Physical activity recognition from accelerometer data using a multi-scale ensemble method

Accurate and detailed measurement of an individual's physical activity is a key requirement for helping researchers understand the relationship between physical activity and health. Accelerometers have become the method of choice for measuring physical activity due to their small size, low cost, convenience and their ability to provide objective information about physical activity. However, interpreting accelerometer data once it has been collected can be challenging. In this work, we applied machine learning algorithms to the task of physical activity recognition from triaxial accelerometer data. We employed a simple but effective approach of dividing the accelerometer data into short non-overlapping windows, converting each window into a feature vector, and treating each feature vector as an i.i.d training instance for a supervised learning algorithm. In addition, we improved on this simple approach with a multi-scale ensemble method that did not need to commit to a single window size and was able to leverage the fact that physical activities produced time series with repetitive patterns and discriminative features for physical activity occurred at different temporal scales.

The effect of prolonged reading on visual functions and reading performance in students with low vision

This study is the first to investigate the effect of prolonged reading on reading performance and visual functions in students with low vision. The study focuses on one of the most common modes of achieving adequate magnification for reading by students with low vision, their close reading distance (proximal or relative distance magnification). Close reading distances impose high demands on near visual functions, such as accommodation and convergence. Previous research on accommodation in children with low vision shows that their accommodative responses are reduced compared to normal vision. In addition, there is an increased lag of accommodation for higher stimulus levels as may occur at close reading distance. Reduced accommodative responses in low vision and higher lag of accommodation at close reading distances together could impact on reading performance of students with low vision especially during prolonged reading tasks. The presence of convergence anomalies could further affect reading performance. Therefore, the aims of the present study were 1) To investigate the effect of prolonged reading on reading performance in students with low vision 2) To investigate the effect of prolonged reading on visual functions in students with low vision. This study was conducted as cross-sectional research on 42 students with low vision and a comparison group of 20 students with normal vision, aged 7 to 20 years. The students with low vision had vision impairments arising from a range of causes and represented a typical group of students with low vision, with no significant developmental delays, attending school in Brisbane, Australia. All participants underwent a battery of clinical tests before and after a prolonged reading task. An initial reading-specific history and pre-task measurements that included Bailey-Lovie distance and near visual acuities, Pelli-Robson contrast sensitivity, ocular deviations, sensory fusion, ocular motility, near point of accommodation (pull-away method), accuracy of accommodation (Monocular Estimation Method (MEM)) retinoscopy and Near Point of Convergence (NPC) (push-up method) were recorded for all participants. Reading performance measures were Maximum Oral Reading Rates (MORR), Near Text Visual Acuity (NTVA) and acuity reserves using Bailey-Lovie text charts. Symptoms of visual fatigue were assessed using the Convergence Insufficiency Symptom Survey (CISS) for all participants. Pre-task measurements of reading performance and accuracy of accommodation and NPC were compared with post-task measurements, to test for any effects of prolonged reading. The prolonged reading task involved reading a storybook silently for at least 30 minutes. The task was controlled for print size, contrast, difficulty level and content of the reading material. Silent Reading Rate (SRR) was recorded every 2 minutes during prolonged reading. Symptom scores and visual fatigue scores were also obtained for all participants. A visual fatigue analogue scale (VAS) was used to assess visual fatigue during the task, once at the beginning, once at the middle and once at the end of the task. In addition to the subjective assessments of visual fatigue, tonic accommodation was monitored using a photorefractor (PlusoptiX CR03™) every 6 minutes during the task, as an objective assessment of visual fatigue. Reading measures were done at the habitual reading distance of students with low vision and at 25 cms for students with normal vision. The initial history showed that the students with low vision read for significantly shorter periods at home compared to the students with normal vision. The working distances of participants with low vision ranged from 3-25 cms and half of them were not using any optical devices for magnification. Nearly half of the participants with low vision were able to resolve 8-point print (1M) at 25 cms. Half of the participants in the low vision group had ocular deviations and suppression at near. Reading rates were significantly reduced in students with low vision compared to those of students with normal vision. In addition, there were a significantly larger number of participants in the low vision group who could not sustain the 30-minute task compared to the normal vision group. However, there were no significant changes in reading rates during or following prolonged reading in either the low vision or normal vision groups. Individual changes in reading rates were independent of their baseline reading rates, indicating that the changes in reading rates during prolonged reading cannot be predicted from a typical clinical assessment of reading using brief reading tasks. Contrary