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Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 5 uppsatser.

Tse Chui-ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 130-146). / Abstracts in English and Chinese. / Acknowledgments --- p.i / Abstract --- p.ii / 論文撮要 --- p.iii / List of Figures and Tables --- p.iv / Abbreviations --- p.vi / Table of contents --- p.vii / Chapter Chapter I --- Introduction --- p.1 / Chapter 1.1 --- Literature review --- p.1 / Chapter 1.1.1 --- An overview of IGFs --- p.3 / Chapter 1.1.2 --- Molecular biology of IGFs --- p.5 / Chapter 1.1.2.1 --- IGF-I and IGF-II genes and mRNAs --- p.5 / Chapter 1.1.2.2 --- Amino acid sequences of IGF-II --- p.8 / Chapter 1.1.2.3 --- Imprinting of IGF-II --- p.12 / Chapter 1.1.3 --- IGF distribution in tissues and body fluids --- p.14 / Chapter 1.1.3.1 --- IGF in serum --- p.14 / Chapter 1.1.3.2 --- IGF binding proteins --- p.16 / Chapter 1.1.4 --- IGF receptors --- p.19 / Chapter 1.1.4.1 --- Structures of the IGF receptors --- p.20 / Chapter 1.1.4.2 --- Ligand binding of the IGF receptors --- p.21 / Chapter 1.1.4.3 --- Signal transduction and biological response --- p.22 / Chapter 1.1.5 --- Biological effects of IGF --- p.24 / Chapter 1.1.6 --- Expression of recombinant IGF --- p.28 / Chapter 1.2 --- Rationale and Objective --- p.29 / Chapter Chapter II --- Methodology --- p.33 / Chapter 2.1 --- Design of degenerate primers --- p.33 / Chapter 2.2 --- Cloning --- p.35 / Chapter 2.2.1 --- DNA extraction from agarose gel --- p.35 / Chapter 2.2.2 --- Linearization and dephosphorylation of plasmid DNA --- p.35 / Chapter 2.2.3 --- Blunt-end ligation of amplicon with linearized plasmid --- p.36 / Chapter 2.2.4 --- T/A ligation of amplicon with linearized plasmid --- p.37 / Chapter 2.2.5 --- Sticky end ligation of foreign DNA with linearized plasmid --- p.37 / Chapter 2.2.6 --- Preparation of competent of E. coli stain DHI5α cells --- p.38 / Chapter 2.2.7 --- Transformation of plasmid vector into competent cells (heat-shock/ electroporation) --- p.39 / Chapter 2.2.8 --- "Spread single colony, PCR check clone and inoculation" --- p.40 / Chapter 2.2.9 --- Small scale alkali preparation of plasmid DNA --- p.41 / Chapter 2.2.10 --- Large scale preparation of plasmid DNA --- p.41 / Chapter 2.2.11 --- Nucleotide sequencing --- p.41 / Chapter 2.2.11.1 --- Manual sequencing --- p.41 / Chapter 2.2.11.2 --- PCR sequencing --- p.43 / Chapter 2.3 --- Northern blot --- p.45 / Chapter 2.4 --- Preparation of radio-labeled probe and hybridization of radio-labeled probe to nylon immobilized nucleic acid --- p.46 / Chapter 2.5 --- RACE --- p.48 / Chapter 2.5.1 --- Design of gene-specific primer --- p.51 / Chapter 2.5.2 --- First strand cDNA synthesis --- p.51 / Chapter 2.5.3 --- TdT tailing of cDNA --- p.52 / Chapter 2.6 --- Poly-A tract extraction --- p.53 / Chapter 2.7 --- Tissue distribution of mRNA --- p.53 / Chapter 2.7.1 --- Tissue preparation --- p.53 / Chapter 2.7.2 --- Total RNA extraction --- p.54 / Chapter 2.7.3 --- Formaldehyde agarose gel electrophoresis of RNA --- p.54 / Chapter 2.8 --- RNAse protection assay --- p.55 / Chapter 2.8.1 --- Antisense probe generation --- p.56 / Chapter 2.8.2 --- Preparation of the sample RNA --- p.58 / Chapter 2.8.3 --- Hybridization --- p.58 / Chapter 2.8.4 --- RNase digestion of hybridized probe and sample RNA --- p.59 / Chapter 2.8.5 --- Preparation of radioactive marker --- p.60 / Chapter 2.8.6 --- Separation and detection of protected fragments --- p.60 / Chapter 2.8.7 --- Data processing and statistical analysis --- p.61 / Chapter 2.9 --- Injection of GH --- p.62 / Chapter 2.10 --- Recombinant protein expression --- p.62 / Chapter 2.10.1 --- Plasmid construction --- p.62 / Chapter 2.10.2 --- Expression --- p.63 / Chapter 2.11 --- Resolution of proteins on SDS-PAGE --- p.63 / Chapter 2.12 --- Purification --- p.64 / Chapter 2.13 --- Western transfer --- p.64 / Chapter 2.14 --- Immunodetection --- p.65 / Chapter Chapter III --- Results & Discussion --- p.67 / Chapter 3.1 --- Isolation and characterization of IGF-II cDNA and its gene organization --- p.67 / Chapter 3.1.1 --- Introduction --- p.67 / Chapter 3.1.2 --- Results --- p.68 / Chapter 3.1.2.1 --- Generation of a fragment of the common carp IGF-II cDNA by PCR --- p.68 / Chapter 3.1.2.2 --- Isolation of the full length common carp IGF-II cDNA by RACE. --- p.69 / Chapter 3.1.2.3 --- Nucleotide sequence analysis --- p.74 / Chapter 3.1.2.4 --- Relationship of common carp IGF-II to common carp IGF-I and insulin --- p.78 / Chapter 3.1.2.5 --- Confirmation of the presence of IGF-II in common carp --- p.79 / Chapter 3.1.2.6 --- Multiple mRNA forms of common carp IGF-I and IGF-II --- p.80 / Chapter 3.1.2.7 --- Gene organization of the common carp IGF-II gene --- p.83 / Chapter 3.1.3 --- Discussion --- p.86 / Chapter 3.2 --- Tissue specific distribution of IGF-I and IGF-II mRNA and their hormonal regulation --- p.90 / Chapter 3.2.1 --- Introduction --- p.90 / Chapter 3.2.2 --- Results --- p.94 / Chapter 3.2.2.1 --- RNase protection assay measurement of tissue mRNA levels in juvenile and adult common carp --- p.94 / Chapter 3.2.2.2 --- Expression of IGF-II mRNA during larval development --- p.99 / Chapter 3.2.2.3 --- Effect of GH on IGF-I and IGF-II mRNA levels in brain and Hver of juvenile common carp --- p.102 / Chapter 3.2.3 --- Discussion --- p.106 / Chapter 3.3 --- Recombinant common carp IGF-II expressed in E. coli --- p.110 / Chapter 3.3.1 --- Introduction --- p.110 / Chapter 3.3.2 --- Results --- p.112 / Chapter 3.3.2.1 --- Product of recombinant common carp IGF-II --- p.112 / Chapter 3.3.2.2 --- Purification of common carp IGF-II --- p.115 / Chapter 3.3.2.3 --- Immunodetection --- p.117 / Chapter 3.3.3 --- Discussion --- p.118 / Chapter Chapter IV --- General conclusion --- p.120 / Appendix: Reagents --- p.124 / Reference list --- p.130

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

Tse Chui-ling. / "July 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 172-185). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web.

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

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 96-116).

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed Oct. 13, 2008). Includes bibliographical references (p. 56-64).