Inteins are autocatalytic protein domains that splice out of the nascent polypeptide shortly after translation, requiring no co-factors to facilitate splicing. There is an intein coding sequence within the Prp8 gene of Cryptococcus neoformans, a human pathogen that causes cryptococcosis in immunocompromised people. The intein, Cne PRP8, is a drug target as Prp8 is a central component of the spliceosome and thus believed to be essential to the fungus. Improved knowledge of the intein and its requirements for splicing can contribute to design of a screening system and the search for an inhibitor of intein splicing. Purification of Cne PRP8 for crystallisation was performed using either an N- or a C-terminal His�Tag�, where the N-terminal His�Tag� was removed by 3C protease prior to crystallisation trials. C-terminally His�Tagged� Cne PRP8 formed the largest crystals. The crystals were triangular plates with stepped faces. A 2.8 Å data set was collected with an R[merge] of 0.151 and a mosaicity of 2.1�. A smaller crystal gave a 3.6 Å data set with an R[merge] of 0.085 and a mosaicity of 1.5�. Molecular replacement was not sufficient to solve the structure, likely because the data were weak and the molecules in the asymmetric unit too numerous. Purified Cne PRP8 was additionally shown by circular dichroism to lack regular secondary structure, suggesting that regions of Cne PRP8 could be natively unstructured. Cne PRP8 was expressed as a fusion protein between Haemophilus influenzae trigger factor (HiTF) and a chitin binding domain (CBD). Antibodies to the different parts of the fusion protein facilitated the observation of splicing ability by western blotting. From this it was determined that Cne PRP8 is capable of splicing in a foreign protein context. Context is important, with maximum splicing occurring when Cne PRP8 has two native N-terminal extein residues and one native C-terminal extein residue. The first residue and the last two residues of Cne PRP8 are essential for splicing; additionally the conserved threonine (T62) and histidine (H65) were shown to be catalytically important. Also required for splicing are arginine 154, tyrosine 162, and aspartate 166. Leucine 161 undergoes ~50% splicing when mutated to alanine, and tryptophan 151 undergoes limited C-terminal cleavage, but no splicing, when mutated to alanine. Tryptophan 151 was identified as a potentially crucial residue, which may function to prevent C-terminal cleavage before the N-terminal rearrangements have taken place. Overall it appears that Cne PRP8 residues that are more diverged from the general intein consensus are less essential for splicing. Wild type Cne PRP8 is insensitive to zinc inhibition in vivo. It is also unresponsive to cadmium, calcium, cobalt, lithium, magnesium, manganese and nickel. However, a partially splicing-deficient mutant exhibited further inhibition in response to zinc and cadmium. This mutant also showed a limited increase in splicing efficiency in response to temperatures lower than 37�C. This study has identified critical residues, in addition to those at the splice junctions necessary for catalysis, which participate in splicing intermediates.

Interleukin (IL)-3, IL-5 and granulocyte-macrophage-colony-stimulating factor (GM-CSF) are cytokines that affect the growth, survival and differentiation of many cells within the haematopoietic system. The functions of these factors are mediated by membrane bound receptor complexes that are composed of specific ligand binding subunits (α)and a common signal transducing subunit(hβc). Constitutively activated mutants of hβc have been previously identified that are able to confer factor-independent signalling in a number of haematopoietic cell lines (including FDC-P1 and FDB-1). These activated mutants fall into two classes defined by the location of the mutation and their biochemical and leukaemogenic properties. In particular, the transmembrane mutant, V449E, causes an acute myeloid leukaemia in vivo, whereas the extracellular mutants (FI∆ or I374N) cause chronic myeloproliferative disorders. The work described in this thesis used the activated hβc mutants to uncover novel transcriptional events induced by the GM-CSF/IL-3/IL-5 receptor complex and to define pathways associated with proliferation and differentiation. Large-scale gene expression profiling techniques were used to investigate the genes involved in these biological processes in the murine myelomonocytic cell line FDC-P1, and the bi-potent FDB-1 myeloid cell line, which are responsive to IL-3 and GM-CSF. Membrane arrays were used to identify differences in gene expression between I374N and V449E expressing FDC-P1 cells. This technique revealed that the gene Ptpmt1 was differentially expressed between V449E and I374N, which was subsequently confirmed by Northern blotting. This finding suggested that the phosphatase encoded by Ptpmt1 may be involved in the different outcomes induced by these two hβc mutants. Northern analysis also revealed Ptpmt1, Nab1 and Ddx26b to be regulated in response to human GM-CSF in FDC-P1 cells expressing human GM CSFα and hβc. A large-scale cDNA microarray experiment was also performed to identify genes that are selectively expressed during differentiation of FI∆ expressing FDB-1 cells, compared to proliferating V449E expressing FDB-1 cells over 24 hours. A comprehensive analysis approach was adopted to examine the microarray data and identify differentially expressed genes. Among the genes displaying differential expression were Btg1, S100a9, Cd24, and Ltf found to be differentiation-associated and Bnip3, Cd34, Myc, Nucleophosmin, and Nucleostemin found to be proliferation-associated. Hipk1, Klf6, Sp100, and Sfrs3 were also identified as potential transcriptional regulators during growth and differentiation. Northern analysis was used to confirm differences in expression for these 13 genes between FI∆ and V449E expressing FDB-1 cells. Eleven of the 13 genes examined were confirmed to be differentially expressed between FI∆ and V449E expressing FDB-1 cells over 24 hours. Furthermore, six genes (Btg1, Hipk1, Cd24, Cd34, Klf6 and Nucleostemin) examined over 72 hours revealed differences in gene expression at early (6-12 hours) and late (48-72 hours) time points. Cell surface expression of CD24 protein was also shown to be induced upon FI∆ expression or GM-CSF induced differentiation of FDB-1 cells, consistent with elevated levels of Cd24 mRNA in FI∆ cells over time. Based on their confirmed gene expression differences seen on the microarrays and Northern analysis, four genes (Btg1, Cd24, Klf6 and Nucleostemin) were selected for over-expression analysis in FDC-P1 or FDB-1 cells, in order to gain insights into the function of these genes. Optimisation of the retroviral infection process was performed so that the role of these genes in proliferation and differentiation could be investigated in the FDB-1 model. Such preliminary functional experiments in FDB-1 cells will enable prioritisation of the genes for further analysis of their function in primary cells. Thus, the work in this thesis describes the first use of microarrays to identify gene expression differences between hβc mutants with differential activities affecting myeloid growth and differentiation. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1284103 / Thesis (PhD)-- School of Medicine, 2005

