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The development of the cardiovascular system, including the heart and circulating blood within a vascular network, relies on mesoderm-derived cells to contribute to the development of both cardiac and hematopoietic tissues. This dissertation focuses on exploring the roles of crip2, downstream of the transcription factor Nkx2.5 established from an RNA sequencing dataset, in cardiac and hematopoietic development using the zebrafish model. In Chapter 2, we investigate the influence of Crip proteins on the development of the zebrafish heart. Congenital heart defects (CHDs), affecting approximately 1% of live births, arise from structural anomalies during heart development primarily caused by genetic mutations. While there isn’t just one driver of CHDs, transcription factors such as Nkx2.5, play a pivotal role in guiding cardiac morphogenesis. NKX2-5-associated CHDs often involve outflow tract (OFT) malformations. The development of the heart involves two progenitor cell populations, the first heart field (FHF) and second heart field (SHF), contributing to the linear heart tube and subsequent growth. Despite understanding the role of Nkx2.5, the spatiotemporal mechanisms directed by Nkx factors in SHF progenitor specification, proliferation, and identity maintenance remain elusive. This study aims to uncover novel effectors of Nkx transcriptional regulation, using RNA sequencing on dissected wild-type and nkx2.5-/- zebrafish hearts at 26 hours post fertilization (hpf). This work focuses on a LIM domain protein, cysteine rich intestinal protein 2 (crip2), identified as a mis-regulated gene in nkx2.5-deficient embryos, and we explore its role downstream of nkx genes in SHF-derived arterial pole formation. While crip2 is abundantly expressed in the developing heart, the family member crip3 also shows a mild expression pattern. Loss-of-function mutations in crip2 and crip3 (referred to as cripDM) reveal normal cardiac chamber specification. Atrioventricular canal morphology remains unaffected in cripDM embryos. The OFT in cripDM embryos displays a significant dilation, accompanied by increased ltbp3 expression. Surprisingly, the smooth muscle cell population of the OFT does not explain the size increase. This research expands our understanding of OFT development, highlighting the multi-layered contributions of various cell types and factors. In Chapter 3, we further examine the role of crip2 in the development of hematopoietic stem cells given its endothelial expression pattern. Hematopoietic stem and progenitor cells (HSPCs) have multilineage potential and can sustain long-term self-renewal. The ability to derive patient-specific HSCs in culture has immense therapeutic potential to overcome the shortage of compatible donors for HSC transplantations. However, differentiation protocols largely fail to produce long-lived HSCs from human pluripotent stem cells. Understanding the complex genetic networks and signaling pathways required to generate HSCs will facilitate clinical applications in patients. The hemogenic endothelium (HE) is a specialized niche of endothelial cells within the ventral portion of the dorsal aorta that gives rise to HSPCs during the definitive wave of hematopoiesis in the zebrafish embryo. Our data reveal that crip2 has a previously unrecognized function in establishing the proper endothelial cell environment for HSPC specification. CripDM embryos exhibit decreased emergence of HSCs by 26 hpf. Loss of HSPCs in the cripDM results in decreased erythroid, myeloid, and lymphoid lineage production between 30 -72 hpf; these perturbations in the hematopoietic lineages recover by 96 hpf. To decipher the spatiotemporal mechanisms underlying the cripDM phenotype, we performed single cell RNA (scRNA) sequencing of sorted, Kdrl:mCherry+ cells at 30 hpf. Our analysis reveals upregulation of genes essential for vascular development and mis-regulation of Notch signaling pathways in the cripDM embryos. Building on these data, our ongoing studies aim to investigate how crip2 regulates the endothelial niche of the ventral aorta to produce HSCs early in definitive hematopoiesis. We anticipate that our insights will inform potential therapeutic interventions for improvements of human HSC production in vitro.

