School of Agriculture, Food and Ecosystem Sciences - Theses
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ItemPlant-based expression of recombinant Lol p 1 protein( 2005)Type 1 allergic diseases such as allergic rhinitis, hay fever, conjunctivitis, allergic asthma and allergic dermatitis, represent a major health problem in industrialized countries, affecting more than 25% of the world's population. The symptoms of allergic diseases can be controlled by pharmacotherapy and the pharmaceutical industry has focused on the treatment of the symptoms rather than addressing underlying causative factors. Specific immunotherapy introduced in 1911, is the only causative approach to type 1 allergy. However, this form of immunotherapy is associated with the risks of the development of a potentially life-threatening systemic allergic response, known as anaphylaxis. In recent year new approaches to allergy immunotherapy including the use of recombinant pollen allergens, have been proposed and are being evaluated to offer an effective alternative. Expression of allergen in a bacterial system such as E. coli is easy to handle, cost effective and can produce a high yield of recombinant protein. However, over production of foreign proteins in E. coli often results in protein mis-folding and segregation into insoluble aggregates as inclusion bodies, which can lead to production of biologically inactive proteins. On the other hand, expression in plants offers a safe and cost effective system without the potential of contamination from infectious pathogens. The study described in this thesis focuses on the expression of a ryegrass pollen allergen, Lol p 1 in plants. Lol p 1 is a group 1 allergen with molecular weights ranging from 28 to 35 KDa. Tobacco, broccoli (Brassica oleracea var. italica), and rice were used with the aim to study heterologous expression system for Lol p 1. Analysis of soluble protein from tobacco leaves indicated Lol p I expression in the range of 1.30 - 4.49 ng/?g protein. Use of plastid target signal increased Lol p 1 expression to 5.91 - 9.25 ng/?g protein. Rice seed specific promoter, GIuB-1 was used to direct the expression of Lol p 1 into tobacco seeds. Analysis of seeds from transgenic tobacco indicated Lol p 1 expression in a range of 0.17 - 1.60 ng/?g protein. Furthermore, Lol p 1 expressed in seeds was found to be stable after storage for a period of 3 months when tested at 4C, and 25C. Development of efficient methods for regeneration of viable shoots from cultured tissue is vital for successful plant genetic engineering. For this purpose, regeneration potentials from different types of seedling explants from broccoli (Brassica oleracea var. italica) were investigated. Regeneration efficiencies for large cotyledons, small cotyledons, petioles, upper hypocotyls, lower hypocotyls, roots were 100%, 100%, 42.22%, 95.55%, 88.88%, and 88.88% respectively. The average shoot number per explant in large cotyledons, small cotyledons, petiole, upper hypocotyls, lower hypocotyls and roots were 8.36, 6.07, 1.11, 5.32, 4.22, and 2.87 respectively. Successful expression of Lol p 1 in Brassicas oleracea var. italica was obtained using Agrobacterium-mediated transformation. Analysis of soluble protein from broccoli florets indicated Lol p 1 expression in a range of 0.34 - 1.24 ng/gg proteins. Lol p 1 was also successfully expressed in rice using Agrobacterium-mediated transformation. Rice seed specific promoter, GluB-1 was used to drive Lol p 1 expression. Analysis of seeds from transgenic rice indicated Lol p 1 protein expression in a range of 13.34 - 60.92 ng/?g proteins. Furthermore, recombinant Lol p 1 in transgenic rice grain was found to be stable for a period of 3 months tested at 4C, 25, and 37C. In summary, the present study provides a first data on successful recombinant Lol p 1 expression in plants potentially offering an alternative system of recombinant allergen production for diagnostics and other uses.
