Thesis (Ph. D.)--University of Oregon, 2003. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 101-117). Also available for download via the World Wide Web; free to University of Oregon users.

Thesis (Ph. D.)--Hong Kong University of Science and Technology, 2003. / Includes bibliographical references (leaves 242-266). Also available in electronic version. Access restricted to campus users.

Chromosome replication and cell division of Escherichia coli are coordinated with growth such that wild-type cells divide once and only once after each replication cycle. Two components of this coordination are the SOS system and nucleoid occlusion. The SOS regulon expresses DNA repair genes after DNA damage and delays FtsZ-ring formation and cell division to enhance survival. Nucleoid occlusion prevents cell division over un-replicated nucleoids, a process partially dependent on the SlmA protein. Z-ring formation is shown here to be dependent on DNA replication by an additional mechanism, independent of the SOS regulon and of the SlmA protein and which acts by preventing FtsZ-ring formation when replication is perturbed. Replication dependent Z-ring formation (RDZ) was shown to be SOS-independent by the fact that FtsZ-rings were inhibited, after replication blockage, in a lexA1 mutant and in strains containing a null allele of sulA or the ftsZ/sulB103 mutation. SlmA protein-independence was shown by the fact that FtsZ-rings were also inhibited in lexA1 [Delta]slmA double mutants after replication blockage. This SOS- and SlmA-independent mechanism functions effectively in cells growing slowly with only one replicating chromosome and also in cells growing rapidly with multi-fork replication and after replication inhibition by different methods - chemical inhibitors and a temperature-sensitive polymerization mutation. / text

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Cyclin B1 is a cell cycle protein typically associated with the regulation of cellular division (mitosis). Previous studies in this laboratory involving preimplantation mouse embryos found that cyclin B1, or a cyclin B 1-related protein, were present at both G1 and G2/M phase of the cell cycle. Not only was cyclin Bi detected during G1 phase in this study, it was found to be present in higher concentrations at G1 phase through the first three cell cycles. These findings were unexpected, because most of the literature suggests that cyclin B1 is normally degraded during G1 phase. Using immunoprecipitation and immunoblot techniques, a more detailed study of cyclin B1 expression was inititated. Using two different primary antibodies direct against cyclin B1, a 48.97 kDa protein band, which is believed to be cyclin B1, was detected at both G1 and G2/M phases in 1-cell mouse embryos. Using another antibody directed against Cdk1, the kinase that forms a complex with cyclin B1 in order to direct the G2/M transition, a 37 kDa protein band was also detected at both G1 and G2/M phases in 1-cell mouse embryos. In order to determine whether cyclin B1 was present as a complex with Cdk1, immunoblotting with the anti-Cdk1 antibody. Again, a 37kDa protein band was detected at both G1 and G2/M phases. Finally, in order to determine whether the cyclin B1/Cdk1 complex exists in its active form, histone kinase assays were performed using anti-cyclin B1 immunoprecipitates. Kinase activity was detected in immunoprecipitates collected from G2/M phase 1-cell embryos, but no kinase activity was detected from immunoprecipitates collected from G1 phase 1-cell embryos. These data indicate that cyclin B1 and Cdk1 are present and exist as a complex in both G1 and G2/M phases of 1-cell mouse embryos, although the complex only appears to be active at the G2/M phase. / Department of Biology

