DOCSLIB.ORG

ARHGAP4 IS a SPATIALLY REGULATED RHOGAP THAT INHIBITS NIH/3T3 CELL MIGRATION and DENTATE GRANULE CELL AXON OUTGROWTH by DANIEL L

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
ARHGAP4 IS a SPATIALLY REGULATED RHOGAP THAT INHIBITS NIH/3T3 CELL MIGRATION and DENTATE GRANULE CELL AXON OUTGROWTH by DANIEL L ARHGAP4 IS A SPATIALLY REGULATED RHOGAP THAT INHIBITS NIH/3T3 CELL MIGRATION AND DENTATE GRANULE CELL AXON OUTGROWTH By DANIEL LEE VOGT Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Neuroscience CASE WESTERN RESERVE UNIVERSITY August, 2007 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of Daniel Lee Vogt ______________________________________________________ candidate for the Ph.D. degree *. (signed) (chair of the committee)________________________________ Stefan Herlitze ________________________________________________Alfred Malouf Robert Miller ________________________________________________ ________________________________________________Thomas Egelhoff ________________________________________________Susann Brady-Kalnay ________________________________________________ (date) _______________________6-21-2007 *We also certify that written approval has been obtained for any proprietary material contained therein. ii Copyright © 2007 by Daniel Lee Vogt All rights reserved iii Table of contents Page # Title page i Table of contents iv List of figures vii Abstract 1 Chapter one: General introduction 2 Hippocampal axon pathways and development 3 Guidance cues in hippocampal axon outgrowth 6 Slit/Robo 7 Semaphorins, plexins and neuropilins 8 Ephrins and ephs 11 Other guidance cues in the hippocampus 13 GTPases: structure and function of ras superfamily members 15 Ras GTPases 17 Ran GTPases 18 Arf GTPases 18 Rab GTPases 19 Rho GTPases 20 GTPase regulatory proteins and the role of GAPs 23 RasGAP family functions, localizations and binding partners 24 RanGAP family 25 ArfGAP family 26 iv RabGAP family 26 RhoGAP family 27 GAP protein mutations and developmental consequences 29 Regulation of GAP proteins 29 Phosphorylation 30 Protein-protein interactions 31 Other types of regulation 32 Actin and microtubule dynamics in growth cone and fibroblast motility 33 General actin dynamics 33 General microtubule dynamics 36 Coordinated signaling regulated by Rho-family GTPases 40 Cell migration and functional parallels to growth cone guidance 41 Previous work on ARHGAP4 45 Conserved domains of ARHGAP4 48 FCH domain, extended FCH domain and ARNEY domain 48 RhoGAP domain 51 SH3 domain 52 Proline rich (PxxP) domains 52 Research goals 53 Figures 55 Chapter two: ARHGAP4 is a spatially regulated GTPase activating protein (GAP) expressed in NIH/3T3 fibroblasts and dentate granule neurons 71 Summary 72 v Introduction 72 Materials and methods 74 Results 82 Discussion 89 Figures 97 Chapter three: ARHGAP4 is an inhibitor of NIH/3T3 cell motility and Dentate granule cell axon outgrowth 123 Summary 124 Introduction 124 Materials and methods 126 Results 130 Discussion 133 Figures 139 Chapter four: Mechanistic insights into the function of ARHGAP4 155 Summary 156 Introduction 156 Materials and methods 158 Results 161 Discussion 165 Figures 169 Chapter five: General Discussion 179 Chapter six: Bibliography 195 vi List of figures Figure Page # 1.1 Hippocampal anatomy and pathways 55 1.2 Common guidance cues and receptors found in the hippocampus utilize various GTPases to effect growth cone guidance 57 1.3 The GTPase cycle and role of GAPs, GEFs and GDIs 59 1.4 Rho-family GAP proteins and their domain assortments 61 1.5 Actin and microtubule cytoskeletons in migrating cells and growth cones 63 1.6 ARHGAP4’s amino-terminus contains FCH, extended FCH and ARNEY domains 65 1.7 ARHGAP4 GAP domain alignment 67 1.8 ARHGAP4 SH3 domain alignment 69 2.1 ARHGAP4 constructs used in transfections and bacterial protein expression 97 2.2 