Changes in the epigenetic landscape are widespread in neoplasia, with de novo methylation and histone repressive marks commonly occurring in association with gene silencing. However, understanding the dynamics of epigenetic changes is often hindered due to the absence of adequate in vitro model systems that accurately reflect events occurring in vivo. Human mammary epithelial cells (HMECs) grown under standard culture conditions enter a growth arrest termed selection, but a subpopulation is able to escape from arrest and continue to proliferate. These cells, called post-selection cells, have many of the hallmarks seen in the earliest lesions of breast cancer, including transcriptional silencing and hypermethylation of the p16INK4A tumour suppressor gene. The overall aim of my thesis was to use post-selection HMECs as model system to identify and dissect the mechanism involved in early epigenetic aberrations. Firstly, using a microarray approach, I found that multiple members of the TGF-β signalling pathway were concordantly suppressed in post-selection cells, and this was associated with functional disruption of the TGF-β pathway. Interestingly, concordant gene suppression was not associated with aberrant DNA methylation, but with repressive chromatin remodelling. Secondly, to further understand the mechanism underpinning epigenetic silencing, I demonstrated using laser capture technology, that p16INK4A silencing is a precursor to DNA methylation and histone remodelling. Thirdly, I found that individual post-selection HMEC strains during the early passages shared a common 'wave' pattern of regional-specific methylation within the p16INK4A CpG island. Interestingly, the 'wave' pattern of early de novo methylation correlated with the apparent footprint of nucleosomes within the p16INK4A CpG island. Lastly, to further characterise the properties of the HMEC culture system, I demonstrated that post-selection cells do not possess a natural tumour-inducing property when transplanted into the mammary fat pad of immunocompromised mice. However, post-selection HMECs were associated with high expression of a variety of stem/progenitor markers, as well as stem/progenitor associated polycomb genes, thus demonstrating that these cells share some common features of stem/progenitor cells. The research presented in this thesis demonstrate that epigenetic changes occur early in the growth of post-selection HMECs and many of these changes are common in breast cancer.

An in vitro model of the follicle-associated epithelia that overlie the Peyer's patches of the small intestine was developed and validated to examine the mechanisms of mucosal antigen sampling. This model displays many phenotypic and physiological characteristics of M cells including apical expression of [alpha]5[beta]l integrin and enhanced energy dependent participate transport. CD4+ T-cells were shown to be an important influence on the development of Mlike cells. The model was used to examine the M cell mediated uptake of several putative whole-cell killed bacterial vaccines. Greater numbers of non-typeable Haemophilus influenzae NTHi 289, NTHi 2019, Escherichia coli 075 HMN and Streptococcus pneumoniae were transported by model M cells compared to control Caco-2 enterocyte-like cells. Studies in isolated murine intestine segments confirmed the selective uptake of NTHi 289 and Escherichia coli demonstrating that intestinal mucosal sampling of these antigens is performed by M cells. Pseudomonas aeruginosa was not absorbed as whole cell bacteria but as soluble antigen, as indicated by the presence of bacterial DNA in the cytoplasm of epithelial cells. These results suggest that bacteria such as NTHi and E. coli are sampled by the mucosal immune system in a different manner to that of bacteria such as Pseudomonas. A number of potential cell surface receptors were investigated to identify which molecules are responsible for intestinal uptake whole-cell killed bacteria. Immunofluorescence studies detected the presence of toll-like receptor-2, toll-like receptor-4, PAF-R and [alpha]5[beta]l integrin on in vitro M-like cell cultures. Examinations of murine intestine confirmed the presence of TLR-4 and PAF-R. TLR-4 was found in small quantities and on M cells. In contrast to the M cell model, TLR-2 expression in the murine intestine was sparse. Receptor inhibition experiments provided evidence for the involvement of TLR-4, PAF-R and [alpha]5[beta]l integrin in M cell uptake of killed bacteria both in vitro and in vivo. This thesis has contributed valuable information regarding the mechanisms of uptake of whole-cell killed bacteria by the intestinal mucosal immune system. For the first time, M cell sampling of whole-cell killed bacteria has been demonstrated. Furthermore, the receptors involved in these processes have been identified. This information will be of great use in the development and optimisation of new oral vaccines.

