Neurogenesis occurs in exclusive regions in the adult nervous system, the subventricular zone and dentate gyrus in the brain, and olfactory epithelium (OE) in the periphery. Cell replacement after death or injury, occurs to varying degrees in neural tissue, and is thought to be dependent upon the biological responses of stem and/or progenitor cells. Despite the progress made to identify adult OE and central nervous system (CNS) progenitors and lineage trace their progeny, our spatial and temporal understanding of embryonic OE neuroglial progenitors has been stalled by the paucity of identifiable genes able to distinguish individual candidate progenitors. In the developing CNS, radial glia serve as both neural progenitors and scaffolding for migrating neuroblasts and are identified by the expression of a select group of antigens, including nestin. Here, I show that the embryonic OE contains a novel radial glial-like progenitor (RGLP) that is not detected in adult OE. RGLPs express the radial glial antigens nestin, GLAST and RC2, but not brain lipid binding protein (BLBP), which, distinct from CNS radial glia, is instead found in olfactory ensheathing cells, a result confirmed using lineage tracing with BLBP-cre mice. Nestin-cre-mediated lineage tracing with three different reporters reveals that only a subpopulation of nestin-expressing RGLPs activate the “CNS-specific” nestin regulatory elements, and produce spatially restricted neurons in the OE and vomeronasal organ. The dorsal-medial restriction of transgene-activating cells is also seen in the embryonic OE of Nestin-GFP transgenic mice, where GFP is found in a subpopulation of GFP+ Mash1+ neuronal progenitors, despite the fact that endogenous nestin expression is found in RGLPs throughout the OE. In vitro, embryonic OE progenitors produce three biologically distinct colony subtypes, that when generated from Nestin-cre/ZEG mice, produce GFP+ neurons, recapitulating their in vivo phenotype, and are enriched for the most neurogenic colony subtype. Neurogenesis in vitro is driven by the proliferation of nestin+ progenitors in response to FGF2. I thus provide evidence for a novel neurogenic precursor, the RGLP of the OE, that can be regulated by FGF2, and provide the first evidence for intrinsic differences in the origin and spatiotemporal potential of distinct progenitors during OE development.

Various members of the insulin-like growth factor (IGF) family of growth factors are highly expressed in bone tissue and are vitally important for the normal development and function of bone. Recent studies have shown that IGF-I can associate with the extra-cellular matrix proteins vitronectin (VN) and fibronectin (FN) via IGF binding protein-5 (IGFBP-5). Furthermore, when these complexes are pre-bound to a tissue culture surface they can stimulate enhanced responses in epithelial cell types in vitro. More recently, transforming growth factor-beta 1 (TGF-β1), epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) have also been shown to interact with VN and to elicit functional responses in various cell types. Taken together, these findings indicate that exploitation of the adhesive properties of these ECM proteins might allow immobilisation of various growth factors at the culture surface. This may provide a novel means of coating engineered biomaterial constructs with agents which can elicit specific functional effects in therapeutically important cells, such as those used in cell-based therapeutics for the replacement and / or regeneration of damaged bone tissue. Since both VN and FN are also important matrix components of bone, this study sought to investigate the hypothesis that select pre-bound combinations of these matrix proteins and growth factors could also stimulate functional responses in bone cells and the therapeutically important so called mesenchymal stem cells. Thus it is reported here that pre-bound combinations of VN, IGFBP-5 and IGF-I or FN IGFBP-5 and IGF-I significantly stimulate cell migration in the osteoblast-like SaOS-2 cells. While, VN, IGFBP-5 and IGF-I stimulated cell proliferation over 72 hr, FN, IGFBP-5 and IGF-I did not. Moreover, I found that VN, IGFBP-5 and IGF-I could facilitate alkaline phosphatase (ALP) expression in SaOS-2 cells. VN, FN and EGF on the other hand could sustain SaOS-2 cells for up to 12 days in culture, but could not sustain ALP expression; hence it is possible that these cells may have entered a state of quiescence in response to this treatment. Extending these studies to cells derived from clinical samples, pre-bound combinations of VN / IGFBP-5 / IGF-I were not able to support initiation of human mesenchymal stem cell (hMSC) cultures. Nevertheless, VN alone in serum free media stimulated substantial metabolic activity and protein synthesis in hMSCs once the cultures were established. Moreover, the addition of IGFBP-3 or -5 together with IGF-I can enhance the response to levels equivalent to that observed with 10% FCS. I also report that the responses to VN and TGF-β1 are synergistic and stimulate greater hMSC metabolic activity than 10% FCS. Interestingly, hMSCs cultured in IGF-I or TGF-β1 and low concentrations of VN aggregated, an effect that was not observed when higher concentrations of VN were used. I hypothesise that this aggregation effect was due to endogenous protease activity, and therefore examined MMP-2 and 9 activity in hMSC conditioned media. Both pro-MMP-2 and pro-MMP-9 were constitutively expressed by hMSCs but there was no evidence of the active forms in the conditioned media, indicating that neither IGF-I nor TGF-β1 affect MMP-2 or -9 expression or activation in serum-free media. However, hMSC conditioned media could degrade IGFBP-5, suggesting that there is proteolytic activity within the conditioned media which may impact on the function of ECM / growth factor components in serum-free media settings. Thus, while ECM and growth factors may stimulate desirable responses in therapeutically important cells in serum-free culture, the role that endogenously expressed proteases have on the efficacy of such media supplements needs to be examined closely. Taken together, the studies reported in this thesis provide proof of principle data indicating that select combinations of ECM proteins and growth factors could be utilised in bone tissue engineering applications. This may be achieved for example, as a biomaterial coating, or could form the basis of a viable alternative media supplement for the serum-free culture of hMSCs.

