Expression profiling of cord blood neutrophil in response to bacterial lipopolysaccharide and peptidoglycan stimulations.

• Other Authors: Fong, Oi Ning.
• Format: Others
• Language: English; Chinese
• Published: 2009
• Subjects: Neutrophils; Fetal blood; Endotoxins; Peptidoglycans; Fetal Blood; Lipopolysaccharides > genetics; Peptidoglycan > genetics
• Online Access: http://library.cuhk.edu.hk/record=b5896564; http://repository.lib.cuhk.edu.hk/en/item/cuhk-326906

Fong, Oi Ning. === Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. === Includes bibliographical references (leaves 170-195). === Abstracts in English and Chinese. === Abstract --- p.i === Acknowledgements --- p.vi === Contents --- p.viii === List of Abbreviations --- p.xii === Chapter CHAPTER ONE --- Introduction --- p.1 === Chapter 1.1 --- Bacterial Infection in Neonates --- p.1 === Chapter 1.2 --- Gram-positive and Gram-negative Bacterial Cell Wall --- p.3 === Chapter 1.2.1 --- Gram-negative Bacterial Cell Wall Component - Lipopolysaccharide --- p.3 === Chapter 1.2.2 --- Gram-positive Bacterial Cell Wall Component - Peptidoglycan --- p.4 === Chapter 1.3 --- Differential Host Response against Gram-specific Bacterial Infection --- p.6 === Chapter 1.4 --- Role of Neutrophils in Host Defense against Bacterial Infection --- p.8 === Chapter 1.4.1 --- Recognition of Bacterial Components --- p.9 === Chapter 1.4.2 --- Neutrophil Functions --- p.10 === Chapter 1.5 --- Expression Profiling of Activated Neonatal Neutrophils --- p.15 === Chapter CHAPTER TWO --- Objectives --- p.26 === Chapter CHAPTER THREE --- Materials and Methodology --- p.27 === Chapter 3.1 --- Overview of the Experimental Procedure --- p.27 === Chapter 3.2 --- Cord Blood Sample Collection --- p.28 === Chapter 3.3 --- Cord Blood Neutrophil Isolation --- p.30 === Chapter 3.3.1 --- Isolation of Neutrophils --- p.30 === Chapter 3.3.2 --- Analysis of Neutrophil Purity by Flow Cytometry --- p.31 === Chapter 3.3.3 --- Cell Viability Test by Trypan Blue Exclusion Assay --- p.31 === Chapter 3.4 --- In Vitro Stimulation of Neutrophils by LPS or PGN --- p.33 === Chapter 3.5 --- Total RNA and Protein Isolation --- p.34 === Chapter 3.5.1 --- Total RNA Isolation --- p.34 === Chapter 3.5.2 --- Protein Isolation --- p.35 === Chapter 3.6 --- Preparation of Total RNA Samples for Expression Profiling and Quantitative Real Time Polymerase Chain Reaction (qPCR) --- p.37 === Chapter 3.6.1 --- DNase Treatment --- p.37 === Chapter 3.6.2 --- Total RNA Cleanup --- p.37 === Chapter 3.6.3 --- Purity Assessment of the Purified Total RNA Sample --- p.38 === Chapter 3.6.4 --- Integrity Assessment of the Purified Total RNA Sample --- p.39 === Chapter 3.6.5 --- Assessment of Tumor Necrosis Factor Alpha (TNF-α) mRNA Expression Level in Neutrophils --- p.42 === Chapter 3.7 --- Determination of the PGN Concentration for Neutrophil Activation --- p.43 === Chapter 3.8 --- "Expression Profiling of the LPS, PGN Stimulated or Unstimulated CB Neutrophils" --- p.44 === Chapter 3.8.1 --- cRNA Preparation and Array Hybridization --- p.44 === Chapter 3.8.2 --- Expression Profiling Data Analysis --- p.46 === Chapter 3.9 --- Validation of Candidate Genes Using qPCR --- p.48 === Chapter 3.10 --- Gram-Negative Bacterial Endotoxin Assay --- p.50 === Chapter CHAPTER FOUR --- LPS Stimulation Induced Transcriptional Changes in Cord Blood Neutrophils --- p.61 === Chapter 4.1 --- Result --- p.61 === Chapter 4.1.1 --- Gene