban design is one of the key responses to the need to better manage urban stormwater runoff, the objectives of which go beyond rapid and efficient conveyance. Underpinned by the concepts of sustainable urban development, water sensitive urban design has proven to be an efficient and environmentally-friendly approach to urban stormwater management, with the necessary technical know-how and skills already available. However, large-scale implementation of water sensitive urban design is still lacking in Australia due to significant impediments and negative perceptions. Identification of the issues, barriers and drivers that affect sustainability outcomes of urban stormwater management is one of the first steps towards encouraging the wide-scale uptake of water sensitive urban design features which integrate sustainable urban stormwater management. This chapter investigates key water sensitive urban design perceptions, drivers and barriers in order to improve sustainable urban stormwater management efforts.

Preface to special issue on digital human modelling for vehicle design and manufacturing

The automotive industry has been the focus of digital human modeling (DHM) research and application for many years. In the highly competitive marketplace for personal transportation, the desire to improve the customer’s experience has driven extensive research in both the physical and cognitive interaction between the vehicle and its occupants. Human models provide vehicle designers with tools to view and analyze product interactions before the first prototypes are built, potentially improving the design while reducing cost and development time. The focus of DHM research and applications began with prediction and representation of static postures for purposes of driver workstation layout, including assessments of seat adjustment ranges and exterior vision. Now DHMs are used for seat design and assessment of driver reach and ingress/egress. DHMs and related simulation tools are expanding into the cognitive domain, with computational models of perception and motion, and into the dynamic domain with models of physical responses to ride and vibration. Moreover, DHMs are now widely used to analyze the ergonomics of vehicle assembly tasks. In this case, the analysis aims to determine whether workers can be expected to complete the tasks safely and with good quality. This preface provides a review of the literature to provide context for the nine new papers presented in this special issue.

Resilience by design: A participatory approach to designing an interactive digital application for promoting children's resilience

The promotion of resilience (the capacity of an individual or community to bounce back and recover from adversity) has become an important area of public health. In recent years it has expanded into the digital domain, and many online applications have been developed to promote children's resilience. In this study, it is argued that the majority of existing applications are limited because they take a didactic approach, and conceive of interaction as providing navigational choices. Because they simply provide information about resilience or replicate offline, scenario-based strategies, the understanding of resilience they provide is confined to a few, predetermined factors. In this study I propose a new, experiential approach to promoting resilience digitally. I define resilience as an emergent, situated and context-specific phenomenon. Using a Participatory Design model in combination with a salutogenic (strength-based) health methodology, this project has involved approximately 50 children as co-designers and co-researchers over two years. The children have contributed to the design of a new set of interactive resilience tools, which facilitate resilience promotion through dialogic and experiential learning. The major outcomes of this study include a new methodology for developing digital resilience tools, a new set of tools that have been developed and evaluated in collaboration with children and a set of design principles to guide future development. Beyond these initial and tangible outcomes, this study has also established that the benefits of introducing Participatory Design into a health promoting model rests primarily in the change of the role of children from "users" of technology and education to co-designers, where they assume a leadership role in both designing the tools and in directing their resilience learning.

A practical signal processing approach for condition monitoring of low speed machinery using Peak-Hold-Down-Sample algorithm

A simple and effective down-sample algorithm, Peak-Hold-Down-Sample (PHDS) algorithm is developed in this paper to enable a rapid and efficient data transfer in remote condition monitoring applications. The algorithm is particularly useful for high frequency Condition Monitoring (CM) techniques, and for low speed machine applications since the combination of the high sampling frequency and low rotating speed will generally lead to large unwieldy data size. The effectiveness of the algorithm was evaluated and tested on four sets of data in the study. One set of the data was extracted from the condition monitoring signal of a practical industry application. Another set of data was acquired from a low speed machine test rig in the laboratory. The other two sets of data were computer simulated bearing defect signals having either a single or multiple bearing defects. The results disclose that the PHDS algorithm can substantially reduce the size of data while preserving the critical bearing defect information for all the data sets used in this work even when a large down-sample ratio was used (i.e., 500 times down-sampled). In contrast, the down-sample process using existing normal down-sample technique in signal processing eliminates the useful and critical information such as bearing defect frequencies in a signal when the same down-sample ratio was employed. Noise and artificial frequency components were also induced by the normal down-sample technique, thus limits its usefulness for machine condition monitoring applications.

Performing design: game design, practice, praxis and the theatre of the impressed

This paper is a reflection on a design teaching project that endeavours to establish a culture of critical design thinking in a tertiary game design course. In the first instance, the ‘performing design’ project arose as a response to contemporary issues and tensions in the Australian games industry and game design education, in essence, the problem of how to scaffold undergraduate students from their entry point as ‘players’ (the impressed) into becoming designers. The performing design project therefore started as a small-scale intervention to inspire reflection in a wider debate that includes: the potential evolution of the contemporary games industry; the purpose of game design education; and the positioning of game design as a design discipline. Our position is that designing interactive playful works or games is victim of a tendency to simplify the discipline and view it from either the perspective of science or art. In this paper we look at some of the historical discussions on the distinct identity of games. Then we present an overview of the typical state of play in contemporary game design education which inspires the performing design project as an intervention or teaching technique. This leads us to question understandings of education and training and creativity and innovation. Finally we reflect on insights arising from the performing design project which lead us to support Archer’s call for a ‘third area’ that balances the monolithic practices of the two major academic disciplines.

