The Hippo pathway is a master regulator of cell proliferation and organ size, namely through regulation of transcriptional co-activators YAP and TAZ which bind TEAD1-4 transcription factors. The Hippo effector YAP is dysregulated in many human solid tumours including rhabdomyosarcoma and oesophageal cancer. Additionally, persistent hyperactivity of YAP in activated but not quiescent satellite cells can give rise to embryonal rhabdomyosarcoma. However, the question of exactly how YAP acts as an oncogene and actively gives rise to tumour progression in these cancers remains unknown. In this thesis I characterised the mechanisms which determine the functional role of YAP in driving instability in the genome. Secondly, lentiviral mediated knockdown of YAP is performed to determine and investigate its effect on tumorigenesis. Thirdly, gene sets from constitutive YAP S127A induced mouse ERMS tumours subjected to array-CGH were further analysed. Finally, I cloned chicken Yap1, Tead1 and Fstl5 to identify its role during chick embryonic development, by the retroviral mediated loss of function approach. The results demonstrated that constitutive YAP S127A expression in-vitro as well as in-vivo induces chromosomal instability by increasing the rate of mitotic chromosome segregation errors and copy number alterations of oncogenes and other cancer related genes. Recurrent copy number gains of the p53 inhibitor Mdm2 were observed in YAP S127A-driven ERMS tumours. Moreover, lentiviral mediated YAP knockdown showed significant reduction in proliferation, migration and invasion as well as transformation potential in human cultured cancer cells. Moreover, retroviral YAP S127A expression during early stages of chick embryo development did not lead to an overt phenotype and showed poor survival. Additionally, I have cloned RCAS-RNAi vectors to study the loss of function effect on Hippo targets and Fstl5 during chicken embryo development. Collectively, my data provides insight into the mechanisms with which YAP could drive tumorigenesis and that YAP knockdown can be considered a potential therapeutic target to reduce cancer progression.

Leung Yee Fong, Vivian. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 159-169). / Acknowledgments --- p.i / Legend for figures --- p.ii / Legend for tables --- p.v / List of abbreviations --- p.vii / Abstract --- p.viii / Chapter Ch 1 --- Introduction --- p.1 / Chapter 1.1 --- Embryology --- p.1 / Chapter 1.1.1 --- Embryology of amniotic cavity --- p.1 / Chapter 1.1.2 --- Embryology of kidney and bladder --- p.3 / Chapter Ch 2 --- Background: What is already known about amniotic fluid volume? --- p.7 / Chapter 2.1 --- Normal physiology --- p.7 / Chapter 2.1.1 --- The origin of amniotic fluid: Where does it come from? --- p.8 / Chapter 2.1.2 --- Where does the amniotic fluid go? How reabsorbed? --- p.14 / Chapter 2.1.3 --- How is amniotic fluid volume controlled? --- p.18 / Chapter 2.2 --- Abnormal physiology --- p.26 / Chapter 2.2.1 --- Too much liquor: polyhydramnios --- p.26 / Chapter 2.2.2 --- Too little liquor: oligohydramnios --- p.28 / Chapter 2.2.3 --- Diseases and gender differences that may be related to parity and amniotic fluid volume --- p.30 / Chapter 2.3 --- Techniques of measuring amniotic fluid volume --- p.32 / Chapter 2.3.1 --- History --- p.32 / Chapter 2.3.2 --- Current most popular technique: amniotic fluid index --- p.38 / Chapter 2.4 --- Summary of what is known and not yet known about amniotic fluid volume --- p.48 / Chapter Ch 3 --- Aims of this