Quantitative proteomic approach to study subcellular localization of membrane proteins

As proteins within cells are spatially organized according to their role, knowledge about protein localization gives insight into protein function. Here, we describe the LOPIT technique (localization of organelle proteins by isotope tagging) developed for the simultaneous and confident determination of the steady-state distribution of hundreds of integral membrane proteins within organelles. The technique uses a partial membrane fractionation strategy in conjunction with quantitative proteomics. Localization of proteins is achieved by measuring their distribution pattern across the density gradient using amine-reactive isotope tagging and comparing these patterns with those of known organelle residents. LOPIT relies on the assumption that proteins belonging to the same organelle will co-fractionate. Multivariate statistical tools are then used to group proteins according to the similarities in their distributions, and hence localization without complete centrifugal separation is achieved. The protocol requires approximately 3 weeks to complete and can be applied in a high-throughput manner to material from many varied sources.

The Australian screen producer in transition

The role of the screen producer is ramifying. Not only are there numerous producer categories, but the screen producer function is also found on a continuum across film, television, advertising, corporate video, and the burgeoning digital media sector. In recent years, fundamental changes to distribution and consumption practices and technologies should have had a correlate impact on screen production practices and on the role of existing screen producers. At the same time, new and recent producers are learning and practicing their craft in a field that has already been transformed by digitisation and media convergence. Our analysis of the work, experience and outlook of screen producers in this chapter is based on data collected in the Australian Screen Producer Survey (ASPS), a nation-wide survey conducted by the ARC Centre of Excellence for Creative Industries and Innovation, the media marketing firm Bergent Research, and the Centre for Screen Business at the Australian Film, Television and Radio School (AFTRS) in 2008/09 and 2011. We analyse the results to better understand the practice of screen production in a period of industry transition, and to recognise the persistence of established production cultures that serve to distinguish different industry sectors.

Australian Screen Producers Survey : online interactive

There is growing scholarly interest in the everyday work undertaken by screen producers in part prompted by disciplinary shifts (the ‘material turn’, the rise of creative industries research) and in part by major transformations in the business of media production and consumption in recent years. However, the production cultures and motivations of screen producers, particularly those working in emergent online and convergent media markets, remain poorly understood. The 2012 Australian Screen Producer survey, building upon research undertaken in the Australian Screen Content Producer Survey conducted in 2009, was a nation-wide survey-based study of screen content producers working in four industry segments: film, television, corporate and new media production. The broad objectives of the 2012 Australian Producer Survey study were to: • Provide deeper and more detailed analysis into the nature of digital media producers and their practices and how these findings compare to the practices of established screen media producers; • Interrogate issues around the pace of industry change, industry sentiment and how producers are adapting to a changing marketplace; and • Offer insight into the transitional pathways of established media producers into production for digital media markets. The Australian Screen Producer Survey Online Interactive provides users (principally filmmakers, scholars and policymakers) with direct access to raw survey data through an interactive website that allows them to customise queries according to particular interests. The Online Interactive therefore provides customisable findings – unlike ‘static’ research outputs – delineating the practices, attitudes, strategies, and aspirations of screen producers working in feature film, television and corporate production as well as those operating in an increasingly convergent digital media marketplace. The survey was developed by researchers at the ARC Centre of Excellence for Creative Industries and Innovation (CCI), Queensland University of Technology, Deakin University, the Centre for Screen Business at Australian Film Television and Radio School (AFTRS) and was undertaken in association with Bergent Research. The Online Interactive website (http://screenproducersurvey.com/) was developed with support from the Centre for Memory Imagination and Invention (CMII).

Promoting active lifestyles in young children: investigating mothers’ decisions about their child’s physical activity and screen time behaviours

Objectives Given increasing trends of obesity being noted from early in life and that active lifestyles track across time, it is important that children at a very young age be active to combat a foundation of unhealthy behaviours forming. This study investigated, within a theory of planned behaviour (TPB) framework, factors which influence mothers’ decisions about their child’s 1) adequate physical activity (PA) and 2) limited screen time behaviours. Methods Mothers (N = 162) completed a main questionnaire, via on-line or paper-based administration, which comprised standard TPB items in addition to measures of planning and background demographic variables. One week later, consenting mothers completed a follow-up telephone questionnaire which assessed the decisions they had made regarding their child’s PA and screen time behaviours during the previous week. Results Hierarchical multiple regression analyses revealed support for the predictive model, explaining an overall 73% and 78% of the variance in mothers’ intention and 38% and 53% of the variance in mothers’ decisions to ensure their child engages in adequate PA and limited screen time, respectively. Attitude and subjective norms predicted intention in both target behaviours, as did intentions with behaviour. Contrary to predictions, perceived behavioural control (PBC) in PA behaviour and planning in screen time behaviour were not significant predictors of intention, neither was PBC a predictor of either behaviour. Conclusions The findings illustrate the various roles that psycho-social factors play in mothers’ decisions to ensure their child engages in active lifestyle behaviours which can help to inform future intervention programs aimed at combating very young children’s inactivity.