to previous reports the silent reading rates of the students with low vision were significantly lower than their oral reading rates, although oral and silent reading was assessed using different methods. Although the visual acuity, contrast sensitivity, near point of convergence and accuracy of accommodation were significantly poorer for the low vision group compared to those of the normal vision group, there were no significant changes in any of these visual functions following prolonged reading in either group. Interestingly, a few students with low vision (n =10) were found to be reading at a distance closer than their near point of accommodation. This suggests a decreased sensitivity to blur. Further evaluation revealed that the equivalent intrinsic refractive errors (an estimate of the spherical dioptirc defocus which would be expected to yield a patient’s visual acuity in normal subjects) were significantly larger for the low vision group compared to those of the normal vision group. As expected, accommodative responses were significantly reduced for the low vision group compared to the expected norms, which is consistent with their close reading distances, reduced visual acuity and contrast sensitivity. For those in the low vision group who had an accommodative error exceeding their equivalent intrinsic refractive errors, a significant decrease in MORR was found following prolonged reading. The silent reading rates however were not significantly affected by accommodative errors in the present study. Suppression also had a significant impact on the changes in reading rates during prolonged reading. The participants who did not have suppression at near showed significant decreases in silent reading rates during and following prolonged reading. This impact of binocular vision at near on prolonged reading was possibly due to the high demands on convergence. The significant predictors of MORR in the low vision group were age, NTVA, reading interest and reading comprehension, accounting for 61.7% of the variances in MORR. SRR was not significantly influenced by any factors, except for the duration of the reading task sustained; participants with higher reading rates were able to sustain a longer reading duration. In students with normal vision, age was the only predictor of MORR. Participants with low vision also reported significantly greater visual fatigue compared to the normal vision group. Measures of tonic accommodation however were little influenced by visual fatigue in the present study. Visual fatigue analogue scores were found to be significantly associated with reading rates in students with low vision and normal vision. However, the patterns of association between visual fatigue and reading rates were different for SRR and MORR. The participants with low vision with higher symptom scores had lower SRRs and participants with higher visual fatigue had lower MORRs. As hypothesized, visual functions such as accuracy of accommodation and convergence did have an impact on prolonged reading in students with low vision, for students whose accommodative errors were greater than their equivalent intrinsic refractive errors, and for those who did not suppress one eye. Those students with low vision who have accommodative errors higher than their equivalent intrinsic refractive errors might significantly benefit from reading glasses. Similarly, considering prisms or occlusion for those without suppression might reduce the convergence demands in these students while using their close reading distances. The impact of these prescriptions on reading rates, reading interest and visual fatigue is an area of promising future research. Most importantly, it is evident from the present study that a combination of factors such as accommodative errors, near point of convergence and suppression should be considered when prescribing reading devices for students with low vision. Considering these factors would also assist rehabilitation specialists in identifying those students who are likely to experience difficulty in prolonged reading, which is otherwise not reflected during typical clinical reading assessments.

Dichotomy in the design studio : adapting to new blended learning environments

In a study aimed at better understanding how staff and students adapt to new blended studio learning environments (BSLE’s), a group of 165 second year architecture students at a large school of architecture in Australia were separated into two different design studio learning environments. 70% of students were allocated to a traditional studio design learning environment (TSLE) and 30% to a new, high technology embedded, prototype digital learning laboratory. The digital learning laboratory was purpose designed for the case-study users, adapted Student-Centred Active Learning Environment for Undergraduate Programs (SCALE-UP) principles, and built as part of a larger university research project. The architecture students attended the same lectures, followed the same studio curriculum and completed the same pieces of assessment; the only major differences were the teaching staff and physical environment within which the studios were conducted. At the end of the semester, the staff and students were asked to complete a questionnaire about their experiences and preferences within the two respective learning environments. Following this, participants were invited to participate in focus groups, where a synergistic approach was effected. Using a dual method qualitative approach, the questionnaire and survey data were coded and extrapolated using both thematic analysis and grounded theory methodology. The results from these two different approaches were compared, contrasted and finally merged, to reveal six distinct emerging themes, which were instrumental in offering resistance or influencing adaptation to, the new BLSE. This paper reports on the study, discusses the major contributors to negative resistance and proposes points for consideration, when transitioning from a TSLE to a BLSE.

