DOCSLIB.ORG

Dissertation Nicolette Mamant

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Dissertation Nicolette Mamant Charakterisierung des Aktivierungsmechanismus der HtrA-Protease DegP von E. coli Inaugural-Dissertation zur Erlangung des Doktorgrades Dr. rer. nat. der Fakultät für Biologie und Geografie an der Universität Duisburg-Essen vorgelegt von Nicolette Mamant aus Essen Gutachter: Prof. Dr. M. Ehrmann, Prof. Dr. B. Siebers, Prof. Dr. H. de Groot Datum der mündlichen Prüfung: 17.12.2009 Teile dieser Arbeit sind in folgenden Veröffentlichungen enthalten: Hauske, P., Mamant, N., Hasenbein, S., Nickel, S., Ottmann, C., Clausen, T., Ehrmann, M. und Kaiser, M. (2009) Peptidic small molecule activators of the stress sensor DegS. Mol. BioSyst. , 5(9): 980-985. Meltzer, M., Hasenbein, S., Mamant, N., Merdanovic, M., Poepsel, S., Hauske, P., Kaiser, M., Huber, R., Krojer, T., Clausen, T. und Ehrmann, M. (2009) Structure, function and regulation of the conserved serine proteases DegP and DegS of E. coli. Res. Microbiol., 2009. Meltzer, M., Hasenbein, S., Hauske, P., Kucz, N., Merdanovic, M., Grau, S., Beil, A., Jones, D., Krojer, T., Clausen, T., Ehrmann, M. und Kaiser, M. (2008) Allosteric activation of HtrA protease DegP by stress signals during bacterial protein quality control. Angew. Chem. Int. Ed. Engl., 47 : 1332-1334; Angew. Chem., 120 : 1352-1355. Kucz, N., Meltzer, M. und Ehrmann, M. (2006) Periplasmic proteases and protease inhibitors. The Periplasm , ASM Press, ed. M. Ehrmann: 150-170. In Vorbereitung: Merdanovic, M., Meltzer, M., Mamant, N., Pöpsel, S., Beil, A., Soerensen, R., Hauske, P., Kaiser, M., Nagel-Steger, L., Sickmann, A., Huber, R., Clausen, T. und Ehrmann, M. Determinants of structural and functional plasticity of a widely conserved protease chaperone machine. Verzeichnisse 3 INHALTSVERZEICHNIS ABBILDUNGSVERZEICHNIS .............................................................................................7 TABELLENVERZEICHNIS ..................................................................................................9 ABKÜRZUNGSVERZEICHNIS..........................................................................................10 1 ZUSAMMENFASSUNG ...............................................................................................12 2 EINLEITUNG ................................................................................................................14 2.1 Escherichia coli ........................................................................................................... 14 2.1.1 E. coli als Modellorganismus................................................................................... 14 2.1.2 Die Morphologie von E. coli ....................................................................................14 2.2 Proteinqualtitätskontrolle..........................................................................................17 2.2.1 Chaperone.................................................................................................................17 2.2.2 Proteasen ..................................................................................................................19 2.3 Serinproteasen ............................................................................................................20 2.4 HtrA-Proteasen...........................................................................................................22 2.5 Die periplasmische Serinprotease DegP................................................................... 25 2.5.1 Die Regulation der degP -Expression in der Zelle....................................................25 2.5.2 Die Struktur von DegP .............................................................................................28 2.5.3 Die Doppelfunktion von DegP als Protease und Chaperon .....................................31 2.6 PDZ-Domänen ............................................................................................................33 2.6.1 Die Rolle von PDZ-Domänen in HtrA-Proteasen....................................................37 2.6.2 Die PDZ-Domänen von DegP..................................................................................40 2.7 Die allosterische Aktivierung der HtrA-Protease DegS..........................................42 2.8 Zielsetzung der Arbeit................................................................................................45 3 MATERIAL UND METHODEN ................................................................................. 46 3.1 Allgemeines .................................................................................................................46 3.2 Bakterienstämme........................................................................................................46 3.3 Vektoren und Plasmide..............................................................................................46 3.4 Oligonukleotide...........................................................................................................48 3.5 Antiseren .....................................................................................................................49 3.6 Proteine und Enzyme .................................................................................................49 3.7 Peptide .........................................................................................................................51 3.8 DNA- und Proteinstandards......................................................................................51 3.9 Reaktions- und Nachweis-Kits ..................................................................................52 3.10 Chemikalien ................................................................................................................52 