The studies described herein focused on the use of intermittent halogenation to control biofouling of water intake systems by the zebra mussel, Dreissena polymorpha, the comparative response of zebra mussels and the Asian clam, Corbicula fluminea, to a surfactant -based chemical control agent, the nontarget impact associated with the control agent, and the use of the Asian clam as a biomonitor of the control agent. Effects of intermittent (2-4 hr/day) treatments with chlorine or bromine at levels of 0.5 and 1.0 mg/L (total residual oxidant) upon settling of zebra mussel veligers were examined in studies conducted in a field laboratory on western Lake Erie. Veliger densities in the water column at the field site peaked at 530/L, while mussel densities on settling monitors reached 147,083/m² over the course of the study period. Zebra mussel settling in test systems treated with the halogens was reduced by as much as 91 % in comparison with controls, although mussel densities of up to 6,044/m² still occurred. Treated mussels which remained settled had growth rates similar to controls, and reached 2-4 mm length over 30 days. The intermittent halogen treatments had no significant impact on either adult zebra mussels or Asian clams. The studies indicate that while the treatment regimes may reduce zebra mussel densities within intake systems, the threat of eventual fouling due to cumulative settling remains. / Ph. D.

This study consisted of a site characterization followed by a biomonitoring study utilizing the zebra mussel, <i>Dreissena polymorpha</i>, at the Times Beach Confined Disposal Facility (CDF), located in Buffalo, New York. Concentrations of the selected contaminants (polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and the following metals: arsenic (As), chromium (Cr), barium (Ba), mercury (Hg), cadmium (Cd), lead (Pb), selenium (Se) and silver (Ag), were at or below detection limits in the water column. In the sediment toxicant concentrations were as high as 549 mg/kg for total PAHs, 9 mg/kg for PCB Aroclor 1248, and 54, 99, 6, 355, 637, and 16 mg/kg for the metals: As, Ba, Cd, Cr, Pb, and Hg respectively. To predict contaminant bioavailability, elutriate and whole sediment toxicity tests were performed utilizing the cladoceran, <i>Daphnia magna</i>. The whole sediment tests showed a significant impact. Control survival was 84%, while the sediment treatment survival range was 1-7%. Mean control reproduction was 86.8 neonates, whereas treatment group reproduction ranged from 1.4 to 9.0. Zebra mussels, placed both in the water column (Upper) and at the sediment level (Lower), survived the 34-day exposure. Contaminants which significantly accumulated in zebra mussel tissue during the exposure period (mg/kg) were total PAHs (6.58), fluoranthene (1.23), pyrene (1.08), chrysene (0.98), benzo(a)anthracene (0.60), PCB Aroclor 1248 (1.64), As (0.97), Cr (2.87), and Ba (7.00). The accumulation of benzo(a)anthracene was statistically higher in the Upper mussels; however, this did not occur for any other toxicant. Accumulation of these contaminants in zebra mussel tissue represent a potential hazard to organisms (ie. fish and birds) which feed on them. / Master of Science

Craniofacial malformations arise from developmental defects in the head, face, and neck and account for one third of congenital defects at birth. Clinical phenotypes such as DiGeorge Syndrome, the most common microdeletion condition, illustrate a developmental link between cardiovascular and craniofacial morphogenesis. Moreover, recent fate mapping studies in mice and zebrafish support this notion through identification of a multipotent progenitor in the cardiopharyngeal field that gives rise to the heart, branchiomeric muscles, and pharyngeal arch (PA) arteries. NKX2-5 is a key cardiac transcription factor associated with human congenital heart disease and mouse models of Nkx2-5 deficiency highlight critical roles in cardiac development. In zebrafish, nkx2.5 and nkx2.7 are paralogous genes in the NK4 family expressed in cardiomyocytes and PAs. Despite the shared cellular origins of cardiac and craniofacial tissues, the function of NK4 factors in head and neck patterning has not been elucidated. Here, we demonstrate that Nkx2.7 serves as a previously unappreciated, crucial regulator of craniofacial muscle and cartilage formation. Our studies reveal a unique requirement for nkx2.7 in PA1- and PA2-derived branchiomeric muscle and cartilage elements for which nkx2.5 cannot compensate. Moreover, molecular evolutionary analysis of NK4 genes reveals that nkx2.5 and nkx2.7 are ohnologs resulting from two rounds of vertebrate whole genome duplications with an early split between them, underscoring the concept that these genes play independent roles during development. The distinct mechanistic function of nkx2.7 is elucidated by cell counting experiments that uncover the requirement of nkx2.7 in specification of PA1 and PA2 branchiomeric muscle progenitors. Furthermore, single cell RNA-sequencing performed on microdissected PA tissues from wild-type and nkx2.7-/- embryos identifies decreased expression of the ventral neural crest gene signature essential for cartilage and jaw joint morphogenesis. Together, our studies shed light on an evolutionarily conserved, unique function of Nkx2.7 in vertebrate craniofacial development and have the potential to advance our understanding of the etiologies and therapeutic interventions for patients with congenital deformities of the head and neck.

