The manipulation of apoptosis-related genes to generate resistance to Fusarium wilt and water stress in banana

Bananas are susceptible to a diverse range of biotic and abiotic stresses, many of which cause serious production constraints worldwide. One of the most destructive banana diseases is Fusarium wilt caused by the soil-borne fungus, Fusarium oxysporum f. sp. cubense (Foc). No effective control strategy currently exists for this disease which threatens global banana production. Although disease resistance exists in some wild bananas, attempts to introduce resistance into commercially acceptable bananas by conventional breeding have been hampered by low fertility, long generation times and association of poor agronomical traits with resistance genes. With the advent of reliable banana transformation protocols, molecular breeding is now regarded as a viable alternative strategy to generate disease-resistant banana plants. Recently, a novel strategy involving the expression of anti-apoptosis genes in plants was shown to result in resistance against several necrotrophic fungi. Further, the transgenic plants showed increased resistance to a range of abiotic stresses. In this thesis, the use of anti-apoptosis genes to generate transgenic banana plants with resistance to Fusarium wilt was investigated. Since water stress is an important abiotic constraint to banana production, the resistance of the transgenic plants to water stress was also examined. Embryogenic cell suspensions (ECS) of two commercially important banana cultivars, Grand Naine (GN) and Lady Finger (LF), were transformed using Agrobacterium with the anti-apoptosis genes, Bcl-xL, Bcl-xL G138A, Ced-9 and Bcl- 2 3’ UTR. An interesting, and potentially important, outcome was that the use of anti-apoptosis genes resulted in up to a 50-fold increase in Agrobacterium-mediated transformation efficiency of both LF and GN cells over vector controls. Regenerated plants were subjected to a complete molecular characterisation in order to detect the presence of the transgene (PCR), transcript (RT-PCR) and gene product (Western blot) and to determine the gene copy number (Southern blot). A total of 36 independently-transformed GN lines (8 x Bcl-xL, 5 x Bcl-xL G138A, 15 x Ced-9 and 8 x Bcl-2 3’ UTR) and 41 independently-transformed LF lines (8 x Bcl-xL, 7 x BclxL G138A, 13 x Ced-9 and 13 x Bcl-2 3’ UTR) were identified. The 41 transgenic LF lines were multiplied and clones from each line were acclimatised and grown under glasshouse conditions for 8 weeks to allow monitoring for phenotypic abnormalities. Plants derived from 3 x Bcl-xL, 2 x Ced-9 and 5 x Bcl-2 3’ UTR lines displayed a variety of aberrant phenotypes. However, all but one of these abnormalities were off-types commonly observed in tissue-cultured, non-transgenic banana plants and were therefore unlikely to be transgene-related. Prior to determining the resistance of the transgenic plants to Foc race 1, the apoptotic effects of the fungus on both wild-type and Bcl-2 3’ UTR-transgenic LF banana cells were investigated using rapid in vitro root assays. The results from these assays showed that apoptotic-like cell death was elicited in wild-type banana root cells as early as 6 hours post-exposure to fungal spores. In contrast, these effects were attenuated in the root cells of Bcl-2 3’ UTR-transgenic lines that were exposed to fungal spores. Thirty eight of the 41 transgenic LF lines were subsequently assessed for resistance to Foc race 1 in small-plant glasshouse bioassays. To overcome inconsistencies in rating the internal (vascular discolouration) disease symptoms, a MatLab-based computer program was developed to accurately and reliably assess the level of vascular discolouration in banana corms. Of the transgenic LF banana lines challenged with Foc race 1, 2 x Bcl-xL, 3 x Ced-9, 2 x Bcl-2 3’ UTR and 1 x Bcl-xL G138A-transgenic line were found to show significantly less external and internal symptoms than wild-type LF banana plants used as susceptible controls at 12 weeks post-inoculation. Of these lines, Bcl-2 3’ UTR-transgenic line #6 appeared most resistant, displaying very mild symptoms similar to the wild-type Cavendish banana plants that were included as resistant controls. This line remained resistant for up to 23 weeks post-inoculation. Since anti-apoptosis genes have been shown to confer resistance to various abiotic stresses in other crops, the ability of these genes to confer resistance against water stress in banana was also investigated. Clonal plants derived from each of the 38 transgenic LF banana plants were subjected to water stress for a total of 32 days. Several different lines of transgenic plants transformed with either Bcl-xL, Bcl-xL G138A, Ced-9 or Bcl-2 3’ UTR showed a delay in visual water stress symptoms compared with the wild-type control plants. These plants all began producing new growth from the pseudostem following daily rewatering for one month. In an attempt to determine whether the protective effect of anti-apoptosis genes in transgenic banana plants was linked with reactive oxygen species (ROS)-associated programmed cell death (PCD), the effect of the chloroplast-targeting, ROS-inducing herbicide, Paraquat, on wild-type and transgenic LF was investigated. When leaf discs from wild-type LF banana plants were exposed to 10 ìM Paraquat, complete decolourisation occurred after 48 hours which was confirmed to be associated with cell death and ROS production by trypan blue and 3,3-diaminobenzidine (DAB) staining, respectively. When leaf discs from the transgenic lines were exposed to Paraquat, those derived from some lines showed a delay in decolourisation, suggesting only a weak protective effect from the transgenes. Finally, the protective effect of anti-apoptosis genes against juglone, a ROS-inducing phytotoxin produced by the causal agent of black Sigatoka, Mycosphaerella fijiensis, was investigated. When leaf discs from wild-type LF banana plants were exposed to 25 ppm juglone, complete decolourisation occurred after 48 hours which was again confirmed to be associated with cell death and ROS production by trypan blue and DAB staining, respectively. Further, TdT-mediated dUTP nick-end labelling (TUNEL) assays on these discs suggested that the cell death was apoptotic. When leaf discs from the transgenic lines were exposed to juglone, discs from some lines showed a clear delay in decolourisation, suggesting a protective effect. Whether these plants are resistant to black Sigatoka is unknown and will require future glasshouse and field trials. The work presented in this thesis provides the first report of the use of anti-apoptosis genes as a strategy to confer resistance to Fusarium wilt and water stress in a nongraminaceous monocot, banana. Such a strategy may be exploited to generate resistance to necrotrophic pathogens and abiotic stresses in other economically important crop plants.

