Chow, Wing Shan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 118-129). / Abstract also in Chinese. / Abstract --- p.i / 摘要 --- p.iv / Acknowledgements --- p.viii / Table of Contents --- p.ix / List of Tables --- p.xiii / List of Figures --- p.xv / List of Abbreviations --- p.xviii / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Pesticide contaminations in the environment --- p.1 / Chapter 1.2 --- Pesticides --- p.1 / Chapter 1.2.1 --- Usage of pesticide in the world --- p.1 / Chapter 1.2.2 --- Organochlorine (OC) pesticides --- p.3 / Chapter 1.2.3 --- Organophosphate (OP) pesticides --- p.4 / Chapter 1.2.4 --- Carbamate pesticides: --- p.6 / Chapter 1.2.5 --- Pyrethroid pesticides: --- p.6 / Chapter 1.3 --- Toxicological model: Zebrafish --- p.7 / Chapter 1.4 --- Biomarkers --- p.9 / Chapter 1.4.1 --- Cytochrome P450 1A (CYP1A) --- p.12 / Chapter 1.4.2 --- Cytochrome P450 3A65 (CYP3A65) --- p.14 / Chapter 1.4.3 --- Biomarker for estrogenicity - Vitellogenin (VTG1) --- p.15 / Chapter 1.4.4 --- Catalase (CAT) and Glutathione S-transferase (GST) --- p.18 / Chapter 1.4.4.1 --- Catalase (CAT) --- p.18 / Chapter 1.4.4.2 --- Glutathion S-transferase (GST) --- p.19 / Chapter 1.4.5 --- Multiple Drug Resistance (MDR1) --- p.20 / Chapter 1.4.6 --- Acetylcholinesterase (AChE) --- p.21 / Chapter 1.5 --- Objectives of this study --- p.26 / Chapter Chapter 2 --- "Toxicity assay and biomarker studies on zebrafish embryo-larvae exposed to organochlorine pesticides: endosulfan, heptachlor and methoxychlor" --- p.28 / Chapter 2.1 --- Introduction --- p.28 / Chapter 2.2 --- Materials and methods --- p.30 / Chapter 2.2.1 --- Chemicals tested --- p.30 / Chapter 2.2.2 --- Zebrafish cultivation and egg production --- p.30 / Chapter 2.2.3 --- Determination of 96h-EC50 and 96h-LC50 of organochlorine pesticides and bisphenol-A for zebrafish embryo-larvae --- p.31 / Chapter 2.2.4 --- Pesticide exposure for determination of mRNA levels of biomarkers --- p.31 / Chapter 2.2.5 --- Extraction of total RNA from the exposed embryo-larvae samples --- p.32 / Chapter 2.2.6 --- Reverse Transcription --- p.33 / Chapter 2.2.7 --- Quantifications of mRNA levels by qPCR --- p.35 / Chapter 2.2.7.1 --- Primer design --- p.35 / Chapter 2.2.7.2 --- Validation of qPCR conditions --- p.36 / Chapter 2.2.7.3 --- Quantification of biomarker gene expression levels in zebrafish embryo-larvae --- p.42 / Chapter 2.2.8 --- Statistical analysis --- p.43 / Chapter 2.3. --- Results --- p.44 / Chapter 2.3.1 --- Toxicities of OC pesticides and bisphenol-A --- p.44 / Chapter 2.3.2 --- Effects of OC pesticides and bisphenol-A on biomarker gene expression levels --- p.44 / Chapter 2.4. --- Discussions --- p.60 / Chapter 2.4.1 --- Toxicities of OC pesticides and bisphenol-A --- p.60 / Chapter 2.4.2 --- Effects of OC pesticides on CYP1A gene expression --- p.61 / Chapter 2.4.3 --- Effects of OC pesticides on CYP3A65 gene expression --- p.61 / Chapter 2.4.4 --- Effects of OC pesticides on VTG1 gene expression --- p.63 / Chapter 2.4.5 --- Effects of OC pesticides on MDR1 gene expression --- p.64 / Chapter 2.5 --- Conclusion --- p.65 / Chapter Chapter 3 --- "Toxicity assay and biomarker studies on zebrafish embryo-larvae exposed to a organochlorine pesticide, chlorpyrifos" --- p.66 / Chapter 3.1 --- Introduction --- p.66 / Chapter 3.2 --- Materials and methods --- p.68 / Chapter 3.2.1 --- Chemicals tested --- p.68 / Chapter 3.2.2 --- Zebrafish cultivation and egg production --- p.68 / Chapter 3.2.3 --- Determination of 96h-EC50 and 96h-LC50 of chlorpyrifos for zebrafish embryo-larvae --- p.68 / Chapter 3.2.4 --- Pesticide exposure for determination of mRNA levels of biomarkers --- p.68 / Chapter 