Thesis (Ph.D.) - University of Queensland, 2004. / Includes bibliography.

Thesis (Ph.D.) - University of Queensland, 2004. / Includes bibliography.

Thesis (M.Phil.) - University of Queensland, 2005. / Includes bibliographical references.

Thesis (M.S.)--Northern Michigan University, 2009. / Bibliography: leaves 35-36.

Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Thesis (M.Phil.) - University of Queensland, 2005. / Includes bibliography.

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

The intestinal barrier represents a complex environment, composed of different physical barriers and immune cells, which act to prevent the entrance of potentially harmful enteric pathogens and to maintain gut tolerance to food antigens and commensal bacteria. Thus, cross-talk between the different components of the intestinal barrier such as the mucus layer, dendritic cells (DC) and intestinal intraepithelial lymphocytes (IELs) may be important in maintenance of gut homeostasis. This thesis investigates how different components of the intestinal barrier regulate immune responses in the gut. Thus, expression of the transmembrane receptor integrin αvβ8 on DCs is shown to be required for the development of a specific IEL subset marked by expression of CD4 and CD8αα, suggesting that intestinal DC play important roles in regulating the IEL compartment. Moreover, considering that intestinal DCs are likely in close contact with intestinal mucus, it was hypothesized that interactions between DCs and mucins, the predominant proteins that form the mucus layer, may modulate DC function. To test this hypothesis, intestinal mucin was purified and used to treat human monocyte-derived DCs. It was found that that expression of the chemokine IL-8 and co-stimulatory DC markers CD86 and CD83 are significantly upregulated on human DCs in the presence of intestinal mucins. Additionally, IL-8 produced by mucin-treated DCs is able to recruit neutrophil-like cells in transmigration assays. These effects were not due to mucin sample contaminants such as LPS, DNA or contaminant proteins. Instead, mucin glycans seem to be important for the induction of these effects on moDCs. Thus, in contrast to recent published results, intestinal mucins appear capable of inducing important pro-inflammatory functions in DC. To investigate whether mucins modulated DCs found in the intestinal environment, intestinal mucins were used to treat murine intestinal DCs, and gene changes explored using microarray analysis. It was found that, amongst several genes modulated in intestinal DC, up-regulation of the mucosal cytokine IL-22 was induced by intestinal mucin. Therefore, interactions between different components of the intestinal barrier might be crucial for maintaining gut homeostasis. Understanding how different components of the intestinal barrier system work together to maintain homoeostasis may identify pathways that can be targeted to restore this balance in inflammatory disorders such as inflammatory bowel disease.

Despite the importance of proper synaptogenesis in the CNS, the molecular mechanisms that regulate the formation and development of synapses remain poorly understood. Indeed, the mechanisms through which initial synaptic contacts are established and modified during synaptogenesis have not been fully determined and a precise understanding of these mechanisms may shed light on synaptic development, plasticity and many CNS developmental diseases. The development and formation of spiny synapses has been thought to occur via filopodia shortening followed by the recruitment of proper postsynaptic proteins, however the precise function of filopodia remains controversial. Thus the goal of this study was to investigate the dynamics of dendritic filopodia and determine their role in the development of synaptic contacts. We initially define and characterize short lipidated motifs that are sufficient to induce process outgrowth. Indeed, the palmitoylated protein motifs of GAP-43 and paralemmin are sufficient to induce filopodial extensions in heterologous cells and to increase the number of filopodia and dendritic branches in neurons. We showed that the morphological changes induced by these FIMs (filopodia inducing motifs) require on-going protein palmitoylation and are modulated by a specific GTPase, Cdc42, that regulates actin dynamics. We also show that their function is palmitoylation dependent and is dynamically regulated by reversible protein palmitoylation. Significantly, our work suggests a general role for those palmitoylated motifs in the development of structures important for synapse formation and maturation. We combined several approaches to monitor the formation and development of filopodia. We show that filopodia continuously explore the environment and probe for appropriate contacts with presynaptic partners. We find that shortly after establishing a contact with axons, filopodia induce the recruitment of presynaptic elements. Remarkably, we find that expression of acylated motifs or the constitutively active form of cdc-42 enhances filopodia number and motility, but reduces the recruitment of synaptophysin positive presynaptic elements and the probability of forming stable axo-dendritic contacts. We provide evidence for the rapid transformation of filopodia to spines within hours of imaging live neurons and reveal potential molecules that accelerate this process. / Medicine, Faculty of / Graduate

Dendritic cells (DCs) are specialized members of the innate immune system that are responsible for the initiation of primary adaptive immune responses whose purpose is to resolve infection and inflammation. During most viral infections, mature dendritic cells present critical viral antigens to naïve T-cells within secondary lymphoid organs, resulting in the generation of an antigen-specific adaptive immune response and clearance of the virus. During infection with HIV-1 however, the virus is not cleared and a chronic systemic infection develops characterized by immune dysfunction, CD4+ T-cell depletion, systemic inflammation, and opportunistic infections. A growing body of evidence indicates that HIV-1 subversion of DCs contributes to both HIV-1 pathologies and viral dissemination. A number of similar effects by accessory HIV-1 peptides on DC physiology have also been reported. In vitro studies demonstrate that HIV-1 inhibits DC maturation and function. Ex vivo studies on the other hand describe partially mature, dysfunctional DCs collecting in secondary lymphoid organs. In vitro studies examining the effects of HIV-1-Tat and HIV-1-Vpr have described opposing effects on DC maturation. Therefore we undertook experiments to comprehensively describe the effects of HIV-1 and the Tat and Vpr accessory peptides on DC maturation and function. To understand the contributions of individual viral proteins to DC dysfunction we infected DCs with a dual tropic HIV-1 and examined phenotypic and functional changes after maturation with inflammatory cytokines. Following this we examined the influence of exogenous and endogenous HIV-1-Tat and HIV-1-Vpr on MDDC maturation and function using recombinant proteins and deletion mutant lab adapted HIV-1 strains. Live dual tropic HIV-1 was found to selectively inhibit aspects of phenotypic maturation as well as antigen capture and presentation functions. MDDC MAPK responsiveness to bacterial LPS remained intact however. Exogenous accessory HIV-1 Tat and Vpr did not affect MDDC phenotype but inhibited dextran endocytosis and viral peptide presentation. HIV-1-gp120 increased iMDDC maturation while blunting cytokine induced decreases in MDDC antigen capture abilities. The deletion of HIV-1-Tat did not affect MDDC phenotype, but was found to affect antigen capture decreases by R5 tropic HIV-1BaL. Deletion of HIV-1-Vpr likewise did not affect MDDC phenotype, however it was found to be influential in HIV-1 induced decreases in MDDC antigen presentation to autologous T-cells. These accumulated results indicate that HIV-1 subverts DC maturation and function through whole virus effects and individual accessory peptide influences. Understanding the mechanisms of DC dysfunction in HIV infection may provide some insight into infection prevention strategies and therapies leading to adaptive immune system activation and viral clearance.