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Chemokines are responsible for directing leukocyte migration and triggering firm arrest by activating integrins on leukocytes. It is now apparent that chemokines have critical biological roles beyond chemo-attraction. Throughout this thesis, I describe the importance of the CCL5/CCR5 axis in the context of the immune response and cancer biology. Specifically, CCL5 invokes dose-dependent distinct signalling events downstream of CCR5 activation in T cells. I show that nM concentrations of CCL5 mediate CD4+ T cell migration that is partially dependent on mTOR activation. CCL5 induces phosphorylation and de-activation of the repressor 4E-BP1, resulting in its dissociation from the eukaryotic initiation factor-4E to initiate protein translation. I provide evidence that CCL5 initiates rapid translation of cyclin D1 and MMP-9, known mediators of cell migration. The data demonstrated that up-regulation of chemotaxis-related proteins may “prime” T cells for efficient migration. During an immune response, recently recruited T cells are exposed to high CCL5 concentrations. The propensity of CCL5 to form higher-order aggregates at high, µM concentrations, prompted studies to investigate their effects on T cell function. I show that at these high doses, CCL5 induces apoptosis in PM1.CCR5 and MOLT4.CCR5 T cell lines. CCL5-induced cell death involves the cytosolic release of cytochrome c and caspase-9/-3 activation. Furthermore, I identified Tyrosine-339 as a critical residue within CCR5, suggesting that tyrosine phosphorylation signalling events are important in CCL5-mediated apoptosis. Our data suggest that CCL5-induced cell death, in addition to Fas/FasL mediated events, may contribute to clonal deletion of T cells during an immunological response. I subsequently examined the possible pathological consequence of aberrant CCL5/CCR5 signalling in breast cancer. Exogenous CCL5 enhances MCF-7.CCR5 proliferation, which is abolished by anti-CCR5 antibody and rapamycin. CCL5 induces the formation of the eIF4F translation initiation complex, and mediates a rapid up-regulation of cyclin D1, c-Myc and Dad-1 protein expression. Thus, our data demonstrate the potential for breast cancer cells to exploit downstream CCL5/CCR5 signalling pathways for their proliferative and survival advantage. Taken altogether, each of these studies reinforces the notion that chemokines are not only potent chemotactic mediators, but are key effectors in diverse developmental, immunological and pathological processes.

Plants in the field are exposed to multiple stresses and their response to these various stresses determines their capacity to survive. Plants can use multiple signaling pathways and signals to mediate their response / for example, at least four different signal pathways have been identified for water-deficit stress (Shinozaki and Yamaguchi-Shinozaki, 1997 / Xiong et al., 2002). Different forms of stress may activate or utilize the same components, including proteins and other signaling molecules. Signaling molecules such as jasmonic acid (JA) are involved in multiple stress response and development in plants (Creelman and Mullet, 1995, 1997 / Turner et al., 2002). However it is the specific combination of various components of the signaling network coupled with spatial and temporal factors that allows the plant to mount a directed response to any given stress factors. Systemic defense responses thus provide an attractive model for the study of cell-to-to cell signal transduction pathways that operates over long distances (Lucas and Lee, 2004).<br /> <br /> Cellular and physiological evidence suggest the presence of a novel class of systemic mobile plant molecule that is recognized by antibodies against vertebrate atrial natriuretic peptides (ANPs). It has been demonstrated that a recombinant Arabidopsis thaliana natriuretic peptide analogue (AtPNP-A) molecule can induce osmoticumdependent water uptake into protoplast at nanomolar concentrations thus affecting cell volume and hence plant growth. In this study we confirm that active recombinant protein causes swelling in Arabidopsis mesophyll cell protoplasts (MCPs).

Dictyostelium, is a simple eukaryote that multiplies as separate amoebae. However when nutrients are no longer available it embarks on a developmental programme in which the amoebae collect together by chemotaxis and the resulting aggregates eventually transform into fruiting bodies consisting of a cluster of spores held up on a cellular stalk. The entire process of development normally takes about 24 hours. However there are mutants, termed rapidly developing mutants (rde) which complete development in about two-thirds of this time. RdeA null mutants have been reported to have elevated levels of cyclic AMP that may lead to increased activity of the enzyme, cAMP dependent protein kinase (PKA). I started my work by measuring total cAMP levels in an rdeA mutant along with an aca-/rdeA- double mutant that is expected to have very low level of cAMP due to the absence of the adenylyl cyclase, AC A. Two Dictyostelium adenylyl cyclases were known at the beginning of my work; one is AC A the aggregative enzyme, and the other ACG, expressed only during spore germination. Contrary to expectation, I detected cAMP in aca-/rdeA cells. This raised the question of which enzyme was responsible for producing this cAMP. In collaboration with Dr.Pauline Schaap, I discovered a novel adenylyl cyclase that I initially detected in rdeA and regA mutants but not in wild-type cells. The product of the rdeA gene, RDEA was thought to be an H2-module histidine phosphotrasferase of the kind acting in multi-step phosphorelays. Similarly REGA was believed to be a response regulator associated with a cAMP-phosphodiesterase. It had been proposed that RDEA phosphorylates REGA in a multi-step phosphorelay and it had been shown that it is the phosphorylated form of REGA that is active as a cAMP-PDE. I therefore thought that cAMP produced by the novel AC could be protected in rdeA mutants by the absence of the REGA cAMP-PDE activity and this idea was supported by my finding that the enzyme activity could also be detected in wild-type (aca-) cells when REGA-PDE was inhibited by IBMX. In order to investigate further the proposed phosphorelay model, I tested for possible interaction between RDEA and REGA using the yeast two-hybrid system and also measured intracellular cAMP-phosphodiesterase activity in rdeA and regA mutants. I found that the interaction between RDEA and REGA appeared to be too transient to be detected in the two-hybrid system. In addition rdeA and regA mutants seemed to have levels of intracellular cAMP-phosphodiesterase activity similar to wild type. However REGA-PDE activity measured specifically by immuno-precipitation was completely absent in the regA mutant. It therefore appeared that there is another intracellular cAMP-phosphodiesterase, in addition to the REGA PDE, in Dictyostelium and that the latter cannot be easily detected in total cell lysates. One possible explanation is that the novel adenylyl cyclase exists together with REGA in a complex (that may also include PKA) and that REGA PDE preferentially destroys the cAMP made by the novel adenylyl cyclase. I conclude that rdeA and regA mutants may develop rapidly due to high PKA activity induced by the accumulation of cAMP made by the novel AC when the REGA cAMP-PDE activity is absent.

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