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.

Preparation and characterisation of tri-calcium phosphate scaffolds with tunnel-like macro-pores for bone tissue engineering

Calcium Phosphate ceramics have been widely used in tissue engineering due to their excellent biocompatibility and biodegradability. In the physiological environment, they are able to gradually degrade, absorbed and promote bone growth. Ultimately, they are capable of replacing damaged bone with new tissue. However, their low mechanical properties limit calcium phosphate ceramics in load-bearing applications. To obtain sufficient mechanical properties as well as high biocompatibility is one of the main focuses in biomaterials research. Therefore, the current project focuses on the preparation and characterization of porous tri-calcium phosphate (TCP) ceramic scaffolds. Hydroxapatite (HA) was used as the raw material, and normal calcium phosphate bioglass was added to adjust the ratio between calcium and phosphate. It was found that when 20% bioglass was added to HA and sintered at 1400oC for 3 hours, the TCP scaffold was obtained and this was confirmed by X-ray diffraction (XRD) analysis. Test results have shown that by applying this method, TCP scaffolds have significantly higher compressive strength (9.98MPa) than those made via TCP powder (<3MPa). Moreover, in order to further increase the compressive strength of TCP scaffolds, the samples were then coated with bioglass. For normal bioglass coated TCP scaffold, compressive strength was 16.69±0.5MPa; the compressive strength for single layer mesoporous bioglass coated scaffolds was 15.03±0.63MPa. In addition, this project has also concentrated on sizes and shapes effects; it was found that the cylinder scaffolds have more mechanical property than the club ones. In addition, this project performed cell culture within scaffold to assess biocompatibility. The cells were well distributed in the scaffold, and the cytotoxicity test was performed by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di- phenytetrazoliumromide (MTT) assay. The Alkaline Phosphatase (Alp) activity of human bone marrow mesenchymal stem cell system (hBMSCs) seeded on scaffold expressed higher in vitro than that in the positive control groups in osteogenic medium, which indicated that the scaffolds were both osteoconductive and osteoinductive, showing potential value in bone tissue engineering.

The importance of organisational culture to improve a project selection process : case of public infrastructure project in Indonesia

Project selection is a decision-making process that is not merely influenced by technical aspects but also by the people who involved in the process. Organisational culture is described as a set of values and norms that are shared by people within the organisation that affects the way they interact with each other and with stakeholders from outside the organisation. The aim of this paper is to emphasize the importance of organisational culture on improving the quality of decisions in the project selection process, in addition to the influence of technical aspects of a project. The discussion is based on an extensive literature review and, as such, represents the first part of a research agenda investigating the impact of organisational culture on the project selection process applicable specifically to road infrastructure contracts. Four existing models of organisational culture (Denison 1990; Cameron and Quinn 2006; Hofstede 2001; Glaser et al 1987) are discussed and reviewed in view of their use in the larger research project to investigate the impact of culture on identified critical elements of decision-making. An understating of the way organisational culture impacts on project selection will increase the likelihood in future of relevant government departments selecting projects that achieve their stated organisational goals.

The impact of organisational culture on the project selection process : The case of public infrastructure project in Indonesia

Project selection is a complex decision-making process as it involves multiple objectives, constraints and stakeholders. Understanding the organisation, in particular organisational culture, is an essential stage in improving decision-making process. The influences of organisational culture on decision-making can be seen in the way people work as a team, act and cooperate in their teamwork to achieve the set goals, and also in how people think, prioritize and decide. This paper aims to give evidence of the impact of organisational culture on the decision-making process in project selection, in the Indonesian context. Data was collected from a questionnaire survey developed based on the existing models of organisational culture (Denison 1990, Hofstede 2001, and Glaser et al 1987). Four main cultural traits (involvement, consistency, mission and power-distance) were selected and employed to examine the influence of organisational culture on the effectiveness of decision-making in the current Indonesian project selection processes. The results reveal that there are differences in organisational cultures for two organisations in three provinces. It also suggests that organisational culture (particularly the traits of ‘involvement’, ‘consistency’ and ‘mission’) contribute to the effectiveness of decision-making in the selected cases.

Psychedelic Culture

This exhibition catalogue essay provides an introduction to psychedelic culture during the postwar period. It describes the early use of LSD in psychiatric circles and its conception as a psychotomimetic substance. It then considers its use by literary figures such as Aldous Huxley and followers of the Beat Generation. Timothy Leary's role as an LSD philosopher is also explained as is the rise of the Hippies and the ensuing counterculture. This culture produced a range of cultural forms such as music, fashion, graphic design and other visual arts that were informed by hallucinations experienced under the influence of LSD. It concludes with a description of the end of the Hippie movement in the 1970s.

