Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / Paracrine factors secreted by the oocyte (oocyte-secreted factors, OSFs) regulate a broad range of cumulus cell functions. The capacity of oocytes to regulate their own microenvironment by OSFs may in turn contribute to oocyte developmental competence. The aim of this thesis was to examine whether cumulus cells exhibit a low incidence of apoptosis due to their close association with oocytes and their exposure to OSFs, and to investigate if OSFs have a direct influence on bovine oocyte developmental competence during in vitro maturation (IVM). This thesis includes a series of studies designed to examine by various means the nature of the paracrine network of bone morphogenetic proteins (BMPs) and their binding proteins involved in the regulation of cumulus cell apoptosis. OSFs, in particular BMP- 15 and BMP-6, but not growth differentiation factor 9 (GDF-9), reduced apoptosis of cumulus cells by following a gradient from the site of the oocytes. Morever, follistatin and a BMP6 neutralizing antibody, which antagonized the anti-apoptotic effects of BMP15 and BMP6, respectively, whether alone or combined, blocked ~50% of the antiapoptotic actions of oocytes. These results demonstrated that OSFs, particularly BMP-15 and BMP-6, maintain the low incidence of apoptosis by establishing a localized gradient of bone morphogenetic proteins. Results from the present thesis also demonstrated that OSFs enhance oocyte developmental competence during IVM, whether in their native form as an uncharacterized mix of growth factors secreted by the oocyte, throughout the oocyte maturation period, or as exogenous BMP-15 and GDF-9, during the first 9 hour of IVM. Also, OSFs improved embryo quality as evident by increased blastocyst total and trophectoderm cell numbers. These results were further verified in neutralization experiments of the exogenous growth factors and of the native OSFs. Follistatin and the kinase inhibitor SB-431542, which antagonize BMP-15 and GDF-9, respectively, neutralized the stimulatory effects of the exogenous growth factors, and impaired the developmental competence of control cumulus-oocyte complexes (COCs). The work presented in this thesis has provided multiple lines of evidence that OSFregulation of the COC microenvironment is an important determinant of cumulus cell apoptosis and oocyte developmental programming. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1260892 / Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2006

Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / Paracrine factors secreted by the oocyte (oocyte-secreted factors, OSFs) regulate a broad range of cumulus cell functions. The capacity of oocytes to regulate their own microenvironment by OSFs may in turn contribute to oocyte developmental competence. The aim of this thesis was to examine whether cumulus cells exhibit a low incidence of apoptosis due to their close association with oocytes and their exposure to OSFs, and to investigate if OSFs have a direct influence on bovine oocyte developmental competence during in vitro maturation (IVM). This thesis includes a series of studies designed to examine by various means the nature of the paracrine network of bone morphogenetic proteins (BMPs) and their binding proteins involved in the regulation of cumulus cell apoptosis. OSFs, in particular BMP- 15 and BMP-6, but not growth differentiation factor 9 (GDF-9), reduced apoptosis of cumulus cells by following a gradient from the site of the oocytes. Morever, follistatin and a BMP6 neutralizing antibody, which antagonized the anti-apoptotic effects of BMP15 and BMP6, respectively, whether alone or combined, blocked ~50% of the antiapoptotic actions of oocytes. These results demonstrated that OSFs, particularly BMP-15 and BMP-6, maintain the low incidence of apoptosis by establishing a localized gradient of bone morphogenetic proteins. Results from the present thesis also demonstrated that OSFs enhance oocyte developmental competence during IVM, whether in their native form as an uncharacterized mix of growth factors secreted by the oocyte, throughout the oocyte maturation period, or as exogenous BMP-15 and GDF-9, during the first 9 hour of IVM. Also, OSFs improved embryo quality as evident by increased blastocyst total and trophectoderm cell numbers. These results were further verified in neutralization experiments of the exogenous growth factors and of the native OSFs. Follistatin and the kinase inhibitor SB-431542, which antagonize BMP-15 and GDF-9, respectively, neutralized the stimulatory effects of the exogenous growth factors, and impaired the developmental competence of control cumulus-oocyte complexes (COCs). The work presented in this thesis has provided multiple lines of evidence that OSFregulation of the COC microenvironment is an important determinant of cumulus cell apoptosis and oocyte developmental programming. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1260892 / Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 2006

Thesis (Ph. D.)--Virginia Commonwealth University, 2007. / Prepared for: Dept. of Biochemistry and Molecular Biology. Bibliography: leaves 132 - 149.

Thesis (Ph. D.)--University of Oregon, 2008. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 43-47). Also available online in ProQuest, free to University of Oregon users.

