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dc.contributor.authorWei-Lin Tuen_US
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dc.description.abstract熱緊迫會造成家禽產業的經濟上損失;下視丘是溫度調控的重要中心,其可以偵測溫度的變化並藉由調整自主神經系統及內分泌系統來調控熱的保持與散失進而調節體溫。本研究之目的為探討急性熱緊迫對蛋用雞型L2品系及肉用雞型B品系台灣土雞母雞下視丘基因及蛋白質表現之影響。12隻30週齡母雞分成四組:熱緊迫處理組在B品系以38°C處理2小時及在L2品系以36°C處理4小時後分別在25°C恢復0小時(H2R0 and H4R0)、2小時(H2R2 and H4R2)或6小時(H2R6 and H4R6)、對照組則維持於25°C。在各時間點採下視丘樣品供RNA及蛋白質分析用。研究結果顯示L2品系及B品系母雞的呼吸速率及體溫在經過熱緊迫處理後都有顯著的上升(P < 0.05)。B品系雞隻下視丘在H2R0、H2R2、及H2R6分別有24、11及25個基因表現量上升,41、15及42個基因表現量下降,這些差異表現基因主要參與細胞過程、代謝過程、定位、多細胞生物過程、發育過程及生物性調控等生物過程。以即時聚合酶鏈鎖反應驗證其中七個基因的表現量,其中六個基因的表現量與微陣列分析的結果相符。二維差異膠體電泳分析結果顯示熱緊迫處理後L2品系雞隻的下視丘有134個蛋白質點之表現具顯著差異(P < 0.05),分別屬於118種不同蛋白質;B品系的台灣土雞母雞的下視丘則有114個蛋白質點之表現具顯著差異,分別屬於71種不同蛋白質,此等差異表現蛋白質主要參與細胞過程、代謝、傳送及細胞物質組織的生物過程。由功能性基因體學的分析結果推測雞隻下視丘因應急性熱緊迫的可能機制為:急性熱緊迫可能導致組織缺氧及蛋白質失去功能,使活性氧化物在粒線體內累積而對下視丘細胞造成損害,此時下視丘細胞透過基因及蛋白表現的改變來維持細胞完整性、修復DNA的損傷及促進細胞分化及增生以舒解急性熱緊迫所造成的不良影響,此細胞保護機制所需之能量可能主要來自於糖解作用。因此,此等差異表現的基因及蛋白質將是雞隻下視丘在熱緊迫反應中所須且獨特的關鍵因子。zh_TW
dc.description.abstractHeat stress causes economic loss in poultry production. The hypothalamus is a crucial center for regulating body temperature, detecting temperature changes, and modulating the autonomic nervous system and the endocrine loop for heat retention and dissipation. The purpose of this study was to investigate the global profile of gene and protein expressions in the hypothalamus of a layer-type L2 strain and a meat-type B strain Taiwan country chickens (TCCs) after acute heat stress. Twelve 30-wk-old hens of TCCs were allocated to four groups. Three heat-stressed groups were subjected to acute heat stress at 38°C for 2 hr in B strain TCCs and 36°C for 4 hr in L2 strain TCCs without recovery (H2R0), with 2 hr of recovery (H2R2), or with 6 hr of recovery (H2R6), respectively. Control hens were maintained at 25°C. The hypothalamus samples were collected for mRNA expression and protein expression analysis at each time point. The results showed that the respiratory rate and body temperature of both strains increased during heat stress (P < 0.05). In B strain TCCs, whole-genome microarrays analysis showed upregulated expression of 24, 11, and 25 genes and downregulation of 41, 15, and 42 genesin H2R0, H2R2, and H2R6 groups, respectively. Most of the differentially expressed genes are involved in biological processes of cellular processes, metabolic processes, localization, multicellular organismal processes, developmental processes, and biological regulation. Six of 7 genes with differential expressions in B strain TCCs by qRT-PCR were consistent with the results of microarray analysis. Results of two-dimensional difference gel electrophoresis revealed 134 protein spots differentially expressed in the hypothalamus of L2 strain TCCs after heat stress (P < 0.05). Peptide mass fingerprinting analysis revealed that these spots belong to 118 distinct proteins. A total of 114 protein spots representing 71 distinct proteins were differentially expressed in the hypothalamus of B strain TCCs after acute heat stress. Most of the differentially expressed proteins are involved in biological processes of cellular processes, metabolism, transport, and cellular component organization. Results of functional genomics analysis suggested that acute heat stress may cause ischemia, protein dysfunction, and the accumulation of reactive oxygen species in mitochondria then cause damage the hypothalamic cells. The changes of transcripts and protein levels within the hypothalamic neuronss reflact a regulatory response to sustain their cellular integrity, DNA damage repairing, and processes of cell division and regeneration to attenuate the detrimental effects by acute heat stress. The major energy supply for these protective actions apparently are derived from glycolysis. Thus the differential gene expressions act as essential and unique key factors in the acute heat stress response of the hypothalamus in chickens.en_US
