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.

Patterns of engagement in workplace learning amongst employees from social sectors

Continuous learning and development has become increasingly important in the information age. However, employees with limited formal education in lower status occupations may be disadvantaged in their opportunities for development, as their jobs tend to require more limited knowledge and skills. In mature age, such workers may be subject to cumulative disadvantage with respect to work related learning and development, as well as negative stereotyping. This thesis concerns work related learning and development from a lifespan development psychology perspective. Development across the lifespan is grounded in biocultural co-constructivism. That is, the reciprocal influences of the individual and environment produce change in the individual. Existing theories and models of adaptive development attempt to explain how developmental resources are allocated across the lifespan. These included the Meta- theory of Selective Optimisation with Compensation, Dual Process Model of Self Regulation, and Developmental Regulation via Optimisation and Primary and Secondary Control. These models were integrated to create the Model of Adaptive Development for Work Related Learning. The Learning and Development Survey (LDS) was constructed to measure the hypothesised processes of adaptive development for work related learning, which were individual goal selection, individual goal engagement, individual goal disengagement, organisational opportunities (selection and engagement), and organisational constraints. Data collection was undertaken in two phases: the pilot study and the main study. The objective of the pilot study was to test the LDS on a target population of 112 employees from a local government organisation. Exploratory factor analysis reduced the pilot version of the survey to 38 items encompassing eight constructs which covered the processes of the model of adaptive development for work related learning. In the main study, the Revised Learning and Development Survey (R-LDS) was administered to another group of 137 employees from the local government organisation, as well as 110 employees from a private healthcare organisation. The purpose of the main study was to validate the R-LDS on two different groups to provide evidence of stability, and compare survey scores according to age and occupational status to determine construct validity. Findings from the main study indicated that only four constructs of the R-LDS were stable, which were organisational opportunities – selection, individual goal engagement, organisational constraints – disengagement and organisational opportunities – engagement. In addition, MANOVA studies revealed that the demographic variables affected organisational opportunities and constraints in the workplace, although individual goal engagement was not influenced by age. The findings from the pilot and main study partially supported the model of adaptive development for work related learning. Given that only four factors displayed adequate reliability in terms of internal consistency and stability, the findings suggest that individual goal selection and individual goal disengagement are less relevant to work related learning and development. Some recent research which emerged during the course of the current study has suggested that individual goal selection and individual goal disengagement are more relevant when goal achievement is impeded by biological constraints such as ageing. However, correlations between the retained factors support the model of adaptive development for work related learning, and represent the role of biocultural co-constructivism in development. Individual goal engagement was positively correlated with both opportunity factors (selection and engagement), while organisational constraints – disengagement was negatively correlated with organisational opportunities – selection. Demographic findings indicated that higher occupational status was associated with more opportunities for development. Age was associated with fewer opportunities or greater constraints for development, especially for lower status workers.

The researcher’s perspective on procuring public works projects

Purpose: As part of a comprehensive research study looking at implementing PPPs, interviews with experienced researchers were conducted to realize their views on private sector involvement in public works projects. Design / methodology / approach: Amongst these interviews, five were launched with academics from Hong Kong and Australia, and two were conducted with Legislative Councillors of the Hong Kong Special Administration Region (HKSAR) government. Findings: The interview findings show that both Hong Kong and Australian interviewees had previously conducted some kind of research in the field of PPP. The interviewees highlighted that “Different risk profiles” and “Private sector more innovative / efficient” were the main differences between projects that were procured by PPP and traditionally. Other differences include risk transfer. In a PPP arrangement the public sector passes on a substantial amount of the project risks to the private sector, whereas in a traditional case the public sector would take the largest responsibility in bearing these risks. Another common feature of the private sector is that they tend to be more efficient and innovative when compared to the public sector hence their expertise is often reflected in PPP projects. The interviewees agreed that the key performance indicators for PPP projects were unique depending on the individual project. The critical success factors mentioned by both groups of interviewees included “Transparent process”, “Project dependent” and “Market need”. Due to the fact that PPP projects tend to be large scaled costly projects, adequate transparency in the process is necessary in order to demonstrate that a fair selection and tendering process is conducted. A market need for the project is also important to ensure that the project will be financially secure and that the private sector can make a reasonable profit to cover their project expenditure. Originality / value: The findings from this study have enabled a comparative analysis between the views of researchers in two completely different jurisdictions. With the growing popularity to implement PPP projects, it is believed that the results presented in this paper would be of interest to the industry at large.