Abstract – The Role of Patched1 in Epidermal Homeostasis Hedgehog (Hh) signalling is a critical pathway involved in the development of many, if not all, organ systems. However the abnormal activation of Hh signalling in fully developed adult organs leads to cancer. Mutation of the Hh signal receptor, Patched1 (Ptc1), causes Naevoid Basal Cell Carcinoma Syndrome, which presents with developmental defects and cancer predisposition. The activation of Hh signalling is seen in a wide range of non-inherited cancer types also, including Medulloblastoma and Basal Cell Carcinoma (BCC) of the skin. BCC is the most common form of human cancer and over 90% of cases are linked to abnormally high Hh signalling. Hh signalling is known to regulate hair follicle morphogenesis during development and more recently has been linked to modulation of the embryonic epidermal stem cell compartment. However both the mechanisms behind this process and the mechanism behind its induction of BCC are still uncharacterised. The aim of this project was to determine the role of Ptc1 in the skin, particularly the adult stem cell compartment, and the role of Hh signalling in BCC formation. The deletion of Ptc1 specifically in the adult epidermis was enabled by the creation of a K14-Cre Recombinase induced Ptc1 Conditional (K14-Cre:Ptc1C/C) transgenic mouse line. Proliferation was increased throughout the epithelia and BCC-like lesions developed within 4 weeks of Ptc1 deletion. This indicates that Hh signalling plays a critical role in repressing cell turnover in the interfollicular epithelium (IFE) and bulge region in the adult despite being previously reported not to play a role in this area. Ptc1 deletion in the epithelia was also found to promote the IFE lineage over hair follicles and expand the expression of many proposed stem cell markers, including K15, Sox9 and p63. K14-Cre:Ptc1C/C transgenic mice also exhibited a severe growth defect, linked to low levels of Igf1 hormone in the serum. Igf1 binding protein alteration in the skin was determined to be the most likely cause and prompted the investigation of Igf axis signalling in Ptc1 deleted epidermis. Insulin-like growth factor binding protein 2 was found to localise to the bulge or stem cell region of the hair follicle, and was increased in K14-Cre:Ptc1C/C epidermis. Igfbp2 was coincident with a loss of PI3K/Akt signal translation. The majority of human BCC samples also expressed Igfbp2 at much higher levels than surrounding normal tissue indicating these results are relevant to the human BCC condition also. Interestingly Hh activation was also shown to increase p38 MAPK throughout the epidermis indicating it is a universal target of Hh signalling in the skin. In summary we have found that Hh signal activation in the epidermis promotes the bulge/stem cell and interfollicular lineages of the skin at the expense of hair follicles. Finally the modulation of PI3K/Akt signalling by Igfbp2 in the bulge is perhaps mediating the effect of Hh signalling via the promotion of the bulge lineage leading to the development of BCC.