Since Shiga-toxigenic Escherichia coli (STEC) strains are not considered to be enteroinvasive, the mechanism(s) by which Shiga toxin (Stx) gains access to the circulation and to target tissues expressing its target receptor Gb3 is crucial to the disease process. There is increasing evidence that by facilitating translocation of Stx across the intestinal epithelium and by transporting bound toxin to remote sites such as the renal endothelium, polymorphonuclear leucocytes (PMNs) play a key role in the pathogenesis of serious STEC disease. Plasma levels of PMN-attracting CXC chemokines such as IL-8 also appear to correlate in humans with the severity of disease. Thus, the capacity of STEC strains to elicit CXC chemokine responses in intestinal epithelial cells may be a crucial step in pathogenesis. In order to determine which STEC factor(s) are responsible for the induction of CXC chemokine responses by intestinal epithelial (HCT-8) cells, a real-time reverse transcription PCR assay was developed to quantitatively measure relative expression of chemokine mRNA for IL-8, ENA-78, GCP-2, MGSA, MIP-2α and MIP-2β. Similarly, a commercially available sandwich ELISA was used to measure levels of IL-8 protein secreted by HCT-8 cells in response to infection with STEC. When HCT-8 cells were infected with the wellcharacterised locus of enterocyte effacement (LEE)-negative O113:H21 strain 98NK2 or the LEE-positive STEC strain EDL933, there were significant differences in the levels of chemokine mRNA and IL-8 protein expression. In particular, the LEE-negative strain 98NK2 induced significantly higher and earlier levels of chemokine mRNAs, including IL-8, MIP-2α and MIP-2β at 1 and 4 h, and ENA-78 at 4 h. However, EDL933 elicited no significant upregulation of any of the chemokine mRNAs at 1 h, and only modest increases in IL-8, MIP-2α and MIP-2β by 4 h, post-infection. These results were confirmed by IL-8 ELISA which showed that 98NK2 elicited significant levels of IL-8 protein by 2 h post-infection, and remained high until 4 h post-infection. In comparison, EDL933 did not elicit significant IL-8 induction over that of control cells, even at 4 h post-infection. When a range of STEC isolates from clinical samples were tested for their capacity to induce chemokine production in HCT-8 cells, highly significant differences were observed between the strains. Infection of HCT-8 cells with a range of LEE-negative STEC strains isolated from patients with severe STEC disease resulted in significantly higher and earlier upregulation of IL-8 and MIP-2α mRNA than that elicited by several LEE-positive STEC strains. Similarly, levels of IL-8 protein in LEE-negative STEC-infected HCT-8 culture supernatants were significantly higher than in LEE-positive STEC-infected culture supernatants. Only one LEE-positive strain, an O26 strain 95ZG1, was capable of inducing chemokine responses comparable to that induced by infection with the LEE-negative STEC strains. These results were also shown not to be attributable to differences in the adherence, initial doses or growth of the strains during the assay, or to a loss of viability of the HCT-8 cells. These results, therefore, suggest that there may be interesting differences in the ability of STEC strains to induce chemokine production in intestinal epithelial cells. The factor(s) that contribute to chemokine induction by epithelial cells in response to STEC were then examined. The difference in responses could not be attributed to the expression or non-expression of LEE genes, the presence or absence of an STEC megaplasmid or to differences in O serogroup. Although purified Stx1 and Stx2 were able to induce IL-8 and MIP-2α mRNA, and IL-8 protein, the levels of chemokine induction in response to wild-type STEC did not correlate with the type or amount of Stx produced by these strains in vitro. Similarly, deletion of the single stx2 gene from 98NK2 had no significant effect on chemokine induction compared to wild-type 98NK2-infected HCT-8 cells. Interestingly, several of the LEE-negative STEC strains eliciting the strongest chemokine responses belonged to flagellar serotype H21. Incubation of HCT-8 cells with purified H21 flagella elicited IL-8 and MIP-2α mRNA responses similar to those seen in the presence of the most potent LEE-negative STEC strains. Deletion of the fliC gene largely abolished the capacity of 98NK2 to elicit IL-8 and MIP-2α mRNA and IL-8 protein responses in HCT-8 cells. Similarly, deletion of both stx2 and fliC from 98NK2 elicited a response similar to that observed with deletion of fliC alone. Flagella were then purified from the high chemokine-inducing STEC strains 95HE4 (O91:H7) and 95ZG1 (O26:H11). Purified H7 and H11 flagella were similarly able to induce both IL-8 and MIP-2α mRNA, and IL-8 protein, in HCT-8 cells at levels similar to their respective wild-type strains. Deletion of fliC from two other STEC strains, 97MW1 (O113:H21) and 86-24 (O157:H7), confirmed that flagellin was responsible for the majority of chemokine responses in these wild-type strains. However, an inability of EDL933 to induce these responses was unexpected and later found to be due to a lack of expression of H7 flagella by this strain. Purified H21 FliC (His6-FliC) alone was able to induce chemokine production (including IL-8, MIP-2α and MIP-2β at 1 and 4 h, and ENA-78 at 4 h) by HCT-8 cells at similar levels to that observed for 98NK2. Taken together, these data suggest that although Stx is capable of inducing CXC chemokine responses, the elevated responses observed in cells infected with certain STEC strains are largely attributable to the production of flagellin. Purified His6-H21 flagellin was also able to induce p38 MAPK activation in vitro and IL-8 and MIP-2α mRNA were superinduced in the presence of both Stx2 and H21 flagellin. Blockade of the p38 pathway with SB203580 resulted in a down-regulation of IL-8 protein levels (by up to 61%) in response to H21 flagellin, but not IL-8 mRNA, suggesting that this inhibition may occur post-transcriptionally. Blocking the ERK and JNK pathways similarly decreased IL-8 secretion in response to H21 flagellin, suggesting that all three MAPK pathways are involved in this response. Indeed, concurrent inhibition of all three pathways resulted in virtually complete inhibition of IL-8 protein production (98%). Transfected HeLa and MDCK cells stably expressing TLR5 activated p38 in the presence of purified H21 flagellin, whereas dominant-negative (DN) TLR5-expressing cells did not, supporting previous studies that show that flagellin acts via TLR5. These data suggest that TLR5 and the p38, ERK and JNK MAPK pathways all play an important role in the response of intestinal epithelial cells to H21 flagellin from STEC, and that the combined effects of Stx and flagellin on host intestinal epithelial cells may result in an augmented inflammatory response. A role for flagellin in virulence was then investigated. BALB/c mice were orally inoculated with wild-type 98NK2 or 98NK2ΔfliC. Of the 16 mice challenged with the wildtype strain 98NK2, 9 (56%) died during the experiment (median survival time 7.6 days). However, only 3 of 16 mice (19%) challenged with 98NK2ΔfliC died (median survival time > 14 days). The difference in survival rate was statistically significant. No significant differences in the level of intestinal colonisation of 98NK2 or 98NK2ΔfliC were observed. Thus, flagellin directly contributes to the virulence of STEC in streptomycin-treated mice. Since the streptomycin-treated mouse is a model for systemic Stx-mediated pathology, these results suggest that the pro-inflammatory effects of flagellin play an important role in the pathogenesis of Stx-mediated STEC disease in vivo. / Thesis (Ph.D.)--School of Molecular and Biomedical Science, 2004.