Expression Profile of CB Neutrophils in Response to LPS Stimulation --- p.61 === Chapter 4.1.1.1 --- Up-regulated Genes in LPS-stimulated CB Neutrophils --- p.61 === Chapter 4.1.1.2 --- Down-regulated Genes in LPS-stimulated CB Neutrophils --- p.62 === Chapter 4.1.1.3 --- Network Analysis of Genes Induced by LPS Stimulation --- p.63 === Chapter 4.2 --- Discussion --- p.64 === Chapter 4.2.1 --- Robust Transcriptional Response in CB Neutrophils --- p.64 === Chapter 4.2.2 --- LPS Modulated Transcriptional Responses --- p.64 === Chapter 4.2.2.1 --- LPS-induced NF-kB Pathway --- p.64 === Chapter 4.2.2.2 --- LPS-induced Expression of Various Transcription Factors --- p.66 === Chapter 4.2.2.3 --- LPS-induced Regulation of Apoptosis --- p.67 === Chapter CHAPTER FIVE --- PGN Stimulation Induced Transcriptional Changes in Cord Blood Neutrophils --- p.83 === Chapter 5.1 --- Result --- p.83 === Chapter 5.1.1 --- Gene Expression Profile of PGN-stimulated CB Neutrophils --- p.83 === Chapter 5.1.2 --- Up-regulated Genes in PGN-stimulated CB Neutrophils --- p.83 === Chapter 5.1.3 --- Down-regulated Genes in PGN-stimulated CB Neutrophils --- p.84 === Chapter 5.1.4 --- Network Analysis of Genes Induced by PGN Stimulation --- p.84 === Chapter 5.2 --- Discussion --- p.86 === Chapter 5.2.1 --- Robust Transcriptional Response in CB Neutrophils --- p.86 === Chapter 5.2.2 --- PGN Modulated Transcriptional Responses --- p.86 === Chapter 5.2.2.1 --- PGN-induced NF-kB Pathway --- p.86 === Chapter 5.2.2.2 --- Possible Role of STAT3 in PGN-stimulated CB Neutrophil --- p.89 === Chapter 5.2.2.3 --- Possible Role of c-Jun in PGN-stimulated CB Neutrophil --- p.90 === Chapter CHAPTER SIX --- Comparison and Validation of LPS- and PGN-activated Transcriptomes in Cord Blood Neutrophils --- p.106 === Chapter 6.1 --- Result --- p.106 === Chapter 6.1.1 --- Comparison of the Transcriptional Changes of LPS- and PGN- stimulated CB Neutrophils --- p.106 === Chapter 6.1.2 --- Common Transcriptional Changes of LPS- and PGN-Stimulated CB Neutrophils --- p.106 === Chapter 6.1.2.1 --- Commonly Up-regulated Genes in LPS- and PGN- Stimulated Neutrophils --- p.107 === Chapter 6.1.2.2 --- Commonly Down-regulated Genes in LPS- and PGN- Stimulated Neutrophils --- p.107 === Chapter 6.1.2.3 --- Network Analysis of Genes Commonly Regulated by LPS and PGN --- p.108 === Chapter 6.1.3 --- Differential Transcriptional Changes of LPS- and PGN- Stimulated CB Neutrophils --- p.108 === Chapter 6.1.4 --- Real Time qPCR Validation of the Expression Levels of Selected Genes --- p.109 === Chapter 6.1.5 --- Expression Changes of the Confirmed Target Genes in Response to High-dose LPS Stimulation --- p.110 === Chapter 6.2 --- Discussion --- p.111 === Chapter 6.2.1 --- Activation of NF-kB and Related Genes by Both LPS- and PGN-stimulation in CB Neutrophils --- p.111 === Chapter 6.2.2 --- Commonly Expressed Genes - Transcription Factor MAFF --- p.112 === Chapter 6.2.3 --- Commonly Expressed Genes - Novel Gene G0S2 --- p.113 === Chapter 6.2.4 --- Suspected Commonly Expressed Genes - Transcription Factor NR4A3 --- p.114 === Chapter 6.2.5 --- Differentially Expressed Genes - Heat Shock Proteins --- p.115 === Chapter 6.2.6 --- Differentially Expressed Genes 226}0ؤ AP-1 Transcription Factor Complex --- p.118 === Chapter 6.2.7 --- Other Differentially Expressed Genes --- p.121 === Chapter CHAPTER SEVEN --- General Discussion and Conclusion --- p.164 === Bibliography --- p.168