Developing a best practice framework for implementing public private partnerships (PPP) in Hong Kong

Public Private Partnership (PPP) is a well established methodology for procuring public works projects. By incorporating the private sector’s expertise, efficiency, innovation, business sense, risk sharing, financing etc. into public works projects, the quality of public services and facilities can be uplifted. Like many jurisdictions, Hong Kong is also keen to take aboard this methodology which is so familiar but yet so distant. Although they have been one of the first jurisdictions to utilise the private sector in public works projects, their comfortable financial reserves has meant that there has been no urge to push the movement until recently. PPP has become increasingly popular amongst governments. The Hong Kong Special Administrative Region (HKSAR) government is no exception. Some of the more active works departments have commissioned studies to investigate the best ways to deliver these projects, others have even trialed the method themselves. The efficiency Unit of the HKSAR government has also become an active arm in conducting research in this area. Although so, the information that is currently available is still very broad. Building from their works there is a need to develop a best practice framework for implementing PPP projects in Hong Kong by incorporating international experiences. To develop a best practice framework will require thorough investigation into the benefits, difficulties and critical success factor of PPP. PPP should also be compared with other procurement methods. In order to do so it is important to clearly understand the local situation by an analysis of projects conducted to date. Lessons learnt can further be derived from other countries and incorporated to those derived locally. Finally the best conditions in terms of project nature, complexity, types, and scales for adopting PPP should be derived. The aim and objectives of this study were achieved via a comprehensive literature review, in-depth case analyses, interview survey with experts from both Hong Kong and overseas, and finally a large scale data collection was conducted via a questionnaire survey with PPP practitioners. These findings were further triangulated before they were used as the basis to form the best practice framework presented in this thesis. The framework was then further validated by PPP experts to ensure it is comprehensive, objective, reliable and practical. This study has presented a methodology that can be adopted for future studies. It has also updated our knowledge on the development trends of PPP as well as opened up the experiences of other jurisdictions. The findings have shown that the local industry is familiar with “what” should be done in PPP projects but they are unsure of “how” these goals can be achieved. This framework has allowed this further knowledge to be delivered to PPP practitioners. As a result, the development of this framework can help to resolve the current economic crisis by encouraging more developments and business opportunities for the private sector. In addition, the correct projects can be delivered by PPP, the advantages of PPP can be maximised, and the general public can benefit from the private sector’s participation.

Bearing-only SLAM : a vision-based navigation system for autonomous robots

To navigate successfully in a previously unexplored environment, a mobile robot must be able to estimate the spatial relationships of the objects of interest accurately. A Simultaneous Localization and Mapping (SLAM) sys- tem employs its sensors to build incrementally a map of its surroundings and to localize itself in the map simultaneously. The aim of this research project is to develop a SLAM system suitable for self propelled household lawnmowers. The proposed bearing-only SLAM system requires only an omnidirec- tional camera and some inexpensive landmarks. The main advantage of an omnidirectional camera is the panoramic view of all the landmarks in the scene. Placing landmarks in a lawn field to define the working domain is much easier and more flexible than installing the perimeter wire required by existing autonomous lawnmowers. The common approach of existing bearing-only SLAM methods relies on a motion model for predicting the robot’s pose and a sensor model for updating the pose. In the motion model, the error on the estimates of object positions is cumulated due mainly to the wheel slippage. Quantifying accu- rately the uncertainty of object positions is a fundamental requirement. In bearing-only SLAM, the Probability Density Function (PDF) of landmark position should be uniform along the observed bearing. Existing methods that approximate the PDF with a Gaussian estimation do not satisfy this uniformity requirement. This thesis introduces both geometric and proba- bilistic methods to address the above problems. The main novel contribu- tions of this thesis are: 1. A bearing-only SLAM method not requiring odometry. The proposed method relies solely on the sensor model (landmark bearings only) without relying on the motion model (odometry). The uncertainty of the estimated landmark positions depends on the vision error only, instead of the combination of both odometry and vision errors. 2. The transformation of the spatial uncertainty of objects. This thesis introduces a novel method for translating the spatial un- certainty of objects estimated from a moving frame attached to the robot into the global frame attached to the static landmarks in the environment. 3. The characterization of an improved PDF for representing landmark position in bearing-only SLAM. The proposed PDF is expressed in polar coordinates, and the marginal probability on range is constrained to be uniform. Compared to the PDF estimated from a mixture of Gaussians, the PDF developed here has far fewer parameters and can be easily adopted in a probabilistic framework, such as a particle filtering system. The main advantages of our proposed bearing-only SLAM system are its lower production cost and flexibility of use. The proposed system can be adopted in other domestic robots as well, such as vacuum cleaners or robotic toys when terrain is essentially 2D.