Proteomic and other analyses to determine the functional consequences of deregulated kallikrein-related peptidase (KLK) expression in prostate and ovarian cancer

Rapidly developing proteomic tools are improving detection of deregulated kallikrein-related peptidase (KLK) expression, at the protein level, in prostate and ovarian cancer, as well as facilitating the determination of functional consequences downstream. Mass spectrometry (MS)-driven proteomics uniquely allows for the detection, identification and quantification of thousands of proteins in a complex protein pool, and this has served to identify certain KLKs as biomarkers for these diseases. In this review we describe applications of this technology in KLK biomarker discovery, and elucidate MS-based techniques which have been used for unbiased, global screening of KLK substrates within complex protein pools. Although MS-based KLK degradomic studies are limited to date, they helped to discover an array of novel KLK substrates. Substrates identified by MS-based degradomics are reported with improved confidence over those determined by incubating a purified or recombinant substrate and protease of interest, in vitro. We propose that these novel proteomic approaches represent the way forward for KLK research, in order to correlate proteolysis of biological substrates with tissue-related consequences, toward clinical targeting of KLK expression and function for cancer diagnosis, prognosis and therapies.

Australian screen content in education research project

Researchers from Queensland University of Technology have teamed up with the Australian Research Council (ARC), Screen Australia, The Australian Broadcasting Corporation (ABC), the Special Broadcasting Service (SBS) and the Australian Children’s Television Foundation (ACTF) to investigate the use of Australian screen content in primary, secondary and tertiary education. Over the next three years (2014-2016), researchers and investigators will undertake a national survey of schools and universities, and will conduct in-depth interviews with hundreds of industry representatives, teachers, principals, librarians and students. Furthermore, new approaches to developing screen content and curricula will be trialled. The project aims to develop a comprehensive picture of why, how, how much and where Australian screen content is used in education.

The SOS screen in Arabidopsis: a search for functions involved in DNA metabolism

The SOS screen, as originally described by Perkins et al. (1999), was setup with the aim of identifying Arabidopsis functions that might potentially be involved in the DNA metabolism. Such functions, when expressed in bacteria, are prone to disturb replication and thus trigger the SOS response. Consistently, expression of AtRAD51 and AtDMC1 induced the SOS response in bacteria, even affecting E. coli viability. 100 SOS-inducing cDNAs were isolated from a cDNA library constructed from an Arabidopsis cell suspension that was found to highly express meiotic genes. A large proportion of these SOS+ candidates are clearly related to the DNA metabolism, others could be involved in the RNA metabolism, while the remaining cDNAs encode either totally unknown proteins or proteins that were considered as irrelevant. Seven SOS+ candidate genes are induced following gamma irradiation. The in planta function of several of the SOS-inducing clones was investigated using T-DNA insertional mutants or RNA interference. Only one SOS+ candidate, among those examined, exhibited a defined phenotype: silenced plants for DUT1 were sensitive to 5-fluoro-uracil (5FU), as is the case of the leaky dut-1 mutant in E. coli that are affected in dUTPase activity. dUTPase is essential to prevent uracil incorporation in the course of DNA replication.

Drawing and writing on the screen

This chapter focuses on the physicality of the iPad as an object, and how that physicality affects the interactions children have with the device generally, and the apps specifically. Thinking about the physicality of the iPad is important because the materials, size, weight and appearance make the iPad quite unlike most other toys and equipment in the kindergarten space. Most strikingly, this physicality does not ‘represent’ the virtual vast dimensions of the iPad brought about through the diverse functions and contents of the apps contained in it. While the iPad is small enough and functional enough to be easily handled and operated even by young children, it is capable of performing highly complex, highly technological tasks that take it beyond its diminutive dimensions. This virtual-actual contrast is interesting to consider in relation to the other resources more commonly found in a kindergarten space. While objects such as toys, bricks, building materials often do prompt the child to imagine and invent beyond the physical boundaries of the toy, they not have the same types of virtual-actual contrasts of a digital device such as the iPad. How then, might children be drawn to the iPad because of its physical, technological and virtual difference? Particularly, how might this virtual-actual difference impact on the physical skills associated with writing and drawing: skills usually learnt through the use of a pencil and paper? While the research project did not set out to compare how digital and paper-based resources affect writing and drawing skills there was great interest to see how young children negotiated drawing and writing on the shiny glass surface of the iPad.