Iron biogeochemistry and associated greenhouse gas evolution in a forested subtropical Australian coastal catchment : Poona Creek, Southeast Queensland

Iron (Fe) is the fourth most abundant element in the Earth’s crust. Excess Fe mobilization from terrestrial into aquatic systems is of concern for deterioration of water quality via biofouling and nuisance algal blooms in coastal and marine systems. Substantial Fe dissolution and transport involve alternate Fe(II) oxidation followed by Fe(III) reduction, with a diversity of Bacteria and Archaea acting as the key catalyst. Microbially-mediated Fe cycling is of global significance with regard to cycles of carbon (C), sulfur (S) and manganese (Mn). However, knowledge regarding microbial Fe cycling in circumneutral-pH habitats that prevail on Earth has been lacking until recently. In particular, little is known regarding microbial function in Fe cycling and associated Fe mobilization and greenhouse (CO2 and CH4, GHG) evolution in subtropical Australian coastal systems where microbial response to ambient variations such as seasonal flooding and land use changes is of concern. Using the plantation-forested Poona Creek catchment on the Fraser Coast of Southeast Queensland (SEQ), this research aimed to 1) study Fe cycling-associated bacterial populations in diverse terrestrial and aquatic habitats of a representative subtropical coastal circumneutral-pH (4–7) ecosystem; and 2) assess potential impacts of Pinus plantation forestry practices on microbially-mediated Fe mobilization, organic C mineralization and associated GHG evolution in coastal SEQ. A combination of wet-chemical extraction, undisturbed core microcosm, laboratory bacterial cultivation, microscopy and 16S rRNA-based molecular phylogenetic techniques were employed. The study area consisted primarily of loamy sands, with low organic C and dissolved nutrients. Total reactive Fe was abundant and evenly distributed within soil 0–30 cm profiles. Organic complexation primarily controlled Fe bioavailability and forms in well-drained plantation soils and water-logged, native riparian soils, whereas tidal flushing exerted a strong “seawater effect” in estuarine locations and formed a large proportion of inorganic Fe(III) complexes. There was a lack of Fe(II) sources across the catchment terrestrial system. Mature, first-rotation plantation clear-felling and second-rotation replanting significantly decreased organic matter and poorly crystalline Fe in well-drained soils, although variations in labile soil organic C fractions (dissolved organic C, DOC; and microbial biomass C, MBC) were minor. Both well-drained plantation soils and water-logged, native-vegetation soils were inhabited by a variety of cultivable, chemotrophic bacterial populations capable of C, Fe, S and Mn metabolism via lithotrophic or heterotrophic, (micro)aerobic or anaerobic pathways. Neutrophilic Fe(III)-reducing bacteria (FeRB) were most abundant, followed by aerobic, heterotrophic bacteria (heterotrophic plate count, HPC). Despite an abundance of FeRB, cultivable Fe(II)-oxidizing bacteria (FeOB) were absent in associated soils. A lack of links between cultivable Fe, S or Mn bacterial densities and relevant chemical measurements (except for HPC correlated with DOC) was likely due to complex biogeochemical interactions. Neither did variations in cultivable bacterial densities correlate with plantation forestry practices, despite total cultivable bacterial densities being significantly lower in estuarine soils when compared with well-drained plantation soils and water-logged, riparian native-vegetation soils. Given that bacterial Fe(III) reduction is the primary mechanism of Fe oxide dissolution in soils upon saturation, associated Fe mobilization involved several abiotic and biological processes. Abiotic oxidation of dissolved Fe(II) by Mn appeared to control Fe transport and inhibit Fe dissolution from mature, first-rotation plantation soils post-saturation. Such an effect was not observed in clear-felled and replanted soils associated with low SOM and potentially low Mn reactivity. Associated GHG evolution post-saturation mainly involved variable CO2 emissions, with low, but consistently increasing CH4 effluxes in mature, first-rotation plantation soil only. In comparison, water-logged soils in the riparian native-vegetation buffer zone functioned as an important GHG source, with high potentials for Fe mobilization and GHG, particularly CH4 emissions in riparian loam soils associated with high clay and crystalline Fe fractions. Active Fe–C cycling was unlikely to occur in lower-catchment estuarine soils associated with low cultivable bacterial densities and GHG effluxes. As a key component of bacterial Fe cycling, neutrophilic FeOB widely occurred in diverse aquatic, but not terrestrial, habitats of the catchment study area. Stalked and sheathed FeOB resembling Gallionella and Leptothrix were limited to microbial mat material deposited in surface fresh waters associated with a circumneutral-pH seep, and clay-rich soil within riparian buffer zones. Unicellular, Sideroxydans-related FeOB (96% sequence identity) were ubiquitous in surface and subsurface freshwater environments, with highest abundance in estuary-adjacent shallow coastal groundwater water associated with redox transition. The abundance of dissolved C and Fe in the groundwater-dependent system was associated with high numbers of cultivable anaerobic, heterotrophic FeRB, microaerophilic, putatively lithotrophic FeOB and aerobic, heterotrophic bacteria. This research represents the first study of microbial Fe cycling in diverse circumneutral-pH environments (terrestrial–aquatic, freshwater–estuarine, surface–subsurface) of a subtropical coastal ecosystem. It also represents the first study of its kind in the southern hemisphere. This work highlights the significance of bacterial Fe(III) reduction in terrestrial, and bacterial Fe(II) oxidation in aquatic catchment Fe cycling. Results indicate the risk of promotion of Fe mobilization due to plantation clear-felling and replanting, and GHG emissions associated with seasonal water-logging. Additional significant outcomes were also achieved. The first direct evidence for multiple biomineralization patterns of neutrophilic, microaerophilic, unicellular FeOB was presented. A putatively pure culture, which represents the first cultivable neutrophilic FeOB from the southern hemisphere, was obtained as representative FeOB ubiquitous in diverse catchment aquatic habitats.