An effective model of using negative relevance feedback for information filtering

Over the years, people have often held the hypothesis that negative feedback should be very useful for largely improving the performance of information filtering systems; however, we have not obtained very effective models to support this hypothesis. This paper, proposes an effective model that use negative relevance feedback based on a pattern mining approach to improve extracted features. This study focuses on two main issues of using negative relevance feedback: the selection of constructive negative examples to reduce the space of negative examples; and the revision of existing features based on the selected negative examples. The former selects some offender documents, where offender documents are negative documents that are most likely to be classified in the positive group. The later groups the extracted features into three groups: the positive specific category, general category and negative specific category to easily update the weight. An iterative algorithm is also proposed to implement this approach on RCV1 data collections, and substantial experiments show that the proposed approach achieves encouraging performance.

The impact of police speed enforcement practices on self-reported speeding: An exploration of the effects of visibility and mobility

Research has highlighted the relationship between vehicle speed and increased crash risk and severity. Evidence suggests that police speed enforcement, in particular speed camera operations, can be an effective tool for reducing traffic crashes. A quantitative survey of Queensland drivers (n = 852) was conducted to investigate the impact of police speed enforcement methods on self-reported speeding behaviour. Results indicate that visible enforcement was associated with significantly greater self-reported compliance than covert operations irrespective of the mobility of the approach, and the effects on behaviour were longer lasting. The mobility of operations appeared to be moderated the visibility of the approach. Specifically, increased mobility was associated with increase reported compliant behaviour, but only for covert operations, and increased longevity of reported compliant behaviour, but only for overt operations. The perceived effectiveness of various speed enforcement approaches are also analysed across a range of driving scenarios. Results are discussed in light of the small effect sizes. Recommendations for policy and future research are presented.

