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.

Concentrations of polybrominated diphenyl ethers (PBDEs) in matched samples of human milk, dust and indoor air

Polybrominated diphenyl ethers (PBDEs) are lipophilic, persistent pollutants found worldwide in environmental and human samples. Exposure pathways for PBDEs remain unclear but may include food, air and dust. The aim of this study was to conduct an integrated assessment of PBDE exposure and human body burden using 10 matched samples of human milk, indoor air and dust collected in 2007–2008 in Brisbane, Australia. In addition, temporal analysis was investigated comparing the results of the current study with PBDE concentrations in human milk collected in 2002–2003 from the same region. PBDEs were detected in all matrices and the median concentrations of BDEs -47 and -209 in human milk, air and dust were: 4.2 and 0.3 ng/g lipid; 25 and 7.8 pg/m3; and 56 and 291 ng/g dust, respectively. Significant correlations were observed between the concentrations of BDE-99 in air and human milk (r = 0.661, p = 0.038) and BDE-153 in dust and BDE-183 in human milk (r = 0.697, p = 0.025). These correlations do not suggest causal relationships — there is no hypothesis that can be offered to explain why BDE-153 in dust and BDE-183 in milk are correlated. The fact that so few correlations were found in the data could be a function of the small sample size, or because additional factors, such as sources of exposure not considered or measured in the study, might be important in explaining exposure to PBDEs. There was a slight decrease in PBDE concentrations from 2002–2003 to 2007–2008 but this may be due to sampling and analytical differences. Overall, average PBDE concentrations from these individual samples were similar to results from pooled human milk collected in Brisbane in 2002–2003 indicating that pooling may be an efficient, cost-effective strategy of assessing PBDE concentrations on a population basis. The results of this study were used to estimate an infant's daily PBDE intake via inhalation, dust ingestion and human milk consumption. Differences in PBDE intake of individual congeners from the different matrices were observed. Specifically, as the level of bromination increased, the contribution of PBDE intake decreased via human milk and increased via dust. As the impacts of the ban of the lower brominated (penta- and octa-BDE) products become evident, an increased use of the higher brominated deca-BDE product may result in dust making a greater contribution to infant exposure than it does currently. To better understand human body burden, further research is required into the sources and exposure pathways of PBDEs and metabolic differences influencing an individual's response to exposure. In addition, temporal trend analysis is necessary with continued monitoring of PBDEs in the human population as well as in the suggested exposure matrices of food, dust and air.

Randomized controlled trial of cognitive-behavioural therapy for coexisting depression and alcohol problems : short-term outcome

AIMS: Alcohol use disorders and depression co-occur frequently and are associated with poorer outcomes than when either condition occurs alone. The present study (Depression and Alcohol Integrated and Single-focused Interventions; DAISI) aimed to compare the effectiveness of brief intervention, single-focused and integrated psychological interventions for treatment of coexisting depression and alcohol use problems. METHODS: Participants (n = 284) with current depressive symptoms and hazardous alcohol use were assessed and randomly allocated to one of four individually delivered interventions: (i) a brief intervention only (single 90-minute session) with an integrated focus on depression and alcohol, or followed by a further nine 1-hour sessions with (ii) an alcohol focus; (iii) a depression focus; or (iv) an integrated focus. Follow-up assessments occurred 18 weeks after baseline. RESULTS: Compared with the brief intervention, 10 sessions were associated with greater reductions in average drinks per week, average drinking days per week and maximum consumption on 1 day. No difference in duration of treatment was found for depression outcomes. Compared with single-focused interventions, integrated treatment was associated with a greater reduction in drinking days and level of depression. For men, the alcohol-focused rather than depression-focused intervention was associated with a greater reduction in average drinks per day and drinks per week and an increased level of general functioning. Women showed greater improvements on each of these variables when they received depression-focused rather than alcohol-focused treatment. CONCLUSIONS: Integrated treatment may be superior to single-focused treatment for coexisting depression and alcohol problems, at least in the short term. Gender differences between single-focused depression and alcohol treatments warrant further study.

