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Call number: LD2668 .T4 1979 R619 / Master of Science

Cheung, Siu Yuen. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 178-200). / Abstracts in English and Chinese. / Abstract --- p.i / Chinese abstract --- p.iii / Acknowledgements --- p.v / Table of contents --- p.vii / Chapter Chapter One --- General Introduction --- p.1 / Chapter 1.1 --- S100 Proteins --- p.1 / Chapter 1.1.1 --- Structure of S100 proteins --- p.2 / Chapter 1.1.2 --- Possible functions of S100 proteins --- p.4 / Chapter 1.1.3 --- Genomic organization of S100 genes --- p.6 / Chapter 1.1.4 --- Clinical importance of S100 proteins --- p.7 / Chapter 1.2 --- S100A1 Protein --- p.8 / Chapter 1.2.1 --- Possible functions of the S100A1 protein --- p.10 / Chapter 1.2.1.1 --- Regulation of cardiac and skeletal muscle contractility --- p.10 / Chapter 1.2.1.2 --- Functional roles in the central nervous system (CNS) --- p.12 / Chapter 1.2.1.3 --- Other possible functions of the S100A1 protein --- p.13 / Chapter 1.2.2 --- S100A1 knockout mice --- p.14 / Chapter 1.2.3 --- Relationships between S100A1 and S100B proteins --- p.16 / Chapter 1.3 --- S100B Protein --- p.18 / Chapter 1.3.1 --- Possible functions of S100B protein --- p.19 / Chapter 1.3.2 --- S100B knockout mice --- p.20 / Chapter 1.4 --- RNA interference --- p.22 / Chapter 1.4.1 --- Mechanisms of RNA interference --- p.24 / Chapter 1.4.2 --- Efficacy and selectivity of siRNA --- p.25 / Chapter 1.4.3 --- siRNA delivery --- p.27 / Chapter 1.5 --- Objective --- p.31 / Figures and legends --- p.34 / Chapter Chapter Two --- S100A1 expression in normal mouse embryos and characterization of S100A1 knockout mouse embryos --- p.40 / Chapter 2.1 --- Introduction --- p.40 / Chapter 2.2 --- Materials and Methods --- p.44 / Chapter 2.2.1 --- Mouse strains --- p.44 / Chapter 2.2.2 --- RNA extraction --- p.46 / Chapter 2.2.3 --- Reverse Transcription-Polymerase Chain Reaction (RT-PCR) --- p.46 / Chapter 2.2.4 --- Protein extraction --- p.48 / Chapter 2.2.5 --- Western blotting --- p.49 / Chapter 2.2.6 --- Immunohistochemical staining --- p.50 / Chapter 2.3 --- Results --- p.53 / Chapter 2.3.1 --- S100A1 mRNA expression in normal mouse embryo --- p.53 / Chapter 2.3.2 --- S100A1 protein expression in normal mouse embryos --- p.55 / Chapter 2.3.2.1 --- Temporal expression of the S100A1 protein --- p.55 / Chapter 2.3.2.2 --- Spatial expression of the S100A1 protein --- p.57 / Chapter 2.3.3 --- Morphological and histological characterization of SI00A1 knockout mouse embryos --- p.60 / Chapter 2.3.4 --- S100B protein expression pattern in Wt and S100A1 KO mouse embryos --- p.62 / Chapter 2.4 --- Discussion --- p.64 / Tables --- p.73 / Figures and legends --- p.76 / Chapter Chapter Three --- Knockdown of S100A1 in S100B in knockout mouse embryos --- p.118 / Chapter 3.1 --- Introduction --- p.118 / Chapter 3.2 --- Materials and Methods --- p.128 / Chapter 3.2.1 --- Mouse strains --- p.128 / Chapter 3.2.2 --- Short-interfering RNA (siRNA) --- p.129 / Chapter 3.2.3 --- In-uterus surgery --- p.130 / Chapter 3.2.4 --- RNA extraction and RT-PCR --- p.132 / Chapter 3.2.5 --- Immunohistochemical staining of S100A1 and S100B --- p.132 / Chapter 3.3 --- Results --- p.133 / Chapter 3.3.1 --- Characterization of S100B knockout mouse embryos --- p.133 / Chapter 3.3.2 --- S100A1 knockdown in S100B wild-type (Wt) mouse embryos --- p.133 / Chapter 3.3.3 --- S100A1 knockdown in S100B knockout (KO) mouse embryos --- p.139 / Chapter 3.4 --- Discussion --- p.146 / Tables --- p.153 / Figures and legends --- p.154 / Chapter Chapter Four --- General Discussion and Conclusions --- p.175 / Reference --- p.178