Internships : effective work integrated learning for law students

Work experience which is integrated into an undergraduate law degree has a vital role to play in assisting law students to develop the skills and attributes they need in order to be effective legal practitioners. Work integrated learning provides a context for students to develop their skills, to see the link between theory and practice and supports students in making the transition from university to practice. The literature in Australian legal education has given little consideration to the design of legal internship subjects (as distinct from legal clinic programs). Accordingly the design of internship subjects needs to be carefully considered to ensure alignment of learning objectives, learning tasks and assessment. This paper will examine the literature relating to internships, particularly in a legal context, and will propose some principles for the design of legal internships. These principles will be considered in light of an evaluation of a newly designed undergraduate legal internship subject.

Within-session variability modelling for factor analysis speaker verification

This work presents an extended Joint Factor Analysis model including explicit modelling of unwanted within-session variability. The goals of the proposed extended JFA model are to improve verification performance with short utterances by compensating for the effects of limited or imbalanced phonetic coverage, and to produce a flexible JFA model that is effective over a wide range of utterance lengths without adjusting model parameters such as retraining session subspaces. Experimental results on the 2006 NIST SRE corpus demonstrate the flexibility of the proposed model by providing competitive results over a wide range of utterance lengths without retraining and also yielding modest improvements in a number of conditions over current state-of-the-art.

An effective approach to verbose queries using a limited dependencies language model

Intuitively, any `bag of words' approach in IR should benefit from taking term dependencies into account. Unfortunately, for years the results of exploiting such dependencies have been mixed or inconclusive. To improve the situation, this paper shows how the natural language properties of the target documents can be used to transform and enrich the term dependencies to more useful statistics. This is done in three steps. The term co-occurrence statistics of queries and documents are each represented by a Markov chain. The paper proves that such a chain is ergodic, and therefore its asymptotic behavior is unique, stationary, and independent of the initial state. Next, the stationary distribution is taken to model queries and documents, rather than their initial distri- butions. Finally, ranking is achieved following the customary language modeling paradigm. The main contribution of this paper is to argue why the asymptotic behavior of the document model is a better representation then just the document's initial distribution. A secondary contribution is to investigate the practical application of this representation in case the queries become increasingly verbose. In the experiments (based on Lemur's search engine substrate) the default query model was replaced by the stable distribution of the query. Just modeling the query this way already resulted in significant improvements over a standard language model baseline. The results were on a par or better than more sophisticated algorithms that use fine-tuned parameters or extensive training. Moreover, the more verbose the query, the more effective the approach seems to become.

