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Investigating α7 nicotinic receptor STAT3 signaling and ... 1743/fulltext.pdf Investigating 7 nicotinic receptor STAT3 signaling and cell-dependent expression Thesis Presented By

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Text of Investigating α7 nicotinic receptor STAT3 signaling and ... 1743/fulltext.pdf Investigating 7...

  • 1

    Investigating 7 nicotinic receptor STAT3

    signaling and cell-dependent expression

    Thesis Presented

    By

    Thomas M. Koperniak

    To

    The Bouve Graduate School of Health Sciences

    in Partial Fulfillment of the Requirements for the

    degree of Doctor of Philosophy in Pharmaceutical Sciences

    with specialization in

    Pharmacology

    NORTHEASTERN UNIVERSITY

    BOSTON, MASSACHUSETTS

    2012

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    Northeastern University

    Bouve Graduate School of Health Sciences

    Thesis title: Investigating 7 nicotinic receptor STAT3 signaling and cell-dependent

    expression

    Author: Thomas M. Koperniak

    Program: Department of Pharmaceutical Sciences

    This project satisfies all research requirements for the Doctoral Degree

    Thesis Committee (Chairman) ___________________________Date________

    ___________________________Date________

    ___________________________Date________

    ___________________________Date________

    ___________________________Date________

    Director of Graduate School ___________________________Date________

    Dean ___________________________Date________

    Copy Deposited at Library ___________________________Date________

  • iii

    Northeastern University

    Bouve Graduate School of Health Sciences

    Thesis title: Investigating 7 nicotinic receptor STAT3 signaling and cell-dependent