3.11 Geräte ..........................................................................................................................52 3.12 Diverses........................................................................................................................53 3.13 Mikrobiologische Methoden......................................................................................54 Verzeichnisse 4 3.13.1 Sterilisation...........................................................................................................54 3.13.2 Bakterienanzucht..................................................................................................54 3.13.2.1 Herstellung von Nährmedien ...........................................................................54 3.13.2.2 Antibiotika........................................................................................................54 3.13.2.3 Andere Medienzusätze .....................................................................................55 3.13.2.4 Anzucht von E. coli ..........................................................................................55 3.13.2.5 Bestimmung der Zelldichte in Flüssigkulturen ................................................55 3.14 Molekularbiologische Methoden...............................................................................55 3.14.1 Präparation von Plasmid-DNA.............................................................................55 3.14.2 Methoden zur Charakterisierung von DNA ......................................................... 55 3.14.2.1 Restriktionshydrolysen von Plasmid-DNA......................................................55 3.14.2.2 Agarosegelelektrophorese ................................................................................56 3.14.2.3 DNA-Konzentrationsbestimmung.................................................................... 56 3.14.2.4 Sequenzierung ..................................................................................................56 3.14.3 Isolierung von DNA-Fragmenten aus Agarosegelen ...........................................57 3.14.4 Ligierung von DNA-Fragmenten......................................................................... 57 3.14.5 Transformation.....................................................................................................57 3.14.5.1 Herstellung elektrokompetenter Zellen............................................................ 57 3.14.5.2 Elektrotransformation.......................................................................................58 3.14.5.3 Herstellung transformationskompetenter Zellen für die Transformation durch Hitzeschock ............................................................................................ 58 3.14.5.4 Transformation der kompetenten Bakterienzellen mit Plasmid-DNA mittels Hitzeschock...........................................................................................59 3.14.6 Polymerase-Kettenreaktion (PCR)....................................................................... 59 3.14.6.1 Kolonie-PCR....................................................................................................60 3.14.6.2 Oligomutagenese..............................................................................................61 3.14.7 Aufreinigung von PCR-Produkten....................................................................... 62 3.14.8 Plasmidkonstruktionen.........................................................................................62 3.14.8.1 Konstruktion der Plasmide pNK11, pNK12, pNK13 und pNK14...................62 3.14.8.2 Konstruktion der Plasmide zur Expression der DegP-Punktmutanten.............63 3.15 Biochemische Methoden ............................................................................................63
Load more

Recommended publications
  • Expression of Membrane Proteins in Escherichia Coli
    From Biogenesis to Over- expression of Membrane Proteins in Escherichia coli Samuel Wagner Stockholm University © Samuel Wagner, Stockholm 2008 ISBN 978-91-7155-594-6, Pages i-81 Printed in Sweden by Universitetsservice AB, Stockholm 2008 Distributor: Department of Biochemistry and Biophysics, Stockholm University To Claudia. Abstract In both pro- and eukaryotes 20-30% of all genes encode α-helical transmem- brane domain proteins, which act in various and often essential capacities. Notably, membrane proteins play key roles in disease and they constitute more than half of all known drug targets. The natural abundance of membrane proteins is in general too low to con- veniently isolate sufficient material for functional and structural studies. Therefore, most membrane proteins have to be obtained through overexpres- sion. Escherichia coli is one of the most successful hosts for overexpression of recombinant proteins, and T7 RNA polymerase-based expression is the major approach to produce recombinant proteins in E. coli. While the pro- duction of soluble proteins is comparably straightforward, overexpression of membrane proteins remains a challenging task. The yield of membrane lo- calized recombinant membrane protein is usually low and inclusion body formation is a serious problem. Furthermore, membrane protein overexpres- sion is often toxic to the host cell. Although several reasons can be postu- lated, the basis of these difficulties is not completely understood. It is gener- ally believed, that the complex requirements of membrane protein biogenesis significantly contribute to the difficulty of membrane protein overexpres- sion. Therefore, an understanding of membrane protein biogenesis is a pre- requisite for understanding membrane protein overexpression and for de- signing rational strategies to improve overexpression yields.
    [Show full text]
  • Role of Regulated Proteolysis in the Communication of Bacteria with the Environment
    fmolb-07-586497 October 11, 2020 Time: 10:35 # 1 REVIEW published: 15 October 2020 doi: 10.3389/fmolb.2020.586497 Role of Regulated Proteolysis in the Communication of Bacteria With the Environment Sarah Wettstadt and María A. Llamas* Department of Environmental Protection, Estación Experimental del Zaidín-Consejo Superior de Investigaciones Científicas, Granada, Spain For bacteria to flourish in different niches, they need to sense signals from the environment and translate these into appropriate responses. Most bacterial signal transduction systems involve proteins that trigger the required response through the modification of gene transcription. These proteins are often produced in an inactive state that prevents their interaction with the RNA polymerase and/or the DNA in the absence of the inducing signal. Among other mechanisms, regulated proteolysis is becoming increasingly recognized as a key process in the modulation of the activity of these signal response proteins. Regulated proteolysis can either produce complete degradation or specific cleavage of the target protein, thus modifying its function. Because proteolysis is a fast process, the modulation of signaling proteins activity by this Edited by: process allows for an immediate response to a given signal, which facilitates adaptation Chew Chieng Yeo, to the surrounding environment and bacterial survival. Moreover, regulated proteolysis Sultan Zainal Abidin University, Malaysia is a fundamental process for the transmission of extracellular signals to the cytosol Reviewed by: through the bacterial membranes. By a proteolytic mechanism known as regulated Iain Lamont, intramembrane proteolysis (RIP) transmembrane proteins are cleaved within the plane of University of Otago, New Zealand the membrane to liberate a cytosolic domain or protein able to modify gene transcription.
    [Show full text]
  • Invariant Chain Complexes and Clusters As Platforms for MIF Signaling
    cells Review Invariant Chain Complexes and Clusters as Platforms for MIF Signaling Robert Lindner Institute of Neuroanatomy and Cell Biology, Hannover Medical School, 30625 Hannover, Germany; [email protected]; Tel.: +49-511-532-2918 Academic Editor: Ritva Tikkanen Received: 8 December 2016; Accepted: 7 February 2017; Published: 10 February 2017 Abstract: Invariant chain (Ii/CD74) has been identified as a surface receptor for migration inhibitory factor (MIF). Most cells that express Ii also synthesize major histocompatibility complex class II (MHC II) molecules, which depend on Ii as a chaperone and a targeting factor. The assembly of nonameric complexes consisting of one Ii trimer and three MHC II molecules (each of which is a heterodimer) has been regarded as a prerequisite for efficient delivery to the cell surface. Due to rapid endocytosis, however, only low levels of Ii-MHC II complexes are displayed on the cell surface of professional antigen presenting cells and very little free Ii trimers. The association of Ii and MHC II has been reported to block the interaction with MIF, thus questioning the role of surface Ii as a receptor for MIF on MHC II-expressing cells. Recent work offers a potential solution to this conundrum: Many Ii-complexes at the cell surface appear to be under-saturated with MHC II, leaving unoccupied Ii subunits as potential binding sites for MIF. Some of this work also sheds light on novel aspects of signal transduction by Ii-bound MIF in B-lymphocytes: membrane raft association of Ii-MHC II complexes enables MIF to target Ii-MHC II to antigen-clustered B-cell-receptors (BCR) and to foster BCR-driven signaling and intracellular trafficking.
    [Show full text]
  • The Role of Proteases in Plant Development
    The Role of Proteases in Plant Development Maribel García-Lorenzo Department of Chemistry, Umeå University Umeå 2007 i Department of Chemistry Umeå University SE - 901 87 Umeå, Sweden Copyright © 2007 by Maribel García-Lorenzo ISBN: 978-91-7264-422-9 Printed in Sweden by VMC-KBC Umeå University, Umeå 2007 ii Organization Document name UMEÅ UNIVERSITY DOCTORAL DISSERTATION Department of Chemistry SE - 901 87 Umeå, Sweden Date of issue October 2007 Author Maribel García-Lorenzo Title The Role of Proteases in Plant Development. Abstract Proteases play key roles in plants, maintaining strict protein quality control and degrading specific sets of proteins in response to diverse environmental and developmental stimuli. Similarities and differences between the proteases expressed in different species may give valuable insights into their physiological roles and evolution. Systematic comparative analysis of the available sequenced genomes of two model organisms led to the identification of an increasing number of protease genes, giving insights about protein sequences that are conserved in the different species, and thus are likely to have common functions in them and the acquisition of new genes, elucidate issues concerning non-functionalization, neofunctionalization and subfunctionalization. The involvement of proteases in senescence and PCD was investigated. While PCD in woody tissues shows the importance of vacuole proteases in the process, the senescence in leaves demonstrate to be a slower and more ordered mechanism starting in the chloroplast where the proteases there localized become important. The light-harvesting complex of Photosystem II is very susceptible to protease attack during leaf senescence. We were able to show that a metallo-protease belonging to the FtsH family is involved on the process in vitro.
