Thesis (M.S.)--West Virginia University, 2002. / Title from document title page. Document formatted into pages; contains vii, 72 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 58-71).

Thesis (M.S.)--West Virginia University, 2008. / Title from document title page. Document formatted into pages; contains vii, 68 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 46-68).

Thesis (M.S.)--West Virginia University, 2006. / Title from document title page. Document formatted into pages; contains vi, 45 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 40-45).

Five new genes apparently involved in the metabolism of cell wall peptidoglycan by Escherichia coli are described. One of these, designated murH, was mapped at 99 min on the E. coli linkage map. The murH1 mutant exhibited temperature- sensitive (ts) growth which was associated with a block in a late step in peptidoglycan synthesis and with peptidoglycan hydrolase-mediated lysis at the restrictive temperature. The murH locus could not be cloned in multicopy vectors but was readily cloned in a single copy phasmid vector derived from phage λ. The instability of murH in multicopy prevented its further characterization. As an alternative approach to characterizing the murH function, extragenic mutations which suppressed the murH1 ts lysis phenotype were isolated. One suppressor mutation, designated smh-A1, (25 min on the genetic linkage map) restored temperature resistance in murH1 mutants but otherwise had no distinguishable phenotype. A second extragenic murH1 suppressor, smhB1 (13 min) conferred a ts lysis phenotype by itself. Interestingly, a combination of murH1 and smhB1 resulted in cosuppression of their lysis phenotypes. The suppressor activities of the smhA1 and smhB1 alleles were relatively specific in that they failed to suppress lysis caused by either mutational (murE or murF) or antibiotic-induced blocks in peptidoglycan synthesis. Two additional ts lysis mutations, lytD1 (mapped at 13 min) and lytE1 (25 min), arose spontaneously in smhB1 and smhA1 backgrounds, respectively. The smhA1 allele suppressed the lysis phenotype of lytE1 but not of lytD1. Furthermore, the combination of smhB1 with either lytD1 or lytE1 resulted in cosuppression of their lysis phenotype. The specificity of the suppressor activities, combined with the similarities in the phenotypes of the mutants representing this collection of loci, suggested functional relationships between the murH, smhA, smhB, lytD, and lytE loci. Four clones which complemented the lytD1 mutation were obtained by screening an E. coli gene library, but it is shown that the complementing activity did not represent the E. coli chromosomal lytD locus. It is shown instead that 2 phage λ genes, identified as cro and cI, accounted for the lytD1 complementing activities in these clones. Evidence is presented which suggests that these clones were derived from phage λ DNA which was fortuitously present as a contaminant in the vector preparation used for construction of the gene library. Since the λ Cro and CI proteins are DNA-binding proteins which bind to identical 17 base-pair recognition sequences (the λ right operator sequences), it is hypothesized that LytD encodes a DNA-binding protein with a similar specificity (i.e., which binds to a λ right operator-like sequence) which regulates, probably negatively, the expression of a gene(s) involved in some way with peptidoglycan hydrolysis. / Graduate

Thesis (Ph. D.)--Illinois State University, 1997. / Title from title page screen, viewed June 13, 2006. Dissertation Committee: Radheshyam K. Jayaswal (chair), Brian J. Wilkinson, Mathew J. Nadakavukaren, Herman E. Brockman, Alan J. Katz. Includes bibliographical references and abstract. Also available in print.

