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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
321

A Molecular and Immunological Investigation of Cellular Responses to Dengue Virus: Identification of Potentially Upregulated Host Genes and the Construction of a Vaccinia Virus Expressing the Dengue 1 Hawaii NS3 Protein

Brown, Jennifer L. 30 March 2000 (has links)
The purpose of this thesis for the degree of Master of Science was to use molecular and immunological techniques to study cellular responses to dengue virus infection. In the initial study, Differential Display was used to compare mRNA expression in dengue-infected K562 cells and mock-infected cells. Cloning and sequencing were then used to identify cellular genes that were potentially up-regulated in response to Dengue virus infection. These genes included bleomycin hydrolase and a dystrophin homologue. The goal of the later part of this research was to construct a recombinant vaccinia virus expressing the dengue 1 Hawaii NS3 protein. Cytotoxic T-lymphocyte assays and protein gel electrophoresis showed that the NS3 protein was being expressed. This construct was then used to study the cytotoxic T-cell response of a dengue 1 vaccine recipient. The results of this study showed that this individual has dengue 1 NS3 specific T-cells and also that this vaccinia virus can be used for subsequent T-cell studies.
322

Evading the anti-tumour immune response : a novel role for Focal Adhesion Kinase

Lund, Thomas Anthony January 2016 (has links)
Here I describe a new function of Focal Adhesion Kinase (FAK) in driving anti-tumour immune evasion. The kinase activity of FAK in squamous cancer cells drives the recruitment of regulatory T-cells (Tregs) by transcriptionally regulating chemokine/cytokine and ligand-receptor networks, including the transcription of CCL5 and TGFβ, which are required for enhanced Treg recruitment. In turn, these changes inhibit antigen-primed cytotoxic CD8+ T-cell activity in the tumour microenvironment, permitting survival and growth of FAK-expressing tumours. I show that immune evasion requires FAK’s catalytic activity, and a small molecule FAK kinase inhibitor, VS-4718, which is currently in clinical development, drives depletion of Tregs and permits CD8+ T-cell-mediated tumour clearance. It is therefore likely that FAK inhibitors may trigger immune-mediated tumour regression, providing previously unrecognized therapeutic benefit.
323

Mechanosensing in Naive CD4+ T cells

Judokusumo, Edward January 2014 (has links)
T cells are key players in adaptive immune response. Originating from the thymus, they seek and eliminate infected cells in various locations of our body. T cells are not anchorage-dependent in nature. However, in our body, cells are constantly under physiological stress. It is not yet known how natural changes in physical environment could affect T cell behaviors. This thesis focuses to study the role, pathway, and main mechanism of rigidity sensing in T cells. Most studies of T cell rigidity sensing have showed that T cell responses are sensitive to external forces. It is unclear whether T cells could generate forces, translate them to biochemical signaling, and regulate their function based on the physical sensing. We tested the idea by developing the use of substrate with varying modulus to analyze the impact of rigidity to T cell activation. We demonstrated that mouse naive CD4+ T cells were capable of sensing and transmitting information from substrate modulus, ultimately affecting the regulation of cytokine secretion, a key indicator of T cell activation. Interestingly, this cytokine secretion correlated with increasing substrate rigidity. This increased cytokine secretion diminished when T cells lost the ability to contract in sensing the underlying substrate rigidity. Contrary to the presumption that T cells are not able to regulate their function based on the forces applied to the environment, our study provides the first demonstration that substrate rigidity has a functional impact to naive CD4+ T cell activation. To understand the translation process from physical to biochemical signaling in T cells, we determined the signaling pathway that regulated T cell rigidity sensing. We found that T cell rigidity sensing was associated with the signaling molecules of the T cell receptor (TCR) complex, the central pathway of T cell response. Analysis of TCR signaling molecules revealed that T cell rigidity sensing was mediated downstream of the early signaling components in the TCR complex. Lastly, we developed a method of combining micron-scale patterning in elastic substrates to determine whether T cell mechanosensing was mediated from local adhestion sites or globally throughout the cell. Circular features of primary signal for naive CD4+ T cells were spatially segregated and patterned on elastic substrates to analyze T cell contractility in generating forces across the segregated primary signals, leading to sustained TCR triggering. We found out that T cell contractility failed to generate forces when the primary signals were arranged in equilateral triangle geometry, leading to loss of TCR triggering. This result shows that T cell rigidity sensing is mediated globally throughout the whole cell rather than locally from adhesion sites. Furthermore, the loss of TCR triggering by T cells when sensing the equilateral triangle geometry in elastic substrates opens up new ideas in characterizing force generation within the cell.
324