3.2.5 --- Extraction of total RNA from the exposed embryo-larvae samples --- p.69 / Chapter 3.2.6 --- Reverse Transcription --- p.69 / Chapter 3.2.7 --- Quantifications of mRNA levels by qPCR --- p.70 / Chapter 3.2.7.1 --- Primer design --- p.70 / Chapter 3.2.7.2 --- Validation of qPCR conditions --- p.70 / Chapter 3.2.7.3 --- Quantification of biomarker gene expression levels in zebrafish embryo-larvae --- p.75 / Chapter 3.2.8 --- Determination of acetylcholinesterase (AChE) activities --- p.76 / Chapter 3.2.9 --- Statistical analysis --- p.77 / Chapter 3.3 --- Results --- p.78 / Chapter 3.3.1 --- Toxicities of chlorpyrifos --- p.78 / Chapter 3.3.2 --- Effects of chlorpyrifos on CAT and GST gene expression levels --- p.81 / Chapter 3.3.3 --- Effects of chlorpyrifos on acetylcholinesterase (AChE) activity --- p.83 / Chapter 3.4 --- Discussions --- p.86 / Chapter 3.4.1 --- Toxicity of chlorpyrifos --- p.86 / Chapter 3.4.2 --- Effect of chlorpyrifos on CAT and GST gene expressions --- p.86 / Chapter 3.4.3 --- Effect of chlorpyrifos on AChE activity --- p.88 / Chapter 3.5 --- Conclusions --- p.89 / Chapter Chapter 4 --- Toxicity assay and biomarker studies on zebrafish embryo-larvae exposed to carbamate and pyrethroid pesticides --- p.90 / Chapter 4.1 --- Introduction --- p.90 / Chapter 4.2 --- Materials and methods --- p.92 / Chapter 4.2.1 --- Chemicals tested --- p.92 / Chapter 4.2.2 --- Zebrafish cultivation and egg production --- p.92 / Chapter 4.2.3 --- Determination of 96h-EC50 and 96h-LC50 of aldicarb and cypermethrin for zebrafish embryo-larvae --- p.92 / Chapter 4.2.4 --- Pesticide exposure for determination of mRNA levels of biomarkers --- p.92 / Chapter 4.2.5 --- Quantification of biomarker gene expression levels in zebrafish embryo- larvae and Determination of acetylcholinesterase (AChE) activity --- p.94 / Chapter 4.2.6 --- Statistical analysis --- p.94 / Chapter 4.3 --- Results --- p.95 / Chapter 4.3.1 --- Toxicities of aldicarb and cypermethrin --- p.95 / Chapter 4.3.2 --- Effects of aldicarb and cypermethrin on CAT and GST gene expression levels.. --- p.99 / Chapter 4.3.3 --- Effects of aldicarb on acetylcholinesterase (AChE) activity --- p.102 / Chapter 4.4 --- Discussion --- p.105 / Chapter 4.4.1 --- Toxicity of aldicarb of cypermethrin --- p.105 / Chapter 4.4.2 --- Effect of aldicarb and cypermethrin on CAT and GST gene expressions --- p.105 / Chapter 4.4.3 --- Effect of aldicarb on AChE activity --- p.107 / Chapter 4.5 --- Conclusion --- p.108 / Chapter Chapter 5 --- General Conclusion --- p.109 / Chapter 5.1 --- Toxicities of pesticides --- p.109 / Chapter 5.2 --- Effects of OC pesticides on biomarker gene expressions --- p.113 / Chapter 5.3 --- "Effects of chlorpyrifos, aldicarb and cypermetrhin on biomarker gene expressions" --- p.116 / Chapter 5.4 --- Effect of chlorpyrifos and aldicarb on AChE activity --- p.116 / References --- p.118

Lau, Shuk Wa. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 84-102). / Abstracts in English and Chinese. / Thesis Committee --- p.i / Abstract (in English) --- p.ii / Abstract (in Chinese) --- p.iv / Acknowledgement --- p.v / Table of content --- p.vi / List of figures --- p.ix / Symbols and abbreviations --- p.x / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Structure of ovarian follicles --- p.1 / Chapter 1.2 --- Folliculogenesis and its control --- p.2 / Chapter 1.2.1 --- Ovarian follicle growth and development --- p.2 / Chapter 1.2.2 --- Follicle recruitment and regulation --- p.4 / Chapter 1.2.3 --- Oocyte maturation and ovulation --- p.9 / Chapter 1.2.4 --- Intercellular communication between oocytes and somatic cells --- p.10 / Chapter 1.3 --- Overview of proteomics --- p.12 / Chapter 1.3.1 --- Two-dimensional gel electrophoresis --- p.13 / Chapter 1.3.2 --- Mass spectrometry --- p.14 / Chapter 1.4 --- Objectives of the study --- p.15 / Chapter Chapter 2 --- Proteomic Analysis of Folliculogenesis in Zebrafish Ovary --- p.19 / Chapter 2.1 --- Introduction --- p.19 / Chapter 2.2 --- Materials and Methods --- p.21 / Chapter 2.2.1 --- Animals --- p.21 / Chapter 2.2.2 --- Isolation of ovarian follicles --- p.21 / Chapter 2.2.3 --- Protein extraction and quantification --- p.22 / Chapter 2.2.4 --- Two-dimensional electrophoresis --- p.23 / Chapter 2.2.5 --- Staining --- p.24 / Chapter 2.2.6 --- In-gel digestion --- p.24 / Chapter 2.2.7 --- Mass spectrometry --- p.25 / Chapter 2.3 --- Results --- p.25 / Chapter 2.3.1 --- Establishment of the protein profiles of different follicle stages --- p.25 / Chapter 2.3.2 --- Mass spectrometry analysis on the differentially expressed proteins --- p.26 / Chapter 2.4 --- Discussion --- p.27 / Chapter Chapter 3 --- Characterization of Y-box Binding Protein 1 (YB-1) in Zebrafish --- p.46 / Chapter 3.1 --- Introduction --- p.46 / Chapter 3.2 --- Materials and Methods --- p.49 / Chapter 3.2.1 --- Animals --- p.49 / Chapter 3.2.2 --- Isolation of ovarian follicles --- p.49 / Chapter 3.2.3 --- Protein extraction and quantification --- p.49 / Chapter 3.2.4 --- SDS polyacrylaminde gel electrophoresis (SDS-PAGE) --- p.50 / Chapter 3.2.5 --- Western blot analysis --- p.50 / Chapter 3.2.6 --- RNA isolation and reverse transcription --- p.51 / Chapter 3.2.7 --- Semi-quantitative RT-PCR quantification of expression --- p.51 / Chapter 3.2.8 --- Data analysis --- p.52 / Chapter 3.2.9 --- Immunohistochemistry --- p.52 / Chapter 3.2.10 --- Cloning of full-length ybl cDNA from zebrafish ovary and construction of recombinant plasmid for expressing ybl --- p.53 / Chapter 3.2.11 --- Expression and purification of recombinant zebrafish YB-1 protein --- p.54 / Chapter 3.2.12 --- Immunoprecipitation --- p.55 / Chapter 3.3 --- Results --- p.58 / Chapter 3.3.1 --- Confirmation of the presence of YB-1 --- p.58 / Chapter 3.3.2 --- Tissue distribution of YB-1 protein and ybl gene expression in zebrafish --- p.58 / Chapter 3.3.3 --- Stage distribution of YB-1 protein and ybl gene expression in ovarian follicles --- p.59 / Chapter 3.3.4 --- Localization of YB-1 protein within the ovarian follicle --- p.59 / Chapter 3.3.5 --- Degradation of YB-1 in the ovary --- p.60 / Chapter 3.3.6 --- Production of recombinant YB-1 (zfYB-1) --- p.60 / Chapter 3.3.7 --- Identification of YB-1 -bound partners --- p.60 / Chapter 3.4 --- Discussion --- p.61 / Chapter Chapter 4 --- General Discussion --- p.77 / References --- p.84

Le virus Epstein-Barr (EBV) est un γ-Herpesvirus infectant plus de 95 % de la population mondiale. Le facteur de transcription viral ZEBRA est responsable de la transition entre phases latentes et lytiques du virus. ZEBRA est une protéine de la famille des protéines bZIP. Elle active les promoteurs des gènes lytiques de l'EBV en se fixant sur des sites ADN spécifiques appelés sites ZREs. ZEBRA fixe préférentiellement certains sites ZREs lorsqu'ils sont méthylés sur leurs motifs CpG et notamment le site cible ZRE2 du promoteur viral du gène précoce BRLF1. Cette capacité particulière est unique à ZEBRA parmi les autres membres de la famille bZIP et s'avère critique pour l'activation du cycle lytique étant donné que le génome de l'EBV est intensivement methylé durant la phase de latence. Nous avons résolu la structure cristallographique du domaine bZIP de ZEBRA en complexe avec le site ZRE2 méthylé. L'analyse structurale corrélée à une étude de mutagenèse et à la détermination des affinités de ZEBRA pour