A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of requirements for the degree of Master of Science. Johannesburg, 2015. / Claudins are a family of proteins that are conserved amongst all vertebrates, they are integral in the formation and maintenance of the tight junctions between epithelial cells. Claudins are implicated in embryo morphogenesis, vertebrate evolution, solute movement, cell-cell adhesion, designation of cellular and tissue identity, and several diseases when mutated. Petromyzon marinus (the sea lamprey) is the most basal extant vertebrate and is a model organism in both developmental and evolutionary biology for this reason. In this study, the expression patterns and functions of novel claudin genes in P. marinus were examined with the aim of discovering more about the role of claudins in vertebrate evolution. Presumptive claudin genes in P. marinus were compared to all known claudins in the NCBI database. Primers were designed against all known P. marinus claudin genes and RT-PCR was performed in order to determine their expression levels at embryonic stages E8 to E18, as well as in adult eye, gill, heart, liver and skin tissues. Probes were designed against Claudin 1a, Claudin 9, Claudin 10 and Claudin 19b and RNA in situ hybridisation was performed on embryos at developmental stages E4 to E31 in order to determine their spatial expression patterns. Areas of common claudin gene expression appear to include the pharyngeal arches, otic placode, neural tube, notochord and ectoderm. Claudin 1a is uniquely expressed in the lamprey migrating neural crest. Morpholino-mediated gene knockouts were performed on P. marinus embryos and the loss of Claudin 19b appears to result in abnormal somite morphogenesis.

Thesis (Ph.D.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / Similarities between patterns of differential gene expression can be used to establish connections between the experimental and biological conditions that give rise to them. The growing volume of gene expression data in repositories such as NCBI's Gene Expression Omnibus (GEO) presents an opportunity to identify such similarities on a large scale across a diverse collection of datasets. In this work, I have developed a pattern-based approach, named openSESAME, to identify datasets enriched in samples displaying coordinate differential expression of a query signature. Importantly, openSESAME performs this search without knowledge of the experimental groups in the datasets being searched, which allows it to identify perturbations of gene expression due to attributes that may not have been recorded. First, I demonstrated the utility of openSESAME using two gene expression signatures to query a set of more than 75,000 human expression profiles obtained from GEO. A query using a signature of estradiol treatment identified experiments in which estrogen signaling was perturbed and also discriminated between estrogen receptor-positive and -negative breast cancers. A second query using a signature of silencing of the transcription factor p63 (a key regulator of epidermal differentiation) identified datasets related to stratified squamous epithelia or epidermal diseases such as melanoma. Next, to improve the utility of openSESAME, I expanded the collection of profiles to include samples from mouse and rat, and automatically translated expression signatures for cross-species queries. Furthermore, I processed the sample annotation associated with these samples in GEO, extracting informative words and phrases and continuous (e.g., age) and categorical (e.g., disease state) variables. I have also recorded sample-specific dates and quality metrics to assess whether batch effects or outliers are affecting individual query results. Finally, I used openSESAME to query this repository with over 800 gene expression signatures from the Broad Institute's Molecular Signatures Database (MSigDB). I then used the scores of the association of each signature with each sample in the repository to build a network of the relatedness of these signatures to each other. This "constellation" of signatures can be used to determine the relationship of a query signature to other biological and experimental perturbations. / 2031-01-02

