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Tie2 is a receptor tyrosine kinase (RTK) involved in numerous aspects of both normal and pathological angiogenesis. Proper functioning of this receptor is essential for normal development of the vasculature in the embryo as well as vessel maintenance and at sites of active angiogenesis in the adult. A growing list of pathological states has been attributed to a disruption of the angiogenic ‘balance’ including psoriasis, arthritis, atherosclerosis and diabetic retinopathy. Elucidating the molecular mechanisms behind this important biological process will provide insight into the various molecules involved as well as provide potential targets for novel angiogenic therapies. In an attempt to better understand the signaling pathways downstream of the Tie2 receptor we have studied tyrosine residues on the receptor believed to play an important role in Tie2 function. Of these, we have identified Y1111 as a negative regulatory site on Tie2. Mutation of this site affects receptor phosphorylation and kinase activity. Furthermore, protease digestion studies indicate that mutation of Y1111 may alter receptor conformation and potentially relieve negative inhibition imparted by the C-tail of Tie2. As well, we examined potential Tie2 downstream binding partners, specifically the novel Grb7 family of proteins. This work describes for the first time tyrosine phosphorylation of Grb14, an adaptor molecule previously shown to bind Tie2 in vitro. Moreover, our data suggests a role for this adaptor in Tie2 signal transduction involving two tyrosine residues in the receptor C-terminal tail; Y1100 and Y1106. These studies provide important insight into both signal transduction downstream of Tie2 as well as help us understand some of the molecular mechanisms behind the intrinsic ability of this RTK to regulate its own activity.

To exit a non-proliferative state and enter cell division, metazoan cells require external signals to facilitate activation and metabolic reprogramming. As cell growth is required before cell division, cells redirect their metabolism for de novo synthesis of cell building blocks, including phospholipids for cell membrane construction. How cells coordinate initial signaling events with metabolism is unknown. Lineage-specific factors transmit activating signals via cell surface receptor-ligand interactions. Among these are PI3K/AKT, MAPK/ERK, and JAK/STAT, all of which have been described to contribute to metabolic regulation. In particular, the signal transducer and activator of transcription (STAT) is a transcription factor with broad roles in cell cycle progression and glucose metabolism. Previous data from our laboratory found that one STAT family member, STAT3, was one of the primary signaling pathways activated when transitioning out of a resting state. Inhibition of STAT3 was found to suppress the initiation of cell growth and citrate levels, a main intermediate for fatty acid synthesis, suggesting a connection to cell metabolism. This thesis investigates the role of STAT3 in the regulation of metabolism in cells transitioning from a resting state to a cell growth state. The first chapter of this thesis provides relevant background information on the metabolic and signaling pathways involved in a resting and cell growth state. It also provides data that supports an important role for STAT3 during initial cell growth. The second chapter demonstrates the importance of STAT3 in multiple cell types using a small molecule inhibitor of STAT3, STAT3 knockdown, and knockout experiments. I also establish a potential link between STAT3 and the metabolic enzyme citrate synthase (CS) for the synthesis of citrate. In the third chapter I show that STAT3 transcriptionally regulates CS through two binding sites, CS1 and CS2. Finally, I determine that CS is essential for initial cell growth and that exogenous citrate can rescue the loss in cell growth and proliferation observed in the CS and STAT3 knockdown cells. Together, these findings describe a novel mechanism for initial cell growth whereby signaling and metabolic events are tightly linked to regulate the transition from a resting state to a state of initial cell growth. These results may uncover new strategies to block the initiation of proliferation in human pathological conditions including tumor recurrence and autoimmunity. / Graduate