Enhancing Student Learning in Large Undergraduate Clasees by Using Audio-visual Case Studies

Critical skills such as identifying and appreciating issues that confront firms engaging in international business, and the ability to undertake creative decision-making, are considered fundamental to the study of International Business. It has been argued that using audio-visual case studies can help develop such skills. However, this is difficult due to a lack of Australian case studies. This paper reviews the literature outlining the advantages believed to result from the use of audio-visual case studies, describes a project implemented in a large cohort of students studying International Business, reports on a pilot evaluation of the project, and outlines the findings and conclusions of the survey.

Crash Risk Assessment with Cooperative Systems

Crash risk is the statistical probability of a crash. Its assessment can be performed through ex post statistical analysis or in real-time with on-vehicle systems. These systems can be cooperative. Cooperative Vehicle-Infrastructure Systems (CVIS) are a developing research avenue in the automotive industry worldwide. This paper provides a survey of existing CVIS systems and methods to assess crash risk with them. It describes the advantages of cooperative systems versus non-cooperative systems. A sample of cooperative crash risk assessment systems is analysed to extract vulnerabilities according to three criteria: market penetration, over-reliance on GPS and broadcasting issues. It shows that cooperative risk assessment systems are still in their infancy and requires further development to provide their full benefits to road users.

Synthesis of 4-arm star poly(L-Lactide) oligomers using an in situ-generated calcium-based initiator

Using an in situ-generated calcium-based initiating species derived from pentaerythritol, the bulk synthesis of well-defined 4-arm star poly(L-lactide) oligomers has been studied in detail. The substitution of the traditional initiator, stannous octoate with calcium hydride allowed the synthesis of oligomers that had both low PDIs and a comparable number of polymeric arms (3.7 – 3.9) to oligomers of similar molecular weight. Investigations into the degree of control observed during the course of the polymerization found that the insolubility of pentaerythritol in molten L-lactide resulted in an uncontrolled polymerization only when the feed mole ratio of L-lactide to pentaerythritol was 13. At feed ratios of 40 and greater, a pseudo-living polymerization was observed. As part of this study, in situ FT-Raman spectroscopy was demonstrated to be a suitable method to monitor the kinetics of the ring-opening polymerization (ROP) of lactide. The advantages of using this technique rather than FT-IR-ATR and 1H NMR for monitoring L-lactide consumption during polymerization are discussed.

Boys 'doing' and 'undoing' media education : new possibilities for theory and practice

