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Semaphorin 5B : an inhibitory transmembrane guidance cue reveals its secretable function Lett, Robyn Lynn Mwuese 2009

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Semaphorin 5B: An Inhibitory Transmembrane Guidance Cue Reveals Its Secretable Function by Robyn Lynn Mwuese Lett  B.Sc. Carleton University, 2001 M.Sc. University of British Columbia, 2003  A THESIS SUBMITTED IN PARTIAL FULFULLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in The Faculty of Graduate Studies (Neuroscience)  The University of British Columbia (Vancouver)  October 2009  © Robyn Lynn Mwuese Lett, 2009  Abstract Corticofugal axons projecting to the thalamus, brainstem and spinal cord must travel the same initial trajectory through the subcortical lateral and medial ganglionic eminences, and are therefore likely subject to the same sets of guidance cues. These cues direct the course of corticofugal axons bringing them to each intermediate target, until they reach the diencephalic-telencephalic boundary, where axons targeting the thalamus turn dorsally and brainstem and spinal cord targeting axons turn ventrally. Many of these guidance cues have been elucidated, yet there are still gaps in our understanding of the formation of the corticofugal projection.  I found that Sema5B expression flanked the presumptive internal capsule during its formation, and was therefore ideally situated both spatially and temporally to act as an instructive cue for descending cortical axons. In Chapter 2, I show that Sema5B is not only capable of inhibiting cortical axons in vitro, but can cause misguidance of cortical axons in slice culture when placed ectopically over normally non-Sema5B expressing regions. In addition, I show that the loss of Sema5B from the neocortical VZ resulted in aberrant penetration of this normally avoided region. Therefore Sema5B is both necessary and sufficient to inhibit the corticofugal projection.  Semaphorins and their plexin receptors are frequently proteolyzed to modulate the elicited responses in navigating growth cones. In Chapter 3 I show that Sema5B cleavage results in an inhibitory fragment that in heterologous cells can produce inhibitory gradients for cortical ii | P a g e  explants in collagen gel co-cultures, and collapse dissociated cortical neuronal growth cones, an effect that can be blocked with a function disrupting antibody to the cell adhesion molecule TAG-1.  This thesis shows that Sema5B, a guidance cue with a hitherto unknown function, is responsible for a very important aspect of cortical development. My work leads to a final proposal that Sema5B is in fact a two-in-one protein with separable inhibitory and alternate complex functions, the implications of which are discussed thoroughly in Chapter 4.  iii | P a g e  Table of Contents Abstract ......................................................................................................................... ii Table of Contents ........................................................................................................ iv List of Tables .............................................................................................................. viii List of Figures .............................................................................................................. ix List of Abbreviations ................................................................................................... xi Preface ....................................................................................................................... xiii Acknowledgements .................................................................................................. xiv Dedication .................................................................................................................. xv Co-authorship Statement ......................................................................................... xvi Chapter 1. Introduction .............................................................................................. 1 1.1 The problem of constructing a mammalian brain................................................................... 1 1.1.1 Why study development of the cerebral cortex? ....................................................... 1 1.1.2 The neural tube ....................................................................................................................... 2 1.1.3 Cortical malformations: early developmental and migration defects ............... 5 1.1.4 Disorders of axon guidance and connectivity............................................................. 8 1.2 The cellular basis of cortical development ............................................................................... 11 1.2.1 Layers of the developing neocortex ............................................................................. 13 1.2.2 Fiber tracts in the developing brain .............................................................................. 17 1.3 Molecular control of axon guidance ........................................................................................... 18 1.3.1 The growth cone .................................................................................................................. 18 1.3.2 Guidance cues, receptors, and effectors ..................................................................... 25 1.3.2.1Morphogens .............................................................................................................. 27 1.3.2.2 Netrins......................................................................................................................... 29 1.3.2.3 Slit and robo ............................................................................................................. 