An investigation into how work-related road safety can be enhanced

Despite the facts that vehicle incidents continue to be the most common mechanism for Australian compensated fatalities and that employers have statutory obligations to provide safe workplaces, very few organisations are proactively and comprehensively managing their work-related road risks. Unfortunately, limited guidance is provided in the existing literature to assist practitioners in managing work-related road risks. The current research addresses this gap in the literature. To explore how work-related road safety can be enhanced, three studies were conducted. Study one explored the effectiveness of a range of risk management initiatives and whether comprehensive risk management practices were associated with safety outcomes. Study two explored barriers to, and facilitators for, accepting risk management initiatives. Study three explored the influence of organisational factors on road safety outcomes to identify optimal work environments for managing road risks. To maximise the research sample and increase generalisability, the studies were designed to allow data collection to be conducted simultaneously drawing upon the same sample obtained from four Australian organisations. Data was collected via four methods. A structured document review of published articles was conducted to identify what outcomes have been observed in previously investigated work-related road safety initiatives. The documents reviewed collectively assessed the effectiveness of 19 work-related road safety initiatives. Audits of organisational practices and process operating within the four researched organisations were conducted to identify whether organisations with comprehensive work-related road risk management practices and processes have better safety outcomes than organisations with limited risk management practices and processes. Interviews were conducted with a sample of 24 participants, comprising 16 employees and eight managers. The interviews were conducted to identify what barriers and facilitators within organisations are involved in implementing work-related road safety initiatives and whether differences in fleet safety climate, stage of change and safety ownership relate to work-related road safety outcomes. Finally, questionnaires were administered to a sample of 679 participants. The questionnaires were conducted to identify which initiatives are perceived by employees to be effective in managing work-related road risks and whether differences in fleet safety climate, stage of change and safety ownership relate to work-related road safety outcomes. Seven research questions were addressed in the current research project. The key findings with respect to each of the research questions are presented below. Research question one: What outcomes have been observed in previously investigated work-related road safety initiatives? The structured document review indicated that initiatives found to be positively associated with occupational road safety both during and after the intervention period included: a pay rise; driver training; group discussions; enlisting employees as community road safety change agents; safety reminders; and group and individual rewards. Research question two: Which initiatives are perceived by employees to be effective in managing work-related road risks? Questionnaire findings revealed that employees believed occupational road risks could best be managed through making vehicle safety features standard, providing practical driver skills training and through investigating serious vehicle incidents. In comparison, employees believed initiatives including signing a promise card commitment to drive safely, advertising the organisation’s phone number on vehicles and consideration of driving competency in staff selection process would have limited effectiveness in managing occupational road safety. Research question three: Do organisations with comprehensive work-related road risk management practices and processes have better safety outcomes than organisations with limited risk management practices and processes? The audit identified a difference among the organisations in their management of work-related road risks. Comprehensive risk management practices were associated with employees engaging in overall safer driving behaviours, committing less driving errors, and experiencing less fatigue and distraction issues when driving. Given that only four organisations participated in this research, these findings should only be considered as preliminary. Further research should be conducted to explore the relationship between comprehensiveness of risk management practices and road safety outcomes with a larger sample of organisations. Research question four: What barriers and facilitators within organisations are involved in implementing work-related road safety initiatives? The interviews identified that employees perceived six organisational characteristics as potential barriers to implementing work-related road safety initiatives. These included: prioritisation of production over safety; complacency towards work-related road risks; insufficient resources; diversity; limited employee input in safety decisions; and a perception that road safety initiatives were an unnecessary burden. In comparison, employees perceived three organisational characteristics as potential facilitators to implementing work-related road safety initiatives. These included: management commitment; the presence of existing systems that could support the implementation of initiatives; and supportive relationships. Research question five: Do differences in fleet safety climate relate to work-related road safety outcomes? The interviews and questionnaires identified that organisational climates with high management commitment, support for managing work demands, appropriate safety rules and safety communication were associated with employees who engaged in safer driving behaviours. Regression analyses indicated that as participants’ perceptions of safety climate increased, the corresponding likelihood of them engaging in safer driving behaviours increased. Fleet safety climate was perceived to influence road safety outcomes through several avenues. Some of these included: the allocation of sufficient resources to manage occupational road risks; fostering a supportive environment of mutual responsibility; resolving safety issues openly and fairly; clearly communicating to employees that safety is the top priority; and developing appropriate work-related road safety policies and procedures. Research question six: Do differences in stage of change relate to work-related road safety outcomes? The interviews and questionnaires identified that participants’ perceptions of initiative effectiveness were found to vary with respect to their individual stage of readiness, with stage-matched initiatives being perceived most effective. In regards to safety outcomes, regression analyses identified that as participants’ progress through the stages of change, the corresponding likelihood of them being involved in vehicle crashes decreases. Research question seven: Do differences in safety ownership relate to work-related road safety outcomes? The interviews and questionnaires revealed that management of road risks is often given less attention than other areas of health and safety management in organisations. In regards to safety outcomes, regression analyses identified that perceived authority and perceived shared ownership both emerged as significant independent predictors of self-reported driving behaviours pertaining to fatigue and distractions. The regression models indicated that as participants’ perceptions of the authority of the person managing road risks increases, and perceptions of shared ownership of safety tasks increases, the corresponding likelihood of them engaging in driving while fatigued or multitasking while driving decreases. Based on the findings from the current research, the author makes several recommendations to assist practitioners in developing proactive and comprehensive approaches to managing occupational road risks. The author also suggests several avenues for future research in the area of work-related road safety.

