Recombinant protein production using a Tobacco yellow dwarf virus-based episomal expression vector : control of Rep activity

Over the past decade, plants have been used as expression hosts for the production of pharmaceutically important and commercially valuable proteins. Plants offer many advantages over other expression systems such as lower production costs, rapid scale up of production, similar post-translational modification as animals and the low likelihood of contamination with animal pathogens, microbial toxins or oncogenic sequences. However, improving recombinant protein yield remains one of the greatest challenges to molecular farming. In-Plant Activation (InPAct) is a newly developed technology that offers activatable and high-level expression of heterologous proteins in plants. InPAct vectors contain the geminivirus cis elements essential for rolling circle replication (RCR) and are arranged such that the gene of interest is only expressed in the presence of the cognate viral replication-associated protein (Rep). The expression of Rep in planta may be controlled by a tissue-specific, developmentally regulated or chemically inducible promoter such that heterologous protein accumulation can be spatially and temporally controlled. One of the challenges for the successful exploitation of InPAct technology is the control of Rep expression as even very low levels of this protein can reduce transformation efficiency, cause abnormal phenotypes and premature activation of the InPAct vector in regenerated plants. Tight regulation over transgene expression is also essential if expressing cytotoxic products. Unfortunately, many tissue-specific and inducible promoters are unsuitable for controlling expression of Rep due to low basal activity in the absence of inducer or in tissues other than the target tissue. This PhD aimed to control Rep activity through the production of single chain variable fragments (scFvs) specific to the motif III of Tobacco yellow dwarf virus (TbYDV) Rep. Due to the important role played by the conserved motif III in the RCR, it was postulated that such scFvs can be used to neutralise the activity of the low amount of Rep expressed from a “leaky” inducible promoter, thus preventing activation of the TbYDV-based InPAct vector until intentional induction. Such scFvs could also offer the potential to confer partial or complete resistance to TbYDV, and possibly heterologous viruses as motif III is conserved between geminiviruses. Studies were first undertaken to determine the levels of TbYDV Rep and TbYDV replication-associated protein A (RepA) required for optimal transgene expression from a TbYDV-based InPAct vector. Transient assays in a non-regenerable Nicotiana tabacum (NT-1) cell line were undertaken using a TbYDV-based InPAct vector containing the uidA reporter gene (encoding GUS) in combination with TbYDV Rep and RepA under the control of promoters with high (CaMV 35S) or low (Banana bunchy top virus DNA-R, BT1) activity. The replication enhancer protein of Tomato leaf curl begomovirus (ToLCV), REn, was also used in some co-bombardment experiments to examine whether RepA could be substituted by a replication enhancer from another geminivirus genus. GUS expression was observed both quantitatively and qualitatively by fluorometric and histochemical assays, respectively. GUS expression from the TbYDV-based InPAct vector was found to be greater when Rep was expected to be expressed at low levels (BT1 promoter) rather than high levels (35S promoter). GUS expression was further enhanced when Rep and RepA were co-bombarded with a low ratio of Rep to RepA. Substituting TbYDV RepA with ToLCV REn also enhanced GUS expression but more importantly highest GUS expression was observed when cells were co-transformed with expression vectors directing low levels of Rep and high levels of RepA irrespective of the level of REn. In this case, GUS expression was approximately 74-fold higher than that from a non-replicating vector. The use of