Thesis (M.S.)--University of Michigan, 2004. / Includes bibliographical references.

published_or_final_version / Medicine / Master / Master of Philosophy

骨形態發生蛋白（BMPs）是調節小鼠發育中肢體的頂外胚層脊（AER）功能和趾間程序性細胞死亡（PCD）的分泌信號。然而這些信號的細胞內第二信使者的身份不明。本研究旨在分析Smad蛋白在受骨形態發生蛋白調節的頂外胚層（AER）功能和趾間程序性細胞死亡的功用和相互作用。 基因剔除骨形態發生蛋白信號元件，包括細胞內第二信使者和骨形態發生蛋白，會導致早期胚胎死亡。本研究採用Cre/loxP系統，選擇性地在小鼠發展中肢體的頂外胚層脊和腹側外胚層剔除Smad1和/或Smad5基因。 本研究採用Smad1和/或Smad5 floxed等位基因和En1[superscript Cre/]⁺ 敲等位基因。 / 單一選擇性地在發展中肢體的頂外胚層脊和腹側外胚層剔除Smad1或Smad5不會導致肢體畸形。然而，同時選擇性剔除Smad1/Smad5會導致帶子手指和腳趾(syndactyly)。帶子手指和腳趾的形成是因趾間程序性細胞死亡減少和趾間細胞異常增生。 Smad1/Smad5雙突變體的細胞跟踪實驗顯視腹側外胚層增厚和原在腹側的外胚層En1[superscript Cre/]⁺後裔細胞出現異位轉移到背側趾間位置。 在分子水平上，Fgf8在Smad1/Smad5雙突變體趾間外胚層的表達延長至胚胎發育的第十三天(E13)。這異位Fgf8表達可作為一個趾間的上皮細胞和間質細胞的生存信號。本研究結果表明，Smad1和Smad5在骨形態發生蛋白信號中擔當必需角色，它們充當頂外胚層脊和腹側外胚層細胞內骨形態發生蛋白信號的細胞內第二信使者，並互補對方的功用。頂外胚層脊和腹側外胚層的Smad1/Smad5信號調節趾間組織萎縮。因Smad1/Smad5雙突變體在趾間的程序性細胞死亡出現缺陷，它將會是一個研究程序性細胞死亡調節機制的重要模型。 / Bone morphogenetic proteins (BMPs) are secreted signals that regulate apical ectodermal ridge (AER) functions and interdigital programmed cell death (PCD) of developing mouse limb. However, the identities of the intracellular mediators of these signals are unknown. The present study aims at investigating the role and interaction of Smad proteins in BMPs-regulated AER functions in limb development. Inactivation of BMP signaling components, including intracellular mediators and BMP ligands, will lead to early embryonic lethality. To circumvent the problem, Cre/loxP system was employed to inactivate Smad1 and/or Smad5 selectively in AER and ventral ectoderm of developing mouse limb. Smad1 or/and Smad5 floxed alleles and an En1[superscript Cre/]⁺ knock-in allele was employed for the study. / Single inactivation of either Smad1 or Smad5 did not result in limb abnormalities. However, the Smad1/Smad5 double mutants exhibited syndactyly due to a reduction in interdigital PCD and an increase in interdigital cell proliferation. Cell tracing experiments in the Smad1/Smad5 double mutants showed that ventral ectoderm became thicker and the descendents of ventral En1[superscript Cre/]⁺ expressing ectodermal cells were located at dorsal interdigital regions. At the molecular level, Fgf8 expression was prolonged in the interdigital ectoderm of embryonic day (E) 13 Smad1/Smad5 double mutants, suggesting that the ectopic Fgf8 expression may serve as a survival signal for interdigital epithelial and mesenchymal cells. The result suggests that Smad1 and Smad5 are required and function redundantly as intra-cellular mediators for BMP signaling in the AER and ventral ectoderm. Smad1/Smad5 signaling in the AER and ventral ectoderm regulates interdigital tissue regression of developing limb. The mutants with defects in interdigital PCD could also serve as a valuable model for investigation of PCD regulation machinery. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Wong, Yuk Lau. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 121-133). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract of thesis in English --- p.i / Abstract of thesis in Chinese --- p.iii / Acknowledgements --- p.iv / Table of Contents --- p.v / List of Figures --- p.xii / List of Tables --- p.xv / List of Abbreviations --- p.xvi / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Limb development: a General Overview --- p.1 / Chapter 1.2 --- Signaling centres and axis determination of developing limb buds --- p.8 / Chapter 1.2.1 --- Apical ectodermal ridge and proximal-distal axis --- p.8 / Chapter 1.2.2 --- The zone of polarizing activity and anterior-posterior axis --- p.8 / Chapter 1.2.3 --- Ectoderm and dorsal-ventral axis --- p.9 / Chapter 1.3 --- Programmed cell death in limb development --- p.10 / Chapter 1.3.1 --- Localization of programmed cell death in developing