Chow Hoi Yee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 127-153). / Abstracts in English and Chinese. / Abstract --- p.iii / Acknowledgments --- p.vi / Abbreviations --- p.vii / Table of contents --- p.viii / List of Figures --- p.xi / List of Tables --- p.xiv / Chapter Chapter One --- Literature Review / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- The life cycle of rice --- p.3 / Chapter 1.3 --- Physiological and molecular studies on rice leaf development --- p.6 / Chapter 1.3.1 --- Physiological study of rice leaf development --- p.6 / Chapter 1.3.1.1 --- Leaf primordium formation and SAM --- p.6 / Chapter 1.3.1.2 --- Leaf expansion and water status --- p.7 / Chapter 1.3.1.3 --- Leaf senescence and phytohormone --- p.8 / Chapter 1.3.1.4 --- Rice leaf and temperature --- p.9 / Chapter 1.3.2 --- Molecular study of rice leaf development / Chapter 1.3.2.1 --- Leaf primordium formation and SAM --- p.10 / Chapter 1.3.2.2 --- Leaf elongation and related genes --- p.13 / Chapter 1.3.2.3 --- Leaf senescence and related genes --- p.13 / Chapter 1.3.2.4 --- Photoreceptor genes --- p.14 / Chapter 1.3.2.5 --- Temperature-related genes --- p.17 / Chapter 1.4 --- Prospectus --- p.18 / Chapter Chapter Two --- Isolation of Genes Differentially Expressed During the Development of Rice by RAP-PCR / Chapter 2.1 --- Introduction --- p.20 / Chapter 2.2 --- Materials and Methods --- p.23 / Chapter 2.2.1 --- Strains and culture conditions --- p.23 / Chapter 2.2.2 --- Isolation of total RNAs --- p.23 / Chapter 2.2.3 --- cDNA Library construction --- p.24 / Chapter 2.2.3.1 --- First strand synthesis --- p.24 / Chapter 2.2.3.2 --- cDNA amplification by LD PCR --- p.25 / Chapter 2.2.3.3 --- Proteinase K digestion --- p.25 / Chapter 2.2.3.4 --- Sfi digestion --- p.26 / Chapter 2.2.3.5 --- cDNA size fractionation by CHROMA-SPIN´ёØ-400 --- p.26 / Chapter 2.2.3.6 --- Ligation of cDNA to λTripEx2vector --- p.26 / Chapter 2.2.3.7 --- Titering the unamplified library --- p.27 / Chapter 2.2.3.8 --- Determining the percentage of recombinant clones --- p.27 / Chapter 2.2.3.9 --- Library amplification --- p.28 / Chapter 2.2.3.10 --- Titering the amplified library --- p.28 / Chapter 2.2.3.11 --- Converting λ TripEx2 recombinant clones to pTripEx2 recombinant plasmids --- p.28 / Chapter 2.2.4 --- RNA fingerprinting by RAP-PCR --- p.29 / Chapter 2.2.5 --- Reverse dot-blot analysis --- p.30 / Chapter 2.2.5.1 --- Membrane preparation --- p.30 / Chapter 2.2.5.2 --- Probe preparation --- p.30 / Chapter 2.2.5.3 --- Hybridization --- p.31 / Chapter 2.2.5.4 --- Stringency washes and chemiluminescent detection --- p.31 / Chapter 2.2.6 --- Sequencing of differentially expressed genes --- p.32 / Chapter 2.2.6.1 --- Extraction of plasmid DNA --- p.32 / Chapter 2.2.6.2 --- DNA cycle sequencing --- p.33 / Chapter 2.3 --- Results --- p.34 / Chapter 2.3.1 --- Total RNA isolation --- p.34 / Chapter 2.3.2 --- cDNA library --- p.37 / Chapter 2.3.3 --- RNA fingerprinting --- p.40 / Chapter 2.3.4 --- Reverse dot-blot analysis --- p.45 / Chapter 2.3.5 --- Sequence analyses --- p.48 / Chapter 2.4 --- Discussion --- p.68 / Chapter Chapter Three --- cDNA Microarray Analysis and expression Pattern Analysis by Northern Blot Hybridization / Chapter 3.1 --- Introduction --- p.71 / Chapter 3.2 --- Materials and Methods --- p.76 / Chapter 3.2.1 --- RNA extraction by RNeasy® Mini Kit for cDNA microarray --- p.76 / Chapter 3.2.2 --- Microarray analysis --- p.76 / Chapter 3.2.2.1 --- Array preparation --- p.76 / Chapter 3.2.2.2 --- Probe preparation --- p.78 / Chapter 3.2.2.3 --- Hybridization --- p.79 / Chapter 3.2.2.4 --- Stringency washes and TSA Detection --- p.79 / Chapter 3.2.2.5 --- Microarray Analyses --- p.80 / Chapter 3.2.3 --- RNA extraction by Tri-reagent for Northern