| 摘要 | 第10-13页 |
| SUMMARY | 第13-16页 |
| LIST OF ABBREVIATIONS | 第17-21页 |
| CHAPTER 1 Introduction | 第21-38页 |
| 1.1 Significance of Bursa of Fabricius in avian immunology | 第21-22页 |
| 1.2 Anatomical features of bursa of Fabricius | 第22页 |
| 1.3 Histological features of bursa of Fabricius | 第22-24页 |
| 1.4 Bursa of Fabricius is the target organ of multiple pathogens | 第24页 |
| 1.5 Salmonella infection and avian lymphoid organs | 第24-25页 |
| 1.6 Pathogenic infections and toll like receptor signaling | 第25-26页 |
| 1.7 Toll like receptor signaling and Chicken immune system | 第26-27页 |
| 1.8 Salmonella LPS and TLR4 activation | 第27-28页 |
| 1.9 Expression of Immune related cells and host immunity | 第28-32页 |
| 1.9.1 Role of mast cells in tissue development and host immunity | 第28-31页 |
| 1.9.2 Role of eosinophils in tissue development and host immunity | 第31-32页 |
| 1.9.3 Role of heterophils in tissue damages during Salmonella LPS stimulation | 第32页 |
| 1.10 Alterations in transcriptional factor NF-κB signaling during LPS stimulation | 第32-33页 |
| 1.11 Cell apoptosis and mechanism of apoptosis during LPS stimulation | 第33-35页 |
| 1.12 Importance and application of RNA-seq technology in transcriptional alterations | 第35-36页 |
| 1.13 Importance of transcriptome analysis in complex disease molecular mechanism | 第36页 |
| 1.14 Application of bioinformatics tools in transcriptome analysis | 第36-37页 |
| 1.15 Conclusion and hypothesis | 第37-38页 |
| CHAPTER 2 Materials and Methods | 第38-63页 |
| 2.1 Animal welfare and ethics statement | 第38页 |
| 2.2 Experimental design and induction of Lipopolysaccharide stimulation | 第38-39页 |
| 2.3 Collection of tissue samples | 第39页 |
| 2.4 Tissue fixation, processing and sectioning for histo-morphological | 第39-40页 |
| 2.4.1 Preparation of tissue fixation solution | 第39页 |
| 2.4.2 Hydration of tissues | 第39页 |
| 2.4.3 Chemical used in tissue processing and embedding | 第39-40页 |
| 2.4.4 Poly-L-Lysine coating on glass slides | 第40页 |
| 2.4.5 Serial tissue sectioning | 第40页 |
| 2.5 Tissue fixation and processing for Scanning Electron Microscope (SEM) | 第40页 |
| 2.6 Hematoxylin and Eosin (H&E) staining | 第40-41页 |
| 2.6.1 Principle | 第40-41页 |
| 2.6.2 Protocol | 第41页 |
| 2.7 Special staining-I (Toluidine blue staining) | 第41-42页 |
| 2.7.1 Principle | 第41页 |
| 2.7.2 Preparation of solutions for Toluidine Blue stain | 第41-42页 |
| 2.7.3 Protocol | 第42页 |
| 2.8 Special staining-II (Lendrum's chromotrope 2R method) | 第42-43页 |
| 2.8.1 Principle | 第42页 |
| 2.8.2 Preparation of Chromotrope 2R stain | 第42页 |
| 2.8.3 Protocol | 第42-43页 |
| 2.9 Immunohistochemical (IHC) staining technique | 第43-45页 |
| 2.9.1 Principle | 第43页 |
| 2.9.2 Preparation of solution for IHC | 第43-44页 |
| 2.9.3 Protocol | 第44-45页 |
| 2.10 In situ cell apoptosis detection technique for ssDNA expression | 第45-46页 |
| 2.11 TUNEL-Apoptosis assay | 第46-47页 |
| 2.11.1 Principle | 第46页 |
| 2.11.2 Protocol | 第46-47页 |
| 2.12 Semi-quantitative analysis of expression of cellular proteins | 第47页 |
| 2.13 Statistical analysis | 第47页 |
| 2.14 Extraction of protein | 第47-48页 |
| 2.14.1 Tissue grinding in steel bead beater using Trizol reagent | 第47-48页 |
| 2.14.2 Protein isolation by tri-reagent | 第48页 |
| 2.14.3 Protein precipitation | 第48页 |
| 2.14.4 Protein wash | 第48页 |
| 2.14.5 Protein solubilization | 第48页 |
| 2.15 Determination of protein concentration by BCA protein assay | 第48-50页 |
| 2.15.1 BCA Protein Assay Kit | 第49页 |
| 2.15.2 The BCA-Protein Reaction Scheme | 第49页 |
| 2.15.3 BCA Kit components | 第49页 |
| 2.15.4 BCA working fluid | 第49页 |
| 2.15.5 Protocol (96 well plates measuring technique) | 第49页 |
| 2.15.6 Preparation of Standard Curve | 第49-50页 |
| 2.16 SDS-PAGE and Western blotting experiments | 第50-53页 |
| 2.16.1 Solutions | 第50-51页 |
| 2.16.2 Preparations of SDS-Polyacrylamide gels | 第51-52页 |
| 2.16.3 Mounting of acrilamide gel in the electrophoresis apparatus | 第52页 |
| 2.16.4 Western blot analysis | 第52页 |