Facilitating Malaysia towards innovative society : arguing the case for open access policy

The Malaysian National Innovation Model blueprint states that there is an urgent need to pursue an innovation-oriented economy to improve the nation’s capacity for knowledge, creativity and innovation. In nurturing a pervasive innovation culture, the Malaysian government has declared the year 2010 as an Innovative Year whereby creativity among its population is highly celebrated. However, while Malaysian citizens are encouraged to be creative and innovative, scientific data and information generated from publicly funded research in Malaysia is locked up because of rigid intellectual property licensing regimes and traditional publishing models. Reflecting on these circumstances, this paper looks at, and argue why, scientific data and information should be made available, accessible and re-useable freely to promote the grassroots level of innovation in Malaysia. Using innovation theory as its platform of argument, this paper calls for an open access policy for publicly funded research output to be adopted and implemented in Malaysia. Simultaneously, a normative analytic approach is used to determine the types of open access policy that ought to be adopted to spur greater innovation among Malaysians.

Supply chain sustainability : a relationship management approach moderated by culture and commitment

This paper explores the nature of relationship management on construction projects in Australia and examines the effects of culture, by means of Schwarz’s value survey, on relationships under different contract strategies. The research was based on the view that the development of a sustainable supply chain depends on the transfer of knowledge and capabilities from the larger players in the supply chain through collaboration brought about by relationship management. The research adopted a triangulated approach in which quantitative data were collected by questionnaire, interviews were conducted to explore and enrich the quantitative data and case studies were undertaken in order to illustrate and validate the fi ndings. The aim was to investigate how values and attitudes enhance or reduce the incorporation of the supply chain into the project. From the research it was found that the degree of match and mismatch between values and contract strategy impacts commitment and the engagement and empowerment of the supply chain.

Safety culture research in the construction sector : interlocked projects in safety competency and safety effectiveness indicators

Safety culture is a concept that has long been accepted in high risk industries such as aviation, nuclear industries and mining, however, considerable research is now being undertaken within the construction sector, with varying levels of success. The current paper discusses three recent interlocked projects that have had some success in the Australian construction industry. The first project examined the development and implementation of a safety competency framework targeted at safety critical positions across first tier construction organisations. Combining qualitative and quantitative methods, the project: developed a matrix of safety critical positions (n=11) and safety managements tasks (SMTs; n=39); mapped the process steps for their acquisition and ongoing development; detailed the knowledge, skills and behaviours required for all SMTs; and outlined organisational cultural outcomes that could be anticipated in a successful implementation of the framework. The second project extended research on safety competency and leadership to develop behavioural guidelines for leaders to drive safety culture change down to second tier companies. This was designed to assist smaller construction companies to customise their own competency framework and develop implementation guidelines that match their aspirations and resources. The third interlocked project explored the use of safety effectiveness indicators (SEIs) as an industry-relevant assessment tool for reducing risk on construction sites. With direct linkages to safety competencies and safety management tasks, the SEIs are the next step towards an integrated safety cultural approach to safety and extend the concept of positive performance indicators (PPIs) by providing a valid, reliable, and user friendly measurement platform. Taken together, the results of the interlocked projects suggest that safety culture research has many potential benefits for the construction industry, particularly when research is conducted in partnership with industry stakeholders. Suggestions are made for future research, including further application and testing of the safety competency framework and aligning SEIs across construction projects of varying size, location and design.

Controlling cell-material interactions with polymer nanocomposites by use of surface modifying additives

Polymer nanocomposites (NC) are fabricated by incorporating well dispersed nanoscale particles within a polymer matrix. This study focuses on elastomeric polyurethane (PU) based nanocomposites, containing organically modified silicates (OMS), as bioactive materials. Nanocomposites incorporating chlorhexidine diacetate as an organic modifier (OM) were demonstrated to be antibacterial with a dose dependence related to both the silicate loading and the loading of OM. When the non-antibacterial OM dodecylamine was used, both cell and platelet adhesion were decreased on the nanocomposite surface. These results suggest that OM is released from the polymer and can impact on cell behaviour at the interface. Nanocomposites have potential use as bioactive materials in a range of biomedical applications.

Brisbane : urban construction, suburban dreaming

Brisbane, the capital city of Queensland, has catapulted from a small, provincial town to a larger metropolis within two decades from the inception of urban renewal in 1992. Once a low-density suburban city, its inner-city and some suburban areas are now medium to high density, with the rise in apartment buildings creating new and denser modes of living. This article suggests that urbanism demands different habits of living from suburbanism and examines the relationship between the material and representational city to explore the ways in which promotions of the “new” Brisbane during its early urban renewal period reproduces the ethos of suburban living.