Development in the annual killifish Austrofundulus limnaeus can follow two distinct developmental trajectories. Typical development includes the entrance of embryos into a state of metabolic and developmental arrest termed diapause. Alternately, embryos can escape diapause and develop directly without pause. These two trajectories are characterized by differences in the rate and timing of developmental, morphological, and physiological traits. Insulin and Insulin-like growth factor (IGF) signaling (IIS) is known to regulate entrance into diapause in a variety of invertebrates. In this thesis I explore the possible role of IGFs in the regulation of development and diapause in embryos of A. limnaeus. Here I report stage-specific expression of IGF-I and II proteins and their associated mRNA transcripts. Patterns of IGF-I protein expression are consistent with IGF signaling playing a major role in supporting the escape trajectory. In addition, treatment of embryos with a potent inhibitor of the IGF-I receptor (IGF1R) mimics the diapause developmental pattern even under conditions that should favor direct development. Evaluation of mRNA gene expression patterns in the two developmental trajectories suggests a role for IGF-I signaling through the RAS-MAPK-ERK pathway, which may be promoting the escape phenotype. Additionally, IGF-I activity may be enhanced in escape trajectory embryos though upregulation of IGF binding protein 2 (IGFBP-2) mRNA. These data suggest a major role for IGF signaling in the promotion of the escape trajectory, and thus we predict that specific mechanisms are in place in diapause-bound embryos that block IGF signaling and thus promote entrance into diapause. The data presented here suggest that blocking IGF signaling is critical for induction of diapause, but also suggests that other signaling pathways are likely also at play. Other pathways such at the TGF-beta signaling molecules and SMAD pathway, may also be involved in the direct regulation of the diapause phenotype, as has been shown for other animal models of developmental arrest.

Cited2 is a member of the Cited gene family, which has no homology to any other genes. It encodes a transcriptional co-factor that is expressed during early heart formation (cardiogenesis). Embryos lacking Cited2 display a range of cardiac defects including bilaterally identical atria, aortic arch abnormalities, rotation of the aorta and pulmonary artery, and malseptation of the cardiac chambers. The latter results in communication between the aorta and pulmonary artery, the aorta and both ventricles, and the atria and ventricles (with themselves and each other). Cardiogenesis is complex, and requires many different cell types and processes to occur correctly. Some of these cells and processes are external to the primary heart. For example, once the initial muscle cells of the heart form a tube, cells from other regions such as the secondary heart field (adjacent mesoderm) and cardiac neural crest (ectoderm) migrate into this tube, and are required for the formation of additional muscle cells and septa. Furthermore, cardiogenesis also requires correct left-right patterning of the embryo to be established prior to heart formation. To understand the developmental origins of the cardiac defects observed in Cited2-null embryos, the expression pattern of Cited2 and the anatomy of Cited2-null embryo hearts were studied. Subsequently, the expression of genes required for left-right patterning were studied in both Cited2-null and Cited2 conditionally-deleted embryos. This demonstrated that Cited2 may be required in many, possibly all, of the processes required for cardiogenesis. Next this study focused on the role of Cited2 in patterning the left-right axis of the embryo. Firstly, Cited2 was found to regulate the expression of the master regulator of left-right patterning (Nodal). Secondly, Cited2 was shown to regulate the expression of the left-specific transcription factor Pitx2 independently of Nodal. Thirdly, gene expression and conditional deletions of Cited2 suggested that Cited2 might regulate left-right patterning in the paraxial mesoderm, a tissue which has not previously been shown to regulate the left-right axis in the mouse. Lastly, an argument is made suggesting the possibility that all the cardiac defects found in Cited2-null embryos may directly or indirectly stem from a failure of correct left-right patterning.

Cited2 is a member of the Cited gene family, which has no homology to any other genes. It encodes a transcriptional co-factor that is expressed during early heart formation (cardiogenesis). Embryos lacking Cited2 display a range of cardiac defects including bilaterally identical atria, aortic arch abnormalities, rotation of the aorta and pulmonary artery, and malseptation of the cardiac chambers. The latter results in communication between the aorta and pulmonary artery, the aorta and both ventricles, and the atria and ventricles (with themselves and each other). Cardiogenesis is complex, and requires many different cell types and processes to occur correctly. Some of these cells and processes are external to the primary heart. For example, once the initial muscle cells of the heart form a tube, cells from other regions such as the secondary heart field (adjacent mesoderm) and cardiac neural crest (ectoderm) migrate into this tube, and are required for the formation of additional muscle cells and septa. Furthermore, cardiogenesis also requires correct left-right patterning of the embryo to be established prior to heart formation. To understand the developmental origins of the cardiac defects observed in Cited2-null embryos, the expression pattern of Cited2 and the anatomy of Cited2-null embryo hearts were studied. Subsequently, the expression of genes required for left-right patterning were studied in both Cited2-null and Cited2 conditionally-deleted embryos. This demonstrated that Cited2 may be required in many, possibly all, of the processes required for cardiogenesis. Next this study focused on the role of Cited2 in patterning the left-right axis of the embryo. Firstly, Cited2 was found to regulate the expression of the master regulator of left-right patterning (Nodal). Secondly, Cited2 was shown to regulate the expression of the left-specific transcription factor Pitx2 independently of Nodal. Thirdly, gene expression and conditional deletions of Cited2 suggested that Cited2 might regulate left-right patterning in the paraxial mesoderm, a tissue which has not previously been shown to regulate the left-right axis in the mouse. Lastly, an argument is made suggesting the possibility that all the cardiac defects found in Cited2-null embryos may directly or indirectly stem from a failure of correct left-right patterning.

Call number: LD2668 .T4 1966 N761 / Master of Science

published_or_final_version / Obstetrics and Gynaecology / Master / Master of Philosophy

No description available.