different terminators, namely CaMV 35S and Nos terminators, in InPAct vectors was found to influence GUS expression. In the presence of Rep, GUS expression was greater using pInPActGUS-Nos rather than pInPActGUS-35S. The only instance of GUS expression being greater from vectors containing the 35S terminator was when comparing expression from cells transformed with Rep, RepA and REnexpressing vectors and either non-replicating vectors, p35SGS-Nos or p35SGS-35S. This difference was most likely caused by an interaction of viral replication proteins with each other and the terminators. These results indicated that (i) the level of replication associated proteins is critical to high transgene expression, (ii) the choice of terminator within the InPAct vector may affect expression levels and (iii) very low levels of Rep can activate InPAct vectors hence controlling its activity is critical. Prior to generating recombinant scFvs, a recombinant TbYDV Rep was produced in E. coli to act as a control to enable the screening for Rep-specific antibodies. A bacterial expression vector was constructed to express recombinant TbYDV Rep with an Nterminal His-tag (N-His-Rep). Despite investigating several purification techniques including Ni-NTA, anion exchange, hydrophobic interaction and size exclusion chromatography, N-His-Rep could only be partially purified using a Ni-NTA column under native conditions. Although it was not certain that this recombinant N-His-Rep had the same conformation as the native TbYDV Rep and was functional, results from an electromobility shift assay (EMSA) showed that N-His-Rep was able to interact with the TbYDV LIR and was, therefore, possibly functional. Two hybridoma cell lines from mice, immunised with a synthetic peptide containing the TbYDV Rep motif III amino acid sequence, were generated by GenScript (USA). Monoclonal antibodies secreted by the two hybridoma cell lines were first screened against denatured N-His-Rep in Western analysis. After demonstrating their ability to bind N-His-Rep, two scFvs (scFv1 and scFv2) were generated using a PCR-based approach. Whereas the variable heavy chain (VH) from both cell lines could be amplified, only the variable light chain (VL) from cell line 2 was amplified. As a result, scFv1 contained VH and VL from cell line 1, whereas scFv2 contained VH from cell line 2 and VL from cell line 1. Both scFvs were first expressed in E. coli in order to evaluate their affinity to the recombinant TbYDV N-His-Rep. The preliminary results demonstrated that both scFvs were able to bind to the denatured N-His-Rep. However, EMSAs revealed that only scFv2 was able to bind to native N-His-Rep and prevent it from interacting with the TbYDV LIR. Each scFv was cloned into plant expression vectors and co-bombarded into NT-1 cells with the TbYDV-based InPAct GUS expression vector and pBT1-Rep to examine whether the scFvs could prevent Rep from mediating RCR. Although it was expected that the addition of the scFvs would result in decreased GUS expression, GUS expression was found to slightly increase. This increase was even more pronounced when the scFvs were targeted to the cell nucleus by the inclusion of the Simian virus 40 large T antigen (SV40) nuclear localisation signal (NLS). It was postulated that the scFvs were binding to a proportion of Rep, leaving a small amount available to mediate RCR. The outcomes of this project provide evidence that very high levels of recombinant protein can theoretically be expressed using InPAct vectors with judicious selection and control of viral replication proteins. However, the question of whether the scFvs generated in this project have sufficient affinity for TbYDV Rep to prevent its activity in a stably transformed plant remains unknown. It may be that other scFvs with different combinations of VH and VL may have greater affinity for TbYDV Rep. Such scFvs, when expressed at high levels in planta, might also confer resistance to TbYDV and possibly heterologous geminiviruses.

