Protein complementation assay as a display system for screening protein libraries in the intracellular environment

A wide range of screening strategies have been employed to isolate antibodies and other proteins with specific attributes, including binding affinity, specificity, stability and improved expression. However, there remains no high-throughput system to screen for target-binding proteins in a mammalian, intracellular environment. Such a system would allow binding reagents to be isolated against intracellular clinical targets such as cell signalling proteins associated with tumour formation (p53, ras, cyclin E), proteins associated with neurodegenerative disorders (huntingtin, betaamyloid precursor protein), and various proteins crucial to viral replication (e.g. HIV-1 proteins such as Tat, Rev and Vif-1), which are difficult to screen by phage, ribosome or cell-surface display. This study used the β-lactamase protein complementation assay (PCA) as the display and selection component of a system for screening a protein library in the cytoplasm of HEK 293T cells. The colicin E7 (ColE7) and Immunity protein 7 (Imm7) *Escherichia coli* proteins were used as model interaction partners for developing the system. These proteins drove effective β-lactamase complementation, resulting in a signal-to-noise ratio (9:1 – 13:1) comparable to that of other β-lactamase PCAs described in the literature. The model Imm7-ColE7 interaction was then used to validate protocols for library screening. Single positive cells that harboured the Imm7 and ColE7 binding partners were identified and isolated using flow cytometric cell sorting in combination with the fluorescent β-lactamase substrate, CCF2/AM. A single-cell PCR was then used to amplify the Imm7 coding sequence directly from each sorted cell. With the screening system validated, it was then used to screen a protein library based the Imm7 scaffold against a proof-of-principle target. The wild-type Imm7 sequence, as well as mutants with wild-type residues in the ColE7- binding loop were enriched from the library after a single round of selection, which is consistent with other eukaryotic screening systems such as yeast and mammalian cell-surface display. In summary, this thesis describes a new technology for screening protein libraries in a mammalian, intracellular environment. This system has the potential to complement existing screening technologies by allowing access to intracellular proteins and expanding the range of targets available to the pharmaceutical industry.

Vitronectin modulates human mesenchymal stem cell response to insulin-like growth factor-I and transforming growth factor beta 1 in a serum-free environment

The use of animal sera for the culture of therapeutically important cells impedes the clinical use of the cells. We sought to characterize the functional response of human mesenchymal stem cells (hMSCs) to specific proteins known to exist in bone tissue with a view to eliminating the requirement of animal sera. Insulin-like growth factor-I (IGF-I), via IGF binding protein-3 or -5 (IGFBP-3 or -5) and transforming growth factor-beta 1 (TGF-beta(1)) are known to associate with the extracellular matrix (ECM) protein vitronectin (VN) and elicit functional responses in a range of cell types in vitro. We found that specific combinations of VN, IGFBP-3 or -5, and IGF-I or TGF-beta(1) could stimulate initial functional responses in hMSCs and that IGF-I or TGF-beta(1) induced hMSC aggregation, but VN concentration modulated this effect. We speculated that the aggregation effect may be due to endogenous protease activity, although we found that neither IGF-I nor TGF-beta(1) affected the functional expression of matrix metalloprotease-2 or -9, two common proteases expressed by hMSCs. In summary, combinations of the ECM and growth factors described herein may form the basis of defined cell culture media supplements, although the effect of endogenous protease expression on the function of such proteins requires investigation.

Facial feature detection for in-car environment

Acoustically, vehicles are extremely noisy environments and as a consequence audio-only in-car voice recognition systems perform very poorly. Seeing that the visual modality is immune to acoustic noise, using the visual lip information from the driver is seen as a viable strategy in circumventing this problem. However, implementing such an approach requires a system being able to accurately locate and track the driver’s face and facial features in real-time. In this paper we present such an approach using the Viola-Jones algorithm. Using this system, we present our results which show that using the Viola-Jones approach is a suitable method of locating and tracking the driver’s lips despite the visual variability of illumination and head pose.

Testing the effectiveness of a Practice Development intervention on changing the culture of evidence based practice in an acute care environment