limb bud --- p.10 / Chapter 1.3.2 --- Functions of programmed cell death in limb development --- p.11 / Chapter 1.4 --- Regulation of programmed cell death in developing mouse limb --- p.12 / Chapter 1.4.1 --- Tissues of limb bud involved in regulation of programmed cell death-question to be answered --- p.12 / Chapter 1.4.2 --- Molecular regulation of programmed cell death in developing mouse limb --- p.12 / Chapter 1.4.3 --- Programmed cell death machinery --- p.14 / Chapter 1.5 --- BMPs signaling and limb development --- p.16 / Chapter 1.5.1 --- Functions of BMPs signaling in limb development --- p.16 / Chapter 1.5.2 --- BMPs signaling cascade --- p.17 / Chapter 1.5.3 --- BMPs signaling and programmed cell death --- p.20 / Chapter 1.5.4 --- Intra-cellular mediators of BMPs signaling in limb programmed cell death-question to be answered --- p.21 / Chapter 1.6 --- Scope of the Thesis --- p.22 / Chapter 1.6.1 --- Central aim of the project --- p.22 / Chapter 1.6.2 --- Specific objectives --- p.24 / Chapter Chapter 2 --- Syndactyly in mice lacking Smad1/Smad5 signaling in ventral ectoderm: Implication for its functions in limb development / Chapter 2.1 --- Confirmation of expression of En1[superscript Cre/]⁺ knock-in allele in AER and ventral ectoderm of developing limb bud --- p.27 / Chapter 2.1.1 --- Introduction --- p.27 / Chapter 2.1.2 --- Material --- p.28 / Chapter 2.1.2.1 --- Mouse lines --- p.28 / Chapter 2.1.2.2 --- Chemicals --- p.28 / Chapter 2.1.3 --- Breeding strategy and methodology --- p.28 / Chapter 2.1.4 --- Result --- p.30 / Chapter 2.1.5 --- Discussion --- p.30 / Chapter 2.2 --- En1[supercript Cre/]⁺ inactivation of the Smad1 and Smad5 conditional alleles in the AER and limb ventral ectoderm --- p.33 / Chapter 2.2.1 --- Introduction --- p.33 / Chapter 2.2.2 --- Material --- p.33 / Chapter 2.2.2.1 --- Mouse lines --- p.33 / Chapter 2.2.2.2 --- Antibodies and chemicals --- p.33 / Chapter 2.2.3 --- Methodology --- p.34 / Chapter 2.2.4 --- Results and discussion --- p.36 / Chapter 2.3 --- Single inactivation of Smad1 or Smad5 conditional null allele in the AER and ventral limb ectoderm does not result in observable limb abnormalities --- p.40 / Chapter 2.3.1 --- Introduction --- p.40 / Chapter 2.3.2 --- Material --- p.40 / Chapter 2.3.2.1 --- Mouse lines --- p.40 / Chapter 2.3.2.2 --- Chemicals --- p.41 / Chapter 2.3.3 --- Breeding strategy and methodology --- p.41 / Chapter 2.3.4 --- Results and discussion --- p.41 / Chapter 2.4 --- Inactivation of both Smad1/Smad5 signaling in the limb AER and ventral ectoderm results in interdigital tissue regression defects --- p.45 / Chapter 2.4.1 --- Introduction --- p.45 / Chapter 2.4.2 --- Material --- p.45 / Chapter 2.4.2.1 --- Mouse lines --- p.45 / Chapter 2.4.2.2 --- Chemicals --- p.45 / Chapter 2.4.3 --- Breeding strategies and methodology --- p.45 / Chapter 2.4.4 --- Results --- p.46 / Chapter 2.4.5 --- Discussion --- p.48 / Chapter 2.5 --- Smad1/Smad5 signaling in the limb AER and ventral ectoderm is required for regulating interdigital cell death and cell proliferation --- p.56 / Chapter 2.5.1 --- Introduction --- p.56 / Chapter 2.5.2 --- Material --- p.56 / Chapter 2.5.3 --- Methodology --- p.57 / Chapter 2.5.3.1 --- Cell death assays --- p.57 / Chapter 2.5.3.2 --- Cell proliferation assays --- p.58 / Chapter 2.5.3.3 --- Statistical analysis --- p.59 / Chapter 2.5.4 --- Result --- p.59 / Chapter 2.5.5 --- Discussion --- p.60 / Chapter 2.6 --- Fgf8 is up-regulated at the interdigital distal ectoderm and serves as survival signal for interdigital mesenchyme upon inactivation of the Smad1/Smad5 signaling in AER and ventral ectoderm --- p.66 / Chapter 2.6.1 --- Introduction --- p.66 / Chapter 2.6.2 --- Material --- p.66 / Chapter 2.6.2.1 --- Material for preparation of