In Canada, colorectal cancer is the second leading cause of cancer death for both men and women. There are many different factors that contribute to the progression and spread of the disease. However, increasing evidence now suggests that the tumor microenvironment plays a paramount role in these processes. CD26 is a multifunctional, cell-surface glycoprotein that has intrinsic enzyme activity, binds adenosine deaminase and interacts with the extracellular matrix. Through its various functions it serves to constrain cancer progression. For example, it is known to cleave CXCL12, the ligand for CXCR4. The CXCL12:CXCR4 axis is normally involved in cancer metastasis by promoting cancer cell migration, invasion and proliferation. Down-regulation of CD26 is observed in certain cancers - this has been shown in vitro to occur in response to certain soluble mediators. The first part of this study looked at the effects of glucose and its metabolic product lactate on CD26 expression in colorectal carcinoma cells. Our study showed that CD26 expression is lower in cancer cells that are grown in low-glucose, high-lactate conditions, which replicates the situation within a tumor. The second part of this study examined the effect of adenosine, a purine nucleoside, on colorectal carcinoma cells and supportive stromal cells - cancer-associated HS675.T fibroblasts (CAFs) and Met-5a mesothelial cells. Adenosine increased the proliferation of CAFs and increased CXCL12 mRNA in both stromal cell lines. It also increased MMP-13 mRNA in stromal cells as well as colorectal cancer cells, suggesting that adenosine may promote progression and metastasis through various mechanisms. The last section focused on the ability of cellular products and 3-dimensional tissue topology to coordinate and affect the behaviour of the different cell populations. Here we show that secretory products from colorectal cancer cells promote CAF proliferation but inhibit mesothelial cell proliferation, and are also able to modulate MMP-13 expression. Finally, certain responses are enhanced in multicellular spheroids. In conclusion, the tumor microenvironment represents a major consideration in the treatment of solid tumors. Our data suggest that various soluble mediators, such as adenosine, may have therapeutic implications in cancer treatment and might represent novel targets for future research.

Proton nuclear magnetic resonance (NMR) spectroscopy has been used to investigate the interaction of yeast phosphoglycerate kinase (PGK) with its phosphate containing substrates, ATP and 3-phosphoglycerate (3-PG). The application of one-dimensional and, for the first time, two-dimensional proton NMR techniques to this large protein has enabled specific resonance assignments to be made. Assignment has been aided by the investigation of specifically deuterated protein and site-specific mutant forms of the protein, including the isolated N- and C-domains. The effects of ATP and 3-PG binding on the proton NMR spectrum of yeast PGK have been characterised and the assigned resonances used as local probes of structural and dynamic changes. Two binding sites have been determined for the nucleotide substrate, ATP, the occupancies of which are dependent on Mg²⁺ concentration. One site corresponds to the catalytic site determined crystallographically. A single binding site was found for 3-PG. This binding was shown to cause highly specific conformational changes throughout the N-domain and the interdomain region, which involve the relative movement of at least three α-helices. Investigation of 3-PG binding to several site-specific mutant forms of yeast PGK revealed a critical role for arginine 168 in the propagation of these changes. The general binding of anions to yeast PGK was investigated using the paramagnetic probes [Cr(CN)₆]^3- and [Fe(CN)₆]^3-, and the diamagnetic anion [Co(CN)₆]^3-. The primary anion binding site was determined from [Cr(CN)₆]^3- broadening data and found to share some side-chains involved in 3-PG binding, namely histidine 62 and arginine 168. Evidence for a secondary anion site was found. The anion binding data is discussed in view of the complex activation/inhibition effects of anions on the catalytic activity. Investigation of the isolated N- and C-domains showed that both can fold independently and confirmed that the C-domain is a nucleotide binding domain. It appears that the presence of the interdomain residues and/or the C-terminal peptide are necessary for 3-PG binding to the N-domain. This work shows that the specificity of the substrates is in binding, as expected, but also in the motions induced in the protein as a whole.