Blot hybridization --- p.81 / Chapter 3.2.4 --- RNA fragmentation by formaldehyde gel electrophoresis --- p.81 / Chapter 3.2.5 --- Northern blotting --- p.82 / Chapter 3.2.6 --- Preparation of probe --- p.83 / Chapter 3.2.7 --- Hybridization and stringency washes --- p.84 / Chapter 3.2.8 --- Stripping and reprobing of Northern Blot --- p.85 / Chapter 3.3 --- Results --- p.86 / Chapter 3.3.1 --- Total RNA isolation --- p.86 / Chapter 3.3.2 --- Microarray analysis --- p.86 / Chapter 3.3.3 --- Normalization of microarray data --- p.91 / Chapter 3.3.4 --- Detection of probe labeling efficiency --- p.92 / Chapter 3.3.5 --- Northern blot analysis --- p.100 / Chapter 3.3.5.1 --- Genes expressed highly in primordia stage --- p.100 / Chapter 3.3.5.2 --- Genes with low expression in primordia stage --- p.101 / Chapter 3.3.5.3 --- Genes with high expression in half expanded stage --- p.101 / Chapter 3.3.5.4 --- Genes steadily increased in expression throughout the development --- p.103 / Chapter 3.3.5.5 --- Genes with low expression in fully expanded stage --- p.104 / Chapter 3.4 --- Discussion --- p.114 / Chapter Chapter four --- General Discussion --- p.122 / References --- p.127

Mak Wai Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 95-115). / Abstracts in English and Chinese. / Declaration --- p.i / Abstract --- p.ii / Acknowledgements --- p.viii / Table of Contents --- p.ix / List of Tables --- p.xiii / List of Figures --- p.xiv / List of Abbreviations --- p.xvi / Chapter Chapter 1 --- Introduction and Literature Review / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- Endocrine control of reproduction in crustaceans --- p.2 / Chapter 1.3 --- The X-organ sinus gland (XOSG) complex --- p.3 / Chapter 1.3.1 --- CHH/MIH/GIH family of neuropeptides --- p.4 / Chapter 1.3.2 --- Gonad inhibiting hormone (GIH) --- p.6 / Chapter 1.3.3 --- Molt inhibiting hormone (MIH) --- p.7 / Chapter 1.3.4 --- Crustaceans hyperglycemic hormone (CHH) --- p.9 / Chapter 1.3.5 --- The chormatophorotropins (RPCH and PDH neuropeptide family) --- p.10 / Chapter 1.4 --- Other mechanisms of reproduction control --- p.11 / Chapter 1.4.1 --- Methyl farnesoate (MF) and mandibular organ inhibiting hormone (MOIH) --- p.11 / Chapter 1.4.2 --- Gonad stimulating hormone (GSH) --- p.13 / Chapter 1.4.3 --- Serotonin (5HT) --- p.13 / Chapter 1.4.4 --- Dopamine --- p.14 / Chapter 1.4.5 --- Enkephalin (ENK) --- p.15 / Chapter 1.4.6 --- 17β-Estradiol --- p.16 / Chapter 1.4.7 --- Progesterone --- p.17 / Chapter 1.5 --- Androgenic hormone (AH) --- p.18 / Chapter 1.6 --- Objective and methodology of present research --- p.19 / Chapter 1.7 --- Reproductive biology of the shrimp Metapenaeus ensis --- p.20 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Introduction --- p.26 / Chapter 2.2 --- Extraction of eyestalk RNA from the shrimp --- p.27 / Chapter 2.3 --- Construction of the eyestalk cDNA library --- p.28 / Chapter 2.3.1 --- First-strand cDNA synthesis --- p.28 / Chapter 2.3.2 --- cDNA amplification by LD PCR --- p.29 / Chapter 2.3.3 --- Proteinase K digestion --- p.29 / Chapter 2.3.4 --- Sfi I digestion --- p.30 / Chapter 2.3.5 --- cDNA size fractionation --- p.30 / Chapter 2.3.6 --- Ligation of cDNA to λ TriplEx2 Vector --- p.31 / Chapter 2.3.7 --- Packaging --- p.31 / Chapter 2.3.8 --- Titering the unamplified library --- p.32 / Chapter 2.3.9 --- Library amplification --- p.33 / Chapter 2.3.10 --- Titering the amplified library --- p.34 / Chapter 2.4 --- Mass Excision of the eyestalk cDNA library --- p.34 / Chapter 2.5 --- PCR screening of inserted sequence --- p.35 / Chapter 2.6 --- RNA arbitrarily primed polymerase chain reaction (RAP-PCR) --- p.35 / Chapter 2.7 --- Dot blot hybridization --- p.37 / Chapter 2.7.1 --- Probe Labelling --- p.37 / Chapter 2.7.2 --- Dotting of membrane --- p.38 / Chapter 2.7.3 --- Hybridization --- p.38 / Chapter 2.7.4 --- Washing and chemiluminescent detection --- p.39 / Chapter 2.7.5 --- Probe stripping for re-hybridization --- p.40 / Chapter 2.8 --- Sequencing --- p.40 / Chapter 2.9 --- BLAST search --- p.41 / Chapter 2.10 --- Northern blot analysis --- p.41 / Chapter 2.10.1 --- Probe labelling --- p.41 / Chapter 2.10.2 --- RNA formaldehyde denaturing gel electrophoresis --- p.41 / Chapter 2.10.3 --- Northern blot --- p.42 / Chapter 2.10.4 --- Pre-hybridization --- p.43 / Chapter 2.10.5 --- Hybridization --- p.43 / Chapter 2.10.6 --- Washing and chemiluminescent detection --- p.43 / Chapter 2.11 --- Real-time RT-PCR --- p.44 / Chapter 2.11.1 --- DNaseI treatment --- p.44 / Chapter 2.11.2 --- First strand synthesis --- p.44 / Chapter 2.11.3 --- Primer design and verification --- p.45 / Chapter 2.11.4 --- Real-time PCR --- p.45 / Chapter 2.11.5 --- Statistical analysis --- p.45 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Experimental animals --- p.50 / Chapter 3.2 --- Total RNA extraction --- p.50 / Chapter 3.3 --- Library construction --- p.50 / Chapter 3.4 --- PCR screening of inserted sequences --- p.50 / Chapter 3.5 --- RNA arbitrarily primed polymerase chain reaction (RAP-PCR) --- p.51 / Chapter 3.6 --- Dot blot hybridization --- p.51 / Chapter 3.7 --- Sequencing and BLAST search --- p.52 / Chapter 3.8 --- Northern blot analysis --- p.53 / Chapter 3.8.1 --- Housekeeping gene - elongation factor la --- p.53 / Chapter 3.8.2 --- Insulin-like growth factor binding protein (IGFBP) --- p.54 / Chapter 3.8.3 --- Arrestin --- p.54 / Chapter 3.8.4 --- Opsin --- p.54 / Chapter 3.9 --- Real-time RT-PCR --- p.55 / Chapter 3.9.1 --- β-Actin --- p.55 / Chapter 3.9.2 --- Farnesoic acid O-methyltranferase (FAMeT) --- p.56 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Application of RAP-PCR and dot blot analysis in identifying differential expressed genes --- p.79 / Chapter 4.1.1 --- Abundant genes --- p.79 / Chapter 4.1.2 --- Appearance of 16S rRNA in eyestalk cDNA library of M. ensis --- p.80 / Chapter 4.1.3 --- False positive --- p.80 / Chapter 4.2 --- Investigation on common housekeeping genes --- p.81 / Chapter 4.3 --- Potential functions of identified differential expressed genes in reproduction of shrimp --- p.82 / Chapter 4.3.1 --- Arrestin --- p.84 / Chapter 4.3.2 --- Opsin --- p.86 / Chapter 4.3.3 --- Insulin-like growth factor binding protein (IGFBP) --- p.88 / Chapter 4.3.4 --- β-Actin --- p.89 / Chapter 4.4 --- Investigation for farnesoic acid O-methyltransferase (FAMeT) in shrimp --- p.89 / Chapter Chapter 5 --- General Conclusion --- p.91 / References --- p.95 / Appendix --- p.116

Post-transcriptional control of gene expression is critical for normal cellular function and viability. Poly(A)-binding protein (PABP) 1 is the prototypical member of a family of RNA-binding proteins which are key post-transcriptional regulators. PABP1 is multifunctional, acting as a primary determinant of translation efficiency and mRNA stability, regulating the fate of specific mRNAs, and participating in microRNAmediated regulation and nonsense-mediated mRNA decay. As well as binding various mRNAs, PABP1 achieves its multifunctionality through protein-protein interactions with numerous PABP-interacting motif (PAM)-2 motif-containing protein partners. These have been identified to bind the same site within the C-terminal PABC domain, therefore it is unclear how different PABP1 functions are coordinated. Recently, PABP1 was found to exhibit extensive post-translational modification (PTM), including putative lysine acetylation/methylation switches, which were suggested as a potential mechanism by which interactions with different PAM2 motifcontaining proteins may be regulated. In particular, in silico molecular modelling of the acetylation or dimethylation of the position 606 lysine residue (Lys606) within the PABC domain, using available structures of PABC in complex with PAM2 peptides of eukaryotic release factor (eRF)-3a and PABP-interacting protein (Paip)-2, suggested that modification of this residue, which is critical in PABC-PAM2 interactions, may differentially affect these PABP1 interactions. To