Development of a high-level gene expression system for the production of bioplastics in sugarcane

Despite various approaches, the production of biodegradable plastics such as polyhydroxybutyrate (PHB) in transgenic plants has met with limited success due largely to low expression levels. Even in the few instances where high levels of protein expression have been reported, the transgenic plants have been stunted indicating PHB is phytotoxic (Poirier 2002). This PhD describes the application of a novel virus-based gene expression technology, termed InPAct („In Plant Activation.), for the production of PHB in tobacco and sugarcane. InPAct is based on the rolling circle replication mechanism by which circular ssDNA viruses replicate and provides a system for controlled, high-level gene expression. Based on these features, InPAct was thought to represent an ideal system to enable the controlled, high-level expression of the three phb genes (phbA, phbB and phbC) required for PHB production in sugarcane at a preferred stage of plant growth. A Tobacco yellow dwarf virus (TbYDV)-based InPAct-phbA vector, as well as linear vectors constitutively expressing phbB and phbC were constructed and different combinations were used to transform tobacco leaf discs. A total of four, eight, three and three phenotypically normal tobacco lines were generated from discs transformed with InPAct-phbA, InPAct-phbA + p1300-TaBV P-phbB/phbC- 35S T, p1300-35S P-phbA-NOS T + p1300-TaBV P-phbB/phbC-35S T and InPAct-GUS, respectively. To determine whether the InPAct cassette could be activated in the presence of the TbYDV Rep, leaf samples from the eight InPActphbA + p1300-TaBV P-phbB/phbC-35S T plants were agroinfiltrated with p1300- TbYDV-Rep/RepA. Three days later, successful activation was indicated by the detection of episomes using both PCR and Southern analysis. Leaf discs from the eight InPAct-phbA + p1300-TaBV P-phbB/phbC-35S T transgenic plant lines were agroinfiltrated with p1300-TbYDV-Rep/RepA and leaf tissue was collected ten days post-infiltration and examined for the presence of PHB granules. Confocal microscopy and TEM revealed the presence of typical PHB granules in five of the eight lines, thus demonstrating the functionality of InPActbased PHB production in tobacco. However, analysis of leaf extracts by HPLC failed to detect the presence of PHB suggesting only very low level expression levels. Subsequent molecular analysis of three lines revealed low levels of correctly processed mRNA from the catalase intron contained within the InPAct cassette and also the presence of cryptic splice sites within the intron. In an attempt to increase expression levels, new InPAct-phb cassettes were generated in which the castorbean catalase intron was replaced with a synthetic intron (syntron). Further, in an attempt to both increase and better control Rep/RepA-mediated activation of InPAct cassettes, Rep/RepA expression was placed under the control of a stably integrated alc switch. Leaf discs from a transgenic tobacco line (Alc ML) containing 35S P-AlcR-AlcA P-Rep/RepA were supertransformed with InPAct-phbAsyn or InPAct-GUSsyn using Agrobacterium and three plants (lines) were regenerated for each construct. Analysis of the RNA processing of the InPAct-phbAsyn cassette revealed highly efficient and correct splicing of the syntron, thus supporting its inclusion within the InPAct system. To determine the efficiency of the alc switch to activate InPAct, leaf material from the three Alc ML + InPAct-phbAsyn lines was either agroinfiltrated with 35S P-Rep/RepA or treated with ethanol. Unexpectedly, episomes were detected not only in the infiltrated and ethanol treated samples, but also in non-treated samples. Subsequent analysis of transgenic Alc ML + InPAct-GUS lines, confirmed that the alc switch was leaky in tissue culture. Although this was shown to be reversible once plants were removed from the tissue culture environment, it made the regeneration of Alc ML + InPAct-phbsyn plant lines extremely difficult, due to unintentional Rep expression and therefore high levels of phb expression and phytotoxic PHB production. Two Alc ML + InPAct-phbAsyn + p1300-TaBV P-phbB/phbC-35S T transgenic lines were able to be regenerated, and these were acclimatised, alcohol-treated and analysed. Although episome formation was detected as late as 21 days post activation, no PHB was detected in the leaves of any plants using either microscopy or HPLC, suggesting the presence of a corrupt InPAct-phbA cassette in both lines. The final component of this thesis involved the application of both the alc switch and the InPAct systems to sugarcane in an attempt to produce PHB. Initial experiments using transgenic Alc ML + InPAct-GUS lines indicated that the alc system was not functional in sugarcane under the conditions tested. The functionality of the InPAct system, independent of the alc gene switch, was subsequently examined by bombarding the 35S Rep/RepA cassette into leaf and immature leaf whorl cells derived from InPAct-GUS transgenic sugarcane plants. No GUS expression was observed in leaf tissue, whereas weak and irregular GUS expression was observed in immature leaf whorl tissue derived from two InPAct- GUS lines and two InPAct-GUS + 35S P-AlcR-AlcA P-GUS lines. The most plausible reason to explain the inconsistent and low levels of GUS expression in leaf whorls is a combination of low numbers of sugarcane cells in the DNA replication-conducive S-phase and the irregular and random nature of sugarcane cells bombarded with Rep/RepA. This study details the first report to develop a TbYDV-based InPAct system under control of the alc switch to produce PHB in tobacco and sugarcane. Despite the inability to detect quantifiable levels of PHB levels in either tobacco or sugarcane, the findings of this study should nevertheless assist in the further development of both the InPAct system and the alc system, particularly for sugarcane and ultimately lead to an ethanol-inducible InPAct gene expression system for the production of bioplastics and other proteins of commercial value in plants.

