Amendments page is pasted onto the front end paper. / Includes bibliographical references (28 leaves). / 1v. : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Shows that the NKT cells and the thymus-independent ?ga?s/?gb?s T cells present in athymic rats are phenotypically and functionally related. Raises the possibility that thymus-independent ?ga?s/?gb?s T cells are distinct from conventional T cells and that their functions in normal individuals are regulatory, as has been suggested for NKT cells. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1999

Amendments page is pasted onto the front end paper. / Includes bibliographical references (28 leaves). / 1v. : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Shows that the NKT cells and the thymus-independent ?ga?s/?gb?s T cells present in athymic rats are phenotypically and functionally related. Raises the possibility that thymus-independent ?ga?s/?gb?s T cells are distinct from conventional T cells and that their functions in normal individuals are regulatory, as has been suggested for NKT cells. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1999

Murine embryonic stem cells (ESCs) are self-renewing, pluripotent cells able to differentiate into cells of all three germ layers. Pluripotency and self-renewal are maintained primarily by the core transcriptional factors Nanog, Oct4 and Sox2, but require the cooperation of other factors and coregulators and an efficient telomere maintenance mechanism. In mammals, telomere maintenance is achieved via a telomerase reverse transcriptase (Tert) that acts together with an RNA component (Terc). Maintenance of functional telomeres is essential to allow ESC proliferation, nevertheless if and how it is involved in the achievement and preservation of cell differentiation is still unknown. Here, we used Tert deficient mouse ESCs to elucidate the role of telomere length in differentiation. We found that Tert-/- ESCs with critically short telomeres are delayed, but still capable, to achieve differentiation after leukemia inhibitory factor (LIF) withdrawal and all-trans retinoic acid (ATRA) treatment, but failed to maintain it after LIF re-introduction to the growth medium. Telomere shortening effect on differentiation was accompanied by pluripotency gene dysregulation (e.g. Nanog overexpression), DNA hypomethylation and epigenetic disorders. This phenotype of metastable differentiation could be rescued by telomere lengthening via re-introduction of Tert, depletion of Nanog via short hairpin RNA, or via enforced expression of the de novo DNA methyltransferase 3b. These results reveal an unanticipated role of telomeres in the epigenetic regulation of gene expression and cell fate determination during physiological or pathological processes.

The fall in circulating oestrogen (E2) after the menopause leads to an increased rate of bone remodelling, excessive osteoclast activity and a greater fracture risk. Until recently hormone replacement therapy (HRT) was prescribed to post-menopausal women to prevent bone loss, however HRT is associated with an elevated incidence of cardiovascular disease, stroke and cancer. These side-effects led to an interest in naturally occurring compounds with oestrogenic action such as phytoestrogens (PEs), which are non-steroidal-plant derived compounds. Human trials and animal studies suggest a beneficial effect of PEs on bone mass, although their ability to modify osteoclast formation in response to key inflammatory cytokines has not been examined. The aim of the following studies was to determine the effect of physiologically relevant concentrations of genistein, coumestrol and daidzein on TNF-α-induced osteoclast formation, osteoblasts differentiation and T cell activity. Genistein (10-7 M), daidzein (10-5 M), and coumestrol (10-7 M) significantly reduced TNF-α-induced TRAP positive osteoclast formation and bone resorption, which was prevented by the E2 antagonist ICI 182,780. The suppressive action on osteoclast formation was associated with a significant reduction in TNF-α-induced c-fos and NFATc1 mRNA expression and NFATc1 nuclear translocation. Constitutive c-fos expression prevented the inhibitory action of PEs on osteoclast differentiation, resorption and NFATc1 expression. The effect of PEs, in the presence or absence of the anabolic nutritional factor zinc, on osteoblasts differentiation and bone nodule formation was examined in-vitro. Coumestrol (10-5 to 10-7 M), daidzein (10-5 to 10-6 M) and genistein (10-5 M) enhanced bone nodule formation and ALP activity in human osteoblasts, and this effect was significantly augmented in the presence of zinc (10-5 M). Furthermore, PEs and zinc increased Runx2 mRNA expression and Zn2+ augmented the inhibitory effect of PEs on RANKL/OPG ratio. This suggests that in addition to the direct inhibitory effect on osteoclast formation PEs also in-directly reduce the osteoblastsic stimulus for osteoclast formation and promote bone formation. E2 deficiency is thought to promote osteoclastogenesis by modifying Thelper1 (Th1) cell proliferation and inflammatory cytokine production in particular TNF-α. I therefore examined the effect of PEs on T cell proliferation and inflammatory cytokine production. All PEs prevented the augmentative effect of con A stimulated T cells on osteoclast formation in co-culture. However the mechanism of action varied, genistein reduced con A stimulated TNF-α, IL-1β and RANKL expression with little effect on viability, coumestrol decreased cell viability and TNF-α expression whereas the inhibitory effect of daidzein was mediated via suppression of viable T cell number. This study provides novel evidence that PEs have multiple effects on bone cell activity, directly inhibiting TNF-α-induced osteoclast formation, reducing the osteoblasts and T cell derived stimulus for osteoclast formation and augmenting osteoblasts differentiation and bone formation. Thus, PEs have a potential role in the treatment of post-menopausal osteoporosis and inflammatory skeletal disorders and that the beneficial effect noted in previous studies is mediated through multiple mechanisms.