30 1.3.2.4 Ephs and ephrins .................................................................................................... 31 1.4 Semaphorins are the largest family of guidance cues ......................................................... 32 1.4.1 Proteolytic regulation of semaphorins ........................................................................ 34 1.4.2 Semaphorin receptors ........................................................................................................ 35 1.4.2.1 Neuropilins ................................................................................................................ 35 1.4.2.2 Plexins ......................................................................................................................... 38 1.4.2.3 Other receptors ....................................................................................................... 39 1.4.3 Semaphorin receptor signaling ...................................................................................... 40 1.4.4 Class 5 semaphorins ............................................................................................................ 42 1.5 Corticofugal pathfinding as a model of complex axon guidance ................................... 46  iv | P a g e  1.6 Hypotheses and thesis objectives ............................................................................................... 50 1.7 References............................................................................................................................................. 52  Chapter 2. Semaphorin 5B is a novel inhibitory cue for corticofugal axons ......... 70 2.1 Introduction ......................................................................................................................................... 70 2.2 Methods ................................................................................................................................................. 72 2.2.1 Animals ..................................................................................................................................... 72 2.2.2 Stable cell lines ...................................................................................................................... 73 2.2.3 in situ hybridization ............................................................................................................. 73 2.2.4 in vitro co-cultures ................................................................................................................ 74 2.2.5 Organotypic slice cultures ................................................................................................ 75 2.2.6 Preparation of shRNA vectors and validation............................................................ 77 2.2.7 ex vivo electroporation ....................................................................................................... 78 2.3 Results .................................................................................................................................................... 79 2.3.1 Semaphorin 5B expression during telencephalic development........................ 79 2.3.2 Sema5B is repulsive to cortical projections in vitro ................................................. 84 2.3.3 Sema5B is repulsive to descending projection within organotypic slices ..... 87 2.3.4 Sema5B is not repulsive to the dorsal thalamocortical projection ................... 90 2.3.5 Loss of Sema5B expression from the neocortical VZ results in corticofugal axon pathfinding errors ............................................................................................. 92 2.4 Discussion ............................................................................................................................................. 97 2.4.1 Exogenous Sema5B is inhibitory to cortical axons in vitro ................................... 98 2.4.2 Sema5B is sufficient to inhibit cortical axons in situ................................................ 99 2.4.3 Thalamic axons are non-responsive to Sema5B .................................................... 100 2.4.4 Sema5B is a necessary inhibitory component of the descending cortical projection ..................................................................................................................... 102 2.5 References.......................................................................................................................................... 105  Chapter 3. The transmembrane Semaphorin 5B can function as a diffusible inhibitory guidance cue .................................................................... 109 3.1 Introduction ...................................................................................................................................... 109 3.2 Methods .............................................................................................................................................. 111 3.2.1 Animals .................................................................................................................................. 111 3.2.2 Generation of Sema5B polyclonal antibodies ........................................................ 111 v|Page  3.2.3 Sema5B peptide array blotting protocol.................................................................. 111 3.2.4 Brain immunohistochemistry ....................................................................................... 112 3.2.5 Cell lines and media ......................................................................................................... 112 3.2.6 Collapse assays and antibody blockade ................................................................... 