published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

Trained male cyclists (6 in study 1, 5 in study 2) cycled to exhaustion (EXH) at 75% of VO₂ max twice; once in the non-supplemented (NS) state and once in the vitamin B-6 (B-6)(20 mg PN) supplemented (S) state. The diet contained 2.3 mg B-6 in study 1 and 1.9 mg B-6 in study 2. Urine was collected during each dietary period. During each exercise (EX) test, blood was drawn prior to (PRE), one hour during (DX), immediately after (POST) and one hour after (POST 60) EX and sweat was collected. Compared to baseline (PRE) levels, plasma pyridoxal 5'-phosphate (PLP) and vitamin B-6 (PB-6) concentrations increased at DX, decreased at POST, and decreased below PRE at POST 60 in the NS and S states. EX to EXH in the S state resulted in a greater increase in PLP DX in study 1 (31% increase vs. 16%) and PB-6 in study 2 (25% increase vs. 11%) as compared to the NS state. Red blood cell (RBC) PLP significantly increased from POST to POST 60 in the S state in study 2. The excretion of urinary 4-pyridoxic acid (4-PA) and urinary B-6 (UB-6) was not significantly altered by EX to EXH. The mean excretion of 4-PA was significantly greater in the NS state in study 2 (7.98 ±1.83 mmol/d) as compared to the excretion in study 1 (6.20 ±0.93 mmol/d), whereas the excretion was significantly greater in the S state in study 1 (92.2 ±8.69 mmol/d) compared to the excretion in study 2 (82.7 ±6.16 mmol/d). The percent of B-6 intake excreted as UB-6 (6% in study 1 and 10% in study 2) was significantly different between the studies in the NS state. Vitamin B-6 supplementation did not significantly alter the rise in growth hormone (hGH) concentration seen with EX to EXH. The loss of B-6 in sweat with EX to EXH was not altered by B-6 supplementation. The loss of B-6 in sweat ranged from 0.0011 mmol to 0.0039 mmol. Therefore, EX to EXH in the B-6 S state resulted in a greater increase in plasma PLP and PB-6 DX as compared to the NS state. The decrease in PB-6 and PLP at POST 60 in the S state coincided with a significant increase in RBC PLP, suggesting the movement of B-6 from the plasma into the RBC at POST 60. EX to EXH and B-6 supplementation did not alter the excretion of 4-PA or UB-6 suggesting that B-6 metabolism was unchanged. The loss of B-6 in sweat was comparable to previously reported values and was not altered by B-6 supplementation. B-6 supplementation did not alter the changes in hGH resulting from EX to EXH alone. / Graduation date: 1995

This study examines how Bovine Somatotropin (BST) may impact Arizona dairy producers. The results of dairy scientists experimenting with BST are summarized in terms of reported milk yields and possible changes in feeding and herd management. Dairy enterprize budgets representative of Arizona are constructed to examine how income statements may change if BST is approved. The effects of increased milk supply on Arizona milk prices are estimated using the institutional structure of the Central Arizona Order and the United Dairyman of Arizona. Results of experiments with BST in Arizona are used to generate net returns at several rates of adoption under changing milk prices for three dairy farms in Arizona.

Com o objetivo de estudar os efeitos do uso da Somatotropina Recombinante Bovina (rBST) no lipidograma e constituição do leite de bubalinos foram utilizadas 28 búfalas divididas em dois grupos: Grupo 1 constituído de 14 búfalas nas quais foi realizada uma aplicação de 500 mg de rBST e Grupo 2 - constituído de 14 búfalas que não receberam qualquer tratamento hormonal (grupo controle). Durante o experimento, os animais tiveram amostras de plasma e soro sanguíneo, bem como de leite colhidas periodicamente nos seguintes momentos: dia da aplicação da rBST, 1º, 3º, 5º, 7º, 10º e 14º dia após a aplicação da rBST. A fim de minimizar possíveis influências da fase da lactação, todas as búfalas utilizadas no experimento estarvam entre 100 e 200 dias de lactação e com produção de leite variando entre 5 e 10 litros por dia. O lipidograma foi avaliado por meio da determinação dos teores séricos de colesterol, triglicérides, ácidos graxos não esterificados e β-HBO e teores plasmáticos de glicose, enquanto a constituição do leite de búfalas foi avaliada por meio de determinação dos teores lácteos de gordura, proteína, lactose, sólidos totais e do número de células somáticas. Durante a avaliação dos resultados obtidos podemos notar que a aplicação da somatotropina recombinante bovina não influenciou os teores séricos de NEFA, colesterol e triglicérides bem como os plasmáticos de glicose. No entanto os teores séricos de β-HBO foram afetados pelo tratamento instituído, pois verificou-se que, a partir do 1º dia após o início do tratamento hormonal, os teores séricos de -HBO no grupo tratado com rBST eram entre 0,21 e 0,55 mg/dL maiores do que os encontrados no grupo controle. Quando avaliamos a composição do leite temos que os resultados nos permitem afirmar que o tratamento instituído não interfere nas concentrações de gordura, lactose, sólidos totais e no número de células somáticas. Nos primeiros dias após a aplicação da rBST ocorria uma diminuição dos teores lácteos de proteína, sendo verificado que no 3º dia após o início do tratamento hormonal os valores lácteos de proteína encontrados no grupo tratado com rBST (3,59 ± 0,22 g/dL) eram significativamente menores do que os observados no grupo controle (3,89 ± 0,22 g/dL). A partir do 5º dia após o início do tratamento hormonal, os valores de proteína passam a oscilar sem que qualquer diferença estatística entre o grupo tratado e controle possam ser observados. / With the aim to evaluate the influence of recombinant bovine somatotropin (rBST) in the lipid profile and milk composition of lactating Murrah water buffaloes, we collected and analyzed samples of blood serum and plasma and milk of clinically healthy animals. We determined the concentrations of cholesterol, triglycerides, nonesterified fatty acids (NEFA), -hydroxibutyrate (-HBO) and glucose from the blood samples. Milk samples were collected after milking and the following parameters were evaluated: fat, protein, lactose, total solids and somatic cell count. The influence of rBST was studied through 28 animals divided in 2 experimental groups: Experimental Group 14 animals that received a single application of 500mg rBST; Control Group - 14 animals that didn´t receive any hormonal treatment. Blood and milk samples were collected in the following moments: day of rBST application, 1st, 3rd, 5th, 7th, 10th and 14th day after the rBST application. To minimize the influence of lactation, the samples were collected between 100-200 days of lactation and the milk yields per cow range from 5 10 Liters. According to the results of this research, we concluded that one single application of rBST didn´t affect the NEFA, cholesterol, triglycerides and glucose blood levels. The serum β-HBO concentration was influenced by the hormonal treatment. In the experimental group, after 24 hours from the treatment, the β-HBO level ranged 0.21 - 0.55 mg/dL and it was greater than that of control group. The fat, lactose, total solids and somatic cell count were not influenced by the rBST treatment. In the first days after the application of rBST, the milk protein decreased gradually reaching its low level in the 3rd day after the application (3.59 ± 0.22 g/dL) and was significant lower than that of control group (3.89 ± 0.22 g/dL). After the 5th day, the milk protein values oscillated without any statistical difference between both groups.

Hui Fong Fong Liza. / Thesis submitted in: December 2002. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 130-182). / Abstracts in English and Chinese. / Chapter I --- Title page --- p.I / Chapter II --- Thesis committee --- p.II / Chapter III --- Acknowledgements --- p.III / Chapter IV --- Abstract (Chinese version) --- p.IV / Chapter V --- Abstract (English version) --- p.VII / Chapter VI --- Table of contents --- p.X / Chapter VII --- List of figures --- p.XIV / Chapter VIII --- List of table --- p.XVIII / Chapter Chapter 1: --- General introduction --- p.1 / Chapter Chapter 2: --- Literature review --- p.5 / Chapter 2.1. --- Gill --- p.5 / Chapter 2.2. --- Chloride cells (Mitochondria-rich cells) --- p.6 / Chapter 2.2.1. --- Ion extrusion by fish in seawater --- p.9 / Chapter 2.2.2. --- Ion uptake by fish in hypo-osmotic condition --- p.12 / Chapter 2.3. --- Sparus sarba (Silver seabream) --- p.14 / Chapter 2.4. --- Sodium-potassium adenosinetriphosphatase (Na+-K+-ATPase) --- p.15 / Chapter 2.4.1. --- Na+-K+-ATPase α-subunit --- p.17 / Chapter 2.4.2. --- Na+-K+-ATPase β-subunit --- p.18 / Chapter 2.4.3. --- Regulation of Na+-K+-ATPase --- p.20 / Chapter 2.5. --- Hormones --- p.21 / Chapter 2.5.1. --- Growth hormone-prolactin family --- p.21 / Chapter 2.5.2. --- Structure of hormones --- p.22 / Chapter 2.5.2.1. --- Structure of growth hormone and prolactin in fish --- p.22 / Chapter 2.5.2.2. --- Structure of insulin-like growth factors in fish --- p.26 / Chapter 2.5.2.3. --- Structure of Cortisol in fish --- p.27 / Chapter 2.5.3. --- Regulation of hormones --- p.28 / Chapter 2.5.3.1. --- Regulation of growth hormone in fish --- p.28 / Chapter 2.5.3.2. --- Regulation of prolactin in fish --- p.32 / Chapter 2.5.3.3. --- Regulation of insulin-like growth factor-I in fish --- p.33 / Chapter 2.5.3.4. --- Regulation of Cortisol in fish --- p.33 / Chapter 2.5.4. --- Functions of hormones --- p.33 / Chapter 2.5.4.1. --- Functions of growth hormone in fish --- p.33 / Chapter 2.5.4.2. --- Functions of prolactin in fish --- p.39 / Chapter 2.5.4.3. --- Functions of insulin-like growth factor-I in fish --- p.44 / Chapter 2.5.4.4. --- Functions of Cortisol in fish --- p.45 / Chapter 2.5.4.5. --- "Combined effects of GH, IGF-I, PRL and Cortisol" --- p.49 / Chapter 2.6. --- Salinity effects on Na+-K+-ATPase expression --- p.52 / Chapter Chapter 3: --- In vitro effect of hormones on branchial Na+-K+- ATPase expression in marine teleost Sparus sarba --- p.58 / Chapter 3.1. --- Abstract --- p.58 / Chapter 3.2. --- Introduction --- p.60 / Chapter 3.3. --- Materials and methods --- p.62 / Chapter 3.3.1. --- Overall experimental design --- p.62 / Chapter 3.3.2. --- Fish preparation --- p.62 / Chapter 3.3.3. --- Tissue sampling --- p.62 / Chapter 3.3.4. --- RNA extraction and dot blot analysis --- p.63 / Chapter 3.3.5. --- Protein extraction --- p.65 / Chapter 3.3.6. --- Protein quantification --- p.65 / Chapter 3.3.7. --- Na+-K+-ATPase activity --- p.65 / Chapter 3.3.8. --- Protein gel electrophoresis and immunoblotting (Western blotting) --- p.66 / Chapter 3.3.9. --- Statistical analysis --- p.67 / Chapter 3.4. --- Results --- p.69 / Chapter 3.4.1. --- Dot blot analysis of Na+-K+-ATPase mRNA subunits --- p.69 / Chapter 3.4.2. --- Analysis of Na+-K+-ATPase protein α-subunit --- p.81 / Chapter 3.4.3. --- Analysis of Na+-K+-ATPase activity --- p.87 / Chapter 3.5. --- Discussion --- p.92 / Chapter 3.5.1. --- Effects of rbGH and rbIGF-I on Na+-K+-ATPase expression --- p.92 / Chapter 3.5.2. --- Effects of oPRL on Na+-K+-ATPase expression --- p.102 / Chapter 3.5.3 --- Effects of Cortisol on Na+-K+-ATPase expression --- p.104 / Chapter 3.6. --- Conclusion --- p.108 / Chapter Chapter 4: --- In vivo effect of salinity on branchial Na+-K+-ATPase expression in marine teleost Sparus sarba --- p.109 / Chapter 4.1. --- Abstract --- p.109 / Chapter 4.2. --- Introduction --- p.110 / Chapter 4.3. --- Materials and methods --- p.112 / Chapter 4.3.1. --- Overall experimental design --- p.112 / Chapter 4.3.2. --- Fish preparation --- p.112 / Chapter 4.3.3. --- Tissue sampling --- p.113 / Chapter 4.3.4. --- "RNA extraction, dot blot analysis, protein extraction, quantification, Na+-K+-ATPase activity, protein gel electrophoresis and immunoblotting (Western blotting)" --- p.113 / Chapter 4.3.5. --- Statistical analysis --- p.114 / Chapter 4.4. --- Results --- p.114 / Chapter 4.4.1. --- Dot blot analysis of Na+-K+-ATPase mRNA subunits --- p.114 / Chapter 4.4.2. --- Analysis of Na+-K+-ATPase protein a-subunit --- p.114 / Chapter 4.4.3. --- Analysis of Na+-K+-ATPase activity --- p.115 / Chapter 4.5. --- Discussion --- p.120 / Chapter 4.6. --- Conclusion --- p.125 / Chapter Chapter 5: --- General discussion and conclusion --- p.126 / References --- p.130

Fung Chun Kit. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 117-139). / Abstracts in English and Chinese. / Declaration of Originality --- p.i / Acknowledgements --- p.ii / Abstract --- p.iii / 摘要 --- p.v / List of figures --- p.vi / List of tables --- p.viii / List of abbreviations --- p.ix / Chapter Chapter I --- General introduction --- p.1 / Chapter 1.1 --- Different fish habitats with various salinities --- p.1 / Chapter 1.2 --- Osmotic challenges faced by teleosts --- p.2 / Chapter 1.3 --- High ionic strength results in DNA damage and excess water gain --- p.3 / Chapter 1.4 --- Osmoregulatory organs and mechanisms for osmotic balance --- p.4 / Chapter 1.5 --- Different tolerance to salinity changes --- p.8 / Chapter 1.6 --- Effective communication among osmoregulatory organs --- p.9 / Chapter 1.7 --- Introduction to GH and PRL --- p.9 / Chapter 1.8 --- Structure of the GHR and PRLR --- p.10 / Chapter 1.9 --- Hypoosmoregulatory action of GH/IGF-I axis in teleosts --- p.11 / Chapter 1.10 --- Hyperosmoregulatory action of PRL in teleosts --- p.11 / Chapter Chapter II --- Research rationale --- p.13 / Chapter 2.1 --- Physiological importance of osmoregulation in fish --- p.13 / Chapter 2.1.1 --- Energy metabolism --- p.13 / Chapter 2.1.2 --- Growth --- p.14 / Chapter 2.1.3 --- Immunity --- p.14 / Chapter 2.1.4 --- Reproduction --- p.15 / Chapter 2.2 --- Aquaculture importance --- p.15 / Chapter 2.3 --- Unknown molecular regulatory mechanism of hormones during salinity changes in fish --- p.16 / Chapter 2.4 --- Animal model --- p.17 / Chapter Chapter III --- In vivo studies of sbGHR and sbPRLR expression in osmoregulatory organs in response to salinity changes --- p.18 / Chapter 3.1 --- Introduction --- p.18 / Chapter 3.1.1 --- Dynamic change of GH level during salinity changes --- p.18 / Chapter 3.1.2 --- Dynamic change of PRL level during salinity changes --- p.19 / Chapter 3.1.3 --- In vitro studies of GH and PRL release from teleost pituitary in response to extracellular osmolality changes --- p.20 / Chapter 3.1.4 --- Biological actions of GH and PRL through the GHR and PRLR --- p.21 / Chapter 3.2 --- Materials and methods --- p.23 / Chapter 3.3 --- Results --- p.28 / Chapter 3.4 --- Discussion --- p.36 / Chapter 3.4.1 --- Plasma osmolality change during salinity changes --- p.36 / Chapter 3.4.2 --- Gene expression after HSW exposure --- p.38 / Chapter 3.4.3 --- Ionic mediators of the gene expression --- p.43 / Chapter 3.4.4 --- Gene expression after BW exposure --- p.44 / Chapter 3.4.5 --- Dynamic changes of the GHR and PRLR in response to salinity changes --- p.45 / Chapter 3.4.6 --- Regulation of the gene expression in response to salinity changes --- p.46 / Chapter Chapter IV --- Gene expression of sbGHR in liver during salinity changes --- p.49 / Chapter 4.1 --- Introduction --- p.49 / Chapter 4.1.1 --- Responses of the somatotropic axis to salinity changes in fish --- p.49 / Chapter 4.2 --- Materials and methods --- p.52 / Chapter 4.3 --- Results --- p.56 / Chapter 4.4 --- Discussion --- p.60 / Chapter 4.4.1 --- Inhibition of GHR and IGF-I gene expression in liver during HSW exposure --- p.60 / Chapter 4.4.2 --- Downregulation of GHR gene expression by hyperosmotic stress --- p.62 / Chapter 4.4.3 --- Growth retardation of fish during hyperosmotic environment --- p.64 / Chapter Chapter V --- Gene expression studies of sbPRLR in gill organ culture --- p.68 / Chapter 5.1 --- Introduction --- p.68 / Chapter 5.1.1 --- Functions of gill in fish osmoregulation --- p.68 / Chapter 5.1.2 --- Gill culture as a model for osmoregulation studies --- p.69 / Chapter 5.2 --- Materials and methods --- p.70 / Chapter 5.3 --- Results --- p.71 / Chapter 5.4 --- Discussion --- p.73 / Chapter Chapter VI --- Regulation of gene expression of sbGHR in liver during hyperosmotic stress: promoter studies --- p.75 / Chapter 6.1 --- Introduction --- p.75 / Chapter 6.1.1 --- What is a promoter? --- p.75 / Chapter 6.1.2 --- Promoter studies of GHR gene --- p.76 / Chapter 6.2 --- Materials and methods --- p.78 / Chapter 6.3 --- Results --- p.85 / Chapter 6.4 --- Discussion --- p.104 / Chapter Chapter VII --- General discussion and future perspectives --- p.111 / References --- p.117

Submitted by Ubirajara Cruz (ubirajara.cruz@gmail.com) on 2017-06-07T16:23:22Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Diego Andres Velasco Acosta.pdf: 1957910 bytes, checksum: 57eabc538f89e509cac0a69175bd4efb (MD5) / Approved for entry into archive by Aline Batista (alinehb.ufpel@gmail.com) on 2017-06-08T13:04:26Z (GMT) No. of bitstreams: 2 Diego Andres Velasco Acosta.pdf: 1957910 bytes, checksum: 57eabc538f89e509cac0a69175bd4efb (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-06-08T13:04:26Z (GMT). No. of bitstreams: 2 Diego Andres Velasco Acosta.pdf: 1957910 bytes, checksum: 57eabc538f89e509cac0a69175bd4efb (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2016-01-12 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / O balanço energético negativo (BEN) está associado com alterações na fertilidade de vacas leiteiras, portanto diferentes estratégias nutricionais e farmacológicas vêm sendo utilizadas para minimizar os efeitos negativos do BEN, visando aumentar a fertilidade. A aplicação pré-parto de somatotropina recombinante bovina (rbST) pode levar a uma redução da concentração sérica dos ácidos graxos não esterificados (AGNE) e β-hidroxibutirato (BHBA) no período pós-parto. Além disso têm sido demonstrado efeito significativo no desenvolvimento folicular ovariano pós-parto. Da mesma forma, estratégias nutricionais e o manejo da alimentação durante o período pré e pós-parto têm um impacto na saúde, produtividade e fertilidade da vaca leiteira. Estudos recentes mostram que a adição de aminoácidos na dieta durante o período de transição de vacas leiteiras pode ter efeitos benéficos no metabolismo, contribuindo para melhorar a performance reprodutiva. Diante desta problemática, esta tese apresenta três artigos, cujos objetivos foram: 1) determinar o efeito da administração pré-parto de somatotropina em novilhas leiteiras sobre marcadores do BEN, retomada da atividade ovariana e produção de leite. 2) Determinar o efeito da suplementação de metionina e colina protegidas da degradação ruminal, isoladas e em combinação, sobre o potencial esteroidogenico do folículo dominante da primeira onda folicular pós-parto de vacas leiteiras. 3) Determinar o efeito da suplementação de metionina e colina protegidas da degradação ruminal, isoladas e em combinação, sobre o desenvolvimento embrionário e nível de metilação global de DNA do embrião. O tratamento com somatotropina pré-parto em novilhas leiteiras aumentou a concentração de IGF-I intrafolicular e a expressão do receptor de LH e da proteína reguladora aguda da esteroidegênese em células foliculares do folículo dominante da primeira onda pós-parto. Essas mudanças foram associadas a um aumento da concentração de estradiol sérica e intrafolicular, o que pode, potencialmente, aumentar a chance de ovulação da primeira onda folicular pós-parto. Entretanto, somatotropina não afetou outros parâmetros metabólicos e produtivos no período pós-parto recente. A suplementação com metionina, colina ou ambas durante o período de transição parece não ter efeito sobre o desenvolvimento folicular pós-parto da primeira onda em vacas da raça Holandês. No entanto, a suplementação apenas com metionina durante o período de transição aumenta a expressão de 3β-HSD nas células foliculares. Além disso, a suplementação com metionina e colina induziu a uma down regulation dos genes pró-inflamatórios, indicando um menor processo inflamatório nas células foliculares. Em relação ao desenvolvimento embrionário, a suplementação com metionina durante o período de transição reduziu a metilação global do DNA e aumentou o acúmulo de lipídios nos embriões. A utilização de estratégias nutracêuticas durante o período de transição aumentaram o potencial esteroidogenico do folículo dominante da primeira onda folicular pós-parto e aumentou a expressão de genes relacionados a esteroidogenese, assim como uma redução na expressão de genes pró inflamatórios, indicando efeitos benéficos na fertilidade de bovinos leiteiros. Além disso, a suplementação de metionina parece aumentar a capacidade de sobrevivência dos embriões, desde que há fortes indícios de que as reservas lipídicas endógenas servem como um substrato energético. / The negative energy balance (NEB) is associated with changes in the fertility of dairy cows, therefore different nutritional and pharmacological strategies are being used to minimize the negative effects of the NEB in order to increase fertility. Pre-partum recombinant bovine somatotropin (rbST) administration