Investigation of highly regulated promoters for the production of recombinant proteins in plants

Plants have been identified as promising expression systems for the commercial production of recombinant proteins. Plant-based protein production or “biofarming” offers a number of advantages over traditional expression systems in terms of scale of production, the capacity for post-translation processing, providing a product free of contaminants and cost effectiveness. A number of pharmaceutically important and commercially valuable proteins, such as antibodies, biopharmaceuticals and industrial enzymes are currently being produced in plant expression systems. However, several challenges still remain to improve recombinant protein yield with no ill effect on the host plant. The ability for transgenic plants to produce foreign proteins at commercially viable levels can be directly related to the level and cell specificity of the selected promoter driving the transgene. The accumulation of recombinant proteins may be controlled by a tissue-specific, developmentally-regulated or chemically-inducible promoter such that expression of recombinant proteins can be spatially- or temporally- controlled. The strict control of gene expression is particularly useful for proteins that are considered toxic and whose expression is likely to have a detrimental effect on plant growth. To date, the most commonly used promoter in plant biotechnology is the cauliflower mosaic virus (CaMV) 35S promoter which is used to drive strong, constitutive transgene expression in most organs of transgenic plants. Of particular interest to researchers in the Centre for Tropical Crops and Biocommodities at QUT are tissue-specific promoters for the accumulation of foreign proteins in the roots, seeds and fruit of various plant species, including tobacco, banana and sugarcane. Therefore this Masters project aimed to isolate and characterise root- and seed-specific promoters for the control of genes encoding recombinant proteins in plant-based expression systems. Additionally, the effects of matching cognate terminators with their respective gene promoters were assessed. The Arabidopsis root promoters ARSK1 and EIR1 were selected from the literature based on their reported limited root expression profiles. Both promoters were analysed using the PlantCARE database to identify putative motifs or cis-acting elements that may be associated with this activity. A number of motifs were identified in the ARSK1 promoter region including, WUN (wound-inducible), MBS (MYB binding site), Skn-1, and a RY core element (seed-specific) and in the EIR1 promoter region including, Skn-1 (seed-specific), Box-W1 (fungal elicitor), Aux-RR core (auxin response) and ABRE (ABA response). However, no previously reported root-specific cis-acting elements were observed in either promoter region. To confirm root specificity, both promoters, and truncated versions, were fused to the GUS reporter gene and the expression cassette introduced into Arabidopsis via Agrobacterium-mediated transformation. Despite the reported tissue-specific nature of these promoters, both upstream regulatory regions directed constitutive GUS expression in all transgenic plants. Further, similar levels of GUS expression from the ARSK1 promoter were directed by the control CaMV 35S promoter. The truncated version of the EIR1 promoter (1.2 Kb) showed some differences in the level of GUS expression compared to the 2.2 Kb promoter. Therefore, this suggests an enhancer element is contained in the 2.2 Kb upstream region that increases transgene expression. The Arabidopsis seed-specific genes ATS1 and ATS3 were selected from the literature based on their seed-specific expression profiles and gene expression confirmed in this study as seed-specific by RT-PCR analysis. The selected promoter regions were analysed using the PlantCARE database in order to identify any putative cis elements. The seed-specific motifs GCN4 and Skn-1 were identified in both promoter regions that are associated with elevated expression levels in the endosperm. Additionaly, the seed-specific RY element and the ABRE were located in the ATS1 promoter. Both promoters were fused to the GUS reporter gene and used to transform Arabidopsis plants. GUS expression from the putative promoters was consitutive in all transgenic Arabidopsis tissue tested. Importantly, the positive control FAE1 seed-specific promoter also directed constitutive GUS expression throughout transgenic Arabidopsis plants. The constitutive nature seen in all of the promoters used in this study was not anticipated. While variations in promoter activity can be caused by a number of influencing factors, the variation in promoter activity observed here would imply a major contributing factor common to all plant expression cassettes tested. All promoter constructs generated in this study were based on the binary vector pCAMBIA2300. This vector contains the plant selection gene (NPTII) under the transcriptional control of the duplicated CaMV 35S promoter. This CaMV 35S promoter contains two enhancer domains that confer strong, constitutive expression of the selection gene and is located immediately upstream of the promoter-GUS fusion. During the course of this project, Yoo et al. (2005) reported that transgene expression is significantly affected when the expression cassette is located on the same T-DNA as the 35S enhancer. It was concluded, the trans-acting effects of the enhancer activate and control transgene expression causing irregular expression patterns. This phenomenon seems the most plausible reason for the constitutive expression profiles observed with the root- and seed-specific promoters assessed in this study. The expression from some promoters can be influenced by their cognate terminator sequences. Therefore, the Arabidopsis ARSK1, EIR1, ATS1 and ATS3 terminator sequences were isolated and incorporated into expression cassettes containing the GUS reporter gene under the control of their cognate promoters. Again, unrestricted GUS activity was displayed throughout transgenic plants transformed with these reporter gene fusions. As previously discussed constitutive GUS expression was most likely due to the trans-acting effect of the upstream CaMV 35S promoter in the selection cassette located on the same T-DNA. The results obtained in this study make it impossible to assess the influence matching terminators with their cognate promoters have on transgene expression profiles. The obvious future direction of research continuing from this study would be to transform pBIN-based promoter-GUS fusions (ie. constructs containing no CaMV 35S promoter driving the plant selection gene) into Arabidopsis in order to determine the true tissue specificity of these promoters and evaluate the effects of their cognate 3’ terminator sequences. Further, promoter truncations based around the cis-elements identified here may assist in determining whether these motifs are in fact involved in the overall activity of the promoter.

Inventing future schools

A group of Australian researchers and designers have been working on ways to imagine, demonstrate and accelerate the use of ICT that extend learning relationships and environments to include the classroom, home and local community. These learning projects aim to transform how students identify and interact with learning, subject areas, teachers, other students, family, organisations and more broadly how learning tools can create connections that permeate students' life worlds now and in the future.---------- It is our intention that such demonstrators must - Be simple, flexible, scalable and adaptive - Result in increased confidence in the use of ICT for both students and teachers - Offer opportunities for personalized learning - Promote new and effective learning partnerships between students, teachers and families. - Extend the learning experience to include other environments both local and virtual. - Inspire further innovation - Provide solutions to current limitations---------- Presenting Innovation in Practice - Innovative ICT projects currently being used by students in schools, at home and in the community - Stories of use from teacher, student, and other stakeholder perspectives - Lessons learnt so far: a design perspective - Surprising and inspiring opportunities

Bioactive inorganic materials/alginate composite microspheres with controllable drug-delivery ability

Alginate microspheres are considered a promising material as a drug carrier in bone repair due to excellent biocompatibility, but their main disadvantage is low drug entrapment efficiency and non-controllable release. The aim of this study was to investigate the effect of incorporating mesoporous bioglass (MBG), non-mesoporous bioglass (BG) or hydroxyapatite (HAp) into alginate microspheres on their drug-loading and release properties. X-ray diffraction (XRD), transmission electron microscopy (TEM), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and atomic emission spectroscopy (AES) were used to analyse the composition, structure and dissolution of bioactive inorganic materials and their microspheres. Dexamethasone (DEX)-loading and release ability of four microspheres were tested in phosphate buffered saline with varying pHs. Results showed that the drug-loading capacity was enhanced with the incorporation of bioactive inorganic materials into alginate microspheres. The MBG/Alginate microspheres had the highest drug loading ability. DEX release from alginate microspheres correlated to the dissolution of MBG, BG and HAp in PBS, and that the pH was an efficient factor in controlling the DEX release; a high pH resulted in greater DEX release, whereas a low pH delayed DEX release. In addition, MBG/alginate, BG/alginate and HAp/alginate microspheres had varying apatite-formation and dissolution abilities, which indicate that the composites would behave differently with respect to bioactivity. The study suggests that microspheres made of a composite of bioactive inorganic materials and alginate have a bioactivity and degradation profile which greatly improves their drug delivery capacity, thus enhancing their potential applications as bioactive filler materials for bone tissue regeneration.

A multicast routing scheme for efficient safety message dissemination in VANET

Vehicular ad hoc network (VANET) is a wireless ad hoc network that operates in a vehicular environment to provide communication between vehicles. VANET can be used by a diverse range of applications to improve road safety. Cooperative collision warning system (CCWS) is one of the safety applications that can provide situational awareness and warning to drivers by exchanging safety messages between cooperative vehicles. Currently, the routing strategies for safety message dissemination in CCWS are scoped broadcast. However, the broadcast schemes are not efficient as a warning message is sent to a large number of vehicles in the area, rather than only the endangered vehicles. They also cannot prioritize the receivers based on their critical time to avoid collision. This paper presents a more efficient multicast routing scheme that can reduce unnecessary transmissions and also use adaptive transmission range. The multicast scheme involves methods to identify an abnormal vehicle, the vehicles that may be endangered by the abnormal vehicle, and the latest time for each endangered vehicle to receive the warning message in order to avoid the danger. We transform this multicast routing problem into a delay-constrained minimum Steiner tree problem. Therefore, we can use existing algorithms to solve the problem. The advantages of our multicast routing scheme are mainly its potential to support various road traffic scenarios, to optimize the wireless channel utilization, and to prioritize the receivers.

Delamination of kaolinite - potassium acetate intercalates by ball-milling

Structural changes in intercalated kaolinite after wet ball-milling were examined by scanning electron microscopy (SEM), X-ray diffraction (XRD), specific surface area (SSA) and Fourier Transform Infrared spectroscopy (FTIR). The X-ray diffraction pattern at room temperature indicated that the intercalation of potassium acetate into kaolinite causes an increase of the basal spacing from 0.718 to 1.42 nm, and with the particle size reduction, the surface area increased sharply with the intercalation and delamination by ball-milling. The wet ball-milling kaolinite after intercalation did not change the structural order, and the particulates have high aspect ratio according SEM images.

Lateral distortional buckling design rules for back to back LiteSteel beams

A new cold-formed steel beam, known as the LiteSteel Beam (LSB), has the potential to transform the low-rise building industry. The new beam is effectively a channel section with two rectangular hollow flanges and a slender web, and is manufactured using a simultaneous cold-forming and electric resistance welding process. Research into the flexural behaviour of single LSB members showed that the LSBs are susceptible to lateral distortional buckling effects and their moment capacities are significantly reduced for intermediate spans. Build-up LSB sections are expected to improve their flexural capacity and to enhance their applications. They are also likely to mitigate the detrimental effects of lateral distortional buckling observed with single LSB members of intermediate spans. However, the behaviour of build up beams is not well understood. Currently available design rules were found to be inadequate to predict the member moment capacities of back to back LSBs. Therefore a research project based on both experimental and numerical studies was undertaken to investigate the flexural behaviour of back to back LSBs with various longitudinal connection spacings under a uniform moment. New design rules were developed using the moment capacity data obtained using finite element analyses and experimental tests. This paper presents the details of the development of design rules for the back to back LSB sections.

An application of near-infrared and mid-infrared spectroscopy to the study of natural halotrichites : halotrichite, apjohnite and wupatkiite

Near-infrared (NIR) and Fourier transform infrared (FTIR) spectroscopy have been used to determine the mineralogical character of isomorphic substitutions for Mg2+ by divalent transition metals Fe, Mn, Co and Ni in natural halotrichite series. The minerals are characterised by d-d transitions in NIR region 12000-7500 cm-1. NIR spectrum of halotrichite reveals broad feature from 12000 to 7500 cm-1 with a splitting of two bands resulting from ferrous ion transition 5T2g ® 5Eg. The presence of overtones of OH- fundamentals near 7000 cm-1 confirms molecular water in the mineral structure of the halotrichite series. The appearance of the most intense peak at around 5132 cm-1 is a common feature in the three minerals and is derived from combination of OH- vibrations of water molecules and 2 water bending modes. The influence of cations like Mg2+, Fe2+, Mn2+, Co2+, Ni2+ shows on the spectra of halotrichites. Especially wupatkiite-OH stretching vibrations in which bands are distorted conspicuously to low wave numbers at 3270, 2904 and 2454 cm-1. The observation of high frequency 2 mode in the infrared spectrum at 1640 cm-1 indicates coordination of water molecules is strongly hydrogen bonded in natural halotrichites. The splittings of bands in 3 and 4 (SO4)2- stretching regions may be attributed to the reduction of symmetry from Td to C2v for sulphate ion. This work has shown the usefulness of NIR spectroscopy for the rapid identification and classification of the halotrichite minerals.

Wide-angle visual feature matching for outdoor localization

Wide-angle images exhibit significant distortion for which existing scale-space detectors such as the scale-invariant feature transform (SIFT) are inappropriate. The required scale-space images for feature detection are correctly obtained through the convolution of the image, mapped to the sphere, with the spherical Gaussian. A new visual key-point detector, based on this principle, is developed and several computational approaches to the convolution are investigated in both the spatial and frequency domain. In particular, a close approximation is developed that has comparable computation time to conventional SIFT but with improved matching performance. Results are presented for monocular wide-angle outdoor image sequences obtained using fisheye and equiangular catadioptric cameras. We evaluate the overall matching performance (recall versus 1-precision) of these methods compared to conventional SIFT. We also demonstrate the use of the technique for variable frame-rate visual odometry and its application to place recognition.

A simple and systematic approach to assigning Denavit-Hartenberg Parameters

This paper presents a simple and intuitive approach to determining the kinematic parameters of a serial-link robot in Denavit– Hartenberg (DH) notation. Once a manipulator’s kinematics is parameterized in this form, a large body of standard algorithms and code implementations for kinematics, dynamics, motion planning, and simulation are available. The proposed method has two parts. The first is the “walk through,” a simple procedure that creates a string of elementary translations and rotations, from the user-defined base coordinate to the end-effector. The second step is an algebraic procedure to manipulate this string into a form that can be factorized as link transforms, which can be represented in standard or modified DH notation. The method allows for an arbitrary base and end-effector coordinate system as well as an arbitrary zero joint angle pose. The algebraic procedure is amenable to computer algebra manipulation and a Java program is available as supplementary downloadable material.

Optimising figure of merit for phonetic spoken term detection

This paper introduces a novel technique to directly optimise the Figure of Merit (FOM) for phonetic spoken term detection. The FOM is a popular measure of sTD accuracy, making it an ideal candiate for use as an objective function. A simple linear model is introduced to transform the phone log-posterior probabilities output by a phe classifier to produce enhanced log-posterior features that are more suitable for the STD task. Direct optimisation of the FOM is then performed by training the parameters of this model using a non-linear gradient descent algorithm. Substantial FOM improvements of 11% relative are achieved on held-out evaluation data, demonstrating the generalisability of the approach.

Robust image hashing using higher order spectral features

Robust image hashing seeks to transform a given input image into a shorter hashed version using a key-dependent non-invertible transform. These image hashes can be used for watermarking, image integrity authentication or image indexing for fast retrieval. This paper introduces a new method of generating image hashes based on extracting Higher Order Spectral features from the Radon projection of an input image. The feature extraction process is non-invertible, non-linear and different hashes can be produced from the same image through the use of random permutations of the input. We show that the transform is robust to typical image transformations such as JPEG compression, noise, scaling, rotation, smoothing and cropping. We evaluate our system using a verification-style framework based on calculating false match, false non-match likelihoods using the publicly available Uncompressed Colour Image database (UCID) of 1320 images. We also compare our results to Swaminathan’s Fourier-Mellin based hashing method with at least 1% EER improvement under noise, scaling and sharpening.

Introduction and Epilogue to Special Issue: Re-conceiving the artful in management development and education

The editorial focus of this issue is on artful, aesthetic and artistic endeavours in management. Being artful is not about arts-based quick fixes. In the context of this Special Issue, to be artful is to transform self through profound learning experiences that expand human consciousness, often facilitated by artistic processes. In management education and development this suggests a shift from instrumental management towards a paradigm of artful creation. Why the arts and artfulness? And why now? In what ways can the arts inform, inspire and leverage management development and education?

Novel analytical and numerical methods for solving fractional dynamical systems

During the past three decades, the subject of fractional calculus (that is, calculus of integrals and derivatives of arbitrary order) has gained considerable popularity and importance, mainly due to its demonstrated applications in numerous diverse and widespread fields in science and engineering. For example, fractional calculus has been successfully applied to problems in system biology, physics, chemistry and biochemistry, hydrology, medicine, and finance. In many cases these new fractional-order models are more adequate than the previously used integer-order models, because fractional derivatives and integrals enable the description of the memory and hereditary properties inherent in various materials and processes that are governed by anomalous diffusion. Hence, there is a growing need to find the solution behaviour of these fractional differential equations. However, the analytic solutions of most fractional differential equations generally cannot be obtained. As a consequence, approximate and numerical techniques are playing an important role in identifying the solution behaviour of such fractional equations and exploring their applications. The main objective of this thesis is to develop new effective numerical methods and supporting analysis, based on the finite difference and finite element methods, for solving time, space and time-space fractional dynamical systems involving fractional derivatives in one and two spatial dimensions. A series of five published papers and one manuscript in preparation will be presented on the solution of the space fractional diffusion equation, space fractional advectiondispersion equation, time and space fractional diffusion equation, time and space fractional Fokker-Planck equation with a linear or non-linear source term, and fractional cable equation involving two time fractional derivatives, respectively. One important contribution of this thesis is the demonstration of how to choose different approximation techniques for different fractional derivatives. Special attention has been paid to the Riesz space fractional derivative, due to its important application in the field of groundwater flow, system biology and finance. We present three numerical methods to approximate the Riesz space fractional derivative, namely the L1/ L2-approximation method, the standard/shifted Gr¨unwald method, and the matrix transform method (MTM). The first two methods are based on the finite difference method, while the MTM allows discretisation in space using either the finite difference or finite element methods. Furthermore, we prove the equivalence of the Riesz fractional derivative and the fractional Laplacian operator under homogeneous Dirichlet boundary conditions – a result that had not previously been established. This result justifies the aforementioned use of the MTM to approximate the Riesz fractional derivative. After spatial discretisation, the time-space fractional partial differential equation is transformed into a system of fractional-in-time differential equations. We then investigate numerical methods to handle time fractional derivatives, be they Caputo type or Riemann-Liouville type. This leads to new methods utilising either finite difference strategies or the Laplace transform method for advancing the solution in time. The stability and convergence of our proposed numerical methods are also investigated. Numerical experiments are carried out in support of our theoretical analysis. We also emphasise that the numerical methods we develop are applicable for many other types of fractional partial differential equations.