Preliminary study on bridge health prediction using Dynamic Objective Oriented Bayesian Network (DOOBN)

The availability of bridges is crucial to people’s daily life and national economy. Bridge health prediction plays an important role in bridge management because maintenance optimization is implemented based on prediction results of bridge deterioration. Conventional bridge deterioration models can be categorised into two groups, namely condition states models and structural reliability models. Optimal maintenance strategy should be carried out based on both condition states and structural reliability of a bridge. However, none of existing deterioration models considers both condition states and structural reliability. This study thus proposes a Dynamic Objective Oriented Bayesian Network (DOOBN) based method to overcome the limitations of the existing methods. This methodology has the ability to act upon as a flexible unifying tool, which can integrate a variety of approaches and information for better bridge deterioration prediction. Two demonstrative case studies are conducted to preliminarily justify the feasibility of the methodology

Intrinsic kinetics of titania photocatalysis : simplified models for their investigation

Even though titanium dioxide photocatalysis has been promoted as a leading green technology for water purification, many issues have hindered its application on a large commercial scale. For the materials scientist the main issues have centred the synthesis of more efficient materials and the investigation of degradation mechanisms; whereas for the engineers the main issues have been the development of appropriate models and the evaluation of intrinsic kinetics parameters that allow the scale up or re-design of efficient large-scale photocatalytic reactors. In order to obtain intrinsic kinetics parameters the reaction must be analysed and modelled considering the influence of the radiation field, pollutant concentrations and fluid dynamics. In this way, the obtained kinetic parameters are independent of the reactor size and configuration and can be subsequently used for scale-up purposes or for the development of entirely new reactor designs. This work investigates the intrinsic kinetics of phenol degradation over titania film due to the practicality of a fixed film configuration over a slurry. A flat plate reactor was designed in order to be able to control reaction parameters that include the UV irradiance, flow rates, pollutant concentration and temperature. Particular attention was paid to the investigation of the radiation field over the reactive surface and to the issue of mass transfer limited reactions. The ability of different emission models to describe the radiation field was investigated and compared to actinometric measurements. The RAD-LSI model was found to give the best predictions over the conditions tested. Mass transfer issues often limit fixed film reactors. The influence of this phenomenon was investigated with specifically planned sets of benzoic acid experiments and with the adoption of the stagnant film model. The phenol mass transfer coefficient in the system was calculated to be km,phenol=8.5815x10-7Re0.65(ms-1). The data obtained from a wide range of experimental conditions, together with an appropriate model of the system, has enabled determination of intrinsic kinetic parameters. The experiments were performed in four different irradiation levels (70.7, 57.9, 37.1 and 20.4 W m-2) and combined with three different initial phenol concentrations (20, 40 and 80 ppm) to give a wide range of final pollutant conversions (from 22% to 85%). The simple model adopted was able to fit the wide range of conditions with only four kinetic parameters; two reaction rate constants (one for phenol and one for the family of intermediates) and their corresponding adsorption constants. The intrinsic kinetic parameters values were defined as kph = 0.5226 mmol m-1 s-1 W-1, kI = 0.120 mmol m-1 s-1 W-1, Kph = 8.5 x 10-4 m3 mmol-1 and KI = 2.2 x 10-3 m3 mmol-1. The flat plate reactor allowed the investigation of the reaction under two different light configurations; liquid and substrate side illumination. The latter of particular interest for real world applications where light absorption due to turbidity and pollutants contained in the water stream to be treated could represent a significant issue. The two light configurations allowed the investigation of the effects of film thickness and the determination of the catalyst optimal thickness. The experimental investigation confirmed the predictions of a porous medium model developed to investigate the influence of diffusion, advection and photocatalytic phenomena inside the porous titania film, with the optimal thickness value individuated at 5 ìm. The model used the intrinsic kinetic parameters obtained from the flat plate reactor to predict the influence of thickness and transport phenomena on the final observed phenol conversion without using any correction factor; the excellent match between predictions and experimental results provided further proof of the quality of the parameters obtained with the proposed method.