Investigation of highly regulated promoters for the production of recombinant proteins in plants

Plants have been identified as promising expression systems for the commercial production of recombinant proteins. Plant-based protein production or “biofarming” offers a number of advantages over traditional expression systems in terms of scale of production, the capacity for post-translation processing, providing a product free of contaminants and cost effectiveness. A number of pharmaceutically important and commercially valuable proteins, such as antibodies, biopharmaceuticals and industrial enzymes are currently being produced in plant expression systems. However, several challenges still remain to improve recombinant protein yield with no ill effect on the host plant. The ability for transgenic plants to produce foreign proteins at commercially viable levels can be directly related to the level and cell specificity of the selected promoter driving the transgene. The accumulation of recombinant proteins may be controlled by a tissue-specific, developmentally-regulated or chemically-inducible promoter such that expression of recombinant proteins can be spatially- or temporally- controlled. The strict control of gene expression is particularly useful for proteins that are considered toxic and whose expression is likely to have a detrimental effect on plant growth. To date, the most commonly used promoter in plant biotechnology is the cauliflower mosaic virus (CaMV) 35S promoter which is used to drive strong, constitutive transgene expression in most organs of transgenic plants. Of particular interest to researchers in the Centre for Tropical Crops and Biocommodities at QUT are tissue-specific promoters for the accumulation of foreign proteins in the roots, seeds and fruit of various plant species, including tobacco, banana and sugarcane. Therefore this Masters project aimed to isolate and characterise root- and seed-specific promoters for the control of genes encoding recombinant proteins in plant-based expression systems. Additionally, the effects of matching cognate terminators with their respective gene promoters were assessed. The Arabidopsis root promoters ARSK1 and EIR1 were selected from the literature based on their reported limited root expression profiles. Both promoters were analysed using the PlantCARE database to identify putative motifs or cis-acting elements that may be associated with this activity. A number of motifs were identified in the ARSK1 promoter region including, WUN (wound-inducible), MBS (MYB binding site), Skn-1, and a RY core element (seed-specific) and in the EIR1 promoter region including, Skn-1 (seed-specific), Box-W1 (fungal elicitor), Aux-RR core (auxin response) and ABRE (ABA response). However, no previously reported root-specific cis-acting elements were observed in either promoter region. To confirm root specificity, both promoters, and truncated versions, were fused to the GUS reporter gene and the expression cassette introduced into Arabidopsis via Agrobacterium-mediated transformation. Despite the reported tissue-specific nature of these promoters, both upstream regulatory regions directed constitutive GUS expression in all transgenic plants. Further, similar levels of GUS expression from the ARSK1 promoter were directed by the control CaMV 35S promoter. The truncated version of the EIR1 promoter (1.2 Kb) showed some differences in the level of GUS expression compared to the 2.2 Kb promoter. Therefore, this suggests an enhancer element is contained in the 2.2 Kb upstream region that increases transgene expression. The Arabidopsis seed-specific genes ATS1 and ATS3 were selected from the literature based on their seed-specific expression profiles and gene expression confirmed in this study as seed-specific by RT-PCR analysis. The selected promoter regions were analysed using the PlantCARE database in order to identify any putative cis elements. The seed-specific motifs GCN4 and Skn-1 were identified in both promoter regions that are associated with elevated expression levels in the endosperm. Additionaly, the seed-specific RY element and the ABRE were located in the ATS1 promoter. Both promoters were fused to the GUS reporter gene and used to transform Arabidopsis plants. GUS expression from the putative promoters was consitutive in all transgenic Arabidopsis tissue tested. Importantly, the positive control FAE1 seed-specific promoter also directed constitutive GUS expression throughout transgenic Arabidopsis plants. The constitutive nature seen in all of the promoters used in this study was not anticipated. While variations in promoter activity can be caused by a number of influencing factors, the variation in promoter activity observed here would imply a major contributing factor common to all plant expression cassettes tested. All promoter constructs generated in this study were based on the binary vector pCAMBIA2300. This vector contains the plant selection gene (NPTII) under the transcriptional control of the duplicated CaMV 35S promoter. This CaMV 35S promoter contains two enhancer domains that confer strong, constitutive expression of the selection gene and is located immediately upstream of the promoter-GUS fusion. During the course of this project, Yoo et al. (2005) reported that transgene expression is significantly affected when the expression cassette is located on the same T-DNA as the 35S enhancer. It was concluded, the trans-acting effects of the enhancer activate and control transgene expression causing irregular expression patterns. This phenomenon seems the most plausible reason for the constitutive expression profiles observed with the root- and seed-specific promoters assessed in this study. The expression from some promoters can be influenced by their cognate terminator sequences. Therefore, the Arabidopsis ARSK1, EIR1, ATS1 and ATS3 terminator sequences were isolated and incorporated into expression cassettes containing the GUS reporter gene under the control of their cognate promoters. Again, unrestricted GUS activity was displayed throughout transgenic plants transformed with these reporter gene fusions. As previously discussed constitutive GUS expression was most likely due to the trans-acting effect of the upstream CaMV 35S promoter in the selection cassette located on the same T-DNA. The results obtained in this study make it impossible to assess the influence matching terminators with their cognate promoters have on transgene expression profiles. The obvious future direction of research continuing from this study would be to transform pBIN-based promoter-GUS fusions (ie. constructs containing no CaMV 35S promoter driving the plant selection gene) into Arabidopsis in order to determine the true tissue specificity of these promoters and evaluate the effects of their cognate 3’ terminator sequences. Further, promoter truncations based around the cis-elements identified here may assist in determining whether these motifs are in fact involved in the overall activity of the promoter.