The effective and the evocative : reflecting on practice-led research approaches in art and design

Early in the practice-led research debate, Steven Scrivener (2000, 2002) identified some general differences in the approach of artists and designers undertaking postgraduate research. His distinctions centered on the role of the artefact in problem-based research (associated with design) and creative-production research (associated with artistic practice). Nonetheless, in broader discussions on practice-led research, 'art and design' often continues to be conflated within a single term. In particular, marked differences between art and design methodologies, theoretical framing, research goals and research claims have been underestimated. This paper revisits Scrivener's work and establishes further distinctions between art and design research. It is informed by our own experiences of postgraduate supervision and research methods training, and an empirical study of over sixty postgraduate, practice-led projects completed at the Creative Industries Faculty of QUT between 2002 and 2008. Our reflections have led us to propose that artists and designers work with differing research goals (the evocative and the effective, respectively), which are played out in the questions asked, the creative process, the role of the artefact and the way new knowledge is evidenced. Of course, research projects will have their own idiosyncrasies but, we argue, marking out the poles at each end of the spectrum of art and design provides useful insights for postgraduate candidates, supervisors and methodologists alike.

Intelligent prognostics of machinery health utilising suspended condition monitoring data

The ability to forecast machinery failure is vital to reducing maintenance costs, operation downtime and safety hazards. Recent advances in condition monitoring technologies have given rise to a number of prognostic models for forecasting machinery health based on condition data. Although these models have aided the advancement of the discipline, they have made only a limited contribution to developing an effective machinery health prognostic system. The literature review indicates that there is not yet a prognostic model that directly models and fully utilises suspended condition histories (which are very common in practice since organisations rarely allow their assets to run to failure); that effectively integrates population characteristics into prognostics for longer-range prediction in a probabilistic sense; which deduces the non-linear relationship between measured condition data and actual asset health; and which involves minimal assumptions and requirements. This work presents a novel approach to addressing the above-mentioned challenges. The proposed model consists of a feed-forward neural network, the training targets of which are asset survival probabilities estimated using a variation of the Kaplan-Meier estimator and a degradation-based failure probability density estimator. The adapted Kaplan-Meier estimator is able to model the actual survival status of individual failed units and estimate the survival probability of individual suspended units. The degradation-based failure probability density estimator, on the other hand, extracts population characteristics and computes conditional reliability from available condition histories instead of from reliability data. The estimated survival probability and the relevant condition histories are respectively presented as “training target” and “training input” to the neural network. The trained network is capable of estimating the future survival curve of a unit when a series of condition indices are inputted. Although the concept proposed may be applied to the prognosis of various machine components, rolling element bearings were chosen as the research object because rolling element bearing failure is one of the foremost causes of machinery breakdowns. Computer simulated and industry case study data were used to compare the prognostic performance of the proposed model and four control models, namely: two feed-forward neural networks with the same training function and structure as the proposed model, but neglected suspended histories; a time series prediction recurrent neural network; and a traditional Weibull distribution model. The results support the assertion that the proposed model performs better than the other four models and that it produces adaptive prediction outputs with useful representation of survival probabilities. This work presents a compelling concept for non-parametric data-driven prognosis, and for utilising available asset condition information more fully and accurately. It demonstrates that machinery health can indeed be forecasted. The proposed prognostic technique, together with ongoing advances in sensors and data-fusion techniques, and increasingly comprehensive databases of asset condition data, holds the promise for increased asset availability, maintenance cost effectiveness, operational safety and – ultimately – organisation competitiveness.