In vitro GAP assay of WT and R562A ARHGAP4 GAP domains 99 2.3 Endogenous ARHGAP4 localizes to the leading edge of NIH/3T3 cells 101 2.4 Amino acids 1-71 are necessary and sufficient to target ARHGAP4 to the tips of NIH/3T3 cell cytoplasmic extensions 103 2.5 ARHGAP4 is enriched in the mossy fiber axons of the hippocampus 105 2.6 Endogenous ARHGAP4 is enriched in mossy fiber growth cones 107 2.7 ARHGAP4 expressed proteins localize to growth cones via amino acids 1-71 109 2.8 Amino acids 1-71 are necessary and sufficient to target ARHGAP4 to growth cones 111 vii 2.9 Amino acids 1-71 can associate indirectly but do not bind microtubules in an in vitro microtubule cosedimentation assay 113 2.10 Leading edge distribution of microtubules and F-actin in the presence of nocodazole or cytochalasin-D 115 2.11 Full length ARHGAP4 (1-965) leading edge distribution in the presence of nocodazole or cytochalasin-D 117 2.12 1-770 leading edge distribution in the presence of nocodazole or cytochalasin-D 119 2.13 72-965 leading edge distribution in the presence of nocodazole or cytochalasin-D 121 3.1 Wound assay model system and quantification of cell migration in individually transfected cells 139 3.2 siRNA mediated knockdown of ARHGAP4 increases cell migration in NIH/3T3 cells 141 3.3 ARHGAP4 inhibits NIH/3T3 cell migration via its GAP domain 143 3.4 ARHGAP4 inhibits axon outgrowth from dentate explant cultures through its FCH, GAP and SH3 domains 145 3.5 Dentate explant axon outgrowth is inhibited by ARHGAP4 in an FCH, GAP and SH3 domain manner 147 3.6 Dentate explant astrocyte outgrowth is not altered by ARHGAP4 149 3.7 ARHGAP4 inhibits axon outgrowth in dissociated granule cells in a GAP dependent manner 151 3.8 ARHGAP4 significantly inhibits axon outgrowth in dissociated granule cells viii in a GAP dependent manner 153 4.1 ARHGAP4 amino-terminal protein design and expression 169 4.2 Amino acids 1-289 are the minimal unit required to inhibit NIH/3T3 cell migration 171 4.3 Amino acids 1-71 and 1-289 of ARHGAP4 result in different actin phenotypes in migrating NIH/3T3 cells 173 4.4 Full length ARHGAP4 overexpression decreases levels of GTP-bound RhoA in NIH/3T3 cells 175 4.5 Trends in axon outgrowth and branching are altered in DRGs expressing the R562A mutant on laminin and aggrecan 177 5.1 Model of ARHGAP4 activity 193 ix ARHGAP4 is a spatially regulated RhoGAP that inhibits NIH/3T3 cell migration and dentate granule cell axon outgrowth Abstract By DANIEL LEE VOGT Cell migration and axonal growth cone guidance are tightly regulated events that share many similarities. While some factors are not always shared between migrating cells and growth cones, the signaling events that are required for cell migration and growth guidance each utilize GTPases to regulate the actin and microtubule cytoskeletons. GTPases are expressed ubiquitously but signal in discrete subcellular locales to control directed cell migration and growth cone guidance. This strict control of GTPase signaling is controlled by GAPs, GEFs and GDIs, which can assemble signaling complexes and localize to unique regions due to an array of conserved functional domains. The Rho-family GAP ARHGAP4 contains FCH and SH3 domains, as well as other conserved domains of unknown function. The FCH domain has been attributed to binding actin, microtubules and lipids, but there is no consensus on its function. Here we show that ARHGAP4 localizes to the leading edges of NIH/3T3 fibroblasts and to growth cones of dentate granule neurons, via its FCH domain. Overall, ARHGAP4 inhibits NIH/3T3 cell migration and dentate granule cell axon outgrowth in a GAP dependent manner, and this inhibition is temporally and spatially regulated by ARHGAP4’s conserved functional domains. 