The need to monitor fresh water ecosystems for pollution is increasing, as is the need to develop a biomarker sensitive to a range of environmental insults. Recently, heat shock proteins have been identified as possible biomarkers of environmental contamination. However, evaluation as to their use as a biomarker of metal contamination in fish species endemic to Southern Africa is limited. The purpose of this study was to identify what members of the 70 kDa family of heat shock proteins (Hsp70) were present in the liver of Oreochromis mossambicus, and if their accumulation was altered after short-term (96 hour) exposure to aqueous copper. In addition to copper exposure, the effect of acclimation media was also examined. Tissue-level analysis was done by means of histological examination so as to determine if alterations in the accumulation of the Hsp70 family had a marked effect on the structural integrity of the liver. Specimens of Oreochromis mossambicus acclimated in either aged tap water or borehole water were placed in flow-through systems and exposed to either 10% or 20% of the LC50 value of cupric chloride for a duration of 96 hours. Control groups were run in conjunction with the exposure groups so as to set control values by which to compare. Heat shock protein analysis was done by Western blotting after separation of hepatic proteins by SDS-PAGE. For the purpose of histological analysis, representative samples were randomly selected. Analysis of the hepatic heat shock protein 70 family identified the presence of three (3) members, each of a different molecular weight. These included members of 70 kDa (Hsp70), 74 kDa (Hsp74) and 76 kDa (Hsp76). In addition to these findings, it was found that Oreochromis mossambicus accumulated high levels of particular members of the heat shock protein 70 family under unstressed conditions, affording the fish adaptability to environmental extremes. Furthermore, individuals acclimated in aged tap water showed decreased Hsp76 accumulation after exposure to sub-lethal copper concentrations, whereas those individuals acclimated in borehole water retained relatively high levels of Hsp76. Additionally, it was shown that the hepatic structure deteriorated in those individuals acclimated to the aged tap water after copper exposure, with observed increases in vacuolation, number of macrophage centres present and the occurrence of intracellular golden-brown granules. However, there was little change from the already-altered hepatic structure of those individuals acclimated in borehole water, with conspicuous golden-brown granules the most obvious histopathological condition present. Histological examination therefore proved to supplement the heat shock protein results obtained. This study thus concluded that a decrease in the accumulation of the Hsp70 family resulted in a negative organismal response, initiating deleterious alterations in the hepatic structure. Additionally, this study concluded that past water quality has a marked effect on a given biomarker response, and should be taken into careful consideration when conducting biomarker studies. / Prof. J.H.J. Van Vuren

Sequences coding for three related 16 kd heat shock proteins (hsps) of the nematode Caenorhabditis elegans were isolated and characterized. The extensive accumulation of hsp16 mRNA during heat stress facilitated the identification of two cDNAs, CEHS48 and CEHS41, which encoded hsp16 variants. These plasmids were selected by their ability to hybridize to mRNA which directed the synthesis of hspl6 in vitro, and were further characterized by sequence analysis. Two-dimensional gel electrophoresis of hspl6 synthesized in vitro from mRNA selected by hybridization to either of the cDNAs under conditions of low stringency revealed the existence of at least five electrophoretic variants with significantly different isoelectric points. The above cDNAs were used as specific probes to isolate recombinant bacteriophage containing C. elegans genomic DNA. Overlapping phage clones were used to define a region of approximately 30 kilobases. The genes coding for hsp16-48, previously identified by cDNA cloning, and for another 16 kd hsp designated hspl6-l were characterized by DNA sequencing. These two genes were arranged in a head-to-head orientation. Both the coding and flanking regions of these genes were located within a 1.9 kb region which was duplicated exactly to form a perfect 3.8 kb inverted repeat structure. This structure ended in unusual G + C-rich sequences 24 bp in length. The identity of the two arms of the inverted repeat at the nucleotide sequence level implied that the duplication event may have occurred relatively recently in evolution. Alternatively, gene conversion between the two modules could have maintained homology between the two gene pairs. Comparison of the hsp16-48 gene with its corresponding cDNA revealed the presence of a single, short intron. An intron of comparable length and in an analogous position was also found in the hsp16-1 gene. The introns separated variable and conserved regions within the amino acid sequences of the encoded heat shock proteins. A domain of approximatey 80 amino acids is contained within the conserved second exon and is homologous to a similar region in the small hsps of Drosophila, Xenopus, soybean and man as well as the a-crystallin protein of the vertebrate lens. Each hsp16 gene contained a TATA box upstream of the start of transcription. Promoter sequences, which have been shown to be required for heat inducibility in various systems, were located upstream of either TATA box Northern blot analysis showed that the hsp16-48 and hsp16-1 genes are expressed at levels approximately 20 - 40 fold lower than two closely related genes, hsp16-41 and hsp16-2, upon temperature elevation. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

No description available.