Enhancing digital road map with lane details extracted from large-scale stereo aerial imagery using object-oriented image analysis

Precise, up-to-date and increasingly detailed road maps are crucial for various advanced road applications, such as lane-level vehicle navigation, and advanced driver assistant systems. With the very high resolution (VHR) imagery from digital airborne sources, it will greatly facilitate the data acquisition, data collection and updates if the road details can be automatically extracted from the aerial images. In this paper, we proposed an effective approach to detect road lane information from aerial images with employment of the object-oriented image analysis method. Our proposed algorithm starts with constructing the DSM and true orthophotos from the stereo images. The road lane details are detected using an object-oriented rule based image classification approach. Due to the affection of other objects with similar spectral and geometrical attributes, the extracted road lanes are filtered with the road surface obtained by a progressive two-class decision classifier. The generated road network is evaluated using the datasets provided by Queensland department of Main Roads. The evaluation shows completeness values that range between 76% and 98% and correctness values that range between 82% and 97%.

A phenomenological study of contemplative experiences : implications for interior design

This research reports on a project concerned with the relationship between the person and the environment in the context of achieving a contemplative or existential state – a state which can be experienced either consciously or subconsciously. The need for such a study originated with the desire to contribute to the design of multicultural spaces which could be used for a range of activities within the public and the personal arena, activities including contemplation, meditation and prayer. The concept of ‘sacred’ is explored in the literature review and in primary interviews with the participants of this study. Given that the word ‘sacred’ is highly value-laden and potentially alienating for some people, it was decided to use the more accessible term ‘contemplative’. The outcomes of the study inform the practice of interior design and architecture which tends currently to neglect the potential for all spaces to be existentially meaningful. Informed by phenomenological methodology, data were collected from a diverse group of people, using photo-elicitation and interviews. The technique of photo-elicitation proved to be highly effective in helping people reveal their everyday lived experience of contemplative spaces. Reflective analysis (Van Manen 2000) was used to explore the data collected. The initial stage of analysis produced three categories of data: varying conceptions of contemplation, aspects of the person involved in the contemplation, and aspects of environment involved in contemplation. From this, it was found that achieving a state of contemplation involves both the person and the environment in a dialectic process of unfolding. The unfolding has various physical, psycho-social, and existential dimensions or qualities which operate sequentially and simultaneously. Two concepts emerged as being central to unfolding: ‘Cleansing’ and ‘Nothingness’. Unfolding is found to comprise the Core; Distinction; Manifestation; Cleansing; Creation; and Sharing. This has a parallel with Mircea Eliade’s (1959) definition of sacred as something that manifests itself as different from the profane. The power of design, re-contextualization through utility and purpose, and the existential engagements between the person and environment are used as a basis for establishing the potential contribution of the study to interior design. In this way, the study makes a contribution to our understanding of how space and its elements inspire, support and sustain person environment interaction – particularly at the existential level – as well as to our understanding of the multi-dimensional and holistic nature of this interaction. In addition, it points to the need for a phenomenological re-conceptualisation of the design/client relationship. In summary, the contributions of this research are: the exploration of contemplative experience as sacred experience; an understanding of the design of space as creating engagement between person and environment; a rationale for the introduction of a phenomenological approach to the relationship between designer and clients; and raising awareness of the spiritual in a holistic approach to design.

Reciprocity : where art meets the community : action research in response to artistic encounters and relationships

This practice-led research project examines some of the factors and issues facing artists working in the public domain who wish to engage with the community as audience. Using the methodology of action research, the three major creative projects in this study use art as a socio-political tool with the aim of providing an effective vehicle for broadening awareness, understanding forms of social protest and increasing tolerance for diversity. The three projects: Floodline November 7, 2004, Look in, Look out, and The Urban Terrorist Project, dealt with issues of marginalisation of communities, audiences and graffiti artists respectively. The artist/researcher is outlined as both creator and collaborator in the work. Processes included ephemeral elements, such as temporary installation and performance, as well as interactive elements that encouraged direct audience involvement as part of the work. In addition to the roles of creator and collaborator, both of which included audience as well as artist, the presence of an outside entity was evident. Whether local, legal authorities or prevailing attitudes, outside entities had an unavoidable impact on the processes and outcomes of the work. Each project elicited a range of responses from their respective audiences; however, the overarching concept of reciprocity was seen to be the crucial factor in conception, artistic methods and outcomes.