1 Chapter 1 General Introduction 2 Unraveling the intricacies of neural networks and their functions during development and in the adult has been an ongoing task since the infancy of developmental biology. Deciphering how neurons migrate, send axons and dendrites to their proper targets and communicate with the vast array of non-neuronal cells is still not well understood. Although many signaling events have been characterized and candidate proteins identified, the detailed mechanisms whereby all the players in neuronal development and maturation are brought together is still far from complete. The mechanisms that regulate axon outgrowth and guidance require tight regulation of cytoskeletal elements, and these mechanisms are shared among many migratory cells as well. Conserved families of proteins that include guidance and growth cues with their respective receptors, actin and microtubule regulatory proteins, GTPases and their regulators the GEFs, GAPs and GDIs, are all involved in guiding an axon or migrating cell to its determined location. These signaling cascades require spatial and temporal control to utilize the potential of the GTPases, and regulatory proteins must have functional domains that allow for spatial control and assembly with signaling components. The RhoGAP family of proteins have diverse tissue expression, subcellular location and signaling partners, and achieve this through a myriad of functional domains. ARHGAP4 is a RhoGAP that inhibits axon outgrowth and cell motility, and whose actions are temporally and spatially regulated by its conserved functional domains. Hippocampal axon pathways and development One of the classic areas studied for axon outgrowth is the hippocampus. With its stereotyped pattern of axonal fibers, adult neurogenesis and axon outgrowth, and rich 3 history of electrophysiological mapping of circuits, the hippocampus has been an excellent model to study axon outgrowth and regeneration. The hippocampus arises from the invaginating dorsal midline of the telencephalon, and its initial formation is dependent on the patterning of the dorsal telencephalon (Theil et al., 1999). The mature hippocampus
Load more

Recommended publications
  • Supplementary Materials: Evaluation of Cytotoxicity and Α-Glucosidase Inhibitory Activity of Amide and Polyamino-Derivatives of Lupane Triterpenoids
    Supplementary Materials: Evaluation of cytotoxicity and α-glucosidase inhibitory activity of amide and polyamino-derivatives of lupane triterpenoids Oxana B. Kazakova1*, Gul'nara V. Giniyatullina1, Akhat G. Mustafin1, Denis A. Babkov2, Elena V. Sokolova2, Alexander A. Spasov2* 1Ufa Institute of Chemistry of the Ufa Federal Research Centre of the Russian Academy of Sciences, 71, pr. Oktyabrya, 450054 Ufa, Russian Federation 2Scientific Center for Innovative Drugs, Volgograd State Medical University, Novorossiyskaya st. 39, Volgograd 400087, Russian Federation Correspondence Prof. Dr. Oxana B. Kazakova Ufa Institute of Chemistry of the Ufa Federal Research Centre of the Russian Academy of Sciences 71 Prospeсt Oktyabrya Ufa, 450054 Russian Federation E-mail: [email protected] Prof. Dr. Alexander A. Spasov Scientific Center for Innovative Drugs of the Volgograd State Medical University 39 Novorossiyskaya st. Volgograd, 400087 Russian Federation E-mail: [email protected] Figure S1. 1H and 13C of compound 2. H NH N H O H O H 2 2 Figure S2. 1H and 13C of compound 4. NH2 O H O H CH3 O O H H3C O H 4 3 Figure S3. Anticancer screening data of compound 2 at single dose assay 4 Figure S4. Anticancer screening data of compound 7 at single dose assay 5 Figure S5. Anticancer screening data of compound 8 at single dose assay 6 Figure S6. Anticancer screening data of compound 9 at single dose assay 7 Figure S7. Anticancer screening data of compound 12 at single dose assay 8 Figure S8. Anticancer screening data of compound 13 at single dose assay 9 Figure S9. Anticancer screening data of compound 14 at single dose assay 10 Figure S10.