can lead to a reduction in serum non-esterified fatty acids (NEFA) and β-hydroxybutyrate (BHBA) in the postpartum period, in addition to improving postpartum ovarian follicular development. Similarly nutritional strategies during the pre and postpartum period have a positive impact on health, productivity and fertility of dairy cows. Recent studies show that the use of amino acids in the diet during the transition period may have beneficial effects in metabolism. Faced with this problem, this thesis presents three papers whosethe objectives were: 1) Determine the effect of pre-partum administration of somatotropin in dairy heifers on the markers of NEB, resumption of ovarian activity and milk production. 2) Determine the effect of methionine, choline supplementation isolated and in combination on esteroidogenic potential of the fist postpartum dominant follicle. 3) Determine the effect of methionine, choline supplementation isolated and in combination on embryo development and global DNA methylation. Pre-partum somatotropin treatment in dairy heifers increased intrafollicular IGF-I and mRNA expression of LH receptor and steroidogenic acute regulatory protein in follicular cells of the first postpartum dominant follicle. These changes were associated to increased intrafollicular and serum estradiol concentration, which can potentially increase the chance of ovulation of the first postpartum follicular wave. Despite that, somatotropin did not affect other metabolic and productive parameters in the early postpartum period. Supplementing methionine, choline or both during the transition period seemed to have no effect on postpartum follicular development of the first postpartum wave in Holstein cows. However, supplementing methionine, during the transition period increased follicular cells 3β-HSD expression, methionine and choline supplementation also induced a down regulation of pro-inflammatory genes, indicating a lower inflammatory processes in follicular cells. Regarding to embryo development, supplementation of methionine, only during the transition period reduce global DNA methylation, moreover, supplemented methionine increase lipid accumulation in preimplantation embryos. The use of nutraceutical strategies during the transition period increased the steroidogenic potential of the first postpartum dominant follicle and increase the expression of genes related steroidogenesis also a down regulation of proinflammatory genes indicating beneficial effects on fertility in dairy cattle. Moreover, the methionine supplementation seems to impact the preimplantation embryo in a way that enhances its capacity for survival since there is strong evidence that endogenous lipid reserves serve as an energy substrate.

Ng, Siu Fung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 206-211). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.iii / 摘要 --- p.v / Table of contents --- p.vii / List of figures --- p.xv / List of tables --- p.xix / List of abbreviations --- p.xx / Chapter / Chapter 1. --- Introduction / Chapter 1.1 --- Growth hormone --- p.1 / Chapter 1.1.1 --- Historic discovery of growth hormone --- p.1 / Chapter 1.1.2 --- Structural and functional study of GH --- p.1 / Chapter 1.1.2.1 --- Molecular evolution of GH --- p.1 / Chapter 1.1.2.2 --- Two-dimensional and three dimensional structures --- p.5 / Chapter 1.1.2.3 --- Heterogeneity of GH --- p.8 / Chapter 1.1.2.4 --- Regulation and secretion pattern of GH --- p.9 / Chapter 1.1.2.5 --- Circulation of GH in blood --- p.11 / Chapter 1.1.2.6 --- Biological activity of GH in human --- p.12 / Chapter 1.2 --- GH receptor and signal transduction --- p.12 / Chapter 1.3 --- GH disorder --- p.15 / Chapter 1.4 --- Treatment for GH disorder --- p.16 / Chapter 1.5 --- GH assay --- p.17 / Chapter 1.6 --- Aims of study --- p.19 / Chapter 2. --- SUMO-hGH expression vector construction / Chapter 2.1 --- Introduction --- p.21 / Chapter 2.2 --- Fusion partner - SUMO --- p.23 / Chapter 2.3 --- Materials --- p.24 / Chapter 2.3.1 --- Reagents for bacterial culture --- p.24 / Chapter 2.3.2 --- Reagents for agarose gel electrophoresis --- p.26 / Chapter 2.3.3 --- 2'-deoxyribonucleoside 5'-triphosphate mix for polymerase chain