113 3.2.7 Three dimensional collagen co-cultures .................................................................. 114 3.3 Results ................................................................................................................................................. 115 3.3.1 Endogenous expression of Sema5B ........................................................................... 115 3.3.2 Recombinant Sema5B is secreted and collapses cortical neuron growth cones .............................................................................................................. 118 3.3.3 Recombinant Sema5B creates an inhibitory gradient ........................................ 120 3.3.4 Anti-TAG-1 antibodies block the collapsing response of Sema5B ................. 120 3.4 Discussion .......................................................................................................................................... 123 3.4.1 Transmembrane Sema5B is proteolytically processed in mouse cortex...... 123 3.4.2 Sema5B N- and C-terminals have distinct yet overlapping localizations .... 124 3.4.3 Cells expressing Sema5B generate inhibitory gradients.................................... 125 3.4.4 TAG-1 is a likely part of a multimeric receptor complex for Sema5B ............ 127 3.4.5 Conclusions ......................................................................................................................... 128 3.5 References.......................................................................................................................................... 129  Chapter 4. Summary and discussion of results ...................................................... 131 4.1 Summary of findings ...................................................................................................................... 131 4.2 Principles of inhibitory guidance .............................................................................................. 132 4.2.1 Transmembrane Sema5B is proteolyzed ................................................................. 135 4.2.2 Sema5B undergoes multiple cleavages to differentially regulate inhibition ...................................................................................................................... 140 4.2.3 Redefining the corticofugal model – Sema5B as a secreted inhibitor ........................................................................................................................ 147 4.2.4 Evidence against the handshake hypothesis ......................................................... 152 4.2.5 A receptor for Sema5B? .................................................................................................. 154 4.3 Not just another semaphorin repellant .................................................................................. 158 4.3.1 The benefits of a two-in-one protein ......................................................................... 158 4.3.2 Left behind – functional implications if the isolated membranebound TSRs .................................................................................................................. 161 4.5 References.......................................................................................................................................... 164  vi | P a g e  Appendix A. Testing guidance cues: common function assays............................ 170 A.1 Three-dimensional repulsion assays for secreted guidance cues ................................ 170 A.2 Two-dimensional assays for substrate bound guidance cues....................................... 170 A.3 Organotypic slice cultures provide a physiologically relevant ex vivo model ........ 171 A.4 References ......................................................................................................................................... 174  Appendix B. Supplemental data for chapters 3 and 4 .......................................... 175 B.1 The secreted version of Sema5B and Sema5B-HA have similar properties in situ . 175 B.2 Additional evidence that Sema5B is an inhibitor for TAG-1 expressing axons .............................................................................................................................. 175 B.3 References ......................................................................................................................................... 179 Appendix C. UBC research ethics board certificates ............................................. 180  vii | P a g e  List of Tables 1.1 Percent sequence identities between class 5 semaphorins .............................................. 45  viii | P a g e  List of Figures 0.1 Coronal section of E14.5 mouse head ........................................................................................ xv Chapter 1 1.1 Neurulation – formation of the neural tube ............................................................................. 3 1.2 The neural tube in cross-section ................................................................................................... 4 1.3 Differentiation of the anterior neural tube in schematic ..................................................... 6 1.4 Cross-sectional anatomy of the telencephalon ..................................................................... 12 1.5 Development of the neocortex ................................................................................................... 14 1.6 Tangential migration in the developing telencephalon .................................................... 16 1.7 Types of tracts in the telencephalon ......................................................................................... 18 1.8 The growth cone and its guidance ............................................................................................. 