A meta-study of SAP financials in the Queensland Government

This thesis consists of three related studies: an ERP Major Issues Study; an Historical Study of the Queensland Government Financial Management System; and a Meta-Study that integrates these and other related studies conducted under the umbrella of the Cooperative ERP Lifecycle Knowledge Management research program. This research provides a comprehensive view of ERP lifecycle issues encountered in SAP R/3 projects across the Queensland Government. This study follows a preliminary ERP issues study (Chang, 2002) conducted in five Queensland Government agencies. The Major Issues Study aims to achieve the following: (1) identify / explicate major issues in relation to the ES life-cycle in the public sector; (2) rank the importance of these issues; and, (3) highlight areas of consensus and dissent among stakeholder groups. To provide a rich context for this study, this thesis includes an historical recount of the Queensland Government Financial Management System (QGFMS). This recount tells of its inception as a centralised system; the selection of SAP and subsequent decentralisation; and, its eventual recentralisation under the Shared Services Initiative and CorpTech. This historical recount gives an insight into the conditions that affected the selection and ongoing management and support of QGFMS. This research forms part of a program entitled Cooperative ERP Lifecycle Knowledge Management. This thesis provides a concluding report for this research program by summarising related studies conducted in the Queensland Government SAP context: Chan (2003); Vayo et al (2002); Ng (2003); Timbrell et al (2001); Timbrell et al (2002); Chang (2002); Putra (1998); and, Niehus et al (1998). A study of Oracle in the United Arab Emirates by Dhaheri (2002) is also included. The thesis then integrates the findings from these studies in an overarching Meta-Study. The Meta-Study discusses key themes across all of these studies, creating an holistic report for the research program. Themes discussed in the meta-study include common issues found across the related studies; knowledge dynamics of the ERP lifecycle; ERP maintenance and support; and, the relationship between the key players in the ERP lifecycle.

Validation of FHWA crash models for rural intersections - Lessons learned

A national-level safety analysis tool is needed to complement existing analytical tools for assessment of the safety impacts of roadway design alternatives. FHWA has sponsored the development of the Interactive Highway Safety Design Model (IHSDM), which is roadway design and redesign software that estimates the safety effects of alternative designs. Considering the importance of IHSDM in shaping the future of safety-related transportation investment decisions, FHWA justifiably sponsored research with the sole intent of independently validating some of the statistical models and algorithms in IHSDM. Statistical model validation aims to accomplish many important tasks, including (a) assessment of the logical defensibility of proposed models, (b) assessment of the transferability of models over future time periods and across different geographic locations, and (c) identification of areas in which future model improvements should be made. These three activities are reported for five proposed types of rural intersection crash prediction models. The internal validation of the model revealed that the crash models potentially suffer from omitted variables that affect safety, site selection and countermeasure selection bias, poorly measured and surrogate variables, and misspecification of model functional forms. The external validation indicated the inability of models to perform on par with model estimation performance. Recommendations for improving the state of the practice from this research include the systematic conduct of carefully designed before-and-after studies, improvements in data standardization and collection practices, and the development of analytical methods to combine the results of before-and-after studies with cross-sectional studies in a meaningful and useful way.

Multiple camera management using wide baseline matching

Camera calibration information is required in order for multiple camera networks to deliver more than the sum of many single camera systems. Methods exist for manually calibrating cameras with high accuracy. Manually calibrating networks with many cameras is, however, time consuming, expensive and impractical for networks that undergo frequent change. For this reason, automatic calibration techniques have been vigorously researched in recent years. Fully automatic calibration methods depend on the ability to automatically find point correspondences between overlapping views. In typical camera networks, cameras are placed far apart to maximise coverage. This is referred to as a wide base-line scenario. Finding sufficient correspondences for camera calibration in wide base-line scenarios presents a significant challenge. This thesis focuses on developing more effective and efficient techniques for finding correspondences in uncalibrated, wide baseline, multiple-camera scenarios. The project consists of two major areas of work. The first is the development of more effective and efficient view covariant local feature extractors. The second area involves finding methods to extract scene information using the information contained in a limited set of matched affine features. Several novel affine adaptation techniques for salient features have been developed. A method is presented for efficiently computing the discrete scale space primal sketch of local image features. A scale selection method was implemented that makes use of the primal sketch. The primal sketch-based scale selection method has several advantages over the existing methods. It allows greater freedom in how the scale space is sampled, enables more accurate scale selection, is more effective at combining different functions for spatial position and scale selection, and leads to greater computational efficiency. Existing affine adaptation methods make use of the second moment matrix to estimate the local affine shape of local image features. In this thesis, it is shown that the Hessian matrix can be used in a similar way to estimate local feature shape. The Hessian matrix is effective for estimating the shape of blob-like structures, but is less effective for corner structures. It is simpler to compute than the second moment matrix, leading to a significant reduction in computational cost. A wide baseline dense correspondence extraction system, called WiDense, is presented in this thesis. It allows the extraction of large numbers of additional accurate correspondences, given only a few initial putative correspondences. It consists of the following algorithms: An affine region alignment algorithm that ensures accurate alignment between matched features; A method for extracting more matches in the vicinity of a matched pair of affine features, using the alignment information contained in the match; An algorithm for extracting large numbers of highly accurate point correspondences from an aligned pair of feature regions. Experiments show that the correspondences generated by the WiDense system improves the success rate of computing the epipolar geometry of very widely separated views. This new method is successful in many cases where the features produced by the best wide baseline matching algorithms are insufficient for computing the scene geometry.

