Includes list of publications produced during the period of candidature / Includes bibliographical references (leaves 254-274) / xiv, 274 leaves : ill. (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 Animal Science, 2003

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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

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

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Insulin sensitivity has been positively correlated with a healthy and extended lifespan, while insulin resistance, decreased insulin sensitivity, has been linked to aging and is the main indicative of type 2 diabetes. Growth Hormone Receptor/ Binding Protein Knockout mice (GHRKO), although obese, are characterized by high insulin sensitivity and a prolonged lifespan. Due to the absence of growth hormone receptors (GHR), growth hormone (GH) is unable to activate its downstream pathway. Interestingly, the secretory activity of visceral fat in GHRKO mice is altered stimulating insulin sensitivity. In this study, we transplanted normal (N) mice with GHRKO visceral fat pads to determine the role of visceral fat developed with the absence of GH signaling on the insulin-signaling pathway in animals with physiologically normal GH action. We found that the visceral fat transplant (VFT) helped the normal mice gain the beneficial effects of fat developed in the absence of GH and caused improvement of their whole body insulin sensitivity when comparing with sham-operated mice and with mice that received visceral fat from N animals. In presented study, RT-PCR was used to determine the levels of hepatic mRNA expression between three experimental groups including Normal-sham mice (N-S), normal mice transplanted with visceral fat from normal animals (N-N), and normal mice receiving visceral fat from GHRKO mice (N-KO). Additionally, Western Blot and ELISA were used to determine the level of total and phosphorylated proteins. By studying the effect of visceral fat transplant from GHRKO or N mice on the whole body insulin signaling in N male mice, and testing different genes expression and proteins quantification, we can shed light on the mechanism by which white adipose tissue (WAT) regulates whole body insulin sensitivity and longevity as well as understanding the role of WATs in development of diabetes and the process behind insulin resistance.

A poluição atmosférica de São Paulo (SP) pode provocar alterações cardiovasculares em seres humanos e animais experimentais, com maior vulnerabilidade em crianças e fetos. O mecanismo fisiopatológico que explicaria a relação entre a exposição aos poluentes e doenças cardiovasculares não está totalmente estabelecido, sendo que o estresse oxidativo pode estar ligado ao dano e morte celular. Há evidências de que o dano oxidativo pelo mecanismo da peroxidação lipídica pode estar relacionado às causas de diversas cardiovasculopatias. Estudamos o efeito da exposição ao ar ambiental nos níveis de peroxidação lipídica no coração de camundongos nos períodos pré e pós-natal. Os animais foram mantidos em duas câmaras de exposição, uma recebendo ar ambiente e outra ar filtrado, em quatro diferentes grupos: 1) LL: gestados e crescidos em câmara com ar filtrado, 2) PP: gestados e crescidos em câmara com ar poluído de SP, 3) PL: gestados na câmara poluída e crescidos na limpa, e 4) LP: gestados na câmara limpa e crescidos na poluída. A peroxidação lipídica do miocárdio foi avaliada tanto pelo método TBA como por imunohistoquímica para 15-F2t-isoprostano. As concentrações de malondialdeído (MDA, indicador de peroxidação lipídica) foi maior nos animais PP quando comparados aos LL (p = 0,004) e PL (p = 0,026), e não mostrou diferença significativa em relação ao grupo LP (p = 0,894). Os valores de MDA para animais PL e LP mostraram-se equivalentes (p = 0,168). Chama a atenção que o grupo PL apresentou um valor de MDA maior que o LL (p = 0,026). A fração de volume de miocárdio marcada imunohistoquimicamente para 15- F2t-isoprostano apresentou valores maior em PL (p = 2,884x10-5), LP (p = 6,632x10-6) e PP (p = 5,45x10-8) que em LL. O valor de PP foi maior que os de PL e LP (p = 3,661x10-4 e 1,058x10-3, respectivamente), sendo esses últimos equivalentes entre si (p = 0,624). A análise ultra-estrutural mostrou de maneira consistente a presença de lisossomos secundários contendo estruturas lipídicas membranosas nos grupos LP e PP. A porcentagem média de arteríolas com área entre 200 e 1000 ?m² em relação ao número total de vasos de cada grupo foi maior no grupo PP do que nos grupos LL e PL (p=0,0387 e p=0,0362, respectivamente). Estes resultados sugerem que existem altos níveis de peroxidação lipídica no tecido cardíaco dos animais expostos ao ar ambiental de SP. Chama a atenção o fato de que a exposição intra-uterina ter implicado em níveis maiores de estresse oxidativo na fase adulta, mesmo com a melhoria das condições ambientais. Comparam-se estes achados no miocárdio a outros resultados da literatura. / I t is well known that air pollution exposure in São Paulo can elicit cardiovascular injuries in humans and experimental animals and that children and fetuses appear to be particularly vulnerable. However, the mechanisms involved in this cardiovascular damage are not well understood. It has been suggested that the oxidative stress generated by air pollution exposure can trigger tissue injury. There is evidence supporting the idea that injury caused by lipid peroxidation may be related to the causes of several cardiovascular diseases. The aim of this study was to investigate the effects of prenatal and postnatal exposure to urban air pollution on the myocardium lipid peroxidation levels of adult mice. Myocardium lipid peroxidation was determined by the TBA method and by the detection of 15-F2t-isoprostan by immunohistochemical technique. The animals were placed in two chambers: one received air that passed through an air filter (clean) and the second received ambient air (polluted), according to four different exposure procedures: 1) Clean (CC): prenatal and postnatal in the clean chamber (control group), 2) Polluted (PP): prenatal and posnatal in the polluted chamber, 3) Polluted-clean (PC): prenatal in the polluted and posnatal in the clean chamber and 4) Clean-polluted (CP): prenatal in the clean and posnatal in the polluted chamber. The concentration of of malondialdehyde (MDA, a indicator of lipid peroxidation) was higher in group PP compared to CC (p = 0.004) and to PC (p = 0.026), and was not different of group CP (p = 0.894). The values of MDA for groups PC and CP turned to be equal (p = 0.168). Interestingly, group PC had a higher value of MDA than group CC (p = 0.026). The volume fraction of myocardium with detection of 15-F2tisoprostane is higher in PC (p = 2.884x10-5), CP (p = 6.632x10-6) and PP (p = 5.45x10-8) than in CC. The value of PP in higher than those of PC and CP (p = 3.661x10-4 and 1.058x10-3, respectively), while the latter two were equal to each other (p = 0.624). ). The mean ratio of arterioles wiht lumen area between 200 and 1000?m² to the total number of vessels in each group was higher in PP than in CC and PC (p=0.0387 and p=0.0362, respectively). These results, which suggest that exposure to air pollution is associated to higher levels of lipid peroxidation in the myocardium, are compared to other results previously published about respiratory and reproductive alterations related to pollution. Interestingly, the increased levels of lipid peroxidation in the PC group gives evidence that the prenatal exposure to urban air pollution can be linked to cardiovascular effects in adult life.