ses sites ADN cibles, nous permet de proposer une hypothèse quant au mécanisme de fixation préférentielle de l'ADN méthylé par le facteur de transcription ZEBRA. En parallèle, nous avons débuté un criblage à haut débit de composés chimiques pouvant inhiber la fixation à l'ADN de ZEBRA et par conséquent, l'induction du cycle lytique. Ce travail améliore considérablement notre compréhension du mécanisme d'induction du cycle lytique par ZEBRA et aidera potentiellement au développement de nouveaux agents thérapeutiques contre les pathologies associées à l'EBV.

<p>The development of the avian embryo is dependent of heat provisioning from the parents. To increase the heat transfer to a cooled egg the Zebra finch females develop a brood patch. Mild cooling generally constricts the blood vessels but the Arterio-venous anastomoses (AVA) in the brood patch in birds dilate. This is called cold-induced vasodilation CIVD. The Zebra finches were anesthetized with isoflurane and the brood patch was stimulated with a cooling probe set at 20-21 °C. Differences in the vascular changes to cooling in broody and non- broody birds were studied by comparing males and broody females. The brood patch skin was cooled, but no cold-induced vasodilation (CIVD) was documented for the males or the broody females. Isoflurane anesthesia depresses the sympathetic nervous system activity and the results support that the mechanism for CIVD in the brood patch of Zebra finches depends on a neural pathway, but does not exclude a local non-neural mechanism.</p>

<p>During the breeding season Zebra finch, Taeniopygia guttata, females develops a brood patch on the ventral surface which facilitates heat exchange between the incubating bird and the egg. The brood patch has to be sensitive to changes in temperature, so that the eggs can be kept at an optimal temperature for embryo development. If the egg temperature drops it has to be re-warmed. Mild cooling of the brood patch has been shown to cause cold induced vasodilation, but the responsible mechanism for this is not known. In this study we investigated if known thermoreceptors, TRPV3 and TRPV4, could be involved in the alteration of blood flow. To activate TRPV3 and TRPV4 two agonists, carvacrol and 4α-PDD respectively, were applied on the brood patch. Changes in skin temperature and vascularity were then examined. The results obtained did not reveal any changes in the vascularity. Temperature changes in the skin that could be caused by an alteration in blood flow did not significantly change either. Still, a role of these channels in the brood patch cannot be excluded.</p>

During the breeding season Zebra finch, Taeniopygia guttata, females develops a brood patch on the ventral surface which facilitates heat exchange between the incubating bird and the egg. The brood patch has to be sensitive to changes in temperature, so that the eggs can be kept at an optimal temperature for embryo development. If the egg temperature drops it has to be re-warmed. Mild cooling of the brood patch has been shown to cause cold induced vasodilation, but the responsible mechanism for this is not known. In this study we investigated if known thermoreceptors, TRPV3 and TRPV4, could be involved in the alteration of blood flow. To activate TRPV3 and TRPV4 two agonists, carvacrol and 4α-PDD respectively, were applied on the brood patch. Changes in skin temperature and vascularity were then examined. The results obtained did not reveal any changes in the vascularity. Temperature changes in the skin that could be caused by an alteration in blood flow did not significantly change either. Still, a role of these channels in the brood patch cannot be excluded.