The purpose of this study is to investigate how secondary school media educators might best meet the needs of students who prefer practical production work to ‘theory’ work in media studies classrooms. This is a significant problem for a curriculum area that claims to develop students’ media literacies by providing them with critical frameworks and a metalanguage for thinking about the media. It is a problem that seems to have become more urgent with the availability of new media technologies and forms like video games. The study is located in the field of media education, which tends to draw on structuralist understandings of the relationships between young people and media and suggests that students can be empowered to resist media’s persuasive discourses. Recent theoretical developments suggest too little emphasis has been placed on the participatory aspects of young people playing with, creating and gaining pleasure from media. This study contributes to this ‘participatory’ approach by bringing post structuralist perspectives to the field, which have been absent from studies of secondary school media education. I suggest theories of media learning must take account of the ongoing formation of students’ subjectivities as they negotiate social, cultural and educational norms. Michel Foucault’s theory of ‘technologies of the self’ and Judith Butler’s theories of performativity and recognition are used to develop an argument that media learning occurs in the context of students negotiating various ‘ethical systems’ as they establish their social viability through achieving recognition within communities of practice. The concept of ‘ethical systems’ has been developed for this study by drawing on Foucault’s theories of discourse and ‘truth regimes’ and Butler’s updating of Althusser’s theory of interpellation. This post structuralist approach makes it possible to investigate the ways in which students productively repeat and vary norms to creatively ‘do’ and ‘undo’ the various media learning activities with which they are required to engage. The study focuses on a group of year ten students in an all boys’ Catholic urban school in Australia who undertook learning about video games in a three-week intensive ‘immersion’ program. The analysis examines the ethical systems operating in the classroom, including formal systems of schooling, informal systems of popular cultural practice and systems of masculinity. It also examines the students’ use of semiotic resources to repeat and/or vary norms while reflecting on, discussing, designing and producing video games. The key findings of the study are that students are motivated to learn technology skills and production processes rather than ‘theory’ work. This motivation stems from the students’ desire to become recognisable in communities of technological and masculine practice. However, student agency is not only possible through critical responses to media, but through performative variation of norms through creative ethical practices as students participate with new media technologies. Therefore, the opportunities exist for media educators to create the conditions for variation of norms through production activities. The study offers several implications for media education theory and practice including: the productive possibilities of post structuralism for informing ways of doing media education; the importance of media teachers having the autonomy to creatively plan curriculum; the advantages of media and technology teachers collaborating to draw on a broad range of resources to develop curriculum; the benefits of placing more emphasis on students’ creative uses of media; and the advantages of blending formal classroom approaches to media education with less formal out of school experiences.

Intercomparison study of six HTDMAs: results and recommendations

We report on an inter-comparison of six different hygroscopicity tandem differential mobility analysers (HTDMAs). These HTDMAs are used worldwide in laboratories and in field campaigns to measure the water uptake of aerosol particles and were never intercompared. After an investigation of the different design of the instruments with their advantages and inconveniencies, the methods for calibration, validation and analysis are presented. Measurements of nebulised ammonium sulphate as well as of secondary organic aerosol generated from a smog chamber were performed. Agreement and discrepancies between the instrument and to the theory are discussed, and final recommendations for a standard instrument are given, as a benchmark for laboratory or field experiments to ensure a high quality of HTDMA data.

