No description available.

No description available.

No description available.

Molecular-targeting therapeutics directed towards growth factor receptors have become promising interventions in cancer. They include the family of mammalian receptor tyrosine kinases such as epidermal growth factor, TrkA and insulin. In particular, the insulin receptor (IR) is one of the most well-known members of the RTK family of receptors playing a role in cancer. IRs are covalently-linked heterodimers of αβ subunits on the cell membrane in the absence of insulin. The IR signaling pathways are initially triggered by insulin binding to the α subunits followed by the interaction of β subunits and ATP. The parameter(s) controlling IR activation remains unknown. Here, we report a membrane receptor signaling platform initiated by insulin binding to its receptor to induce Neu1 in live HTC-IR and MiaPaCa-2 cell lines. Microscopy colocalization and co-immunoprecipitation analyses reveal that Neu1 and MMP9 form a complex with naïve and insulin-treated receptors. Tamiflu (neuraminidase inhibitor), galardin and piperazine (broad range MMP inhibitors), MMP9 specific inhibitor and anti-Neu1 antibody blocked Neu1 activity associated with insulin stimulated live cells. Moreover, Tamiflu, anti-Neu1 antibody, and MMP9 specific inhibitor blocked insulin induced insulin receptor substrate-1 phosphorylation (p-IRS1). The previous findings reveal a molecular organizational signaling platform of Neu1 and MMP-9 crosstalk in alliance with insulin receptors. It proposes that insulin binding to the receptor induces MMP9 to activate Neu1, which hydrolyzes α-2,3 sialic acid in removing steric hindrance to generate a functional receptor. The results predict a prerequisite desialylation process by activated Neu1. A complete understanding of IR activation and the role of sialic acids in the iii signaling pathways may provide a therapeutic strategy in the prevention of different diseases such as diabetes mellitus and cancer. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2013-02-20 11:27:44.861

Schizophrenia is characterized by positive and negative symptoms along with cognitive symptoms that include impairment in working memory (WM). WM is the storage of relevant information for short intervals of time to guide thoughts and actions. The neural correlate of WM is thought to be the persistent activity exhibited during the retention interval of WM tasks. Persistent activity is hypothesized to be mediated by the activation of NMDA receptors (NMDAR) within recurrent neuronal circuits. Consistent with this hypothesis, studies with healthy humans and monkeys have shown that the administration of the NMDAR antagonist ketamine induces memory-load dependent deficits in WM, along with increasing response time. In parallel to this, the pathophysiology of schizophrenia has been hypothesized to rest on the hypofunction of NMDAR. Previous studies in humans indicate that blockade of NMDAR induces schizophrenia-like symptoms. In addition, symptoms of schizophrenia patients are alleviated with sub-chronic treatments focusing on the activation of the NMDAR co-agonist site. Based on these observations, I tested the hypothesis that increasing the activation of NMDAR with co-agonist stimulation has beneficial effects on WM. D-serine (100mg/kg/day-6 weeks) was orally administered to two female macaque monkeys performing a visual sequential comparison task (VCST), which allows the manipulation of memory load. In this task, the monkeys had to identify the location of a colour change within an array of 2 to 5 coloured stimuli following a retention interval of 1 second. I hypothesized that sub-chronic treatment with D-serine produces a gradual improvement in the monkeys’ performance on the VSCT. Specifically, I predicted that the improvement would scale with memory load due to increased demands on WM resources at higher loads. Contrary to my hypothesis, D-serine produced minute changes in response accuracy, which were not memory load-dependent. Also, the response latency of the monkeys was found to increase, which is commonly observed following NMDAR antagonist treatments. These findings suggest that D-serine has a limited role in increasing the activation of NMDARs to improve WM per se. The beneficial effects reported by NMDAR co-agonists in schizophrenic patients could be a general reduction in cognitive symptoms, not specifically related to WM. / Thesis (Master, Neuroscience Studies) -- Queen's University, 2013-10-01 17:45:20.643

In diabetes the body lacks the mechanism for producing insulin. This disease is one of the most prevalent in the world, causing a tremendous loss of health, life and economy. Thus, there is a need for developing novel therapies effective in control of diabetes. In an effort to develop such a therapy we have targeted G-protein coupled receptors (GPCRs) to stimulate 13-cells for insulin secretion. GPCRs are membrane bound receptors which respond to a variety of external signals and mediate intracellular signal Stransduction. GPCRs, therefore, are the targets of many current therapeutic drugs. The objective of this study was to generate chimeric receptors containing portions of two closely related GPCRs to identify domains important in binding various ligands to stimulate increased secretion of insulin by f3-cells of the pancreas. In this collaborative research with Kelly Wilbur of Eli Lilly, domains of receptors GPR40 and GPR41 were exchanged at different regions to construct two chimeric receptors (GPR40.431_41.459 and GPR40.567 41.547) using two separate cloning steps to insert these fragments sequentially into the cloning vector, pcDNA3.1. Construction of the chimeric receptors was carefully planned to include specific amino acid residues important in ligand binding. Priority was given to locate the joining section of the two receptor portions at the transmembrane region and to maintain full length of the receptor. This was to maintain the integrity of external and internal loops of the receptors important in ligand binding and signal transduction. Following transformation of the chimeras into E. coli to obtain sufficient DNA, construction of the desired chimeric receptors was verified by agarose gel electrophoresis for size and by PCR for the presence of the correct portions of each receptor. The two constructs were sent to Eli Lilly for sequencing. One construct was found to be appropriately constructed (GPR40.431_GPR41.459) but the other one was unstable and had undergone recombination as is often seen in cloned membrane proteins which can be toxic to E. coli. In the future, Human Embryonic Kidney cells will be transfected with the chimeric receptor and a FLIPR analysis will be performed to assess the activity of the receptor when stimulated by ligands of interest to Eli Lilly. Construction of additional chimeras will be needed in the future to fully understand the specific regions responsible for ligand binding and activation of GPR40 to aid in the design of drugs to stimulate insulin secretion by 03-cells. / Department of Biology

