School of Botany - Theses
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ItemEstablishing and elucidating conditional genetic manipulation of the malaria parasite, Plasmodium falciparum( 2013)Methods to alter gene function in Plasmodium are barely out of infancy, with much growth occurring within the past five years via adaptation of protocols established in other species. In this thesis, a method where the parasite’s innate ability to control gene expression in response to environmental stimuli, and a synthetic small molecule regulated protein stability system were applied to change the phenotypic expression of introduced transgenes. In chapter 2, we aim to control activity of a temperature sensitive mutant FLP recombinase protein. Our goal was to induce FLP activity and thus change the open reading frame of a GFP expressing plasmid to mCherry using single site recombination. Dual transgenic parasites were placed in a variety of conditions to favour FLP activity and to prevent negative selection. No evidence of conditional genetic recombination or phenotypic change from green to cherry was observed. Direct expression of FLP during a short period of drug selection produced a mixed population of GFP and mCherry genotypes. The activity of FLP is inefficient and was deemed unsuccessful for our goal of raising transgenic parasites poised for conditional genetic recombination. A homogenic population of mCherry genotypes by successful recombination of the GFP ORF was achieved after drug-cycled expression of evolved FLP (FLPe). Chapter 3 focuses on turning the Plasmodium specific phenomenon of altering rRNA expression profiles in response to fluctuations of temperature and glucose concentration into a tool suitable for conditional gene expression. We copied the promoter region of the S2-Type rRNA gene and measured its ability to express luciferase in altered environmental conditions. A low level of basal activity was observed during the intra-erythrocytic developmental cycle, with expression peaking at levels similar to the weak chloroquine transporter (CRT) promoter during the trophozoite stage. The S2-Type rRNA promoter region did not produce a notable increase of luciferase transcription or activity in response to environmental change. Use of the S2-Type rRNA promoter to express FLP at the enzymatic favourable temperature of 26°C did not result in genetic recombination. The tools developed are capable of expressing transgenes, however their ability to conditionally alter the gene product is limited.
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ItemThe ecology and ecophysiology of Caulerpa in Port Phillip Bay( 2012)While recognized as a key ecosystem component since at least the 1960s, Caulerpa has not received the attention that Port Phillip Bay (PPB) sediment, pelagic, reef and seagrass ecosystems have. Contemporary data on native PPB Caulerpa populations are a notable omission from the global literature on the genus. The genus Caulerpa has been heavily researched on a global scale for at least the past two decades, mostly due to the introduction of two species to several ecosystems overseas and in Australia. Here current Caulerpa species distributions were documented and modeling used to elucidate correlations with environmental variables. The ecophysiology of Caulerpa remotifolia was investigated using PAM fluorometry due to its broad environmental distribution. Current Caulerpa species distributions and abundance in western Port Phillip Bay were found to be consistent with historical records. Cross-validated Boosted Regression Tree models applied to data on three Caulerpa species distributions were able to model strong relationships between distributions and predictor variables (giving high correlations (0.66-0.80) and ROC scores (0.86-0.97)). Environmental variables describing substrate availability were consistently the most influential predictors of distributions while light and nutrient availability were also important. Caulerpa longifolia was mostly confined to the area adjacent to the Western Treatment Plant wastewater discharges; an area also having high Caulerpa species richness. Caulerpa remotifolia was the most widely distributed in both environmental and physical space; over most of its range it appears to be epizoic on the benthic ascidian Pyura dalbyi. Caulerpa sedoides was mostly confined to the deeper areas; their fine organically rich sediments indicating a role for it in sediment biogeochemistry. Caulerpa remotifolia was found to have substantial physiological plasticity over daily and seasonal variations in light availability. Despite low light availability and temperature in winter this species was able to achieve photosynthetic rates only slightly lower than in summer. Nutrient status appears to reflect C. remotifolia light harvesting efficiency suggesting a role for nutrients in its acclimation to light availability. Physiological plasticity of C. remotifolia’s photosynthetic apparatus explains some of its success in Port Phillip Bay. The prevalence of Caulerpa in the PPB ecosystem warrants substantial further investigation, especially regarding its role or services to the ecosystem, temporal dynamics and ecophysiology. This study provides suggestions of avenues for such investigation.
