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.

Challenges facing sustainable housing in Saudi Arabia : a current study showing the level of public awareness

Sustainable housing implementation requires strong support from the public, government and the housing industry. Lack of public awareness and understanding of the language and the meaning of sustainable housing may cause lack of public support. Salama stated that "sustainability or sustainable design is simply a rephrasing of some of the forgotten values of traditional architecture and urbanism"(Salama 2007). This exploratory paper examines public awareness of sustainable housing in Saudi Arabia. In developing countries, like Saudi Arabia, which have been experiencing a rapid rate of urbanisation, sustainable concept intervention is essential due to the scarcity of resources (Reffat 2004a). Sustainable building methods include the full use of the site design, passive solar design, natural light and ventilation. This paper reports on an exploratory survey on understanding the potential of the implementation of sustainable housing in Saudi Arabia. The main problem is that more than half of respondents were not aware of sustainable housing. Thus, one of the recommendations from the survey is to educate the public by using local media to inform people of the benefits of sustainable implementation to both new and existing housing stock.

Making the case for low cost railway level crossings

This paper discusses the challenges of making a case for the adoption of low cost railway level crossings in Australia. Several issues are discussed in this paper including legal issues associated with the treatment of low-exposure passive crossings with low cost level crossing warning devices (LCLCWDs); principles of operation and deployment for LCLCWDs; and technical and human factors aspects of safety and availability. The Cooperative Research Centre (CRC) for Rail Innovation’s affordable level crossings project aims to address a number of these technical and human factors issues through research and field trials.

Barriers to information transfer for multi-trauma patients upon discharge from the Emergency Department in a tertiary level hospital

Purpose: Communication is integral to effective trauma care provision. This presentation will report on barriers to meaningful information transfer for multi-trauma patients upon discharge from the Emergency Department (ED) to the care areas of Intensive Care Unit, High Dependency Unit, and Perioperative Services. This is an ongoing study at one tertiary level hospital in Queensland. Method: This is a multi-phase, mixed method study. In Phase 1 data were collected about information transfer. This Phase was initially informed by a comprehensive literature review, then via focus groups, chart audit, staff survey and review of national and international trauma forms. Results: The barriers identified related to nursing handover, documented information, time inefficiency, patient complexity and stability and time of transfer. Specifically this included differences in staff expectations and variation in the nursing handover processes, no agreed minimum dataset of information handed over, missing, illegible or difficult to find information in documentation (both medical and nursing), low compliance with some forms used for documentation. Handover of these patients is complex with information coming from many sources, dealing with issues is more difficult for these patients when transferred out of hours. Conclusions and further directions: This study investigated the current communication processes and standards of information transfer to identify barriers and issues. The barriers identified were the structure used for documentation, processes used (e.g. handover), patient acuity and time. This information is informing the development, implementation and evaluation of strategies to ameliorate the issues identified.

In search of the antecendents for risky driving at railway level crossings

This paper discusses human factors issues of low cost railway level crossings in Australia. Several issues are discussed in this paper including safety at passive level railway crossings, human factors considerations associated with unavailability of a warning device, and a conceptual model for how safety could be compromised at railway level crossings following prolonged or frequent unavailability. The research plans to quantify safety risk to motorists at level crossings using a Human Reliability Assessment (HRA) method, supported by data collected using an advanced driving simulator. This method aims to identify human error within tasks and task units identified as part of the task analysis process. It is anticipated that by modelling driver behaviour the current study will be able to quantify meaningful task variability including temporal parameters, between participants and within participants. The process of complex tasks such as driving through a level crossing is fundamentally context-bound. Therefore this study also aims to quantify those performance-shaping factors that contribute to vehicle train collisions by highlighting changes in the task units and driver physiology. Finally we will also consider a number of variables germane to ensuring external validity of our results. Without this inclusion, such an analysis could seriously underestimate the probabilistic risk assessment.

Effective adsorption of sodium dodecylsulfate (SDS) by hydrocalumite (CaAl-LDH-Cl) induced by self-dissolution and re-precipitation mechanism

Hydrocalumite (CaAl-LDH-Cl) were synthesized through a rehydration method involving a freshly prepared tricalcium aluminate (C3A) with CaCl2 solution. To understand the intercalation behaviour of sodium dodecylsulfate (SDS) with CaAl-LDH-Cl, X-ray diffraction (XRD), Fourier transform infrared (FTIR), scanning electron microscopy (SEM), transmission electron microscope (TEM), X-ray photoelectron spectroscopy (XPS), inductively coupled plasma-atomic emission spectrometer (ICP) and elemental analysis have been undertaken. The sorption isotherms with SDS reveal that the maximum sorption amount of SDS by CaAl-LDH-Cl could reach 3.67 mmol•g-1. The results revealed that CaAl-LDH-Cl holds a self-dissolution property, about 20-30% of which is dissolved. And the dissolved Ca2+, Al3+ ions are combined with SDS to form CaAl-SDS or Ca-SDS precipitation. It has been highlighted that the composition of resulting products is strongly dependent upon the SDS concentration. With increasing SDS concentrations, the main resulting product changes from CaAl-SDS to Ca-SDS, and the value of interlayer spacing increased to 3.27 nm.