Pathways and mechanisms for human memory T cell differentiation and maintenance have largely been inferred from studies of peripheral blood, though the majority of T cells are found in lymphoid and mucosal sites. We present here a novel, multidimensional, quantitative analysis of human T cell compartmentalization and maintenance over six decades of life in blood, lymphoid and mucosal tissues obtained from 56 individual organ donors. Our results reveal that the distribution and tissue residence of naïve, central and effector memory, and terminal effector subsets is contingent on both differentiation state and tissue localization. Moreover, T cell homeostasis driven by cytokine or TCR-mediated signals is dependent on CD4+ or CD8+ T cell lineage, subset differentiation and tissue localization, and cannot be inferred from blood. Our data provide an unprecedented spatial and temporal map of human T cell compartmentalization and maintenance, supporting new pathways for human T cell fate determination and homeostasis. Memory T cells can remain in tissues as non-circulating, resident memory populations, which provide optimal protection against infection at barrier surfaces. Lung-resident memory T cells (TRM) mediate in situ protection to respiratory pathogens, though mechanisms for their maintenance and retention are unknown. Through whole transcriptome profiling, we identify a cohesive network of genes enriched in lung CD4+ TRM, including Itgad (CD11d), Cd69, and IFN-associated responders. We find that upregulation of CD11d enhances CD69 expression through type I IFN signaling downstream of homotypic cell adhesion, and is required for optimal T cell differentiation and lung retention. Moreover, blockade of IFNαR1 reduces CD11d expression and retention of influenza-generated lung TRM, suggesting that CD11d-dependent type I IFN signaling promotes TRM establishment. Our results implicate CD11d and type I IFN in retaining lung CD4+ TRM cells, and identify potential targets for modulating tissue immunity.

In mammalian development, endoderm formation occurs in two phases and the fate of these populations is different. In the blastocyst, inner cell mass (ICM) cells generate the primitive endoderm (PrE), which will give rise to the extra-embryonic parietal (PE) and visceral endoderm (VE). Hematopoietically expressed homeobox (Hhex) protein is initially expressed throughout the PrE and subsequently becomes restricted to the anterior visceral endoderm (AVE), one of two important early embryonic signalling centres in the mouse. During gastrulation a second wave of endoderm differentiation occurs, the definitive endoderm (DE), generating the foregut. Immediately following the induction of DE, regional identity is initially established in the anterior region with the expression of Hhex. One of the earliest specification events in this lineage is the specification of anterior fate by Hhex, this time in a second signalling centre, the anterior definitive endoderm (ADE). The ADE is both important for embryonic patterning, and as the precursor population for differentiating to the foregut and its derivatives the thyroid, liver and pancreas. The literature surrounding these early embryonic patterning events is covered in depth in chapter 1. Embryonic stem cells (ESCs) are normal cell lines derived from the mammalian blastocyst at the time that it is making PrE. A number of laboratories have generated protocols to make endoderm from ESCs and in my thesis I define approaches to distinguish between PrE and DE. I generated a new ESC reporter line utilising a gene normally expressed in both the PrE and later in hepatic endoderm; this reporter contains a GFP in the first exon of the Hnf4α locus. This was combined with a second fluorescent reporter containing DSRed in the Hhex locus. This cell line is described and characterised in chapter 3. As Hnf4α is initially expressed in PrE prior to Hhex, but in the DE following Hhex, I was able to use the temporal expression of this reporter to distinguish the induction of PrE from DE. As Activin and Wnt are known to induce endoderm from ESCs, I was then able to ask what sort of endoderm the combination of these two signals induced. In chapter 4 I found that normal ESCs would readily differentiate to iPrE in the presence of Activin and Wnt3a. While this has not been described previously, my analysis suggests that ESC protocols applying these cytokines directly to ESCs have produced PrE. Given that ESCs are derived from the blastocyst, the generation of iPrE from Wnt3a/Activin treatment fits with developmental paradigms. However, Act/Wnt3a is used routinely on Human ESCs (hESCs) and so I attempted to reconcile these observations. HESCs, while derived from the blastocyst, appear to progress developmentally in vitro, to a stage closer to the epiblast, immediately prior to gastrulation. I therefore assessed the effect of Activin and Wnt3a on mouse stem cell lines derived from the epiblast (Epiblast Stem Cells, EpiSCs), that are grown under similar conditions to hESCs. When Wnt3a/Act is applied to these cells I found that they made DE rather than PrE, which I describe in chapter 4. Taken together my observations suggest that Act/Wnt3a are general endoderm inducers that induce context specific differentiation in vitro. The cell type derived in response to this treatment depends on the developmental stage of the starting stem cell culture. During the course of this work, I also observed that PrE was growing under Activin/Wnt3a treatment. As a number of cell culture systems have been established that reflect PE, but not truly bipotent PrE, I investigated the conditions under which PrE can be expanded. In chapter 5 I characterize a new PrE culture system, in which bipotent extra-embryonic endoderm can be expanded indefinitely in culture. I also explore a bit more precisely the nature of the starting cells that initially become exposed to Activin/Wnt3a treatment. Previous work has extensively characterized the existence of a primed population of PrE in ESC culture and in chapter 6 I explore the existence of a primed DE population in EpiSC culture. Taken together, my thesis is the first demonstration that Activin/Wnt3a can induce different endoderm populations in different embryonic stem cell populations. It underlies the notion that the evolutionary origin of both cell types is the same and that the pathways evolved for extra-embryonic development in mammals just exploit the ancient modes of germ layer specification that evolved with gastrulation.