study --- p.49 / Chapter Ch4 --- Method --- p.50 / Chapter 4.1 --- Equipment --- p.50 / Chapter 4.2 --- Subject selection criteria --- p.50 / Chapter 4.2.1 --- Criteria --- p.50 / Chapter 4.2.2 --- Total number of subjects studied --- p.51 / Chapter 4.2.3 --- Total number of subjects selected fulfilling all criteria --- p.51 / Chapter 4.2.4 --- Subject preparation --- p.52 / Chapter 4.3 --- Technique --- p.53 / Chapter 4.3.1 --- "Standard measurement of BPD， AC, FL and EFW" --- p.53 / Chapter 4.3.2 --- Standard measurement of Doppler --- p.54 / Chapter 4.3.3 --- Amniotic fluid index --- p.55 / Chapter 4.3.4 --- Bladder volume --- p.59 / Chapter 4.3.5 --- Fetal renal pelvis --- p.61 / Chapter 4.3.6 --- Intra-observer error techniques and calculation --- p.63 / Chapter 4.4 --- Techniques used in analysis --- p.65 / Chapter Ch5 --- Results --- p.67 / Chapter 5.1 --- Fetal parameters --- p.68 / Chapter 5.1.1 --- Fetal biparietal diameter (BPD) --- p.68 / Chapter 5.1.2 --- Fetal abdominal circumference (AC) --- p.69 / Chapter 5.1.3 --- Fetal femur length (FL) --- p.70 / Chapter 5.1.4 --- Pulsatility index values of umbilical artery --- p.71 / Chapter 5.1.5 --- Birth weight (BW) --- p.74 / Chapter 5.1.6 --- Estimated fetal weight --- p.76 / Chapter 5.2 --- Amniotic fluid index --- p.79 / Chapter 5.2.1 --- Amniotic fluid index-overall --- p.79 / Chapter 5.2.2 --- Amniotic fluid index-male and female --- p.81 / Chapter 5.2.3 --- The ten segments of amniotic fluid index distribution --- p.83 / Chapter 5.2.4 --- Amniotic fluid index relationship to estimated fetal weight --- p.86 / Chapter 5.2.5 --- Amniotic fluid index with gravidity and parity --- p.89 / Chapter 5.2.6 --- Amniotic fluid index with estimated fetal weight of different parity (best fit line) for both male and female --- p.93 / Chapter 5.3 --- Fetal urinary bladder volume (BV) --- p.96 / Chapter 5.3.1 --- Bladder volume-overall --- p.96 / Chapter 5.3.2 --- Bladder volume-male and female --- p.97 / Chapter 5.3.3 --- Bladder volume with estimated fetal weight- overall --- p.100 / Chapter 5.3.4 --- Bladder volume with estimated fetal weight in both male and female --- p.101 / Chapter 5.3.5 --- Bladder volume with gravidity and parity --- p.103 / Chapter 5.3.6 --- Bladder volume with amniotic fluid index --- p.105 / Chapter 5.4 --- Anteroposterior diameter of the fetal renal pelvis --- p.106 / Chapter 5.5 --- Hydronephrosis index values --- p.107 / Chapter Ch 6 --- Discussion --- p.108 / Chapter 6.1 --- Review of the study --- p.108 / Chapter 6.2 --- Discussion on subject --- p.111 / Chapter 6.2.1 --- Gestational age chosen --- p.111 / Chapter 6.2.2 --- Subject preparation --- p.112 / Chapter 6.3 --- Discussion of method --- p.114 / Chapter 6.3.1 --- Equipment --- p.114 / Chapter 6.3.2 --- Technique --- p.117 / Chapter 6.4 --- Discussion on results --- p.128 / Chapter 6.4.1 --- Normality of population --- p.128 / Chapter 6.4.2 --- Low birth weight/ IUGR in Chinese and Caucasian --- p.129 / Chapter 6.4.3 --- Cut-off points to detect oligohydramnios and polyhydramnios --- p.132 / Chapter 6.4.4 --- Amniotic fluid index-relationship with fetal weight --- p.143 / Chapter 6.4.5 --- Amniotic fluid index-relationship to parity --- p.145 / Chapter 6.4.6 --- "Relationship between gender, estimated fetal weight and amniotic fluid index" --- p.147 / Chapter 6.4.7 --- Parity and cut-off points for oligohydramnios and polyhydramnios --- p.150 / Chapter 6.4.8 --- Relationship of amniotic fluid volume to urinary function --- p.152 / Chapter Ch 7 --- Conclusions --- p.157 / References --- p.159

