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.

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.

'It had to be my choice' Indigenous smoking cessation and negotiations of risk, resistance and resilience

While Australia is considered a world leader in tobacco control, smoking rates within the Aboriginal and Torres Strait Islander population have not declined at the same rate. This failure highlights an obvious shortcoming of mainstream anti-smoking efforts to effectively understand and engage with the socio-cultural context of Indigenous smoking and smoking cessation experiences. The purpose of this article is to explore the narrative accounts of 20 Indigenous ex-smokers within an urban community and determine the motivators and enablers for successful smoking cessation. Our findings indicated that health risk narratives and the associated social stigma produced through anti-smoking campaigns formed part of a broader apparatus of oppression among Indigenous people, often inspiring resistance and resentment rather than compliance. Instead, a significant life event and supportive relationships were the most useful predictors of successful smoking cessation acting as both a motivator and enabler to behavioural change. Indigenous smoking cessation narratives most commonly involved changing and reordering a person’s life and identity and autonomy over this process was the critical building block to reclaiming control over nicotine addiction. Most promisingly, at an individual level, we found the important role that individual health professionals played in encouraging and supporting Indigenous smoking cessation through positive rather than punitive interactions. More broadly, our findings highlighted the central importance of resilience, empowerment, and trust within health promotion practice.

Emergent participant interaction

Emergence has the potential to effect complex, creative or open-ended interactions and novel game-play. We report on research into an emergent interactive system. This investigates emergent user behaviors and experience through the creation and evaluation of an interactive system. The system is +-NOW, an augmented reality, tangible, interactive art system. The paper briefly describes the qualities of emergence and +-NOW before focusing on its evaluation. This was a qualitative study with 30 participants conducted in context. Data analysis followed Grounded Theory Methods. Coding schemes, induced from data and external literature are presented. Findings show that emergence occurred in over half of the participants. The nature of these emergent behaviors is discussed along with examples from the data. Other findings indicate that participants found interaction with the work satisfactory. Design strategies for facilitating satisfactory experience despite the often unpredictable character of emergence, are briefly reviewed and potential application areas for emergence are discussed.

Open in art, nature and emergence

The interactive art system +-now is modelled on the openness of the natural world. Emergent shapes constitute a novel method for facilitating this openness. With the art system as an example, the relationship between openness and emergence is discussed. Lastly, artist reflections from the creation of the work are presented. These describe the nature of open systems and how they may be created.

Emergence in interactive art

This thesis is concerned with creating and evaluating interactive art systems that facilitate emergent participant experiences. For the purposes of this research, interactive art is the computer based arts involving physical participation from the audience, while emergence is when a new form or concept appears that was not directly implied by the context from which it arose. This emergent ‘whole’ is more than a simple sum of its parts. The research aims to develop understanding of the nature of emergent experiences that might arise during participant interaction with interactive art systems. It also aims to understand the design issues surrounding the creation of these systems. The approach used is Practice-based, integrating practice, evaluation and theoretical research. Practice used methods from Reflection-in-action and Iterative design to create two interactive art systems: Glass Pond and +-now. Creation of +-now resulted in a novel method for instantiating emergent shapes. Both art works were also evaluated in exploratory studies. In addition, a main study with 30 participants was conducted on participant interaction with +-now. These sessions were video recorded and participants were interviewed about their experience. Recordings were transcribed and analysed using Grounded theory methods. Emergent participant experiences were identified and classified using a taxonomy of emergence in interactive art. This taxonomy draws on theoretical research. The outcomes of this Practice-based research are summarised as follows. Two interactive art systems, where the second work clearly facilitates emergent interaction, were created. Their creation involved the development of a novel method for instantiating emergent shapes and it informed aesthetic and design issues surrounding interactive art systems for emergence. A taxonomy of emergence in interactive art was also created. Other outcomes are the evaluation findings about participant experiences, including different types of emergence experienced and the coding schemes produced during data analysis.