The molecular mechanics of bacterial cell division remain one of the most fundamental unsolved problems in bacterial cell biology. During bacterial cytokinesis, bacteria divide symmetrically to give rise to two identical daughter cells. This tightly regulated process is orchestrated by an assembly of essential cell division proteins that form a supramolecular structure known as the divisome. The divisome, which forms at the cell centre, is responsible for the invagination and fusion of the cell’s membrane and peptidoglycan layers. The Escherichia coli divisome is comprised of at least ten essential proteins whose individual functions are mostly unknown. These divisomal proteins are recruited in a semi-hierarchical order, with the early recruits being predominantly cytoplasmic and the later recruits being predominantly extracytoplasmic or multi-pass transmembrane proteins. DivIB and its ortholog FtsQ are essential members of the divisome in Gram-positive and Gram-negative bacteria, respectively. DivIB is a bitopic membrane protein composed of an N-terminal cytoplasmic domain, a single-pass transmembrane domain, and a C-terminal extracytoplasmic region comprised of three separate protein domains. The α domain is located next to the transmembrane segment and is a polypeptide-transport-associated (POTRA) domain. The β domain comprises approximately half of the extracytoplasmic region, and has a unique three-dimensional fold. The most C-terminal domain, the γ domain, is relatively unstructured. This protein has been proposed to play a role in septal peptidoglycan cross-linking or lengthening. The primary aims of these studies were to further characterise the structure and function of the bacterial cell division protein DivIB as well as investigate the interactions this protein has with the other divisomal proteins. It was anticipated that the knowledge gained should aid in the development of antimicrobials that target this protein’s function or protein-protein interactions. A molecular dissection approach was used to determine which of DivIB’s domains are essential for its recruitment to incipient division sites and for its cell division functions. It was determined that DivIB has three molecular epitopes that mediate its localisation to division septa; two epitopes are encoded within the extracytoplasmic region while the third is located in the transmembrane domain. It is proposed that these epitopes represent sites of interaction with other divisomal proteins, and this information was used to develop a model of the way in which DivIB and FtsQ are integrated into the divisome. Remarkably, two of the three DivIB localisation epitopes are dispensable for vegetative cell division; this suggests that the divisome is assembled using a complex network of protein-protein interactions, many of which are redundant and likely to be individually nonessential. Yeast and bacterial two-hybrid studies have revealed that most of these proteins have multiple binding partners, making it difficult to pinpoint epitopes that mediate the interaction between pairs of interacting proteins. Recently, a heterologous septal targeting approach was introduced to study the interaction between Bacillus subtilis divisomal proteins in E. coli. This technique allows the interaction between pairs of divisomal proteins to be studied in vivo without the complications caused by other interacting proteins. This approach was used to perform a molecular dissection of the interaction between B. subtilis DivIB and the divisomal transpeptidase PBP 2B. Although both proteins have septal localisation determinants in their transmembrane domains, it was found that these regions do not mediate their interaction. Rather, it was shown that DivIB interacts with PBP 2B through its extracytoplasmic region. Dissection studies revealed that all three extracytoplasmic domains of DivIB are necessary for interaction with PBP 2B, suggesting that the two proteins make multiple interactions, each of which is not strong enough in isolation to mediate formation of a stable complex. Finally, it was shown that E. coli FtsQ localises to the division septum in B. subtilis but cannot complement a divIB null. Multi-angle laser light scattering (MALLS) analysis revealed that the extracytoplasmic domain of Geobacillus stearothermophilus DivIB is predominantly monomeric at high concentrations. This indicated that if DivIB does exist as a dimer in vivo, it dimerises through its cytoplasmic or transmembrane region. In vitro observations suggest the C-terminal residues of DivIB may play a role in peptidoglycan binding. Finally, attempts were made to determine the three-dimensional structure of the complete extracytoplasmic domain of DivIB. Although it proved impossible to determine the structure using NMR spectroscopy, crystals were obtained under many different crystallisation conditions. Despite diffracting to 3.5 Å, we were unable to solve the protein structure using X-ray crystallography. However, this work has laid the groundwork for future attempts at solving the structure of this protein using X-ray crystallography.

Errata is pasted onto back end paper. / Includes bibliographical references (26 leaves). / 115, [68] leaves, [8] 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. / This study entailed work towards the isolation of the pbl gene and preliminary characterisation of a candidate pbl transcript. Plasmid rescue of the genomic DNA flanking the inserted P element led to the isolation of a third candidate p61 cDNA, the 1A cDNA. This data suggests that the IA cDNA is encoded by the p61 gene. / Thesis (Ph.D.)--University of Adelaide, Dept. of Genetics, 1998

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.