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.

Preparation of future weather data to study the impact of climate change on buildings

The dynamic interaction between building systems and external climate is extremely complex, involving a large number of difficult-to-predict variables. In order to study the impact of climate change on the built environment, the use of building simulation techniques together with forecast weather data are often necessary. Since most of building simulation programs require hourly meteorological input data for their thermal comfort and energy evaluation, the provision of suitable weather data becomes critical. In this paper, the methods used to prepare future weather data for the study of the impact of climate change are reviewed. The advantages and disadvantages of each method are discussed. The inherent relationship between these methods is also illustrated. Based on these discussions and the analysis of Australian historic climatic data, an effective framework and procedure to generate future hourly weather data is presented. It is shown that this method is not only able to deal with different levels of available information regarding the climate change, but also can retain the key characters of a “typical” year weather data for a desired period.

The effectiveness of marketing communication within the immersive environment

Immersive environments are part of a recent media innovation that allow users to become so involved within a computer-based simulated environment that they feel part of that virtual world (Grigorovici, 2003). A specific example is Second Life, which is an internet-based, three-dimensional immersive virtual world in which users create an online representation of themselves (an avatar) to play games and interact socially with thousands of people simultaneously. This study focuses on Second Life as an example of an immersive environment, as it is the largest adult freeform virtual world, home to 12 million avatars (IOWA State University, 2008). Already in Second Life there are more than 100 real-life brands from a range of industries, including automotive, professional services, and consumer goods and travel, among others (KZero, 2007; New Business Horizons, 2009). Compared to traditional advertising media, this interactive media can immerse users in the environment. As a result of this interactivity, users can become more involved with a virtual environment, resulting in prolonged usage over weeks, months and even years. Also, it can facilitate presence. Despite these developments, little is known about the effectiveness of marketing messages in a virtual world context. Marketers are incorporating products into Second Life using a strategy of online product placement. This study, therefore, explores the perceived effectiveness of online product placement in Second Life in terms of effects on product/brand recall, purchase intentions and trial. This research examines the association between individuals’ involvement with Second Life and online product placement effectiveness, as well as the relationship between individuals’ Second Life involvement and the effectiveness of online product placement. In addition, it investigates the association of immersion and product placement involvement. It also examines the impact of product placement involvement on online product placement effectiveness and the role of presence in affecting this relationship. An exploratory study was conducted for this research using semi-structured in-depth interviews face-to-face, email-based and in-world. The sample comprised 24 active Second Life users. Results indicate that product placement effectiveness is not directly associated with Second Life involvement, but rather effectiveness is impacted through the effect of Second Life involvement on product placement involvement. A positive relationship was found between individuals’ product placement involvement and online product placement effectiveness. Findings also indicate that online product placement effectiveness is not directly associated with immersion. Rather, it appears that effectiveness is impacted through the effect of immersion on product placement involvement. Moreover, higher levels of presence appear to have a positive impact on the relationship between product placement involvement and product placement effectiveness. Finally, a model was developed from this qualitative study for future testing. In terms of theoretical contributions, this study provides a new model for testing the effectiveness of product placement within immersive environments. From a methodological perspective, in-world interviews as a new research method were undertaken. In terms of a practical contribution, findings identified useful information for marketers and advertising agencies that aim to promote their products in immersive virtual environments like Second Life.

Technical report : acoustic analysis of the natural environment

This technical report is concerned with one aspect of environmental monitoring—the detection and analysis of acoustic events in sound recordings of the environment. Sound recordings offer ecologists the potential advantages of cheaper and increased sampling. An acoustic event detection algorithm is introduced that outputs a compact rectangular marquee description of each event. It can disentangle superimposed events, which are a common occurrence during morning and evening choruses. Next, three uses to which acoustic event detection can be put are illustrated. These tasks have been selected because they illustrate quite different modes of analysis: (1) the detection of diffuse events caused by wind and rain, which are a frequent contaminant of recordings of the terrestrial environment; (2) the detection of bird calls using the spatial distribution of their component events; and (3) the preparation of acoustic maps for whole ecosystem analysis. This last task utilises the temporal distribution of events over a daily, monthly or yearly cycle.