    [Show full text]
  • Active Rac1 Detection Kit 2012 09/20
    Active Rac1 Detection Kit 1 Kit Orders n 877-616-CELL (2355) (30 immunoprecipitations) [email protected] Support n 877-678-TECH (8324) [email protected] Web n www.cellsignal.com rev. 09/24/20 #8815 For Research Use Only. Not For Use In Diagnostic Procedures. Species Cross-Reactivity: H, M Components Ship As: 11894S Item # Kit Quantity Storage Temp Description: The Active Rac1 Detection Kit provides all GTPgS 11521 1 X 50 µl –80°C reagents necessary for measuring activation of Rac1 GTPase in the cell. GST-PAK1-PBD fusion protein is used to bind GDP 11522 1 X 50 µl –80°C the activated form of GTP-bound Rac1, which can then be Components Ship As: 11894S Item # Kit Quantity Storage Temp immunoprecipitated with glutathione resin. Rac1 activation levels are then determined by western blot using a Rac1 GST-Human PAK1-PBD 8659 1 X 600 µg –20°C Mouse mAb. Rac1 Mouse mAb 8631 1 X 50 µl –20°C Specificity/Sensitivity: Active Rac1 Detection Kit detects Components Ship As: 11860S Item # Kit Quantity Storage Temp endogenous levels of GTP-bound (active) Rac1 as shown in Lysis/Binding/Wash Buffer 11524 1 X 100 mL 4°C Figure 1. This kit detects proteins from the indicated species, as determined through in-house testing, but may also detect Glutathione Resin 11523 1 X 3 ml 4°C homologous proteins from other species. SDS Sample Buffer 11525 1 X 1.5 ml 4°C Background: The Ras superfamily of small GTP-binding Spin Cup and Collection Tubes 11526 1 X 30 vial RT proteins (G proteins) comprise a large class of proteins (over 150 members) that can be classified into at least five families based on their sequence and functional similarities: 1 2 3 4 Figure 1.
    [Show full text]
  • Universidade Estadual De Campinas Instituto De Biologia
    UNIVERSIDADE ESTADUAL DE CAMPINAS INSTITUTO DE BIOLOGIA VERÔNICA APARECIDA MONTEIRO SAIA CEREDA O PROTEOMA DO CORPO CALOSO DA ESQUIZOFRENIA THE PROTEOME OF THE CORPUS CALLOSUM IN SCHIZOPHRENIA CAMPINAS 2016 1 VERÔNICA APARECIDA MONTEIRO SAIA CEREDA O PROTEOMA DO CORPO CALOSO DA ESQUIZOFRENIA THE PROTEOME OF THE CORPUS CALLOSUM IN SCHIZOPHRENIA Dissertação apresentada ao Instituto de Biologia da Universidade Estadual de Campinas como parte dos requisitos exigidos para a obtenção do Título de Mestra em Biologia Funcional e Molecular na área de concentração de Bioquímica. Dissertation presented to the Institute of Biology of the University of Campinas in partial fulfillment of the requirements for the degree of Master in Functional and Molecular Biology, in the area of Biochemistry. ESTE ARQUIVO DIGITAL CORRESPONDE À VERSÃO FINAL DA DISSERTAÇÃO DEFENDIDA PELA ALUNA VERÔNICA APARECIDA MONTEIRO SAIA CEREDA E ORIENTADA PELO DANIEL MARTINS-DE-SOUZA. Orientador: Daniel Martins-de-Souza CAMPINAS 2016 2 Agência(s) de fomento e nº(s) de processo(s): CNPq, 151787/2F2014-0 Ficha catalográfica Universidade Estadual de Campinas Biblioteca do Instituto de Biologia Mara Janaina de Oliveira - CRB 8/6972 Saia-Cereda, Verônica Aparecida Monteiro, 1988- Sa21p O proteoma do corpo caloso da esquizofrenia / Verônica Aparecida Monteiro Saia Cereda. – Campinas, SP : [s.n.], 2016. Orientador: Daniel Martins de Souza. Dissertação (mestrado) – Universidade Estadual de Campinas, Instituto de Biologia. 1. Esquizofrenia. 2. Espectrometria de massas. 3. Corpo caloso.