Incorporation of nanoparticles (NPs) into consumer products is on the rise and human exposure to NPs is unavoidable. Currently, there is insufficient data to assess the safety of nanoparticles. I conducted a series of five studies using the zebrafish model to determine which NP components (i.e., core material or surface functionalization) contribute to biological responses and how ionic strength influences these results. The first study employed a systematic, rapid embryonic zebrafish assay to identify specific responses to precisely engineered lead sulfide (PbS-NPs) and gold nanoparticles (AuNPs) functionalized with different surface ligands. Lead sulfide nanoparticles functionalized with either 3-mercaptopropanesulfane (MT) or sodium 2,3-dimercaptopropanesulfonate (DT) ligands with nearly identical core sizes caused differential responses at the same concentration. I determined that the different responses were because MT-functionalized NPs released more soluble lead ions than DT-functionalized NPs due to different decomposition and oxidation rates. The second study investigated the different biological responses of three NPs identified during toxicity screening of a gold nanoparticle library. AuNPs functionalized with 2-mercaptoethanesulfonic acid (MES), N,N,N-trimethylammoniumethanethiol (TMAT), or 2-(2-(2-mercaptoethoxy)ethoxy)ethanol (MEEE), induced differential biological responses in embryonic zebrafish at the same concentration. Exposure to MES-AuNPs induced sublethal effects, while TMAT-AuNPs were embryo-lethal and MEEE-AuNPs were benign. Gold tissue concentration was confirmed to be similar in exposed embryos using inductively coupled-mass spectrometry. Microarrays were used to gain insight to the causes of the different responses. This approach identified that MES- and TMAT-AuNPs perturbed inflammatory and immune responses. These differential biological responses may be due to misregulated transport mechanisms causing numerous downstream defects unique to each surface functional group‟s property. In the next study, I tested the long-term consequences of developmental exposure to TMAT-, MES, and MEEE-AuNPs, and showed that MES- and TMAT-AuNPs affected larval behavior that persisted into adulthood. During the course of these investigations, I found that high ion concentration in exposure solutions results in NP agglomeration, presenting a problem for NP testing in the zebrafish model. For the fourth study, I focused on solving this by determining that zebrafish can be raised in nearly ion-free media without adverse consequences. When 3-MPA-AuNPs were dispersed in this new low ionic media, I observed adverse responses in the embryonic zebrafish toxicity assay, but not when the NPs were suspended in high ionic media. Thus, I demonstrated that the media greatly influences both agglomeration rates and biological responses, but most importantly, that the zebrafish is insensitive to external ions. The fifth study focused on the adverse response observed when embryonic zebrafish were exposed to 3-MPA-AuNPs. Exposed larvae failed to respond to a touch in the caudal fin at 120 hours post fertilization (hpf). Addition of a neuromuscular stimulus, nicotine, revealed the exposed embryos were not paralyzed, but experienced a reduction in axonal projections. A global genomic analysis (RNA-seq) using embryos exposed to 3-MPA-AuNP and MEEE-AuNPs (non-toxic control) from 6 to 120 hpf suggested that neurophysiological and signal transduction processes were perturbed. Functional analysis of the data led to the hypothesis that the most elevated gene, early growth response 1 (EGR-1), impacts axonogenesis in the caudal fin, interfering with glutaminergic synapses and preventing the connection of sensory neurons and touch perception. Although MEEE-AuNPs did not cause morphological defects, the RNA-seq analysis identified that these NPs perturbed immune and inflammatory system processes. Collectively, these results suggest that surface functional groups drive the differential responses to nanomaterials. The five studies summarized here confirm that a systems toxicological approach using the zebrafish model enables the rapid identification of structure-activity relationships, which will facilitate the design of safer nano-containing products. / Graduation date: 2013

University of Macau / Institute of Chinese Medical Sciences