Technical report : acoustic analysis of the natural environment

This technical report is concerned with one aspect of environmental monitoring—the detection and analysis of acoustic events in sound recordings of the environment. Sound recordings offer ecologists the potential advantages of cheaper and increased sampling. An acoustic event detection algorithm is introduced that outputs a compact rectangular marquee description of each event. It can disentangle superimposed events, which are a common occurrence during morning and evening choruses. Next, three uses to which acoustic event detection can be put are illustrated. These tasks have been selected because they illustrate quite different modes of analysis: (1) the detection of diffuse events caused by wind and rain, which are a frequent contaminant of recordings of the terrestrial environment; (2) the detection of bird calls using the spatial distribution of their component events; and (3) the preparation of acoustic maps for whole ecosystem analysis. This last task utilises the temporal distribution of events over a daily, monthly or yearly cycle.

Wandering and the physical environment

Background/Rationale Guided by the need-driven dementia-compromised behavior (NDB) model, this study examined influences of the physical environment on wandering behavior. Methods Using a descriptive, cross-sectional design, 122 wanderers from 28 long-term care (LTC) facilities were videotaped 10 to 12 times; data on wandering, light, sound, temperature and humidity levels, location, ambiance, and crowding were obtained. Associations between environmental variables and wandering were evaluated with chi-square and t tests; the model was evaluated using logistic regression. Results In all, 80% of wandering occurred in the resident’s own room, dayrooms, hallways, or dining rooms. When observed in other residents’ rooms, hallways, shower/baths, or off-unit locations, wanderers were likely (60%-92% of observations) to wander. The data were a good fit to the model overall (LR [logistic regression] χ2 (5) = 50.38, P < .0001) and by wandering type. Conclusions Location, light, sound, proximity of others, and ambiance are associated with wandering and may serve to inform environmental designs and care practices.

The design environment

This chapter provides an overview of the Editor, which is the core modeling component of the YAWL system. Specifically, it shows how to specify control-flow, data and resourcing requirements in a YAWL workflow model. Moreover, it describes the error reporting capabilities of the Editor and the features of its interchange format.

Ecological sustainable development: a study of the obstacles in Australia's construction industry

Ecological sustainable development (ESD), defined as that which meets the needs of the present without compromising the ability of future generations to meet their own needs, has much to offer in enhancing the quality of life of people and maintaining the environment for future generations by reducing the pollution of water, air and land, minimizing the destruction of irreplaceable ecosystems and cutting down the amount of toxic materials released. However, there is still much to do to achieve full implementation world-wide. This paper reports on three factors-design, attitudes and financial constraints - that are likely barriers to the implementation of ESD within the built environment in Australian industry. A postal questionnaire survey is described aimed at soliciting views on detailed aspects of the factors. This shows that ESD in the Australian built environment has also not been successfully implemented. The main reason is found to be due to the perceived costs involved - the cost of using environmental materials being a predominant factor. The design of ESD, being more sophisticated, also is perceived as involving stakeholders in more expense. There also appears to be a lack of knowledge and a lack of specialised and interdisciplinary design teams available in the Australian context.

Effectiveness of constructability concept in the provision of infrastructure assets

The concept of constructability is to use construction knowledge and experience during all phases of a project, particularly in the earliest phases of planning and design. It facilitates project objectives before delivery stage, and decreases unnecessary costs during construction phase. Despite the extensive use, constructability concept fails to address many issues related to Operation and Maintenance (O&M) of construction projects. Extending constructability concept, to include the O&M issues, could lead to the projects that are not fitted for construction purposes only, but also fitted for use. This study reviews the literature of constructability implementation, its benefits and shortcomings during the infrastructure life cycle, as well as the delivery success factors of infrastructure projects. This contributes to the propose need of a model to improve the effectiveness and efficiency of infrastructure project by extending the concept of constructability to include O&M. Development of such a model can facilitate post-occupancy stakeholders’ participation in a constructability program. It will help infrastructure owners eliminate project reworks, and improve O&M effectiveness and efficiency.