La compréhension des mécanismes moléculaires de différentes pathologies a permis la caractérisation de gènes et de protéines impliqués dans la pathogénèse et l'identification de cibles thérapeutiques intracellulaires. La nature hydrophobique de la membrane cellulaire empêche le passage des médicaments dans les cellules. Les Cell-Penetrating Peptides (CPP) ou domaines de transduction protéiques (PTD) sont des peptides qui permettent l'internalisation de macromolécules hydrophiles in cellulo et in vivo. Un nouveau peptide issu du facteur de transcription ZEBRA du virus Epstein-Barr, et qui possède des propriétés de transduction a été caractérisé récemment dans notre laboratoire. Des études par mutagénèse de délétion de la protéine ZEBRA ont permis d'identifier la région d'acides aminés (nommé ainsi MD) impliquée dans la pénétration cellulaire. Ce peptide traverse les membranes des cellules de mammifères par un mécanisme de translocation directe, même lorsqu'il est fusionné à des molécules telles que la protéine reportrice eGFP. Le mécanisme de pénétration directe représente un grand avantage pour les applications thérapeutiques: les molécules cargos peuvent être internalisées directement dans le cytoplasme cellulaire sans dégradation et sous une forme biologiquement active. L'objectif de cette thèse est d'étudier les propriétés de pénétration cellulaire du peptide MD et d'évaluer ses applications thérapeutiques comme système de vectorisation des protéines. Ce travail est structuré en trois parties. La première partie porte sur l'étude de l'optimisation de la séquence peptidique MD par réduction de taille et l'évaluation du rôle de sa composition en acides aminés dans le processus de translocation à travers la membrane cellulaire. Cette étude a conduit à l'identification d'une séquence plus courte MD (MD11) possédant une efficacité et un mécanisme de translocation inchangés. La deuxième partie décrit une approche thérapeutique basée sur MD11 visant à la complémentation protéique d'un dysfonctionnement identifiée dans la plupart des cancers. Les cellules tumorales présentent des altérations dans la machinerie de traduction résultant dans une prolifération cellulaire incontrôlée. Parmi les différents facteurs intervenant dans la régulation de ce processus, le facteur eucaryote d'initiation 3 (eIF3) contribue à l'oncogenèse et au maintien de l'état cancéreux. Ce complexe est composé de 13 sous-unités, désignées eIF3 a-m. L'expression de certaines sous-unités est altérée dans plusieurs cancers, et en particulier la sous-unité f (eIF3f) est significativement diminuée dans le mélanome, les cancers du pancréas, de la vulve, du sein, de l'intestin et de l'ovaire. L'expression ectopique par transfection transitoire du gène eIF3f inhibe la synthèse protéique et induit l'apoptose dans le mélanome et dans les cellules cancéreuses pancréatiques. A partir de ces observations, nous avons développé une approche thérapeutique innovante pour le traitement des cancers dans lesquels la protéine manquante eIF3f est produite sous forme recombinante fusionnée à la séquence de MD11, et ensuite internalisée dans les cellules cibles tumorales. Ces résultats démontrent que le système de transfert de eIF3f basé sur MD11 représente une stratégie efficace pour supprimer la prolifération des cellules tumorales. La dernière partie de cette thèse explore la propriété de pénétration de MD11 dans les cellules de levure, et en particulier dans le champignon pathogène Candida albicans. Les résultats obtenus démontrent la polyvalence de MD11, qui fonctionne comme vecteur de protéines à activité biologique aussi bien dans la levure que dans les cellules de mammifères. Le potentiel de MD11 comme système de transport et de relargage des protéines a donc été établis, toutefois