Theoretical and numerical investigation of plasmon nanofocusing in metallic tapered rods and grooves

Effective focusing of electromagnetic (EM) energy to nanoscale regions is one of the major challenges in nano-photonics and plasmonics. The strong localization of the optical energy into regions much smaller than allowed by the diffraction limit, also called nanofocusing, offers promising applications in nano-sensor technology, nanofabrication, near-field optics or spectroscopy. One of the most promising solutions to the problem of efficient nanofocusing is related to surface plasmon propagation in metallic structures. Metallic tapered rods, commonly used as probes in near field microscopy and spectroscopy, are of a particular interest. They can provide very strong EM field enhancement at the tip due to surface plasmons (SP’s) propagating towards the tip of the tapered metal rod. A large number of studies have been devoted to the manufacturing process of tapered rods or tapered fibers coated by a metal film. On the other hand, structures such as metallic V-grooves or metal wedges can also provide strong electric field enhancements but manufacturing of these structures is still a challenge. It has been shown, however, that the attainable electric field enhancement at the apex in the V-groove is higher than at the tip of a metal tapered rod when the dissipation level in the metal is strong. Metallic V-grooves also have very promising characteristics as plasmonic waveguides. This thesis will present a thorough theoretical and numerical investigation of nanofocusing during plasmon propagation along a metal tapered rod and into a metallic V-groove. Optimal structural parameters including optimal taper angle, taper length and shape of the taper are determined in order to achieve maximum field enhancement factors at the tip of the nanofocusing structure. An analytical investigation of plasmon nanofocusing by metal tapered rods is carried out by means of the geometric optics approximation (GOA), which is also called adiabatic nanofocusing. However, GOA is applicable only for analysing tapered structures with small taper angles and without considering a terminating tip structure in order to neglect reflections. Rigorous numerical methods are employed for analysing non-adiabatic nanofocusing, by tapered rod and V-grooves with larger taper angles and with a rounded tip. These structures cannot be studied by analytical methods due to the presence of reflected waves from the taper section, the tip and also from (artificial) computational boundaries. A new method is introduced to combine the advantages of GOA and rigorous numerical methods in order to reduce significantly the use of computational resources and yet achieve accurate results for the analysis of large tapered structures, within reasonable calculation time. Detailed comparison between GOA and rigorous numerical methods will be carried out in order to find the critical taper angle of the tapered structures at which GOA is still applicable. It will be demonstrated that optimal taper angles, at which maximum field enhancements occur, coincide with the critical angles, at which GOA is still applicable. It will be shown that the applicability of GOA can be substantially expanded to include structures which could be analysed previously by numerical methods only. The influence of the rounded tip, the taper angle and the role of dissipation onto the plasmon field distribution along the tapered rod and near the tip will be analysed analytically and numerically in detail. It will be demonstrated that electric field enhancement factors of up to ~ 2500 within nanoscale regions are predicted. These are sufficient, for instance, to detect single molecules using surface enhanced Raman spectroscopy (SERS) with the tip of a tapered rod, an approach also known as tip enhanced Raman spectroscopy or TERS. The results obtained in this project will be important for applications for which strong local field enhancement factors are crucial for the performance of devices such as near field microscopes or spectroscopy. The optimal design of nanofocusing structures, at which the delivery of electromagnetic energy to the nanometer region is most efficient, will lead to new applications in near field sensors, near field measuring technology, or generation of nanometer sized energy sources. This includes: applications in tip enhanced Raman spectroscopy (TERS); manipulation of nanoparticles and molecules; efficient coupling of optical energy into and out of plasmonic circuits; second harmonic generation in non-linear optics; or delivery of energy to quantum dots, for instance, for quantum computations.