20 1.9 Control of outgrowth in mediated by the Rho family GTPases ....................................... 24 1.10 The classical guidance cue families and their receptors................................................... 28 1.11 The semaphorin family and receptors ..................................................................................... 37 1.12 Corticofugal and thalamocortical connectivity .................................................................. 47 Chapter 2 2.1 Early embryonic expression of Sema5B..................................................................................... 81 2.2 Sema5B is expressed in regions of the telencephalon avoided by corticothalamic axons ......................................................................................................................................... 83 2.3 Dorsal and lateral cortical axons avoid Sema5B-expressing cells in vitro .................... 85 2.4 Ectopic Sema5B is sufficient to cause aberrant pathfinding of CTAs ............................. 89 2.5 Dorsal thalamic axons do not avoid Sema5B either in vitro or organotypic slice culture ....................................................................................................................................... 91 2.6 Effective knock down of Sema5B with shRNA vectors......................................................... 93 2.7 Loss of Sema5B in organotypic slice culture causes DiI-labeled cortical fibers to misproject into the subventricular and ventricular zones of the pallium....... 95  Chapter 3 3.1 Sema5B expression in the embryonic mouse neocortex ................................................ 117 3.2 Recombinant Sema5B is secreted and collapses cortical neurons .............................. 119 3.3 Sema5B creates inhibitory gradients in a 3D collagen matrix ....................................... 121 3.4 Antibodies to TAG-1 attenuate the collapsing effects of Sema5B-HA........................ 122 Chapter 4 4.1 Sequence analysis of Sema5B supports the endogenous cleavage hypothesis .... 138 4.2 Time course of semaphorin-induced cortical growth cone collapse .......................... 145 4.3 Initial guidance of the corticofugal projection .................................................................... 149 4.4 Mutations in corticofugal and thalamocortical pathfinding .......................................... 151 4.5 TAG-1 and Sema5B expression in the developing mouse spinal cord ....................... 156 4.6 Divergently localized fragments of proteolyzed Sema5B in the cortical layers ...... 160 ix | P a g e  4.7 Sema5B-TSRs as a permissive substrate for corticofugal axon extension ................. 163 Appendix A A.1 Assays for the evaluation of guidance cue function ......................................................... 173 Appendix B B.1 Sema5B in the spinal cord reflects regions avoided by TAG-1 fibers .......................... 176 B.2 Sema5B-TSRs as a permissive substrate for corticofugal axon extension ................. 179  x|Page  List of Abbreviations Anatomical/Developmental Terms CFA .................... corticofugal axons CGE.................... caudal ganglionic eminence CNS ................... central nervous system CP....................... cortical plate CSB .................... corticostriatal boundary CR ...................... Cajal-Retzius neuron dctx ................... dorsal cortex Dien .................. diencephalon DTB.................... dien-telencephalic boundary dTh .................... dorsal thalamus E.......................... embryonic day (gestational) HEK293 ............ human embryonic kidney cells Hind .................. hindbrain hipp................... hippocampus IPC ..................... Intermediate progenitor cell IZ ........................ intermediate zone LGE .................... lateral ganglionic eminence LMS ................... lateral migratory stream N ......................... notochord NC ...................... neural crest NE ...................... neuroepithelium  NT ...................... neural tube MB ..................... midbrain Mesen............... mesencephalon Meten ............... metencephalon MGE................... medial ganglionic eminence Myelen ............. myelencephalon MZ ..................... marginal zone PNS .................... peripheral nervous system PP ....................... preplate Prosen .............. prosencephalon RG ...................... radial glial cell Rhomben ........ rhombencephalon RMS ................... rostral migratory stream SC ....................... spinal cord SPL ..................... subplate layer SVZ .................... subventricular zone TCA .................... thalamocortical axons Tel/telen .......... telencephalon vlctx .................. ventrolateral cortex vTh .................... ventral thalamus VZ....................... ventricular zone  Molecular Terms ADAM ............... a disintegrin and metalloprotease ADF ................... actin depolymerizing factor BMP ................... bone morphogenic protein CAM .................. cell adhesion molecule cAMP ................ cyclic adenosine monophosphate Cdk .................... cyclin dependent kinase cGMP ................ cyclic guanosine monophosphate CRMP ................ collapse response mediator protein  CSPG ................. chondroitin sulfate proteoglycan CXCR ................. alpha chemokine receptor dcc ..................... deleted in colorectal cancer DCX ................... doublecortin DiI ...................... 1,1’,dioctodecyl-3,3,3’,3’tetramethyl-indocarbocyanine perchlorate DNT1................. Drosophila neurotrophic dpp .................... decapentaplegic ERM ................... “ezrin, radixin, moesin” F-actin .............. filamentous actin xi | P a g e  FGF .................... fibroblast growth factor GABA ................ γ-aminobutyric acid GDP ................... guanosine diphosphate GPI ..................... glycosylphosphatidylinositol GSK-3 ................ glycogen synthase kinase-3 GTP .................... guanine triphosphate HGF ................... hepatocyte growth factor HSPG................. heparan sulfate proteoglycan Ig ........................ immunoglobulin domain Lis1 .................... lissencephaly-1 MET mesenchymal-epithelial transition factor MLC ................... myosin light-chain MLCP ................ myosin light-chain phosphatase MLCK ................ myosin light-chain kinase MMP ................. matrix metalloproteinase MRS ................... MET-related sequence MT...................... microtubule MT-MMP.......... membrane-type MMP NARC ................ neural apoptosis-regulated convertase NGF ................... nerve growth factor NP ...................... neuropilin PAK .................... p21 related kinase Pax6 .................. paired-box transcription factor-6  PC....................... proprotein convertase PCSK.................. PC substilin-kexin-like PDZ ................... “post-synaptic density-95, discs large, zona occludens-1” domain PI3K ................... phosphoinositide 3-kinase PKA .................... protein kinase A PSI ...................... “plexin-semaphorin............................ integrin” Robo ................. roundabout ROCK................. rho-activated kinase RTK .................... receptor tyrosine kinase Ryk ..................... related to receptor tyrosine kinase Sema ................. semaphorin SH ...................... sarcoma homology domain shh..................... sonic hedgehog SF ....................... scatter factor SFR..................... scatter factor receptor TAG-1 ............... transient axonal guidance-1 TGF-β ................ transforming growth factor-β trk ...................... tropomyosin-related kinase TSP..................... thrombospondin TSR..................... thrombospondin repeat Wnt.................... wingless Unc .................... uncoordinated  xii | P a g e  Preface Semaphorins are typically abbreviated as “Sema”, but the capitalization changes depending on the organism, and if it is necessary, the organism of origin is indicated by the first letter of the organism’s name. Invertebrate semaphorins are not capitalized; therefore, Drosophila semaphorin 1a would appear as d-sema-1a, for example. Vertebrate semaphorins are capitalized in the first letter. For example, murine semaphorin 5B would be abbreviated as mSema5B. For simplicity’s sake, I have simply referred to mSema5B as Sema5B throughout the text herein. Please not that all figure drawings/diagrams in this thesis (including adaptations) are original designs under copyright  2009 by Robyn Lynn Mwuese Lett, and are not to be used or reproduced without permission of the author.  xiii | P a g e  Acknowledgements First and foremost I want to thank Tim O'Connor for letting me take up space in his lab for two whole degrees. I really appreciate all your input and effort, particularly in these last months; it has been a pleasure. One of the things I have always enjoyed, for better or worse, is the candor between us and the faith that you have shown in my intellect. I have striven in this, my second degree in your lab, to really earn respect, earn my place, and to become a real scientist. I would like to think that under your tutelage I have managed this. I hope I have made you proud; for I am proud to be a graduate of the O'Connor lab. Thank you also to my committee, Drs Vanessa Auld, Wolfram Tetzlaff, and Alaa El-Husseini. Alaa's unfortunate passing on Dec 23, 2007 was a shock, and all who knew him have felt his loss. I was glad to have had him on my committee. Vanessa and Wolf, you have been most indispensable as counselors and mentors. Thank you so much for your help, guidance, and the second shot that I really and truly needed, even if it wasn't deserved until now. To my lab mates present and past – thanks for being my colleagues and friends. Ana, you were an amazing mentor and friend to me. I am a different scientist for having worked with you. You are the kind of scientist anyone could aspire to and I have immense respect for you. Kenny, you are a great friend and colleague that I could always count on for a non-emotional view, and I hope to know you for years to come. Winnie; you did everything and managed everything. You have my endless gratitude for your endless help during my time in the O'Connor lab. I would like to mention Kristen B(ond) as an indispensable additional collaborator for my second paper. It has been a pleasure working with you. Finally, Andrew Tait; we’ve come a long way from the time you declared your grade 7 crush... You are one of my oldest friends and a wonderful colleague, and I love our coffee break discussions. Dad, Mom, Ryan, Tara, and Grandma, I swear I almost quit science for art three times. But each one of you kept me going and made sure I never gave up. I put you all through so much, and you were all there for me, always. You have supported me emotionally, physically, financially, and intellectually. Dad, you were like a second supervisor. I sometimes found it easier to take criticism from you than anyone else in science – and as it turns out, you were right! Mom, I loved your visits and our wonderful conversations on work, life, and love, especially in the last two agonizing years when I was so down and needed you so much. Grandma, I always look forward to our exclusive book club meetings – I think I’m becoming more like you with each passing year. Ryan and Tara: I am so fortunate to be living in the same city as my sibs. It has been awesome! Ryan, my partner in killing time and aliens; I love that I have gotten to spend so much quality time with you recently. It has been nice to know I can always count on you for anything! Tara, I feel like I owe you so much…tu es ma petite soeur et ma grande soeur a la meme fois. N’oublie-pas qu’il saurait jamais personne qui peut tu remplacer. You might be a cancer but you are also a rock (star!). And to the new addition, Lana; I hope you can read this one day! Mmmm-ahhh! I love you all so, so much!!! Theunis my lieflung; Ek weet dit was n lang en moeilike pad en soms baie stresvol tussen ons, nietemin, jy was my minnaar en my beste vriend. Elke pot het sy dacsl; jy is mein. My liefde is vir jou, vir altyd.  