Thesis (Ph. D.)--Drexel University, 2006. / Includes abstract and vita. Includes bibliographical references (leaves 83-89).

Understanding the behavior of a cell requires that its molecular constituents, such as mRNA or protein levels, be profiled quantitatively. Typically, these measurements are performed in bulk and represent values aggregated from thousands of cells. Insights from such data can be very useful, but the loss of single-cell resolution can prove misleading for heterogeneous tissues and in diseases like cancer. Recently, technological advances have allowed us to profile multiple cellular parameters simultaneously at single-cell resolution, for thousands to millions of cells. While this provides an unprecedented opportunity to learn new biology, analyzing such massive and high-dimensional data requires efficient and accurate computational tools to extract the underlying biological phenomena. Such methods must take into account biological properties such as non-linear dependencies between measured parameters. In this dissertation, I contribute to the development of tools from harmonic analysis and computational geometry to study the shape and geometry of single-cell data collected using mass cytometry and single-cell RNA sequencing (scRNA-seq). In particular, I focus on diffusion maps, which can learn the underlying structure of the data by modeling cells as lying on a low-dimensional phenotype manifold embedded in high dimensions. Diffusion maps allow non-linear transformation of the data into a low-dimensional Euclidean space, in which pairwise distances robustly represent distances in the high-dimensional space. In addition to the underlying geometry, this work also attempts to study the shape of the data using archetype analysis. Archetype analysis characterizes extreme states in the data and complements traditional approaches such as clustering. It facilitates analysis at the boundary of the data enabling potentially novel insights about the system. I use these tools to study how the negative costimulatory molecules Ctla4 and Pdcd1 affect T-cell differentiation. Negative costimulatory molecules play a vital role in attenuating T-cell activation, in order to maintain activity within a desired physiological range and prevent autoimmunity. However, their potential role in T cell differentiation remains unknown. In this work, I analyze mass cytometry data profiling T cells in control and Ctla4- or Pdcd1-deficient mice and analyze differences using the tools above. I find that genetic loss of Ctla4 constrains CD4+ T-cell differentiation states, whereas loss of Pdcd1 subtly constrains CD8+ T-cell differentiation states. I propose that negative costimulatory molecules place limits on maximal protein expression levels to restrain differentiation states. I use similar approaches to study breast cancer cells, which are profiled using scRNA-seq as they undergo the pathological epithelial-to-mesenchymal transition (EMT). For this work, I introduce Markov Affinity based Graph Imputation of Cells (MAGIC), a novel algorithm designed in our lab to denoise and impute sparse single-cell data. The mRNA content of each cell is currently massively undersampled by scRNA-seq, resulting in 'zero' expression values for the majority of genes in a large fraction of cells. MAGIC circumvents this problem by using a diffusion process along the data to share information between similar cells and thereby denoise and impute expression values. In addition to MAGIC, I apply archetype analysis to study various cellular stages during EMT, and I find novel biological processes in the previously unstudied intermediate states. The work presented here introduces a mathematical modeling framework and advanced geometric tools to analyze single-cell data. These ideas can be generally applied to various biological systems. Here, I apply them to answer important biological questions in T cell differentiation and EMT. The obtained knowledge has applications in our basic understanding of the process of EMT, T cell biology and in cancer treatment.