In this age of evidence-based practice, nurses are increasingly expected to use research evidence in a systematic and judicious way when making decisions about patient care practices. Clinicians recognise the role of research when it provides valid, realistic answers in practical situations. Nonetheless, research is still perceived by some nurses as external to practice and implementing research findings into practice is often difficult. Since its conceptual platform in the 1960s, the emergence and growth of Nursing Development Units, and later, Practice Development Units has been described in the literature as strategic, organisational vehicles for changing the way nurses think about nursing by promoting and supporting a culture of inquiry and research-based practice. Thus, some scholars argue that practice development is situated in the gap between research and practice. Since the 1990s, the discourse has shifted from the structure and outcomes of developing practice to the process of developing practice, using a Practice Development methodology; underpinned by critical social science theory, as a vehicle for changing the culture and context of care. The nursing and practice development literature is dominated by descriptive reports of local practice development activity, typically focusing on reflection on processes or outcomes of processes, and describing perceived benefits. However, despite the volume of published literature, there is little published empirical research in the Australian or international context on the effectiveness of Practice Development as a methodology for changing the culture and context of care - leaving a gap in the literature. The aim of this study was to develop, implement and evaluate the effectiveness of a Practice Development model for clinical practice review and change on changing the culture and context of care for nurses working in an acute care setting. A longitudinal, pre-test/post-test, non-equivalent control group design was used to answer the following research questions: 1. Is there a relationship between nurses' perceptions of the culture and context of care and nurses' perceptions of research and evidence-based practice? 2. Is there a relationship between engagement in a facilitated process of Practice Development and change in nurses' perceptions of the culture and context of care? 3. Is there a relationship between engagement in a facilitated process of Practice Development and change in nurses' perceptions of research and evidence-based practice? Through a critical analysis of the literature and synthesis of the findings of past evaluations of Nursing and Practice Development structures and processes, this research has identified key attributes consistent throughout the chronological and theoretical development of Nursing and Practice Development that exemplify a culture and context of care that is conducive to creating a culture of inquiry and evidence-based practice. The study findings were then used in the development, validation and testing of an instrument to measure change in the culture and context of care. Furthermore, this research has also provided empirical evidence of the relationship of the key attributes to each other and to barriers to research and evidence-based practice. The research also provides empirical evidence regarding the effectiveness of a Practice Development methodology in changing the culture and context of care. This research is noteworthy in its contribution to advancing the discipline of nursing by providing evidence of the degree to which attributes of the culture and context of care, namely autonomy and control, workplace empowerment and constructive team dynamics, can be connected to engagement with research and evidence-based practice.

Impromptu : an interactive programming environment for composition and performance

Interactive development environments are making a resurgence. The traditional batch style of programming, edit -> compile -> run, is slowly being reevaluated by the development community at large. Languages such as Perl, Python and Ruby are at the heart of a new programming culture commonly described as extreme, agile or dynamic. Musicians are also beginning to embrace these environments and to investigate the opportunity to use dynamic programming tools in live performance. This paper provides an introduction to Impromptu, a new interactive development environment for musicians and sound artists.

Making the processes of designing explicit within an information technology environment

In this paper, technology is described as involving processes whereby resources are utilised to satisfy human needs or to take advantage of opportunities, to develop practical solutions to problems. This study, set within one type of technology context, information technology, investigated how, through a one semester undergraduate university course, elements of technological processes were made explicit to students. While it was acknowledged in the development and implementation of this course that students needed to learn technical skills, technological skills and knowledge, including design, were seen as vital also, to enable students to think about information technology from a perspective that was not confined and limited to `technology as hardware and software'. This paper describes how the course, set within a three year program of study, was aimed at helping students to develop their thinking and their knowledge about design processes in an explicit way. An interpretive research approach was used and data sources included a repertory grid `survey'; student interviews; video recordings of classroom interactions, audio recordings of lectures, observations of classroom interactions made by researchers; and artefacts which included students' journals and portfolios. The development of students' knowledge about design practices is discussed and reflections upon student knowledge development in conjunction with their learning experiences are made. Implications for ensuring explicitness of design practice within information technology contexts are presented, and the need to identify what constitutes design knowledge is argued.

Simulation and enhancement of a cardiovascular device test rig

Cardiovascular assist devices are tested in mock circulation loops (MCLs) prior to animal and clinical testing. These MCLs rely on characteristics such as pneumatic parameters to create pressure and flow, and pipe dimensions to replicate the resistance, compliance and fluid inertia of the natural cardiovascular system. A mathematical simulation was developed in SIMULINK to simulate an existing MCL. Model validation was achieved by applying the physical MCL characteristics to the simulation and comparing the resulting pressure traces. These characteristics were subsequently altered to improve and thus predict the performance of a more accurate physical system. The simulation was successful in simulating the physical mock circulation loop, and proved to be a useful tool in the development of improved cardiovascular device test rigs.

A practical approach for reliability prediction of pipeline systems

Pipelines play an important role in the modern society. Failures of pipelines can have great impacts on economy, environment and community. Preventive maintenance (PM) is often conducted to improve the reliability of pipelines. Modern asset management practice requires accurate predictability of the reliability of pipelines with multiple PM actions, especially when these PM actions involve imperfect repairs. To address this issue, a split system approach (SSA) based model is developed in this paper through an industrial case study. This new model enables maintenance personnel to predict the reliability of pipelines with different PM strategies and hence effectively assists them in making optimal PM decisions.

Young Children and the Environment : Early Learning for Sustainability

This world-first text book on early childhood edcuation for sustainabilty tackles one of the biggest contemporary issues of our times - the changing environmment - and demonstrates how early education can contribute to sustainable living. An essential text for students in early childhood teacher education and a practcial resource for child care practitioners and primary school teachers, it is designed to promote edcuation for sustainabilty from birth to eight years. the text refers to national and international initiatives such as 'sustainable Schools', 'Child Friendly Cities' and 'Health Promoting Schools' and explores their existing and potential links with early childhood education. Groundbreaking content draws on recent literature in the areas of organisational, educational and cultural chnage, and environmental sustainabilty. Specific chapters explore ethical challenges and the use of ICT to advance learning. Case studies and vignettes exemplify leadership in practice and 'provocations' are integrated throughout to inspire new ways of thinking about the environment, the wider world, young children and the transformative power of early edcuation.