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ItemBiotechnology of grass pollen allergens for improved hay fever immunotherapy( 2002)This thesis describes the application of biotechnology to the generation of improved immunotherapeutic reagents, and novel methods for the delivery of such reagents, for the treatment of grass pollen allergy. Using site-directed mutagenesis, nine variants of the major rye grass pollen allergen, Lol p 5, were developed and tested for reduced IgE-reactivity. The results show that alteration of as few as one amino acid residue can reduce the human IgE-reactivity of the rLol p 5 protein. Furthermore, variant 9, which possessed four internal amino acid substitutions and was five amino acid residues shorter than rLol p 5 at the carboxyl-terminal, showed reductions in IgE-reactivity, the ability to induce histamine release from basophils and immediate skin reactivity. This protein also had similar secondary structural properties to rLol p 5 and retained the ability to stimulate T cell-proliferation. Low IgE-binding variants of the group 1 allergen from rye grass pollen were also generated. Site-directed mutagenesis of rLol p 1 showed that replacement of the cysteine residue at amino acid position 77 could significantly reduce the IgE-reactivity of this protein. However, the greatest reduction in IgE-reactivity of rLol p 1 was effected by the generation of truncation variants lacking internal and carboxyl-terminal regions which represented mapped linear IgE epitopes of LoI p 1. Hypoallergenic forms of the two major rye grass pollen allergens may aid the development of safer reagents for the treatment of grass pollen allergy. The mucosal administration of immunotherapeutic reagents for the treatment of human diseases has been shown to offer numerous immunological advantages and pose fewer risks than parenteral immunization. To demonstrate the applicability of mucosal immunization for the delivery of immunotherapeutic reagents for the treatment of pollen allergy, rLol p 5 and its hypoallergenic variant were orally administered to naive mice. Both proteins were shown to induce systemic immune responses when delivered directly into the stomach. However, the hypoallergenic variant was shown to induce a reduced systemic IgE response but a similar total IgG response in mice, as compared to mice administered rLol p 5. Both proteins also induced the production of systemic antibodies that could block an interaction between rLol p 5 and human IgE antibodies and which could recognize pollen proteins from other grass species. rLoi p 5 and its hypoallergenic variant also induced comparable levels of mucosal IgA antibodies after repeated immunization. As an innovative method of vaccine production and delivery, recent advances in recombinant DNA technology and plant transformation techniques have allowed proteins from numerous human pathogens to be expressed in transgenic plants. In an attempt to develop a novel method of recombinant protein expression for the production and delivery of grass pollen allergy immunotherapy, rLol p 1 and rLol p 5 were recombinantly expressed following Agrobacterium-mediated transformation of tobacco. Tobacco-expressed recombinant grass pollen allergens were shown to possess similar IgE binding epitopes to native rye grass pollen proteins and E. coli expressed rLol p 5.
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ItemDevelopment of a technique to overcome the problems associated with high dieldrin concentration in soil( 1995)Degradation of dieldrin in soils was studied in laboratory experiments. Application of powdered zinc in dilute acetic acid, nickel chloride in sodium borohydride and potassium tertiary butoxide in dimethyl sulphoxide to pots containing solonized brown soil, podzol, red brown earth, grey clay, alluvial and krasnozem soils had little effect on the rate of degradation. Application of potassium tertiary butoxide in dimethyl sulphoxide solution to soil samples containing dieldrin achieved a rapid decrease in pesticide concentration in some cases. However, the undesirable effects of these chemicals on soil fauna and flora and on soil physical properties indicate that their use in the field could not be recommended. Adsorption of dieldrin (as illustrated by the Freundlich constant) was greater in red brown earth and grey clay soils than in the solonized brown soil. The rate of dieldrin desorption from these soils was found to be in the order solonized brown soil > red brown earth > grey clay. Dieldrin uptake by plants was determined, with carrots being found to absorb the greatest quantities of dieldrin. All plant species took up considerably more dieldrin when grown on sand than on clay. Lupin translocated very little dieldrin to the plant tops. The amounts of insecticide translocated to the plant tops were not proportional to the amounts of insecticide present in soil. Brown coal was found to be a very good dieldrin adsorbent. Addition of brown coal in the proportion 4:1 soil:brown coal reduced the uptake of dieldrin by carrots from contaminated soil to acceptable levels. Both mice and sheep did not show any obvious symptoms of ill-health after being fed diets containing 10 ?g g-1 dieldrin plus 10% brown coal for 16 weeks. Animals fed diets containing 10% brown coal and 10 ?g g-1 dieldrin had lower concentrations of the insecticide in the internal organs. The concentration of dieldrin in the kidney fat of sheep fed this diet was above the maximum limit suggested to apply to animals raised for human consumption.
