Thesis (Ph. D.)--University of Chicago, Committee on Neurobiology, August 1999. / Includes bibliographical references. Also available on the Internet.

Thesis (Ph.D.)--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 March 10, 2009) Includes bibliographical references.

Thesis (M.S.)--Missouri State University, 2009. / "May 2009." Includes bibliographical references (leaves 42-44). Also available online.

No description available.

No description available.

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

Chemokine-induced macrophage recruitment into the vascular wall is an early pathological event in the progression of atherosclerosis. Macrophage activation and chemotaxis during cell recruitment are mediated by chemokine ligation of multiple G- protein coupled receptors. The Regulator of G-Protein Signalling-l (RGS-l) acts to down-regulate the response to sustained chemokine stimulation. Studies in this laboratory have shown Rgsl is up-regulated in atherosclerotic ApoE1- mice in association with atherosclerotic plaque progression and published findings have reported that RGS 1 is highly expressed in leukocytes. However an in vivo role for RGS-l in macrophage function or in atherosclerosis has not been investigated. This thesis aimed to address the hypothesis that RGS 1 has an important role in atherosclerosis and modulates the inflammatory response by controlling chemokine signalling and macrophage chemotaxis to atherosclerotic plaques. To investigate the role of RGS 1 in macrophage function and the development of atherosclerosis, Rgsrl- mice were characterised on the ApoE1- background. Flow cytometric analysis of leukocytes in blood, spleen and bone marrow indicated Rgsrl- ApoE1- mice had no significant differences in the numbers of monocytes or lymphocytes compared to ApoE1- mice. Rgsl was found to be highly expressed in macrophages from ApoE1- mice compared to B-Iymphocytes, where it has a non-redundant role, and other cells involved in plaque formation. Furthermore, Rgsl is up-regulated with monocyte- macrophage activation by innate stimuli. For the first time, RGS 1 'was shown to affect chemokine receptor signalling in macrophages in vitro. RgsrlApoE1- macrophages showed significantly enhanced chemotaxis to CCL2, CCL3 and CCLS and impaired homologous desensitisation to the chemokine CCLS in comparison to ApoE1- cells. To determine the role of RGS-l in leukocyte trafficking and atherosclerosis, a detailed atherosclerosis study was carried out. RgsrlApoE1- mice had significantly less lesion formation in the aortic roots at 9-weeks and in the aorta at 16-weeks on a chow diet in comparison to ApoE1- mice. This was accompanied with decreased macrophage content in the aortic root at 9-weeks. To further investigate aortic leukocyte recruitment, an Angiotensin IT-induced model of acute vascular inflammation was used. At 9 weeks of age, Rgsrl-ApoE1- mice had significantly less aortic CD4S+ leukocytes and cons' myeloid cells recruited to the aorta in comparison to ApoE1- mice. Collectively, these findings identify a new role for RGS-l in macrophage function and support a role for RGS-l in leukocyte recruitment and retention in the initial stages of atherosclerotic plaque formation. These results identify RGS 1 as a novel target for the treatment of acute vascular inflammation and early atherosclerosis.