Mobile phone use while driving : an investigation of the beliefs influencing drivers’ hands-free and hand-held mobile phone use.

This study explored the psychological influences of hands-free and hand-held mobile phone use while driving. Participants were 796 Australian drivers aged 17 to 76 years who owned mobile phones. A cross-sectional survey assessed frequency of calling and text messaging while driving (overall, hands-free, hand-held) as well as drivers’ behavioural, normative, and control beliefs relating to mobile phone use while driving. Irrespective of handset type, 43% of drivers reported answering calls while driving on a daily basis, followed by making calls (36%), reading text messages (27%), and sending text messages (18%). In total, 63.9% of drivers did not own hands-free kits and, of the drivers that owned hand-free kits, 32% did not use it most or all of the time. Significant differences were found in the behavioural, normative, and control beliefs of frequent and infrequent users of both types of handset while driving. As expected, frequent users reported more advantages of, more approval from others for, and fewer barriers that would prevent them from, using either a hands-free or a hand-held mobile phone while driving than infrequent users. Campaigns to reduce mobile phone use while driving should attempt to minimise the perceived benefits of the behaviour and highlight the risks of this unsafe driving practice.

Behaviour and design of cold-formed steel compression members at elevated temperatures

Cold-formed steel members have been widely used in residential, industrial and commercial buildings as primary load bearing structural elements and non-load bearing structural elements (partitions) due to their advantages such as higher strength to weight ratio over the other structural materials such as hot-rolled steel, timber and concrete. Cold-formed steel members are often made from thin steel sheets and hence they are more susceptible to various buckling modes. Generally short columns are susceptible to local or distortional buckling while long columns to flexural or flexural-torsional buckling. Fire safety design of building structures is an essential requirement as fire events can cause loss of property and lives. Therefore it is essential to understand the fire performance of light gauge cold-formed steel structures under fire conditions. The buckling behaviour of cold-formed steel compression members under fire conditions is not well investigated yet and hence there is a lack of knowledge on the fire performance of cold-formed steel compression members. Current cold-formed steel design standards do not provide adequate design guidelines for the fire design of cold-formed steel compression members. Therefore a research project based on extensive experimental and numerical studies was undertaken at the Queensland University of Technology to investigate the buckling behaviour of light gauge cold-formed steel compression members under simulated fire conditions. As the first phase of this research, a detailed review was undertaken on the mechanical properties of light gauge cold-formed steels at elevated temperatures and the most reliable predictive models for mechanical properties and stress-strain models based on detailed experimental investigations were identified. Their accuracy was verified experimentally by carrying out a series of tensile coupon tests at ambient and elevated temperatures. As the second phase of this research, local buckling behaviour was investigated based on the experimental and numerical investigations at ambient and elevated temperatures. First a series of 91 local buckling tests was carried out at ambient and elevated temperatures on lipped and unlipped channels made of G250-0.95, G550-0.95, G250-1.95 and G450-1.90 cold-formed steels. Suitable finite element models were then developed to simulate the experimental conditions. These models were converted to ideal finite element models to undertake detailed parametric study. Finally all the ultimate load capacity results for local buckling were compared with the available design methods based on AS/NZS 4600, BS 5950 Part 5, Eurocode 3 Part 1.2 and the direct strength method (DSM), and suitable recommendations were made for the fire design of cold-formed steel compression members subject to local buckling. As the third phase of this research, flexural-torsional buckling behaviour was investigated experimentally and numerically. Two series of 39 flexural-torsional buckling tests were undertaken at ambient and elevated temperatures. The first series consisted 2800 mm long columns of G550-0.95, G250-1.95 and G450-1.90 cold-formed steel lipped channel columns while the second series contained 1800 mm long lipped channel columns of the same steel thickness and strength grades. All the experimental tests were simulated using a suitable finite element model, and the same model was used in a detailed parametric study following validation. Based on the comparison of results from the experimental and parametric studies with the available design methods, suitable design recommendations were made. This thesis presents a detailed description of the experimental and numerical studies undertaken on the mechanical properties and the local and flexural-torsional bucking behaviour of cold-formed steel compression member at ambient and elevated temperatures. It also describes the currently available ambient temperature design methods and their accuracy when used for fire design with appropriately reduced mechanical properties at elevated temperatures. Available fire design methods are also included and their accuracy in predicting the ultimate load capacity at elevated temperatures was investigated. This research has shown that the current ambient temperature design methods are capable of predicting the local and flexural-torsional buckling capacities of cold-formed steel compression members at elevated temperatures with the use of reduced mechanical properties. However, the elevated temperature design method in Eurocode 3 Part 1.2 is overly conservative and hence unsuitable, particularly in the case of flexural-torsional buckling at elevated temperatures.

Determining the psychosocial predictors of living, living-related, and posthumous organ donation