A study into the permeability and compressibility of Australian bagasse pulp

This is an experimental study into the permeability and compressibility properties of bagasse pulp pads. Three experimental rigs were custom-built for this project. The experimental work is complemented by modelling work. Both the steady-state and dynamic behaviour of pulp pads are evaluated in the experimental and modelling components of this project. Bagasse, the fibrous residue that remains after sugar is extracted from sugarcane, is normally burnt in Australia to generate steam and electricity for the sugar factory. A study into bagasse pulp was motivated by the possibility of making highly value-added pulp products from bagasse for the financial benefit of sugarcane millers and growers. The bagasse pulp and paper industry is a multibillion dollar industry (1). Bagasse pulp could replace eucalypt pulp which is more widely used in the local production of paper products. An opportunity exists for replacing the large quantity of mainly generic paper products imported to Australia. This includes 949,000 tonnes of generic photocopier papers (2). The use of bagasse pulp for paper manufacture is the main application area of interest for this study. Bagasse contains a large quantity of short parenchyma cells called ‘pith’. Around 30% of the shortest fibres are removed from bagasse prior to pulping. Despite the ‘depithing’ operations in conventional bagasse pulp mills, a large amount of pith remains in the pulp. Amongst Australian paper producers there is a perception that the high quantity of short fibres in bagasse pulp leads to poor filtration behaviour at the wet-end of a paper machine. Bagasse pulp’s poor filtration behaviour reduces paper production rates and consequently revenue when compared to paper production using locally made eucalypt pulp. Pulp filtration can be characterised by two interacting factors; permeability and compressibility. Surprisingly, there has previously been very little rigorous investigation into neither bagasse pulp permeability nor compressibility. Only freeness testing of bagasse pulp has been published in the open literature. As a result, this study has focussed on a detailed investigation of the filtration properties of bagasse pulp pads. As part of this investigation, this study investigated three options for improving the permeability and compressibility properties of Australian bagasse pulp pads. Two options for further pre-treating depithed bagasse prior to pulping were considered. Firstly, bagasse was fractionated based on size. Two bagasse fractions were produced, ‘coarse’ and ‘medium’ bagasse fractions. Secondly, bagasse was collected after being processed on two types of juice extraction technology, i.e. from a sugar mill and from a sugar diffuser. Finally one method of post-treating the bagasse pulp was investigated. The effects of chemical additives, which are known to improve freeness, were also assessed for their effect on pulp pad permeability and compressibility. Pre-treated Australian bagasse pulp samples were compared with several benchmark pulp samples. A sample of commonly used kraft Eucalyptus globulus pulp was obtained. A sample of depithed Argentinean bagasse, which is used for commercial paper production, was also obtained. A sample of Australian bagasse which was depithed as per typical factory operations was also produced for benchmarking purposes. The steady-state pulp pad permeability and compressibility parameters were determined experimentally using two purpose-built experimental rigs. In reality, steady-state conditions do not exist on a paper machine. The permeability changes as the sheet compresses over time. Hence, a dynamic model was developed which uses the experimentally determined steady-state permeability and compressibility parameters as inputs. The filtration model was developed with a view to designing pulp processing equipment that is suitable specifically for bagasse pulp. The predicted results of the dynamic model were compared to experimental data. The effectiveness of a polymeric and microparticle chemical additives for improving the retention of short fibres and increasing the drainage rate of a bagasse pulp slurry was determined in a third purpose-built rig; a modified Dynamic Drainage Jar (DDJ). These chemical additives were then used in the making of a pulp pad, and their effect on the steady-state and dynamic permeability and compressibility of bagasse pulp pads was determined. The most important finding from this investigation was that Australian bagasse pulp was produced with higher permeability than eucalypt pulp, despite a higher overall content of short fibres. It is thought this research outcome could enable Australian paper producers to switch from eucalypt pulp to bagasse pulp without sacrificing paper machine productivity. It is thought that two factors contributed to the high permeability of the bagasse pulp pad. Firstly, thicker cell walls of the bagasse pulp fibres resulted in high fibre stiffness. Secondly, the bagasse pulp had a large proportion of fibres longer than 1.3 mm. These attributes helped to reinforce the pulp pad matrix. The steady-state permeability and compressibility parameters for the eucalypt pulp were consistent with those found by previous workers. It was also found that Australian pulp derived from the ‘coarse’ bagasse fraction had higher steady-state permeability than the ‘medium’ fraction. However, there was no difference between bagasse pulp originating from a diffuser or a mill. The bagasse pre-treatment options investigated in this study were not found to affect the steady-state compressibility parameters of a pulp pad. The dynamic filtration model was found to give predictions that were in good agreement with experimental data for pads made from samples of pretreated bagasse pulp, provided at least some pith was removed prior to pulping. Applying vacuum to a pulp slurry in the modified DDJ dramatically reduced the drainage time. At any level of vacuum, bagasse pulp benefitted from chemical additives as quantified by reduced drainage time and increased retention of short fibres. Using the modified DDJ, it was observed that under specific conditions, a benchmark depithed bagasse pulp drained more rapidly than the ‘coarse’ bagasse pulp. In steady-state permeability and compressibility experiments, the addition of chemical additives improved the pad permeability and compressibility of a benchmark bagasse pulp with a high quantity of short fibres. Importantly, this effect was not observed for the ‘coarse’ bagasse pulp. However, dynamic filtration experiments showed that there was also a small observable improvement in filtration for the ‘medium’ bagasse pulp. The mechanism of bagasse pulp pad consolidation appears to be by fibre realignment. Chemical additives assist to lubricate the consolidation process. This study was complemented by pulp physical and chemical property testing and a microscopy study. In addition to its high pulp pad permeability, ‘coarse’ bagasse pulp often (but not always) had superior physical properties than a benchmark depithed bagasse pulp.