Architecture post mortem : the diastolic architecture of decline, dystopia, and death

Architecture Post Mortem surveys architecture’s encounter with death, decline, and ruination following late capitalism. As the world moves closer to an economic abyss that many perceive to be the death of capital, contraction and crisis are no longer mere phases of normal market fluctuations, but rather the irruption of the unconscious of ideology itself. Post mortem is that historical moment wherein architecture’s symbolic contract with capital is put on stage, naked to all. Architecture is not irrelevant to fiscal and political contagion as is commonly believed; it is the victim and penetrating analytical agent of the current crisis. As the very apparatus for modernity’s guilt and unfulfilled drives-modernity’s debt-architecture is that ideological element that functions as a master signifier of its own destruction, ordering all other signifiers and modes of signification beneath it. It is under these conditions that architecture theory has retreated to an “Alamo” of history, a final desert outpost where history has been asked to transcend itself. For architecture’s hoped-for utopia always involves an apocalypse. This timely collection of essays reformulates architecture’s relation to modernity via the operational death-drive: architecture is but a passage between life and death. This collection includes essays by Kazi K. Ashraf, David Bertolini, Simone Brott, Peggy Deamer, Didem Ekici, Paul Emmons, Donald Kunze, Todd McGowan, Gevork Hartoonian, Nadir Lahiji, Erika Naginski, and Dennis Maher. Contents: Introduction: ‘the way things are’, Donald Kunze; Driven into the public: the psychic constitution of space, Todd McGowan; Dead or alive in Joburg, Simone Brott; Building in-between the two deaths: a post mortem manifesto, Nadir Lahiji; Kant, Sade, ethics and architecture, David Bertolini; Post mortem: building deconstruction, Kazi K. Ashraf; The slow-fast architecture of love in the ruins, Donald Kunze; Progress: re-building the ruins of architecture, Gevork Hartoonian; Adrian Stokes: surface suicide, Peggy Deamer; A window to the soul: depth in the early modern section drawing, Paul Emmons; Preliminary thoughts on Piranesi and Vico, Erika Naginski; architectural asceticism and austerity, Didem Ekici; 900 miles to Paradise, and other afterlives of architecture, Dennis Maher; Index.

Responding to the 5Rs : an alternate perspective of Slowmation

This paper is a response to Hoban and Neilsen's (2010) Five Rs model for understanding how learners engage with slowmation. An alternative model (the Learning MMAEPER Model) that builds on the 5Rs model is explained in terms of its use in secondary science preservice teacher education. To probe into the surface and deep learning that can occur during the creation of a slowmation, the learning and relearning model is explored in terms of learning elements. This model can assist teachers to monitor the learning of their students and direct them to a deeper understanding of science concepts.

Re-targeting choreographic enquiry through motion capture

This creative work is the outcome of preliminary experiments through practice aiming to explore the collaboration of a Dancer/choreographer with an Animator, along with enquiry into the intergeneration of motion capture technologies within the work-flow. The animated visuals derived from the motion capture data is not aimed at just re-targeting of movement from one source to another but looks at describing the thought and emotions of the choreographed dance through visual aesthetics.

The use of CCTV to police public spaces : a case of big brother or big friend?

This paper considers the role of CCTV (closed circuit television) in the surveillance, policing and control of public space in urban and rural locations, specifically in relation to the use of public space by young people. The use of CCTV technology in public spaces is now an established and largely uncontested feature of everyday life in a number of countries and the assertion that they are essentially there for the protection of law abiding and consuming citizens has broadly gone unchallenged. With little or no debate in the U.K. to critique the claims made by the burgeoning security industry that CCTV protects people in the form of a ‘Big Friend’, the state at both central and local levels has endorsed the installation of CCTV apparatus across the nation. Some areas assert in their promotional material that the centre of the shopping and leisure zone is fully surveilled by cameras in order to reassure visitors that their personal safety is a matter of civic concern, with even small towns and villages expending monies on sophisticated and expensive to maintain camera systems. It is within a context of monitoring, recording and control procedures that young people’s use of public space is constructed as a threat to social order, in need of surveillance and exclusion which forms a major and contemporary feature in shaping thinking about urban and rural working class young people in the U.K. As Loader (1996) notes, young people’s claims on public space rarely gain legitimacy if ‘colliding’ with those of local residents, and Davis (1990) describes the increasing ‘militarization and destruction of public space’, while Jacobs (1965) asserts that full participation in the ‘daily life of urban streets’ is essential to the development of young people and beneficial for all who live in an area. This paper challenges the uncritical acceptance of widespread use of CCTV and identifies its oppressive and malevolent potential in forming a ‘surveillance gaze’ over young people (adapting Foucault’s ‘clinical gaze’c. 1973) which can jeopardise mental health and well being in coping with the ‘metropolis’, after Simmel, (1964).

