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.

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

Bibliography : leaves 79-101 / xix, 101, [54] leaves, [12] 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, 1992

Impaired implantation and placental development have been implicated in several disorders of pregnancy such as unexplained miscarriage, preeclampsia, and intrauterine growth retardation. Insulin-Like Growth Factor (IGF)-II has previously been shown to promote blastocyst development and placental growth and function. We were interested in how IGF-II interacts with other factors throughout blastocyst development, implantation and placentation in the mouse to improve pregnancy outcome. In vitro embryo culture increases the risk of pregnancy complications associated with poor placentation. Recent research has focussed on optimising the culture conditions to more resemble that of the in vivo environment. IGF-II, Urokinase Plasminogen Activator (uPA) and Plasminogen individually have all been shown to be important for embryo development. However, it is likely that a combination of factors is required to counteract the negative effects of in vitro culture. Here we show that IGF-II, uPA and Plasminogen, in combination, significantly improve mouse blastocyst hatching rates and implantation rates on day 8 and doubles the number of mothers that are pregnant after embryo transfer. Following implantation, IGF-II is suggested to play a role in promoting placental development and function. We demonstrate that IGF-II is co-localised with both IGF receptors throughout early pregnancy in trophoblasts and in the developing blood vessels and adjacent stromal cells in the mesometrial decidua. This suggests that IGF-II may play a role in both decidual angiogenesis and placentation. We suggest that perhaps murine trophoblasts secrete molecules such as IGF-II to promote angiogenesis in the decidua early in pregnancy to compensate for their shallow invasion and allow for adequate trophoblast remodelling later in pregnancy. The first trimester human placenta experiences a low oxygen environment. The Hypoxia-Inducible Factors (HIFs) mediate the response to low oxygen, inducing genes such as IGF-II. Currently, the role of oxygen in mouse placentation, the mechanisms by which HIFs promote placentation or their interaction with IGF-II in the placenta is unknown. Here, we demonstrate that the early mouse implantation site is exposed to low oxygen levels similar to those seen in humans and expresses HIF-1 protein. We were interested then in the interaction between IGF-II, oxygen and HIFs in trophoblasts in vitro. Prolonged exposure to low oxygen reduced trophoblast outgrowth, and increased Tpbp mRNA levels, suggesting commitment to the spongiotrophoblast lineage. Interestingly, we found that antisense (as) Hif-1 may mediate the response to prolonged hypoxia in murine trophoblasts. Importantly, Hif-1 and Hif-2 were differentially regulated by oxygen and IGF-II in cultured trophoblast cells suggesting a novel interaction between IGF-II and oxygen. In conclusion, it appears that IGF-II is a central growth factor which interacts with other molecules to regulate a wide variety of process in early pregnancy to promote blastocyst development, implantation and placentation. The results outlined in this thesis demonstrate a novel interaction between IGF-II, uPA and Plasminogen in promoting blastocyst development and implantation which may be used to improve pregnancy outcome following ART. In addition, we have also identified a novel interaction between IGF-II, oxygen and the HIF system which may regulate trophoblast function. This has important implications not only for placental research, but also for cancer research. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1326731 / Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2008

Impaired implantation and placental development have been implicated in several disorders of pregnancy such as unexplained miscarriage, preeclampsia, and intrauterine growth retardation. Insulin-Like Growth Factor (IGF)-II has previously been shown to promote blastocyst development and placental growth and function. We were interested in how IGF-II interacts with other factors throughout blastocyst development, implantation and placentation in the mouse to improve pregnancy outcome. In vitro embryo culture increases the risk of pregnancy complications associated with poor placentation. Recent research has focussed on optimising the culture conditions to more resemble that of the in vivo environment. IGF-II, Urokinase Plasminogen Activator (uPA) and Plasminogen individually have all been shown to be important for embryo development. However, it is likely that a combination of factors is required to counteract the negative effects of in vitro culture. Here we show that IGF-II, uPA and Plasminogen, in combination, significantly improve mouse blastocyst hatching rates and implantation rates on day 8 and doubles the number of mothers that are pregnant after embryo transfer. Following implantation, IGF-II is suggested to play a role in promoting placental development and function. We demonstrate that IGF-II is co-localised with both IGF receptors throughout early pregnancy in trophoblasts and in the developing blood vessels and adjacent stromal cells in the mesometrial decidua. This suggests that IGF-II may play a role in both decidual angiogenesis and placentation. We suggest that perhaps murine trophoblasts secrete molecules such as IGF-II to promote angiogenesis in the decidua early in pregnancy to compensate for their shallow invasion and allow for adequate trophoblast remodelling later in pregnancy. The first trimester human placenta experiences a low oxygen environment. The Hypoxia-Inducible Factors (HIFs) mediate the response to low oxygen, inducing genes such as IGF-II. Currently, the role of oxygen in mouse placentation, the mechanisms by which HIFs promote placentation or their interaction with IGF-II in the placenta is unknown. Here, we demonstrate that the early mouse implantation site is exposed to low oxygen levels similar to those seen in humans and expresses HIF-1 protein. We were interested then in the interaction between IGF-II, oxygen and HIFs in trophoblasts in vitro. Prolonged exposure to low oxygen reduced trophoblast outgrowth, and increased Tpbp mRNA levels, suggesting commitment to the spongiotrophoblast lineage. Interestingly, we found that antisense (as) Hif-1 may mediate the response to prolonged hypoxia in murine trophoblasts. Importantly, Hif-1 and Hif-2 were differentially regulated by oxygen and IGF-II in cultured trophoblast cells suggesting a novel interaction between IGF-II and oxygen. In conclusion, it appears that IGF-II is a central growth factor which interacts with other molecules to regulate a wide variety of process in early pregnancy to promote blastocyst development, implantation and placentation. The results outlined in this thesis demonstrate a novel interaction between IGF-II, uPA and Plasminogen in promoting blastocyst development and implantation which may be used to improve pregnancy outcome following ART. In addition, we have also identified a novel interaction between IGF-II, oxygen and the HIF system which may regulate trophoblast function. This has important implications not only for placental research, but also for cancer research. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1326731 / Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2008