Gene expression is regulated at various levels, such as transcription, RNA transport and translation. Additionally, it has been shown that chromatin structure, location and dynamics also have an important role in gene expression control. While active gene regions are strongly associated with an open chromatin structure at the surface of the chromosome territory (CT) and a location in the nuclear interior, inactive gene regions seem to be related with a closed structure within the CT and a position at the nuclear periphery. However, it is still unclear how these features are regulated. Importantly, malfunction of gene regulation can impact on health and longevity. Therefore, the aim of this project was to investigate the correlation of gene expression and chromatin organisation both in single gene loci and the MHC gene cluster. The MHC locus has the highest gene density in mammalian cells and contains genes that can be reprogrammed by pro-inflammatory cytokines. The original goal of this project was to label the MHC locus by the Lac operator/repressor (LacO/LacI) approach in order to study chromatin dynamics in living cells using labelled CTs as reference for genome mobility. The thymidine analogue EdU, that can be used to label CTs, was analysed for its effects on cell cycle progression and survival, and revealed to have a strong negative impact on the cells' well-being. In the end, the LacO/LacI-recognition system for live-cell imaging did not succeed, thus FISH analyses were carried out to study chromatin dynamics in snap-shots. The location and structure of the hybridised gene regions were analysed in response to gene activation and inactivation during ESC differentiation to neuroepithelial progenitors (NPs). Single-gene focused experiments were performed using the cell line specific genes, Oct4 and Sox1, together with Gapdh as a housekeeping gene. Even though, the results showed less changes between the days of differentiation on the Gapdh locus, the gene expression profiles for the cell line specific genes did not match with the hypothesised chromatin organisation (see above). However, investigations on the gene-dense MHC locus showed structural chromatin changes that correlated with the activation of genes in this region. Interestingly, ESC treated with TNFalpha were unable to activate NF-kappaB signalling, probably due to the lack of a functional IKK complex. In summary, this project was focussing on the regulation of gene expression by the chromatin architecture and revealed complex chromatin dynamics that are likely to be affected by the sum of genes in a genome region, rather than a single gene.

Memory T cells are a critical component of immunological memory, which provides long-lasting immunological protection. These cells are characterized by a lower response threshold, rapid effector cytokine production, and prolonged longevity, and thus allow organisms to respond to pathogens more rapidly and effectively. However, the mechanisms that regulate the generation, function, homeostasis and tissue targeting of memory CD4+ T cells are not clear. This body of work investigated post-effector requirement for T-bet expression in determining the circulating and tissue resident memory CD4+ T cell fate and the implications of early T-bet deletion on lung CD4+ TRM development. We used mouse models with conditional expression of T-bet to delete T-bet in CD4+ T cells after priming and effector differentiation to analyze the development of resultant memory CD4+ T cells. We found that T-bet-ablation following cell priming and Th1 polarization did not impair the ability of Th1 effector cells to produce high levels of IFN-γ production, and moreover, there were dramatic increases in IL-2 production, suggesting post-effector T-bet expression is not required for functional maintenance in effector cells. Memory CD4+ T cells that developed from T-bet ablated effector cells after transfer to lymphocyte deficient RAG1/2-/- hosts or intact congenic hosts had increased persistence, and they maintained lower but substantial levels of IFN-γ and higher IL-2 production. We found elevation of IL-17 production and RORγt expression in T-bet ablated memory CD4+ T cells, and transcriptome analysis further showed that these cells upregulated genes expressed by other CD4+ T cell subsets, including Foxp3 and GATA3, indicating greater functional plasticity of T-bet-ablated memory CD4+ T cells. Increased localization of T-bet-ablated memory CD4+ T cells in the lung resident niche was found only in RAG1/2-/- hosts but not in congenic hosts, indicating the importance of the tissue environment in the development of TRM cells. Using antigen specific T-bet+/- OT-II and T-bet-/- OT-II cells, we found that T-bet+/- OT-II cells had increased persistence while T-bet-/- OT-II cells had decreased persistence compared with the wild type OT-II cells after PR8-OVA influenza virus infection. However, both T-bet+/- and T-bet-/- OT-II cells had normal TRM formation. Collectively, our results reveal the roles of T-bet in regulating the generation, function, maintenance and tissue targeting of memory CD4+ T cells.