DIG-labeled RNA probe --- p.66 / Chapter 2.6.2.2 --- Materials for whole-mount and section in-situ hybridization --- p.67 / Chapter 2.6.3 --- Methodology --- p.67 / Chapter 2.6.3.1 --- Preparation of DIG-labeled RNA probe --- p.67 / Chapter 2.6.3.1.1 --- Transformation of DNA into competent cells --- p.67 / Chapter 2.6.3.1.2 --- Preparation of recombinant plasmid --- p.68 / Chapter 2.6.3.1.2.1 --- Birnboim and Doly method --- p.68 / Chapter 2.6.3.1.2.2 --- QIAGEN column method --- p.68 / Chapter 2.6.3.1.3 --- Preparation of linearized recombinant plasmid for riboprobe preparation --- p.70 / Chapter 2.6.3.1.4 --- Preparation of DIG-labelled riboprobes for in situ hybridization --- p.70 / Chapter 2.6.3.2 --- Whole-mount in situ hybridization --- p.72 / Chapter 2.6.3.3 --- Section in situ hybridization --- p.73 / Chapter 2.6.4 --- Results --- p.74 / Chapter 2.6.5 --- Discussion --- p.75 / Chapter 2.7 --- Mesenchymal BMP signals are not altered in the developing autopod of the Smad1/Smad5 mutants --- p.81 / Chapter 2.7.1 --- Introduction --- p.81 / Chapter 2.7.2 --- Material --- p.81 / Chapter 2.7.3 --- Methodology --- p.81 / Chapter 2.7.4 --- Result and discussion --- p.81 / Chapter 2.8 --- Inactivation of Smad1/Smad5 in AER and ventral ectoderm results in postaxial polydactyly --- p.87 / Chapter 2.8.1 --- Introduction --- p.87 / Chapter 2.8.2 --- Material --- p.87 / Chapter 2.8.3 --- Methodology --- p.87 / Chapter 2.8.4 --- Results and discussion --- p.88 / Chapter 2.9 --- Smad1/Smad5 inactivation in the limb ventral ectoderm resulted in ventral ectoderm thickening and ectopic En1-expressing cells and their descendents in the dorsal interdigital ectoderm --- p.91 / Chapter 2.9.1 --- Introduction --- p.91 / Chapter 2.9.2 --- Material p91 / Chapter 2.9.3 --- Methodology --- p.92 / Chapter 2.9.3.1 --- Breeding scheme --- p.92 / Chapter 2.9.3.2 --- X-gal stainin --- p.92 / Chapter 2.9.3.3 --- Immunofluorescence --- p.93 / Chapter 2.9.4 --- Results --- p.93 / Chapter 2.9.5 --- Discussion --- p.94 / Chapter 2.10 --- Inactivation of both Smad1 and Smad5 in ventral limb ectoderm does not cause defects in dorsal-ventral patterning --- p.99 / Chapter 2.10.1 --- Introduction --- p.99 / Chapter 2.10.2 --- Material --- p.99 / Chapter 2.10.3 --- Methodology --- p.99 / Chapter 2.10.1 --- Whole-mount in situ hybridization --- p.99 / Chapter 2.10.2 --- Histological analysis --- p.99 / Chapter 2.10.4 --- Results --- p.100 / Chapter 2.10.5 --- Discussion --- p.101 / Chapter 2.11 --- Contribution of present study --- p.104 / Chapter Chapter 3 --- Proteomic analysis on developing limbs of Smad1/Smad5 knock-out mutant: potential protein candidates that regulate cell death and cell proliferation / Chapter 3.1 --- Introduction --- p.106 / Chapter 3.2 --- Materials --- p.106 / Chapter 3.3 --- Methodology / Chapter 3.3.1 --- Protein sample preparation of limb --- p.107 / Chapter 3.3.2 --- Protein quantification --- p.107 / Chapter 3.3.3 --- 2D gel electrophoresis --- p.108 / Chapter 3.3.4 --- Image analysis --- p.109 / Chapter 3.3.5 --- In gel digestion and MALDI-TOF MS --- p.109 / Chapter 3.4 --- Results --- p.110 / Chapter 3.5 --- Discussion --- p.111 / Chapter Chapter 4 --- General Discussion and Future Direction / Chapter 4.1 --- The intracellular signaling components of BMP signaling --- p.115 / Chapter 4.2 --- The programmed cell death machinery in BMPs-regulated interdigital programmed cell death --- p.117 / Chapter 4.3 --- The possible involvement of reactive oxygen species in BMPs- regulated interdigital programmed cell death --- p.118 / Chapter 4.4 --- Interaction of BMP signaling and retinoic acid signaling --- p.119 / Chapter 4.5 --- Potential candidate genes regulated by Smad1/Smad5 signaling --- p.119 / Bibliography --- p.121 / Publication --- p.134