by Pui-kuen Lee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 168-182). / ACKNOWLEDGMENT --- p.I / ABSTRACT --- p.II / TABLE OF CONTENTS --- p.IV / ABBREVIATIONS --- p.X / LIST OF FIGURES --- p.XII / LIST OF TABLES --- p.XIV / Chapter CHAPTER1 --- INTRODUCTION AND BACKGROUND --- p.1 / Chapter 1.1 --- ROLE OF CYTOKINES IN MOUSE EMBRYONIC DEVELOPMENT --- p.1 / Chapter 1.1.1 --- Why mouse model --- p.1 / Chapter 1.1.2 --- Embryonic development of mouse --- p.1 / Chapter 1.1.3 --- An overview of cytokines --- p.4 / Chapter a. --- Classes of cytokines --- p.5 / Chapter i) --- Growth factors --- p.5 / Chapter ii) --- Interleukins --- p.7 / Chapter iii) --- Colony-stimulating factors --- p.9 / Chapter iv) --- Interferons --- p.10 / Chapter v) --- Tumor necrosis factor --- p.11 / Chapter b. --- Cytokine networks --- p.12 / Chapter c. --- Role of cytokines in the whole organism --- p.13 / Chapter 1.1.4 --- Cytokine and receptor gene expression in mouse embryonic development --- p.15 / Chapter a. --- Murine embryonic stem cell model --- p.15 / Chapter b. --- Leukemia Inhibitory Factor (LIF) in mouse embryos --- p.16 / Chapter c. --- IL-6 in mouse embryo --- p.19 / Chapter d. --- Ciliary Neurotrophic Factor (CNTF) in mouse embryo --- p.19 / Chapter e. --- TNF-a and TNF-β in mouse embryos --- p.20 / Chapter f. --- TGF-a in mouse embryos --- p.20 / Chapter g. --- TGF-P in mouse embryos --- p.20 / Chapter h. --- Stem cell factor / c-kit --- p.21 / Chapter i. --- Other cytokines in mouse embryos --- p.22 / Chapter j. --- Cytokine receptors --- p.24 / Chapter 1.2 --- NEUROPOIETIC CYTOKINES --- p.28 / Chapter 1.2.1 --- Family members --- p.28 / Chapter 1.2.2 --- Shared signal transducer gpl30 --- p.29 / Chapter 1.2.3 --- "LIF, CNTF and OSM inhibit differentiation of embryonic stem cells" --- p.31 / Chapter 1.3 --- BIOLOGY OF ONCOSTATIN M (OSM) --- p.33 / Chapter 1.3.1 --- Physical properties of OSM --- p.33 / Chapter 1.3.2 --- Biological activities of OSM --- p.34 / Chapter 1.3.3 --- Molecular aspect of OSM --- p.35 / Chapter 1.4 --- AIMS OF THE STUDY --- p.38 / Chapter CHAPTER2 --- CYTOKINE GFNE EXPRESSION DURING MOUSE EMBRYONIC DEVELOPMENT --- p.40 / Chapter 2.1 --- INTRODUCTION --- p.40 / Chapter 2.1.1 --- Rationale --- p.40 / Chapter 2.1.2 --- Design of primers --- p.43 / Chapter 2.2 --- MATERIALS --- p.44 / Chapter 2.2.1 --- Chemicals and Reagents --- p.44 / Chapter 2.2.2 --- Enzymes --- p.45 / Chapter 2.2.3 --- Buffers --- p.45 / Chapter 2.2.4 --- Solutions --- p.47 / Chapter 2.2.5 --- Probe labeling and detection kits --- p.48 / Chapter 2.2.6 --- Primers and internal probes --- p.49 / Chapter 2.3 --- METHODS --- p.52 / Chapter 2.3.1 --- Preparation of total RNA from mouse embryos at different stages --- p.52 / Chapter a. --- Mice dissection for embryo --- p.52 / Chapter b. --- Guanidinium thiocyanate cell lysate --- p.52 / Chapter c. --- Isolation of RNA by centrifugation through CsCl gradient --- p.53 / Chapter d. --- Spectrophotometric determination of RNA amount --- p.54 / Chapter 2.3.2 --- Preparation of embryo sections --- p.54 / Chapter 2.3.3 --- Primers and internal probes --- p.55 / Chapter 2.3.4 --- Cytokine mRNA Phenotyping by Reverse transcription-Polymerase chain reaction --- p.56 / Chapter a. --- Reverse transcription (First strand cDNA synthesis) --- p.56 / Chapter b. --- Polymerase chain reaction (PCR) --- p.56 / Chapter 2.3.5 --- Analysis of PCR products with agarose gel electrophoresis --- p.57 / Chapter 2.3.6 --- Analysis of PCR products with