No description available.

P2X7 receptors belong to a family of membrane bound ion channels which are activated by extracellular ATP, resulting in the opening of a non-selective cation channel. After prolonged or repeated exposure to agonist, functional and cellular changes can occur, including the formation of a large pore, cell lysis and the release of mature, biologically active interleukin-1β. It is this diversity of functions that underlies the significance of this receptor in pain processing. P2X7 receptors are expressed on microglia, which when activated, release a host of mediators which contribute to central sensitisation, a phenomenon associated with neuropathic pain. The role of P2X7 receptors in the activation of microglia is less well established and is the main subject of this thesis. Before considering the interaction between P2X7 receptors and microglia, the first aim was to establish whether P2X7 receptors played a role in a pathological process known to be associated with microglial activation. An additional aim was to establish whether the site of action was in the central nervous system (CNS), where microglia are located. These aims were accomplished using a surgery-based rat model of neuropathic pain, the chronic constriction injury (CCI) model, and by comparing the effects of different P2X7 receptor antagonists when dosed peripherally or directly into the spinal cord. The results indicated that P2X7 receptor antagonists produced efficacy in the CCI model via a mechanism located in the CNS. To further investigate the association between P2X7 receptors and microglia, a different experimental paradigm was explored. Chronically dosed morphine is known to activate microglia, the consequence of which is thought to underlie morphine tolerance and reduced morphine analgesia. By administering a P2X7 receptor antagonist to CCI-operated rats treated with chronic morphine, the interaction between the P2X7 receptor and morphine tolerance and analgesia was explored. The results showed that P2X7 receptor antagonism delayed morphine tolerance and increased the efficacy of low doses of morphine, suggesting an association between P2X7 receptors and microglia. It was intended to confirm the interaction between a P2X7 receptor antagonist and morphine in another neuropathic pain model, namely varicella zoster virus-induced neuropathy. However due to a lack of reproducibility, this model was not used for pharmacological studies. Having demonstrated an association between P2X7 receptor antagonist and morphine in a chronic pain setting, studies were initiated to explore whether this interaction occurred in other morphine-related behaviours. The effect on body weight, motor coordination and single dosed morphine-induced analgesia was assessed in rats co-administered with P2X7 receptor antagonist and morphine. Results demonstrated that the blockade of P2X7 receptors enhanced morphine acute dose-induced analgesia, but had no influence on motor-impairment and body weight. The final part of the thesis used immunohistochemical and molecular techniques to confirm that microglia played a role in established allodynia induced by CCI-surgery and that P2X7 receptors directly influenced microglia-activation. In conclusion, the data in this thesis has illustrated an association between centrally activated P2X7 receptors and microglia, as well as an association between the P2X7 receptor and morphine-induced tolerance and analgesia. It is possible that co-administration of a P2X7 receptor antagonist with morphine could reduce the effective dose of morphine clinically, thereby reducing the side effects of this commonly used analgesic.

No description available.

Roundabout (Robo) receptors were first identified in neurons as guidance molecules, however growing evidence suggests that they also play a role in other cells. The aim of this thesis was to characterise the expression and function of a novel endothelial specific member of this family, Robo4. This study revealed that Robo4 is expressed primarily in vessels but also differentially expressed in tumour vessels. Interestingly Robo4 was primarily located within cytoplasmic vesicles coated with clathrin, suggesting that the presence of Robo4 on the cell surface is being tightly regulated. Overexpression of Robo4 induced filopodia and pseudopodia formation and actin re-organisation into stress fibres. It co-localised with actin and tubulin suggesting an important interaction between Robo4 and the cytoskeleton. Robo4's function in endothelial cells was directly investigated using two approaches, overexpression using adenovirus and knockdown using small interfering RNA. Functional cell-based assays revealed that disrupting Robo4's level of expression negatively affects endothelial cell functions that are required during angiogenesis, such as proliferation, migration and tubulogenesis. Overexpression of a truncated version of Robo4, which lacks the C-terminus, provided clues regarding Robo4's function. The intracellular domain is critical for Robo4's localisation and its association with the cytoskeleton. It is also required for pseudopodia formation. Other findings include possible cleavage of Robo4 and Robo4 homodimerisation and heterodimerisation with Robo1. Taken together, the findings presented in this study strongly suggest a role for Robo4 in endothelial cell guidance. Cell guidance during angiogenesis is poorly understood therefore the identification of a new molecule potentially involved in this mechanism will hopefully help elucidate the process.