Includes bibliographical references (p. 85-99).

Thesis (Ph. D.)--University of Oregon, 2000. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 106-112). Also available for download via the World Wide Web; free to University of Oregon users.

Thesis (M. Phil.)--University of Hong Kong, 2010. / Includes bibliographical references (leaves 96-103). Also available in print.

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Despite recognition of aldehyde dehydrogenase (Aldh) as a surrogate marker in isolating primitive hematopoietic stem and progenitor cells (HSPC) [1], its role in HSPC regulation, particularly during embryonic development, remains unclear. In this study, we examined the role of Aldh during embryonic hematopoiesis in zebrafish, which has emerged as a model for hematopoietic studies. [2] Wild--?type and transgenic [Tg(gata1:gfp),Tg(fli1:gfp)] zebrafish embryos were microinjected with anti--?sense morpholinos (MO) at 1--?cell to 4--?cell stage and evaluated by morphology, flow cytometry, in situ hybridization (ISH) and Q-RT-PCR. In addition, human CD34+ cells, which were enriched with hematopoietic stem cells (HSC), were isolated from umbilical cord blood samples for analysis of ALDH16A1 expression. It was subsequently compared with CD34- cells which were devoid of HSC activity. When aldh16a1 was knocked down by anti-sense morpholino (the embryos were referred herewith aldh16a1MO embryos), gene expression associated with erythropoiesis was significantly reduced at 18hpf .(gata1:0.70±0.03fold; p=0.002) (α-embryonic hemoglobin: 0.48±0.04fold; p=0.003) (β-embryonic hemoglobin: 0.56±0.03fold; p=0.001). Angiogenesis was also perturbed at 48 and 72hpf. Furthermore, human ALDH16A1 was significantly upregulated (4.79±1.00fold; p=0.00006) in CD34+ (enriched with HSC) as compared to CD34- (devoid of HSC) populations in umbilical cord blood. Aldh16a1 is important for the maintenance of primitive hematopoiesis at early (18hpf) and angiogenesis at later (48,72 hpf) embryonic stages. As angiogenesis plays an important role in pathophysiology of malignancies, novel therapy against ALDH16A1 might be exploited in therapeutic intervention in cancer treatment. Moreover, a specific role of zebrafish aldh16a1 in primitive erythropoiesis and a higher level of ALDH16A1 expression in human HSC-enriched cells suggested a conserved mechanism whereby ALDH regulates hematopoiesis. / published_or_final_version / Medicine / Master / Master of Research in Medicine

Prostanoid receptors (PG-Rs: prostaglandin D, E, F, prostacyclin and thromboxane receptors (DP, EP1-4, FP, IP and TP)) are known to mediate a diverse range of biological responses, such as cardiovascular homeostasis, nociception and reproduction, via binding to their respective ligands belonging to the five classes of prostanoids (PGs: class D, E, F, I and thromboxane). The majority of these findings were reported in mammals, and despite suggestive evidence provided by previous pharmacological and physiological studies in non-mammalian vertebrates, investigations on the mechanisms behind actions of PGs were impeded by the limited information on their receptors. In the present study, the full-length cDNAs of chicken (c-) and zebrafish (z-) prostanoid receptors – cEP3, cFPs, zEP1s and zFP – were identified from respective adult ovaries and their tissue distribution examined by RT-PCR. A novel middle-truncated splice variant, cFPb, which lacks 107 amino acids between transmembrane domains 4 and 6 but otherwise identical to cFPa was first identified. Three isoforms of zEP1 – zEP1a, zEP1b, zEP1c – were found, which might have subfunctionalized in their ligand binding and G protein coupling specificity, in addition to differential tissue distribution. Using various luciferase reporter systems (pGL3-CRE, pGL-NFAT-RE, pGL4-SRE), all the cloned receptors, except cFPb, were shown to potentially couple to intracellular cAMP, Ca2+, and/or MAPK signaling pathways. Owing to the proposed roles of PGs and its potential regulation by and/or on EGFR ligands and gonadotropins in mammals and chicken, genes involved in regulation of PG functions at various levels, including biosynthesis (COX1, COX2, mPGES1, mPGES2 and cPGES), availability (PGT) and signaling (cEPs and cFPs), were also characterized in granulosa cells during hen follicular development. Lastly, using our experimental data and systematic sequence retrieval from available databases, the PG receptor cascades from representative vertebrate species were pooled and analysed using phylogenetic analyses and synteny studies. Three putative clusters (IP-like, EP4-like and EP1-like cluster) were found in lamprey genome; meanwhile, only one PG-R-like cluster was identified from the Cephalochordate lancelet (amphioxus) genome. This concurs with the 1-2-4 rule proposed in first round/second round (1R/2R) whole genome duplication in which the missing lamprey cluster was presumably lost secondarily. With support from conserved orthologs-localization, the four PG-R paralogs (proto-EP4, proto-IP/EP2/DP, proto-TP/FP/EP1 & putative proto-EP3 genes) in the ancestral vertebrates might have further diversified via either localized- (e.g. EP2 and DP) or chromosomal segmental duplication (e.g. EP1, FP and TP) which resulted in the present array of vertebrate PGRs. Additional paralogs (e.g. EP1 and EP4) were identified from fishes, by which molecular dating coincide with and hint of their origins whence the ancient fishspecific whole genome duplication (3R) occurred ~350 million years ago. The present study offers the first glimpse and a better understanding of the roles of the PG-Rs and presents a higher resolution to the evolutionary history of each PG-R family member, consolidating that particular care has to be taken when studying non-mammalian PG-R functions in which some members are absent or present in multiples and propel the investigation of adaptational changes in the coding sequence during evolution of vertebrate PG-Rs. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Bisphenol A (BPA) is an alkylphenol compound used in plastic manufacturing, which enters the aquatic environment through wastewater treatment plants and landfill leachates. Exposure of fish to BPA results in developmental defects, decreased hatching, and increased occurrence of the egg yolk precursor protein vitellogenin in male fish. BPA is an estrogenic like compound (xenoestrogen) that can bind and activate the nuclear estrogen receptors, ERα and ERβ, causing changes in gene transcription (genomic mechanism). However, the ability of BPA to activate rapid estrogen signaling and to disrupt nongenomic physiological responses to estrogens is not known. One important nongenomic estrogen action in fish is to maintain meiotic arrest of oocytes and to prevent precocious oocyte maturation through activation of the transmembrane G-protein coupled estrogen receptor (Gper). Binding of estrogens to Gper on zebrafish oocytes results in rapid activation of epidermal growth factor receptor (Egfr) signaling and subsequent Mapk3/1 activation. We show here that BPA and three structurally-related chemicals, tetrachlorobisphenol A, tetrabromobisphenol A and nonylphenol, can mimic estrogen by activating this mechanism of meiotic arrest through Gper in zebrafish (Danio rerio). BPA bound to zebrafish Gper and inhibited spontaneous oocyte maturation (OM) of denuded oocytes. Treatment of oocytes with Actinomycin D did not block the effects of BPA, suggesting that this inhibition of OM is through a nongenomic mechanism. Incubation of oocytes with a selective GPER antibody and the specific GPER antagonist G-15 blocked the effects of BPA on OM, further suggesting that BPA inhibition of OM is through its interaction with the receptor. Various inhibitors of the EGFR pathway were utilized to determine if the inhibition of OM by BPA is mediated through this mechanism. BPA activation of the Egfr pathway resulted in Mapk3/1 (also known as Erk 1/2) phosphorylation. The results show that BPA disrupts oocyte maturation through a novel mechanism involving activation of a Gper/Egfr/Mapk3/1 pathway with potential adverse impacts on reproductive success. / text