Southern blotting --- p.58 / Chapter a. --- DNA transfer from gel to nylon membrane --- p.58 / Chapter b. --- Probe labeling --- p.61 / Chapter c. --- Prehybridization --- p.61 / Chapter d. --- Hybridization --- p.62 / Chapter e. --- Detection of DIG-labeled probe --- p.62 / Chapter 2.3.7 --- Cycle titration of PCR and dot blotting of regulatory cytokine mRNA --- p.63 / Chapter a. --- Cycle titration of PCR --- p.63 / Chapter b. --- Dot blotting --- p.63 / Chapter 2.4 --- RESULTS --- p.65 / Chapter 2.4.1 --- Sagittal sections of mouse embryos --- p.65 / Chapter 2.4.2 --- Preparation of total RNA --- p.69 / Chapter 2.4.3 --- Cytokine mRNA phenotyping --- p.71 / Chapter a. --- Southern hybridization for 'no expression' cytokines --- p.74 / Chapter b. --- Consistent' and 'regulatory ´ة cytokines in embryo and placenta --- p.79 / Chapter 2.5 --- DISCUSSION --- p.95 / Chapter 2.5.1 --- Isolation of embryo RNA by guanidinium thiocyanate/ cesium chloride centrifugation --- p.95 / Chapter 2.5.2 --- mRNA Quantitation --- p.96 / Semi-quantitative PCR --- p.98 / Chapter 2.5.3 --- Cytokine mRNA phenotyping by RT-PCR --- p.99 / Chapter a. --- Reverse Transcription --- p.99 / Chapter b. --- GAPDH as a control for normalization --- p.100 / Chapter c. --- PCR for cytokine transcripts --- p.101 / Chapter 2.5.4 --- Cytokines and receptors in embryonic development --- p.103 / Chapter 2.5.4.1 --- Cytokines in hematopoietic development of mouse fetus --- p.104 / Chapter 2.5.4.2 --- Other cytokines --- p.113 / Chapter 2.5.5 --- Expression Pattern in placenta: maternal and fetal communication --- p.116 / Chapter CHAPTER3 --- MOLECULAR ANALYSTS OF MOUSE ONCOSTATIN M --- p.117 / Chapter 3.1 --- INTRODUCTION --- p.117 / Chapter 3.2 --- MATERIALS --- p.121 / Chapter 3.2.1 --- Chemicals and Reagents --- p.121 / Chapter 3.2.2 --- Enzymes --- p.121 / Chapter 3.2.3 --- Buffers --- p.122 / Chapter 3.2.4 --- Solutions --- p.122 / Chapter 3.2.5 --- Culture media --- p.124 / Chapter 3.2.6 --- Competent cell --- p.125 / Chapter 3.2.7 --- DNA materials --- p.125 / Chapter 3.2.8 --- Primers --- p.126 / Chapter 3.3 --- METHODS --- p.127 / Chapter 3.3.1 --- Primers and internal probes --- p.127 / Chapter 3.3.2 --- Cloning of human Oncostatin M exon 2 and exon 3 by PCR --- p.127 / Chapter 3.3.3 --- Subcloning of human OSM exons 2 and 3 into pUC18 --- p.128 / Chapter a. --- Preparation of human OSM exons and plasmid --- p.128 / Chapter i) --- Purification of PCR products --- p.128 / Chapter ii) --- T4 DNA polymerase ´بblunt-end´ة reaction for PCR products --- p.129 / Chapter iii) --- Sma I digestion of pUC18 --- p.129 / Chapter b. --- Ligation --- p.129 / Chapter c. --- Preparation of competent cell --- p.130 / Chapter d. --- Transformation --- p.131 / Chapter e. --- Screening of recombinants by PCR --- p.131 / Chapter f. --- Screening of recombinants by restriction enzyme digestion --- p.132 / Chapter i) --- Preparation of plasmids --- p.132 / Chapter ii) --- Double restriction enzymes digestion of pUC18 --- p.133 / Chapter 3.3.4 --- Verification of the clones of human OSM exons 2 and 3 by cycle sequencing --- p.135 / Chapter 3.3.5 --- Purification of human OSM exons from plasmid for making probe --- p.136 / Chapter 3.3.6 --- Southern blotting --- p.136 / Chapter a. --- Probe making and labeling --- p.136 / Chapter b. --- Preparation of mouse genomic DNAs --- p.137 / Chapter c. --- DNA transfer --- p.138 / Chapter i) --- Digestion of genomic DNA with restriction endonucleases --- p.138 / Chapter ii) --- Gel electrophoresis and DNA