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.

Thesis (Ph. D.)--University of Wyoming, 2006. / Title from PDF title page (viewed on Dec. 17, 2007). Includes bibliographical references (p. 94-118).

Thesis (M.S.)--University of Delaware, 2007. / Principal faculty advisor: Eric B. Kmiec, Dept. of Biological Sciences. Includes bibliographical references.

Thesis (Ph.D.)--University of Delaware, 2006. / Principal faculty advisor: Melinda K. Duncan, Dept. of Biological Sciences. Includes bibliographical references.

Thesis (M. Med. Sc.)--University of Hong Kong, 2006.

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