examine the role of the Lys606 modification as a molecular switch to dictate PABC-mediated protein-protein interactions, site-specifically acetylated recombinant PABC domain was generated using cutting–edge amber codon suppression recoding technology. Following sequential purification by affinity, ion exchange and size exclusion chromatography, recombinant PABC protein quality was analysed by biophysical approaches such as thermal denaturation assay (TDA), dynamic light scattering (DLS), circular dichroism (CD) and liquid chromatography mass spectrometry (LCMS). Biochemical and biophysical analysis of PABC-PAM2 interactions was subsequently undertaken using GST-pulldown analysis, with the well characterised Paip2 protein, and Surface Plasmon Resonance (SPR) using PAM2 peptides of eRF3, Paip2 and trinucleotide repeat-containing (Tnrc) 6C (or GW182) proteins. These revealed that PABC Lys606 acetylation significantly increased the affinity and increased the association rate for eRF3 peptide. In contrast, effects on Paip2 peptide binding were less suggestive. Furthermore, although approaches to decipher the biological relevance of Lys606 and its modifications within cells are in their infancy, they reflect the complexity of studying PTM function in vitro. Overall, these data provide support for the hypothesis that Lys606 modification status confers selectivity between PABP1 protein partners suggesting a potential mechanism for how its multi-functionality may be coordinated.

The successive activation of tissue-specific genes during cellular differentiation is orchestrated by the formation of transcriptional complexes consisting of cellspecific and ubiquitous transcription factors. Understanding the molecular events associated with normal megakaryocyte (Mk) differentiation is an issue of central importance to haematology. The aims of this study were therefore to: (i) define the transcription factors responsible for regulating the expression of Mkspecific genes such as Glycoprotein IX, (ii) identify the protein partners of such important Mk-regulatory transcription factors and (iii) examine the mechanisms utilised by these factors to regulate gene expression. First, the regulatory elements in the GPIX promoter required for basal and inducible expression were examined in megakaryoblastic Dami cells stimulated to undergo differentiation. The resulting data suggested that an Ets site in the GPIX promoter binding the Ets-family member Fli-1 was crucial in regulating both constitutive and inducible GPIX expression. Second, a two-hybrid screen of a K-562 cDNA library was used to identify transcription factors that interacted with Fli-1 and were potential regulators of Mk development. Results of this screen identified a novel protein-protein interaction with GATA-1, a previously well-characterised zinc finger transcription factor also implicated in erythroid and Mk development. Mapping of the domains required for the interaction show that the zinc fingers of GATA-1 interact with the Ets domain of Fli-1. The biological significance of the Fli-1/GATA-1 interaction was demonstrated in transient transfection assays, which resulted in synergistic activation of Mkspecific promoters. Analysis of Fli-1 and GATA-1 expression in a series of erythroleukaemic and megakaryoblastic cell lines demonstrated that the Fli- 1/GATA-1 combination correlates with a Mk-phenotype. Moreover, expression of Fli-1 in K-562 cells (a line rich in GATA-1 but normally lacking Fli-1) induces endogenous GPIX expression. Quantitative mobility shift assays reveal that Fli- 1 and GATA-1 exhibit cooperative DNA-binding in which the binding of GATA-1 to DNA is increased approximately 26 fold in the presence of Fli-1. This data provides a mechanism for the observed transcriptional synergy. In conclusion, this work suggests that Fli-1 and GATA-1 work together through protein-protein interaction and cooperative DNA-binding to activate the expression of genes associated with the terminal differentiation of Mks.