Bayesian methodology incorporating expert judgment for ranking countermeasure effectiveness under uncertainty: Example applied to at grade railroad crossings in Korea

This paper describes the formalization and application of a methodology to evaluate the safety benefit of countermeasures in the face of uncertainty. To illustrate the methodology, 18 countermeasures for improving safety of at grade railroad crossings (AGRXs) in the Republic of Korea are considered. Akin to “stated preference” methods in travel survey research, the methodology applies random selection and laws of large numbers to derive accident modification factor (AMF) densities from expert opinions. In a full Bayesian analysis framework, the collective opinions in the form of AMF densities (data likelihood) are combined with prior knowledge (AMF density priors) for the 18 countermeasures to obtain ‘best’ estimates of AMFs (AMF posterior credible intervals). The countermeasures are then compared and recommended based on the largest safety returns with minimum risk (uncertainty). To the author's knowledge the complete methodology is new and has not previously been applied or reported in the literature. The results demonstrate that the methodology is able to discern anticipated safety benefit differences across candidate countermeasures. For the 18 at grade railroad crossings considered in this analysis, it was found that the top three performing countermeasures for reducing crashes are in-vehicle warning systems, obstacle detection systems, and constant warning time systems.

Effect of clear zone widths on run-off-road crash outcomes

Current guidelines on clear zone selection and roadside hazard management adopt the US approach based on the likelihood of roadside encroachment by drivers. This approach is based on the available research conducted in the 1960s and 70s. Over time, questions have been raised regarding the robustness and applicability of this research in Australasia in 2010 and in the Safe System context. This paper presents a review of the fundamental research relating to selection of clear zones. Results of extensive rural highway statistical data modelling suggest that a significant proportion of run-off-road to the left casualty crashes occurs in clear zones exceeding 13 m. They also show that the risk of run-off-road to the left casualty crashes was 21% lower where clear zones exceeded 8 m when compared with clear zones in the 4 – 8 m range. The paper discusses a possible approach to selection of clear zones based on managing crash outcomes, rather than on the likelihood of roadside encroachment which is the basis for the current practice. It is expected that this approach would encourage selection of clear zones wider than 8 m when the combination of other road features suggests higher than average casualty crash risk.

Critical success factors of project management for Brunei construction projects : improving project performance

The problem of delays in the construction industry is a global phenomenon and the construction industry in Brunei Darussalam is no exception. The goal of all parties involved in construction projects – owners, contractors, engineers and consultants in either the public or private sector is to successfully complete the project on schedule, within planned budget, with the highest quality and in the safest manner. Construction projects are frequently influenced by either success factors that help project parties reach their goal as planned, or delay factors that stifle or postpone project completion. The purpose of this research is to identify success and delay factors which can help project parties reach their intended goals with greater efficiency. This research extracted seven of the most important success factors according to the literature and seven of the most important delay factors identified by project parties, and then examined correlations between them to determine which were the most influential in preventing project delays. This research uses a comprehensive literature review to design and conduct a survey to investigate success and delay factors and then obtain a consensus of expert opinion using the Delphi methodology to rank the most needed critical success factors for Brunei construction projects. A specific survey was distributed to owners, contractors and engineers to examine the most critical delay factors. A general survey was distributed to examine the correlation between the identified delay factors and the seven most important critical success factors selected. A consensus of expert opinion using the Delphi methodology was used to rank the most needed critical success factors for Brunei building construction. Data was collected and evaluated by statistical methods to identify the most significant causes of delay and to measure the strength and direction of the relationship between critical success factors and delay factors in order to examine project parties’ evaluation of projects’ critical success and delay factors, and to evaluate the influence of critical success factors on critical delay factors. A relative importance index has been used to determine the relative importance of the various causes of delays. A one and two-way analysis of variance (ANOVA) has been used to examine how the group or groups evaluated the influence of the critical success factors in avoiding or preventing each of the delay factors, and which success factors were perceived as most influential in avoiding or preventing critical delay factors. Finally the Delphi method, using consensus from an expert panel, was employed to identify the seven most critical success factors used to avoid the delay factors, and thereby improve project performance.