The role of adenosine as an endogenous neuromodulator is well established, but the mechanism(s) mediating the extensive modulatory and regulatory actions of adenosine have not yet been fully elucidated. In fact, although adenosine, through activation of adenosine A1 and A2A receptors, has been demonstrated as neuroprotective or neurodegenerative, respectively, little is known about the mechanism by which adenosine mediates these actions. In the hippocampus, essential physiological processes rely on adenosine signaling, including regulation of long-term potentiation (LTP) and long-term depression (LTD). Neuromodulation by adenosine is dominantly inhibitory in the hippocampus, mediated by the abundant and high-affinity adenosine A1 receptor. In ischemia and hypoxia, A1 receptor activation induces rapid synaptic depression which is mediated by multiple signaling pathways including the induction of excitatory AMPA glutamate receptor internalization, which inhibits synaptic transmission in the hippocampus. Considerable effort has been devoted to investigating the role of adenosine in ischemic stroke, due to the fact that in cerebral ischemia or hypoxia, extracellular levels of adenosine increase dramatically. This thesis explores the functional consequences of adenosine signaling in hypoxia and ischemia, which mediate GluA1 AMPA receptor subunit internalization. Three major serine/threonine protein phosphatases (PPs), PP1, PP2A, and PP2B are investigated and shown to mediate A1 receptor-mediated GluA1 internalization in hypoxic conditions in the rat hippocampus. Further experiments demonstrate the role of adenosine A2A receptors in potentiating hippocampal synaptic transmission in reperfusion by increasing GluA1 surface expression through increased phosphorylation of regulatory C-terminal phosphorylation sites of GluA1. The mechanism of extracellular adenosine regulation by equilibrative nucleoside transporters (ENTs) and casein kinase 2 (CK2) are examined and shown to interact in hypoxia/reperfusion experiments on hippocampal slices. Finally, using a pial vessel disruption (PVD) permanent focal cortical ischemia stroke model, experiments demonstrate increased adenosine tone in the hippocampus, which mediates increased adenosine-induced synaptic depression. CK2 inhibition was also neuroprotective after 20min hypoxia. This shows that adenosine tone is increased in the hippocampus after a small cortical stroke, implying a potential global effect of focal ischemia. Together, these studies further reveal the paramount role of adenosine as a neuromodulator in the hippocampus during neuronal insults, furthering our understanding of the mechanism of neuronal death in hypoxic and ischemic conditions.The role of adenosine as an endogenous neuromodulator is well established, but the mechanism(s) mediating the extensive modulatory and regulatory actions of adenosine have not yet been fully elucidated. In fact, although adenosine, through activation of adenosine A1 and A2A receptors, has been demonstrated as neuroprotective or neurodegenerative, respectively, little is known about the mechanism by which adenosine mediates these actions. In the hippocampus, essential physiological processes rely on adenosine signaling, including regulation of long-term potentiation (LTP) and long-term depression (LTD). Neuromodulation by adenosine is dominantly inhibitory in the hippocampus, mediated by the abundant and high-affinity adenosine A1 receptor. In ischemia and hypoxia, A1 receptor activation induces rapid synaptic depression which is mediated by multiple signaling pathways including the induction of excitatory AMPA glutamate receptor internalization, which inhibits synaptic transmission in the hippocampus. Considerable effort has been devoted to investigating the role of adenosine in ischemic stroke, due to the fact that in cerebral ischemia or hypoxia, extracellular levels of adenosine increase dramatically. This thesis explores the functional consequences of adenosine signaling in hypoxia and ischemia, which mediate GluA1 AMPA receptor subunit internalization. Three major serine/threonine protein phosphatases (PPs), PP1, PP2A, and PP2B are investigated and shown to mediate A1 receptor-mediated GluA1 internalization in hypoxic conditions in the rat hippocampus. Further experiments demonstrate the role of adenosine A2A receptors in potentiating hippocampal synaptic transmission in reperfusion by increasing GluA1 surface expression through increased phosphorylation of regulatory C-terminal phosphorylation sites of GluA1. The mechanism of extracellular adenosine regulation by equilibrative nucleoside transporters (ENTs) and casein kinase 2 (CK2) are examined and shown to interact in hypoxia/reperfusion experiments on hippocampal slices. Finally, using a pial vessel disruption (PVD) permanent focal cortical ischemia stroke model, experiments demonstrate increased adenosine tone in the hippocampus, which mediates increased adenosine-induced synaptic depression. CK2 inhibition was also neuroprotective after 20min hypoxia. This shows that adenosine tone is increased in the hippocampus after a small cortical stroke, implying a potential global effect of focal ischemia. Together, these studies further reveal the paramount role of adenosine as a neuromodulator in the hippocampus during neuronal insults, furthering our understanding of the mechanism of neuronal death in hypoxic and ischemic conditions.

Thesis (Ph. D.)--Georgia State University, 2007. / Title from file title page. Chun Jiang, committee chair; Teryl Frey, Deborah Baro, Delon Barfuss, committee members. Electronic text (168 p. : ill. (some col.)) : digital, PDF file. Description based on contents viewed Jan. 30, 2008. Includes bibliographical references (p. 146-151).

Thesis (Ph.D.)--McMaster University, 2002. / Advisor: R.S. Gupta. Includes bibliographical references. Also available via World Wide Web.

Thesis (Ph.D.)--McMaster University, 2002. / Advisor: R.S. Gupta. Includes bibliographical references. Also available via World Wide Web.

Thesis (Ph.D. in Molecular Biology) -- University of Colorado at Denver and Health Sciences Center, 2006. / Typescript. Includes bibliographical references (leaves 67-74). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

Dissertation (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Vita. Bibliography: p. 92-107.

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

Thesis (M.S.)--University of Missouri-Columbia, 2008. / 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 August 28, 2008) Vita. Includes bibliographical references.