I just want to design a sexy flying car! Teaching design-led innovation to designers

Many international management programs have capitalised on the value design can have upon potential business solutions and strategies (Martin, 2009 & Brown, 2008) as well as many international design programs introducing designers to business theory and curriculum (Manzini & Rizzo, 2011). This paper presents the findings from structured interviews with undergraduate design students and design industry professionals. Current literature surrounding design led innovation and the role designers’ play within it is also discussed and the challenges facing designers in this emerging design era are presented. The findings from this study indicate that most designers enter an undergraduate program not wanting to become the business leaders of tomorrow. Instead, they enter in the hope they can humbly help people and to make a difference in the world. There are contentions with this perspective, felt by industry, academia and students around why designers need to be taught business theory content. This paper provides the first step to overcoming this challenge by providing insight into the attitudes, perceptions and challenges designers are facing with this new design era.

Design Integration Workshops 2012

Businesses will learn how design integration can increase growth and productivity, and gain a sustainable competitive advantage. This hands on two-day workshop will demystify design thinking and introduce you to both theory and practice for the latest, world-class design integration methods. Design integration will transform your business and boost resilience in the face of current global, social and economic challenges.

First year design "Visualisation II": The hybridisation of analogue and digital tools

This paper discusses first year students’ responses and outcomes to the integration of digital technologies in their second semester foundational visualisation class; ‘Visualisation II’. As the second class in the Visualisation series, previous analogue knowledge taught in ‘Visualisation I’ is compounded with new digital technologies establishing the introduction to a myriad of hybrid visualisation tools and techniques for design exploration and design artefact. This research examines the differentiation between analogue and digital design, common precedents of the two, and reflects upon the environment and class structure with the learning experiences and confidence of surveyed participants.

Analyzing the literature for the link between the conservative Islamic culture of Saudi Arabia and the design of sustainable housing

Saudi Arabia experiences housing shortage for mid and low-income families, which is caused by rapid population growth. This condition is worsened by the fact that the current housing supply has problems in meeting both sustainable requirements and cultural needs of those families. This paper aims to investigate the link between the unique conservative Saudi culture and the design of sustainable housing, while keeping the housing cost affordable for mid and low-income families. The paper is based on a review of literatures on the issues of the Islamic culture and how can they be integrated into the design process of a Saudi house. Findings from literature reveiw suggest several design requirements for accommodating the conservative Saudi Culture in low cost sustainable houses. Such requirements include the implementation of proper usage of windows, and house orientation with a courtyard inside rather than facing the main street will provide natural ventilation while maintaining privacy. The main contribution to the body of knowledge is that this is a new approach to sustainable housing in Saudi Arabia considering not only energy use and architectural design issues but also socio-cultural issues as an essential part of sustainability.

Couture Academy Exhibition : a curation of the story behind a couture design

Research background: The general public is predominantly unaware of the complexities and skills involved in the fashion supply chain (design, manufacture and retail) of couture/bespoke garments. As cited in McMahon and Morley (2011) “While a high price tag is widely accepted as a necessary element of luxury products (Fionda &Moore, 2009) this must be accompanied by a story that gives the items intrinsic as well as extrinsic value (Keller, 2009). Research question: Is it possible to simulate a fashion couture studio environment in a non-traditional public space in order to produce and promote the processes involved in couture designs; each with their own story and aligned to the aesthetic of six collaborating high profile couture fashion retailers? Research contribution: The Couture Academy project allowed the team to curate the story behind the couture design and supply chain process. It was an experimental, curated, ‘hot-house’ fashion design project undertaken in real time to create one-off couture garments, inspired by key seasonal fashion trends as determined by leading Westfield retailers. The project was industry based, with Westfield Chermside as the launch pad for six QUT fashion students to experiment with design nuances aligned to renowned national fashion industry retailers; Cue, Dissh, Kitten D'Amour, Mombasa and Pink Mint. Industry mentors were assigned to each student designer, in order to heighten the design challenge. The exhibition consisted of a pop-up couture workshop based at Westfield Chermside. A complete fashion studio (sewing machines, pattern-cutting tables and mannequins) was set up for a seven day period in the foyer of the shopping centre with the public watching as the design process unfolded in real-time. The final design outcomes were paraded at the Southbank Precinct to a prominent industry and media panel, with the winner receiving a $2000 prize to fund a research trip to an international fashion capital of their choice. Research significance: This curated fashion project was funded by Westfield Group Australia. "It was the most successful season launch Westfield Chermside has ever had from both an average volume for exposure perspective, and in terms of the level of engagement with retailers and shoppers," said Laura Walls, Westfield Public Relations Consultant. Significant media coverage was generated; including three full pages of editorial in Brisbane’s Sunday Mail, with an estimated publicity value of $95,000. And public exposure through the live project/exhibition was estimated at 7,000 people over the 7 days.