Professional education in built environment and design

Early and rich conversations with a range of stakeholders – academics, professionals and graduates in their early years of practice – quickly clarified that the singular challenge for most parties centres on the ways in which courses prepare graduates for the pace, diversity and flux of contemporary professional practice.---------In pursuing understanding of this central challenge this study has focused on new graduates in BED disciplines by canvassing their views and those of two other major stakeholder groups (academic staff and professional practitioners in the disciplines studied). The first crucial years of a young graduate’s life in the workforce are shaped by a number of factors including the quality of the transition-to-work experience. The quality of this life-shaping transition is dependent on a range of factors including the ways in which graduates are educated in universities, their personal developmental characteristics and those of the professional people around them and the preparedness of workplaces and other professional groups to guide new recruits through the transition experience. This study makes recommendations about how the variations in transition experience, resulting from the vagaries of all these factors across a range of worksites, may be better understood, perhaps normalised, and, at least, supported. . Early and rich conversations with a range of stakeholders – academics, professionals and graduates in their early years of practice – quickly clarified that the singular challenge for most parties centres on the ways in which courses prepare graduates for the pace, diversity and flux of contemporary professional practice. The study proceeded through literature review, focus group interviews, national online survey and workshops. Through all these methods a number of challenges and factors essential to the transition experience, and the quality of education which precedes it, were identified. Firstly the study found further evidence of the importance of higher-order graduate capabilities, namely, the development of judgment, critical enquiry and strategic thinking. Alongside these capabilities the importance of the development of emotional intelligence, particularly interpersonal and social skills, was stressed by all stakeholders. At the time of writing the global economic crisis was providing challenges to the sector and its young graduates. This phenomenon proved the value of the development of resilience and persistence in graduates, the education system was called upon by all stakeholders as a place where the future-proofing of neophytes would ensure that the unknown challenges of the future could also be confronted. The study found that the challenges of transition to work are best supported by authentic undergraduate experiences both on and off campus, inside and outside classrooms, and that commencing professional life is made easier for new graduates when university courses and workplace settings develop, sustain and support high standards and high expectations of students. All these findings indicate the importance of stakeholder expectations, roles and responsibilities in respect of the transition-to-work experience. Whilst full agreement about how these things should occur is not necessary, a process (amongst stakeholders) which seeks value alignment around transition through discussion, debate and agenda-setting would probably assist to address what is seen as a major challenge in built environment and design education.

Soft-biometrics : unconstrained authentication in a surveillance environment

Soft biometrics are characteristics that can be used to describe, but not uniquely identify an individual. These include traits such as height, weight, gender, hair, skin and clothing colour. Unlike traditional biometrics (i.e. face, voice) which require cooperation from the subject, soft biometrics can be acquired by surveillance cameras at range without any user cooperation. Whilst these traits cannot provide robust authentication, they can be used to provide coarse authentication or identification at long range, locate a subject who has been previously seen or who matches a description, as well as aid in object tracking. In this paper we propose three part (head, torso, legs) height and colour soft biometric models, and demonstrate their verification performance on a subset of the PETS 2006 database. We show that these models, whilst not as accurate as traditional biometrics, can still achieve acceptable rates of accuracy in situations where traditional biometrics cannot be applied.

Communication of business process models via virtual environment simulations

Games and related virtual environments have been a much-hyped area of the entertainment industry. The classic quote is that games are now approaching the size of Hollywood box office sales [1]. Books are now appearing that talk up the influence of games on business [2], and it is one of the key drivers of present hardware development. Some of this 3D technology is now embedded right down at the operating system level via the Windows Presentation Foundations – hit Windows/Tab on your Vista box to find out... In addition to this continued growth in the area of games, there are a number of factors that impact its development in the business community. Firstly, the average age of gamers is approaching the mid thirties. Therefore, a number of people who are in management positions in large enterprises are experienced in using 3D entertainment environments. Secondly, due to the pressure of demand for more computational power in both CPU and Graphical Processing Units (GPUs), your average desktop, any decent laptop, can run a game or virtual environment. In fact, the demonstrations at the end of this paper were developed at the Queensland University of Technology (QUT) on a standard Software Operating Environment, with an Intel Dual Core CPU and basic Intel graphics option. What this means is that the potential exists for the easy uptake of such technology due to 1. a broad range of workers being regularly exposed to 3D virtual environment software via games; 2. present desktop computing power now strong enough to potentially roll out a virtual environment solution across an entire enterprise. We believe such visual simulation environments can have a great impact in the area of business process modeling. Accordingly, in this article we will outline the communication capabilities of such environments, giving fantastic possibilities for business process modeling applications, where enterprises need to create, manage, and improve their business processes, and then communicate their processes to stakeholders, both process and non-process cognizant. The article then concludes with a demonstration of the work we are doing in this area at QUT.