Currently, the intensive chemotherapy remains the first line treatment for B cell acute lymphoblastic leukemia (B-ALL). Although these regimens have significantly improved patient outcomes, their use is associated with debilitating morbidities and fatal relapses, highlighting the great need in new agents that target essential survival signals in leukemia. Thus, the overall goal of my project was to gain insights into the signaling abnormalities that regulate aberrant proliferation and survival of B-ALL cells in an effort to identify novel targets in this malignancy. This study demonstrated that pre-B cell receptor (pre-BCR)-independent spleen tyrosine kinase (SYK) activity was required for the survival and proliferation of a p53-/-PrkdcSCID/SCID mouse model of B-ALL. I extended this discovery to human disease, demonstrating that SYK was activated in primary B-ALL, independent of the pre-BCR expression. The small molecule SYK inhibitor fostamatinib (fosta) significantly attenuated proliferation of 79 primary diagnostic B-ALL samples at clinically achievable concentrations. Importantly, fosta treatment reduced dissemination of engrafting B-ALL cells into the spleen, liver, kidney and central nervous system (CNS) in a NOD.Prkdcscid/scidIl2rgtm1Wjl/SzJ xenotransplant model of B-ALL. Analysis of signaling abnormalities using a high-throughput phospho-flow cytometry platform demonstrated that pediatric and adult B-ALL samples exhibit variable basal activation of BCR, iii PI3K/AKT/mTOR, MAPK and JAK/STAT pathways. Importantly, we identified that fosta-mediated inhibition of SYK, PLC2, CRKL and EIF4E phosphorylation in B-ALL was predictive of its anti-leukemic activity, and was distinct from the cellular actions of other small molecule inhibitors of key nodal signaling pathways. Examination of molecular mechanism of fosta action by gene expression profiling revealed transcriptional effects of fosta treatment that included, most notably, potent inhibition of pathways involved in lymphocyte activation and inflammation. In conclusion, this study demonstrates that SYK signaling is crucial for B-ALL survival and provides detailed characterization of cellular and molecular mechanisms of fosta action in B-ALL. These data argue in favor of testing small molecule SYK inhibitors in pediatric and adult B-ALL.

Many environmental stresses result in increased generation of active oxygen species (AOS) in plant cells, leading to the induction of protective mechanisms. In this study, signalling components linking AOS perception to downstream responses were examined, with particular emphasis on H₂O₂ signalling. All AOS investigated had an early [Ca²⁺]_cyt peak in common, but differed in other aspects of their Ca²⁺ signatures, indicating that the plant is able to discriminate between different types of AOS. An early event in AOS signal transduction may involve changes in the cellular redox balance as reduction of glutathione levels prior to stress application increased the height of the first [Ca²⁺]_cyt peak. Inhibiting or enhancing the height of the H₂O₂-triggered Ca²⁺ signature lead to inhibition or enhancement of GST1 and APX1 induction, respectively, demonstrating that the Ca²⁺ signature is required for induction of genes encoding antioxidant enzymes. OX1, encoding a putative ser/thr kinase, was shown to be involved in signal transduction in response to H₂O₂-generating stresses. Transcript levels of OX1 were increased upon treatment with H₂O₂ and a range of abiotic and biotic stresses as well as ABA, all of which have been shown to result in H₂O₂ accumulation. Inhibition of stress-induced [Ca²⁺]_cyt elevations inhibited OX1 induction, placing the OX1 kinase downstream of Ca²⁺ in the signalling chain. OX1 is required for full activation of AtMPKS and AtMPK6 in response to ozone fumigation, indicating that OX1 functions upstream of these MAP kinases. An ox1 null-mutant displayed enhanced susceptibility to infection with a virulent Peronospora parasitica isolate as well as reduced induction of several defence genes. In addition, the ox1 mutant exhibited shorter root hairs and an early flowering phenotype. AOS treatment induced several genes encoding AtERF transcription factors, but did not have an effect on other members of this family. Induction occurred in an ethylene-independent but Ca²⁺-dependent manner.