On importer trust and commitment : a comparative study of two developing countries

Purpose: Relationship trust and commitment are two key dimensions of international exchanges. Both have been extensively investigated from an exporter (as opposed to importer) perspective in developed country (as opposed to developing country) contexts. To address these gaps, this study aims to develop a model of antecedents and outcomes of importer trust and commitment in two developing countries.---------- Design/methodology: The authors test the proposed model using data from Chile and Bangladesh. Hypotheses were tested using structural equation modeling (SEM).---------- Findings: SEM analysis revealed that most of the hypotheses were supported in both the Bangladeshi and Chilean context. The findings of this study also suggest that the effects of importer transaction-specific investments on importer commitment are distinct in the Bangladeshi context. Practical implications: Practically, these results show that trust and commitment are essential for enhancing importer relationship performance in developing countries. Importer trust in a foreign supplier is effective when suppliers are competent and provide relatively superior facilities, as opposed to opportunistic proclivity. Importer commitment to a foreign supplier is stronger when importers perceive that the foreign supplier is not opportunistic, but is knowledgeable and experienced with the importer market, and they perceive that it is an advantage importing from that supplier. Cultural similarity between importers and foreign suppliers improves importer trust in both countries. However, importer commitment in Chile increases with importer transaction-specific investment, but this is not found to be the case in Bangladesh.---------- Originality/value: This study contributes to the importer-exporter exchange relationship literature by testing a model of antecedents and outcomes of importer trust and commitment. The tested model is one of few that considers developing country contexts and incorporates two novel antecedents of trust and commitment: importer knowledge and experience, and supplier resource competency.

Quality of cause-of-death reporting using ICD-10 drowning codes : a descriptive study of 69 countries

Background: The systematic collection of high-quality mortality data is a prerequisite in designing relevant drowning prevention programmes. This descriptive study aimed to assess the quality (i.e., level of specificity) of cause-of-death reporting using ICD-10 drowning codes across 69 countries.---------- Methods: World Health Organization (WHO) mortality data were extracted for analysis. The proportion of unintentional drowning deaths coded as unspecified at the 3-character level (ICD-10 code W74) and for which the place of occurrence was unspecified at the 4th character (.9) were calculated for each country as indicators of the quality of cause-of-death reporting.---------- Results: In 32 of the 69 countries studied, the percentage of cases of unintentional drowning coded as unspecified at the 3-character level exceeded 50%, and in 19 countries, this percentage exceeded 80%; in contrast, the percentage was lower than 10% in only 10 countries. In 21 of the 56 countries that report 4-character codes, the percentage of unintentional drowning deaths for which the place of occurrence was unspecified at the 4th character exceeded 50%, and in 15 countries, exceeded 90%; in only 14 countries was this percentage lower than 10%.---------- Conclusion: Despite the introduction of more specific subcategories for drowning in the ICD-10, many countries were found to be failing to report sufficiently specific codes in drowning mortality data submitted to the WHO.

The return of a forgotten polymer : Polycaprolactone in the 21st century

During the resorbable-polymer-boom of the 1970s and 1980s, polycaprolactone (PCL) was used in the biomaterials field and a number of drug-delivery devices. Its popularity was soon superseded by faster resorbable polymers which had fewer perceived disadvantages associated with long term degradation (up to 3-4 years) and intracellular resorption pathways; consequently, PCL was almost forgotten for most of two decades. Recently, a resurgence of interest has propelled PCL back into the biomaterials-arena. The superior rheological and viscoelastic properties over many of its aliphatic polyester counterparts renders PCL easy to manufacture and manipulate into a large range of implants and devices. Coupled with relatively inexpensive production routes and FDA approval, this provides a promising platform for the production of longer-term degradable implants which may be manipulated physically, chemically and biologically to possess tailorable degradation kinetics to suit a specific anatomical site. This review will discuss the application of PCL as a biomaterial over the last two decades focusing on the advantages which have propagated its return into the spotlight with a particular focus on medical devices, drug delivery and tissue engineering.