certaines améliorations en ce qui concerne la formulation des protéines de fusion et des études in vivo doivent être réalisées afin de valider son efficacité thérapeutique. / In recent years, the understanding of disease molecular mechanisms has led to the identification of genes and proteins that are altered in disease state and many therapeutic targets have been found located within cells. The protective and hydrophobic nature of plasma membrane prevents therapeutic drugs from entering cells. Cell-penetrating peptides (CPPs) or protein transduction domains (PTDs) have emerged as a group of non-invasive delivery vectors for various hydrophilic macromolecules, and several in vitro and in vivo applications as pharmaceutical carriers have been reported. A novel cell-penetrating peptide deriving from the Epstein-Barr virus ZEBRA transcription factor has been recently characterized in our laboratory. A reductionist study of full-length ZEBRA protein has allowed to identify the amino acid region (named as Minimal Domain, MD) implicated in cellular uptake. This peptide is able to cross the mammalian cell membranes via a direct translocation mechanism even when fused to cargo molecules such as eGFP reporter protein. The direct penetration mechanism represents a great advantage for therapeutic applications as the cargo molecules can be directly delivered into cells cytoplasm in a biological active form. The aim of this thesis is to explore the cell-penetrating properties of the MD peptide and evaluate its applications as therapeutic protein delivery system. This work is structured in three parts.The first part describes the study on the optimization of MD peptide sequence by size-reduction and the evaluation of its amino acid composition role in the translocation process across the cell membrane. This study has led to the identification of a shorter MD sequence (MD11) with unvaried mechanism of translocation. The second section describes a MD11-based therapeutic approach aiming at repair a dysfunction of the protein synthesis identified in most cancers. The regulation of the protein synthesis has a crucial role in governing the eukaryotic cell growth and subtle defects in the translational machinery can alter the cellular physiology and lead to cell malignancy. Among the different factors intervening in the regulation of this process, the eukaryotic initiation factor 3 (eIF3) contributes to oncogenesis and maintenance of the cancer state. This complex is composed of 13 subunits (designated eIF3 a-m). The expression of eIF3 subunits is altered in several cancers, and in particular the f subunit (eIF3f) is significantly down-regulated in pancreas, vulva, breast, melanoma, ovary and small intestine tumors. The eIF3f ectopic expression by transient gene transfection inhibits cellular protein synthesis and induces apoptosis in melanoma and pancreatic cancer cells. Starting from these observations, we developed an innovative therapeutic approach for cancer treatment in which the missing eIF3f protein is produced in vitro in fusion to MD11, and delivered to cells. These results have demonstrated that the MD11- based eIF3f transfer system may represent a powerful strategy to suppress the tumor-cell proliferation. The last part of this thesis explores the cell-penetrating property of MD11 in yeast cells, and in particular in the pathogenic fungus Candida albicans. The presented results demonstrate the versatility of MD11, functioning as vectors in both yeast and mammalian cells and as carrier for proteins with biological activity.The MD11 potential as protein delivery system is evident; however some improvements regarding the fusion protein formulation and in vivo studies should be realized to validate the effectiveness of its therapeutic application.