研究背景： / 肝癌是全球常見的惡性腫瘤之一，世界上每年大約有50萬死亡病例，並且呈逐年上升之勢， 是全球第3位的腫瘤死亡原因。慢性乙型和丙型肝炎病毒感染是肝癌的主要成因。肝癌惡性程度高、預後差，並且目前的治療手段非常有限，術後易復發和轉移，迄今尚無正式獲准有效治療藥物。現階段，治療肝癌的主要方法是手術切除，但是隨之引起的併發症以及較高的復發機率嚴重影響了治療的療效，大大降低肝癌病人的存活期。 / 研究目的： / 分析TDP對肝癌細胞和肝腫瘤旁細胞生長的影響；分析TDP抑癌的分子靶標蛋白及其分子機理；驗證TDP對肝癌動物模型的抑制效果。開發一種新型有效的肝癌治療藥物。 / 研究方法： / 首先用MTT法從102種來源於嶺南山竹子的純複合物中分離出了TDP,它是一種甾醇類化合物。採用MTT法檢測TDP對腫瘤細胞生長的影響；流式細胞實驗驗證TDP能否引起腫瘤細胞的凋亡；採用蛋白組學和質譜分析找出TDP抑癌的分子靶標；進一步的蛋白功能增加和缺失實驗證明Hsp27的功能和作用；生物資訊學驗證HSP27和TDP的作用結果；最後利用動物模型驗證TDP對肝腫瘤的治療效果。 / 結果： / TDP不但能效率極高的抑制肝癌細胞的生長而且可以大量誘發肝癌細胞的凋亡，而對正常的肝癌旁細胞沒有影響。二維電泳以及質譜分析TDP處理的肝癌細胞對比DMSO處理的肝癌細胞發現了具有不同表達水準的18種蛋白，Hsp27是其中一個在TDP誘導下調變化倍數較大並且與細胞凋亡有密切關係的蛋白，Hsp27的過表達以及Knock-down都充分驗證了TDP通過調節Hsp27的表達參與了依賴於caspase的線粒體凋亡途徑，在Western Blotting以及RT-PCR中得到了充分的驗證。生物資訊學預測TDP可以與Hsp27結合，實驗結果表明TDP可以誘導Hsp27的聚集並導致功能喪失。動物實驗腫瘤生長結果以及免疫組化結果證明，TDP可以在很大程度上對肝癌有抑製作用。 / 結論： / 本研究首次表明，TDP如果不是完全的,最起碼也是部分通過誘導依賴於caspase的線粒體凋亡的途徑來抑制肝癌細胞的增值和分化, 具有明顯的抗腫瘤的功效，特別是對Hsp27高表達的腫瘤細胞有比較明顯的作用，是一種值得繼續深入研究的有較高潛在價值的藥物。 / Background： / Hepatocellular carcinoma (HCC), the most common primary hepatic malignancy, is a global public health problem that accounts for approximately 500,000 deaths annually. Chronic hepatitis B and hepatitis C infections are the major risk factors for the development of HCC. Due to the high rate of these infections, the incidence of HCC remains alarmingly high globally. Although great advances have been made in HCC treatment, poor prognosis and high risk of recurrence have been major challenges to patients. Currently, surgical resection is the main treatment option for HCC patients; however, complications arising from surgery can threaten its therapeutic effect and patients’ survival. / Objectives： / To characterize the functions of 1,3,5-trihydroxy-13,13-dimethyl-2H-pyran [7,6-b]Xanthone (TDP) in cell proliferation of HepG2 cells; to discover the molecular target genes and elucidate the underlying molecular mechanism of TDP; to examine the in vivo function of TDP in a nude mouse tumor model of HCC. Finally, to investigate TDP’s potential as an anti-HCC drug candidate. / Methods and Results： / In this study, we discovered that TDP, isolated from the Chinese medicinal herb, Garcinia oblongifolia, strongly inhibited cell growth and induced caspase-dependent mitochondrial apoptosis in HCC, as evidenced from MTT assay and flow cytometry analysis. Two-dimensional gel electrophoresis and mass spectrometry-based comparative proteomics were applied to find the molecular targets of TDP in HCC cells, and eighteen proteins were identified with altered expression, with Hsp27 protein being one of the proteins most significantly down-regulated by TDP. Further Hsp27-siRNA knockdown and Lenti-Hsp27 overexpression studies found that Hsp27 was involved in TDP induced mitochondrial apoptosis, with bioinformatics predictions and biological results revealing that TDP might cause Hsp27 protein form dimer and consequent degradation via the ubiquitin-proteasome system. Finally, subcutaneously injecting cancer cells with Hsp27 expression vector into the dorsal flank of nude mice tumor model also demonstrated the suppressive effect of TDP on HCC. / Conclusions： / In summary, our study discovered that TDP, a natural xanthone, was a potent inhibitor of Hsp27 in HCC. TDP inhibited cell growth and induced apoptosis by inducing Hsp27 degradation, which stimulated mitochondrial cytochrome C release which resultantly activated caspase-3 and caspase-9. These data combined with the results of the animal model strongly supported TDP’s potential as a novel anti-cancer drug candidate, especially for cancers with an abnormally high expression of Hsp27. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Fu, Weiming. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 111-151). / Abstract also in Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.iiv / Acknowledgment --- p.vi / Publications --- p.viii / List of Contents --- p.ix / List of Tables --- p.xii / List of Figures --- p.xiii / List of Abbreviations --- p.xv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- General Introduction --- p.1 / Chapter 1.1.1 --- Overview of HCC --- p.1 / Chapter 1.1.2 --- Epidemiology of HCC in China and Hong Kong --- p.3 / Chapter 1.2 --- Etiology of HCC --- p.7 / Chapter 1.2.1 --- Cirrhosis --- p.8 / Chapter 1.2.2 --- HBV infection --- p.9 / Chapter 1.2.3 --- HCV infection --- p.10 / Chapter 1.2.4 --- Viral Co-Infection --- p.11 / Chapter 1.2.5 --- Fatty Liver Disease and Cryptogenic Cirrhosis --- p.12 / Chapter 1.2.6 --- Alcohol --- p.13 / Chapter 1.2.7 --- Iron --- p.13 / Chapter 1.2.8 --- Aflatoxin --- p.14 / Chapter 1.2.9 --- Others --- p.14 / Chapter 1.3 --- Diagnosis of HCC --- p.14 / Chapter 1.4 --- Prognosis of HCC --- p.17 / Chapter 1.5 --- Treatment of HCC --- p.19 / Chapter 1.5.1. --- Early stage --- p.19 / Chapter 1.5.2. --- Intermediate and advanced stage --- p.24 / Chapter 1.5.3. --- Terminal stage --- p.28 / Chapter 1.6 --- Signaling pathways in HCC --- p.28 / Chapter 1.6.1 --- Proliferation signaling pathways --- p.29 / Chapter 1.6.2 --- Signaling pathways frequently dysregulated in HCC --- p.30 / Chapter 1.6.3 --- Pathways involved in liver development and cell differentiation --- p.34 / Chapter 1.6.4 --- Inflammation pathways involved in hepatocarcinogenesis --- p.35 / Chapter 1.6.5 --- Pathways involved in neoangiogenesis --- p.37 / Chapter 1.6.6 --- The P53 tumor suppressor --- p.38 / Chapter 1.6.7 --- Heat shock proteins in HCC --- p.39 / Chapter 1.7 --- The roles of microRNAs in liver cancer progression --- p.42 / Chapter 1.8 --- TCM in the treatment of HCC --- p.45 / Chapter 1.8.1 --- Introduction --- p.45 / Chapter 1.8.2 --- Garcinia --- p.49 / Chapter 1.9 --- Objectives of the study --- p.51 / Chapter Chapter 2 --- Materials and Methods --- p.52 / Chapter 2.1 --- Preparation of the pure compounds --- p.52 / Chapter 2.2 --- Liver cell lines and tissue culture --- p.52 / Chapter 2.3 --- Human tissue samples --- p.52 / Chapter 2.4 --- Cell viability assessment with MTT assay --- p.53 / Chapter 2.5 --- Apoptosis analysis --- p.53 / Chapter 2.6 --- Two-dimensional electrophoresis (2-DE), protein visualization and image analysis --- p.54 / Chapter 2.6.1 --- Materials --- p.54 / Chapter 2.6.2 --- Protein extraction --- p.54 / Chapter 2.6.3. --- 2-DE protein profiling --- p.55 / Chapter 2.6.4. --- Gel staining and image analysis --- p.55 / Chapter 2.6.5. --- In-gel protein digestion with trypsin --- p.56 / Chapter 2.6.6. --- MALDI-TOF mass spectrometric analysis --- p.56 / Chapter 2.6.7. --- Database search --- p.57 / Chapter 2.7.1 --- Sample preparation --- p.58 / Chapter 2.7.2 --- SDS-PAGE --- p.58 / Chapter 2.7.3 --- Protein transfer --- p.58 / Chapter 2.7.4 --- Blocking --- p.59 / Chapter 2.7.5 --- Incubation with primary and secondary antibodies --- p.59 / Chapter 2.7.6 --- Proteins Visualization --- p.59 / Chapter 2.8 --- Real-time PCR --- p.60 / Chapter 2.9 --- Vector construction and lentivirus production --- p.61 / Chapter 2.9.1 --- Lenti-vector construction for Hsp27 expression --- p.61 / Chapter 2.9.2 --- Lentivirus production --- p.62 / Chapter 2.9.3 --- Lentivirus infection --- p.63 / Chapter 2.10 --- SiRNAs transfection. --- p.63 / Chapter 2.11 --- Identification of potential protein targets for TDP --- p.64 / Chapter 2.12 --- In Vivo Tumorigenesis --- p.64 / Chapter 2.13 --- Assay of chaperone activity of Hsp27 using lysozyme as substrate --- p.65 / Chapter 2.14 --- Mitochondria and cytosolic proteins preparation --- p.66 / Chapter 2.15 --- Immunohistochemistry (IHC) --- p.67 / Chapter 2.15.1 --- Preparation of paraffin tissue sections --- p.67 / Chapter 2.15.2 --- Immunostaining --- p.67 / Chapter 2.16 --- Methodology of this study --- p.68 / Chapter 2.17 --- Statistical analysis --- p.68 / Chapter CHAPTER 3 --- Results --- p.69 / Chapter 3.1 --- Introduction --- p.69 / Chapter 3.2 --- TDP significantly suppressed cell growth and induced apoptosis in HCC cells. --- p.69 / Chapter 3.2.1 --- TDP was identified from 102 pure compounds by using MTT assay --- p.69 / Chapter 3.2.2 --- TDP significantly suppressed HCC cell growth --- p.73 / Chapter 3.2.3 --- TDP induced the apoptosis of HCC cells --- p.74 / Chapter 3.3 --- Study of the molecular mechanism of TDP on HCC --- p.76 / Chapter 3.3.1 --- The comparative proteomic profiling --- p.76 / Chapter 3.3.2 --- Hsp27 was one of the molecular targets of TDP in HepG2 cells. --- p.80 / Chapter 3.3.3 --- TDP induced apoptosis through the caspase-dependent mitochondrial pathway. --- p.82 / Chapter 3.3.4 --- Hsp27 involved in the mitochondrial apoptosis induced by TDP --- p.84 / Chapter 3.3.5 --- Enforced Hsp27 overexpression rescued the mitochondrial apoptosis induced by TDP in HepG2 cells --- p.87 / Chapter 3.3.6 --- The possible regulatory signaling by TDP --- p.91 / Chapter 3.4 --- TDP directly targeted Hsp27 and destroyed its chaperone action --- p.92 / Chapter 3.5 --- Degradation of Hsp27 aggregation stimulated by TDP was mediated by ubiquitin-proteasome system (UPS) pathway --- p.96 / Chapter 3.6 --- Nude mice model demonstrated the suppressive effect of TDP on HCC --- p.97 / Chapter Chapter 4 --- Discussion and Conclusions --- p.100 / Chapter 4.1 --- Discussion --- p.100 / Chapter 4.2 --- Conclusion --- p.110 / Reference --- p.111

by Lo Ka-Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 175-197). / Chapter I --- Title page --- p.i / Chapter II --- Thesis committee --- p.ii / Chapter II --- Acknowledgment --- p.iii-iv / Chapter III --- Abstract --- p.v-vi / Chapter IV --- Table of content --- p.vii-xiv / Chapter V --- List of figures --- p.xv-xviii / Chapter VI --- List of tables --- p.xix-xx / Forewords: / Overall objectives --- p.1 / Introduction on the fish used in this research study --- p.2 / Chapter Chapter 1: --- Literature Review on Biomarkers of Stress in Teleosts --- p.5 / Chapter 1.1 --- Definition of stress --- p.7 / Chapter 1.2 --- Classification of stress indicators --- p.8 / Chapter 1.2.1 --- Primary stress indicators --- p.8 / Chapter 1.2.1.1 --- Molecular stress indicators --- p.8 / Chapter 1.2.1.2 --- Hormonal stress indicators --- p.9 / Chapter (I) --- Corticosteroid --- p.9 / Chapter (II) --- Catecholamines --- p.11 / Chapter 1.2.2 --- Secondary stress indicators --- p.12 / Chapter 1.2.2.1 --- Metabolic changes --- p.12 / Chapter (I) --- Glucose metabolism --- p.13 / Chapter (II) --- Lactic acid --- p.14 / Chapter 1.2.2.2 --- Osmoregulatory changes --- p.15 / Chapter 1.2.2.3 --- Haematological changes --- p.16 / Chapter 1.2.2.4 --- Reproductive changes --- p.17 / Chapter 1.2.3 --- Tertiary stress indicators --- p.18 / Chapter 1.2.3.1 --- Histopathological indicators --- p.18 / Chapter 1.2.3.2 --- Ecological indicators --- p.19 / Chapter 1.3 --- Recent trends on the study of biomarkers --- p.20 / Chapter 1.3.1 --- Use of detoxification enzymes for specific indication of toxic pollutants in aquatic environment --- p.20 / Chapter 1.3.1.1 --- Metallothioneins (MTs) --- p.20 / Chapter 1.3.1.2 --- Cytochrome P450 monoxygenase (CYP450) --- p.21 / Chapter 1.3.2 --- Use of HSP 70 as a biomarker in teleost --- p.22 / Chapter 1.4 --- Future perspectives on the study of biomarkers in fish --- p.24 / Chapter Chapter 2: --- Literature Review on Heat Shock Proteins (HSPs) --- p.28 / Chapter 2.1 --- General Characteristics of HSPs --- p.29 / Chapter 2.1.1 --- HSP90 family --- p.30 / Chapter 2.1.2 --- HSP70 family --- p.31 / Chapter 2.1.3 --- HSP60 family (Chaperonin-60) --- p.32 / Chapter 2.1.4 --- Low-molecular weight HSPs (HSP20) --- p.33 / Chapter 2.2 --- Structure of HSP70 encoding gene --- p.33 / Chapter 2.2.1 --- General characteristics of HSP70-encoding gene --- p.33 / Chapter 2.2.2 --- Heat shock transcription factor (HSF) --- p.35 / Chapter 2.2.3 --- Heat shock elements (HSE) --- p.35 / Chapter 2.3 --- Stress-mediated control of HSP70 transcription --- p.36 / Chapter 2.4 --- Characterization of HSP70 expression in teleost --- p.38 / Chapter 2.4.1 --- Tissues-specific expression of HSP70 in teleost --- p.39 / Chapter 2.4.2 --- Inter-relationship of HSP70 expression with seasonal variation and thermotolerance of teleost --- p.40 / Chapter 2.4.3 --- Induction of HSP70 in teleost upon acute thermal stress --- p.41 / Chapter 2.4.4 --- Induction of HSP70 in teleost by non-thermal stressors --- p.43 / Chapter 2.4.4.1 --- Heavy metal-induced HSP70 expression --- p.43 / Chapter 2.4.4.2 --- Handling stress-induced HSP70 expression --- p.43 / Chapter Chapter 3: --- "Induction of HSP70 in blood cells of seabream, Sparus sarba subjected to in vivo and in vitro thermal stress" --- p.48 / Chapter 3.1 --- Introduction --- p.49 / Chapter 3.2 --- Materials and Methods --- p.52 / Chapter 3.2.1 --- Overall experimental design --- p.52 / Chapter 3.2.2 --- Fish --- p.53 / Chapter 3.2.3 --- Blood sampling --- p.53 / Chapter 3.2.4 --- Preparation of blood cells --- p.54 / Chapter 3.2.5 --- Thermal stress regimes --- p.54 / Chapter 3.2.5.1 --- Time couse of HSP70 induction profile in blood cells after in vitro exposure to thermal stress --- p.54 / Chapter 3.2.5.2 --- HSP70 synthesis in blood cells taken from fish subjected to in vivo hyper- thermic stress --- p.55 / Chapter 3.2.5.3 --- Transcriptional inhibitory effect of actinomycin D on the