Dishevelled (Dvl) proteins are key transducers of Wnt signaling and are encoded by members of a multi-gene family in vertebrates. We report here divergent, tissue-specific expression patterns for all three Dvl genes in Xenopus embryos, which contrast dramatically with their expression in the mouse. Moreover, we find that the expression patterns of Dvl genes in the chick diverge significantly from those of Xenopus. In addition, in hemichordates, one of the outgroups to chordates, we find that the one Dvl gene is dynamically expressed in a tissue-specific manner. Using knockdowns, we find that Dvl1 and Dvl2 are required for early neural crest specification and for somite segmentation. Most strikingly, we report a novel role for Dvl3 in the maintenance of differentiated muscle and the development of the Xenopus sclerotome. Together, these data demonstrate that that the expression patterns and developmental functions of specific Dvl genes have diverged significantly during chordate evolution. The planar cell polarity (PCP) signaling pathway is essential for embryonic development because it governs diverse cellular behaviors, and the "core PCP" proteins, such as Dishevelled and Frizzled, have been extensively characterized. By contrast, the "PCP effector" proteins, such as Intu and Fuz, remain largely unstudied. These proteins are essential for PCP signaling, but they have never been investigated in a mammal and their cell biological activities remain entirely unknown. We report here that Fuz mutant mice display neural tube defects, polydactyly, and skeletal dysmorphologies that stem from defective ciliogenesis. Using bioinformatics and imaging of an in vivo mucociliary epithelium, we establish a central role for Fuz in membrane trafficking, showing that Fuz is essential for apical trafficking of ciliogenesis factors in ciliated cells and also for exocytosis in secretory cells. We identify a novel, Rab-related small GTPase as an interaction partner for Fuz, and this GTPase also is essential for ciliogenesis and secretion. These results are significant because they provide novel insights into the mechanisms by which developmental regulatory systems like PCP signaling interface with fundamental cellular systems such as the vesicle trafficking machinery. / text

published_or_final_version / Dentistry / Master / Master of Orthodontics

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

Introduction Globesity, the worldwide obesity epidemic, represents a major threat and public health burden. An uncontrolled expansion of the adipose tissue followed by a chronic low-grade inflammation leads to the dysfunction of the adipose organ resulting in obesity and its associated metabolic complications. Uncovering the mechanisms of adipogenesis, the development of adipocytes, therefore strikes as a key strategy in combating the disease. MicroRNAs (miRs), a class of small non-coding RNAs, have emerged in recent years as crucial modulators of diverse biological processes such as cell proliferation, differentiation, and signal transduction emphasizing their large potential as targets. Numerous miRs have been associated with the adipose tissue and metabolism and their dysregulation has repeatedly been linked to diseases including diabetes and obesity. This study aimed to investigate the role of miR-34a, an obesity-related miR, in the regulation of pre-adipocyte differentiation. Materials and Methods Mouse 3T3-L1 pre-adipocytes were employed as an in vitro system to study adipogenesis. Oil Red O staining served to evaluate the degree of adipogenesis and the over-expression of miR-34a in adipocytes was achieved by a lentiviral system. MiR and messenger RNA (mRNA) levels were analysed using TaqMan and SYBR Green-based quantitative real time PCR (qPCR) respectively. Results The expression of miR-34a was substantially down-regulated upon treatment of differentiation medium for two days and remained significantly low during the differentiation period compared with undifferentiated pre-adipocytes. Lentivirus-mediated over-expression of miR-34a successfully up-regulated miR-34a. Higher levels of miR-34a in turn mitigated adipogenesis as evidenced by blunted Oil Red O staining. This observation was found to be in good agreement with the qPCR analysis, which showed a down-regulation of several key adipogenic markers. Conclusion The down-regulation of miR-34a is required during pre-adipocyte differentiation for the efficient proceedings of the adipogenic programme. Further investigation is needed to evaluate the potential therapeutic implication of miR-34a-based treatment in managing obesity. / published_or_final_version / Medicine / Master / Master of Medical Sciences