by Suen Yick-keung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1991. / Bibliography: leaves 163-170. / Abstract --- p.i / Acknowledgements --- p.v / Abbreviations --- p.vi / Objective of the study --- p.vii / Contents --- p.viii / Chapter Section 1 --- Introduction / Chapter 1. --- Roles of macrophges in immune system --- p.3 / Chapter 2. --- Special features of macrophages --- p.6 / Chapter 3. --- Transmembrane signalling in mammalian cells --- p.9 / Chapter 4. --- Transmembrane signalling in macrophages --- p.32 / Chapter 5. --- Choice of the macrophages for the study --- p.40 / Chapter Section 2 --- Materials and Methods / Materials / Chapter 1. --- Animals --- p.44 / Chapter 2. --- Chemicals --- p.44 / Chapter 3. --- Reagents --- p.45 / Methods / Chapter 1. --- pell culture --- p.47 / Chapter 2. --- 3H-thymidine incorporation --- p.48 / Chapter 3. --- Cytosolic free calcium determination --- p.48 / Chapter 4. --- Intracellular pH mesurement --- p.50 / Chapter 5. --- Determination of membrane potential --- p.50 / Chapter 6. --- Determination of phagocytic activity --- p.51 / Chapter 7. --- Cell adhesion assay --- p.52 / Chapter 8. --- Statistical analysis --- p.52 / Chapter Section 3 --- Results / Chapter 1. --- Effect of membrane potential on cell proliferation in PU5-1.8 cells and bone marrow-derived macrophages / Evidence for induction of cell proliferation mediated by membrane depolarization in PU5-1.8 cells --- p.53 / Evidence of an array of agonists on cell proliferation and membrane potential in PU5-1.8 cells --- p.57 / Interrelationship between membrane potential and FCS-mediated proliferation in PU5-1.8 cells --- p.61 / Cytosolic alkalinization induces membrane depolarization in PU5-1.8 cells --- p.64 / Suppression of membrane depolarization and cell proliferation by protein kinase C activation --- p.66 / Effect of membrane potentials on cell proliferation in bone marrow-derived macrophages --- p.68 / Chapter 2. --- Intracellular signals for the regulation of phagocytosis in PU5-1.8 cells / Phagocytosis of unopsonized yeast in PU5-1.8 cells --- p.71 / Effect of membrane potential on phagocytosis in PU5-1.8 cells --- p.73 / Changes in phagocytic activities in PU5-1.8 cells by activation of protein kinase C --- p.82 / Effects of protein kinase C on membrane potential- induced enhancement of phagocytosis in PU5-1.8 cells --- p.84 / Phagocytosis in PU5-1.8 cells requires assembly of microtubule --- p.90 / Effects of intracellular calcium and cAMP on phagocytosis in PU5-1.8 cells --- p.93 / Acidic intracellular pH enhances phagocytosis in PU5-1.8 cells --- p.98 / Chapter 3. --- Effects of various agonists on phagocytosis of yeast in PU5-1.8 cells / Effect of chemotactic peptide N-formyl- methionyl-leucyl-phenylalanine (FMLP) on phagocytosis in PU5-1.8 cells --- p.100 / Effects of lipopolysaccharide (LPS) on phagocytosis in PU5-1.8 cells --- p.105 / Effects of concanavalin A (Con A) on phagocytosis in PU5-1.8 cells --- p.109 / Effect of complement components on phagocytosis in PU5-1.8 cells --- p.113 / Chapter 4. --- Signal pathways for the regulation of cell adhesion on plastic surface in PU5-1.8 cells / Adhesion of PU5-1.8 cells on plastic surface --- p.119 / Effects of membrane potential on cell adhesion on plastic surface in PU5-1.8 cells --- p.121 / Effects of activation of protein kinase C on cell adhesion on plastic surface in PU5-1.8 cells --- p.125 / Effects of intracellular calcium and cAMP on adhesion on plastic surface in PU5-1.8 cells --- p.129 / In vivo cell adhesion of PU5-1.8 cells in Balb/c mice --- p.133 / Chapter 5. --- Effects of various agonists on the cell adhesion on plastic surface in PU5-1.8 cells / Dose dependent of various agonists against the cell adhesion ability of PU5-1.8 cells --- p.141 / Chapter 6. --- Cell adhesion to plastic surface in bone marrow-derived macrophages / Membrane potentials control the adhesiveness of bone marrow-derived macrophages (BMDM0) to plastic surface --- p.143 / Effects of phorbol ester PMA on cell adhesion to plastic surface in bone marrow-derived macrophages --- p.143 / "Effects of cAMP, [Ca2+ ]i and microtubule assembly on cell adhesion to plastic surfacein bone marrow-derived macrophages" --- p.146 / Chapter Section 4 --- Discussion / Chapter 1. --- Effects of membrane potential on cell proliferation in PU5-1.8 cells and bone marrow-derived macrophages --- p.148 / Chapter 2. --- Intracellular signals for the regulation of phagocytosis in PU5-1.8 cells --- p.151 / Chapter 3. --- Signal pathways for the regulation of cell adhesion on plastic surface in PU5-1.8 cells and bone marrow-derived macrophages --- p.158 / Chapter 4. --- General discussion --- p.161 / Chapter Section 5. --- Bibliography / References --- p.163

by Kong Siu-Kai. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1989. / Bibliography: leaves 313-340.