Since the early 1990s, the concept of substantial equivalence has been a guiding principle of the Canadian Food Inspection Agency and Health Canada's regulatory approach toward products of plant biotechnology destined for the food and livestock feed markets. To assess substantial equivalence in terms of chemical composition, genetically modified (GM) plants are compared to conventional counterparts at the level of macro- and micro-nutrients, allergens and toxicants. Such targeted comparative analyses are limited in their scope and their capacity to detect unintended changes in chemical composition. There is a need to develop more effective testing protocols to improve the substantial equivalence assessment of GM crops. The objective of this thesis was to explore high-density oligoarrays as tools to assess substantial equivalence of Roundup Ready(TM) soybean. Three conventional and two GM soybean varieties were selected according to the similarity of their performance in field trials. Total RNA was extracted from first trifoliate leaves harvested from soybean plants grown in a controlled environment until the V2 stage. To annotate the 37 776 soybean probesets present on the multi-organism Soybean Affymetrix GeneChip(TM), consensus sequences were aligned with TIGR Soybean Gene Index tentative consensus sequences using BLASTN. After redefining the chip description file to exclude non-soybean probesets, the effects of three different normalization methods (Robust Multichip Average (RMA), Microarray Analysis Suite (MAS 5.0) and Model-Based Expression Index) were compared and Significance Analysis of Microarrays (SAM for R-Bioconductor) was applied to detect differential gene expression between conventional and GM soybean varieties. Eleven candidate genes were selected for further studies.

Potato leafroll virus (PLRV) is one of the most destructive potato viruses in South Africa. In order to establish resistance against PLRV in commercial potato cultivars, the coat protein (CP) gene of the virus was previously isolated, cloned and subcloned into the plant expression vector pBI121 in both the sense and antisense orientations (BURGER, unpublished results). The pBI121 constructs containing the PLRV-CP gene were subsequently transferred to Agrobacterium tumefaciens LBA 4404 in a triparental mating process with the helper plasmid pRK2013. Two A. tumefaciens- mediated transformation methods for potatoes were investigated, viz. vacuum infiltration and leaf disk transformation. In addition, optimal transformation and regeneration conditions were identified for potato cultivars Late Harvest and BP[1] In total, 27 transgenic potato lines containing the PLRV-CP, β-glucoronidase (GUS) and nptII (neomycin phosphotransferase II) trans genes were generated under kanamycin selection. Transgenic plants grown in the glasshouse appeared to be phenotypically normal, and no differences in ploidy level in comparison to non-transformed plants could be established. Stable transgene insertion into the genome of the transgenic plants was verified using PCR and Southern blot analysis. Expression of the GUS transgene was investigated using a fluorometric assay (JEFFERSON et al. 1987), and it was found that orientation of the inserted PLRV-CP gene upstream from the GUS gene had a direct influence on the levels of GUS expression. The expression of the PLRV-CP gene was analysed using DAS-ELISA and immunoblot detection. Coat protein could not be detected in either assay. RNA dot blots were used successfully to show PLRV-CP expression in transgenic potato plants at the mRNA level. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1995.

For the expression of transgenes in plant cells, appropriate promoter sequences have to be introduced upstream of the gene to ensure efficient transcription. Tissue- or signal-responsive promoters are in high demand in practical plant biotechnology. The present study sought to characterise the activities of two promoters in sugarcane, namely the UBI (ubiquitin) promoter and the SUC-1 promoter (UBI linked in tandem to the cauliflower mosaic virus 35S promoter). It was hypothesised that the activity of UBI would be maintained or even increased under conditions of environmental stress, since it is well documented that ubiquitin is a stress-related protein. A further hypothesis was that SUC-1 might enhance overall gene expression since the CaMV 35S component is a constitutive promoter widely and successfully used in plant transformation. Plants of the sugarcane variety NC0310, containing the cry1A(c) (Bt) gene from Bacillus thuringiensis, were used as models in a system in which the plants were stressed by withholding water supply in a controlled manner. Since large numbers of clones of both transgenic and wild-type plants were needed for the water stress and expression experiments, three micropropagation techniques, namely, shoot tip-, callus- and node culture, were optimised and compared. The objective was to propagate genetically stable plants rapidly. Compared to shoot tip culture, node and callus culture proved slow and inefficient. Shoot tip culture was thus chosen as the most suitable for the regeneration of experimental material. Relative Water Content (RWC) determination, leaf elongation measurements and Infra Red Gas Analysis (IRGA) were compared in order to find the most appropriate method of measuring plant water status. In addition to being destructive, no observable differences were evident between the control (non-stressed) and water-stressed plants when using RWC as a measure. Results obtained from leaf elongation measurements compared favourably to the more sophisticated IRGA readings, showing that leaf elongation is as sensitive a measure of water stress. On the basis of preliminary studies with untransformed plants using the latter two techniques, water regimes for stress-induction in the final experiments were designed. Leaf elongation measurements, which are simple and non-destructive, were ultimately chosen to measure plant water status. In the final water stress experiment non-transgenic NCo310 and clonal populations of six transformants were used (three containing the UBI promoter; three the SUC-1 promoter). Exactly half of the plants of each type were stressed by withholding water supply, while the other half (controls) were watered manually twice a day. Leaf elongation measurements were made at the same time daily on the third youngest leaf of 6 plants from each population per treatment. At the same time, leaf samples were taken daily for molecular analysis. The stress regime led to marked differences in leaf elongation between control and water-stressed plants. In terms of physiological response (leaf rolling and senescing), plants containing the SUC-1 promoter appeared least affected. The reverse transcription-polymerase chain reaction (RT-PCR) and Northern hybridisation were used to assay UBI and SUC-1 activity. RT-PCR revealed that both promoters drove Bt gene expression in controls and experimentals throughout the stress period, although differences in signal intensity were not observed. The extent of expression occurring in each type of plant was revealed in Northern blots probed with two genic sequences (1) the transgene and (2) sugarcane EST ME42, homologous to heat shock protein 82 in rice. Individual transformants showed overall levels of transgene expression that were variable, possibly due to insert position in the plant genome, as well as variations in relation to the application of stress. SUC-1 seemed superior to UBI in terms of driving transgene expression under stressful environmental conditions, since UBI promoter activity appeared to decrease under stress, while SUC-1 promoter activity remained constant. In addition to the expected 2.0 kb Bt transcript, transcripts of smaller than expected size were also obtained, leading to the suggestion of premature polyadenylation signals in the coding region of the wild-type Bt234 gene. Upon inspection of the transgene sequence, a number of motifs rarely present in plant genes were observed, namely A/T rich sequences, ATTTA motifs and numerous potential polyadenylation sites. / Thesis (M.Sc.)-University of Natal, Durban, 1999.