Endochondral bone development depends on the progression of chondrocyte proliferation, hypertrophy and terminal differentiation, which requires precise transcriptional regulation and signaling coordination. Disturbance of this process would disrupt chondrocyte differentiation and lead to chondrodysplasias. In cells, a highly conserved mechanism, ER stress signaling, has been developed to sense the protein load and maintain the cellular homeostasis. In humans, mutations in COL10A1 induce ER stress and result in metaphyseal chondrodysplasia type Schmid (MCDS). Previous analysis of a MCDS mouse model (13deltg mouse) had revealed a novel mechanism of chondrocyte adaptation to ER stress. The hypertrophic chondrocytes survive ER stress by reverting to a pre-hypertrophic like state (Tsang et al., 2007). To dissect the underlying mechanisms that coordinate chondrocyte survival, reverted differentiation and adaptation to ER stress, different chondrocyte populations in the wild type and 13del growth plates were fractionated for global gene expression analyses. The genome-wide expression profiles of proliferating chondrocytes, prehypertrophic chondrocytes, hypertrophic chondrocytes and terminally differentiated chondrocytes in the wild type growth plate provide molecular bases to understand the processes underlying both physiological and pathological bone growth. Systematic analyses of these transcriptomic data revealed the gene expression patterns and correlation in the dynamics of endochondral ossification. Genes associated with sterol metabolism and cholesterol biosynthesis are enriched in the prehypertrophic chondrocytes. Selected genes (Wwp2, Zbtb20, Ppa1 and Ptgis) that may potentially contribute to endochondral ossification were identified differentially expressed in the growth plate. Bioinformatics approaches were applied to predict regulatory networks in chondrocytes at different differentiation stages, implying the essential and dominant roles of Sox9 in coordination of stage specific gene expression. We further confirmed that Sox9 directly regulates the transcription of Cyr61, Lmo4, Ppa1, Ptch1 and Trps1, suggesting that Sox9 integrates different steps of chondrocyte differentiation via regulation of its target genes and partially crosstalk with IHH signaling pathway. The information on gene expression and regulation from physiological growth plate provides important basis to understand the molecular defects of chondrodysplasia. The hypertrophic zone in 13del growth plate was fractionated into upper, middle and lower parts for microarray profiling, corresponding for the onset of ER stress, onset of reverted differentiation and adaptation phase. Comparative transcriptomics of wild type and 13del growth plates revealed genes related to glucose, amino acid and lipid metabolisms are up regulated in response to ER stress. Fgf21 was identified as a novel ER stress inducible factor regulated by ATF4. Removal of Fgf21 results in increasing cell apoptosis in 13del hypertrophic zone without affecting the reverted differentiation process. Up regulation of genes expression related to hypoxic stress (Slc2a1, Hyou1, Stc2 and Galectin3) in 13del hypertrophic chondrocytes suggested that survival and adaptation of chondrocytes to ER stress involve cross-regulation by other stress pathways. Our findings have provided a new insight into the mechanisms that facilitate chondrocyte survival under ER stress in vivo, and propose the integrative effects of hypoxic stress pathway during the stress adaptation process, which broaden the molecular horizons underlying chondrodysplasias caused by protein folding mutations. / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

In recent years, great advances have been made in the field of stem cell differentiation. Seminal insights in the area of developmental biology and tissue regeneration have made ex vivo differentiated cells a realistic alternative for transplantation applications. The recent application of these murine-based insights to human systems has paved new paths in autoimmune disease, chemotherapy, and immuno-deficiency research. Such strides would eliminate the hurdles associated with adoptive transfer including limited availability of transplantable cells, site morbidity, difficulties in cell isolation and expansion lag time. Current approaches in ex vivo hematopoiesis and T cell differentiation have begun to explore the effects of biomaterials on differentiation efficiency. These approaches, however, have not fully studied the quantitative effects of biomaterials and their properties on hematopoietic and T cell differentiation generation. Our goal was to design biomaterials whose properties could be tailored to improve differentiation efficiencies in T cell differentiation. Our work is dedicated to fabricating and characterizing Notch ligand functionalized microbeads for T cell differentiation applications. Our work has shown stable functionalization of Notch ligands on microbeads that can be quantitatively varied to achieve optimal Notch signaling. We have also demonstrated limited cellular toxicity and effective Notch signaling upon exposure to Notch ligand functionalized beads. Finally, we have successfully differentiated T cell progenitors from hematopoietic stem cells using the functionalized microbeads. As a side study, we have fabricated and characterized polymeric PLA scaffolds that were systematically varied and studied for their effects on hematopoietic differentiation efficiency. Insights gained from these studies should provide a better understanding of the microenvironmental signals in hematopoiesis and aid in the development of efficient technologies for the production of hematopoietic progenitors and T cells for therapeutic applications.

published_or_final_version / abstract / toc / Paediatrics / Master / Master of Philosophy

published_or_final_version / Anatomy / Master / Master of Philosophy

No description available.

Cell image analysis of lymphocytes from (1) control and (2) killed coccidioides immitis arthrospore immunized guinea pigs was performed. Lymphocytes from peripheral blood and lymph nodes were obtained two and tree weeks after immunization from each group of animals. Histograms from peripheral lymphocytes taken at two weeks from immunized animals showed no significant difference from those of the control animals. A significant difference, however, was seen in the conturs of the histograms of the cells taken at three weeks from four of six immunized animals. These histograms extended into the higher optical density range when compared to histograms of control animals. Smaller nuclear areas were also found in the majority of three week peripheral cells. In material from the nodes of immunized animals two weeks after immunization, the ratio of densely staining to lightly staining cells reverses from 3:1 to .7:1. Samples taken from nodes three weeks after immunization show no such differences in their cellular composition between control and immunized animals.