The role of intestinal mononuclear phagocytes in control of mucosal T cell homeostasis

Panea, Casandra M. January 2016 (has links)
The intestine is constantly exposed to a wide variety of dietary antigens, commensal bacteria and pathogens, toward which it has evolved complex immune responses to protect the host. The intestinal immune system relies on innate immune cells, such as mononuclear phagocytes (MNPs), that include dendritic cells (DCs), monocytes (Mo) and macrophages (Mfs), to sense and respond to luminal and mucosal challenges. MNPs are essential players as they instruct adaptive immune cells, in particular T cells, to discriminate between innocuous and harmful antigens. Generation of different CD4 T cell responses to commensal and pathogenic bacteria is crucial for maintaining a healthy gut environment, but the associated cellular mechanisms are poorly understood. Lamina propria (LP) T helper 17 (Th17) cells participate in mucosal protection and are induced by epithelium-associated commensal segmented filamentous bacteria (SFB). Several reports suggest that the cytokine environment induced by gut bacteria is sufficient to drive LP Th17 cell differentiation. In this context, intestinal DCs are proposed to facilitate the conversion of naïve CD4 T cells to Th17 cells within gut-draining lymph nodes. Whether such mechanisms control commensal-mediated Th17 cell differentiation has not been examined. In this work, I explore the mechanisms of induction of Th17 cells by SFB, with a particular focus on the role of antigen-presenting cells in this process. Initiation of CD4 T cell responses requires both major histocompatibility II (MHCII)-mediated antigen presentation and cytokine stimulation, which can be provided by the same or different subsets of intestinal MNPs. To test the requirement for either function in the induction of Th17 cells by SFB, we analyzed the role of SFB-induced cytokine environment in driving Th17 cell differentiation of non-SFB transgenic CD4 T cells. We find that although the cytokine environment is important, it is not sufficient to promote Th17 cell differentiation of activated CD4 T cells. In fact, we show that MHCII-dependent antigen presentation of SFB antigens by intestinal MNPs is crucial for Th17 cell induction. Expression of MHCII on CD11c+ cells was necessary and sufficient for SFB-induced Th17 cell differentiation. We also show that most SFB-induced Th17 cells respond to SFB antigens, which stressed that they carry T cell receptors that recognize SFB moieties. SFB primed and induced Th17 cells locally in the LP and Th17 cell induction occurred normally in mice lacking secondary lymphoid organs. Our results outline the complex role of MNPs in the regulation of intestinal Th17 cell homeostasis, and we investigated the contribution of individual subsets to SFB-specific Th17 cell differentiation. Although the role of DCs in initiating T cell responses is well appreciated, how Mfs contribute to the generation of CD4 T cell responses to intestinal microbes is unclear. To this end, I examined the role of mucosal DCs and Mfs in Th17 induction by SFB in vivo. Employing DC and Mf subset-specific depletion and gain-of-function mouse models, I show that Mfs, and not conventional CD103+ DCs, are essential for generation of SFB-specific Th17 responses. Thus, Mfs drive mucosal T cell responses to certain commensal bacteria.
325

Biological Insights from Geometry and Structure of Single-Cell Data

Sharma, Roshan January 2019 (has links)
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.
326