A greenhouse experiment was carried out to screen for potential effects of transgenic aspen over-expressing a hybrid poplar leaf polyphenol oxidase gene on rhizosphere communities. Heterotrophic plate counts and cultivation-independent methods were used to compare bacterial and fungal populations associated with transgenic PPO over-expressing and unmodified control trees. Total community DNA extracted from rhizosphere soils was used to establish Iibraries containing partial gene sequences that were PCR-amplified from community members, and putative taxonomy was assigned to clones based on similarity to reference sequences. Gene libraries for the bacterial component of the rhizosphere were established using partial 16S rRNA and chaperonin-60 gene sequences, and the fungal community was characterized based on partial 18S rRNA gene sequences. Phylogenetic analysis revealed that bacterial 16S gene libraries were dominated by Alphaproteobacterial sequences, and the CPN-60 gene libraries were dominated by members of the Bacteroidetes/Chlorobi group, illustrating the biases potentially incurred by using a single gene locus to profile microbial diversity. In both CPN-60 and 16S rRNA libraries, only minor components of the bacterial community differed between transgenic and unmodified trees. Comparisons based on library coverage indicated that changes in bacterial community structure between transgenic and unmodified trees were minor in comparison to differences observed between individual trees of the same type, and no significant differences in terms of bacterial species diversity were revealed by the calculated diversity, dominance and evenness indices. In comparison to the bacterial gene libraries, higher coverage of the underlying population was achieved in the fungal 18S libraries. Members of the Zygomycota, Chytridiomycota, Ascomycota, and Basidiomycota were recovered from both libraries. Dominant groups of fungi associated with each tree type were highly similar, although there were some qualitative differences in the recovery of less abundant fungi as a result of the underlying heterogeneity of the fungal population. No clear differences in terms of fungal species richness were associated with transgenic or unmodified trees, although control libraries were characterized by a slightly higher level of dominance. In general, the methods employed revealed only minor differences between the bacterial and fungal communities associated with transgenic and unmodified trees, suggesting that impacts of the transgenic plants on the rhizosphere community were minimal.

Many environmental stresses result in increased generation of active oxygen species (AOS) in plant cells, leading to the induction of protective mechanisms. In this study, signalling components linking AOS perception to downstream responses were examined, with particular emphasis on H₂O₂ signalling. All AOS investigated had an early [Ca²⁺]_cyt peak in common, but differed in other aspects of their Ca²⁺ signatures, indicating that the plant is able to discriminate between different types of AOS. An early event in AOS signal transduction may involve changes in the cellular redox balance as reduction of glutathione levels prior to stress application increased the height of the first [Ca²⁺]_cyt peak. Inhibiting or enhancing the height of the H₂O₂-triggered Ca²⁺ signature lead to inhibition or enhancement of GST1 and APX1 induction, respectively, demonstrating that the Ca²⁺ signature is required for induction of genes encoding antioxidant enzymes. OX1, encoding a putative ser/thr kinase, was shown to be involved in signal transduction in response to H₂O₂-generating stresses. Transcript levels of OX1 were increased upon treatment with H₂O₂ and a range of abiotic and biotic stresses as well as ABA, all of which have been shown to result in H₂O₂ accumulation. Inhibition of stress-induced [Ca²⁺]_cyt elevations inhibited OX1 induction, placing the OX1 kinase downstream of Ca²⁺ in the signalling chain. OX1 is required for full activation of AtMPKS and AtMPK6 in response to ozone fumigation, indicating that OX1 functions upstream of these MAP kinases. An ox1 null-mutant displayed enhanced susceptibility to infection with a virulent Peronospora parasitica isolate as well as reduced induction of several defence genes. In addition, the ox1 mutant exhibited shorter root hairs and an early flowering phenotype. AOS treatment induced several genes encoding AtERF transcription factors, but did not have an effect on other members of this family. Induction occurred in an ethylene-independent but Ca²⁺-dependent manner.

