The ability of tumor necrosis factor (TNF) and lipopolysaccharide (LPS) to stimulate gene expression in bone marrow-derived macrophages (BMM) was first compared. It is demonstrated that they stimulated gene expression through distinct signal transduction pathways and that TNF stimulated gene expression through a protein kinase C (PKC)-dependent signal transduction pathway. The effect of the intracellular parasite of macrophages Leishmania donovani in BMM was then investigated. It is demonstrated that L. donovani impaired c-fos and TNF gene expression through two distinct mechanisms. The first one is indomethacin-reversible, and the second one involves the inhibition of diacylglycerol-induced PKC-dependent gene expression. A purified cell surface glycoconjugate of the parasite, termed lipophosphoglycan, selectively inhibited PKC-dependent gene expression in BMM. While the translocation of PKC from the cytosol to the membrane was normal, total cellular PKC enzyme activity was inhibited in the U937 human monocyte cell line pretreated with lipophosphoglycan.

Living cells keep sensing the changes in their environments, mostly, via cell surface receptors for different ligands. Attachment-dependent cells are sensitive to alterations in extracellular matrix (ECM). ECM is not only required for cell survival, but also prerequisite for epidermal growth factor (EGF) to stimulate cell proliferation. The receptors for the majority of ECM components are integrins and the receptor for EGF is EGF receptor (EGFR). When bound by their ligands, integrins and EGFR induce signal transduction cascades composed of alternative pathways. A quantitative assessment of relative contributions of alternative pathways to one final cell signaling will help understand designing principles of the network. Unfortunately, a methodology for such assessment is still not available, partly because of lack of relatively mature mathematical models. On the other hand, in most biochemical cascades, existence of alternative pathways increases the complexity and thus the robustness of networks. The relationships between the topology and robustness of large-scale biochemical networks have been studied intensively recently. In small-scale networks, while feedback has been revealed as an important contributor for adaptation and robustness, the quantitative correlation between the topology/pathway redundancy of small networks and their robustness remains unknown. / In this thesis, apoptosis of bovine mammary gland epithelial cells was demonstrated to be induced when fibronectin, one of the major components of ECM, was degraded by overexpressed tPA via two potential ways: deprivation of attachment and the effects of fibronectin fragments. Secondly, a mathematical model for EGFR activation of the MAPK cascade, in which alternative pathways exist, was explored and it was found that the Shc-dependent pathway is both redundant and dominant. We hypothesize that the Shc-dependent pathway is important for EGFR to compete with other receptors, which need Shc to transduce cell signals; and this pathway is not aimed to increase the robustness of the EGFR cascade. Finally, for the general importance of alternative pathways to the network topology and robustness, several concepts have been proposed to decompose and quantitatively characterize the networks. We demonstrate that the pathnet score is a better assessment for robustness than the molecular connectivity.

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.

3BP2 was originally identified through its interaction with the ABL kinase. Fusion of ABL with the BCR gene forms the BCR-ABL onco-protein, which is causative in Chronic Myeloid Leukemia (CML) and acute lymphoid leukemia (ALL). Due to the ability of 3BP2 to regulate ABL activity in osteoblasts, we hypothesize that 3BP2 modulates BCR-ABL signalling. Overexpression of 3BP2 in the CML-T1 cell line produced a marked decrease in global tyrosine phosphorylation. 3BP2 overexpression also resulted in a significant increase in CML-T1 cell growth, accompanied by altered ERK1/2, AKT, SYK, LYN, HCK, and CBL phosphorylation and expression. A phospho-SRC family protein and a 116 kDa phospho-protein were identified as 3BP2 interaction partners in response to BCR-ABL activation. BCR-ABL bone marrow transplantation (BMT) models in 3bp2-/- mice exhibit accelerated disease compared to wild-type mice, with altered leukemic phenotype. In conclusion, 3BP2 is able to modulate signalling through BCR-ABL and affect BCR-ABL-induced disease outcome.

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).

Following starvation, Dictyostelium discoideum cells aggregate, a response that requires chemotaxis to cyclic AMP (cAMP) and the relay of the cAMP signal by the activation of adenylyl cyclase (ACA). Insertional inactivation of the rasG gene resulted in delayed aggregation and a partial inhibition of early gene expression, suggesting that RasG does have a role in early development. When the responses of rasG⁻ cells to cAMP were compared with the responses of rasC⁻ strain, these studies revealed that signal transduction through RasG is more important in chemotaxis and early gene expression, but that signal transduction through RasC is more important in ACA activation. Characterization of a rasC⁻/rasG⁻ mutant revealed that both cAMP chemotaxis and adenylyl cyclase (ACA) activation were negligible in this strain. The ectopic expression of carA from the actin 15 promoter restored early developmental gene expression to the rasC⁻/rasG⁻ strain, rendering it suitable for an analysis of cAMP signal transduction. Since there was negligible signaling through either the cAMP chemotactic pathway or the adenylyl cyclase activation pathway in this strain, it is clear that RasG and RasC are the only two Ras subfamily proteins that directly control these pathways. The mutational analysis of Switch I and Switch II regions also defined the key residues that generate functional differences between RasC and RasG. Rap1 is also activated in response to cAMP but its position in the signal transduction cascade was clarified by the finding that its activation was totally abolished in rasC⁻/rasG⁻/[act15]:carA and in rasG⁻ cells, but only slightly reduced in rasC⁻ cells. The finding that in vitro guanylyl cyclase activation is also abolished in the rasC/rasG⁻4act15]:carA strain identifies RasG⁻/RasC⁻ as the presumptive monomeric GTPases required for this activation. The phenotypes of the vegetative ras null mutants were also examined. The results indicate that RasG plays an important role in cytokinesis. The partial absence of chemotaxis to folate in rase cells compared to the total absence of chemotaxis to folate in rasC⁻/rasG⁻, and rasC⁻/rasG⁻/[act15]:carA cells suggests a compensatory role of RasC for RasG during this process, a similar phenomenon to that observed for cAMP chemotaxis by aggregating cells.

Thesis (M.S.)--University of Missouri-Columbia, 2007. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on April 1, 2008) Includes bibliographical references.

Thesis (Ph.D.)--Tufts University, 1999. / Adviser: Marc d'Alarcao. Submitted to the Dept. of Chemistry. Includes bibliographical references (leaves 123-128). Access restricted to members of the Tufts University community. Also available via the World Wide Web;

Thesis (Ph.D.)--Ohio University, November, 2007. / Title from PDF t.p. Includes bibliographical references.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 5 uppsatser.