Bioinformatic searching on the zebrafish genome indicates that there are five members of this hormone family (namely GH, SLalpha, SLbeta, PRL1 and PRL2) and four receptors (namely GHR1, GHR2, PRLR1 and PRLR2). However, it should be noted that these ligands and receptors are only named according to their sequence homology with those in other species. There is so far no systematic study to unravel the relationship among the ligands and receptors. The last point is particularly relevant as some of the ligands and receptors are duplicated in the fish genome. In addition, there is much controversy regarding whether one of the two GHRs is in fact the receptor for SL. A systematic study on the interaction among the ligands and receptors in zebrafish would help to resolve these issues. / In fish, growth hormone (GH), prolactin (PRL) and somatolactin (SL) are members of a gene family of polypeptide hormones which share homology in protein sequence and structure. To date, numerous functions have been attributed to this family of hormones such as growth, immune response, protein metabolism and ion regulation. The biological functions of GHlPRL are mediated through binding of the ligands on their respective receptors. It is believed that this gene family arose as the result of multiple gene duplications and subsequent divergent evolution, co-evolving with their corresponding receptors. Despite the above mentioned similarities in their structures, their cognate receptors and their signaling mechanisms, important differences among this gene family of polypeptide hormones can be recognized in their biological functions. / In the present study, the luciferase reporter assay, His-tag pulldown assay and signaling pathway activation were employed to investigate the interaction among the ligands and their receptors. It was shown that recombinant zebrafish GH, PRLI and PRL2 could only interact with their cognate receptors, i.e. GHRl, GHR2, PRLRI and PRLR2 respectively. In comparison, zebrafish SLalpha and SLbeta could neither interact with GHR1, GHR2, PRLR1 and PRLR2 in the binding study, nor could these two SLs activate the receptor-mediated downstream signaling and transcriptional activities of the four receptors in zebrafish. These data argue against the hypothesis that GHRI is the SL receptor. / The role of PRLR in early development of zebrafish was also explored. Whole mount in situ hybridization (WISH) study showed that PRLR1 was mainly expressed in the pancreas and pronephric duct, while PRLR2 was expressed in the pronephric duct only. In the PRLR1 morpholino (MO) knockdown embryos, the yolk extension (YE), the formation of which was reported to be associated with pronephric duct development, disappeared at 24 hours post fertilization. This phenotype could not be observed in the PRLR2 MO knockdown or control embryos. Real time quantitative RT-PCR and WISH data revealed that several genes expressed in the pronephric duct were up or down-regulated. The protein expression pattern of pronephric duct marker atplal was also affected in the embryos injected with PRLRI MO. In addition, histological studies showed that structure of the pronephric duct was destroyed in the PRLRI MO embryos. These results suggest that PRLRI plays an important role in the development of the pronephric duct in zebrafish embryos. / Chen, Mingliang. / "October 2010." / Adviser: Cheung Wing-Tai. / Source: Dissertation Abstracts International, Volume: 73-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 140-179). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Firstly, a cell line derived from the liver of tilapia, Hepa-T1, was used as a model and exposed to two sub-lethal concentrations of waterborne copper for 96 h. The proteins expressed in Hepa T1 were investigated by differential protein profiling using two-dimensional gel electrophoresis (2-DE). It was found that Cu2+ (120 microM and 300 microM) caused differential expression of 93 different proteins, 18 of which were further verified by real-time quantitative polymerase chain reaction (PCR) analysis. Following analysis with ingenuity pathway software, several proteins were found to be involved in lipid metabolism, tissue connective development and cell cycle control, thus indicating that copper toxicity affects these cellular functions. / Fourthly, to further reveal the mechanism of copper tolerance and sensitivity in tilapia and zebrafish, two important copper transporters (ATP7A & B) and metallothionein (MT) were chosen for studying. Until now, a full length of ATP7A and partial length of ATP7B were obtained in tilapia. Then a real time quantitative PCR was conducted to study the different regulations of these three genes in tilapia and zebrafish. It was found that Cu2+ could induce more MT and ATP7A & B in tilapia than zebrafish both in vivo and in vitro. These results help us to understand that the copper tolerance of tilapia is possibly due to higher expression level of both copper transporters and MT. / Last but not least, I also compared the toxicity and biomarker gene expression in zebrafish exposed to Cu2O nanoparticle (NP) and CuCl2, respectively. It was found that the toxicity of CuCl2 is much higher than that of Cu2O NP. Then seven genes, including MT, ATP7A & B, copper transporter 1 (Ctr1), metal regulatory transcription factor 1 (MTF1), glutathione sulfur transferase (GST), Cu/Zn superoxide dismutase (Cu/Zn SOD), were chosen for studying. It was found that both Cu2O NP and CuCl 2 up-regulated the mRNA levels of MT, Cu/Zn SOD, and Ctr1, ATP7A & 7B, but down-regulated the mRNA levels of GST. Interestingly, the inductions of MT, Ctr1, ATP7A & B in the Cu2O NP exposure groups were much higher than that of CuCl2 exposure groups in vivo . Furthermore, as determined by using Ctr1, ATP7A and ATP7B gene expression, the no observable effect levels (NOELs) of CuCl2 and nano-Cu2O were 11 ppb and 50 ppb, whereas the lowest observable effect levels (LOELs) of CuCl2 and nano-Cu2O were 43 ppb and 125 ppb. (Abstract shortened by UMI.) / Secondly, the high copper contents in the liver of the tilapia make this fish a suitable model for the study of copper binding proteins. Liver was dissected from tilapia injected with Cu2+ and cytosolic fractions were separated by using Superdex 75 column chromatography followed with atomic absorption spectrometry. Fractions containing copper-binding proteins were found in two major peaks, analyzed using differential proteomic approaches, and loaded on a Cu chelating ion-immobilized affinity column (Cu-IMAC). Of the 113 differentially expressed proteins in these two peaks, some well-characterized copper binding proteins were found, including copper transporter ATP7A, cytochrome c oxidase, metallothionein, collagen, catalase, and vitellogenin. These proteins are mainly involved in endocrine disruption, mitochondria dysfunction, ion competition, lipid metabolism, copper transfer, and cytoskeleton disruption. In addition, a more concrete image about copper transportation pathway was hypothesized according to the function of the novel copper binding proteins identified. / The aims of this study are to identify some novel copper binding proteins and proteins related to Cu2+ toxicity or detoxification mechanisms in the tilapia (Oreochromis niloticus) and the zebrafish (Danio rerio) using a proteomic approach, and to reveal the mechanism of copper tolerance and copper sensitivity by comparing the different biochemical responses to copper exposures between the two model species. / Thirdly, zebrafish liver cell line (ZFL) was also used as a model to study the mechanism of copper toxicity. After processing similar experimental procedures of previous Hepa T1 experiment, 72 different proteins were identified to be regulated by Cu2+ (100 microM and 200 microM). More than 50 % of these proteins were also found differentially expressed in the tilapia. The results suggested that the toxicity mechanism between zebrafish and tilapia was generally conserved. Although, in ZFL, the regulation of several proteins, related to ROS effect, mitochondrion copper transportation, and stress response, was quite different from that in tilapia. / Chen, Dongshi. / Adviser: Chun Ung Ming. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 173-190). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Many studies addressing the toxicity of silver nanomaterials have found that smaller sized silver nanoparticles are usually more toxic to organisms and in cell culture than particles of larger sizes yet it is not entirely clear why. We investigated the size dependent toxicity of silver nanoparticles by measuring the response of embryonic zebrafish (Danio rerio) following exposure to a library of thirteen distinct silver nanoparticle size distributions with mean diameters between 8.9 nm and 112.6 nm. Data analysis using dose���response modeling revealed that silver nanoparticles (AgNP) induced embryo toxicity that is dependent on the total surface area and not on the mass or particle number in solution. Included in this study is a comparison between embryo toxicity induced by silver nitrate (AgNO���) and AgNPs for cardiovascular endpoints, as well as an investigation into the influence of the chorion on AgNP toxicity. This study demonstrates the importance of using alternative dose metrics in nanotoxicology, and highlights the value of using the embryonic zebrafish to explore nanomaterial structure activity relationships. / Graduation date: 2013