The advent of microarray technology, allowing measurement of gene expression levels for thousands of genes in parallel, has made possible experiments designed to investigate the genetic control of variation in gene expression level (described in the literature as ???genetical genomics??? or ???eQTL??? experiments). Published results from these studies, in yeast and in mice, show that genetic variation is an important factor in gene regulation, and furthermore that individual polymorphisms modify the expression level of many genes. The concern of this thesis is the bioinformatic analyses of the expression level and genotype data sets that are the raw material for these studies. In particular this thesis addresses the two issues of detection of artefactual effects, and maximizing the information that can be extracted from the data. It is shown that while a polymorphism affecting the expression of many genes may be readily detected, care must be taken to determine whether the detected effect is genuinely one of genetic control of expression level, rather than the effect of correlations in measured expression level not of genetic cause. A significance test is devised to distinguish between these cases. The detection of artefactual correlation is explored further in the reanalysis of the published data from a large yeast study. A critique is given of the permutation method used to ascribe genetic control as the cause of inter gene expression level correlation. The presence of some degree of artefactual correlation is shown, and novel methods are presented for identifying such artefacts. To extend the analyses that may be applied to eQTL data, an algorithm is presented for determining secondary eQTLs for gene expression level (as opposed to a single primary QTL), along with a significance test for the putative QTL found. The technique is demonstrated on a large public data set. In addition to the use for which they are intended, the data sets generated for eQTL studies provide opportunities for additional analyses. In this thesis a method is developed for calculating a genome wide map of meiotic recombination frequency from the genotype data for multiple segregant strains. The method is demonstrated on the published genotype data generated for a large yeast eQTL study.

T cells are at the heart of the adaptive immune response. They mediate many important immunological processes that provide protection against viruses, bacteria and other pathogens. The aim of the work described in this thesis was to use gene expression profiling to gain insights into different aspects of T cell biology. In particular we wanted to examine the mechanisms and identify the genes that underlie T cell effector function. IFN-g-producing Th1 cells are a major effector subset that protects against intracellular pathogens, while Th2 cells produce IL-4, IL-5 and IL-13 and mediate protection against large extracellular pathogens. Microarray profiling of gene expression in mouse and human Th1 and Th2 cells, as well as mouse Tc1 and Tc2 cells, identified a number of novel markers of these T cells which may have important roles in T cell differentiation/function. We found that T cell type, host species and differentiation conditions significantly influenced gene expression profiles generated during T cell polarization. Providing help to B cells for antibody production is the major function of the third effector subset of CD4+ T cells termed T follicular homing or TFH cells. Relatively little is known about the generation of these cells, and the mechanisms of their effector function. Using oligonucleotide microarrays we identified a TFH-specific gene expression signature, which included many novel genes which will undoubtedly enable better identification and characterization of this novel subset. A comprehensive study profiling all the major leukocyte subsets revealed their distinct gene expression signatures and numerous leukocyte subset specific genes. A detailed examination of most major T cell subsets identified distinguishing features of each subset together with gene expression changes associated with T cell activation and exposure to cell culture conditions. In addition, we described a distinctive transcriptional profile for gd T cells and examined the differences between central and effector memory T cells. We also showed that specific gene expression signatures provide a powerful tool for subset classification. Taken together this work provides important insights into T cell differentiation and effector function, and presents a basis for future work examining numerous novel genes relevant to T cell biology.