The worldwide organ shortage occurs despite people’s positive organ donation attitudes. The discrepancy between attitudes and behaviour is evident in Australia particularly, with widespread public support for organ donation but low donation and communication rates. This problem is compounded further by the paucity of theoretically based research to improve our understanding of people’s organ donation decisions. This program of research contributes to our knowledge of individual decision making processes for three aspects of organ donation: (1) posthumous (upon death) donation, (2) living donation (to a known and unknown recipient), and (3) providing consent for donation by communicating donation wishes on an organ donor consent register (registering) and discussing the donation decision with significant others (discussing). The research program used extended versions of the Theory of Planned Behaviour (TPB) and the Prototype/Willingness Model (PWM), incorporating additional influences (moral norm, self-identity, organ recipient prototypes), to explicate the relationship between people’s positive attitudes and low rates of organ donation behaviours. Adopting the TPB and PWM (and their extensions) as a theoretical basis overcomes several key limitations of the extant organ donation literature including the often atheoretical nature of organ donation research, thefocus on individual difference factors to construct organ donor profiles and the omission of important psychosocial influences (e.g., control perceptions, moral values) that may impact on people’s decision-making in this context. In addition, the use of the TPB and PWM adds further to our understanding of the decision making process for communicating organ donation wishes. Specifically, the extent to which people’s registering and discussing decisions may be explained by a reasoned and/or a reactive decision making pathway is examined (Stage 3) with the novel application of the TPB augmented with the social reaction pathway in the PWM. This program of research was conducted in three discrete stages: a qualitative stage (Stage 1), a quantitative stage with extended models (Stage 2), and a quantitative stage with augmented models (Stage 3). The findings of the research program are reported in nine papers which are presented according to the three aspects of organ donation examined (posthumous donation, living donation, and providing consent for donation by registering or discussing the donation preference). Stage One of the research program comprised qualitative focus groups/interviews with university students and community members (N = 54) (Papers 1 and 2). Drawing broadly on the TPB framework (Paper 1), content analysed responses revealed people’s commonly held beliefs about the advantages and disadvantages (e.g., prolonging/saving life), important people or groups (e.g., family), and barriers and motivators (e.g., a family’s objection to donation), related to living and posthumous organ donation. Guided by a PWM perspective, Paper Two identified people’s commonly held perceptions of organ donors (e.g., altruistic and giving), non-donors (e.g., self-absorbed and unaware), and transplant recipients (e.g., unfortunate, and in some cases responsible/blameworthy for their predicament). Stage Two encompassed quantitative examinations of people’s decision makingfor living (Papers 3 and 4) and posthumous (Paper 5) organ donation, and for registering and discussing donation wishes (Papers 6 to 8) to test extensions to both the TPB and PWM. Comparisons of health students’ (N = 487) motivations and willingness for living related and anonymous donation (Paper 3) revealed that a person’s donor identity, attitude, past blood donation, and knowing a posthumous donor were four common determinants of willingness, with the results highlighting students’ identification as a living donor as an important motive. An extended PWM is presented in Papers Four and Five. University students’ (N = 284) willingness for living related and anonymous donation was tested in Paper Four with attitude, subjective norm, donor prototype similarity, and moral norm (but not donor prototype favourability) predicting students’ willingness to donate organs in both living situations. Students’ and community members’ (N = 471) posthumous organ donation willingness was assessed in Paper Five with attitude, subjective norm, past behaviour, moral norm, self-identity, and prior blood donation all significantly directly predicting posthumous donation willingness, with only an indirect role for organ donor prototype evaluations. The results of two studies examining people’s decisions to register and/or discuss their organ donation wishes are reported in Paper Six. People’s (N = 24) commonly held beliefs about communicating their organ donation wishes were explored initially in a TPB based qualitative elicitation study. The TPB belief determinants of intentions to register and discuss the donation preference were then assessed for people who had not previously communicated their donation wishes (N = 123). Behavioural and normative beliefs were important determinants of registering and discussing intentions; however, control beliefs influenced people’s registering intentions only. Paper Seven represented the first empirical test of the role of organ transplant recipient prototypes (i.e., perceptions of organ transplant recipients) in people’s (N = 465) decisions to register consent for organ donation. Two factors, Substance Use and Responsibility, were identified and Responsibility predicted people’s organ donor registration status. Results demonstrated that unregistered respondents were the most likely to evaluate transplant recipients negatively. Paper Eight established the role of organ donor prototype evaluations, within an extended TPB model, in predicting students’ and community members’ registering (n = 359) and discussing (n = 282) decisions. Results supported the utility of an extended TPB and suggested a role for donor prototype evaluations in predicting people’s discussing intentions only. Strong intentions to discuss donation wishes increased the likelihood that respondents reported discussing their decision 1-month later. Stage Three of the research program comprised an examination of augmented models (Paper 9). A test of the TPB augmented with elements from the social reaction pathway in the PWM, and extensions to these models was conducted to explore whether people’s registering (N = 339) and discussing (N = 315) decisions are explained via a reasoned (intention) and/or social reaction (willingness) pathway. Results suggested that people’s decisions to communicate their organ donation wishes may be better explained via the reasoned pathway, particularly for registering consent; however, discussing also involves reactive elements. Overall, the current research program represents an important step toward clarifying the relationship between people’s positive organ donation attitudes but low rates of organ donation and communication behaviours. Support has been demonstrated for the use of extensions to two complementary theories, the TPB and PWM, which can inform future research aiming to explicate further the organ donation attitude-behaviour relationship. The focus on a range of organ donation behaviours enables the identification of key targets for future interventions encouraging people’s posthumous and living donation decisions, and communication of their organ donation preference.