Thesis (M. Phil.)--University of Hong Kong, 2008. / Includes bibliographical references (leaves 113-131) Also available in print.

5-Aminolevulinic acid (ALA), one of the body's naturally occurring molecules in the biosynthetic pathway to haem, is being used in Photodynamic Therapy (PDT), a new approach to the treatment of cancer. It is a highly reactive molecule, which, upon standing in solution, dimerises to give 2,5-di-(beta-carboxyethyl) pyrazine. This reaction, and other similar ones, have been studied in some detail as it remains a major problem in the clinic, lowering the dose of active drug being administered and forming a molecule of which the toxicology is not known. The initial product of dimerisation is a dihydropyrazine, which is immediately oxidised in air to give a pyrazine. The chemistry of dihydropyrazines has also been investigated. The chemical synthesis of ALA itself has been optimised and derivatives of the molecule have been prepared to try to prevent this dimerisation from occurring and, potentially, increase the tissue selectivity of the drug. Twelve derivatives of ALA have been prepared and biologically tested for their activity in PDT.

Diabetic nephropathy (DN) has become the leading cause of end stage renal disease (ESRD) in developed countries, in which over 90% of the diabetes burden is type 2 in origin. Despite the ever advancing knowledge in the pathogenetic mechanisms of DN, a specific treatment is still lacking and management using contemporary approaches or exploiting newly described therapeutic targets are largely unrewarding. BMP-7 has been reported to confer renal protective effects in acute and chronic kidney disease models, but its potential utility in type 2 diabetic nephropathy remains unknown. In this work, therefore, I hypothesized that BMP-7 confers renal protection that will be explore in vitro in AGE-induced tubular epithelial cells, and in vivo in a murine type 2 DN model. Primary human proximal tubular epithelial cells (PTECs) were growth-arrested and exposed to glycated human serum albumin (AGEs) with or without BMP-7. Inhibitors of different signaling pathways were used to dissect the involvement of each pathway. Nine-week-old db/db mice and their db/m littermates underwent uninephrectomy (Unx) or sham operation, and received BMP-7 (300 μg/kg body weight) or vehicle treatment intraperitoneally every other day for 8 weeks before sacrifice. In cultured human PTECs, exposure to AGEs induced overexpression of ICMA-1, MCP-1, IL-8 and IL-6, involving activation of at least p44/42 and p38 MAPK signaling. BMP-7 dose-dependently attenuated AGE-induced up-regulation of ICMA-1, MCP-1, IL-8 and IL-6 at both mRNA and protein levels. Moreover, BMP-7 suppressed AGE-induced p38 and p44/42 MAPK phosphorylation and reactive oxygen species production in PTECs. Compared with vehicle control, Unx db/db mice treated with BMP-7 for 8 weeks had significantly lower urinary albumin-to-creatinine ratio (3549±816.2 μg/mg vs. 8612±2037 μg/mg, p=0.036), serum BUN (33.26±1.09 mg/dL vs. 37.49±0.89 mg/dL, p=0.006), and renal cortical expression of ICMA-1 and MCP-1 at both gene and protein levels. PAS staining of kidney tissue showed less severe tubular damage and interstitial inflammatory cell infiltration in the BMP-7-treated group. In conclusion, this series of experiments demonstrated that BMP-7 attenuates tubular pro-inflammatory responses in diabetic kidney disease by suppressing oxidative stress and multiple signaling pro-inflammatory pathways including p38 and p44/42 MAPK. These observations are largely translated in animals with diabetic kidney inflammation. The potential application of BMP-7 as a therapeutic molecule in diabetic nephropathy warrants further investigation and development. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy