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.

Men's intimate partner abuse and control : reconciling paradoxical masculinities and social contradictions

Intimate partner abuse and control is one of the most common forms of violence against women, and is considered an international problem of social, political, legal and human rights significance. Yet few studies have attempted to understand this problem from the perspective of male perpetrators. This gap is addressed by conducting in-depth interviews with 16 able-bodied men of white European ancestry born and educated in New Zealand or Australia, who have been physically violent and/or emotionally, intellectually, sexually or financially controlling of a live-in female partner. This thesis extends and deepens the dominant ways of thinking about men’s intimate partner abuse by utilising a new theoretical framework compatible with contemporary feminist scholarship. A synthesis of Connell’s theory of masculinities and Bourdieu’s field theory is utilised for the purpose of exploring more nuanced, complex understandings of manliness and men’s relationships with men, women and social structures. Through such an analysis, this thesis finds that men’s perpetration of power and control over women is driven by a need to avoid the stigma of appearing weak. As a consequence, their desire and ability to show love, care and empathy is suppressed in favour of a presumed honourable manliness, and their female partners are used as weapons in the pursuit of symbolic capital in the form of recognition, prestige and acceptance from real and/or imagined men. This research also uncovers the complex interplay between masculine practices and particular social contexts. For example, the norms of practice encountered from those in authority, such as teachers, sports coaches, police, court judges and workplace management, influences the decision making of the men in this study, to use, or not to use, physical violence, psychological abuse and structural control. The principal conclusion is that there is a repertoire of paradoxical masculinities and contradictory social messages available to the men in this study. But gender policing by other men, complicit women and those in authority provides little room for legitimate complexity in masculine practices. Perpetrators in this study reconcile these conflicts of interest by generally avoiding subordinated masculinity and possible ostracism, and instead practicing more heroic hegemonic masculinities by abusing and controlling women and particular other men. This thesis concludes that for intimate partner abuse and control to cease, changes in power structures have to occur at all levels of society.

Adaptive dual control of topic-based information retrieval

Information Retrieval is an important albeit imperfect component of information technologies. A problem of insufficient diversity of retrieved documents is one of the primary issues studied in this research. This study shows that this problem leads to a decrease of precision and recall, traditional measures of information retrieval effectiveness. This thesis presents an adaptive IR system based on the theory of adaptive dual control. The aim of the approach is the optimization of retrieval precision after all feedback has been issued. This is done by increasing the diversity of retrieved documents. This study shows that the value of recall reflects this diversity. The Probability Ranking Principle is viewed in the literature as the “bedrock” of current probabilistic Information Retrieval theory. Neither the proposed approach nor other methods of diversification of retrieved documents from the literature conform to this principle. This study shows by counterexample that the Probability Ranking Principle does not in general lead to optimal precision in a search session with feedback (for which it may not have been designed but is actively used). Retrieval precision of the search session should be optimized with a multistage stochastic programming model to accomplish the aim. However, such models are computationally intractable. Therefore, approximate linear multistage stochastic programming models are derived in this study, where the multistage improvement of the probability distribution is modelled using the proposed feedback correctness method. The proposed optimization models are based on several assumptions, starting with the assumption that Information Retrieval is conducted in units of topics. The use of clusters is the primary reasons why a new method of probability estimation is proposed. The adaptive dual control of topic-based IR system was evaluated in a series of experiments conducted on the Reuters, Wikipedia and TREC collections of documents. The Wikipedia experiment revealed that the dual control feedback mechanism improves precision and S-recall when all the underlying assumptions are satisfied. In the TREC experiment, this feedback mechanism was compared to a state-of-the-art adaptive IR system based on BM-25 term weighting and the Rocchio relevance feedback algorithm. The baseline system exhibited better effectiveness than the cluster-based optimization model of ADTIR. The main reason for this was insufficient quality of the generated clusters in the TREC collection that violated the underlying assumption.