Notch signalling plays a key role in physiological development and tumourigenesis. The recent discovery and characterisation of Notch ligand Delta-like 4 (D114), which is predominantly expressed in endothelial cells, have underscored the role of Notch signalling in angiogenesis. This thesis investigates the regulation and function of D114-Notch signalling in angiogenesis and tumourigenesis. First, the D114-Notch pathway interacted with the cellular hypoxia-sensing pathway. In human umbilical vein endothelial cells (HUVECs), D114 overexpression repressed hypoxic induction, and the repression was mediated by Notch target gene Hey2. In the breast cancer cell line MCF7, hypoxia induced Notch target gene Hey1 and ligand Jagged2 via hypoxia inducible factor 1. The hypoxic induction of Hey1 was also dependent on Notch signalling. Second, D114 expression in HUVECs was up-regulated by several pathways. Notch signalling, activated by receptor overexpression, ligand stimulation or cell-cell contact, induced D114 expression. Treatment with vascular endothelial growth factor (VEGF) or hypoxia also induced D114 expression via Notch signalling. In addition, VEGF promoted Notch signalling and D114 expression in tumours. Third, D114 expression in HUVECs was up-regulated by co-culturing with cancer cells. B16 mouse melanoma cells and human breast cancer cell lines induced D114 expression in HUVECs via Notch signalling. B16 cells also induced D114 expression via soluble factors independent of Notch signalling. Finally, D114-Notch signalling regulated tumour growth in vivo. D114 overexpression in cancer cell lines activated Notch signalling in the stroma of xenograft tumours, and promoted the growth of human U87 (glioblastoma) and PC3 (prostate cancer) xenografts. In addition, D114-overexpressing U87 tumours were resistant to anti- VEGF treatment during later stages; however, they did respond to anti-Notch treatment. Altogether, the D114-Notch pathway is tightly regulated and plays an important role in physiological and tumour angiogenesis. Inhibiting this pathway may be a viable therapy for cancers resistant to VEGF inhibition.

During the present studies we attempted to answer the following questions: (i) Does low temperature alter the phosphorylation level of proteins in isolated nuclei? (ii) Does the nuclear phosphoprotein population change during a prolonged exposure of seedlings to cold? (iii) Do heat-stable proteins accumulate in the nucleus during a prolonged exposure of seedlings to cold? (iv) Are the answers to the above three questions related to freezing tolerance? A possible relationship between the observed cold-induced changes in phosphoproteins and the level of freezing tolerance was explored by comparing the results of experiments conducted on two cultivars (Apica and Trek) of alfalfa (Medicago sativa L.) which markedly differ in their capacity for cold acclimation. / We show that the phosphorylation level of several nuclear proteins is subject to rapid and reversible enhancement by low temperature. Several phosphoproteins were found to be constitutively present in the nucleus of both cultivars. The cold-induced stimulation of the phosphorylation of many of these proteins was much greater in the relatively freezing tolerant cultivar Apica than in the relatively freezing sensitive cultivar Trek. Population of nuclear phosphoproteins was found to be considerably more complex in Apica than in Trek. During a prolonged exposure of the seedlings to 4$ sp circ$C, additional phosphoproteins were imported into the nucleus of Apica seedlings but not those Trek. / Some heat-stable proteins were constitutively present in the nucleus of both cultivars. However during the 4-day cold treatment, a large accumulation of several additional heat-stable proteins was observed in the tolerant, but not the sensitive, cultivar. (Abstract shortened by UMI.)