Aggravation of ADAMTS and MMP production and ERK1/2 pathway in the interaction of osteoarthritic subchondral bone osteoblasts and articular cartilage chondrocytes : a possible pathogenic role in osteoarthritis

Introduction: Degradative enzymes, such as A disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) and matrix metalloproteinases (MMPs), play key roles in osteoarthritis (OA) development. The aim of the present study was to investigate if cross-talk between subchondral bone osteoblasts (SBOs) and articular cartilage chondrocytes (ACCs) in OA alters the expression and regulation of ADAMTS5, ADAMTS4, MMP-1, MMP-2, MMP-3, MMP-8, MMP-9 and MMP-13, and also to test the possible involvement of mitogen activated protein kinase (MAPK) signaling pathway during this process. Methods: ACCs and SBOs were isolated from normal and OA patients. An in vitro co-culture model was developed to study the regulation of ADAMTS and MMPs under normal and OA joint cross-talk conditions. MAPK-ERK inhibitor, PD98059 was applied to delineate the involvement of specific pathway during this interaction process. Results: Indirect co-culture of OA SBOs with normal ACCs resulted in significantly increased expression of ADAMTS5, ADAMTS4, MMP-2, MMP-3 and MMP-9 in ACCs, whereas co-culture of OA ACCs led to increased MMP-1 and MMP-2 expression in normal SBOs. The upregulation of ADAMTS and MMPs under these conditions was correlated with activation of the MAPK-ERK1/2 signaling pathway and the addition of the MAPK-ERK inhibitor, PD98059, reversed the overexpression of ADAMTS and MMPs in co-cultures. Conclusion: In summary, we believe, these results add to the evidence that in human OA, altered bi-directional signals transmitted between SBOs and ACCs significantly impacts the critical features of both cartilage and bone by producing abnormal levels of ADAMTS and MMPs. Furthermore, we have demonstrated for the first time that this altered cross-talk was mediated by the phosphorylation of MAPK-ERK1/2 signaling pathway.

Evaluating the transport impacts of transit oriented developments (TODS)

Sustainable transport has become a necessity instead of an option, to address the problems of congestion and urban sprawl, whose effects include increased trip lengths and travel time. A more sustainable form of development, known as Transit Oriented Development (TOD) is presumed to offer sustainable travel choices with reduced need to travel to access daily destinations, by providing a mixture of land uses together with good quality of public transport service, infrastructure for walking and cycling. However, performance assessment of these developments with respect to travel characteristics of their inhabitants is required. This research proposes a five step methodology for evaluating the transport impacts of TODs. The steps for TOD evaluation include pre–TOD assessment, traffic and travel data collection, determination of traffic impacts, determination of travel impacts, and drawing outcomes. Typically, TODs are comprised of various land uses; hence have various types of users. Assessment of characteristics of all user groups is essential for obtaining an accurate picture of transport impacts. A case study TOD, Kelvin Grove Urban Village (KGUV), located 2km of north west of the Brisbane central business district in Australia was selected for implementing the proposed methodology and to evaluate the transport impacts of a TOD from an Australian perspective. The outcomes of this analysis indicated that KGUV generated 27 to 48 percent less traffic compared to standard published rates specified for homogeneous uses. Further, all user groups of KGUV used more sustainable modes of transport compared to regional and similarly located suburban users, with higher trip length for shopping and education trips. Although the results from this case study development support the transport claims of reduced traffic generation and sustainable travel choices by way of TODs, further investigation is required, considering different styles, scales and locations of TODs. The proposed methodology may be further refined by using results from new TODs and a framework for TOD evaluation may be developed.