    [Show full text]
  • Characterization of the Cpx Response in Vibrio Cholerae
    Characterization of the Cpx Response in Vibrio cholerae by Paula Nicole Acosta Amador A thesis submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology and Biotechnology Department of Biological Sciences University of Alberta © Paula Nicole Acosta Amador, 2015 Abstract The gram negative bacterial cell envelope is composed of the outer membrane, the periplasm and the inner membrane. These compartments are exposed directly to changes in the environment that are sensed and adapted to through different signaling transduction pathways. This often occurs through two-component signal transduction systems (TCS), which are broadly distributed among different bacterial species. The Cpx pathway is a TCS that employs the sensor histidine kinase CpxA and the response regulator CpxR, and regulates crucial adaptations to envelope stress response that affects many functions, including antibiotic resistance, across bacterial species. This system has also been implicated in the regulation of a number of envelope localized virulence determinants across bacterial species. The first goal of this thesis was to characterize the Cpx regulon members in the human pathogen Vibrio cholerae when the Cpx pathway is activated. For this purpose I characterized the transcriptional profile of the pandemic V. cholerae El Tor strain C6706 upon overexpression of cpxR, and the inducing cues that lead to the activation of the Cpx pathway. My data shows that the Cpx regulon of V. cholerae is enriched for genes encoding membrane localized and transport proteins, including a large number of genes known or predicted to be iron-regulated. The V. cholerae Cpx regulon included three strongly Cpx-regulated, putative ferric reductases that are likely directly regulated by CpxR.
    [Show full text]
  • Westerhausen, Geb
    Development of highly sensitive tools to investigate the Salmonella Type III Secretion System Dissertation der Mathematisch-Naturwissenschaftlichen Fakultät der Eberhard Karls Universität Tübingen zur Erlangung des Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) vorgelegt von Sibel Westerhausen, geb. Şeker aus Uşak/Türkei Tübingen 2020 Gedruckt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Eberhard Karls Universität Tübingen. Tag der mündlichen Qualifikation: 28.01.2021 Stellvertretender Dekan: Prof. Dr. József Fortágh 1. Berichterstatter: Prof. Ph.D. Samuel Wagner 2. Berichterstatter: Prof. Dr. Ana J. Garcia-Saéz Table of Contents List of abbreviations .............................................................................................................................. IV Deutsche Zusammenfassung ................................................................................................................. VI Abstract ................................................................................................................................................ VII 1 Introduction ..................................................................................................................................... 1 1.1 General protein transport pathways through the inner membrane........................................... 1 1.1.1 Protein degradation system .............................................................................................. 3 1.2 The type III secretion system .................................................................................................
    [Show full text]
  • New Insight Into Plant Intramembrane Proteases Małgorzata Adamiec
    bioRxiv preprint doi: https://doi.org/10.1101/101204; this version posted January 18, 2017. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. New insight into plant intramembrane proteases Małgorzata Adamiec *, Lucyna Misztal, and Robert Luciński Adam Mickiewicz University, Faculty of Biology, Institute of Experimental Biology, Department of Plant Physiology, ul. Umultowska 89, 61-614 Poznań, Poland. *-corresponding author ABSTRACT The process of proteolysis is a factor involved in control of the proper development of the plant and its responses to a changeable environment. Recent research has shown that proteases are not only engaged in quality control and protein turnover processes but also participate in the process which is known as regulated membrane proteolysis (RIP). Four families of integral membrane proteases, belonging to three different classes, have been identified: serine intramembrane proteases known as rhomboid proteases, site-2 proteases belonging to zinc metalloproteases, and two families of aspartic proteases: presenilins and signal peptide peptidases. The studies concerning intramembrane proteases in higher plants are, however, focused on Arabidopsis thaliana. The aim of the study was to identify and retrieve protein sequences of intramembrane protease homologs from other higher plant species and perform a detailed analysis of their primary sequences as well as their phylogenetic relations. This approach allows us to indicate several previously undescribed issues which may provide important directions for further research. INTRODUCTION Proteolysis is considered as a crucial factor determining the proper development of the plant and its efficient functioning in variable environmental conditions.