Platelet-derived microparticles, released as a result of platelet activation, promote coagulation through the surface exposure of phosphatidylserine, acting as the catalytic site for the conversion of prothrombin to thrombin by the activated coagulation factors X and V. Although elevated numbers of circulating platelet-derived microparticles can be detected in a number of clinical disorders, the methods for the detection of these microparticles are far from standardised. In addition, recent reports have also speculated that not all microparticles may expose phosphatidylserine, demonstrating that the binding of Annexin V, a phosphatidylserine-specific binding protein, is not detectable on a population of microparticles. The initial stage of this thesis was to establish a flow cytometric method for the detection and enumeration of microparticles based on their capacity to bind Annexin V and to utilise this assay to investigate a number of the issues that have limited assay standardisation. The assay could be performed on either stimulated or unstimulated plasma or whole blood samples. Interestingly, plasma microparticle counts were significantly higher than whole blood microparticle counts. The effects of centrifugation alone could not be attributed as the sole source of this discrepancy. The antigenic characteristics of platelet-derived microparticles were also investigated, with platelet-derived microparticles demonstrated to express the platelet glycoproteins CD31, CD41a, CD42a and CD61. Platelet-derived microparticles also expressed CD42b, and this expression was significantly decreased when compared to their progenitor platelets. The expression of the platelet activation markers CD62p, CD63, CD40L and PAC-1 was dependent upon the sample milieu, suggesting that the centrifugation conditions required to generate platelet-poor plasma may lead to artefactual increases in the expression of platelet activation markers. An investigation of the role of the GpIIb/IIIa complex on the formation of platelet-derived microparticles was also performed. A monoclonal antibody to the GpIIb/IIIa complex (Abciximab) significantly inhibited in vitro collagen-stimulated platelet-derived microparticle formation. Interestingly, platelets obtained from two subjects with impaired GpIIb/IIIa activation, demonstrated normal microparticle formation following collagen stimulation, suggesting that the presence of GpIIb/IIIa complex, but not its activation, is required for collagen-induced microparticle formation. A novel mechanism for microparticle formation was also investigated, with platelet-derived microparticles demonstrated to form in response to the sclerosing agents sodium-tetradecyl sulphate and polidocanol. Interestingly, the removal of plasma proteins by the washing of platelets left platelets more susceptible to sclerosant-induced microparticle formation, suggesting that plasma proteins may protect platelets from microparticle formation. The procoagulant activity of platelet-derived microparticles was also investigated using a novel coagulation assay (XACT) specific for the procoagulant phospholipid. An evaluation of this assay demonstrated a significant correlation between Annexin V binding microparticle counts and procoagulant activity in both whole blood and plasma samples. There was more procoagulant activity in whole blood samples than in plasma samples, suggesting that the procoagulant phospholipid activity was also associated with erythrocytes or leukocytes. To further investigate this phenomenon, a whole blood flow cytometric assay was developed to assess Annexin V binding to erythrocytes, leukocytes, platelets and microparticles. This assay demonstrated that a large proportion of Annexin V binding (51.0%) was associated with erythrocytes. Interestingly, a proportion of the Annexin V binding erythrocytes (24.5%) and leukocytes (78.8%) were also associated with platelet CD61 antigen, suggesting that they also bound a platelet or platelet-derived microparticle. The effect of sample anticoagulant on microparticle procoagulant activity was investigated. Microparticle counts were most stable in EDTA anticoagulated samples, but were stable in sodium citrate for up to 15 minutes following sample collection. The procoagulant activity of microparticles was significantly inhibited by EDTA in collagen-stimulated platelet-rich plasma samples, when compared to sodium citrate anticoagulated samples. Although the initial method used to investigate microparticles was based upon their ability to bind Annexin V, it was consistently observed that a large proportion of events in the size region of a microparticle were Annexin V negative. An investigation was therefore commenced into the procoagulant activity of microparticles based on their capacity to bind Annexin V. The presence of Annexin V negative microparticles was confirmed by flow cytometry and the proportion of microparticles that bound Annexin V was dependent upon type of agonist used to stimulate microparticle formation. Varying the assay constituents (calcium concentration / Annexin V concentration / buffer type) did not alter the proportion of Annexin V binding microparticles. When compared to Annexin V positive microparticles, Annexin V negative microparticles expressed significantly higher levels of CD42b on their surface, but possessed significantly decreased expressions of CD62p, and CD63. A significant correlation between the percentage of Annexin V binding and XACT procoagulant activity was found (p=0.03). Furthermore, Annexin V binding inhibited greater than 98% of procoagulant phospholipid activity, suggesting that Annexin V binding was a true reflection of procoagulant activity. Microparticles could be sorted using either a flow cytometric or magnetic sorting strategy. By electron microscopy, Annexin V negative events isolated following magnetic sorting were vesicular structures and not small platelets or the remnants of activated platelets. In summary, this thesis has demonstrated the ability of the flow cytometer and XACT assays to detect microparticles and their procoagulant activity. It has also shown that the use of Annexin V to detect microparticles may warrant further investigation.