    [Show full text]
  • UCSD MOLECULE PAGES Doi:10.6072/H0.MP.A002549.01 Volume 1, Issue 2, 2012 Copyright UC Press, All Rights Reserved
    UCSD MOLECULE PAGES doi:10.6072/H0.MP.A002549.01 Volume 1, Issue 2, 2012 Copyright UC Press, All rights reserved. Review Article Open Access WAVE2 Tadaomi Takenawa1, Shiro Suetsugu2, Daisuke Yamazaki3, Shusaku Kurisu1 WASP family verprolin-homologous protein 2 (WAVE2, also called WASF2) was originally identified by its sequence similarity at the carboxy-terminal VCA (verprolin, cofilin/central, acidic) domain with Wiskott-Aldrich syndrome protein (WASP) and N-WASP (neural WASP). In mammals, WAVE2 is ubiquitously expressed, and its two paralogs, WAVE1 (also called suppressor of cAMP receptor 1, SCAR1) and WAVE3, are predominantly expressed in the brain. The VCA domain of WASP and WAVE family proteins can activate the actin-related protein 2/3 (Arp2/3) complex, a major actin nucleator in cells. Proteins that can activate the Arp2/3 complex are now collectively known as nucleation-promoting factors (NPFs), and the WASP and WAVE families are a founding class of NPFs. The WAVE family has an amino-terminal WAVE homology domain (WHD domain, also called the SCAR homology domain, SHD) followed by the proline-rich region that interacts with various Src-homology 3 (SH3) domain proteins. The VCA domain located at the C-terminus. WAVE2, like WAVE1 and WAVE3, constitutively forms a huge heteropentameric protein complex (the WANP complex), binding through its WHD domain with Abi-1 (or its paralogs, Abi-2 and Abi-3), HSPC300 (also called Brick1), Nap1 (also called Hem-2 and NCKAP1), Sra1 (also called p140Sra1 and CYFIP1; its paralog is PIR121 or CYFIP2). The WANP complex is recruited to the plasma membrane by cooperative action of activated Rac GTPases and acidic phosphoinositides.
    [Show full text]
  • A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus
    Page 1 of 781 Diabetes A Computational Approach for Defining a Signature of β-Cell Golgi Stress in Diabetes Mellitus Robert N. Bone1,6,7, Olufunmilola Oyebamiji2, Sayali Talware2, Sharmila Selvaraj2, Preethi Krishnan3,6, Farooq Syed1,6,7, Huanmei Wu2, Carmella Evans-Molina 1,3,4,5,6,7,8* Departments of 1Pediatrics, 3Medicine, 4Anatomy, Cell Biology & Physiology, 5Biochemistry & Molecular Biology, the 6Center for Diabetes & Metabolic Diseases, and the 7Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202; 2Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, Indianapolis, IN, 46202; 8Roudebush VA Medical Center, Indianapolis, IN 46202. *Corresponding Author(s): Carmella Evans-Molina, MD, PhD ([email protected]) Indiana University School of Medicine, 635 Barnhill Drive, MS 2031A, Indianapolis, IN 46202, Telephone: (317) 274-4145, Fax (317) 274-4107 Running Title: Golgi Stress Response in Diabetes Word Count: 4358 Number of Figures: 6 Keywords: Golgi apparatus stress, Islets, β cell, Type 1 diabetes, Type 2 diabetes 1 Diabetes Publish Ahead of Print, published online August 20, 2020 Diabetes Page 2 of 781 ABSTRACT The Golgi apparatus (GA) is an important site of insulin processing and granule maturation, but whether GA organelle dysfunction and GA stress are present in the diabetic β-cell has not been tested. We utilized an informatics-based approach to develop a transcriptional signature of β-cell GA stress using existing RNA sequencing and microarray datasets generated using human islets from donors with diabetes and islets where type 1(T1D) and type 2 diabetes (T2D) had been modeled ex vivo. To narrow our results to GA-specific genes, we applied a filter set of 1,030 genes accepted as GA associated.