University of Macau / Institute of Chinese Medical Sciences

University of Macau / Institute of Chinese Medical Sciences

University of Macau / Institute of Chinese Medical Sciences

Thesis (Ph.D.)--Mississippi State University. College of Veterinary Medicine. / Title from title screen. Includes bibliographical references.

Thrombocytes are important players in hemostasis. There is still much to be explored regarding the molecular basis of the thrombocyte function. In our previous microarray analysis data, we found IFT122 (an intraflagellar transport protein known to be involved in cilia formation) transcripts in zebrafish thrombocytes. Given recent discoveries of non-ciliary roles for IFTs, we examined the possibility that IFT122 affects thrombocyte function. We studied the role of IFT122 in thrombocyte function. We also found that IFT122 plays a central role in thrombocyte activation initiated by the agonists ADP, collagen, PAR-1 peptide and epinephrine. Although the receptors for ADP, PAR-1 peptide and epinephrine are present in the zebrafish genome, the collagen receptor GPVI was missing. In this study, we identified G6fL as a collagen receptor in zebrafish thrombocytes. Furthermore, IFT knockdown results in reduction in Wnt signaling. The Wnt signaling has been shown to be involved in megakaryocyte proliferation and proplatelets production. Therefore, defects in IFT could lead to thrombocytopenia. Splenectomy is performed in humans to treat such conditions. Therefore, in this study we developed a survival surgery protocol for splenectomy. We have shown that number of thrombocytes and their microparticles increase following splenectomy in zebrafish. Thus overall the studies on thrombocyte function in zebrafish could enhance fundamental knowledge on hemostasis and may provide future target candidates for therapies.