Dendritic cells (DC) are critical to an immune response by functioning as sensors of foreign antigen and presenting antigen to direct T cell responses. Under the influence of Flt3 ligand this heterogeneous group of cells originate from hematopoietic stem cells (HSCs) in the bone marrow and takes up residence in lymphoid organs, skin as well as mucosal surfaces where they are most likely to encounter pathogens. GM-CSF is another cytokine involved in DC development that is specifically involved in the maintenance of skin resident DC. Together Flt3L and GM-CSF as well as their respective receptors, Flt3 and GM-CSFR, are the most important factors identified to date for maintenance of DC homeostasis. The Src-like adaptor proteins, SLAP and SLAP-2, are negative regulators of antigen receptor signaling. SLAP and SLAP2 contain SH3 and SH2 protein interaction modules that facilitate interaction with proline-rich sequences and phosphotyrosine motifs, respectively. Through a unique C-terminal region, SLAP and SLAP2 also interact with the E3 ubiquitin ligase, c-Cbl. SLAP and SLAP2 enhance c-Cbl-mediated ubiquitylation and down-regulation of antigen receptors by binding both activated receptors and c-Cbl. SLAP and SLAP2 have also been shown to function as negative regulators of receptor tyrosine kinases (RTKs) and our group has shown that SLAP and SLAP2 bind to and inhibit the colony-stimulating factor-1 receptor (CSF-1R). In the work presented here, we identify a novel role for SLAP in regulation of the GM-CSF receptor of bone marrow (BM)-derived DC. We show that potentiated GM-CSF signaling, in the absence of SLAP and SLAP2, impairs BM-DC maturation such that these cells express minimal MHCII, secrete low amounts of IL-12 and are functionally impaired in their ability to stimulate T cell responses. SLAP and SLAP2 deficiency also has an effect on Flt3L-derived BM-DC development. For example, SLAP/SLAP2-/- BM-DC numbers are reduced in the presence of Flt3L as compared to wild-type BM-DC. To investigate the mechanism of reduced DC numbers, we examined splenic DC and found that DC numbers were similar in wild-type and SLAP/SLAP2-/- mice. In fact, SLAP/SLAP2-/- mice had proportionally more CD8α+ splenic DC in vivo than wild-type mice. Thus there may be cytokines affected by SLAP/SLAP2-deficiency in our cultures that are either dispensable or compensated for in vivo DC development. The work presented in this thesis has implications for the role of SLAP and SLAP2 in immune response to infections by the regulation of GM-CSFR and Flt3 in maintaining dendritic cell homeostasis.

Dendritic cells (DC) are critical to an immune response by functioning as sensors of foreign antigen and presenting antigen to direct T cell responses. Under the influence of Flt3 ligand this heterogeneous group of cells originate from hematopoietic stem cells (HSCs) in the bone marrow and takes up residence in lymphoid organs, skin as well as mucosal surfaces where they are most likely to encounter pathogens. GM-CSF is another cytokine involved in DC development that is specifically involved in the maintenance of skin resident DC. Together Flt3L and GM-CSF as well as their respective receptors, Flt3 and GM-CSFR, are the most important factors identified to date for maintenance of DC homeostasis. The Src-like adaptor proteins, SLAP and SLAP-2, are negative regulators of antigen receptor signaling. SLAP and SLAP2 contain SH3 and SH2 protein interaction modules that facilitate interaction with proline-rich sequences and phosphotyrosine motifs, respectively. Through a unique C-terminal region, SLAP and SLAP2 also interact with the E3 ubiquitin ligase, c-Cbl. SLAP and SLAP2 enhance c-Cbl-mediated ubiquitylation and down-regulation of antigen receptors by binding both activated receptors and c-Cbl. SLAP and SLAP2 have also been shown to function as negative regulators of receptor tyrosine kinases (RTKs) and our group has shown that SLAP and SLAP2 bind to and inhibit the colony-stimulating factor-1 receptor (CSF-1R). In the work presented here, we identify a novel role for SLAP in regulation of the GM-CSF receptor of bone marrow (BM)-derived DC. We show that potentiated GM-CSF signaling, in the absence of SLAP and SLAP2, impairs BM-DC maturation such that these cells express minimal MHCII, secrete low amounts of IL-12 and are functionally impaired in their ability to stimulate T cell responses. SLAP and SLAP2 deficiency also has an effect on Flt3L-derived BM-DC development. For example, SLAP/SLAP2-/- BM-DC numbers are reduced in the presence of Flt3L as compared to wild-type BM-DC. To investigate the mechanism of reduced DC numbers, we examined splenic DC and found that DC numbers were similar in wild-type and SLAP/SLAP2-/- mice. In fact, SLAP/SLAP2-/- mice had proportionally more CD8α+ splenic DC in vivo than wild-type mice. Thus there may be cytokines affected by SLAP/SLAP2-deficiency in our cultures that are either dispensable or compensated for in vivo DC development. The work presented in this thesis has implications for the role of SLAP and SLAP2 in immune response to infections by the regulation of GM-CSFR and Flt3 in maintaining dendritic cell homeostasis.

