Dendritic morphology and microsegregation in the ternary Fe- 1.6% Mn - 0.1 to 0.8 % C alloys have been investigated by quenching the unidirectionally solidified specimens. The microprobe analysis of these specimens showed that the manganese segregation was significantly controlled by the back diffusion. This back diffusion was extremely high in the case of ferritic solidification whereas only a small rise in Cmin was obtained for the austenitic phase. It was found that the manganese microsegregation between the primary arms was always higher than between the secondary arms. The measured segregation ratios indicated a rise with increasing carbon content for both morphologies. No clear effect of cooling rate on segregation was seen for secondary arms and only a sliqht increase was recorded with increasing the cooling rate for primary arms. Secondary dendrite arms solidified to produce asymmecric distribution profiles (saw-tooth or TGZM effect). Measurements of the secondary dendrite arms during qrowth showed that the rate of the coarsening in these manganese steels was higher than other steels resulting in high homogenization between the arms . No tertiary arms have been observed. The primary arms grew mainly in the so-called 'close packed' arrangement and their spacinq did not chanqe with time. By increasing the qrowth rate and the temperature qradient in the liquid a decrease in primary arm spacings was seen. The results agree well with available experimental data in the literature. The microsegregation calculations obtained from the secondary dendrite arm coarseninq model is in a very good agreement with the experimental measurements. The same model without arm coarsening was applied to different primary arm morphologies and the predictions of these models are also in reasonable agreement with observations.

The rising demand for speciality polymers that possess novel properties has led to an interest in the tailored synthesis of dendritic polymers having highly controlled molecular architectures. Control over size, shape, molecular weight and functionality at the periphery of the molecule was used to design a series of dendrimer wedges with the ultimate aim of enhanced binding of a functional, property modifying unit to a cotton surface in water. Poly (propyleneimine) dendrimer wedges with various foci were produced in a stepwise way via a repetitive reaction sequence using the divergent approach. Initially, long chain aliphatic amines were used as starting materials and optimisation of the reaction conditions produced wedges up to the third generation possessing eight primary amine end groups. Subsequently dendrimer wedges were synthesised from a siloxysilane core up to the second generation. End group modification with amino acid residues was performed at the periphery of the wedges and adsorption studies were carried out to ascertain if enhanced molecular recognition at a cotton surface occurred. Also an investigation of the modification of polar surfaces by amine terminated dendrimers with a siloxysilane unit at the focus was performed.

The purinergic P2X7 receptor (P2X7R), an adenosine triphosphate (ATP)-gated receptor, is widely distributed in a variety of cell types such as neuron cells, immune cells and epithelial cells. P2X7R on cells senses extracellular ATP released from dying cells which then acts as a danger signal and initiates inflammation. Activation of P2X7R results in various downstream events, including Ca2+ influx, nonselective membrane pore formation, cell death, assembly of the inflammasome, and killing of intracellular pathogens. Epithelial cells in the gut also express P2X7R and act as a sentinel that protects against infection and responds to changes in environmental stimuli. However, the role of P2X7R in IECs is poorly defined. Given that infection of pathogens often causes cellular damage and the released ATP may be sensed by P2X7R, we hypothesised that IECs initiate intestinal inflammation via activation of the P2X7R in response to infection. Thus, the aim of this thesis was to characterise the role of P2X7R in the initiation and development of small intestinal inflammation. In order to achieve this aim, we used two parasite-induced murine ileitis models, Toxoplasma gondii (T. gondii) and Trichinella spiralis (T. spiralis), which induce Th1 and Th2 immunity respectively. In the in vivo model of T. gondii infection, we found that P2X7R deficiency was associated with less intestinal epithelial responsiveness to the infection. The P2X7R-/- IECs had reduced CCL5 and CCL20 chemokine expression which was associated with reduced recruitment of CD103+CD11b- dendritic cells (DCs) to the small intestinal epithelium at day 1 post infection (p.i.). This finding was supported by infection of bone marrow chimeras showing a decrease in the recruitment of WT P2X7R+/+ CD103+ DCs to a P2X7R-/- epithelium. To address whether the reduced DC response impacted on development of adaptive immunity, we analysed serum IFN-g and the proportion of splenic IFN-g+CD4+ T cells at day 8 p.i., and showed they were reduced in P2X7R-/- mice. In the in vivo model of T. spiralis infection, P2X7R deficiency was also associated with reduced intestinal epithelial responsiveness to this infection characterised by lower CCL5 expression in IECs. A significant decrease in the recruitment of CD103+CD11b+ DCs at day 2 p.i. was noted in P2X7R-/- animals, and the importance of epithelial P2X7R in DC recruitment was confirmed using bone marrow chimeras. The P2X7R-/- mice, compared with the WT, had delayed progression of small intestinal inflammation, accompanied by a reduction in the percentage of IL-4+CD4+ T cells and IL-4 levels at day 8 p.i. The reduced IL-4 response was associated with a delayed worm expulsion in the P2X7R-/- mice at day 12 p.i. An in vitro study demonstrated that P2X7R blockade using the chemical inhibitor A-740003, significantly decreased CCL5, IL-6 and TNF-a secretion from mouse intestinal epithelial CMT-93 cells in response to T. gondii infection. A similar decrease in the level of CCL5 produced was also observed using primary P2X7R-/- intestinal crypt cells stimulated with lipopolysaccharide (LPS) compared with WT cells. This data indicates a proinflammatory role for P2X7R during infection. Although P2X7R signalling is known to induce the assembly of the inflammasome, IECs did not secrete the inflammasome-associated cytokines IL-1b and IL-18 in response to T. gondii infection. Moreover, P2X7R signalling had no effect on the induction of cell death in T. gondii-infected IECs. Interestingly, there was a novel finding that P2X7R antagonism inhibited T. gondii infectivity in CMT-93 cells. In summary, we have shown that P2X7R signalling mediated CCL5 expression in IECs in response to infection. Epithelial chemokines are important for the initiation of small intestinal inflammation via recruitment of innate cells such as DCs which can then prime for protective adaptive immunity. These results in this thesis improve the understanding of the role of P2X7R in the intestinal immune system and reveal novel roles for epithelial P2X7R. The work suggests the potential of epithelial P2X7R as a target for pharmacological treatment of intestinal inflammatory disorders.