User-generated content and the future of public broadcasting : a case study of the special broadcasting service

This thesis presents a case study of the Special Broadcasting Service documenting the broadcasting challenges posed by user-generated content initiatives and the work-place approach to strategies for participation. Using the action research method, the project findings reveal that limitations to resources and funding determined the scope for innovation and that the practice of executive editorial control over content was considered fundamental to fulfilling the responsibilities of the public service mandate. Media workers were overwhelmingly positive about the enhanced productive capabilities of the audience and willing to facilitate moderated interactions, however the effectiveness of these initiatives differed according to the level of skills required. This thesis demonstrates how participatory initiatives can enhance aspects of the public service remit relating to cultural diversity, the servicing of niche interests, and broader social representation, and help reinvigorate the relevance of public service broadcasting in the digitalised media sphere.

Destination branding : tracking brand equity for a competitive set of near-home destinations

In the emerging literature related to destination branding, little has been reported about performance metrics. The focus of most research reported to date has been concerned with the development of destination brand identities and the implementation of campaigns (see for example, Crockett & Wood 1999, Hall 1999, May 2001, Morgan et al 2002). One area requiring increased attention is that of tracking the performance of destination brands over time. This is an important gap in the tourism literature, given: i) the increasing level of investment by destination marketing organisations (DMO) in branding since the 1990s, ii) the complex political nature of DMO brand decision-making and increasing accountability to stakeholders (see Pike, 2005), and iii) the long-term nature of repositioning a destination’s image in the market place (see Gartner & Hunt, 1987). Indeed, a number of researchers in various parts of the world have pointed to a lack of market research monitoring destination marketing objectives, such as in Australia (see Prosser et. al 2000, Carson, Beattie and Gove 2003), North America (Sheehan & Ritchie 1997, Masberg 1999), and Europe (Dolnicar & Schoesser 2003)...

Assessment of architectural work experience by employers and students

A major element in the architectural work experience program at Queensland University of Technology is the assessment reports provided by students and employers. This paper gives an analysis of assessments submitted during the period 2000 to 2007 as viewed from a practice-base perspective. By comparing the 398 student assessments with 403 employer assessments in five specific categories over an eight year period one is able to obtain a clear understanding of the performance of the program and the relevance of its various sections for its participants that is not always obvious in a yearly analysis. In the major work experience areas there is close agreement between the student and employer assessments. However, the analysis did highlight a misunderstanding of the program’s aims by some participants. Overall the students were very positive about the program and appreciated the opportunity to work on real projects and be given a degree of responsibility for these projects. For Work Integrated Learning (WIL) practitioners this study clearly demonstrates the value of obtaining assessments from students and employers in order to establish the acceptance of a WIL program.

Groundwater Visualisation System (GVS): a software framework for developing low-end, scalable and robust software for 3D visualisation and animation of groundwater conceptual models

Effective management of groundwater requires stakeholders to have a realistic conceptual understanding of the groundwater systems and hydrological processes.However, groundwater data can be complex, confusing and often difficult for people to comprehend..A powerful way to communicate understanding of groundwater processes, complex subsurface geology and their relationships is through the use of visualisation techniques to create 3D conceptual groundwater models. In addition, the ability to animate, interrogate and interact with 3D models can encourage a higher level of understanding than static images alone. While there are increasing numbers of software tools available for developing and visualising groundwater conceptual models, these packages are often very expensive and are not readily accessible to majority people due to complexity. .The Groundwater Visualisation System (GVS) is a software framework that can be used to develop groundwater visualisation tools aimed specifically at non-technical computer users and those who are not groundwater domain experts. A primary aim of GVS is to provide management support for agencies, and enhancecommunity understanding.