Flexible high-power multi DC-DC converters for train systems

This thesis reports on the investigations, simulations and analyses of novel power electronics topologies and control strategies. The research is financed by an Australian Research Council (ARC) Linkage (07-09) grant. Therefore, in addition to developing original research and contributing to the available knowledge of power electronics, it also contributes to the design of a DC-DC converter for specific application to the auxiliary power supply in electric trains. Specifically, in this regard, it contributes to the design of a 7.5 kW DC-DC converter for the industrial partner (Schaffler and Associates Ltd) who supported this project. As the thesis is formatted as a ‘thesis by publication’, the contents are organized around published papers. The research has resulted in eleven papers, including seven peer reviewed and published conference papers, one published journal paper, two journal papers accepted for publication and one submitted journal paper (provisionally accepted subject to few changes). In this research, several novel DC-DC converter topologies are introduced, analysed, and tested. The similarity of all of the topologies devised lies in their ‘current circulating’ switching state, which allows them to store some energy in the inductor, as extra inductor current. The stored energy may be applied to enhance the performance of the converter in the occurrence of load current or input voltage disturbances. In addition, when there is an alternating load current, the ability to store energy allows the converter to perform satisfactorily despite frequently and highly varying load current. In this research, the capability of current storage has been utilised to design topologies for specific applications, and the enhancement of the performance of the considered applications has been illustrated. The simplest DC-DC converter topology, which has a ‘current circulating’ switching state, is the Positive Buck-Boost (PBB) converter (also known as the non-inverting Buck-Boost converter). Usually, the topology of the PBB converter is operating as a Buck or a Boost converter in applications with widely varying input voltage or output reference voltage. For example, in electric railways (the application of our industrial partner), the overhead line voltage alternates from 1000VDC to 500VDC and the required regulated voltage is 600VDC. In the course of this research, our industrial partner (Schaffler and Associates Ltd) industrialized a PBB converter–the ‘Mudo converter’–operating at 7.5 kW. Programming the onboard DSP and testing the PBB converter in experimental and nominal power and voltage was part of this research program. In the earlier stages of this research, the advantages and drawbacks of utilization of the ‘current circulating’ switching state in the positive Buck-Boost converter were investigated. In brief, the advantages were found to be robustness against input voltage and current load disturbances, and the drawback was extra conduction and switching loss. Although the robustness against disturbances is desirable for many applications, the price of energy loss must be minimized to attract attention to the utilization of the PBB converter. In further stages of this research, two novel control strategies for different applications were devised to minimise the extra energy loss while the advantages of the positive Buck-Boost converter were fully utilized. The first strategy is Smart Load Controller (SLC) for applications with pre-knowledge or predictability of input voltage and/or load current disturbances. A convenient example of these applications is electric/hybrid cars where a master controller commands all changes in loads and voltage sources. Therefore, the master controller has a pre-knowledge of the load and input voltage disturbances so it can apply the SLC strategy to utilize robustness of the PBB converter. Another strategy aiming to minimise energy loss and maximise the robustness in the face of disturbance is developed to cover applications with unexpected disturbances. This strategy is named Dynamic Hysteresis Band (DHB), and is used to manipulate the hysteresis band height after occurrence of disturbance to reduce dynamics of the output voltage. When no disturbance has occurred, the PBB converter works with minimum inductor current and minimum energy loss. New topologies based on the PBB converter have been introduced to address input voltage disturbances for different onboard applications. The research shows that the performance of applications of symmetrical/asymmetrical multi-level diode-clamped inverters, DC-networks, and linear-assisted RF amplifiers may be enhanced by the utilization of topologies based on the PBB converter. Multi-level diode-clamped inverters have the problem of DC-link voltage balancing when the power factor of their load closes to unity. This research has shown that this problem may be solved with a suitable multi-output DC-DC converter supplying DClink capacitors. Furthermore, the multi-level diode-clamped inverters supplied with asymmetrical DC-link voltages may improve the quality of load voltage and reduce the level of Electromagnetic Interference (EMI). Mathematical analyses and experiments on supplying symmetrical and asymmetrical multi-level inverters by specifically designed multi-output DC-DC converters have been reported in two journal papers. Another application in which the system performance can be improved by utilization of the ‘current circulating’ switching state is linear-assisted RF amplifiers in communicational receivers. The concept of ‘linear-assisted’ is to divide the signal into two frequency domains: low frequency, which should be amplified by a switching circuit; and the high frequency domain, which should be amplified by a linear amplifier. The objective is to minimize the overall power loss. This research suggests using the current storage capacity of a PBB based converter to increase its bandwidth, and to increase the domain of the switching converter. The PBB converter addresses the industrial demand for a DC-DC converter for the application of auxiliary power supply of a typical electric train. However, after testing the industrial prototype of the PBB converter, there were some voltage and current spikes because of switching. To attenuate this problem without significantly increasing the switching loss, the idea of Active Gate Signalling (AGS) is presented. AGS suggests a smart gate driver that selectively controls the switching process to reduce voltage/current spikes, without unacceptable reduction in the efficiency of switching.