The relative contribution of lymphocytes to hepatic ischemia reperfusion injury

Richards, James Alexander January 2016 (has links)
Background: Hepatic ischemia reperfusion injury (IRI) results from the interruption and then reinstatement of the liver’s blood supply. IRI involves both an ischemic and an immune-mediated reperfusion phase of tissue injury; similar inflammatory events are seen in other forms of acute (sterile) liver injury (ALI), including paracetamol toxicity. Hypothesis Irrespective of the primary insult, common pathways exist in the pathophysiology of the lymphocyte-mediated secondary liver injury. Natural mechanisms exist to limit lymphocyte function and these pathways can be targeted therapeutically by immunomodulatory agents. Aims: 1. To assess the relative importance of different lymphocyte subsets in IRI. 2. To correlate observations in IRI with other models of ALI. 3. To identify possible pharmacological targets. Materials and Methods Three experimental murine models of acute liver injury were utilised to test this hypothesis: murine model of warm hepatic IRI, concanavalin A (con A) hepatitis and paracetamol-induced liver injury. These models were interrogated with a combination of (transgenic and knockout) mouse lines, in vivo antibody depletion and small molecule inhibition. Injury was evaluated primarily in terms of the biochemical marker of liver injury alanine aminotransferase (ALT). Data were correlated with human tissue where possible. Results: T cells (CD3εKO vs WT p=0.010), but not other lymphocyte populations (B cells, NK cells, or other innate lymphoid cells), play a central role in warm hepatic IRI. Programmed Death Receptor-1 (PD-1) is a negative regulator of pro-inflammatory cytokine production by T cells and the absence of PD-1 was associated with significantly worse hepatic IRI (p=0.034), con A hepatitis (p=0.00020) and paracetamol-induced liver injury (p=0.0050). Interferon-γ (IFNγ) and T-box expressed in T cells (T-bet) are important mediators of hepatic IRI (p=0.017) and paracetamol induced liver injury (p=0.0007). The absence of IL-6 was associated with significant protection in paracetamol induced liver injury (p=0.006). The infiltrates within the recipient liver of patients transplanted following paracetamol overdose stain positively for PD-1, IFNγ and T-bet. The Janus family of kinases (JAK) play an important role in the common pathways of cytokine signal transduction. In vivo use of a selective JAK1/JAK2 inhibitor is protective in hepatic IRI (p=0.0014), con A hepatitis (p=0.019) and paracetamol-induced liver injury (p=0.0045). Conclusions: Common pathways appear to exist in the immune-mediated secondary phase of injury in ALI. Targeting these pathways will complement existing (cytoprotective) treatment strategies.
327

Roles of transcription factor T-bet in memory CD4+ T cell generation, function, homeostasis and tissue targeting

Chen, Jun Kui January 2017 (has links)
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.
328

The role of JAK1 and JAK3 in CD8⁺ effector T cells

Rollings, Christina January 2016 (has links)
The aim of this project was to explore the role of the tyrosine kinases JAK1 and JAK3 in cytokine signalling, focusing on interleukin-2 signalling in CD8<sup>+</sup> effector T lymphocytes. Initial experiments compared the effects of the pan JAK1/JAK3 inhibitor tofacitinib, the selective JAK1 inhibitor GSK186, and the selective JAK3 inhibitor GSK192 on IL-2 control of effector CD8+ cytotoxic T cells (CTL). On the basis of these preliminary data, a detailed analysis of the effect of tofacitinib on effector CD8<sup>+</sup> T lymphocytes was performed. Phosphorylation events regulated by tofacitinib were identified using mass spectrometry analysis of SILAC (stable isotope labelling with amino acids in cell culture) labelled CTL. Tofacitinib regulated a selective number of phosphorylation sites, with less than 1.2% of the CTL phosphoproteome significantly regulated by tofacitinib treatment following 4hrs tofacitinib treatment. Proteins with downregulated phosphorylation sites were enriched in functions related to the Jak-STAT signalling, regulation of gene expression, and MAPK signalling cascades. Proteins with upregulated phosphorylations were also enriched in functions related to regulation of gene transcription. The proteome of tofacitinib treated CTL was defined by label free mass spectrometry. Approximately 4.5% of the CTL proteome was significantly regulated following 24 hours tofacitinib treatment, suggesting tofacitinib regulates the expression of a selective subset of proteins. Tofacitinib treatment resulted in the downregulation of proteins involved in ribosome biosynthesis, steroid biosynthesis, regulation of transcription and the cell cycle; and the upregulation of proteins with hydrolase activity, and with roles in the lysosome and extracellular exosomes. The phosphoproteomic and proteomic data demonstrates that JAK kinase dependent IL-2 signalling regulates essential processes in CTL by controlling a selective number of phosphorylation events and proteins. Validation of proteins identified as regulated following tofacitinib treatment identified new targets of IL-2 signalling in CTL, including the transcription factor NFIL3. NFIL3 was shown to be upregulated in CD8<sup>+</sup> T lymphocytes following stimulation with IL-2 and regulated perforin and CD62L expression, suggesting a role in the regulation of CTL effector function.
329