Bibliography: leaves 116-129 / iv, 129 leaves, [23] leaves of plates : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1987

Includes a copy of an article co-authored by the author during the preparation of this thesis. / Bibliography: leaves 132-148. / iv, 148 leaves : ill. (some col.); 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This thesis describes the screening of the sheep wool follicle for expression of Antennapedia-like homeobox genes by RT-PCR and the isolation and characterisation of the Hoxc-13 gene and a novel hoeobox gene, Barx2. / Thesis (Ph.D.)--Adelaide University, Dept. of Animal Science, 2001

Bibliography: leaves 127-151. / xiii, 151, [61] leaves, [19] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / The successful application of genetic engineering in wheat is dependent on the availability of suitable tissue culture and transformation methods. The primary object of this project was the development of these technologies using elite Australian wheat varieties. / Thesis (Ph.D.)--University of Adelaide, Dept. of Plant Science, Waite Campus, 2001?

Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Bibliography: leaves 103-124. In this project, using rice as a model plant, methods have been developed to improve regeneration frequency and to enhance the efficiency of Agrobacterium mediated transformation technique.

<p>This thesis investigates the use of Genetic Programming (GP) to evolve controllers for an autonomous robot.</p><p>GP is a type of Genetic Algorithm (GA) using the Darwinian idea of natural selection and genetic recombination, where the individuals most often is represented as a tree-structure. The GP is used to evolve a population of possible solutions over many generations to solve problems.</p><p>The most common approach used today, to develop controllers for autonomous robots, is to employ a GA to evolve an Artificial Neural Network (ANN). This approach is most often used in simulation only or in conjunction with online evolution; where simulation still covers the largest part of the process.</p><p>The GP has been largely neglected in Behaviour Based Robotics (BBR). The is primarily due to the problem of speed, which is the biggest curse of any standard GP. The main contribution of this thesis is the approach of using a linear representation of the GP in online evolution, and to establish whether or not the GP is feasible in this situation. Since this is not a comparison with other methods, only a demonstration of the possibilities with GP, there is no need for testing the particular test cases with other methods.</p><p>The work in this thesis builds upon the work by Wolfgang Banzhaf and Peter Nordin, and therefore a comparison with their work will be done.</p>

Graduation date: 1984

Single nucleotide polymorphisms (SNPs) are the most common DNA polymorphisms in plant and animal genomes. SNPs were identified in the allele sequences of up to 12 sunflower (Helianthus annuus L.) genotypes for a genome-wide sample of 81 loci originally mapped using restriction fragment length polymorphism (RFLP) markers. The RFLP loci anchor a dense RFLP linkage map and second-generation linkage maps constructed using simple sequence repeats and other high-throughput DNA markers. The goal of this study was to develop high-throughput SNP markers for the cDNA-RFLP loci for linkage mapping, diversity analysis, and molecular breeding and genomics research. The specific objectives were to: (i) develop FP-TDI or fluorescent primer-extension SNP assays for the cDNA-RFLP loci; (ii) validate the SNP markers by screening 12 sequenced (reference) genotypes; and (iii) assess the utility and polymorphism rate of the SNP markers across a genetically diverse panel of unsequenced wild and domesticated sunflower genotypes. SNP markers were successfully developed for 44 of the targeted 49 cDNA-RFLP loci. Thirty-one of the SNP markers were further selected for a diversity analysis across a genetically diverse panel of wild and domesticated sunflower genotypes. The mean heterozygosity (H) score for the loci was 0.41, which nears the maximum H score of 0.5 for biallelic markers. SNPs in this study exhibited polymorphism rates close to those of RFLP and SSR markers in inbred sunflower lines. / Graduation date: 2004