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ItemBayesian modelling of tree mortality( 2012)In this work I aimed to relate two topics of importance in applied ecology and the statistical analysis of ecological data: the challenge of analysing tree mortality data and the use of informative priors in Bayesian ecological models. To achieve these aims I conducted analyses on two separate case studies of tree mortality. The first study concerned the mortality of transplanted eucalypt tree seedlings during an experiment carried out in Southeastern Australia. For the second case study I used data collected over 20 years in a 50 ha tropical forest dynamics plot located in Northern Thailand. A key challenge when modelling death data is how to account for a hazard that varies through time. Ignoring a variable hazard leads to biased estimates of mortality rate. I identified evidence of a declining hazard rate in the eucalypt seedling data. To account for a potential source of bias in estimating the mortality rate of the seedlings I introduced a novel method whereby the expected mortality is a function of the sum of mortality experienced in the past. Bayesian methods have become common in the ecological research literature. Yet ecologists rarely use informative priors in their Bayesian models—a feature which has been shown to increase the precision of model predictions. This reluctance may in part stem from a perception that informative priors could introduce bias and lead to less accurate models, compared with those that use uninformative priors or fit with non-Bayesian methods. To date there have been no comprehensive tests of the effect of informative priors on the accuracy of Bayesian ecological models. Using the eucalypt seedling dataset I demonstrate that incorporating an informative prior based on the results of a pilot study does not negatively affect the accuracy of the model, while increasing the precision and making cost-effective use of the available information. With the tropical forest dataset I tested the effect on model accuracy of informative priors. In the first comprehensive test of this kind, I demonstrate that a properly specified informative prior, in this case based on the correlation between tree growth and mortality, will on average have no effect on the accuracy of models of mortality rate.
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ItemAustralasian Huperzia as potential sources of Huperzine alkaloids( 2010)The Lycopodiaceae is an ancient and cosmopolitan family of fern allies that include an estimated 35 Huperzia species occurring throughout the South-East Asian and Australian region. Thirteen species naturally occur in Australia and are found mainly in the tropical rainforests of far north Queensland. Over the past decade, there has been renewed interest in Huperzia and their respective Huperzine alkaloid concentrations following the discovery of Huperzine A (HupA) and Huperzine B (HupB) alkaloids in H. serrata (Thunb. ex Murray) Trevis. Both alkaloids are of pharmaceutical interest since they are highly selective and potent reversible inhibitors of acetylcholine esterase. Huperzine alkaloid concentrations of Australasian Huperzia have not been well documented, and no prior studies have been undertaken to investigate the amenability of Australasian Huperzia to alternative propagation techniques such as axenic culture. This research presents an extensive screen of 16 Australasian Huperzia species to investigate their Huperzine alkaloid concentrations. HupA (0.032 to 1.012 mg g-1 DW) was detected in ten out of the sixteen Huperzia species examined, while HupB (0.008 to 0.339 mg g-1 DW) was detected in eight. From this extensive study, H. elmeri (Herter) Holub was observed as the species with the greatest potential to yield high Huperzine-containing individuals. In addition, the screen established that Australasian Huperzia generally contain higher HupA levels than H. serrata, the main source of commercial HupA, which on average only contains 0.082 mg g-1 DW HupA. The fractionation and spectrometric analysis of alkaloids as part of the screen led to the discovery of three Huperzine alkaloids co-occurring within the same plant: HupA, HupB and Huperzine C, isolated from an individual of an Australian H. carinata (Desv. Ex Poir.) Trevis. The potential of establishing axenic cultures of Australasian Huperzia was also investigated in this research. Actively growing axenic cultures of H. carinata, H. squarrosa (C.Forster) Trevisan, H. phlegmaria (L.) Rothm. and H. phlegmarioidies (Gaudich.) Rothm., together with callus and cell suspension cultures of H. carinata and H. phlegmaria, were successfully established. The results suggest that culturing in total darkness is essential to allow for optimal callus and cell suspension growth. In addition, this study also investigated the possibilities of germinating various Huperzia spores, by both symbiotic and asymbiotic means. Germination of H. squarrosa spores was achieved by both symbiotic and asymbiotic means, and was only observed in cultures which were kept in the dark, implying that there is a form of photo-inhibition mechanism preventing spores from germinating when they are exposed to light. Beneficial effects of various types of spore treatments prior to sowing, in terms of increased spore germination was also observed. In conclusion, the results presented suggest that Australasian Huperzia are indeed a potentially valuable resource for Huperzine alkaloids. The investigations into the conditions required for the successful introduction and maintenance of Australasian Huperzia in axenic culture has also further extended our understanding of these plants, and their amenability towards axenic culture conditions as a means of alternative propagation.