Competition and innovation : independent production in the Chinese TV industry

Independent television production is recognised for its capacity to generate new kinds of program content, as well as deliver innovation in formats. Globally, the television industry is entering into the post-broadcasting era where audiences are fragmented and content is distributed across multiple platforms. The effects of this convergence are now being felt in China, as it both challenges old statist models and presents new opportunities for content innovation. This thesis discusses the status of independent production in China, making relevant comparisons with independent production in other countries. Independent television production has become an important element in the reform of broadcasting in China in the past decade. The first independent TV production company was registered officially in 1994. While there are now over 4000 independent companies, the term „independent. does not necessarily constitute autonomy. The question the thesis addresses is: what is the status and nature of independence in China? Is it an appropriate term to use to describe the changing environment, or is it a misnomer? The thesis argues that Chinese independents operate alongside the mainstream state-owned system; they are „dependent. on the mainstream. Therefore independent television in China is a relative term. By looking at several companies in Beijing, mainly in entertainment, TV drama and animation, the thesis shows how the sector is injecting fresh ideas into the marketplace and how it plays an important role in improving innovation in many aspects of the television industry. The thesis shows how independent television companies in China are looking to protect their property rights. It demonstrates that far from being at the cutting edge, independents are reliant on a system that has many inbuilt structural problems. The thesis outlines many of the challenges facing 'independents'.

Improving service innovation through structured process-oriented knowledge infrastructure design

Formalised service innovation is a central tenet of enterprise systems lifecycle phases. Event driven process models extended with knowledge objects are found to be not useful in early lifecycle phases. When an upgrade is required, a map of the knowledge infrastructure is needed to better design further service innovation because functional maps no longer adequately describe the context adequately. By looking at formal changes to business processes as service innovations, and recognising the knowledge infrastructure inherent in services generally, changes driven through technology such as ES can be better understood with the application of frameworks such as B-KIDE.

Analysis of productive performance of crop and animal production systems : an integrated analytical framework

This article presents a two-stage analytical framework that integrates ecological crop (animal) growth and economic frontier production models to analyse the productive efficiency of crop (animal) production systems. The ecological crop (animal) growth model estimates "potential" output levels given the genetic characteristics of crops (animals) and the physical conditions of locations where the crops (animals) are grown (reared). The economic frontier production model estimates "best practice" production levels, taking into account economic, institutional and social factors that cause farm and spatial heterogeneity. In the first stage, both ecological crop growth and economic frontier production models are estimated to calculate three measures of productive efficiency: (1) technical efficiency, as the ratio of actual to "best practice" output levels; (2) agronomic efficiency, as the ratio of actual to "potential" output levels; and (3) agro-economic efficiency, as the ratio of "best practice" to "potential" output levels. Also in the first stage, the economic frontier production model identifies factors that determine technical efficiency. In the second stage, agro-economic efficiency is analysed econometrically in relation to economic, institutional and social factors that cause farm and spatial heterogeneity. The proposed framework has several important advantages in comparison with existing proposals. Firstly, it allows the systematic incorporation of all physical, economic, institutional and social factors that cause farm and spatial heterogeneity in analysing the productive performance of crop and animal production systems. Secondly, the location-specific physical factors are not modelled symmetrically as other economic inputs of production. Thirdly, climate change and technological advancements in crop and animal sciences can be modelled in a "forward-looking" manner. Fourthly, knowledge in agronomy and data from experimental studies can be utilised for socio-economic policy analysis. The proposed framework can be easily applied in empirical studies due to the current availability of ecological crop (animal) growth models, farm or secondary data, and econometric software packages. The article highlights several directions of empirical studies that researchers may pursue in the future.

Towards a model of dynamic capabilities in innovation-based competitive strategy : insights from project-oriented service firms

With the growing significance of services in most developed economies, there is an increased interest in the role of service innovation in service firm competitive strategy. Despite growing literature on service innovation, it remains fragmented reflecting the need for a model that captures key antecedents driving the service innovation-based competitive advantage process. Building on extant literature and using thirteen in-depth interviews with CEOs of project-oriented service firms, this paper presents a model of innovation-based competitive advantage. The emergent model suggests that entrepreneurial service firms pursuing innovation carefully select and use dynamic capabilities that enable them to achieve greater innovation and sustained competitive advantage. Our findings indicate that firms purposefully use create, extend and modify processes to build and nurture key dynamic capabilities. The paper presents a set of theoretical propositions to guide future research. Implications for theory and practice are discussed. Finally, directions for future research are outlined.