    [Show full text]
  • Lysophosphatidic Acid Signaling in the Nervous System
    Neuron Review Lysophosphatidic Acid Signaling in the Nervous System Yun C. Yung,1,3 Nicole C. Stoddard,1,2,3 Hope Mirendil,1 and Jerold Chun1,* 1Molecular and Cellular Neuroscience Department, Dorris Neuroscience Center, The Scripps Research Institute, La Jolla, CA 92037, USA 2Biomedical Sciences Graduate Program, University of California, San Diego School of Medicine, La Jolla, CA 92037, USA 3Co-first author *Correspondence: [email protected] http://dx.doi.org/10.1016/j.neuron.2015.01.009 The brain is composed of many lipids with varied forms that serve not only as structural components but also as essential signaling molecules. Lysophosphatidic acid (LPA) is an important bioactive lipid species that is part of the lysophospholipid (LP) family. LPA is primarily derived from membrane phospholipids and signals through six cognate G protein-coupled receptors (GPCRs), LPA1-6. These receptors are expressed on most cell types within central and peripheral nervous tissues and have been functionally linked to many neural pro- cesses and pathways. This Review covers a current understanding of LPA signaling in the nervous system, with particular focus on the relevance of LPA to both physiological and diseased states. Introduction LPA synthesis/degradative enzymes (reviewed in Sigal et al., The human brain is composed of approximately 60%–70% lipids 2005; Brindley and Pilquil, 2009; Perrakis and Moolenaar, by dry weight (Svennerholm et al., 1994). These lipids can be 2014). In view of the broad neurobiological influences of LPA divided into two major pools, structural and signaling, which signaling, its dysregulation may lead to diverse neuropathologies include well-known families such as cholesterol, fatty acids, ei- (Bandoh et al., 2000; Houben and Moolenaar, 2011; Yung et al., cosanoids, endocannabinoids, and prostaglandins (Figure 1).
    [Show full text]
  • Figure S1. Representative Report Generated by the Ion Torrent System Server for Each of the KCC71 Panel Analysis and Pcafusion Analysis
    Figure S1. Representative report generated by the Ion Torrent system server for each of the KCC71 panel analysis and PCaFusion analysis. (A) Details of the run summary report followed by the alignment summary report for the KCC71 panel analysis sequencing. (B) Details of the run summary report for the PCaFusion panel analysis. A Figure S1. Continued. Representative report generated by the Ion Torrent system server for each of the KCC71 panel analysis and PCaFusion analysis. (A) Details of the run summary report followed by the alignment summary report for the KCC71 panel analysis sequencing. (B) Details of the run summary report for the PCaFusion panel analysis. B Figure S2. Comparative analysis of the variant frequency found by the KCC71 panel and calculated from publicly available cBioPortal datasets. For each of the 71 genes in the KCC71 panel, the frequency of variants was calculated as the variant number found in the examined cases. Datasets marked with different colors and sample numbers of prostate cancer are presented in the upper right. *Significantly high in the present study. Figure S3. Seven subnetworks extracted from each of seven public prostate cancer gene networks in TCNG (Table SVI). Blue dots represent genes that include initial seed genes (parent nodes), and parent‑child and child‑grandchild genes in the network. Graphical representation of node‑to‑node associations and subnetwork structures that differed among and were unique to each of the seven subnetworks. TCNG, The Cancer Network Galaxy. Figure S4. REVIGO tree map showing the predicted biological processes of prostate cancer in the Japanese. Each rectangle represents a biological function in terms of a Gene Ontology (GO) term, with the size adjusted to represent the P‑value of the GO term in the underlying GO term database.
    [Show full text]
  • Hras Intracellular Trafficking and Signal Transduction Jodi Ho-Jung Mckay Iowa State University
    Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 2007 HRas intracellular trafficking and signal transduction Jodi Ho-Jung McKay Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Biological Phenomena, Cell Phenomena, and Immunity Commons, Cancer Biology Commons, Cell Biology Commons, Genetics and Genomics Commons, and the Medical Cell Biology Commons Recommended Citation McKay, Jodi Ho-Jung, "HRas intracellular trafficking and signal transduction" (2007). Retrospective Theses and Dissertations. 13946. https://lib.dr.iastate.edu/rtd/13946 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. HRas intracellular trafficking and signal transduction by Jodi Ho-Jung McKay A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Genetics Program of Study Committee: Janice E. Buss, Co-major Professor Linda Ambrosio, Co-major Professor Diane Bassham Drena Dobbs Ted Huiatt Iowa State University Ames, Iowa 2007 Copyright © Jodi Ho-Jung McKay, 2007. All rights reserved. UMI Number: 3274881 Copyright 2007 by McKay, Jodi Ho-Jung All rights reserved. UMI Microform 3274881 Copyright 2008 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O.