Enhancing agrobacterium-mediated transformation efficiency of sugarcane : progress towards an efficient, genotype-independent method

Several key issues need to be resolved before an efficient and reproducible Agrobacterium-mediated sugarcane transformation method can be developed for a wider range of sugarcane cultivars. These include loss of morphogenetic potential in sugarcane cells after Agrobacterium-mediated transformation, effect of exposure to abiotic stresses during in vitro selection, and most importantly the hypersensitive cell death response of sugarcane (and other nonhost plants) to Agrobacterium tumefaciens. Eight sugarcane cultivars (Q117, Q151, Q177, Q200, Q208, KQ228, QS94-2329, and QS94-2174) were evaluated for loss of morphogenetic potential in response to the age of the culture, exposure to Agrobacterium strains, and exposure to abiotic stresses during selection. Corresponding changes in the polyamine profiles of these cultures were also assessed. Strategies were then designed to minimize the negative effects of these factors on the cell survival and callus proliferation following Agrobacterium-mediated transformation. Some of these strategies, including the use of cell death protector genes and regulation of intracellular polyamine levels, will be discussed.

Changing user behaviour using a digital repository system

Griffith University is developing a digital repository system using HarvestRoad Hive software to better meet the needs of academics and students using institutional learning and teaching, course readings, and institutional intellectual capital systems. Issues with current operations and systems are discussed in terms of user behaviour. New repository systems are being designed in such a way that they address current service and user behaviour issues by closely aligning systems with user needs. By developing attractive online services, Griffith is working to change current user behaviour to achieve strategic priorities in the sharing and reuse of learning objects, improved selection and use of digitised course readings, the development of ePrint and eScience services, and the management of a research portfolio service.

The key to successful implementation : project management of sustainable infrastructure provision

Delivering infrastructure projects involves many stakeholders. Their responsibilities and authorities vary over the course of the project lifecycle - from establishing the project parameters and performance requirements, to operating and maintaining the completed infrastructure. To ensure the successful delivery of infrastructure projects, it is important for the project management team to identify and manage the stakeholders and their requirements. This chapter discusses the management of stakeholders in delivering infrastructure projects, from their conception to completion. It includes managing the stakeholders for project selection and involving them to improve project constructability, operability and maintainability.

An evidence-based approach to professional development planning in large state educational systems

This paper presents a systems-level approach for adjudicating the prioritization, selection, and planning of inservcie professional development (PD) for teachers. We present a step-by-step model for documenting and assessing system-wide 'bids' for professional development programs

Development of new evaluation methods for qualitative alternatives, using fuzzy calculations

Evaluation, selection and finally decision making are all among important issues, which engineers face in long run of projects. Engineers implement mathematical and nonmathematical methods to make accurate and correct decisions, whenever needed. As extensive as these methods are, effects of any selected method on outputs achieved and decisions made are still suspicious. This is more controversial and challengeable, where evaluation is made among non-quantitative alternatives. In civil engineering and construction management problems, criteria include both quantitative and qualitative ones, such as aesthetic, construction duration, building and operation costs, and environmental considerations. As the result, decision making frequently takes place among non-quantitative alternatives. It should be noted that traditional comparison methods, including clear-cut and inflexible mathematics, have always been criticized. This paper demonstrates a brief review of traditional methods of evaluating alternatives. It also offers a new decision making method using, fuzzy calculations. The main focus of this research is some engineering issues, which have flexible nature and vague borders. Suggested method provides analyzability of evaluation for decision makers. It is also capable to overcome multi criteria and multi-referees problems. In order to ease calculations, a program named DeMA is introduced.