A common neighbour based two-way collaborative recommendation method

Traditional recommendation methods offer items, that are inanimate and one way recommendation, to users. Emerging new applications such as online dating or job recruitments require reciprocal people-to-people recommendations that are animate and two-way recommendations. In this paper, we propose a reciprocal collaborative method based on the concepts of users' similarities and common neighbors. The dataset employed for the experiment is gathered from a real life online dating network. The proposed method is compared with baseline methods that use traditional collaborative algorithms. Results show the proposed method can achieve noticeably better performance than the baseline methods.

From tiers to tables – enhancing student experience through collaborative learning spaces

BACKGROUND Collaborative and active learning have been clearly identified as ways students can engage in learning with each other and the academic staff. Traditional tier based lecture theatres and the didactic style they engender are not popular with students today as evidenced by the low attendance rates for lectures. Many universities are installing spaces designed with tables for group interaction with evolutions on spaces such as the TEAL (Technology Enabled Active Learning) (Massachusetts Institute of Technology, n.d.) and SCALE-UP (Student-Centred Activities for Large-Enrolment Undergraduate Programs) (North Carolina State University, n.d.) models. Technology advances in large screen computers and applications have also aided the move to these collaborative spaces. How well have universities structured learning using these spaces and how have students engaged with the content, technology, space and each other? This paper investigates the application of collaborative learning in such spaces for a cohort of 800+ first year engineers in the context of learning about and developing professional skills representative of engineering practice. PURPOSE To determine whether moving from tiers to tables enhances the student experience. Does utilising technology rich, activity based, collaborative learning spaces lead to positive experiences and active engagement of first year undergraduate engineering students? In developing learning methodology and approach in new learning spaces, what needs to change from a more traditional lecture and tutorial configuration? DESIGN/METHOD A post delivery review and analysis of outcomes was undertaken to determine how well students and tutors engaged with learning in new collaborative learning spaces. Data was gathered via focus group and survey of tutors, students survey and attendance observations. The authors considered the unit delivery approach along with observed and surveyed outcomes then conducted further review to produce the reported results. RESULTS Results indicate high participation in the collaborative sessions while the accompanying lectures were poorly attended. Students reported a high degree of satisfaction with the learning experience; however more investigation is required to determine the degree of improvement in retained learning outcomes. Survey feedback from tutors found that students engaged well in the activities during tutorials and there was an observed improvement in the quality of professional practice modelled by students during sessions. Student feedback confirmed the positive experiences in these collaborative learning spaces with 30% improvement in satisfaction ratings from previous years. CONCLUSIONS It is concluded that the right mix of space, technology and appropriate activities does engage students, improve participation and create a rich experience to facilitate potential for improved learning outcomes. The new Collaborative Teaching Spaces, together with integrated technology and tailored activities, has transformed the delivery of this unit and improved student satisfaction in tutorials significantly.

A simple test method for profiled steel cladding systems under wind uplift

A new simple test method using small scale models has been developed for testing profiled steel cladding systems under wind uplift/suction forces. This simple method should replace the large scale test method using two-span claddings used at present. It can be used for roof or wall cladding systems fastened with screw fasteners at crests or valleys.