xiv | P a g e  This thesis is dedicated to my grandfather,  Glen Charles Lett.  I think you would have been proud as punch. I miss you.  2009 RLML Figure 0.1 Coronal section of E14.5 mouse head Probes with antibodies to Sem5B (green), β-III tubulin (red), and counterstained with DAPI (in blue). xv | P a g e  Co-authorship Statement Other than the following specific contributions, I performed all experiments and data analysis, prepared all the figures, and wrote the manuscripts. Concepts and experimental design were also my responsibility, with guidance and editing provided by my supervisor, Dr. O’Connor, and a former colleague (not a co-author), Dr. Ana Mingorance-Le Meur. Chapter 2 and 3 are versions of manuscripts that include authors in addition to me and the primary investigator, T.P. O’Connor. Chapter 2 is published at Cerebral Cortex, vol 19, pp14081421. The second author, Dr. Wenyan Wang, was responsible for sub-cloning mSema5B from pBK-CMV into pDisplay, and for generating and maintaining the stable cell lines associated with the paper. She also prepared the shRNA vectors which I had designed, and validated their knock down efficiency in heterologous cells, giving rise to figure 2.6 panels A-C, all of which she provided. In chapter 3, a version of a second submitted manuscript, Dr. Wenyan Wang was the second author. In conjunction with Dr. O’Connor, Dr. Wang devised, produced, and purified the Nterminal and C-terminal antibodies to Sema5B. Kristen Browne, the third author, performed the western blots for figure 3.2, panel A and did the MMP inhibition experiments resulting in figure 3.2 panel H, and prepared all the concentrated media for collapse assays as well.  xvi | P a g e  1|Page  2|Page    3|Page    4|Page  5|Page    6|Page  7|Page  8|Page  9|Page  10 | P a g e  11 | P a g e   12 | P a g e  13 | P a g e    14 | P a g e  15 | P a g e    16 | P a g e  17 | P a g e    18 | P a g e  19 | P a g e    20 | P a g e  21 | P a g e  22 | P a g e  23 | P a g e    24 | P a g e  25 | P a g e  26 | P a g e  27 | P a g e    28 | P a g e  29 | P a g e  30 | P a g e  31 | P a g e  32 | P a g e  33 | P a g e  α  34 | P a g e  35 | P a g e  36 | P a g e    37 | P a g e  38 | P a g e  39 | P a g e  40 | P a g e  41 | P a g e  42 | P a g e  43 | P a g e  44 | P a g e  45 | P a g e  46 | P a g e    47 | P a g e  48 | P a g e  49 | P a g e  50 | P a g e  51 | P a g e  52 | P a g e  53 | P a g e  54 | P a g e  55 | P a g e  56 | P a g e  57 | P a g e  58 | P a g e  59 | P a g e  60 | P a g e  61 | P a g e  62 | P a g e  63 | P a g e  64 | P a g e  65 | P a g e  66 | P a g e  67 | P a g e  68 | P a g e  69 | P a g e  70 | P a g e  71 | P a g e     72 | P a g e      73 | P a g e  ◦  ◦    74 | P a g e  β  75 | P a g e  μ  μ  μ  μ  76 | P a g e  77 | P a g e  β  78 | P a g e  79 | P a g e  80 | P a g e  81 | P a g e  82 | P a g e  83 | P a g e    84 | P a g e  85 | P a g e         86 | P a g e  87 | P a g e  88 | P a g e  89 | P a g e  90 | P a g e  91 | P a g e  92 | P a g e      93 | P a g e  94 | P a g e  95 | P a g e      96 | P a g e  ± ±  ±  ±  ±  ±  97 | P a g e  98 | P a g e  99 | P a g e  100 | P a g e  ň  101 | P a g e  102 | P a g e  103 | P a g e  104 | P a g e  á  105 | P a g e  106 | P a g e  107 | P a g e  108 | P a g e  109 | P a g e  110 | P a g e  111 | P a g e  ü  112 | P a g e  ◦           113 | P a g e  114 | P a g e  115 | P a g e  116 | P a g e  117 | P a g e    ±  ±  118 | P a g e  α  119 | P a g e  ±  ± ±  120 | P a g e  121 | P a g e  122 | P a g e  123 | P a g e  124 | P a g e  125 | P a g e  126 | P a g e  127 | P a g e  128 | P a g e  129 | P a g e  130 | P a g e  131 | P a g e  132 | P a g e  133 | P a g e  134 | P a g e  135 | P a g e  136 | P a g e  137 | P a g e  138 | P a g e  139 | P a g e  140 | P a g e  141 | P a g e  142 | P a g e  143 | P a g e  144 | P a g e  145 | P a g e  146 | P a g e  147 | P a g e  148 | P a g e  149 | P a g e  150 | P a g e  151 | P a g e  152 | P a g e  153 | P a g e  154 | P a g e  155 | P a g e  156 | P a g e  157 | P a g e  158 | P a g e  159 | P a g e  160 | P a g e    161 | P a g e  162 | P a g e  163 | P a g e  164 | P a g e  165 | P a g e  166 | P a g e  167 | P a g e  168 | P a g e  169 | P a g e  170 | P a g e  171 | P a g e  172 | P a g e  173 | P a g e  174 | P a g e  175 | P a g e  176 | P a g e  177 | P a g e  178 | P a g e  179 | P a g e  180 | P a g e  THE UNIVERSITY OF BRITISH COLUMBIA  ANIMAL CARE CERTIFICATE  Investigator or Course Director: Timothy P. O'Connor Department: Cellular & Physiological Sc.  Funding Sources:  181 | P a g e  N/A  The Animal Care Committee has examined and approved the use of animals for the above experimental project. This certificate is valid for one year from the above start or approval date (whichever is later) provided there is no change in the experimental procedures. Annual review is required by the CCAC and some granting agencies.  A copy of this certificate must be displayed in your animal facility.  Office of Research Services and Administration 102, 6190 Agronomy Road, Vancouver, BC V6T 1Z3 Phone: 604-827-5111 Fax: 604-822-5093  182 | P a g e  

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