Latent variable models in statistical genetics

Understanding the complexities that are involved in the genetics of multifactorial diseases is still a monumental task. In addition to environmental factors that can influence the risk of disease, there is also a number of other complicating factors. Genetic variants associated with age of disease onset may be different from those variants associated with overall risk of disease, and variants may be located in positions that are not consistent with the traditional protein coding genetic paradigm. Latent Variable Models are well suited for the analysis of genetic data. A latent variable is one that we do not directly observe, but which is believed to exist or is included for computational or analytic convenience in a model. This thesis presents a mixture of methodological developments utilising latent variables, and results from case studies in genetic epidemiology and comparative genomics. Epidemiological studies have identified a number of environmental risk factors for appendicitis, but the disease aetiology of this oft thought useless vestige remains largely a mystery. The effects of smoking on other gastrointestinal disorders are well documented, and in light of this, the thesis investigates the association between smoking and appendicitis through the use of latent variables. By utilising data from a large Australian twin study questionnaire as both cohort and case-control, evidence is found for the association between tobacco smoking and appendicitis. Twin and family studies have also found evidence for the role of heredity in the risk of appendicitis. Results from previous studies are extended here to estimate the heritability of age-at-onset and account for the eect of smoking. This thesis presents a novel approach for performing a genome-wide variance components linkage analysis on transformed residuals from a Cox regression. This method finds evidence for a dierent subset of genes responsible for variation in age at onset than those associated with overall risk of appendicitis. Motivated by increasing evidence of functional activity in regions of the genome once thought of as evolutionary graveyards, this thesis develops a generalisation to the Bayesian multiple changepoint model on aligned DNA sequences for more than two species. This sensitive technique is applied to evaluating the distributions of evolutionary rates, with the finding that they are much more complex than previously apparent. We show strong evidence for at least 9 well-resolved evolutionary rate classes in an alignment of four Drosophila species and at least 7 classes in an alignment of four mammals, including human. A pattern of enrichment and depletion of genic regions in the profiled segments suggests they are functionally significant, and most likely consist of various functional classes. Furthermore, a method of incorporating alignment characteristics representative of function such as GC content and type of mutation into the segmentation model is developed within this thesis. Evidence of fine-structured segmental variation is presented.

Associations among body size dissatisfaction, perceived dietary control, and diet history in African American and European American women

European American (EA) women report greater body dissatisfaction and less dietary control than do African American (AA) women. This study investigated whether ethnic differences in dieting history contributed to differences in body dissatisfaction and dietary control, or to differential changes that may occur during weight loss and regain. Eighty-nine EA and AA women underwent dual-energy X-ray absorptiometry to measure body composition and completed questionnaires to assess body dissatisfaction and dietary control before, after, and one year following, a controlled weight-loss intervention. While EA women reported a more extensive dieting history than AA women, this difference did not contribute to ethnic differences in body dissatisfaction and perceived dietary control. During weight loss, body satisfaction improved more for AA women, and during weight regain, dietary self-efficacy worsened to a greater degree for EA women. Ethnic differences in dieting history did not contribute significantly to these differential changes. Although ethnic differences in body image and dietary control are evident prior to weight loss, and some change differentially by ethnic group during weight loss and regain, differences in dieting history do not contribute significantly to ethnic differences in body image and dietary control.

On the use of different spatial reference frames for movement control

Previous work has shown that amplitude and direction are two independently controlled parameters of aimed arm movements, and performance, therefore, suffers when they must be decomposed into Cartesian coordinates. We now compare decomposition into different coordinate systems. Subjects pointed at visual targets in 2-D with a cursor, using a two-axis joystick or two single-axis joysticks. In the latter case, joystick axes were aligned with the subjects’ body axes, were rotated by –45°, or were oblique (i.e., one axis was in an egocentric frame and the other was rotated by –45°). Cursor direction always corresponded to joystick direction. We found that compared with the two-axis joystick, responses with single-axis joysticks were slower and less accurate when the axes were oriented egocentrically; the deficit was even more pronounced when the axes were rotated and was most pronounced when they were oblique. This confirms that decomposition of motor commands is computationally demanding and documents that this demand is lowest for egocentric, higher for rotated, and highest for oblique coordinates. We conclude that most current vehicles use computationally demanding man–machine interfaces.