Plastid division, sustained by the equilibrium expression and coordination of plastid division genes is vital for the maintenance of plastid populations in dividing plant cells. Macrochloroplasts (MCP), the occurrence of one or a few chloroplasts per cell is due to the imbalance in the expression of plastid division genes. Because of the MCP size and number it was proposed that they may provide better targets for the plastid transformation than the normal (WT) chloroplasts and result in better plastid transformation frequencies. The objective of this research was to produce transgenic plants containing macrochloroplasts by nuclear transformation and then to use these plants as a model for the development of plastid transformation of crop species. By using AtFtsZ1-1 and AtMinD1 as query sequences in the TIGR (U.S.A) and ASTRA (Australia) Brassica oleracea EST databases, this project resulted in the isolation of cauliflower FtsZ1-1 (EU684588) and MinD (EU684589) genes. In addition, AtFtsZ1-1 was used as a control gene for comparison to the cauliflower FtsZ1-1. Binary vectors were constructed to express these genes in tobacco and cauliflower either by Agrobacterium tumefaciens-mediated or PEG-mediated transformation methods. Transgenic tobacco and cauliflower plants with abnormal chloroplasts (MCP, minichloroplasts, honeycomb or doughnut shaped chloroplasts, uneven surface membrane chloroplasts) were developed. Furthermore, the transgenic tobacco and cauliflower plants were examined by PCR, RT-PCR and Southern blotting. In addition, th ese plants were also analysed for the different abnormal chloroplast phenotypes by fluorescence microscopy. This project also generated the first plastid transformants from macrochloroplast bearing tobacco plants via biolistics. After one round of regeneration homoplasmic plastid transformants were obtained from both WT chloroplast and MCP tobacco plants. The homoplasmic nature of plastid transformants were confirmed by PCR and Southern blotting. Plastid expression of GFP in WT and MCP was confirmed by fluorescence/confocal microscopy and western blot analysis. This project showed for the first time the characterisation of cauliflower FtsZ1-1 and MinD plastid division genes in homologous and heterologous systems (cauliflower and tobacco). Moreover, obtaining homoplasmic plastid transformant shoots from one round of regeneration from the MCP containing tobacco plants is reported for the first time in this study. In addition this study explored the effect of transgene expression level on the chloroplast abnormality, highlighting the importance of analysing transgenic tobacco and cauliflower plants at the protein lev el specifically with regard to plastid division genes. The maintenance of MCP phenotype in the regenerated shoots and the requirement of standardisation of MCP containing plants via biolistics for increasing the plastid transformation frequency were also examined.

It is estimated that over 200,000 Australians have problem or chronic wounds at any one time (Australian Wound Management Association, 2008). Over the past 4 decades while there has been significant advancement in wound care, a high proportion of wounds become chronic. Despite the availability of wound care resources and specialist services, there remains an inconsistency in the management of chronic wounds that impacts both on the quality of life of individuals with chronic wounds and the health care budget (Harding 2002). Using a Grounded theory approach, the aim of this study was to explore and describe the impact of living with a chronic wound and findings indicate that individuals living with a chronic wound are receiving conflicting or inappropriate advice and care. Individuals living with a chronic wound experience a life of uncertainty related to the struggle to endure a wounded body and the layers of professional care they receive. When they are provided with conflicting or inappropriate advice and treatment, inconsistencies of care and poor coordination of care, layers of unnecessary burden are added to their experience. The uncertainty and dissonance individuals are faced with, leads them to question their care, themselves and the expertise and professionalism of their treating health professionals. As a result, they experienced a loss of respect and trust for their treating health professionals and a loss of confidence in their care. Chronic wounds impose of individuals, an intense burden of physical suffering, cause major disruption to the normality of their lives, and often entail a constant personal struggle to secure appropriate care and understanding from their treating health professionals. In order to enable individuals living with chronic wounds to develop appropriate coping strategies, it is essential that health professionals: understand the burden of suffering associated with living with a chronic wound; ensure that they develop and maintain a high level of knowledge with regards to contemporary wound care practices; ensure that their clientele are provided with high quality care information that is based on the best available evidence; ensure continuity of care; and foster quality professional-client relationships that negates the need for individuals to have to constantly question their care.

Diss. (sammanfattning). Umeå : Sveriges lantbruksuniv. / Härtill 4 uppsatser.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2004. / Härtill 4 uppsatser.

Thesis (Ph.D.)--University of Melbourne, School of Forest and Ecosystem Science, 2006. / Typescript. Includes bibliographical references (leaves 184-216).