A human factors investigation into the unavailability of active warnings at railway level crossings

This paper discusses human factors issues of low cost railway level crossings in Australia. Several issues are discussed in this paper including safety at passive level railway crossings, human factors considerations associated with unavailability of a warning device, and a conceptual model for how safety could be compromised at railway level crossings following prolonged or frequent unavailability. The research plans to quantify safety risk to motorists at level crossings using a Human Reliability Assessment (HRA) method, supported by data collected using an advanced driving simulator. This method aims to identify human error within tasks and task units identified as part of the task analysis process. It is anticipated that by modelling driver behaviour the current study will be able to quantify meaningful task variability including temporal parameters, between participants and within participants. The process of complex tasks such as driving through a level crossing is fundamentally context-bound. Therefore this study also aims to quantify those performance-shaping factors that contribute to vehicle train collisions by highlighting changes in the task units and driver physiology. Finally we will also consider a number of variables germane to ensuring external validity of our results. Without this inclusion, such an analysis could seriously underestimate risk.

Charting the system : the integrated master schedule as a multi-level and poly-temporal boundary object in complex projects

This paper investigates the use of visual artifacts to represent a complex adaptive system (CAS). The integrated master schedule (IMS) is one of those visuals widely used in complex projects for scheduling, budgeting, and project management. In this paper, we discuss how the IMS outperforms the traditional timelines and acts as a ‘multi-level and poly-temporal boundary object’ that visually represents the CAS. We report the findings of a case study project on the way the IMS mapped interactions, interdependencies, constraints and fractal patterns in a complex project. Finally, we discuss how the IMS was utilised as a complex boundary object by eliciting commitment and development of shared mental models, and facilitating negotiation through the layers of multiple interpretations from stakeholders.

A multi-level model of leadership in complex project management

Management (or perceived mismanagement) of large-scale, complex projects poses special problems and often results in spectacular failures, cost overruns, time blowouts and stakeholder dissatisfaction. While traditional project management responds with increasingly administrative constraints, we argue that leaders of such projects also need to display adaptive and enabling behaviours to foster adaptive processes, such as opportunity recognition, which requires an interaction of cognitive and affective processes of individual, project, and team leader attributes and behaviours. At the core of this model we propose is an interaction of cognitive flexibility, affect and emotional intelligence. The result of this interaction is enhanced leader opportunity recognition that, in turn, facilitates multilevel outcomes.

How do level of experience, purpose for riding, and preference for facilities affect location of riding?

Characteristics of the road infrastructure affect both the popularity of bicycling and its safety, but comparisons of the safety performance of infrastructure may be confounded by differences in the profiles of cyclists who use them. Data from a survey of 2,532 adult bicycle riders in Queensland, Australia, demonstrated that many riders rode reluctantly in particular locations and that preference for riding location was influenced by degree of experience and riding purpose. Most riders rode most often and furthest per week on urban roads, but approximately one-third of all riders (and more new riders) rode there reluctantly. Almost two-thirds of riders rode on bicycle paths, most by choice, not reluctantly. New riders rode proportionally more on bicycle paths, but continuing riders rode further in absolute terms. Utilitarian riders were more likely to ride on bicycle paths than social and fitness riders and almost all of this riding was by choice. Fitness riders were more reluctant in their use of bicycle paths, but still most of their use was by choice. One-third of the respondents reported riding on the sidewalk (legal in Queensland), with approximately two-thirds doing so reluctantly. The frequency and distance ridden on the sidewalk was less than for urban roads and bicycle paths. Sidewalks and bicycle paths were important facilities for both inexperienced and experienced riders and for utilitarian riding, especially when urban roads were considered a poor choice for cycling.