Empirical Evidence Showing The Relationships Between Three Approaches for Pollution Control

Willingness to pay models have shown the theoretical relationships between the contingent valuation, cost of illness and the avertive behaviour approaches. In this paper, field survey data are used to compare the relationships between these three approaches and to demonstrate that contingent valuation bids exceed the sum of cost of illness and the avertive behaviour approach estimates. The estimates provide a validity check for CV bids and further support the claim that contingent valuation studies are theoretically consistent.

Automated control of a dragline using machine vision

The research described in this paper is directed toward increasing productivity of draglines through automation. In particular, it focuses on the swing-to-dump, dump, and return-to-dig phases of the dragline operational cycle by developing a swing automation system. In typical operation the dragline boom can be in motion for up to 80% of the total cycle time. This provides considerable scope for improving cycle time through automated or partially automated boom motion control. This paper describes machine vision based sensor technology and control algorithms under development to solve the problem of continuous real time bucket location and control. Incorporation of this capability into existing dragline control systems will then enable true automation of dragline swing and dump operations.

Real-time vision, tracking and control

This paper, which serves as an introduction to the mini-symposium on Real-Time Vision, Tracking and Control, provides a broad sketch of visual servoing, the application of real-time vision, tracking and control for robot guidance. It outlines the basic theoretical approaches to the problem, describes a typical architecture, and discusses major milestones, applications and the significant vision sub-problems that must be solved.

Choice of image features for depth-axis control in image based visual servo control

This paper is concerned with choosing image features for image based visual servo control and how this choice influences the closed-loop dynamics of the system. In prior work, image features tend to be chosen on the basis of image processing simplicity and noise sensitivity. In this paper we show that the choice of feature directly influences the closed-loop dynamics in task-space. We focus on the depth axis control of a visual servo system and compare analytically various approaches that have been reported recently in the literature. The theoretical predictions are verified by experiment.

Adaptive depth estimation in image based visual servo control of dynamic systems

This paper considers the question of designing a fully image based visual servo control for a dynamic system. The work is motivated by the ongoing development of image based visual servo control of small aerial robotic vehicles. The observed targets considered are coloured blobs on a flat surface to which the normal direction is known. The theoretical framework is directly applicable to the case of markings on a horizontal floor or landing field. The image features used are a first order spherical moment for position and an image flow measurement for velocity. A fully non-linear adaptive control design is provided that ensures global stability of the closed-loop system. © 2005 IEEE.

Autonomous control of underground mining vehicles using reactive navigation

Describes how many of the navigation techniques developed by the robotics research community over the last decade may be applied to a class of underground mining vehicles (LHDs and haul trucks). We review the current state-of-the-art in this area and conclude that there are essentially two basic methods of navigation applicable. We describe an implementation of a reactive navigation system on a 30 tonne LHD which has achieved full-speed operation at a production mine.

Guided chaos : path planning and control for a UAV-forced landing

In recent years, unmanned aerial vehicles (UAVs) have been widely used in combat, and their potential applications in civil and commercial roles are also receiving considerable attention by industry and the research community. There are numerous published reports of UAVs used in Earth science missions [1], fire-fighting [2], and border security [3] trials, with other speculative deployments, including applications in agriculture, communications, and traffic monitoring. However, none of these UAVs can demonstrate an equivalent level of safety to manned aircraft, particularly in the case of an engine failure, which would require an emergency or forced landing. This may be arguably the main factor that has prevented these UAV trials from becoming full-scale commercial operations, as well as restricted operations of civilian UAVs to only within segregated airspace.

Improved joint control using a genetic algorithm for a humanoid robot

This paper describes experiments conducted in order to simultaneously tune 15 joints of a humanoid robot. Two Genetic Algorithm (GA) based tuning methods were developed and compared against a hand-tuned solution. The system was tuned in order to minimise tracking error while at the same time achieve smooth joint motion. Joint smoothness is crucial for the accurate calculation of online ZMP estimation, a prerequisite for a closedloop dynamically stable humanoid walking gait. Results in both simulation and on a real robot are presented, demonstrating the superior smoothness performance of the GA based methods.