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ItemPhysiology of fibre development in Eucalyptus globulus( 1995)Plantation Eucalypt forests are an important source of wood fibre for pulp and paper manufacture and opportunities exist to enhance the quality of wood grown in these forests by silvicultural and genetic management. However the ability to do this is limited by the relatively poor understanding of the processes of secondary xylem cell production and differentiation and the mechanisms that control these processes. Length is one important fibre dimension affecting paper properties and this project has investigated the within-tree variation in secondary xylem fibre tracheid length (fibre length) in Eucalyptus globulus Labill. Evidence is presented that cambial region gibberellins are a likely causal factor in secondary xylem fibre elongation in Eucalypts. The dimensions of the secondary xylem fibres of woody dicotyledons are determined in part by the dimensions of the fusiform cambial cells from which they are derived and in part by the processes that occur during differentiation. For Eucalyptus globulus, a strong positive correlation (r = 0.757, df = 7, P < 0.05) existed between fibre length and fusiform cambial cell length along the axis of the stem. There also existed a basipetal gradient of increasing secondary xylem fibre elongation during differentiation. Although not statistically significant, the estimated rate of fibre elongation and the time required for differentiating fibres to complete elongation also increased basipetally. Gibberellins GA1, GA4, GA8, GA9, GA19, GA20, GA29, GA44, GA81, indole-3-acetic acid (IAA) and abscisic acid (ABA) were identified in cambial region extracts of Eucalyptus globulus by comparing mass spectra and Kovats retention indices with those of authentic standards. The presence of brassinosteroid-like substances was also indicated by activity in the rice seedling leaf inclination assay. Using [17, 17- 2H2] GAs as internal standards, GA19, GA20 and GA44 were quantified at levels of 2-7 ng (g fresh weight)-1. Other GAs were present at levels < 1 ng (g fresh weight)-1. It seems likely that the early C-13 hydroxylation pathway of GA biosynthesis predominates in Eucalyptus cambial region tissues. Although it has yet to be definitively determined whether these endogenous cambial region GAs arise by in situ biosynthesis and metabolism or by transport from apical meristems and leaves, the presence of high levels of GA44 and GA19 indicate that cambial region tissues can likely perform both early and late oxidative steps in GA biosynthesis. Within-tree gradients in cambial region GA, IAA and ABA levels were also identified. However the interpretation of these gradients is difficult since they vary depending upon how they are expressed, i.e. hormone per g cambial region tissue that gives a concentration or hormone per square cm of stem surface that gives an absolute amount. This problem will be difficult to resolve without a greater understanding of the localisation of cambial region hormones in the various cell types in various stages of development that comprise the cambial region. Localised stem injection of Primo (trinexapac-ethyl), a late stage acylcyclohexanedione type inhibitor of GA biosynthesis, was used to reduce endogenous cambial region GA levels. High doses of Primo also reduced the elongation of secondary xylem fibres and the radial number of primary walled differentiating fibres undergoing elongation. Fibre elongation was positively correlated with endogenous cambial region levels of both GA1 (rs = 0.74, P < 0.01) and GA20 (rs = 0.72, P < 0.01). Correlations between endogenous IAA or ABA and fibre elongation were positive but weak and non significant. Gibberellins may thus be causal factors in secondary xylem fibre elongation in Eucalyptus. However a definite relationship between spatial and temporal variation in cambial region hormone levels and within-tree variation in secondary xylem fibre elongation has yet to be established.