Cardiovascular disease, which involves the cardiac, cerebrovascular and peripheral vascular system, is the major cause of morbidity and mortality in the western world. Changes in the vascular microenvironment trigger cascades of molecular events involving altered signaling, transcription and translation of a gene. The aim of this thesis was to increase our understanding on the molecular regulation of activated vascular smooth muscle cells. The first study looking at PDGF-D expression provides new insights into the regulatory mechanisms controlling the phosphorylation of Sp1. Studies performed identified three amino acids in Sp1 (Thr668, Ser670 and Thr681) that is phosphorylated by PKC-zeta activated by AngII. In the second study, the translational regulatory role of a novel gene YrdC induced by injury was investigated. Current knowledge of translational regulators controlling altered gene expression is little and studies in this thesis shows a splice variant of YrdC playing an important role in controlling mRNA translation and thus protein synthesis in the context of injury. The final study investigated in this study was the increased expression of the apoptotic FasL by the activation of GATA6. Although FasL has been extensively studied over the years, this is the first study linking a GATA factor with FasL in any cell type and provides key insights into the transcriptional events underpinning FasL-dependent SMC apoptosis following exposure to AngII.

Bibliography :leaves 250-302. / xix, 302, [19] leaves, [4] leaves of plates : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Results suggest that IGF-I peptides significantly influence gastrointestinal growth in normal adult and suckling rats and indicate they may have therapeutic implications both in conditions of impaired gut function in the adult gastrointestinal tract and in the treatment of gut disease in the immature intestine. / Thesis (Ph.D.)--University of Adelaide, Dept. of Obstetrics & Gynaecology, 1995?

Addendum pasted onto front end-paper. / Bibliography: leaves 176-216. / x, 216 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / The aim of this project is to determine why LR3IGF-1 has such divergent effects in two different species. The study investigates the endocrine regulation of IGF-I and IGF binding protein-3 (IGFBP-3) in the pig and determines the effects of IGF-I and LR3IGF-I treatment on porcine IGF-I and IGFBP-3 expression at the gene and protein level. / Thesis (Ph.D.)--University of Adelaide, Dept. of Obstetrics and Gynaecology, 1997

Errata tipped inside back end paper. / Bibliography: leaves 150-190. / vi, 190 leaves : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Insulin-like growth factors (IGFs) and inflammatory cytokines (e.g. IL-1) affect cartilage metabolism in opposite ways. The actions of IGFs in biological systems are modulated by locally produced IGF binding proteins (IGFBPs). This thesis investigated the effects of the IGFs and inflammatory cytokines on IGFBPs produced by chondrocytes and the subsequent interplay of these factors on proteoglycan production in vitro. To do this, a primary culture of ovine articular chondrocytes was used as an in vitro experimental model system. It was concluded that the IGFBP-5-mediated decrease in proteoglycan synthesis could be a relevant in vivo mechanism by which IL-1 exerts its catabolic effect and disturbs the balance between the synthesis and degradation of cartilage matrix macromolecules in pathological conditions. / Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine, 1998?

Bibliography: leaves 105-136. / vi, 136, [73] leaves, [13] 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. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1992

Two leaves of errata and addenda pasted into back pages. / Bibliography: leaves 174-208. / xix, 208 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis aimed to determine general roles for insulin-like growth factor binding proteins (IGFBPs) in regulating insulin-like growth factor-I (IGF-I) actions in wound repair. Preliminary experiments sought to characterise alterations to IGF-I levels and IGFBP profiles that may occur during wound repair. The effects that interactions with IGFBPs may have on IGF actions in wounds were addressed. Final experiments aimed to determine whether IGFBP-3 proteolysis observed in the initial work of the thesis acted to increase IGF bioavailablity. The results are discussed. / Thesis (Ph.D.)--University of Adelaide, Dept. of Surgery, 2000