Although deafness is a universal problem, effective treatments have remained elusive. In order to develop potential treatments, an overall understanding of the cellular process of auditory hair cell regeneration, which occurs in fish but not mammals, must be established. A previous microarray analysis and qRT-PCR validation of noise-exposed zebrafish showed that growth hormone (GH) was significantly upregulated during the process of auditory hair cell regeneration. Thus, GH may play an important role during hair cell regeneration. However, cellular effects of exogenous GH in the zebrafish auditory hair cell regeneration have not been examined after noise exposure. To understand the effect of GH in hair cell regeneration, adult zebrafish were exposed to a 150 Hz pure tone at a source level of 179 dB re 1 μPa RMS for 36 hours. Afterward the fish were immediately injected intraperitoneally with carp recombinant GH (20 μg/gram of body mass) or buffer (0.1 M, pH 7.4 phosphate buffer) and then placed in a recovery tank. The effect of GH on apoptosis in fish inner ear end organs were examined using TUNEL-labeling. Cell proliferation was measured by BrdU incorporation assay. Hair cell regeneration was determined by phalloidin-labeling to allow visualization of hair cell stereociliary bundles. After GH injection, the numbers of TUNEL-labeled cells showed a significant decrease in all three inner ear end organs (saccule, lagena, utricle), suggesting GH may suppress hair cell death induced by acoustic trauma. Higher levels of cell proliferation were also observed in the ears of GH-injected fish, indicating that GH is capable of activating cell mitosis in the zebrafish auditory system. Following sound exposure, the GH-injected group exhibited greater numbers of saccular hair cell bundles compared to the buffer-injected group. These results indicate that GH promotes hair cell regeneration following acoustic damage. Future studies are needed to examine the potential therapeutic benefits of GH in the mammalian ear.

Zebra mussels (Dreissena polymorpha) have invaded a great proportion of the waters of the eastern part of North America. This mollusk may be a useful sentinel species for bioaccumulation of heavy metals and organic contaminants in aquatic ecosystems. The zebra mussel's capacity to bioaccumulate cadmium at environmentally relevant exposure and to sequester metals in metal-binding proteins, metallothioneins, was investigated. Elevated (relative to control) concentrations of Cd$ sp{2+}$ were detected in Dreissena exposed to $ ge$2 $ mu$g Cd/liter suggesting that zebra mussels cannot regulate Cd$ sp{2+}$ of trace exposure concentration. More than 85% of the measured Cd$ sp{2+}$ was bound to metallothioneins. / Lesser scaup (Aythya affinis) was assessed as a model species for potential impacts of zebra mussels on higher trophic levels. The feasibility of incubating and rearing scaup in semi-natural conditions was evaluated. A series of biomarkers was assessed in 3 groups of scaup fed a contaminant-free diet or diets containing zebra mussels from the St-Lawrence River or Lake Erie. / Lesser scaup proved to be a suitable species to raise in captivity providing daily water misting through out incubation and strict hygiene conditions during brooding. A hatching success of 89% was achieved. Appropriate housing, feeding and bathing conditions yielded low (3%) post-hatching mortality. / Phagocytosis and respiratory burst activities of heterophils of scaup were suppressed after 6 weeks of feeding on zebra mussels, compared to the control group. These two heterophilic functions were negatively correlated with the incidence of pododermatitis (bacterial feet infection), suggesting a suppression of the non specific immunity. Increased liver/body mass ratio and decreased hepatic vitamin A (retinol and retinyl palmitate) concentrations were observed in scaups fed zebra mussels. Lipid accumulation and glycogen overload were found in the livers of scaups fed mussels from the St-Lawrence River and the Lake Erie groups, respectively. These immunological, biochemical and histopathological biomarkers show promise for monitoring "early" injury and may help in the understanding of health impairment of different species of waterfowl exposed to xenobiotics via contaminated food sources.