Support for energy-oriented design in the Australian context

There is a need for decision support tools that integrate energy simulation into early design in the context of Australian practice. Despite the proliferation of simulation programs in the last decade, there are no ready-to-use applications that cater specifically for the Australian climate and regulations. Furthermore, the majority of existing tools focus on achieving interaction with the design domain through model-based interoperability, and largely overlook the issue of process integration. This paper proposes an energy-oriented design environment that both accommodates the Australian context and provides interactive and iterative information exchanges that facilitate feedback between domains. It then presents the structure for DEEPA, an openly customisable system that couples parametric modelling and energy simulation software as a means of developing a decision support tool to allow designers to rapidly and flexibly assess the performance of early design alternatives. Finally, it discusses the benefits of developing a dynamic and concurrent performance evaluation process that parallels the characteristics and relationships of the design process.

Energy-oriented design tools for collaboration in the cloud

Emerging from the challenge to reduce energy consumption in buildings is the need for energy simulation to be used more effectively to support integrated decision making in early design. As a critical response to a Green Star case study, we present DEEPA, a parametric modeling framework that enables architects and engineers to work at the same semantic level to generate shared models for energy simulation. A cloud-based toolkit provides web and data services for parametric design software that automate the process of simulating and tracking design alternatives, by linking building geometry more directly to analysis inputs. Data, semantics, models and simulation results can be shared on the fly. This allows the complex relationships between architecture, building services and energy consumption to be explored in an integrated manner, and decisions to be made collaboratively.

Enterprise architecture and the integration of service-oriented architecture

In recent years, enterprise architecture (EA) has captured a growing attention as a means to systematically consolidate and interrelate diverse business and IT artefacts in order to provide holistic decision support. The recent popularity of a service-orientation has added “service “and related constructs as a new element that requires consideration within an Enterprise Architecture. Since the emergence of the Service-Oriented Architecture (SOA), many attempts have been made to incorporate SOA artefacts in existing EA frameworks. Yet, the approaches taken to achieve this goal differ substantially for the most commonly used EA frameworks to date. SOA in the context of enterprise architecture is one of the future research challenges. Several authors argue that further research is needed in order to understand how SOA impacts prior enterprise architecture frameworks. This study explores SOA integration within EA, identifies SOA integration approaches within EA and identifies factors that impact SOA integration within Enterprise Architecture.

Preliminary study on bridge health prediction using Dynamic Objective Oriented Bayesian Network (DOOBN)

The availability of bridges is crucial to people’s daily life and national economy. Bridge health prediction plays an important role in bridge management because maintenance optimization is implemented based on prediction results of bridge deterioration. Conventional bridge deterioration models can be categorised into two groups, namely condition states models and structural reliability models. Optimal maintenance strategy should be carried out based on both condition states and structural reliability of a bridge. However, none of existing deterioration models considers both condition states and structural reliability. This study thus proposes a Dynamic Objective Oriented Bayesian Network (DOOBN) based method to overcome the limitations of the existing methods. This methodology has the ability to act upon as a flexible unifying tool, which can integrate a variety of approaches and information for better bridge deterioration prediction. Two demonstrative case studies are conducted to preliminarily justify the feasibility of the methodology

A task-oriented approach to search engine usability studies

Usability is a multi-dimensional characteristic of a computer system. This paper focuses on usability as a measurement of interaction between the user and the system. The research employs a task-oriented approach to evaluate the usability of a meta search engine. This engine encourages and accepts queries of unlimited size expressed in natural language. A variety of conventional metrics developed by academic and industrial research, including ISO standards,, are applied to the information retrieval process consisting of sequential tasks. Tasks range from formulating (long) queries to interpreting and retaining search results. Results of the evaluation and analysis of the operation log indicate that obtaining advanced search engine results can be accomplished simultaneously with enhancing the usability of the interactive process. In conclusion, we discuss implications for interactive information retrieval system design and directions for future usability research. © 2008 Academy Publisher.

Power relations and small and medium-sized enterprise strategies for capturing value in global production networks: visual effects (VFX) service firm in the Hollywood film industry

Power relations and small and medium-sized enterprise strategies for capturing value in global production networks: visual effects (VFX) service firms in the Hollywood film industry, Regional Studies. This paper provides insights into the way in which non-lead firms manoeuvre in global value chains in the pursuit of a larger share of revenue and how power relations affect these manoeuvres. It examines the nature of value capture and power relations in the global supply of visual effects (VFX) services and the range of strategies VFX firms adopt to capture higher value in the global value chain. The analysis is based on a total of thirty-six interviews with informants in the industry in Australia, the United Kingdom and Canada, and a database of VFX credits for 3323 visual products for 640 VFX firms.