Port of Brisbane stormwater quality : stage 2 development of a port specific water quality model

Background: The quality of stormwater runoff from ports is significant as it can be an important source of pollution to the marine environment. This is also a significant issue for the Port of Brisbane as it is located in an area of high environmental values. Therefore, it is imperative to develop an in-depth understanding of stormwater runoff quality to ensure that appropriate strategies are in place for quality improvement, where necessary. To this end, the Port of Brisbane Corporation aimed to develop a port specific stormwater model for the Fisherman Islands facility. The need has to be considered in the context of the proposed future developments of the Port area. ----------------- The Project: The research project is an outcome of the collaborative Partnership between the Port of Brisbane Corporation (POBC) and Queensland University of Technology (QUT). A key feature of this Partnership is that it seeks to undertake research to assist the Port in strengthening the environmental custodianship of the Port area through ‘cutting edge’ research and its translation into practical application. ------------------ The project was separated into two stages. The first stage developed a quantitative understanding of the generation potential of pollutant loads in the existing land uses. This knowledge was then used as input for the stormwater quality model developed in the subsequent stage. The aim is to expand this model across the yet to be developed port expansion area. This is in order to predict pollutant loads associated with stormwater flows from this area with the longer term objective of contributing to the development of ecological risk mitigation strategies for future expansion scenarios. ----------------- Study approach: Stage 1 of the overall study confirmed that Port land uses are unique in terms of the anthropogenic activities occurring on them. This uniqueness in land use results in distinctive stormwater quality characteristics different to other conventional urban land uses. Therefore, it was not scientifically valid to consider the Port as belonging to a single land use category or to consider as being similar to any typical urban land use. The approach adopted in this study was very different to conventional modelling studies where modelling parameters are developed using calibration. The field investigations undertaken in Stage 1 of the overall study helped to create fundamental knowledge on pollutant build-up and wash-off in different Port land uses. This knowledge was then used in computer modelling so that the specific characteristics of pollutant build-up and wash-off can be replicated. This meant that no calibration processes were involved due to the use of measured parameters for build-up and wash-off. ---------------- Conclusions: Stage 2 of the study was primarily undertaken using the SWMM stormwater quality model. It is a physically based model which replicates natural processes as closely as possible. The time step used and catchment variability considered was adequate to accommodate the temporal and spatial variability of input parameters and the parameters used in the modelling reflect the true nature of rainfall-runoff and pollutant processes to the best of currently available knowledge. In this study, the initial loss values adopted for the impervious surfaces are relatively high compared to values noted in research literature. However, given the scientifically valid approach used for the field investigations, it is appropriate to adopt the initial losses derived from this study for future modelling of Port land uses. The relatively high initial losses will reduce the runoff volume generated as well as the frequency of runoff events significantly. Apart from initial losses, most of the other parameters used in SWMM modelling are generic to most modelling studies. Development of parameters for MUSIC model source nodes was one of the primary objectives of this study. MUSIC, uses the mean and standard deviation of pollutant parameters based on a normal distribution. However, based on the values generated in this study, the variation of Event Mean Concentrations (EMCs) for Port land uses within the given investigation period does not fit a normal distribution. This is possibly due to the fact that only one specific location was considered, namely the Port of Brisbane unlike in the case of the MUSIC model where a range of areas with different geographic and climatic conditions were investigated. Consequently, the assumptions used in MUSIC are not totally applicable for the analysis of water quality in Port land uses. Therefore, in using the parameters included in this report for MUSIC modelling, it is important to note that it may result in under or over estimations of annual pollutant loads. It is recommended that the annual pollutant load values given in the report should be used as a guide to assess the accuracy of the modelling outcomes. A step by step guide for using the knowledge generated from this study for MUSIC modelling is given in Table 4.6. ------------------ Recommendations: The following recommendations are provided to further strengthen the cutting edge nature of the work undertaken: * It is important to further validate the approach recommended for stormwater quality modelling at the Port. Validation will require data collection in relation to rainfall, runoff and water quality from the selected Port land uses. Additionally, the recommended modelling approach could be applied to a soon-to-be-developed area to assess ‘before’ and ‘after’ scenarios. * In the modelling study, TSS was adopted as the surrogate parameter for other pollutants. This approach was based on other urban water quality research undertaken at QUT. The validity of this approach should be further assessed for Port land uses. * The adoption of TSS as a surrogate parameter for other pollutants and the confirmation that the <150 m particle size range was predominant in suspended solids for pollutant wash-off gives rise to a number of important considerations. The ability of the existing structural stormwater mitigation measures to remove the <150 m particle size range need to be assessed. The feasibility of introducing source control measures as opposed to end-of-pipe measures for stormwater quality improvement may also need to be considered.