Large arrays and networks of carbon nanotubes : morphology control by process parameters

Large arrays and networks of carbon nanotubes, both single- and multi-walled, feature many superior properties which offer excellent opportunities for various modern applications ranging from nanoelectronics, supercapacitors, photovoltaic cells, energy storage and conversation devices, to gas- and biosensors, nanomechanical and biomedical devices etc. At present, arrays and networks of carbon nanotubes are mainly fabricated from the pre-fabricated separated nanotubes by solution-based techniques. However, the intrinsic structure of the nanotubes (mainly, the level of the structural defects) which are required for the best performance in the nanotube-based applications, are often damaged during the array/network fabrication by surfactants, chemicals, and sonication involved in the process. As a result, the performance of the functional devices may be significantly degraded. In contrast, directly synthesized nanotube arrays/networks can preclude the adverse effects of the solution-based process and largely preserve the excellent properties of the pristine nanotubes. Owing to its advantages of scale-up production and precise positioning of the grown nanotubes, catalytic and catalyst-free chemical vapor depositions (CVD), as well as plasma-enhanced chemical vapor deposition (PECVD) are the methods most promising for the direct synthesis of the nanotubes.

Synthesis of biodegradable polymer-mesoporous silica composite microspheres for DNA prime-protein boost vaccination

DNA vaccines or proteins are capable of inducing specific immunity; however, the translation to the clinic has generally been problematic, primarily due to the reduced magnitude of immune response and poor pharmacokinetics. Herein we demonstrate a composite microsphere formulation, composed of mesoporous silica spheres (MPS) and poly(d,l-lactide-co-glycolide) (PLGA), enables the controlled delivery of a prime-boost vaccine via the encapsulation of plasmid DNA (pDNA) and protein in different compartments. Method with modified dual-concentric-feeding needles attached to a 40 kHz ultrasonic atomizer was studied. These needles focus the flow of two different solutions, which passed through the ultrasonic atomizer. The process synthesis parameters, which are important to the scale-up of composite microspheres, were also studied. These parameters include polymer concentration, feed flowrate, and volumetric ratio of polymer and pDNA-PEI/MPS-BSA. This fabrication technique produced composite microspheres with mean D[4,3] ranging from 6 to 34 μm, depending upon the microsphere preparation. The resultant physical morphology of composite microspheres was largely influenced by the volumetric ratio of pDNA-PEI/MPS-BSA to polymer, and this was due to the precipitation of MPS at the surface of the microspheres. The encapsulation efficiencies were predominantly in the range of 93-98% for pDNA and 46-68% for MPS. In the in vitro studies, the pDNA and protein showed different release kinetics in a 40 day time frame. The dual-concentric-feeding in ultrasonic atomization was shown to have excellent reproducibility. It was concluded that this fabrication technique is an effective method to prepare formulations containing a heterologous prime-boost vaccine in a single delivery system.

Process considerations related to the microencapsulation of plasmid DNA via ultrasonic atomization

An effective means of facilitating DNA vaccine delivery to antigen presenting cells is through biodegradable microspheres. Microspheres offer distinct advantages over other delivery technologies by providing release of DNA vaccine in its bioactive form in a controlled fashion. In this study, biodegradable poly(D,L-lactide-coglycolide) (PLGA) microspheres containing polyethylenimine (PEI) condensed plasmid DNA (pDNA) were prepared using a 40 kHz ultrasonic atomization system. Process synthesis parameters, which are important to the scale-up of microspheres that are suitable for nasal delivery (i.e., less than 20 μm), were studied. These parameters include polymer concentration; feed flowrate; volumetric ratio of polymer and pDNA-PEI (plasmid DNA-polyethylenimine) complexes; and nitrogen to phosphorous (N/P) ratio. PDNA encapsulation efficiencies were predominantly in the range 82-96%, and the mean sizes of the particle were between 6 and 15 μm. The ultrasonic synthesis method was shown to have excellent reproducibility. PEI affected morphology of the microspheres, as it induced the formation of porous particles that accelerate the release rate of pDNA. The PLGA microspheres displayed an in vitro release of pDNA of 95-99% within 30 days and demonstrated zero order release kinetics without an initial spike of pDNA. Agarose electrophoresis confirmed conservation of the supercoiled form of pDNA throughout the synthesis and in vitro release stages. It was concluded that ultrasonic atomization is an efficient technique to overcome the key obstacles in scaling-up the manufacture of encapsulated vaccine for clinical trials and ultimately, commercial applications.