Validating the IS-Impact model : two exploratory case studies in China and Malaysia

For over a decade, IT expenditure in China and Malaysia has shown a significant increase, as organisations in these countries are increasingly dependent on information systems (IS) for achieving strategic advantages and business benefits. However, there have been numerous reports of dissatisfaction with IS, and in some cases the effectiveness of the information systems have yet to be reviewed. Two exploratory case studies reported in this paper are the first phase of an overall research in validating the IS-Impact model introduced by Gable, Sedera and Chan in two countries: China and Malaysia. This validation research aims to produce a standard measuring model across different contexts. The purpose of this paper is to present preliminary findings from two exploratory case studies, attempt to test the feasibility of the research design and to investigate applicability of the IS-Impact model in Chinese and Malaysian organisations. Twenty-nine respondents from a Chinese private company and seventeen respondents from a state government in Malaysia were involved in these studies. Findings indicated that most of existing IS-Impact measures are applicable in the study contexts, however, there are some new measures informed by the respondents. Feedback from the case studies also suggested necessary modifications to the Mandarin instrument.

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.

Anterior vertebral stapling for the fusionless correction of scoliosis

Fusionless scoliosis surgery is an emerging treatment for idiopathic scoliosis as it offers theoretical advantages over current forms of treatment. Currently the treatment options for idiopathic scoliosis are observation, bracing and fusion. While brace treatment is non-invasive, and preserves the growth, motion, and function of the spine, it does not correct deformity and is only modestly successful in preventing curve progression. In adolescents who fail brace treatment, surgical treatment with an instrumented spinal fusion usually results in better deformity correction but is associated with substantially greater risk. Furthermore in younger patients requiring surgical treatment, fusion procedures are known to adversely effect the future growth of the chest and spine. Fusionless treatments have been developed to allow effective surgical treatment of patients with idiopathic scoliosis who are too young for fusion procedures. Anterior vertebral stapling is one such fusionless treatment which aims to modulate the growth of vertebra to allow correction of scoliosis whilst maintaining normal spinal motion The Mater Misericordiae Hospital in Brisbane has begun to use anterior vertebral stapling to treat patients with idiopathic scoliosis who are too young for fusion procedures. Currently the only staple approved for clinical use is manufactured by Medtronic Sofamor Danek (Memphis, TN). This thesis explains the biomechanical and anatomical changes that occur following anterior vertebral staple insertion using in vitro experiments performed on an immature bovine model. Currently there is a paucity of published information about anterior vertebral stapling so it is hoped that this project will provide information that will aid in our understanding of the clinical effects of staple insertion. The aims of this experimental study were threefold. The first phase was designed to determine the changes in the bending stiffness of the spine following staple insertion. The second phase was designed to measure the forces experienced by the staple during spinal movements. The third and final phase of testing was designed to describe the structural changes that occur to a vertebra as a consequence of staple insertion. The first phase of testing utilised a displacement controlled testing robot to compare the change in stiffness of a single spinal motion segment following staple insertion for the three basic spinal motions of flexion-extension, lateral bending, and axial rotation. For the second phase of testing strain gauges were attached to staples and used to measure staple forces during spinal movement. In the third and final phase the staples were removed and a testing specimen underwent micro-computed tomography (CT) scanning to describe the anatomical changes that occur following staple insertion. The displacement controlled testing showed that there was a significant decrease in bending stiffness in flexion, extension, lateral bending away from the staple, and axial rotation away from the staple following staple insertion. The strain gauge measurements showed that the greatest staple forces occurred in flexion and the least in extension. In addition, a reduction in the baseline staple compressive force was seen with successive loading cycles. Micro-CT scanning demonstrated that significant damage to the vertebral body and endplate occurred as a consequence of staple insertion. The clinical implications of this study are significant. Based on the findings of this project it is likely that the clinical effect of the anterior vertebral staple evaluated in this project is a consequence of growth plate damage (also called hemiepiphysiodesis) causing a partial growth arrest of the vertebra rather than simply compression of the growth plate. The surgical creation of a unilateral growth arrest is a well established treatment used in the management of congenital scoliosis but has not previously been considered for use in idiopathic scoliosis.

An adaptable framework for the teaching and assessment of software development across year levels

Few frameworks exist for the teaching and assessment of programming subjects that are coherent and logical. Nor are they sufficiently generic and adaptable to be used outside the particular tertiary institutions in which they were developed. This paper presents the Teaching and Assessment of Software Development (TASD) frame-work. We describe its development and implementation at an Australian university and demonstrate, with examples, how it has been used, with supporting data. Extracts of criteria sheets (grading rubrics) for a variety of assessment tasks are included. The numerous advantages of this new framework are discussed with comparisons made to those reported in the published literature.