synthesis of HSP70 in blood cells subjected to in vitro thermal stress --- p.55 / Chapter 3.2.6 --- Protein analysis --- p.56 / Chapter 3.2.7 --- Gel electrophoresis --- p.57 / Chapter 3.2.8 --- Immunoblotting (Western blot analysis) --- p.57 / Chapter 3.2.9 --- Autroradiography --- p.58 / Chapter 3.3 --- Results --- p.59 / Chapter 3.3.1 --- Time course of HSP70 induction profile in blood cells subjected to in vitro thermal treatments --- p.59 / Chapter 3.3.1.1 --- Results of immunoblotting from blood cells of fish acclimated to 26°C --- p.59 / Chapter 3.3.1.2 --- Results of immunoblotting in blood cells from 18°C-acclimated fish --- p.60 / Chapter 3.3.1.3 --- Results of immunoblotting in blood cells from fish acclimated to 20°C --- p.60 / Chapter 3.3.1.4 --- 35S-methionine labelling of de novo protein synthesis in blood cells of fish acclimated to 15 and 20°C --- p.61 / Chapter 3.3.2 --- Blood cell HSP70 levels in 20°C-acclimated fish subjected to in vivo hyperthermic stress --- p.61 / Chapter 3.3.3 --- Transcriptional inhibitory effect of actinomycin D on HSP70 induction in blood cells subjected to in vitro thermal stress --- p.62 / Chapter 3.4 --- Discussions --- p.60 / Chapter 3.4.1 --- Characteristics of HSP70 induction in blood cells of seabream subjected to in vitro temperature stress --- p.72 / Chapter 3.4.1.1 --- Induction profile of HSP70 in blood cells --- p.72 / Chapter 3.4.1.2 --- Time course ofHSP70 induction in blood cells --- p.75 / Chapter 3.4.1.3 --- Effect of acclimation temperature of fish on the induction of HSP70 --- p.76 / Chapter 3.4.2 --- Comparison of HSP70 induction in in vitro and in vivo thermal treatment on blood cells --- p.78 / Chapter 3.4.3 --- "Effect of transcriptional inhibitor, actinomycin D, on the de novo synthesis of HSP70" --- p.80 / Chapter 3.5 --- Conclusions --- p.70 / Chapter Chapter 4: --- "Effects of seasonal variation and transportation stress on level of HSP70, serum glucose and serum Cortisol in seabream, Sparus sarba" --- p.86 / Chapter 4.1 --- Introduction --- p.87 / Chapter 4.2 --- Materials and methods --- p.90 / Chapter 4.2.1 --- Overall experimental design --- p.90 / Chapter 4.2.2 --- Fish and blood sampling --- p.91 / Chapter 4.2.3 --- Preparation of blood samples --- p.92 / Chapter 4.2.4 --- Determination of HSP70 levels in blood cells sampled from seabream upon different seasons --- p.92 / Chapter 4.2.5 --- Immunoblotting analysis --- p.92 / Chapter 4.2.6 --- Enzyme-linked Immnosorbent Assay (ELISA) --- p.93 / Chapter 4.2.7 --- Measurement of serum parameter in seabream --- p.95 / Chapter 4.2.7.1 --- Serum glucose --- p.95 / Chapter 4.2.7.2 --- Serum Cortisol --- p.96 / Chapter 4.3 --- Results --- p.96 / Chapter 4.3.1 --- Determination of HSP70 levels in blood cells sampled from seabream in different seasons --- p.96 / Chapter 4.3.1.1 --- Immunoblotting analysis --- p.96 / Chapter 4.3.1.2 --- Enzyme-linked immunosorbent assay (ELISA) --- p.96 / Chapter 4.3.2 --- Serum analysis of seabream sampled from fish farm in different seasons --- p.98 / Chapter 4.3.2.1 --- Serum glucose --- p.98 / Chapter 4.3.2.2 --- Serum Cortisol --- p.99 / Chapter 4.4 --- Discussions --- p.117 / Chapter 4.4.1 --- Characterization of HSP70 expression in blood cells of seabream --- p.117 / Chapter 4.4.2 --- Dynamicity of HSP70 