The fishtown series

Fishtown is a series of mediated animated works which embody artistic conceptions of ambience and explore the interplay between foreground and background. The series draws upon a representation of natural patterns and rhythms in the ambient environment and is produced using a hybrid style of animation process that incorporates motion capture, dynamics and keyframe animation to construct a biomemtic peripheral rhythm. The display of the work is a crucial part of the project, and contributes a considerable amount to the reception of the work. Based on the ambient conceptions defined by Cage, Eno and Bizzocchi, ambient animation should incorporate some form of ambient display. As Eno (1978) states, it should be as ignorable as it is interesting. The ultimate intention is to place the work outside the gallery setting, to provide a more neutral ambient setting for the viewing of the work, and therefore the use of an ambient display is necessary if the work is to be situated in an ambient setting. Craig Walsh is a contemporary artist producing work for large scale projections in ambient settings. Completing Walsh's masterclass in 2011 (Tanawha Arts and Ecology Centre) has been an important factor in arriving at a strategy for the display of the Fishtown series. The most recent work in the Fishtown series was developed during a residency at the Crane Arts studios in Philadelphia USA in August 2012, and is comprised of a screen based animated work, utilizing large scale digital projection. Documentation of this work can be found at the Crane Arts Residency Website: http://cranearts.qcagriffith.com/crane-arts-residency-chris-denaro

Imaging affect : abstraction and the echo of the unknowable

As an artist my primary interest is in the abstract, that is in images of the imageless. I am curious about the emergence of pictorial significance and content from this unknowable space. To speak of the significance of an imageless image is also to speak of its affect. I aim to explore this both theoretically and practically. Theoretically I will explore affect through the late work of Lyotard and his notion of the affect-phrase. This is an under-examined aspect of Lyotard and demarcates a valuable way to look at the origins, impact and ramifications of affect for art. Practically I will apply these understandings to the development of my own creative work which includes both painting and digital work. My studio practice moves towards exploring the unfamiliar through the powerful and restless silence of affect.In this intense space each work or body of work 'leaks' into the next occasioning a sense of borderlessness, or of uncertainty. This interpenetration and co-mingling of conceptual and material terrains combines to present temporal and spatial slippages evident within the works themselves and their making, but it is also evident in bodies of work across the chronology of their making. Through a mapping of my own painting and digital arts practice and the utilisation of Lyotard’s notion of the affect -phrase I aim to describe the action of this ‘charged emptiness’ on creativity and explore and explain its significance on that we call image and its animation of what we call critical discourse.

Shift

Shift was an exhibition held in October 2008, and was the culmination of a 10 month artist in residence held at Metro Arts, Brisbane in 2008. A number of works were produced and exhibited, and were a response to the ambient urban landscape of inner city Brisbane. The research component contributes to the discussion of the form and display of digital animation, and builds upon strategies of presentation developed from series of works completed in 2005-2007 as part of an MA (research) at QUT, Brisbane. For the two week exhibition, one interactive kiosk and several large scale digital prints were produced, and also a site specific digital animation sequence was projected onto urban landscape features next to the gallery.

One night stand : only you

Technical: This looped video work is made up from a number of still images animated in a sequence, not intended to be a smooth action, but syncopated. The gaps in real time suggest blinking, lapses in technology, warps in time and distance. The still images have been manipulated in photoshop and imported into an animation software program, showing subtle emphasis on various aspects of the features of the face and location from the screen shots. Content: Obsession is one of the most constant expressions of love, whether it is a negative attribute, such as stalking, or the need to see or stroke the beloved, or telling and re-telling the story of first meeting. “like the beat, beat, beat of the tom tom when the jungle shadows fall like the tick, tick, tock of the stately clock as it stands against the wall like the drip, drip drip of the rain drops when the summer shower’s through a voice within me keeps repeating you, you, you… only you...” (Cole Porter, Night and Day) My desire to immerse myself in my newly-met adult daughter as a reality is as obsessive as any new parent. The primary means of contact is video cam, which has become a kind of surveillance for me. I stare at her mouth as it moves, her ear as she moves her head, her smile, her grimaces, her high cheekbones, her eyes that are like mine, the shape of her head, her teeth that are like her father’s… hundreds of candid pictures screen shot over a year and a half were the source of the video images.

Nucleation experiments in a microgravity environment

A simple experimental apparatus is described in which a wide variety of vapor phase nucleation studies of refractory materials could be performed aboard NASA's KC-135 Research Aircraft. The chief advantage of a microgravity environment for these studies is the expected absence of thermally driven convective motions in the gas. The absence of convection leads to much more accurate knowledge of both the temperature distribution in the system and the time evolution of the refractory vapor concentration as a function of distance from the crucible.The evolution of the apparatus will be described as more experience is gained with the microgravity environment. Such experiments will be used to prepare for similar ones carried out aboard either the shuttle or Space Station where considerably longer duration experiments are possible.