Cellular and molecular mechanisms of postembryonic muscle fibre recruitment were investigated in zebrafish (Danio rerio L.), a standard animal model for developmental and genetic studies. Distinct cellular mechanisms of postembryonic muscle fibre recruitment in fast and slow myotomal muscles were found. In slow muscle, three overlapping waves of stratified hyperplasia (SH) from distinct germinal zones sequentially contributed to a slow and steady increase in fibre number (FN) through the life span. In fast muscle, SH only contributed to an initial increase of FN in early larvae. Strikingly, mosaic hyperplasia (MH) appeared in late larvae and early juveniles and remained active until early adult stages, accounting for >70% of the final fibre number (FFN). The molecular regulation of postembryonic muscle fibre recruitment was then studied by characterising myospryn and cee, two strong candidate genes previously identified from a large scale screen for genes differentially expressed during the transition from hyperplastic to hypertrophic muscle phenotypes. Zebrafish myospryn contained very similar functional domains to its mammalian orthologues, which function to bind to other proteins known to regulate muscle dystrophy. Zebrafish myospryn also shared a highly conserved syntenic genomic neighbourhood with other vertebrate orthologues. As in mammals, zebrafish myospryn were specifically expressed in striated muscles. Zebrafish cee was a single-copy gene, highly conserved among metazoans, ubiquitously expressed across tissues, and did not form part of any wider gene family. Its protein encompassed a single conserved domain (DUF410) of unknown function although knock-down of cee in C. elegans and yeast have suggested a role in regulating growth patterns. Both myospyrn and cee transcripts were up-regulated concomitant with the cessation of postembryonic muscle fibre recruitment in zebrafish, indicating a potential role in regulating muscle growth. Furthermore, a genome-wide screen of genes involved in the regulation of postembryonic muscle fibre recruitment was performed using microarray. 85 genes were found to be consistently and differentially expressed between growth stages where muscle hyperplasia was active or inactive, including genes associated with muscle contraction, metabolism, and immunity. Further bioinformatic annotation indicated these genes comprised a complex transcriptional network with molecular functions, including catalytic activity and protein binding as well as pathways associated with metabolism, tight junctions, and human diseases. Finally, developmental plasticity of postembryonic muscle fibre recruitment to embryonic temperature was characterised. It involved transient effects including the relative timing and contribution of SH and MH, plus the rate and duration of fibre production, as well as a persistent alteration to FFN. Further investigation of FFN of fish over a broader range of embryonic temperature treatments (22, 26, 28, 31, 35°C) indicated that 26°C produced the highest FFN that was approximately 17% greater than at other temperatures. This finding implies the existence of an optimal embryonic temperature range for maximising FFN across a reaction norm. Additionally, a small but significant effect of parental temperature on FFN (up to 6% greater at 24 and 26°C than at 31°C) was evident, suggesting some parental mechanisms can affect muscle fibre recruitment patterns of progeny. This work provides a comprehensive investigation of mechanisms underlying postembryonic muscle fibre recruitment and demonstrates the power of zebrafish as an ideal teleost model for addressing mechanistic and practical aspects of postembryonic muscle recruitment, especially the presence of all major phases of muscle fibre production in larger commercially important teleost species.

Environmental toxicants are ubiquitous throughout the environment as a result of human activity. Among these toxicants, environmental estrogens are a category of particular concern due to their environmental prevalence and potency in altering reproductive traits. While many studies have addressed the detrimental effects of environmental estrogens on both aquatic and terrestrial organisms, few have analyzed the potential for these compounds to alter mitochondrial function. Mitochondria are the primary energy-generating system for all eukaryotic life, supporting all aspects of development, metabolism, and growth. Each cell within the body contains many mitochondria which in turn contain multiple copies of their own DNA genome, mitochondrial DNA (mtDNA). Mutations in mtDNA are responsible for a wide range of human diseases such as metabolic syndromes, cancers, and obesity. Among these mitochondrial diseases many are characterized by increased levels of heteroplasmy, multiple mitochondrial DNA haplotypes within an individual. Increased heteroplasmy can alter normal mitochondrial function and influence disease initiation and progression. To date, no studies have investigated the effects of synthetic estrogens on mitochondrial genome stability. Synthetic estrogens have the capacity to bind to estrogen receptors and initiate estrogenic responses through translocation into the mitochondrion. Despite our knowledge about the relationship of heteroplasmy and disease, we still do not have a complete grasp of the mechanisms of heteroplasmic induction. Here we report our analysis of the effects of 17α-ethynylestradiol (EE2) exposure in three studies to investigate its effect on mitochondrial genome stability. Data analysis reveals a statistically significant relationship between EE2 exposure and increased heteroplasmic frequency.