Fish-scale bio-inspired multifunctional ZnO nanostructures

Scales provide optical disguise, low water drag and mechanical protection to fish, enabling them to survive catastrophic environmental disasters, predators and microorganisms. The unique structures and stacking sequences of fish scales inspired the fabrication of artificial nanostructures with salient optical, interfacial and mechanical properties. Herein, we describe fish-scale bio-inspired multifunctional ZnO nanostructures that have similar morphology and structure to the cycloid scales of the Asian Arowana. These nanostructured coatings feature tunable light refraction and reflection, modulated surface wettability and damage-tolerant mechanical properties. The salient properties of these multifunctional nanostructures are promising for applications in: - (i) optical coatings, sensing or lens arrays for use in reflective displays, packing, advertising and solar energy harvesting; - (ii) self-cleaning surfaces, including anti-smudge, anti-fouling and anti-fogging, and self-sterilizing surfaces, and; - (iii) mechanical/chemical barrier coatings. This study provides a low-cost and large-scale production method for the facile fabrication of these bio-inspired nanostructures and provides new insights for the development of novel functional materials for use in 'smart' structures and applications.

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.

Development and evaluation of hydrocarbon sorbent materials with the aid of chemometrics

Hydrocarbon spills on roads are a major safety concern for the driving public and can have severe cost impacts both on pavement maintenance and to the economy through disruption to services. The time taken to clean-up spills and re-open roads in a safe driving condition is an issue of increasing concern given traffic levels on major urban arterials. Thus, the primary aim of the research was to develop a sorbent material that facilitates rapid clean-up of road spills. The methodology involved extensive research into a range of materials (organic, inorganic and synthetic sorbents), comprehensive testing in the laboratory, scale-up and field, and product design (i.e. concept to prototype). The study also applied chemometrics to provide consistent, comparative methods of sorbent evaluation and performance. In addition, sorbent materials at every stage were compared against a commercial benchmark. For the first time, the impact of diesel on asphalt pavement has been quantified and assessed in a systematic way. Contrary to conventional thinking and anecdotal observations, the study determined that the action of diesel on asphalt was quite rapid (i.e. hours rather than weeks or months). This significant finding demonstrates the need to minimise the impact of hydrocarbon spills and the potential application of the sorbent option. To better understand the adsorption phenomenon, surface characterisation techniques were applied to selected sorbent materials (i.e. sand, organo-clay and cotton fibre). Brunauer Emmett Teller (BET) and thermal analysis indicated that the main adsorption mechanism for the sorbents occurred on the external surface of the material in the diffusion region (sand and organo-clay) and/or capillaries (cotton fibre). Using environmental scanning electron microscopy (ESEM), it was observed that adsorption by the interfibre capillaries contributed to the high uptake of hydrocarbons by the cotton fibre. Understanding the adsorption mechanism for these sorbents provided some guidance and scientific basis for the selection of materials. The study determined that non-woven cotton mats were ideal sorbent materials for clean-up of hydrocarbon spills. The prototype sorbent was found to perform significantly better than the commercial benchmark, displaying the following key properties: • superior hydrocarbon pick-up from the road pavement; • high hydrocarbon retention capacity under an applied load; • adequate field skid resistance post treatment; • functional and easy to use in the field (e.g. routine handling, transportation, application and recovery); • relatively inexpensive to produce due to the use of raw cotton fibre and simple production process; • environmentally friendly (e.g. renewable materials, non-toxic to environment and operators, and biodegradable); and • rapid response time (e.g. two minutes total clean-up time compared with thirty minutes for reference sorbents). The major outcomes of the research project include: a) development of a specifically designed sorbent material suitable for cleaning up hydrocarbon spills on roads; b) submission of patent application (serial number AU2005905850) for the prototype product; and c) preparation of Commercialisation Strategy to advance the sorbent product to the next phase (i.e. R&D to product commercialisation).