Enhancing the standard operating range of a placido disk videokeratoscope for corneal surface estimation

Abstract—Corneal topography estimation that is based on the Placido disk principle relies on good quality of precorneal tear film and sufficiently wide eyelid (palpebral) aperture to avoid reflections from eyelashes. However, in practice, these conditions are not always fulfilled resulting in missing regions, smaller corneal coverage, and subsequently poorer estimates of corneal topography. Our aim was to enhance the standard operating range of a Placido disk videokeratoscope to obtain reliable corneal topography estimates in patients with poor tear film quality, such as encountered in those diagnosed with dry eye, and with narrower palpebral apertures as in the case of Asian subjects. This was achieved by incorporating in the instrument’s own topography estimation algorithm an image processing technique that comprises a polar-domain adaptive filter and amorphological closing operator. The experimental results from measurements of test surfaces and real corneas showed that the incorporation of the proposed technique results in better estimates of corneal topography, and, in many cases, to a significant increase in the estimated coverage area making such an enhanced videokeratoscope a better tool for clinicians.

Assessment of tear film surface quality using dynamic-area high-speed videokeratoscopy

A new method for noninvasive assessment of tear film surface quality (TFSQ) is proposed. The method is based on high-speed videokeratoscopy in which the corneal area for the analysis is dynamically estimated in a manner that removes videokeratoscopy interference from the shadows of eyelashes but not that related to the poor quality of the precorneal tear film that is of interest. The separation between the two types of seemingly similar videokeratoscopy interference is achieved by region-based classification in which the overall noise is first separated from the useful signal (unaltered videokeratoscopy pattern), followed by a dedicated interference classification algorithm that distinguishes between the two considered interferences. The proposed technique provides a much wider corneal area for the analysis of TFSQ than the previously reported techniques. A preliminary study with the proposed technique, carried out for a range of anterior eye conditions, showed an effective behavior in terms of noise to signal separation, interference classification, as well as consistent TFSQ results. Subsequently, the method proved to be able to not only discriminate between the bare eye and the lens on eye conditions but also to have the potential to discriminate between the two types of contact lenses.

Protein complementation assay as a display system for screening protein libraries in the intracellular environment

A wide range of screening strategies have been employed to isolate antibodies and other proteins with specific attributes, including binding affinity, specificity, stability and improved expression. However, there remains no high-throughput system to screen for target-binding proteins in a mammalian, intracellular environment. Such a system would allow binding reagents to be isolated against intracellular clinical targets such as cell signalling proteins associated with tumour formation (p53, ras, cyclin E), proteins associated with neurodegenerative disorders (huntingtin, betaamyloid precursor protein), and various proteins crucial to viral replication (e.g. HIV-1 proteins such as Tat, Rev and Vif-1), which are difficult to screen by phage, ribosome or cell-surface display. This study used the β-lactamase protein complementation assay (PCA) as the display and selection component of a system for screening a protein library in the cytoplasm of HEK 293T cells. The colicin E7 (ColE7) and Immunity protein 7 (Imm7) *Escherichia coli* proteins were used as model interaction partners for developing the system. These proteins drove effective β-lactamase complementation, resulting in a signal-to-noise ratio (9:1 – 13:1) comparable to that of other β-lactamase PCAs described in the literature. The model Imm7-ColE7 interaction was then used to validate protocols for library screening. Single positive cells that harboured the Imm7 and ColE7 binding partners were identified and isolated using flow cytometric cell sorting in combination with the fluorescent β-lactamase substrate, CCF2/AM. A single-cell PCR was then used to amplify the Imm7 coding sequence directly from each sorted cell. With the screening system validated, it was then used to screen a protein library based the Imm7 scaffold against a proof-of-principle target. The wild-type Imm7 sequence, as well as mutants with wild-type residues in the ColE7- binding loop were enriched from the library after a single round of selection, which is consistent with other eukaryotic screening systems such as yeast and mammalian cell-surface display. In summary, this thesis describes a new technology for screening protein libraries in a mammalian, intracellular environment. This system has the potential to complement existing screening technologies by allowing access to intracellular proteins and expanding the range of targets available to the pharmaceutical industry.