T cell-dependent immune responses are highly sensitive to suppression by exposure to the environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), yet direct effects of TCDD on T cells have been difficult to demonstrate. Because the activation of naive T cells is initiated by dendritic cells (DC), the studies presented in this dissertation were designed to test the hypothesis that TCDD affects these antigen-presenting cells in a manner which ultimately results in suppressed T cell activation. The expression of numerous cell surface proteins known to be important in signaling T cells to proliferate and differentiate was evaluated on splenic DC from C57B1/6 and Balb/c mice. The production of IL-12 and the ability of DC to activate allogeneic and antigen-specific T cells were also tested. Contrary to expectation, exposure to TCDD resulted in enhanced expression of several accessory molecules including B7-2, CD40, ICAM-1, CD24 and the major histocompatibility complex (MHCII). In contrast, expression of LFA-1 was significantly decreased on DC from TCDD-treated mice. These effects were dose-dependent, persisted for at least 14 days, and did not occur in aryl hydrocarbon receptor (AhR)-deficient mice. Interestingly, TCDD treatment also decreased the numbers of DC recovered from the spleen by day 7 following exposure in C57B1/6 mice and by day 3 in Balb/c animals. When T cells were cultured with DC from TCDD-treated mice, the proliferative response of the T cells and the production of IL-2, IL-4, and IFN-�� was not suppressed but instead tended to be increased. DC production of IL-12 was also enhanced. Furthermore, TCDD did not interfere with the ability of DC to internalize latex beads or to activate antigen-specific T cells, suggesting that uptake and processing of antigen by DC is not impaired by TCDD. AhR message was detected in splenic DC and AhR protein was found in two DC cell lines, indicating that DC may be directly affected by TCDD. Taken together, these results suggest that TCDD provides an activation stimulus to DC and may lead to their premature deletion. The relationship between these effects and TCDD-induced immune suppression remains to be determined. / Graduation date: 2001

CD45 is a leukocyte specific protein tyrosine phosphatase present on the surface of all nucleated, hematopoietic cells. Despite its well-characterized role in antigen receptor signaling, little is known about its function in cell types like dendritic cells (DCs). DCs are crucial to the immune response both for its initiation and for its suppression. In this dissertation, the effects of the lack of CD45 on dendritic cell development and function were studied. The most important finding was that the lack of CD45 had a differential impact on the proinflammatory cytokine profiles elicited in DCs by different TLR agonists. TLR4 ligation led to a decrease in proinflammatory cytokine and IFNβ production whereas stimulation through TLR2 or TLR9 increased cytokine production. This suggests CD45 may be acting as a negative regulator of MyD88-dependent cytokine signaling and a positive regulator of the Trif pathway. The absence of CD45 caused alterations in the phosphotyrosine levels of several Src family kinases including Lyn. In CD45-/- DCs, Lyn was not activated upon LPS stimulation and several substrates of Lyn that appear as negative regulators in the MyD88-dependent pathway of TLR4 signaling are also not phosphorylated, providing evidence that CD45 may be a negative regulator of this pathway. The absence of CD45 in TLR activated DCs had an effect on the IFNγ secretion by CD4+ T cells and NK cells, consistent with the cytokine profiles of the DCs These data demonstrate that modulation of TLR signaling by CD45, in DCs, has the ability to impact the development of the adaptive immune response. The absence of CD45 in mice did not result in increased survival upon challenge with a high dose of LPS. Serum TNFα levels were increased in the CD45-/- mice and they showed more severe symptoms of septic shock. However, the CD45-/- mice were also found to have an increase in the number of peritoneal macrophages. Overall this study shows that CD45 does play an important role in cell types other than lymphocytes. CD45 is a regulator of TLR-mediated cytokine secretion in DCs and thus directs the outcome of the adaptive immune response.