reaction --- p.26 / Chapter 2.3.4 --- Sonication buffer --- p.26 / Chapter 2.3.5 --- Modified solubilization buffer --- p.27 / Chapter 2.3.6 --- Reagents for sodium dodecylsulphate polyacrylamide gel electrophoresis --- p.27 / Chapter 2.4 --- Methods --- p.29 / Chapter 2.4.1 --- General techniques in molecular cloning of hGH gene --- p.29 / Chapter 2.4.2 --- Expression of SUMO-hGH fusion protein - small scale --- p.42 / Chapter 2.4.3 --- General protein analysis --- p.43 / Chapter 2.5 --- Results --- p.45 / Chapter 2.5.1 --- Molecular cloning of hGH gene into expression vector --- p.45 / Chapter 2.5.2 --- Expression of SUMO-hGH --- p.46 / Chapter 2.5.3 --- Modification of the expression conditions --- p.46 / Chapter 2.6 --- Discussion --- p.50 / Chapter 2.6.1 --- Expression vector --- p.53 / Chapter 2.6.2 --- Protein expression --- p.53 / Chapter 2.7 --- Conclusion --- p.54 / Chapter 3. --- SUMO-hGH purification and downstream processing / Chapter 3.1 --- Introduction --- p.55 / Chapter 3.2 --- Immobilized-metal affinity chromatography --- p.55 / Chapter 3.3 --- SUMO protease --- p.57 / Chapter 3.4 --- Materials --- p.59 / Chapter 3.4.1 --- Reagents for IMAC purification of SUMO-hGH fusion protein --- p.59 / Chapter 3.4.2 --- Reagents for IMAC purification of mature rhGH --- p.60 / Chapter 3.4.3 --- Reagents for Western blotting --- p.60 / Chapter 3.4.4 --- Gel filtration running buffer --- p.62 / Chapter 3.5 --- Methods --- p.62 / Chapter 3.5.1 --- Purification of SUMO-hGH fusion protein by Ni2+-NTA affinity chromatography --- p.62 / Chapter 3.5.2 --- Cleavage of His-SUMO fusion partner to generate mature rhGH --- p.63 / Chapter 3.5.3 --- Purification of mature rhGH by 2nd round of Ni2+-NTA affinity chromatography --- p.64 / Chapter 3.5.4 --- Purification of rhGH by size exclusion chromatography - gel filtration chromatography --- p.64 / Chapter 3.5.5 --- General protein analysis --- p.65 / Chapter 3.6 --- Results --- p.67 / Chapter 3.6.1 --- Purification of SUMO-hGH fusion protein by Ni2+-NTA affinity chromatography --- p.67 / Chapter 3.6.2 --- Cleavage of His-SUMO fusion partner to generate mature rhGH --- p.69 / Chapter 3.6.3 --- Digestion efficiency of different constructs of SENP1C --- p.73 / Chapter 3.6.4 --- Purification of mature rhGH by 2nd round of Ni2+-NTA affinity chromatography --- p.77 / Chapter 3.6.5 --- Purification of rhGH by size exclusion chromatography -gel filtration chromatography --- p.78 / Chapter 3.7 --- Discussion --- p.81 / Chapter 3.7.1 --- Purification of SUMO-hGH fusion protein by Ni2+-NTA affinity chromatography --- p.81 / Chapter 3.7.2 --- Cleavage of His-SUMO fusion partner to generate mature rhGH --- p.82 / Chapter 3.7.3 --- Purification of mature rhGH by 2nd round of Ni2+-NTA affinity chromatography --- p.82 / Chapter 3.7.4 --- Purification of rhGH by size exclusion chromatography -gel filtration chromatography --- p.85 / Chapter 3.8 --- Conclusion --- p.85 / Chapter 4. --- Fermentation expression of SUMO-hGH and scale-up of downstream process / Chapter 4.1 --- Introduction --- p.86 / Chapter 4.2 --- Bioreactor system for E.coli host cultivation --- p.87 / Chapter 4.3 --- Mechanical cell disruption for cell --- p.88 / Chapter 4.4 --- rhGH binding assay --- p.88 / Chapter 4.5 --- Materials --- p.89 / Chapter 4.5.1 --- Reagents for bacterial culture by fermenter --- p.89 / Chapter 4.5.2 --- Reagents for HEK293 Hi cultivation --- p.91 / Chapter 4.5.3 --- Reagents for Dual-Luciferase® Reporter Assay System --- p.92 / Chapter 4.5.4 --- Reagents for silver stain of SDS-PAGE mini-gel --- p.93 / Chapter 4.6 --- Methods --- p.94 / Chapter 4.6.1 --- Bioreactor system and fixed volume fed-batch fermentation --- p.94 / Chapter 4.6.2 --- Large scale mechanically disruption of cell membrane --- p.97 / Chapter 4.6.3 --- Downstream processing of SUMO-hGH --- p.97 / Chapter 4.6.4 --- Culture of HEK293 Hi cells --- p.97 / Chapter 4.6.5 --- Dual-Luciferase® Reporter Assay System --- p.98 / Chapter 4.6.6 --- Silver staining of SDS-PAGE mini-gels --- p.101 / Chapter 4.7 --- Results --- p.101 / Chapter 4.7.1 --- Fed-batch fermentation of E. coli BL21 --- p.101 / Chapter 4.7.2 --- Comparison on disruption methods and the purification of SUMO-hGH from cell