    [Show full text]
  • Mapping of Quantitative Trait Loci for Milk Yield Traits on Bovine Chromosome 5 in the Fleckvieh Cattle
    From the Department of Veterinary Sciences Faculty of Veterinary Medicine Ludwig-Maximilians-Universität München Arbeit angefertigt unter der Leitung von Univ. Prof. Dr. Dr. habil. Martin Förster Mapping of Quantitative Trait Loci for Milk Yield Traits on Bovine Chromosome 5 in the Fleckvieh Cattle Inaugural–Dissertation For the attainment of Doctor Degree in Veterinary Medicine From the Faculty of Veterinary Medicine of the Ludwig-Maximilians-Universität München by Ashraf Fathy Said Awad from Sharkia- Egypt München 2011 Gedruckt mit Genehmigung der Tierärztlichen Fakultät der Ludwig–Maximilians–Universität München Dekan: Univ. Prof. Dr. Braun Berichterstatter: Univ. Prof. Dr. Dr. habil Förster Korreferent: Univ. Prof. Dr. Mansfeld Tag der Promotion: 12. February 2011 This work is dedicated to My Parents, my wife and my lovely daughters; Sama, Shaza, Hana CONTENTS CONTENTS ABBREVIATION……………………………………………………………… IV CHAPTER 1: GENERAL INTRODUCTION……………………………….. 1 CHAPTER 2: REVIEW OF LITERATURE………………………………… 3 2.1. DNA Markers……………………………………………………….. 3 2.1.1. Microsatellites………………………………………………………….. 3 2.1.2. Single Nucleotide Polymorphism (SNPs)…………………………… 4 2.2. Mapping of Quantitative Trait Loci (QTL)…………………….. 5 2.2.1. QTL Mapping Designs………………………………………………... 6 2.2.1.1. Daughter Design………………………………………………... 6 2.2.1.2. Granddaughter Design………………………………………… 7 2.2.1.3. Complex Pedigree Design…………………………………….. 9 2.2.2. QTL Mapping Strategies……………………………………………… 10 2.2.2.1. Candidate Gene Approach……………………………………. 10 2.2.2.2. Genome Scan Approach……………………………………… 11 2.3. Principles of Linkage Mapping…………………………………. 12 2.4. QTL Fine Mapping………………………………………………… 14 2.4.1. Linkage Disequilibrium……………………………………………… 15 2.4.2. Combined Linkage Disequilibrium and Linkage (LDL) Mapping… 17 2.5. Identification of Candidate Genes……………………………… 18 2.6.
    [Show full text]
  • Supplemental Information
    Supplemental information Dissection of the genomic structure of the miR-183/96/182 gene. Previously, we showed that the miR-183/96/182 cluster is an intergenic miRNA cluster, located in a ~60-kb interval between the genes encoding nuclear respiratory factor-1 (Nrf1) and ubiquitin-conjugating enzyme E2H (Ube2h) on mouse chr6qA3.3 (1). To start to uncover the genomic structure of the miR- 183/96/182 gene, we first studied genomic features around miR-183/96/182 in the UCSC genome browser (http://genome.UCSC.edu/), and identified two CpG islands 3.4-6.5 kb 5’ of pre-miR-183, the most 5’ miRNA of the cluster (Fig. 1A; Fig. S1 and Seq. S1). A cDNA clone, AK044220, located at 3.2-4.6 kb 5’ to pre-miR-183, encompasses the second CpG island (Fig. 1A; Fig. S1). We hypothesized that this cDNA clone was derived from 5’ exon(s) of the primary transcript of the miR-183/96/182 gene, as CpG islands are often associated with promoters (2). Supporting this hypothesis, multiple expressed sequences detected by gene-trap clones, including clone D016D06 (3, 4), were co-localized with the cDNA clone AK044220 (Fig. 1A; Fig. S1). Clone D016D06, deposited by the German GeneTrap Consortium (GGTC) (http://tikus.gsf.de) (3, 4), was derived from insertion of a retroviral construct, rFlpROSAβgeo in 129S2 ES cells (Fig. 1A and C). The rFlpROSAβgeo construct carries a promoterless reporter gene, the β−geo cassette - an in-frame fusion of the β-galactosidase and neomycin resistance (Neor) gene (5), with a splicing acceptor (SA) immediately upstream, and a polyA signal downstream of the β−geo cassette (Fig.