Customer satisfaction survey of the facilities provided by office building “X” in Surabaya

Many high-rise office buildings have been built in Surabaya. The investors have provided complimentary facilities to satisfy their tenants. However, not all given facilities has satisfied the tenants. The purpose of this study is to find out the level of tenant satisfaction in office “X” to the existing facilities and to suggest additional required facilities. Although office “X” is offered the highest rental rate and has known as a prestigious place in Surabaya, only location and public transport have satisfied the Indonesian tenants. Meanwhile, the multi National companies have not satisfied for any existing facilities. Additional ATM facilities and presentable cafeteria, improvement of service and the security system are required by tenants.

Characterisation of mesenchymal cells from osteophytes in osteoarthritis

Osteophytes form through the process of chondroid metamorphosis of fibrous tissue followed by endochondral ossification. Osteophytes have been found to consist of three different mesenchymal tissue regions including endochondral bone formation within cartilage residues, intra-membranous bone formation within fibrous tissue and bone formation within bone marrow spaces. All these features provide evidence of mesenchymal stem cells (MSC) involvement in osteophyte formation; nevertheless, it remains to be characterised. MSC from numerous mesenchymal tissues have been isolated but bone marrow remains the “ideal” due to the ease of ex vivo expansion and multilineage potential. However, the bone marrow stroma has a relatively low number of MSC, something that necessitates the need for long-term culture and extensive population doublings in order to obtain a sufficient number of cells for therapeutic applications. MSC in vitro have limited proliferative capacity and extensive passaging compromises differentiation potential. To overcome this barrier, tissue derived MSC are of strong interest for extensive study and characterisation, with a focus on their potential application in therapeutic tissue regeneration. To date, no MSC type cell has been isolated from osteophyte tissue, despite this tissue exhibiting all the hallmark features of a regenerative tissue. Therefore, this study aimed to isolate and characterise cells from osteophyte tissues in relation to their phenotype, differentiation potential, immuno-modulatory properties, proliferation, cellular ageing, longevity and chondrogenesis in in vitro defect model in comparison to patient matched bone marrow stromal cells (bMSC). Osteophyte derived cells were isolated from osteophyte tissue samples collected during knee replacement surgery. These cells were characterised by the expression of cell surface antigens, differentiation potential into mesenchymal lineages, growth kinetics and modulation of allo-immune responses. Multipotential stem cells were identified from all osteophyte samples namely osteophyte derived mesenchymal stem cells (oMSC). Extensively expanded cell cultures (passage 4 and 9 respectively) were used to confirm cytogenetic stability and study signs of cellular aging, telomere length and telomerase activity. Cultured cells at passage 4 were used to determine 84 pathway focused stem cell related gene expression profile. Micro mass pellets were cultured in chondrogenic differentiation media for 21 days for phenotypic and chondrogenic related gene expression. Secondly, cell pellets differentiated overnight were placed into articular cartilage defects and cultured for further 21 days in control medium and chondrogenic medium to study chondrogenesis and cell behaviour. The surface antigen expression of oMSC was consistent with that of mesenchymal stem cells, such as lacking the haematopoietic and common leukocyte markers (CD34, CD45) while expressing those related to adhesion (CD29, CD166, CD44) and stem cells (CD90, CD105, CD73). The proliferation capacity of oMSC in culture was superior to that of bMSC, and they readily differentiated into tissues of the mesenchymal lineages. oMSC also demonstrated the ability to suppress allogeneic T-cell proliferation, which was associated with the expression of tryptophan degrading enzyme indoleamine 2,3 dioxygenase (IDO). Cellular aging was more prominent in late passage bMSC than in oMSC. oMSC had longer telomere length in late passages compared with bMSC, although there was no significant difference in telomere lengths in the early passages in either cell type. Telomerase activity was detectable only in early passage oMSC and not in bMSC. In osteophyte tissues telomerase positive cells were found to be located peri vascularly and were Stro-1 positive. Eighty-four pathway-focused genes were investigated and only five genes (APC, CCND2, GJB2, NCAM and BMP2) were differentially expressed between bMSC and oMSC. Chondrogenically induced micro mass pellets of oMSC showed higher staining intensity for proteoglycans, aggrecan and collagen II. Differential expression of chondrogenic related genes showed up regulation of Aggrecan and Sox 9 in oMSC and collagen II in bMSC. The in vitro defect models of oMSC in control medium showed rounded and aggregated cells staining positively for proteoglycan and presence of some extracellular matrix. In contrast, defects with bMSC showed fragmentation and loss of cells, fibroblast-like cell morphology staining positively for proteoglycans. For defects maintained in chondrogenic medium, rounded, aggregated and proteoglycan positive cells were found in both oMSC and bMSC cultures. Extracellular matrix and cellular integration into newly formed matrix was evident only in oMSC defects. For analysis of chondrocyte hypertrophy, strong expression of type X collagen could be noticed in the pellet cultures and transplanted bMSC. In summary, this study demonstrated that osteophyte derived cells had similar properties to mesenchymal stem cells in the expression of antigen phenotype, differential potential and suppression of allo-immune response. Furthermore, when compared to bMSC, oMSC maintained a higher proliferative capacity due to a retained level of telomerase activity in vitro, which may account for the relatively longer telomeres delaying growth arrest by replicative senescence compared with bMSC. oMSC behaviour in defects supported chondrogenesis which implies that cells derived from regenerative tissue can be an alternative source of stem cells and have a potential clinical application for therapeutic stem cell based tissue regeneration.