氫化酶作為一種催化劑，能催化氫分子成為質子及電子的相互轉換。 [鎳鐵]- 氫化酶散播最廣的一種氫化酶，從古菌到細菌都能找到 [鎳鐵]- 氫化酶。完整成熟的 [鎳鐵]-氫化酶需要插入鐵、氰化物、一氧化碳以及鎳到它的催化核心。這複雜的過程需要其它由若干 hyp 基因編譯的輔助蛋白酶的幫助，其中蛋白HypA 與 HypB 負責將鎳運送到[鎳鐵] -氫化酶的催化核心。敲除了 hypA 或hypB 基因的細菌株缺失[鎳鐵] -氫化酶的活性，如在生長介質裡添補鎳可恢復部份[鎳鐵] -氫化酶的活性。當HypB 與鳥嘌呤核苷酸結合時會變成蛋白二聚體。對比HypB 脫輔基蛋白及與HypB 與鳥嘌呤三核苷酸類似物的蛋白複合物的晶體結構可發現，HypB 透過一個保守賴氨酸殘基( Archaeoglobus fulgidus HypB 的殘基 148 )組成分子間鹽橋以構成蛋白二聚體。Escherichia coli 的體內實驗顯示，此保守賴氨酸殘基對活性氫化酶的製造起必要的作用，反映由此殘基所構成的鹽橋對HypB 功能的重要性。此外，本研究展示了A. fulgidusHypA 及 HypB 蛋白之間的相互作用。通過在A. fulgidus HypB 上進行系統性的突變，發現HypB 利用其GTP 酶域上的一段氨基端區域與HypA 相互作用。跟據這個結果，我們進而在E. coli HypB 上發現了兩個保守的非極性殘基與HypA 相互作用。當以丙氨酸取代在HypB 上的這兩個非極性殘基時，HypB 無法激活E. coli 中的氫化酶，導置降低的氫化酶活性，這表明了HypA 和HypB 的相互作用對[鎳鐵] -氫化酶成熟過程的必要性。 / Hydrogenases catalyze the inter-conversion of molecular hydrogen into protons and electrons. [NiFe]-hydrogenase is the most widely distributed hydrogenases, which is found in organisms ranging from archaea to bacteria. Maturation of [NiFe]-hydrogenase requires the insertion of iron, cyanide and carbon monoxide, followed by nickel, to the catalytic core of the enzyme. The maturation process of hydrogenase is a complicated procedure, which requires many accessory proteins encoded by hyp genes. HypA and HypB participate in the nickel delivery step to the catalytic core of hydrogenase, which is supported by the fact that strain deficient in hypA or hypB gene lack hydrogenase activity which can be recovered partially by elevating nickel content in the medium. HypB is capable to form dimer in solution upon guanine nucleotide binding. By comparing the crystal structures of HypB in dimer and monomer form, an important lysine residue (residue 148 in A. fulgidus HypB) which is required to form an intermolecular salt bridge during GTP-dependent dimerization, has been identified. Substitution of this lysine resiue with alanie would break HypB dimer in vitro. In vivo complementation study in E. coli showed that the corresponding lysine residue in E. coli HypB is required for active hydrogenase production indicating the importance of this intermolecular salt bridge to the biological function of HypB. Besides, interaction between A. fulgidus HypA and HypB are demonstrated in this work. By making systematic mutation to A. fulgidus HypB, the N‐terminal region of the GTPase‐domain has been identified to be important for its interaction with HypA. Further mutagenesis study has been done on E. coli HypB and two conserved non‐polar residues responsible for interaction with HypA have been identified. Alanine substitution of these conserved non‐polar residues result in HypB mutants which failed to rescue hydrogenase activity in vivo in E. coli showing that HypA/HypB interaction is required for hydrogenase maturation. / Detailed summary in vernacular field only. / Chan, Kwok Ho. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 88-95). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Chapter Chapter 1 --- Introduction: Hydrogenase biosynthesis requires insertion of nickel facilitated by protein HypA and HypB --- p.1 / Chapter 1.1 --- What is hydrogenase? --- p.1 / Chapter 1.2 --- [NiFe] hydrogenase contains a complex catalytic core composed of metal atoms and diatomic ligands --- p.2 / Chapter 1.3 --- The [NiFe] catalytic core --- p.4 / Chapter 1.4 --- Building the catalytic [NiFe] core --- p.4 / Chapter 1.5 --- Nickel insertion into the hydrogenase precursor involves the proteins HypB, HypA and SlyD --- p.7 / Chapter 1.5.1 --- Protein HypB --- p.7 / Chapter 1.5.2 --- Protein HypA --- p.11 / Chapter 1.5.3 --- Protein SlyD --- p.12 / Chapter 1.6 --- Objectives - How HypB dimerization and HypA/HypB interaction are involved in hydrogenase maturation process? --- p.13 / Chapter Chapter 2 --- A conserved Lys residue is required for GTP-dependent dimerization and hydrogenase maturation --- p.17 / Chapter 2.1 --- Introduction --- p.17 / Chapter 2.2 --- Materials and Methods --- p.22 / Chapter 2.2.1 --- Recombinant Plasmid Construction --- p.22 / Chapter 2.2.2 --- HypB mutant construction by site-directed Mutagenesis --- p.22 / Chapter 2.2.3 --- Protein Expression and purification --- p.23 / Chapter 2.2.4 --- HypB protein purification --- p.23 / Chapter 2.2.5 --- Analytical gel filtration chromatography coupled with Light Scattering (SEC/LS) --- p.24 / Chapter 2.2.6 --- Nucleotide binding affinity determination --- p.25 / Chapter 2.2.7 --- GTPase activity determination --- p.26 / Chapter 2.2.8 --- Sample preparation for hydrogenase activity assay --- p.26 / Chapter 2.2.9 --- Hydrogenase activity determination --- p.27 / Chapter 2.3 --- Results --- p.29 / Chapter 2.3.1 --- AfHypB undergoes GTP-dependent dimerization --- p.29 / Chapter 2.3.2 --- Analysis of Structural difference between the apo form and GTP S-bound form suggests a mechanism of GTP-dependent dimerization for HypB --- p.30 / Chapter 2.3.3 --- Lys-148 is essential for GTP-dependent dimerization --- p.31 / Chapter 2.3.4 --- Disruption of dimerization by K148 mutation did not affect nucleotide binding and GTP hydrolysis activity significantly --- p.32 / Chapter 2.3.5 --- The conserved lysine residue is required for hydrogenase maturation in E. coli --- p.33 / Chapter 2.4 --- Discussion --- p.45 / Chapter 2.4.1 --- A conserved intermolecular salt‐bridge is required for GTP-dependent dimerization of HypB and hydrogenase maturation --- p.45 / Chapter 2.4.2 --- The extra metal binding site at the dimeric interface of HypB may provide a mechanism of why GTP-dependent dimerization is essential to Ni insertion --- p.46 / Chapter Chapter 3 --- N-terminal region of GTPase‐domain of HypB is required for interaction with HypA --- p.51 / Chapter 3.1 --- Introduction --- p.51 / Chapter 3.2 --- Methods and materials --- p.53 / Chapter 3.2.1 --- Recombinant Plasmid Construction --- p.53 / Chapter 3.2.2 --- HypB variant construction by site‐directed Mutagenesis --- p.53 / Chapter 3.2.3 --- Protein Expression --- p.54 / Chapter 3.2.4 --- Tag‐free AfHypA and AfHypB purification --- p.54 / Chapter 3.2.5 --- Analytical size exclusion chromatography coupled with Light Scattering --- p.54 / Chapter 3.2.6 --- GST pull‐down of GST‐AfHypA and AfHypB --- p.55 / Chapter 3.2.7 --- Tandem affinity pull‐down of GST‐EcHypA and His‐SUMO‐EcHypB --- p.55 / Chapter 3.2.8 --- GST pull‐down of GST‐EcHypA and His‐SUMO‐EcHypB --- p.56 / Chapter 3.2.9 --- Hydrogenase activity determination --- p.57 / Chapter 3.3 --- Results --- p.58 / Chapter 3.3.1 --- HypA and HypB from A. fulgidus form 1:1 heterodimer in solution --- p.58 / Chapter 3.3.2 --- The N‐terminal regions upstream of the first helix of A. fulgidus HypB is required for HypA-HypB interaction --- p.59 / Chapter 3.3.3 --- Two conserved hydrophobic residues on HypB from E. coli are required to interact with HypA --- p.60 / Chapter 3.3.4 --- HypA-HypB interaction is required for hydrogenase maturation in E. coli --- p.62 / Chapter 3.4 --- Discussion --- p.73 / Chapter 3.4.1 --- The N‐terminal region of the GTPase domain is required for interaction with HypA and hydrogenase maturation in E. coli --- p.73 / Chapter 3.4.2 --- Location of interaction site on HypB reveals possible role for HypA/HypB interaction --- p.74 / Chapter 3.4.3 --- Mode of specific interaction with HypA: Interaction via a disordered region implies a coupled folding and binding process --- p.75 / Chapter Chapter 4 --- Conclusion and Future Perspectives --- p.80 / Chapter A1.1 --- Summary of findings in this work --- p.80 / Chapter A1.2 --- Implications in hydrogenase maturation --- p.81 / Chapter A1.3 --- Questions unresolved --- p.82 / Chapter 4.3.1 --- Factors that activate GTPase activity of HypB are still elusive --- p.82 / Chapter 4.3.2 --- How nickel delivery is regulated by HypA/HypB complex is still unclear --- p.83 / References --- p.88 / Chapter Appendix 1 --- Preliminary results of HypA/HypB protein complex structural study --- p.96 / Chapter A1.1 --- Structural study may provide invaluable insights to the role of HypA‐HypB interaction --- p.96 / Chapter A1.2 --- X‐ray crystallography as an approach to determine HypA/HypB complex structure --- p.96 / Chapter A1.3 --- Initial crystal hits were obtained with purified AfHypA/HypB complex --- p.97 / Chapter Appendix 2 --- Publications associated to the thesis --- p.100 / Chapter Appendix 3 --- Constructs and Primers used --- p.101