    [Show full text]
  • G Protein Regulation of MAPK Networks
    Oncogene (2007) 26, 3122–3142 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc REVIEW G Protein regulation of MAPK networks ZG Goldsmith and DN Dhanasekaran Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA, USA G proteins provide signal-coupling mechanisms to hepta- the a-subunits has been used as a basis for the helical cell surface receptors and are criticallyinvolved classification of G proteins into Gs,Gi,Gq and G12 in the regulation of different mitogen-activated protein families in which the a-subunits that show more than kinase (MAPK) networks. The four classes of G proteins, 50% homology are grouped together (Simon et al., defined bythe G s,Gi,Gq and G12 families, regulate 1991). In G-protein-coupled receptor (GPCR)-mediated ERK1/2, JNK, p38MAPK, ERK5 and ERK6 modules by signaling pathways, ligand-activated receptors catalyse different mechanisms. The a- as well as bc-subunits are the exchange of the bound GDP to GTP in the a-subunit involved in the regulation of these MAPK modules in a following which the GTP-bound a-subunit disassociate context-specific manner. While the a- and bc-subunits from the receptor as well as the bg-subunit. The GTP- primarilyregulate the MAPK pathwaysvia their respec- bound a-subunit and the bg-subunit stimulate distinct tive effector-mediated signaling pathways, recent studies downstream effectors including enzymes, ion channels have unraveled several novel signaling intermediates and small GTPase, thus regulating multiple signaling including receptor tyrosine kinases and small GTPases pathways including those involved in the activation of through which these G-protein subunits positivelyas well mitogen-activated protein kinase (MAPK) modules as negativelyregulate specific MAPK modules.
    [Show full text]
  • Free PDF Download
    Eur opean Rev iew for Med ical and Pharmacol ogical Sci ences 2013; 17: 2318-2322 Molecular changes of mesenchymal stromal cells in response to dexamethasone treatment J.- M. CHEN, X.-P. CUI 1, X.-D. YAO, L .-H. HUANG, H. XU Department of Orthopedics, Fuzhou General Hospital of Nanjing Command, PLA, Clinical Medical College of Fujian Medical University, Fuzhou, People’s Republic of China 1Department of Neurology, Fuzhou General Hospital of Nanjing Command, PLA Clinical Medical College of Fujian Medical University, Fuzhou, People’s Republic of China Jian-Mei Chen and Xiao-Ping Cui should be regarded as co-first authors Abstract. – BACKGROUND: Mesenchymal stem such as repair of impairment of bone, cartilage, cells (MSCs) are multipotent stromal cells that can tendons and other tissues 3. Research on the MSCs differentiate into a variety of cell types. The MSCs culture, proliferation and differentiation under canAbIMe a: ctivated and mobilized if needed. This study aimed to investigate the re - regulation was critical . sponse mechanism of MSCs under Dexametha - Dexamethasone (Dex) is a potent synthetic sone (Dex) treatment by combining MSCs mi - member of the glucocorticoid class of steroid drugs croMarAraTyERaInAdLbSioAiNnfDorMmaEtTicHsOmDSet:hods. that has immunosuppressant and anti-inflammato - We downloaded ry properties 4. It has been widely used as anti-in - the gene expression profile of ratʼs MSCs chal - flammatory and chemotherapeutic agents 5; howev - lenge with or without Dex (GSE3339) from Gene Expression Omnibus database, including 2 Dex er, prolonged use of Dex enhances direct respon - treated samples and 3 untreated samples. The dif - siveness of osteoblasts and ultimately impairs bone ferentially expressed genes (DEGs) were identi - formation, leading to changes in cell number and fied by packages in R language.
    [Show full text]