Streptococcus pneumoniae is a nasopharyngeal commensal in most healthy individuals. However, it can translocate from this niche to deeper tissues, causing diseases such as otitis media, meningitis, sepsis and pneumonia, which are responsible for significant morbidity and mortality worldwide. At the commencement of this work, inherent difficulties in harvesting sufficient bacterial numbers from experimental animals restricted the examination of pneumococcal gene expression during pathogenesis, and thus virulence gene transcription patterns were largely unknown outside of an in vitro environment. This thesis aimed to investigate such transcriptional patterns in vivo, and to hence gain a better understanding of pneumococcal behaviour during colonisation and disease. This work describes refinement of an intranasal S. pneumoniae infection model in CD-1 mice that enables pneumococci to be harvested from multiple niches with low contamination by nasopharyngeal microflora or host tissue, and minimal crosscontamination with circulating pneumococci in the vascular system. The challenge route simulates the acquisition of S. pneumoniae in the human population, and progression to IPD occurs naturally. RNA extraction, enrichment and linear amplification procedures were optimised so that RNA could be obtained from in vivo site in sufficient quantities and with sufficient integrity to be used in semi-quantitative assays. Linear amplification allowed the examination of gene expression in niches where low bacterial numbers had previously prevented such analyses. Real-time RT-PCR and microarray analyses were used to examine bacterial RNA samples recovered from the nasopharynx, lungs, blood and brains of CD-1 mice, providing the first comparative transcriptional data for pneumococci during carriage and disease, within the same animal model. Two pneumococcal serotypes were examined; a type 2 (D39) and a type 6A (WCH16) strain. CbpA, Ply, and SpxB were shown to be important for carriage in both strains, with pneumococci up-regulating the expression of the genes encoding these virulence proteins in the nasopharynx. This provides in vivo evidence supporting the ascribed roles of these proteins in reducing the level of competing microflora and promoting nasopharyngeal adherence. Similarly, D39 nanA and pspA transcription levels were up-regulated in the nasopharynx. The level of pspA mRNA was also higher in the blood than the lungs, suggesting an increased requirement in the bloodstream, where PspA is involved in reducing complement-mediated opsonisation. Despite the antiphagocytic role of the pneumococcal polysaccharide capsule in the bloodstream, D39 cpsA mRNA was present in similar quantities in the nasopharynx, lungs and blood, which may support previous studies indicating post-transcriptional regulation of capsule expression. However, cpsA expression was up-regulated in the blood for WCH16. These results may indicate the existence of strain-specific differences in virulence gene regulation. Microarray analysis of in vivo-harvested S. pneumoniae D39 found that mRNAs encoding components of phosphotransferase systems, CbpA, a putative neuraminidase, and v-type sodium ATP synthase subunits were significantly higher in bacteria involved in carriage than bacteraemia. Conversely, the expression of genes involved in competence, and dinF (present on a competence-induced operon), were up-regulated in the blood compared to the nasopharynx, providing evidence that competence is induced during bacteraemia. Pneumococci also showed increased expression of genes involved in fatty acid metabolism, pgdA, lytB and cbpG in the blood compared to the nasopharynx. This study used a single pneumococcal strain and infection model and, therefore, overcomes inherent issues of serotype/strain- and animal model- specific gene expression that may have complicated interpretation of data in previous studies. This thesis reports some of the first in vivo pneumococcal gene expression data gained using a single animal model and pneumococcal strain. The data reinforce the putative roles of several virulence factors, and provides novel transcription data for pneumococci during carriage. Results suggest the existence of core genes that are essential for infection in multiple pneumococcal serotypes, whereas other genes appear to have strain-specific roles. / http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1287056 / Thesis (Ph.D.)-- University of Adelaide, School of Molecular and Biomedical Science, 2007

Thesis (Ph. D.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.