An ecological assessment of secondary poisoning risk in the Australian sugarcane industry

Rodenticide use in agriculture can lead to the secondary poisoning of avian predators. Currently the Australian sugarcane industry has two rodenticides, Racumin® and Rattoff®, available for in-crop use but, like many agricultural industries, it lacks an ecologically-based method of determining the potential secondary poisoning risk the use of these rodenticides poses to avian predators. The material presented in this thesis addresses this by: a. determining where predator/prey interactions take place in sugar producing districts; b. quantifying the amount of rodenticide available to avian predators and the probability of encounter; and c. developing a stochastic model that allows secondary poisoning risk under various rodenticide application scenarios to be investigated. Results demonstrate that predator/prey interactions are highly constrained by environmental structure. Rodents used crops that provided high levels of canopy cover and therefore predator protection and poorly utilised open canopy areas. In contrast, raptors over-utilised areas with low canopy cover and low rodent densities, but which provided high accessibility to prey. Given this pattern of habitat use, and that industry baiting protocols preclude rodenticide application in open canopy crops, these results indicate that secondary poisoning can only occur if poisoned rodents leave closed canopy crops and become available for predation in open canopy areas. Results further demonstrate that after in-crop rodenticide application, only a small proportion of rodents available in open areas are poisoned and that these rodents carry low levels of toxicant. Coupled with the low level of rodenticide use in the sugar industry, the high toxic threshold raptors have to these toxicants and the low probability of encountering poisoned rodents, results indicate that the risk of secondary poisoning events occurring is minimal. A stochastic model was developed to investigate the effect of manipulating factors that might influence secondary poisoning hazard in a sugarcane agro-ecosystem. These simulations further suggest that in all but extreme scenarios, the risk of secondary poisoning is also minimal. Collectively, these studies demonstrate that secondary poisoning of avian predators associated with the use of the currently available rodenticides in Australian sugar producing districts is minimal. Further, the ecologically-based method of assessing secondary poisoning risk developed in this thesis has broader applications in other agricultural systems where rodenticide use may pose risks to avian predators.

Approaches for calculation of holding costs in the context of greenfield residential development