dc.description.tableofcontents誌謝 i 摘要 ii Abstract iii Table of contents v List of Tables ix List of Figures x Chapter 1. General introduction 1 1.1. The detrimental effects of acute heat stress on Taiwan country chickens (TCCs) 2 1.2. The hypothalamus 2 1.3. Acute heat stress causes the damage to hypothalamus 5 1.4. Functional genomics study on the heat stress response in chickens 8 1.5. Using genetic tools to alleviate heat stress 10 Chapter 2. Materials and Methods 12 2.1. Experimental animals, conditions of acute heat stress 13 2.2. Sample preparation 13 2.3. Microarray analysis of gene expression 14 2.4. Gene network analysis and gene annotation 15 2.5. Validation of gene expression by quantitative real-time polymerase chain reaction (qRT-PCR) 15 2.6. Protein expressions by two-dimensional difference gel electrophoresis (2-D DIGE) analysis 16 2.6.1. Sample preparation for 2-D DIGE analysis 16 2.6.2. 2D-DIGE analysis and image analysis 17 2.6.3. Gel staining for spot picking and protein identification 18 2.7. Annotation of the differentially expressed proteins 20 2.8. Validation of protein expression by Western blotting 20 2.9. Statistical analysis 21 Chapter 3. Profiling of differential gene expression in the hypothalamus of broiler–type Taiwan country chickens in response to acute heat stress 22 3.1. Abstract 23 3.2. Introduction 25 3.3. Results 26 3.3.1. Effects of acute heat stress on gene expression in the hypothalamus of hens of broiler-type B strain TCCs 26 3.3.2. Functional categories of differentially expressed genes in the hypothalamus of hens of broiler-type B strain TCCs after acute heat stress 42 3.3.3. Validation of representative differentially expressed genes in the hypothalamus of hens of broiler-type B strain TCCs after acute heat stress by using qRT-PCR 45 3.4. Discussion 48 3.4.1. Acute heat stress induced the expression of HSP and antioxidative genes in the hypothalamus of hens of broilertype TCCs 48 3.4.2. Acute heat stress affected reproduction- and cell integrity–related genes in the hypothalamus of hens of broilertype B strain TCCs 49 3.4.3. Acute heat stress affected metabolism-related genes in the hypothalamus of hens of broiler-type TCCs 51 3.4.4. A putative mechanism for the detrimental effects of acute heat stress on the hypothalamus of hens of broiler-type TCCs 51 Chapter 4. Changes of protein expression in the hypothalamus of a broiler-type strain Taiwan country chickens in response to acute heat stress 54 4.1. Abstract 55 4.2. Introduction 55 4.3. Results 57 4.3.1. Effects of acute heat stress on hypothalamic protein expression in broiler-type TCC hens 57 4.3.2. Bioinformatics analysis of differential protein expressions in the hypothalamus of broiler-type TCC hens after acute heat stress 60 4.3.3. Validation of differentially expressed proteins in the hypothalamus of TCC hens after acute heat stress 65 4.3.4. Identification of the signal pathways of differentially expressed proteins in the hypothalamus of TCC hens after acute heat stress 67 4.4. Discussion 70 4.4.1. Acute heat stress induces defensive responses in the hypothalamus 70 4.4.2. Acute heat stress causes protein degradation in the hypothalamus 71 4.4.3. Acute heat stress induces oxidative stress in the hypothalamus 72 4.4.4. The putative mechanism of acute heat stress response in the hypothalamus 73 Chapter 5. Annotation of differential protein expression in the hypothalami of layer-type Taiwan country chickens in response to acute heat stress 75 5.1. Abstract 76 5.2. Introduction 78 5.3. Results 79 5.3.1. Effects of acute heat stress on protein expression in the hypothalamus 79 5.3.2. Annotation of differentially expressed proteins 81 5.3.3. Validation of representative differentially expressed proteins in the hypothalamus after acute heat stress through western blot 102 5.4. Discussion 104 5.4.1. Acute heat stress affects the utilization of glucose in the hypothalamus 104 5.4.2. Antioxidant enzymes are upregulated in the hypothalamus after acute heat stress 107 5.4.3. Protective mechanism of hypothalamic cells in response to acute heat stress 107 Chapter 6. General discussion 111 6.1. Acute heat stress affects metabolism–related genes and proteins in the hypothalamus of TCC hens 112 6.2. The detrimental effects of acute heat stress on the hypothalamus of hens of TCCs 114 6.3. The protective mechanism for the detrimental effects of acute heat stress on the hypothalamus of TCC hens 115 6.4. The difference of response to acute heat stress in hypothalamus of layer-type strain and broiler-type strain chickens 119 Chapter 7. General conclusion 122 References 125 Appendices 152zh_TW
dc.subjectgene expressionen_US
dc.subjectprotein expressionen_US
dc.subjectacute heat stressen_US
dc.subjectwhole-genome microarrays analysisen_US
dc.subjecttwo-dimensional difference gel electrophoresisen_US
dc.titleA functional genomics study on the acute heat stress response in the hypothalamus of Taiwan country chickensen_US
dc.typethesis and dissertationen_US
item.openairetypethesis and dissertation-
item.fulltextwith fulltext-
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