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

Three Streptococcal strains: S. milleri P213, S. milleri P35 and S. milleri B200 and three enterococcal strains: E. faecalis 123, E. faecalis 126 and E. faecium were used to test for vancomycin resistance. Two strains were used as reference strains that were already characterized as vancomycin resistant. E. faecium BM4147 was used as a VanA control and E. faecalis ATCC was used as a VanB control. Susceptibility of each strain to this antibiotic was tested by disk-diffusion assay and the MIC values for the strains were found to be between 5 - 10 ug/ml and for the VanA control, the MIC was > 64 ug/ml and for the VanB control was 32 ug/ml. These MIC values indicate that S. milleri P213, S. milleri P35, S. milleri B200, E. faecalis 123, E. faecalis 126, and E. faecium are all of the VanC phenotype. All strains were tested for lysis by means of addition of vancomycin (10 ug/ml) to the bacterial cultures. Lytic curves were constructed and the VanB control was found to be most autolytic upon addition of vancomycin and E. faecalis 123 was the least autolytic. However, under normal conditions in phosphate buffer, lytic curves showed that S. milleri P213 was the most autolytic and the VanA control, the least autolytic. PCR assays were performed to detect specific antibiotic resistant genes. Primers were selected from Dukta-Malen et al., 1995. The VanA primer yielded amplification of 732 bp for only the VanA control DNA and the VanB primer set yielded products for the VanB control DNA. S. milleri P213, P35, B200 and E. faecalis 123 and 126, and E. faecium DNA were amplified with the VanC primers. This supports the results obtained in MIC that these strains are possibly VanC resistant strains. Amplified VanA control and that of E. faecalis 126 were thereafter sequenced. VanA control amplicon was correctly amplified since it showed homology to E. faecium BM4147 as well as the VanB amplicons which was found to be homologous to the transposon Tn1549 found on the well-characterized E. faecalis strain which is known to harbour the VanB vancomycin-resistant genes. Whilst E. faecalis 126 which represented the VanC phenotype showed 96% homology to E. gallinarum BM4147 which is a well-characterized glycopeptide-resistant enterococci belonging to the VanC phenotype. Southern blots were performed using specific primers as a probe to verify whether the gene sequences for the specific genotype were present in these strains and results confirmed those found in the PCR assays and in DNA sequencing. The peptidoglycan precursors of each strain were arrested in vancomycin (20 ug/ml) to block transpeptidation and transglycosylation steps of peptidoglycan synthesis and bacitracin (100 ug/ml) was used to amplify precursors at the transglycosylation step. Precursors were extracted and analysed by reverse-phase HPLC. UDP-MurNAc-tetrapeptides cell wall precursors, which are found abundantly in vancomycin-resistant strains, were found in large proportions in all strains, except in E. faecalis 123 when arrested with vancomycin. This precursor has a noticeably decreased affinity for vancomycin, hence contributing to its resistance. The precursor accumulated when arrested with bacitracin, was, UDPMurNAc-tetrapeptide in all strains except in E. faecalis 126. UDP-MurNAc-pentapeptides were also found in moderate amounts in most strains. The molecular masses of the peptidoglycan precursors obtained from mass spectrometry correctly identified them. This confirmed that the bacterial strains investigated were in fact resistant to the antibiotic vancomycin and this study shows that results obtained from conventional phenotypical screening methods reliably correlated with the genotypes classified using more advanced techniques such as PCR, southern blot/hybridisation and DNA sequencing. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2005.

Indiana University-Purdue University Indianapolis (IUPUI) / Sepsis is a clinical syndrome due to a systemic inflammatory response to severe microbial infection. Little is known about the changes in the bone marrow (BM) and how they affect the hematopoietic response to bacterial infection. Using an animal model of severe sepsis induced by Pseudomonas aeruginosa, we have previously reported that hematopoietic stem cells (HSC) undergo a significant expansion in the BM accompanied with myeloid suppression. This bone marrow response was Toll-like Receptor 4 (TLR4)-dependent. TLR4 is activated by bacterial lipopolysaccharide (LPS) and signals through two major independent downstream molecules: TRIF and MyD88. In the present study, I found that the TLR4/TRIF and the TLR4/MyD88 pathways contribute in a distinct manner to the BM response to P. aeruginosa's LPS. TRIF plays a major role in the expansion of the HSC pool, whereas MyD88 is required for myeloid suppression. Following LPS stimulation, HSCs enter in the cell cycle, expand and exhaust when transplanted in healthy mice. Loss of TRIF rescued completely the long-term engraftment and multilineage reconstitution potential of septic HSCs, but did not affect myeloid differentiation. Conversely, MyD88 deficiency prevented completely the myeloid suppression in the myeloid progenitors, but conferred limited protective effects on the HSC function. It is of great therapeutic value to identify the downstream molecules involved in TLR4/MyD88 dependent myeloid suppression. I found miR-21, a microRNA that is involved in inflammation, was up-regulated upon LPS challenge in a MyD88-dependent manner. However, deletion of miR-21 in the BM did not rescue LPS-induced bone marrow dysfunction, demonstrating that miR-21 is not a critical regulator in these processes. Further studies are warranted to determine the precise molecular mechanisms involved in the complex pathogenesis of BM response to sepsis. Taken together, my results show for the first time that the TLR4/TRIF signaling as a key mediator of HSC damage during acute LPS exposure and that activation of the TLR4/MyD88 signaling pathway play a dominant role in myeloid suppression. These results provide novel insights into our understanding of the molecular mechanisms underlying bone marrow injury during severe sepsis and may lead to the development of new therapeutic approaches in this disease.