blotting --- p.139 / Chapter d. --- Hybridization --- p.139 / Chapter 3.4 --- RESULTS --- p.142 / Chapter 3.4.1 --- Cloning of human OSM exon 2 and exon 3 by PCR --- p.142 / Chapter 3.4.2 --- Subcloning of human OSM exons 2 and 3 into pUC18 --- p.142 / Chapter a. --- Screening of recombinants by PCR --- p.142 / Chapter b. --- Screening of recombinants by restriction enzymes digestion --- p.143 / Chapter 3.4.3 --- Sequence of subcloned exons 2 and3 --- p.147 / Chapter 3.4.4 --- Southern hybridization --- p.149 / Chapter a. --- Genomic DNA preparation --- p.149 / Chapter b. --- Digestion of genomic DNAs --- p.151 / Chapter c. --- Hybridization signal --- p.154 / Chapter 3.5 --- DISCUSSION --- p.158 / Chapter 3.5.1 --- Cross-species hybridization --- p.158 / Chapter 3.5.2 --- Hybridization of human OSM exon fragments against mouse genome --- p.158 / Chapter a. --- hOSM exon 2 as probe --- p.158 / Chapter b. --- hOSM exon 3 as probe --- p.160 / Chapter c. --- Feasibility of using hOSM as probe for fishing out the mOSM gene --- p.160 / Chapter d. --- The cloning of mouse OSM by Yoshimura's group --- p.161 / Chapter CHAPTER4 --- CONCLUSION --- p.162 / Chapter 4.1 --- SUMMARY OF CYTOKINE AND CYTOKINE RECEPTOR GENES EXPRESSION DURING EMBRYONIC DEVELOPMENT --- p.162 / Chapter 4.2 --- FURTHER STUDIES OF THE CYTOKINE ACTIONS ON EMBRYOGENESIS --- p.165 / Chapter 4.3 --- MOLECULAR ANALYSIS OF MOUSE OSM GENE --- p.167 / REFERENCES --- p.168

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Abstract not available

The phylum Priapulida is a small group of marine worms that is allied with the nematodes, kinorhynchs, loriciferans and nematomorphs in a clade called the Cycloneuralia or Introverta. Together with the arthropods they are generally considered to comprise the Ecdysozoa, a clade of moulting animals. A number of recent priapulid species possess features that resemble the predicted Ecdysozoan ancestor. In addition, recent molecular studies have also shown that they are basal within the Ecdysozoa/Cycloneuralia (Garey 2001, Webster et al. 2006). Their putative basal position thus makes priapulids highly interesting research objects for understanding the evolution of Ecdysozoa. Earlier investigations of the early embryology of the priapulid Priapulus caudatus are critically revised with the aid of modern techniques and equipment, confirming earlier studies that the early cleavages are highly symmetrical, total, subequal, radial and stereotypical. New results show that up to the sixth cleavage, the spindles are oriented along the animal/vegetal axis at both poles. This unique cleavage pattern has only limited similarities to other animals. During the sixth cleavage two cells move inwards and gastrulation commences. If the mesoderm is derived from both cells, its origin differs from that of many other protostomes. Two previously undescribed larval stages of P. caudatus; the light bulb shaped hatchling and the first lorica larva are described. The second lorica larva superficially resembles the previously described type 2 lorica larva (Higgins et al 1993). Differences between the second lorica larva and the type 2 lorica larva, with respect to possible ecophenotypical variation and sub-specialization, are described. Preliminary data are presented on musculature development of P. caudatus. Preliminary data have also been obtained on the early development of a second priapulid, Halicryptus spinulosus. Comparison of Halicryptus and Priapulus may help to resolve developmental ground pattern of the priapulids.