Thesis (Ph. D.)--University of Missouri-Columbia, 2007. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on December 12, 2007) Vita. Includes bibliographical references.

Thesis (Ph. D.)--Ohio State University, 2006. / Title from first page of PDF file. Includes bibliographical references (p. 138-153).

Early innate response of intestinal epithelial cells is the first line defense against enteric pathogens. Toxoplasma gondii infections acquired naturally via the peroral route, encounter intestinal epithelial cells early post-infection. Although the population structure of T. gondii is known to be highly clonal, clinical strains of T. gondii have been classified into three genotypes based on their virulence. In this study we investigated whether human intestinal epithelial cell immune response to T. gondii is virulence dependent. We demonstrated distinct virulence of the three T. gondii genotype strains evaluated in human intestinal epithelial cells by their capacity to replicate and induce host cell cytotoxicity. The early host innate mechanisms such as activation of signaling pathways and induction of innate effectors were likewise differentially elicited by the three T. gondii strains. Low levels of TLR dependent NF-kB activation and a failure to rapidly up-regulate innate cytokine and chemokine genes was observed after virulent Type I strain infection. In contrast, early innate response to the less virulent Type II strain was rapid, efficient and led to high levels of IL-8 and IL-6 secretion, whereas response to Type III parasites was intermediate. Early expression of b-defensin 2 gene was suppressed specifically by virulent Type I strain and its activation prior to infection in intestinal epithelial cells led to decreased parasite viability. These findings provide evidence for T. gondii strain virulence dependent down-modulation of early human intestinal epithelial cell innate responses and highlight the importance of these cells in host defense against this infection.