A Bayesian approach for estimating detection times in horses : exploring the pharmacokinetics of a urinary acepromazine metabolite

We describe the population pharmacokinetics of an acepromazine (ACP) metabolite (2-(1-hydroxyethyl)promazine) (HEPS) in horses for the estimation of likely detection times in plasma and urine. Acepromazine (30 mg) was administered to 12 horses, and blood and urine samples were taken at frequent intervals for chemical analysis. A Bayesian hierarchical model was fitted to describe concentration-time data and cumulative urine amounts for HEPS. The metabolite HEPS was modelled separately from the parent ACP as the half-life of the parent was considerably less than that of the metabolite. The clearance ($Cl/F_{PM}$) and volume of distribution ($V/F_{PM}$), scaled by the fraction of parent converted to metabolite, were estimated as 769 L/h and 6874 L, respectively. For a typical horse in the study, after receiving 30 mg of ACP, the upper limit of the detection time was 35 hours in plasma and 100 hours in urine, assuming an arbitrary limit of detection of 1 $\mu$g/L, and a small ($\approx 0.01$) probability of detection. The model derived allowed the probability of detection to be estimated at the population level. This analysis was conducted on data collected from only 12 horses, but we assume that this is representative of the wider population.

The development of a conceptual framework for a district 4-Year Pavement Management Plan, implementation of network-level project screening process

The Texas Department of Transportation (TxDOT) is concerned about the widening gap between preservation needs and available funding. Funding levels are not adequate to meet the preservation needs of the roadway network; therefore projects listed in the 4-Year Pavement Management Plan must be ranked to determine which projects should be funded now and which can be postponed until a later year. Currently, each district uses locally developed methods to prioritize projects. These ranking methods have relied on less formal qualitative assessments based on engineers’ subjective judgment. It is important for TxDOT to have a 4-Year Pavement Management Plan that uses a transparent, rational project ranking process. The objective of this study is to develop a conceptual framework that describes the development of the 4-Year Pavement Management Plan. It can be largely divided into three Steps; 1) Network-Level project screening process, 2) Project-Level project ranking process, and 3) Economic Analysis. A rational pavement management procedure and a project ranking method accepted by districts and the TxDOT administration will maximize efficiency in budget allocations and will potentially help improve pavement condition. As a part of the implementation of the 4-Year Pavement Management Plan, the Network-Level Project Screening (NLPS) tool including the candidate project identification algorithm and the preliminary project ranking matrix was developed. The NLPS has been used by the Austin District Pavement Engineer (DPE) to evaluate PMIS (Pavement Management Information System) data and to prepare a preliminary list of candidate projects for further evaluation.

A fragment of the LG3 peptide of endorepellin is present in the urine of physically active mining workers : a potential marker of physical activity

Biomarker analysis has been implemented in sports research in an attempt to monitor the effects of exertion and fatigue in athletes. This study proposed that while such biomarkers may be useful for monitoring injury risk in workers, proteomic approaches might also be utilised to identify novel exertion or injury markers. We found that urinary urea and cortisol levels were significantly elevated in mining workers following a 12 hour overnight shift. These levels failed to return to baseline over 24h in the more active maintenance crew compared to truck drivers (operators) suggesting a lack of recovery between shifts. Use of a SELDI-TOF MS approach to detect novel exertion or injury markers revealed a spectral feature which was associated with workers in both work categories who were engaged in higher levels of physical activity. This feature was identified as the LG3 peptide, a C-terminal fragment of the anti-angiogenic / anti-tumourigenic protein endorepellin. This finding suggests that urinary LG3 peptide may be a biomarker of physical activity. It is also possible that the activity mediated release of LG3 / endorepellin into the circulation may represent a biological mechanism for the known inverse association between physical activity and cancer risk / survival.

Family-level relationships among the Australasian marsupial “herbivores” (Diprotodontia: Koala, wombats, kangaroos and possums)

The marsupial order Diprotodontia includes 10 extant families, which occupy all terrestrial habitats across Australia and New Guinea and have evolved remarkable dietary and locomotory diversity. Despite considerable attention, the interrelations of these families have for the most part remained elusive. In this study, we separately model mitochondrial RNA and protein-coding sequences in addition to nuclear protein-coding sequences to provide near-complete resolution of diprotodontian family-level phylogeny. We show that alternative topologies inferred in some previous studies are likely to be artifactual, resulting from branch-length and compositional biases. Subordinal groupings resolved herein include Vombatiformes (wombats and koala) and Phalangerida, which in turn comprises Petauroidea (petaurid gliders and striped, feathertail, ringtail and honey possums) and a clade whose plesiomorphic members possess blade-like premolars (phalangerid possums, kangaroos and their allies and most likely, pygmy possums). The topology resolved reveals ecological niche structuring among diprotodontians that has likely been maintained for more than 40 million years.