A proteomic view into infection of greyback canegrubs (Dermolepida albohirtum) by Metarhizium anisopliae

Metarhizium anisopliae is a naturally occurring cosmopolitan fungus infecting greyback canegrubs (Dermolepida albohirtum). The main molecular factors involved in the complex interactions occurring between the greyback canegrubs and M. anisopliae (FI-1045) were investigated by comparing the proteomes of healthy canegrubs, canegrubs infected with Metarhizium and fungus only. Differentially expressed proteins from the infected canegrubs were subjected to mass spectrometry to search for pathogenicity related proteins. Immune-related proteins of canegrubs identified in this study include cytoskeletal proteins (actin), cell communication proteins, proteases and peptidases. Fungal proteins identified include metalloproteins, acyl-CoA, cyclin proteins and chorismate mutase. Comparative proteome analysis provided a view into the cellular reactions triggered in the canegrub in response to the fungal infection at the onset of biological control.

Chroma Key Theatre : an examination of similarities and divergences in the use of Green Screen in two experimental theatre works

This paper examines the frame as it contributes to the debate on contemporary intermedial theatre and performance practices in light of increasing astriction between filmic and theatrical discourses. Informed by Auslander (1999), Lehmann (2006), and Giesekam (2007), and through an extrapolation of the tenets Eckersall, Gretchen and Scheer identify in the theory of New Media Dramaturgy, it will analyse two recent works of experimental theatre-making. RUFF (2013), a New York produced solo performance by one of the world’s leading female performers, explores her experiences of having a stroke. Total Dik! (2013), produced in Brisbane, Australia, is an interdisciplinary collaborative performance that examines aspects of dictatorship. They are clearly very different works yet there are a number of significant theatrical similarities in their use of Chroma Key technology and live compositing as material scenic devices. These works overtly and evocatively draw on the cinematic technique and technology of Chroma Key to augment and reveal the tensions and overlaps in their production processes.

Australian screen content in education

Our presentation today will introduce our current ARC Linkage project on Australian screen content in education. We will then go on to discuss our initial research, which is attempting to quantify the use of screen content in Australian education. The presentation concludes with a brief discussion of some of the emerging video on demand services available to schools and universities.

To see and be seen : cinematic constructions of gender and spectatorship in contemporary screen-based art

To See and Be Seen: Cinematic Constructions of Gender and Spectatorship in Contemporary Screen-Based Art addresses how gendered representation can be structured within visual art practice through a series of creative moving-image works. Using the aesthetic language of French New Wave cinema as its primary point of departure, this research project investigates how gendered representations are constructed by cinematic language. In doing this, it proposes latent possibilities present within the dominant gaze created by patriarchal relations of power. This project, in a series of creative works, demonstrates how the 'masculine' authorial gaze is learnt culturally, and by examining the gendered syntax of film, reveals how this can be recontextualised by the female artist.

H2AX phosphorylation screen of cells from radiosensitive cancer patients reveals a novel DNA double-strand break repair cellular phenotype

BACKGROUND: About 1-5% of cancer patients suffer from significant normal tissue reactions as a result of radiotherapy (RT). It is not possible at this time to predict how most patients' normal tissues will respond to RT. DNA repair dysfunction is implicated in sensitivity to RT particularly in genes that mediate the repair of DNA double-strand breaks (DSBs). Phosphorylation of histone H2AX (phosphorylated molecules are known as gammaH2AX) occurs rapidly in response to DNA DSBs, and, among its other roles, contributes to repair protein recruitment to these damaged sites. Mammalian cell lines have also been crucial in facilitating the successful cloning of many DNA DSB repair genes; yet, very few mutant cell lines exist for non-syndromic clinical radiosensitivity (RS). METHODS: Here, we survey DNA DSB induction and repair in whole cells from RS patients, as revealed by gammaH2AX foci assays, as potential predictive markers of clinical radiation response. RESULTS: With one exception, both DNA focus induction and repair in cell lines from RS patients were comparable with controls. Using gammaH2AX foci assays, we identified a RS cancer patient cell line with a novel ionising radiation-induced DNA DSB repair defect; these data were confirmed by an independent DNA DSB repair assay. CONCLUSION: gammaH2AX focus measurement has limited scope as a pre-RT predictive assay in lymphoblast cell lines from RT patients; however, the assay can successfully identify novel DNA DSB repair-defective patient cell lines, thus potentially facilitating the discovery of novel constitutional contributions to clinical RS.

Exploring parents' beliefs about their young child's physical activity and screen time behaviours

Drawing on the belief-based framework of the theory of planned behaviour, 20 adults living in Queensland, Australia participated in semi-structured interviews to elicit salient beliefs regarding their young child’s physical activity (PA) and screen time behaviours. Data were analysed separately for PA and screen time with a range of beliefs emerging that guided parents’ decisions for these important health behaviours. Underlying advantages (e.g., improve family interactions, improve child behaviour), disadvantages (e.g., mess and noise factor, increase in parental distress), barriers (e.g., lack of time, parental fatigue), and facilitators (e.g., access to parks, social support) to engaging their child in adequate PA and limited screen time emerged. Normative pressures were also identified as affecting parents’ decisions for their child in these contexts. Parents experience unique difficulties in engaging their child in adequate PA and limited screen time that interventions can draw on when designing and implementing programs aimed at modifying these important child health behaviours.