CD4+ lymphocytes (T helper cells) play a crucial role in orchestrating adaptive immune responses. This project aims to characterise the CD4+ cells in zebrafish (Danio rerio) for a better understanding of adaptive immunity in teleost fish. I cloned three CD4 homologues, termed zfCD4 and zfCD4 related (zfCD4rel1 and zfCD4rel2), with both reported previously in other bony fish species. The zfCD4 and zfCD4rels transcripts were detected in immune organs in zebrafish and were most highly expressed in the lymphocyte population. A moderate induction of the zfCD4 and zfCD4rels (and other immune related genes) was seen in kidney, spleen and intestine after poly (I:C) injection. Two antibodies against CD4 and IFN-γ, respectively, have been validated using various immunostaining approaches for further functional studies. The CD4 positive cells ranged from 20-30% of lymphocytes in zebrafish, similar to what is seen in other vertebrates. The expression level of IFN-γ and other Th cell related genes were analysed in immunised fish following re-stimulation with antigen, revealing that in zfCD4+ lymphocytes an increased expression of cytokines and master transcript factors was seen when the same antigen was used for boosting. This is the first demonstration of Th-type responses effected by CD4+ lymphocytes in a poikilotherm. Lastly, I studied an aspect of Treg function in zebrafish, focused on a master transcription factor of Tregs, the FoxP3 gene. Knocking down FoxP3 genes in zebrafish resulted in modulated gene expression of cytokines and transcription factors associated with Th and Treg cells, providing some evidence that the immune tolerance function of Treg cells may exist in teleost fish, with some sub-functionalisation of the two FoxP3 paralogues apparent. This thesis extends our knowledge into teleost adaptive immune responses mediated by CD4+ Th cells and putative FoxP3+ Treg cells and may aid future studies using zebrafish as a model of vertebrate immune function.

No description available.

Canonical Wnt signaling is involved in many aspects of development including axis specification and anterior-posterior neuroectoderm formation during vertebrate embryogenesis. Axin2, a homologue of Axin1, is thought to have a similar regulatory role within the cell, but differences in their expression and binding partners suggest Axin2 is not completely redundant with Axin1. To better understand Axin2 in canonical Wnt signaling, I utilized several approaches to explore its expression and function. In the zebrafish embryo, I found Axin2 is expressed in known active domains of Wnt signaling, suggesting an inducible regulatory role. Additionally, canonical Wnt signaling was sufficient and necessary to induce Axin2 expression and Axin2 was sufficient and necessary to inhibit Wnt signaling. As Wnt signaling is important in development and its dysregulation has been implicated in diseases such as colorectal cancer, this study helps advance our understanding of how Wnt signaling regulates itself through the use of negative feedback inhibitors, such as Axin2.