T cell regulation of acute and chronic viral infection

Christiaansen, Allison Fae 01 May 2016 (has links)
A balanced immune response is required to mediate clearance of a virus infection without immune-mediated disease. CD4 and CD8 T cells are capable of both exerting antiviral effector functions and regulating the immune response. The regulatory T cell (Treg) subset of CD4 T cells helps to modulate immune activation and inflammation. During respiratory syncytial virus (RSV) infection in mice, conventional CD4 T-cell-mediated cytokine production has been shown to contribute to immune-mediated pathology. I demonstrate that Tregs are critical to control immunopathology during RSV infection. This was demonstrated through diphtheria toxin (DT)-mediated Treg elimination in a mouse strain expressing the DT receptor (DTR) under the control of the Foxp3 promoter. However, these mice were unable to maintain extended Treg depletion limiting the effectiveness of this model. In addition, DT-treated wild-type (WT) mice were found to be a necessary control for adverse DT-induced disease. In humans, I have shown that activated Tregs are reduced in the peripheral blood of RSV-infected infants compared to controls. RSV-infected infants also exhibited an increased proinflammatory cytokine response in nasal aspirates. However, the alarmin cytokine IL-33, which has been shown to mediate Treg homeostasis, was the only cytokine that exhibited reduced protein levels in RSV-infected infants compared to controls. Thus, severe RSV infection in infants may be due to lack of proper Treg-mediated immune regulation. Similar to RSV, regulation of the T cell response during chronic viral infection with lymphocytic choriomeningitis virus (LCMV) is vital to prevent immune-mediated pathology. During LCMV and human chronic viral infections, CD4 and CD8 T cells exhibit T cell exhaustion where they lose the ability to exert effector functions. However, a functional CD4 and CD8 T cell response is required for viral clearance. During human chronic viral infection, an association between increased CD4 and CD8 T cell function and enhanced viral control has been identified that can be influenced by genetic factors. I aimed to identify the contribution of the host genetic factors that contribute to enhanced CD8 T cell function and viral control using the LCMV model. I found that increasing the major histocompatibility complex (MHC) diversity resulted in enhanced viral control in both a C57BL and BALB genetic background. Thus, induction of a broader T cell response was associated with enhanced viral control. However, mice expressing a heterozygous MHC on the C57BL background also exhibited mortality following chronic viral infection. Both CD4 and CD8 T cells were shown to contribute to this mortality and exhibited reduced T cell exhaustion during LCMV infection in these mice. Heterozygous MHC expression on the C57BL mouse background was also associated with an increased T helper (Th)-1 skewed CD4 T cell response compared to mice on the BALB background. Furthermore, CD4 T-cell-mediated IFN-γ production contributed to both CD8 T cell effector activity and mortality during chronic LCMV infection. Thus, both T cell epitope diversity and host genetics contribute to LCMV-induced mortality. Collectively, my data highlight both the need for effective immune-meditated viral control and regulation of T-cell-mediated pathology during both acute and chronic viral infections.
330

THE ROLE OF CD8 T CELL IMMUNODOMINANCE AND REGULATORY T CELLS IN NEONATAL IMMUNITY TO INFLUENZA VIRUS

Heil, Luke 01 January 2019 (has links)
Neonates are more susceptible to influenza virus infection than adults, resulting in increased morbidity and mortality as well as delayed clearance of the virus. Efforts to improve influenza infection outcomes in neonates typically center on prevention, although current vaccines fall short of complete protection and can only be administered in humans after 6 months of life. We propose that as the neonatal immune system responds differently than the adult immune system, interventions that are efficacious or tolerable in adults cannot be guaranteed to perform the same in neonates. T cell vaccines that target conserved influenza virus epitopes have been proposed for conferring protection to multiple influenza virus strains, but if T cell vaccines will be used in infants and adults, neonates must be able to respond to the same T cell antigens as adults. Mouse pups responded to influenza virus peptide PA224-233 but not NP366-374 during influenza virus infection in contrast to the codominant adult response. Mice infected as pups also generated diminished T cell memory compared to mice infected as adults and displayed skewed immunodominance during secondary infection. Adult bone marrow derived dendritic cells (BMDCs) improved viral clearance when loaded with influenza virus and promoted NP366-374-specific CD8+ T cell responses in infected pups. BMDC peptide vaccination could stimulate PA224-233-specific but not NP366-374-specific CD8+ T cell responses in pups, but, PA224-233 vaccination offered no protection to pups during lethal infection. These data suggest that altered immunodominance must be considered when stimulating CD8+ T cell responses in adults and neonates. Immaturity and active suppression of immune responses are both factors in neonatal vulnerability to disease. Specifically, active suppression of neonatal immunity by regulatory T cells (Tregs) has been proposed as a driving factor in diminished neonatal immunity, but removing these cells can compromise viral defense or increase deleterious inflammation. Mice that lacked Tregs displayed compromised anti-influenza antibody responses and decreased lymph node responses during influenza virus infection. A high proportion of pup Tregs also expressed Gata3. Transgenic pups with a Treg specific Gata3 knockout displayed an increase in Tbet expression in both conventional and regulatory T cells and an increase in IFNγ producing CD4+ T cells in the lungs during infection. These data suggest that Tregs are required for effective humoral responses to influenza virus and that Gata3 expression influences Treg suppressive function in neonates.

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