Conventional DC (cDC) arise from circulating immediate precursors (pre-cDC), and are currently thought to be terminally differentiated. Here we show that cDC are capable of generating progeny that lost all characteristic features of cDC and aquired regulatory properties. Sorted bone marrow pre-cDCs were cultured on a stromal monolayer in the presence or absence of granulocyte-macrophage colony stimulating factor (GM-CSF). In the absence of GM-CSF, pre-cDC derived DCs gave rise to a homogeneous population of CD11clow MHClow cells (DC-regs) on day 8-10 of culture. DC-regs failed to up-regulate major histocompatibility complex class II (MHCII) and co-stimulatory molecules in response to DC maturation stimuli, were poor stimulators in T cell proliferation assays and suppressed T cell proliferation in cultures containing immuno-stimulatory DC. Co-transfer of DC-regs with DCs in vivo did not inhibit proliferation of T cells. These findings reveal the potential of DCs to generate a regulatory DC population with immunosuppressive properties.

Conventional DC (cDC) arise from circulating immediate precursors (pre-cDC), and are currently thought to be terminally differentiated. Here we show that cDC are capable of generating progeny that lost all characteristic features of cDC and aquired regulatory properties. Sorted bone marrow pre-cDCs were cultured on a stromal monolayer in the presence or absence of granulocyte-macrophage colony stimulating factor (GM-CSF). In the absence of GM-CSF, pre-cDC derived DCs gave rise to a homogeneous population of CD11clow MHClow cells (DC-regs) on day 8-10 of culture. DC-regs failed to up-regulate major histocompatibility complex class II (MHCII) and co-stimulatory molecules in response to DC maturation stimuli, were poor stimulators in T cell proliferation assays and suppressed T cell proliferation in cultures containing immuno-stimulatory DC. Co-transfer of DC-regs with DCs in vivo did not inhibit proliferation of T cells. These findings reveal the potential of DCs to generate a regulatory DC population with immunosuppressive properties.

CD45 is a leukocyte specific protein tyrosine phosphatase present on the surface of all nucleated, hematopoietic cells. Despite its well-characterized role in antigen receptor signaling, little is known about its function in cell types like dendritic cells (DCs). DCs are crucial to the immune response both for its initiation and for its suppression. In this dissertation, the effects of the lack of CD45 on dendritic cell development and function were studied. The most important finding was that the lack of CD45 had a differential impact on the proinflammatory cytokine profiles elicited in DCs by different TLR agonists. TLR4 ligation led to a decrease in proinflammatory cytokine and IFNβ production whereas stimulation through TLR2 or TLR9 increased cytokine production. This suggests CD45 may be acting as a negative regulator of MyD88-dependent cytokine signaling and a positive regulator of the Trif pathway. The absence of CD45 caused alterations in the phosphotyrosine levels of several Src family kinases including Lyn. In CD45-/- DCs, Lyn was not activated upon LPS stimulation and several substrates of Lyn that appear as negative regulators in the MyD88-dependent pathway of TLR4 signaling are also not phosphorylated, providing evidence that CD45 may be a negative regulator of this pathway. The absence of CD45 in TLR activated DCs had an effect on the IFNγ secretion by CD4+ T cells and NK cells, consistent with the cytokine profiles of the DCs These data demonstrate that modulation of TLR signaling by CD45, in DCs, has the ability to impact the development of the adaptive immune response. The absence of CD45 in mice did not result in increased survival upon challenge with a high dose of LPS. Serum TNFα levels were increased in the CD45-/- mice and they showed more severe symptoms of septic shock. However, the CD45-/- mice were also found to have an increase in the number of peritoneal macrophages. Overall this study shows that CD45 does play an important role in cell types other than lymphocytes. CD45 is a regulator of TLR-mediated cytokine secretion in DCs and thus directs the outcome of the adaptive immune response.

Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / This study demonstrates that dendritic cell (DC) defects are common in chronic renal failure, dialysis and transplant recipients and this is likely to contribute to their underlying immune deficiency and high risk of infections and malignancies. Simple and effective clinical strategies (e.g. improvement in dialysis efficiency) which aim to correct DC deficiencies could potentially lead to direct improvement in patient outcome. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1277089 / Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2006

Title page, table of contents and abstract only. The complete thesis in print form is available from the University of Adelaide Library. / This study demonstrates that dendritic cell (DC) defects are common in chronic renal failure, dialysis and transplant recipients and this is likely to contribute to their underlying immune deficiency and high risk of infections and malignancies. Simple and effective clinical strategies (e.g. improvement in dialysis efficiency) which aim to correct DC deficiencies could potentially lead to direct improvement in patient outcome. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1277089 / Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2006