content and thermo- tolerance of fish in different seasons --- p.118 / Chapter 4.4.3 --- Effect of transportation stress on HSP70 induction in blood cells of seabream --- p.121 / Chapter 4.4.4 --- Dynamicity of serum glucose level in seabream subjected to seasonal variations --- p.123 / Chapter 4.4.5 --- Effect of transportation stress on the serum glucose level of seabream in different seasons --- p.124 / Chapter 4.4.6 --- Dynamicity of senam Cortisol level in seabream subjected to seasonal variations --- p.125 / Chapter 4.4.7 --- Effect of transportation stress on the serum Cortisol level of seabream in different seasons --- p.126 / Chapter 4.4.8 --- "Comments on the use of HSP70, serum Cortisol and serum glucose as biomarkersin environmental supervision" --- p.126 / Chapter 4.5 --- Conclusions --- p.129 / Chapter Chapter 5: --- "In vitro and in vivo effects of Cortisol, dexamethasone and adrenaline on the induction of HSP70 in seabream, Sparus sarba" --- p.131 / Chapter 5.1 --- Introduction --- p.132 / Chapter 5.2 --- Materials and methods --- p.133 / Chapter 5.2.1 --- Overall experimental design --- p.133 / Chapter 5.2.2 --- Acclimation of fish and regimes of treatment --- p.133 / Chapter 5.2.3 --- Serum Cortisol and adrenaline analysis --- p.135 / Chapter 5.2.4 --- "Protein analysis, gel electrophoresis, immuno- blotting and ELISA analysis" --- p.136 / Chapter 5.3 --- Results --- p.137 / Chapter 5.3.1 --- "HSP70 level in blood cells treated with Cortisol, dexamethasone and adrenaline in vitro" --- p.137 / Chapter 5.3.2 --- "Serum hormones and HSP70 level in tissues of fish injected with Cortisol, adrenaline and dexamethasone invivo" --- p.137 / Chapter 5.3.2.1 --- Serum Cortisol and adrenaline level of fish after hormone injections --- p.137 / Chapter 5.3.2.2 --- "HSP70 level in blood cells, brain and liver tissue of fish after hormone injections" --- p.138 / Chapter (I) --- Level of HSP70 in blood cells of fish after hormone injections --- p.138 / Chapter 5.4 --- Discussions --- p.156 / Chapter 5.4.1 --- In vitro and in vivo study of the hormonal effect on HSP70 level in blood cells of seabream --- p.156 / Chapter 5.4.2 --- Hypothetical mechanism of hormone-receptor mediated HSP70 regulation --- p.158 / Chapter 5.4.3 --- In vivo study of the hormonal effect on HSP70 level in blood cells of seabream --- p.160 / Chapter 5.4.4 --- In vivo study on the hormonal effect of HSP70 synthesis in liver of seabream --- p.163 / Chapter 5.4.5 --- In vivo study on the hormonal effect of HSP70 synthesis in brain tissue of seabream --- p.165 / Chapter 5.4.6 --- HSP70 level in different tissues of fish in relation to the induction and sensitivity against stress --- p.166 / Chapter 5.5 --- Conclusions --- p.169 / Chapter Chapter 6: --- Summary --- p.171 / References --- p.175

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves 113-139).

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [128]-146).

Thesis (Ph. D.)--West Virginia University, 2005. / Title from document title page. Document formatted into pages; contains vii, 107 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

Thesis (M.S.)--University of Michigan, 1994.

Thesis (Ph.D.)--University of Nevada, Reno, 2004. / Includes bibliographical references. Online version available on the World Wide Web.