No description available.

The excitotoxin domoic acid (DOM) acts at both kainic acid (KA)- and α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-sensitive glutamate receptors. Clinical reports suggest that elderly people are hypersensitive to the neurological effects of DOM intoxication. Young, but not aged hippocampal slices which have been preconditioned with low concentrations of DOM or KA exhibit an acute �tolerance� to subsequent high doses of DOM or KA; application of the selective AMPA agonist fluorowillardiine (FW) fails to induce tolerance to excitotoxins. The aim of this study was to further investigate the molecular mechanism of tolerance induction in vitro, and to examine the ability of compounds to cross-condition against excitotoxic insult. In addition, in vivo techniques were used to explore the age-related susceptibility to the neurological effects of DOM and acute in vivo tolerance induction. Here we show that low doses of �classical� ionotropic kainate receptor agonists and AMPA/kainate receptor antagonists act as net inverse agonists at G-protein coupled receptors, reducing constitutive GTPase activity by up to 73% in the young hippocampus. Further evidence that inverse agonist activity at G-protein coupled receptors is responsible for acute in vitro tolerance induction by kainate receptor agonists and antagonists was also identified because preconditioning with the AMPA receptor antagonist GYKI-52466 significantly inhibited KA-induced population spike suppression in in vitro hippocampal brain slices from both Sprague-Dawley and Wistar rats. The broad-spectrum protein kinase inhibitor H-7 partially blocked tolerance induction when preconditioning occurs in the presence of suggesting that protein kinases are one of the downstream effectors of this phenomenon. Tolerance-inducing compounds are also capable of cross-conditioning against the effects of other excitotoxins; with 250 nM FW suppressed population spike area by only 62.8 � 10.0% at 30 minutes following a 500 nM KA preconditioning dose, compared to almost complete spike suppression within twenty minutes in naive hippocampal brain slices. In vivo experiments indicated that despite aged animals exhibiting significantly higher cumulative behavioural scores in response to i.p. DOM (1 mg kg⁻�; young = 102 � 9, aged = 179 � 19; P < 0.01) in response to DOM after two hours), and that this age-related supersensitivity is due to impaired renal clearance (young serum DOM = 41.5 � 30.3 ng ml⁻�, aged = 813.3 � 804.4 ng ml⁻� following administration of 1 mg kg⁻� DOM after 2.5 hours earlier). Tolerance to high doses of DOM was induced within a matter of minutes following i.p. preconditioning by low dose DOM in vivo. This was evidenced by severe seizure manifestations being almost absent in both young and aged animals, despite occurring frequently in naive animals. Therefore, this study concludes that tolerance is induced by kainate receptor ligands in vitro and in vivo within a matter of minutes, and is the result of a reduction in the turnover of G-protein coupled receptors and protein kinase activation. In addition, the increased sensitivity of aged rats to in vivo DOM is a result of elevated serum DOM concentrations most likely resulting from impaired renal clearance.