published_or_final_version / abstract / Botany / Doctoral / Doctor of Philosophy

Cell cycle progression is the series of steps a cell has to take in order to duplicate its DNA and produce two daughter cells. Correct spatial and temporal coordination of the cell cycle is key for the normal development of any organ or tissue and is stringently controlled during embryogenesis and homeostasis. Misregulation of cell cycle progression is causal in many developmental disorders and diseases such as microcephaly and cancer. Fucci (Fluorescent Ubiquitination based Cell Cycle Indicator) is a system that allows for the visualisation of cell cycle progression by the use of two differently coloured fluorescent probes whose abundance is regulated reciprocally during the cell cycle. The probes contain the E3 ligase recognition domains of Cdt1 and Geminin fused to the fluorophores mCherry (red fluorescence) and mVenus (yellow fluorescence) respectively. Cells are therefore labelled red during G1, yellow in the G1/S transition and green during late S/G2 and M phases of the cell cycle. In order to study development and tissue homoeostasis a Fucci expressing mouse line was developed however this has several key limitations: First, the two Fucci probes are expressed from separate loci complicating mouse colony maintenance. Second, the constructs were not inducible, making it impossible to follow cell cycle progression in specific cell lineages and third the mice were generated by random transgenesis which is prone to silencing and can exhibit variation in expression between different tissues. Here I have characterised an improved version of the original Fucci system known as Fucci2a designed by Dr Richard Mort (University of Edinburgh) to overcome these limitations. The Fucci2a genetic construct contains both Fucci probes fused with the Thosea asigna virus self-cleaving peptide sequence T2A. This allows expression of both probes as a single bicistronic mRNA with subsequent cleavage by ribosomal ‘skipping’ during translation to yield separate proteins. A Fucci2a mouse (R26Fucc2aR) was generated by homologous recombination into the ROSA26 locus using the strong, ubiquitous CAG promoter to drive expression and incorporating a floxed-Neo stop cassette. This allows tissue specific activation by Cre recombinase when combined with a second Cre expressing mouse line. Building on the bicistronic Fucci2a technology I have gone on to develop and characterise four new tricistronic reporter constructs which allow for the dual visualisation of cell cycle progression with apoptosis, cytokinesis and ciliogenesis. In each case an additional fluorescent probe was added to the original Fucci2a construct separated by the self-cleaving peptide P2A and the construct characterised in 3T3 stable cell lines. The combination of a dual cilia and cell cycle reporter construct proved fruitful and I have gone on to investigate the relationship between cell cycle progression and ciliogenesis in 3T3 cells and have generated and characterised the R26Arl13b-Fucci2aR mouse line. I have also illustrated the utility of the R26Fucci2aR mouse for generating quantitative data in development research in two development situations; melanocyte development and lung branching morphogenesis. Melanocytes are specialised melanin producing cells responsible for the pigmentation of the hair, skin and eyes. Their precursors, melanoblasts, are derived from the neural crest where they migrate and proliferate before becoming localised to hair follicles and their study provides a good model for understanding the development of other neural crest derived lineages such as the peripheral nervous system. Using time-lapse imaging of ex vivo skin cultures in which melanoblasts are labelled with the Fucci probes I have characterised melanoblast migration and proliferation. In addition, I have shown that Kit signalling, which is necessary for melanoblast migration and survival, controls melanoblast proliferation in a density dependent manner and that melanoblast migration is more persistent in S/G2/M phases of the cell cycle. Lung branching morphogenesis requires constant proliferation at the apical tip of a growing epithelial branch. Loss of epithelial symmetry through an unidentified mechanism (requiring BMP, FgF10, Shh and Wnt signalling) within a branch is required to initiate branching either latterly from the side of a elongating branch by domain branching or by bifurcation of the tip. In the final section of this thesis I performed a comparative analysis of the behaviour of the developing lung epithelium using proliferative status (Fucci2a expression) to categorise each cell. Using a combination of live imaging and immunohistochemistry I have identified a transition zone 100-150μm from the tip of the branching lung epithelium where epithelial cells become stationary and drop out of the cell cycle corresponding with the onset of proximal bronchial progenitor marker Sox2. A comparative gene expression analysis of the proliferating and non-proliferating regions using Fucci2a to distinguish them has eluded to several interesting genes which could influence branching morphogenesis during lung development.

Includes list of papers and publications by the author Includes bibliographical references (leaves 198-231) Reports on studies aimed at increasing the efficiency of livestock transgenesis programs. Overall the experiments provide an improved basis for understanding the application of animal biotechnology to the pig.

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

Includes list of papers and publications by the author / Includes bibliographical references (leaves 198-231) / [12], vii, 231 leaves, 6 p. of col. plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Reports on studies aimed at increasing the efficiency of livestock transgenesis programs. Overall the experiments provide an improved basis for understanding the application of animal biotechnology to the pig. / Thesis (Ph.D.)--University of Adelaide, Depts. of Obstetrics and Gynaecology and Animal Sciences, 1991

Thesis (Ph. D.)--University of Missouri-Columbia, 1997. / Typescript. Vita. Includes bibliographical references (leaves 109-122). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 1997. / Typescript. Vita. Includes bibliographical references (leaves 109-122). Also available on the Internet.

Thesis (M.A.)--University of Canterbury, 2006. / Typescript (photocopy). Includes bibliographical references (leaves 132-144). Also available via the World Wide Web.

The 1-adrenoceptors (1-ARs) serve as an interface between the sympathetic nervous system and the cardiovascular system where they are mediators of systemic arterial blood pressure, initiators of positive inotropy, and regulators of cellular growth responses. There are three subtypes: 1A-, 1B-, and 1D-ARs. This dissertation research investigated the trafficking properties of the 1-ARs at the cellular level as well as physiological relevance of the 1-ARs at the tissue level. In vitro studies using transiently transfected 1-AR/GFP subtypes revealed distinct basal localization patterns and different agonist-mediated activation and desensitization properties. The 1A- and the 1B-AR/GFP subtypes displayed agonist-mediated receptor redistribution, in which rate and degree of redistribution differed. Additionally, redistribution of either of these two receptor subtypes required arrestin-1, a protein often associated with receptor internalization. In contrast, the 1D-AR/GFP did not require arrestin-1 for maintaining the basal receptor orientation pattern. Although these data increase our knowledge of trafficking properties of the 1-AR subtypes, it is of equal importance to determine the role(s) that each subtype contributes to cardiovascular function. The lack of subtype-selective 1-AR pharmacological agents prompted the use of genetically manipulated mouse models with a systemic overexpression of a constitutively active 1B-AR. Echocardiographic analysis of transgenic hearts indicated both an enlarged left ventricular chamber in the absence of hypertrophy and a depressed cardiac function. From isolated transgenic hearts, experimental results suggested a role for the 1B-AR in attenuating the inotropic responses. However, experiments using isolated thoracic aortae from transgenic animals suggested that the 1B-AR does not participate in vascular smooth muscle contractile responses. Additional studies investigated the role of 1D-AR in cardiovascular function by using animals systemically lacking the 1D-AR subtype. Experimental data suggested an 1D-AR participation in vascular smooth muscle function since the deficiency of the 1D-AR subtype affected vasoconstriction in the coronary arteries but not inotropy in the heart. The data presented in this dissertation research suggest subtype specific differences of 1-ARs in cellular localization, signal regulation, and trafficking. Additionally, the data provide an investigation into the physiologic significance of both the 1B- and the 1D-ARs in cardiovascular tissue.

1 v. / 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, 1994