Recruitment of effector T cells to sites of infection is essential for an effective adaptive immune response. The inflammatory chemokine CCL5/RANTES activates its cognate receptor, CCR5, to initiate cellular functions including chemotaxis. This thesis describes the signaling events invoked by CCL5 and its ability to regulate the energy status of activated T cells. CCL5 treatment in ex vivo activated human T cells induced the activation of AMPK and downstream substrates ACC1, PFKFB2 and GSK-3. Evidence is provided that CCL5 treatment is able to induce glucose uptake in an mTOR-dependent manner. Using 2-deoxy-D-glucose, an inhibitor of glucose uptake, and Compound C, an inhibitor of AMPK, evidence is provided that demonstrate that CCL5-mediated chemotaxis is dependent on metabolic events, since these inhibitors perturb chemotaxis in a dose-dependent manner. Collectively, these studies suggest that CCL5 may also influence the metabolic status of activated T cells by simultaneously activating the AMPK and mTOR pathways.

Recruitment of effector T cells to sites of infection is essential for an effective adaptive immune response. The inflammatory chemokine CCL5/RANTES activates its cognate receptor, CCR5, to initiate cellular functions including chemotaxis. This thesis describes the signaling events invoked by CCL5 and its ability to regulate the energy status of activated T cells. CCL5 treatment in ex vivo activated human T cells induced the activation of AMPK and downstream substrates ACC1, PFKFB2 and GSK-3. Evidence is provided that CCL5 treatment is able to induce glucose uptake in an mTOR-dependent manner. Using 2-deoxy-D-glucose, an inhibitor of glucose uptake, and Compound C, an inhibitor of AMPK, evidence is provided that demonstrate that CCL5-mediated chemotaxis is dependent on metabolic events, since these inhibitors perturb chemotaxis in a dose-dependent manner. Collectively, these studies suggest that CCL5 may also influence the metabolic status of activated T cells by simultaneously activating the AMPK and mTOR pathways.

Bipolar disorder is a psychiatric condition which affects up to 1% of the general population and results in episodes of mania and depression. Molecular biological studies have shown that several components of the signal transduction pathways are affected in bipolar illness. However, the precise systems and components involved in the disorder still remain unknown. Our goal was to identify some of the differences in signal transduction pathways of B-lymphocytes. Using cultured lymphocytes obtained from bipolar patients, we found that there exist no difference in the levels of protein kinase C, Galphas and Galphai proteins, and tubulin between control and bipolar cell lines following stimulation of the PI pathway with the 5-HT 2 receptor agonist, alpha-methyl serotonin. These data are not consistent with previous findings. The lack of a significant difference between control and bipolar with respect to PKC might be due to the fact that we studied a different cell type or to poor stimulation conditions, and/or possibly to a high PKC content in the membrane of these cells, thereby masking the effect of stimulation. The results obtained for the G proteins can be attributed to a lack of effect of the agonist on these proteins which are associated with the adenylate cyclase pathway.

TGFbeta signaling peptides have been shown to play increasingly diverse roles in metazoan development and tissue homeostasis. Negative regulation of TGFbeta ligands such as Nodal can be achieved by physical interactions with inhibitory molecules. Dante is a recently identified putative member of DAN/Cerberus family of TGFbeta inhibitors. Previously shown to be unilaterally expressed on the right side of the mouse node, Dante has been suggested to play a role in Left-Right axis formation possibly by inhibition of Nodal molecules. The aim of this study was to further characterize Dante and to determine whether it can physically interact with Nodal. First, a longer Dante cDNA was isolated in an attempt to clone the full-length transcript. Furthermore, Dante expression pattern was analyzed during murine development and adult tissues. Lastly, co-immunoprecipitation experiments demonstrated that Dante is able to physically interact with Nodal, providing further support for its potential role as a Nodal inhibitor.