Hipopituitarismo congênito é caracterizado por deficiência hormonal múltipla devido a mutações de fatores de transcrição envolvidos na embriogênese hipofisária. As células-tronco estão presentes na hipófise e são caracterizadas por dar origem a uma célula progenitora e uma célula indiferenciada por divisão assimétrica. Estão envolvidas na hipófise em processos de alta demanda metabólica em diferentes fases da vida. Em estudos prévios, observou-se o acúmulo dos marcadores de células-tronco Sox2 e Nr2e1 no camundongo Ames, que apresenta mutação no gene Prop1. O Sox2 é o marcador consenso de células-tronco na hipófise enquanto que o Nr2e1, nunca antes caracterizado na hipófise, é essencial para a manutenção de células-tronco e neogenese no cérebro. A perda de função deste gene pode causar agressão e falta de instinto materno em camundongos. Com isso, o objetivo desse projeto foi utilizar o animal modelo zebrafish para avaliar o papel repressor do gene prop1 e caracterizar o gene nr2e1 bem como, confirmar se o mesmo está envolvido com a diferenciação terminal na hipófise, e sua interferência no comportamento do animal mutado. O zebrafish se encaixa adequadamente nesse projeto pois é de fácil manutenção, econômico e com rápido desenvolvimento. No presente estudo criou-se 2 modelos de zebrafish utilizando-se a técnica de edição genômica conhecida como CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) para nocautear os genes prop1 e nr2e1. Esta técnica permite uma interrupção específica e substituição de bases no genoma, resultando em uma alta especificidade, baixa toxicidade celular e é herdável. O zebrafish homozigoto com mutação no gene nr2e1 se desenvolve e reproduz como o animal controle, porém apresenta um comportamento mais exploratório quando comparado com o animal selvagem e o heterozigoto. A imunofluorescência para o anticorpo Sox2 no animal mutado mostrou se diferente do selvagem, pois apresenta um aumento da expressão temporal e o mesmo não se colocaliza com o Nr2e1. A imunofluorescência feita com os hormônios não se mostrou diferente entre o mutado e o selvagem. Conclui-se diante dos achados de normalidade do desenvolvimento, fertilidade, ausência de co-localização com o gene Sox2 e presença de hormônios como Tsh, Fsh e Gh, que o gene nr2e1 não é crucial na diferenciação terminal na hipófise porém o animal mutado apresenta um comportamento diferente do animal selvagem. Os resultados da caracterização do zebrafish com mutação no gene prop1 ainda estão em andamento devido a dificuldade de se estabelecer essa linhagem / Congenital hypopituitarism is characterized by multiple hormone deficiencies due to mutations in transcription factors involved in pituitary embryogenesis. Stem cells, which by definition can each give rise to a progenitor and an undifferentiated cell by asymmetric division, are present in the pituitary gland and are important during periods of high metabolic demand in different phases of life. In previous studies, the accumulation of the stem cell markers Sox2 and Nr2e1 was observed in the Ames mouse, which harbors a mutation in Prop1. Sox2 is the consensus stem cell marker in the pituitary gland, while the role of Nr2e1 in the pituitary development has not been characterized although it is essential for neural stem cell maintenance and neogenesis in the brain and its loss of function causes pathological aggression and lack of maternal instinct in mice. In this project, the zebrafish animal model was used to characterize the role of nr2e1, to confirm whether this gene can be involved in the pituitary terminal differentiation, and to determine the effects of this gene on animal behavior. The zebrafish is a particularly appropriate model for use in this project because it is easy to maintain, is economical, and has a rapid metabolism and growth rate. In the present study, we created 2 zebrafish models by knocking out prop1 and nr2e1 using the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) genome-editing technique. This technique enables highly specific gene/reading frame interruption and/or base substitution in the genome, with low cellular toxicity and high heritability. Zebrafish with homozygous nr2e1 mutations develop and reproduce similarly to wild-type zebrafish, but present a more exploratory behavioral pattern compared to wild-type and heterozygous zebrafish. Based on immunofluorescence, Sox2 expression was higher in the mutant zebrafish than in the wild type and was not co-localized with Nr2e1 expression. Hormone expression did not differ between wild-type and mutant zebrafish. We conclude that nr2e1 is not crucial in the terminal differentiation of the hormone-forming pituitary gland; however, it induces a distinct behavioral phenotype at the larval stage. Analyses of zebrafish harboring a prop1 mutation are ongoing owing to issues with the establishment of the lineage

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.

No description available.