There are increasing indications that the contribution of holding costs and its impact on housing affordability is very significant. Their importance and perceived high level impact can be gauged from considering the unprecedented level of attention policy makers have given them recently. This may be evidenced by the embedding of specific strategies to address burgeoning holding costs (and particularly those cost savings associated with streamlining regulatory assessment) within statutory instruments such as the Queensland Housing Affordability Strategy, and the South East Queensland Regional Plan. However, several key issues require further investigation. Firstly, the computation and methodology behind the calculation of holding costs varies widely. In fact, it is not only variable, but in some instances completely ignored. Secondly, some ambiguity exists in terms of the inclusion of various elements of holding costs and assessment of their relative contribution. Perhaps this may in part be explained by their nature: such costs are not always immediately apparent. They are not as visible as more tangible cost items associated with greenfield development such as regulatory fees, government taxes, acquisition costs, selling fees, commissions and others. Holding costs are also more difficult to evaluate since for the most part they must be ultimately assessed over time in an ever-changing environment based on their strong relationship with opportunity cost which is in turn dependant, inter alia, upon prevailing inflation and / or interest rates. This paper seeks to provide a more detailed investigation of those elements related to holding costs, and in so doing determine the size of their impact specifically on the end user. It extends research in this area clarifying the extent to which holding costs impact housing affordability. Geographical diversity indicated by the considerable variation between various planning instruments and the length of regulatory assessment periods suggests further research should adopt a case study approach in order to test the relevance of theoretical modelling conducted.

Impact of cognitive load and frustration on drivers’ speech [Abstract]

Secondary tasks such as cell phone calls or interaction with automated speech dialog systems (SDSs) increase the driver’s cognitive load as well as the probability of driving errors. This study analyzes speech production variations due to cognitive load and emotional state of drivers in real driving conditions. Speech samples were acquired from 24 female and 17 male subjects (approximately 8.5 h of data) while talking to a co-driver and communicating with two automated call centers, with emotional states (neutral, negative) and the number of necessary SDS query repetitions also labeled. A consistent shift in a number of speech production parameters (pitch, first format center frequency, spectral center of gravity, spectral energy spread, and duration of voiced segments) was observed when comparing SDS interaction against co-driver interaction; further increases were observed when considering negative emotion segments and the number of requested SDS query repetitions. A mel frequency cepstral coefficient based Gaussian mixture classifier trained on 10 male and 10 female sessions provided 91% accuracy in the open test set task of distinguishing co-driver interactions from SDS interactions, suggesting—together with the acoustic analysis—that it is possible to monitor the level of driver distraction directly from their speech.

ePortfolio use by university students in Australia: A review of the Australian ePortfolio Project

In October 2008, the Australian Learning and Teaching Council (ALTC) released the final report for the commissioned project ePortfolio use by university students in Australia: Informing excellence in policy and practice. The Australian ePortfolio Project represented the first attempt to examine the breadth and depth of ePortfolio practice in the Australian higher education sector. The research activities included surveys of stakeholder groups in learning and teaching, academic management and human resource management, with respondents representing all Australian universities; a series of focus groups and semi-structured interviews which sought to explore key issues in greater depth; and surveys designed to capture students’ pre-course expectations and their post-course experiences of ePortfolio learning. Further qualitative data was collected through interviews with ‘mature users’ of ePortfolios. Project findings revealed that, while there was a high level of interest in the use of ePortfolios in terms of the potential to help students become reflective learners who were conscious of their personal and professional strengths and weaknesses, the state of play in Australian universities was very fragmented. The project investigation identified four individual, yet interrelated, contexts where strategies may be employed to support and foster effective ePortfolio practice in higher education: government policy, technical standards, academic policy, and learning and teaching. Four scenarios for the future were also presented with the goal of stimulating discussion about opportunities for stakeholder engagement. It is argued that the effective use of ePortfolios requires open dialogue and collaboration between the different stakeholders across this range of contexts.

Sources of coding discrepancies in injury morbidity data: Implications for injury surveillance

Objective: To examine the sources of coding discrepancy for injury morbidity data and explore the implications of these sources for injury surveillance.-------- Method: An on-site medical record review and recoding study was conducted for 4373 injury-related hospital admissions across Australia. Codes from the original dataset were compared to the recoded data to explore the reliability of coded data aand sources of discrepancy.---------- Results: The most common reason for differences in coding overall was assigning the case to a different external cause category with 8.5% assigned to a different category. Differences in the specificity of codes assigned within a category accounted for 7.8% of coder difference. Differences in intent assignment accounted for 3.7% of the differences in code assignment.---------- Conclusions: In the situation where 8 percent of cases are misclassified by major category, the setting of injury targets on the basis of extent of burden is a somewhat blunt instrument Monitoring the effect of prevention programs aimed at reducing risk factors is not possible in datasets with this level of misclassification error in injury cause subcategories. Future research is needed to build the evidence base around the quality and utility of the ICD classification system and application of use of this for injury surveillance in the hospital environment.