Technical report : acoustic analysis of the natural environment

This technical report is concerned with one aspect of environmental monitoring—the detection and analysis of acoustic events in sound recordings of the environment. Sound recordings offer ecologists the advantage of cheaper and increased sampling but make available so much data that automated analysis becomes essential. The report describes a number of tools for automated analysis of recordings, including noise removal from spectrograms, acoustic event detection, event pattern recognition, spectral peak tracking, syntactic pattern recognition applied to call syllables, and oscillation detection. These algorithms are applied to a number of animal call recognition tasks, chosen because they illustrate quite different modes of analysis: (1) the detection of diffuse events caused by wind and rain, which are frequent contaminants of recordings of the terrestrial environment; (2) the detection of bird and calls; and (3) the preparation of acoustic maps for whole ecosystem analysis. This last task utilises the temporal distribution of events over a daily, monthly or yearly cycle.

Estimating the latent demand for rail transit : a case study in Perth, Western Australia

This paper presents a study on estimating the latent demand for rail transit in Australian context. Based on travel mode-choice modelling, a two-stage analysis approach is proposed, namely market population identification and mode share estimation. A case study is conducted on Midland-Fremantle rail transit corridor in Perth, Western Australia. The required data mainly include journey-to-work trip data from Australian Bureau of Statistics Census 2006 and work-purpose mode-choice model in Perth Strategic Transport Evaluation Model. The market profile is analysed, such as catchment areas, market population, mode shares, mode specific trip distributions and average trip distances. A numerical simulation is performed to test the sensitivity of the transit ridership to the change of fuel price. A corridor-level transit demand function of fuel price is thus obtained and its characteristics of elasticity are discussed. This study explores a viable approach to developing a decision-support tool for the assessment of short-term impacts of policy and operational adjustments on corridor-level demand for rail transit.

The Brisbane Cordon scheme : part A - preliminary investigation

It could be said that road congestion is one of the most significant problems within any modern metropolitan area. For several decades now, around the globe, congestion in metropolitan areas has been worsening for two main reasons. Firstly, road congestion has significantly increased due to a higher demand for road space because of growth in populations, economic activity and incomes (Hensher & Puckett, 2007). This factor, in conjunction with a significant lack of investment in new road and public transport infrastructure, has seen the road network capacities of cities exceeded by traffic volumes and thus, resulted in increased traffic congestion. This relentless increase in road traffic congestion has resulted in a dramatic increase in costs for both the road users and ultimately the metropolitan areas concerned (Bureau of Transport and Regional Economics, 2007). In response to this issue, several major cities around the world, including London, Stockholm and Singapore, have implemented congestion-charging schemes in order to combat the effects of road congestion. A congestion-charging scheme provides a mechanism for regulating traffic flows into the congested areas of a city, whilst simultaneously generating public revenue that can be used to improve both the public transport and road networks of the region. The aim of this paper was to assess the concept of congestion-charging, whilst reflecting on the experiences of various cities that have already implemented such systems. The findings from this paper have been used to inform the design of a congestion-charging scheme for the city of Brisbane in Australia in a supplementary study (Whitehead, Bunker, & Chung, 2011). The first section of this paper examines the background to road congestion; the theory behind different congestion-charging schemes; and the various technologies involved with the concept. The second section of this paper details the experiences, in relation to implementing a congestion-charging scheme, from the city of Stockholm in Sweden. This research has been crucial in forming a list of recommendations and lessons learnt for the design of a congestion-charging scheme in Australia. It is these recommendations that directly inform the proposed design of the Brisbane Cordon Scheme detailed in Whitehead et al. (2011).