Development of patient-specific computer simulation tools for endoscopic scoliosis surgery

Endoscopic approaches for anterior correction of idiopathic scoliosis are a relatively new surgical technique. This paper describes the development of patient-specific finite element modelling techniques to investigate the biomechanics of single rod anterior scoliosis correction. Spinal geometry is obtained from pre-operative CT scans and material properties for osteo-ligamentous spinal tissues are based on existing literature. The techniques being developed will allow pre-surgical prediction of stresses, forces and deformations in spinal tissues, rods and screws under post-operative physiological loads.

Longing to belong : social inclusion and wellbeing among youth with refugee backgrounds in the first three years in Melbourne, Australia

For young people with refugee backgrounds, establishing a sense of belonging to their family and community, and to their country of resettlement is essential for wellbeing. This paper describes the psychosocial factors associated with subjective health and wellbeing outcomes among a cohort of 97 refugee youth (aged 11-19) during their first three years in Melbourne, Australia. The findings reported here are drawn from the Good Starts Study, a longitudinal investigation of settlement and wellbeing among refugee youth conducted between 2004 and 2008. The overall aim of Good Starts was to identify the psychosocial factors that assist youth with refugee backgrounds in making a good start in their new country. A particular focus was on key transitions: from pre-arrival to Australia, from the language school to mainstream school, and from mainstream school to higher education or to the workforce. Good Starts used a mix of both method and theory from anthropology and social epidemiology. Using standardized measures of wellbeing and generalised estimating equations to model the predictors of wellbeing over time, this paper reports that key factors strongly associated with wellbeing outcomes are those that can be described as indicators of belonging e the most important being subjective social status in the broader Australian community, perceived discrimination and bullying. We argue that settlement specific policies and programs can ultimately be effective if embedded within a broader socially inclusive society - one that offers real opportunities for youth with refugee backgrounds to flourish.

Structural equation model of construction contract dispute potential

This paper presents the results of a structural equation model (SEM) for describing and quantifying the fundamental factors that affect contract disputes between owners and contractors in the construction industry. Through this example, the potential impact of SEM analysis in construction engineering and management research is illustrated. The purpose of the specific model developed in this research is to explain how and why contract related construction problems occur. This study builds upon earlier work, which developed a disputes potential index, and the likelihood of construction disputes was modeled using logistic regression. In this earlier study, questionnaires were completed on 159 construction projects, which measured both qualitative and quantitative aspects of contract disputes, management ability, financial planning, risk allocation, and project scope definition for both owners and contractors. The SEM approach offers several advantages over the previously employed logistic regression methodology. The final set of structural equations provides insight into the interaction of the variables that was not apparent in the original logistic regression modeling methodology.