This thesis aimed to investigate the role of alveolar epithelial cells (AEC) on immune responses against mycobacterial infections, specifically, the role of AEC in modulating macrophage functions through the secretion of broad variety of factors. In paper I, we compared murine AEC with interstitial macrophages (PuM) in their ability to take up and control mycobacterial growth and their capacity as antigen-presenting cells. We found that AEC were able to internalize and control bacterial growth and present antigens to T cells from immunized mice. In addition, both AEC and PuM exhibited distinct patterns of secreted factors, and a more comprehensive profile of AEC responses revealed that AEC were able to secrete different factors important to generate various effects in other cells. Paper II: Since AEC secrete a broad variety of factors, we hypothesized that being in the interface; AEC may play an important role in transmitting signals from the external to the internal compartment and in modulating the activity of PuM. Thus, we prepared AEC-derived media and tested their effect on bacteria and a number of macrophage functions a) migration, b) phagocytosis and control of intracellular bacterial growth, and c) alteration in cell morphology and expression of surface markers. We found that AEC-secreted factors had a dual effect, in one hand controlling bacterial growth and on the other hand increasing macrophage activity. In paper III, we first investigated the responsible mechanisms of intracellular bacterial growth control mediated by AEC-derived media. We found that infected macrophages upon AEC-secreted factors increased the control of intracellular bacterial growth by iNOS-independent pathways. Compared with other macrophage types, PuM, did not control the intracellular bacterial growth upon the well-known potent macrophage activator, IFN-γ. We found that SOCS1 was involved in the un-responsiveness to IFN-γ by PuM to control the intracellular bacterial growth. We suggested that PuM are restricted in their inflammatory responses perhaps for avoiding tissue damage. Overall, the current findings highlight the importance of AEC in the defense against bacterial infection in the lungs by secreting factors involved in activation and differentiation of immune cells such as macrophages. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Manuscript.</p>

Leukocyte transmigration is a critical step of the inflammatory process. In this project I have examined leukocyte responses to transmigration and endothelial responses to both chemical and mechanical stimuli which are known to be involved in leukocyte transmigration. My work has identified ~2500 differentially expressed genes following endothelial exposure to interleukin-1 beta (IL1β). Interestingly, IL1β induces up-regulation of claudin-1 and pre-b-cell colony enhancing factor and down-regulation of claudin-5 and occludin, which are all involved in maintaining endothelial cell-cell junctions. Analysis of endothelial cell (EC) transcriptional changes following neutrophil transmigration found few differentially expressed genes in comparison to IL1β treated ECs; indicating that the effects of transmigration on ECs are minimal in comparison to the global transcriptional changes induced by IL1β. Atherosclerosis, characterized by monocyte accumulation within the vessel lumen, is found in regions of flow reversal and low time averaged oscillatory shear stress. I have examined the effects of this type of shear stress on endothelial cell gene expression. My data indicates that most genes differentially expressed under these conditions are controlled by low average shear stress rather than flow reversal. These differentially expressed genes are involved in regulating the cell cycle and the immune response. My work shows that cell proliferation is increased following exposure to low steady shear stress or exposure to reversing oscillatory flow in comparison to high steady shear stress. Additionally monocyte adhesion is increased following exposure of ECs to reversing oscillatory flow. My work has also examined the impact of transmigration on monocyte gene expression. I have identified genes which are differentially expressed in monocytes by exposure to EC secretions, monocyte/EC contact, and diapedesis. I have also shown that freshly isolated human monocytes have reduced apoptosis following transmigration. Surprisingly, I also found that monocytes had reduced expression of anti-microbial peptides following transmigration. Overall my work identifies important endothelial and leukocyte transcriptional responses to the process of transmigration which extends from cytokine stimulation through diapedesis.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2007. / Title from PDF title page (viewed on Feb. 19, 2010). Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 117-126).