lysate --- p.106 / Chapter 4.7.3 --- Optimization of His-MBP-SENP1C digestion condition --- p.108 / Chapter 4.7.4 --- Optimization of rhGH purification in 2nd round of IMAC --- p.110 / Chapter 4.7.5 --- Characterization of mature rhGH --- p.112 / Chapter 4.8 --- Discussion --- p.116 / Chapter 4.8.1 --- Fed-batch fermentation of E. coli BL21 --- p.118 / Chapter 4.8.2 --- Downstream processing of fermentation culture and characterization of rhGH --- p.120 / Chapter 4.8.3 --- M9 based defined medium fermentation study --- p.122 / Chapter 4.8.4 --- rhGH production yield estimation --- p.128 / Chapter 4.8.5 --- Comparison of our fermentation expression system to the published data --- p.130 / Chapter 4.9 --- Conclusion --- p.132 / Chapter 5. --- His-MBP-SENPIC expression and purification / Chapter 5.1 --- Introduction --- p.133 / Chapter 5.2 --- Materials --- p.134 / Chapter 5.2.1 --- Reagents for bacterial culture --- p.134 / Chapter 5.2.2 --- Reagents for immobilized metal affinity chromatography purification of His-MBP-SENP1C --- p.135 / Chapter 5.3 --- Methods --- p.136 / Chapter 5.3.1 --- Expression of His-MBP-SENP1C --- p.136 / Chapter 5.3.2 --- Semi-purification of His-MBP-SENP1C by Ni2+-NTA affinity chromatography --- p.138 / Chapter 5.4 --- Results --- p.139 / Chapter 5.4.1 --- Expression of His-MBP-SENP1C --- p.139 / Chapter 5.4.2 --- Digestion activity of His-MBP-SENP1C expressed --- p.139 / Chapter 5.5 --- Discussion --- p.141 / Chapter 5.5.1 --- Expression and purification of His-MBP-SENP1C --- p.141 / Chapter 5.5.2 --- His-MBP-SENP1C production yield estimation --- p.143 / Chapter 6. --- Production and characterization of monoclonal antibodies against rhGH / Chapter 6.1 --- Introduction --- p.145 / Chapter 6.2 --- Materials --- p.146 / Chapter 6.2.1 --- Reagents for Sp2/0-Ag14 cultivation --- p.146 / Chapter 6.2.2 --- Reagents for PEG fusion --- p.147 / Chapter 6.2.3 --- Reagents for enzyme linked immunosorbent assay --- p.149 / Chapter 6.2.4 --- Reagents for mAbs purification by HiTrap´ёØ Protein G HP Column --- p.150 / Chapter 6.3 --- Methods --- p.151 / Chapter 6.3.1 --- ELISA --- p.151 / Chapter 6.3.2 --- Immunization --- p.152 / Chapter 6.3.3 --- Culturing of myeloma fusion partner cells --- p.153 / Chapter 6.3.4 --- Isolation of splenocyte --- p.153 / Chapter 6.3.5 --- PEG fusion --- p.154 / Chapter 6.3.6 --- Limiting dilution --- p.155 / Chapter 6.3.7 --- Cryopreservation of hybridoma cell lines --- p.156 / Chapter 6.3.8 --- Mass production of monoclonal antibodies --- p.157 / Chapter 6.3.9 --- Purification of IgG mAbs from ascites --- p.157 / Chapter 6.3.10 --- MAbs isotyping --- p.159 / Chapter 6.3.11 --- Determination of kinetic parameters of mAbs --- p.159 / Chapter 6.4 --- Results --- p.162 / Chapter 6.4.1 --- Production of murine anti-rhGH monoclonal antibodies --- p.162 / Chapter 6.4.2 --- Characterization of anti-rhGH mAbs --- p.170 / Chapter 6.5 --- Discussion --- p.178 / Chapter 6.5.1 --- Mass production of mAbs --- p.179 / Chapter 6.5.2 --- Future works on mAbs --- p.179 / Chapter 6.6 --- Conclusion --- p.181 / Chapter 7. --- Development of sandwich ELISA for rhGH / Chapter 7.1 --- Introduction --- p.182 / Chapter 7.2 --- Materials --- p.184 / Chapter 7.2.1 --- Reagents for sandwich ELISA --- p.184 / Chapter 7.3 --- Methods --- p.184 / Chapter 7.3.1 --- Production of rabbit polyclonal antiserum against rhGH --- p.184 / Chapter 7.3.2 --- Sandwich ELISA --- p.185 / Chapter 7.4 --- Results --- p.186 / Chapter 7.4.1 --- Production of rabbit antiserum against rhGH --- p.186 / Chapter 7.4.2 --- Sandwich ELISA --- p.188 / Chapter 7.4.3 --- Optimization of sandwich ELISA --- p.190 / Chapter 7.4.4 --- Specificity of sandwich ELISA --- p.194 / Chapter 7.4.5 --- Cross reactivity of sandwich ELISA to E.coli cell lysate --- p.196 / Chapter 7.4.6 --- Measurement of SUMO-hGH with sandwich ELISA --- p.198 / Chapter 7.5 --- Discussion --- p.201 / Chapter 7.5.1 --- Application of sandwich ELISA --- p.203 / Chapter 7.5.2 --- Future works on sandwich ELISA --- p.205 / Chapter 7.6 --- Conclusion --- p.205 / References --- p.206 / Appendix - pJ2:G01458 nucleotide sequence --- p.213

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Abstract not available