| 2.16.5 Protocol | 第52-53页 |
| 2.17 Extraction of RNA | 第53-54页 |
| 2.17.1 Tissue grinding in steel bead beater using Trizol reagent | 第53页 |
| 2.17.2 Phase separation | 第53页 |
| 2.17.3 RNA Precipitation | 第53-54页 |
| 2.17.4 RNA wash | 第54页 |
| 2.17.5 RNA Solubilization | 第54页 |
| 2.17.6 RNA quantization and visualization | 第54页 |
| 2.18 Experimental Pipeline for RNA-seq Analysis | 第54-56页 |
| 2.18.1 Preparation of c DNA library and RNA sequencing | 第54-55页 |
| 2.18.2 Protocol | 第55-56页 |
| 2.19 Bioinformatics Analysis of RNA-seq data | 第56-59页 |
| 2.19.1 Data Filtering | 第56-57页 |
| 2.19.2 Reads Mapping | 第57页 |
| 2.19.3 Gene Quantification | 第57-58页 |
| 2.19.4 Screening of DEGs using NOISeq | 第58-59页 |
| 2.20 Gene Ontology Annotation | 第59-60页 |
| 2.21 KEGG Pathway Enrichment Analysis | 第60页 |
| 2.22 Validation of RNA-seq results by real time quantitative PCR (RT-qPCR) | 第60-62页 |
| 2.22.1 RT-qPCR | 第60页 |
| 2.22.2 Elimination of genomic DNA (gDNA) | 第60-61页 |
| 2.22.3 Kit components | 第61页 |
| 2.22.4 Protocol | 第61-62页 |
| 2.22.5 Real-Time PCR- reverse-transcription reaction | 第62页 |
| 2.23 Availability of supporting data | 第62-63页 |
| CHAPTER 3 Results | 第63-107页 |
| 3.1 Bursal atrophy after LPS stimulation | 第63-64页 |
| 3.2 Micro-morphological alterations in chicken Bursa under LPS stress | 第64-65页 |
| 3.3 Ultra-structural alteration in chicken bursa of Fabricius under LPS stress | 第65-66页 |
| 3.4 Effect of LPS stimulation on immune related cells in chicken bursa | 第66-69页 |
| 3.4.1 Effect on Mast Cells | 第66-68页 |
| 3.4.2 Effect on Eosinophils | 第68-69页 |
| 3.5 RNA-Seq data output (Raw and Clean Reads) | 第69页 |
| 3.6 Quantification of Gene Expression | 第69-71页 |
| 3.7 Correlation Analysis of Samples | 第71-72页 |
| 3.8 Alignment Statistics of reads align to reference chicken genome | 第72-73页 |
| 3.9 Screening of Differentially Expressed Genes (DEGs) | 第73-75页 |
| 3.10 Clustering Analysis of DEGs | 第75-76页 |
| 3.11 Statistics of DEGs | 第76-77页 |
| 3.12 Validation of significant DEGs in chicken bursa of Fabricius by RT-qPCR | 第77-78页 |
| 3.13 Gene Ontology (GO) annotation of DEGs | 第78-82页 |
| 3.13.1 GO functional analysis at early time points | 第78-80页 |
| 3.13.2 GO functional analysis at mid time points | 第80-81页 |
| 3.13.3 GO functional analysis at late time points | 第81-82页 |
| 3.14 KEGG Analysis of DEGs | 第82-94页 |
| 3.14.1 KEGG enrichment analysis at early time points | 第84-86页 |
| 3.14.2 Description of DEGs in regulation of actin cytoskeleton pathway | 第86-88页 |
| 3.14.3 KEGG enrichment analysis at mid time points | 第88-89页 |
| 3.14.4 Description of DEGs in phagosome pathway (saline vs LPS) at 36 h | 第89-90页 |
| 3.14.5 KEGG enrichment analysis at late time points | 第90-92页 |
| 3.14.6 Description of DEGs in HTLV-1 infection pathway (saline vs LPS) at 72 h | 第92-94页 |
| 3.15 Selection of candidate genes and identification of important transcriptional factors | 第94-95页 |
| 3.16 Functional pathway analysis | 第95页 |
| 3.17 LPS-TLR/JNK-MAPK signaling pathway may mediate acute bursal atrophy | 第95-96页 |
| 3.18 Effect of LPS stimulation on TLR4 expression (IHC) | 第96-97页 |
| 3.19 Effect of LPS stimulation on TLR4 protein (Western Blot Analysis) | 第97-98页 |
| 3.20 Effect of LPS stimulation on NFκB expression | 第98-99页 |
| 3.21 Determination of TUNEL-apoptosis under LPS stress | 第99-101页 |
| 3.22 Effect of LPS stimulation on PCNA expression | 第101-102页 |
| 3.23 Effect of LPS stimulation on ssDNA expression (Apoptosis) | 第102-103页 |
| 3.24 Reactome Pathway Interaction Analysis | 第103-104页 |
| 3.25 String analysis | 第104-107页 |
| CHAPTER 4 Discussion | 第107-115页 |
| CHAPTER 5 Conclusion and future prospects | 第115-116页 |
| 5.1 Conclusion | 第115页 |
| 5.2 Future prospects | 第115-116页 |
| CHAPTER 6 References | 第116-130页 |
| CHAPTER 7 List of Figures | 第130-132页 |
| CHAPTER 8 List of Tables | 第132-133页 |
| CHAPTER 9 Scientific Contributions | 第133-135页 |
| APPENDIX I Acknowledgements | 第135-137页 |