    expression

    Author: Thomas M. Koperniak

    Program: Department of Pharmaceutical Sciences

    This project satisfies all research requirements for the Doctoral Degree

    Thesis Committee (Chairman) ___________________________Date________

    ___________________________Date________

    ___________________________Date________

    ___________________________Date________

    ___________________________Date________

    Director of Graduate School ___________________________Date________

    Dean ___________________________Date________

    Copy Deposited at Library ___________________________Date________

  • iv

    LIST OF ABBREVIATIONS

    5HT Serotonin

    AA Arachidonic acid

    BGT -Bungarotoxin

    AC Adenylate cyclase

    AChBP Acetylcholine binding protein

    ACh Acetylcholine

    AD Alzheimer’s disease

    ADP Adenosine diphosphate

    ADSS Aged and diluted sidestream smoke

    ANOVA Analysis of variation

    AS Antisense

    ATP Adenosine triphosphate

    BAPTA-AM Bis(2-aminophenoxyl)ethane tetraacetic acid

    BATH-42 BTB-associated Traf homology 42

    BiP Binding immunoglobulin protein

    BLAST Basic Local Alignment Search Tool

    bp Base pairs

    BSA Bovine serum albumin

    CA Constitutively active

    cAMP Cyclic adenosine monophosphate

    CC Coiled-coil domain

    cDNA Complementary DNA

    CMV Cytomegalovirus

    CNS Central nervous system

    CREB cAMP response element-binding

    CTZ Coelenterazine

    DA Dopamine

    DHE Dihydro-beta-erythroidine

    DHS Donor horse serum

    DMEM Dulbecco’s modified Eagle’s medium

    DMSO Dimethyl sulfoxide

    DN Dominant negative

    dsRNA Double stranded RNA

    EGFP Enhanced green fluorescent protein

    EGFR Epidermal growth factor receptor

    EGTA Ethylene glycol tetraacetic acid

    ELISA Enzyme-linked immunosorbent assay

    ER Endoplasmic reticulum

    ERK Extracellular signal-regulated kinase

    FBS Fetal bovine serum

    FDA Food and Drug Administration

    FGF2 Fibroblast growth factor 2

    GABA Gamma-aminobutyric acid

    GAPDH Glyceraldehyde phosphate dehydrogenase

    GFP Green fluorescent protein

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    Glu Glutamate

    HBSS Hank’s balanced salt solution

    HEK Human embryonic kidney

    hRic3 Human Ric3

    HRP Horseradish peroxidase

    Hsp90 Heat shock protein 90

    IBD Inflammatory bowel disease

    IDT Integrated DNA Technologies

    IL6 Interleukin 6

    IP Intraperitoneal

    IP3 Inositol phosphate 3

    JAK2 Janus kinase 2

    KC Keratinocyte

    LBD Ligand binding domain

    LD-PCR Long distance-polymerase chain reaction

    LPS Lipopolysaccharide

    MAPK Mitogen-activated protein kinase

    MIP2 Macrophage inflammatory protein 2

    ML Metridia luciferase

    MLA Methyllycaconitine

    Mnmc Manumycin A

    mRic3 Mouse Ric3

    mRNA Messenger RNA

    mTOR Mammalian target of rapamycin

    NAc Nucleus accumbens

    nAChR Nicotinic acetylcholine receptor

    NBM Nucleus basilis of Meynert

    NFB Nuclear factor kappa B

    NMDA N-Methyl-D-aspartate

    NMJ Neuromuscular junction

    NNK Nicotine-derived nitrosamine ketone

    NNN N'-nitrosonornicotine

    NNR Neuronal nicotinic receptor

    nSCLC Non-small cell lung cancer

    p Probability

    P9KB pREP9-kanamycin-blasticidin

    P9KG pREP9-kanamycin-geneticin

    PBS Phosphate buffered saline

    PJAK2 Phosphorylated JAK2

    PI3K Phosphoinositol-3 kinase

    PKA Protein kinase A

    PLC Phospholipase C

    PM Plasma membrane

    PMSF Phenylmethylsulfonyl fluoride

    PNS Peripheral nervous system

    PPT Pedunculo-pontine nucleus

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    PSTAT3 Phosphorylated STAT3

    RFP Red fluorescent protein

    Ric3 Resistance to inhibitors of cholinesterase-3

    RIPA Radio-immunoprecipitation assay

    RISC RNAi-induced silencing complex

    RNAi RNA interference

    ROS Reactive oxygen species

    RT Reverse transcriptase

    S-rRic3 Serine (absent) rat Ric3

    S+rRic3 Serine (present) rat Ric3

    SCLC Small cell lung cancer

    sdrRic3 Splice-deleted rat Ric3

    SEAP Secreted alkaline phosphatase

    SEM Standard error of the mean

    shRNA Short hairpin RNA

    siRNA Small interfering RNA

    SMART Switching mechanism at 5’ end of RNA template

    SN/SNc Substantia nigra

    SOCS3 Suppressor of cytokine signaling 3

    SS Signal sequence

    STAT3 Signal transducer and activator of transcription-3

    STR Striatum

    TBS Tris buffered saline

    TBS-T Tris buffered saline-Triton

    THAL Thalamus

    TLR Toll-like receptor

    TM Transmembrane domain

    TNF Tumor necrosis factor

    Tyk Tyrosine kinase

    UNC-50 Uncoordinated-like protein 50

    UT Untransfected

    US United States

    Vegf Vascular endothelial growth factor

    VGCC Voltage-gated calcium channel

    VTA Ventral tegmental area

    WT Wild-type

    XIAP X-linked inhibitor of apoptosis protein

  • vii

    ABSTRACT

    Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels

    consisting of a combination of at least 17 possible subunits. Although most receptor subtypes

    are heteromeric, 7 subunits can assemble into homomeric receptors. 7 nicotinic receptors are

    the 2 nd

    most highly expressed nicotinic receptor in the brain, and their high permeability to

    calcium ions allows participation in calcium-dependent processes such as neurotransmitter

    release and immune modulation. Although these receptors have been well-studied, 7-mediated

    STAT3 signaling and 7 maturation remain unclear. The two major hypotheses we are

    investigating stem from these issues. The first hypothesis, regarding 7-mediated STAT3

    activation, is that 7 drives STAT3 signaling independently of calcium ion flow. The second

    hypothesis, regarding 7 maturation, is that rat pituitary-derived GH4C1 cells possess Resistance

    to Inhibitors of Cholinesterase 3 (Ric3) to allow surface 7 expression.

    The major advancement of this project is the development of a novel STAT3-Metridia

    luciferase (ML) signaling assay using heterologous expression systems. To our knowledge, this

    is the only study using heterologous expression systems to investigate such signaling. To study

    7-mediated STAT3 signaling, hu