    [Show full text]
  • Exploring the Regulatory Mechanism of the Notch Ligand Receptor Jagged1 Via the Aryl Hydrocarbon Receptor in Breast Cancer Sean Alan Piwarski
    Marshall University Marshall Digital Scholar Theses, Dissertations and Capstones 2018 Exploring the Regulatory Mechanism of the Notch Ligand Receptor Jagged1 Via the Aryl Hydrocarbon Receptor in Breast Cancer Sean Alan Piwarski Follow this and additional works at: https://mds.marshall.edu/etd Part of the Biological Phenomena, Cell Phenomena, and Immunity Commons, Medical Molecular Biology Commons, Medical Toxicology Commons, and the Oncology Commons EXPLORING THE REGULATORY MECHANISM OF THE NOTCH LIGAND RECEPTOR JAGGED1 VIA THE ARYL HYDROCARBON RECEPTOR IN BREAST CANCER A dissertation submitted to the Graduate College of Marshall University In partial fulfillment of the requirements for the degree of Doctor of Philosophy In Biomedical Sciences by Sean Alan Piwarski Approved by Dr. Travis Salisbury, Committee Chairperson Dr. Gary Rankin Dr. Monica Valentovic Dr. Richard Egleton Dr. Todd Green Marshall University July 2018 APPROVAL OF DISSERTATION We, the faculty supervising the work of Sean Alan Piwarski, affirm that the dissertation, Exploring the Regulatory Mechanism of the Notch Ligand Receptor JAGGED1 via the Aryl Hydrocarbon Receptor in Breast Cancer, meets the high academic standards for original scholarship and creative work established by the Biomedical Sciences program and the Graduate College of Marshall University. This work also conforms to the editorial standards of our discipline and the Graduate College of Marshall University. With our signatures, we approve the manuscript for publication. ii © 2018 SEAN ALAN PIWARSKI ALL RIGHTS RESERVED iii DEDICATION To my mom and dad, This dissertation is a product of your hard work and sacrifice as amazing parents. I could never have accomplished something of this magnitude without your love, support, and constant encouragement.
    [Show full text]
  • Discovery of Molecular Mechanisms Underlying Lysosomal and Mitochondrial Defects in Parkinson’S Disease
    Discovery of Molecular Mechanisms Underlying Lysosomal and Mitochondrial Defects In Parkinson’s Disease Brigitte Phillips Supervisor: Associate Professor Antony Cooper A thesis in fulfillment of the requirements for the degree of Doctor of Philosophy St Vincent’s Clinical School, Faculty of Medicine The University of New South Wales & The Garvan Institute of Medical Research April, 2018 THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet Surname or Family name: Phillips First name: Brigitte Other names/s: Radinovic Abbreviation for degree as given in the University calendar: PhD School: St Vincent’s Clinical School Title: The emerging contributions of the lysosome and mitochondria to Parkinson’s diseases Abstract 350 words maximum Parkinson’s disease (PD) is a common, debilitating neurodegenerative disease yet the causes of cell dysfunction in PD remain unclear. By integrating available patient data with data from an unbiased assessment of proteomic changes in multiple cellular PD models, this study has identified new aspects of mitochondrial and lysosomal dysfunction that likely contribute to PD. Two areas were investigated in detail; the mitochondrial protein CHCHD2 and the V-ATPase complex, which acidifies endolysosomal compartments. CHCHD2 had not been well characterized or associated with PD when identified in this study, however PD-causative variants have since been described and CHCHD2 has recently been proposed to regulate mitochondrial cristae structure and interact with cytochrome c. Data from this thesis extends CHCHD2 dysfunction to sporadic PD patients, where its reduced expression was identified in the brain. In exploring the potential function of CHCHD2, mitochondrial impairment resulted in rapid translational up- regulation of CHCHD2 and its specific accumulation in depolarised mitochondria, suggesting that CHCHD2 plays a targeted role in aiding mitochondrial recovery or quarantining cytochrome c in response to mitochondrial damage.
    [Show full text]
  • Enzymatic Analysis of a Rhomboid Intramembrane Protease Implicates
    Enzymatic analysis of a rhomboid intramembrane SEE COMMENTARY protease implicates transmembrane helix 5 as the lateral substrate gate Rosanna P. Baker*, Keith Young*, Liang Feng†, Yigong Shi†, and Sinisa Urban*‡ *Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, 507 Preclinical Teaching Building, 725 North Wolfe Street, Baltimore, MD 21205; and †Department of Molecular Biology, Lewis Thomas Laboratory, Princeton University, Princeton, NJ 08544 Edited by Douglas C. Rees, California Institute of Technology, Pasadena, CA, and approved March 19, 2007 (received for review January 30, 2007) Intramembrane proteolysis is a core regulatory mechanism of cells scription factors required for fatty acid and sterol biosynthesis in that raises a biochemical paradox of how hydrolysis of peptide humans (10). Recently, intramembrane proteolysis was recon- bonds is accomplished within the normally hydrophobic environ- stituted in vitro with purified RseP from Escherichia coli (11). ment of the membrane. Recent high-resolution crystal structures This achievement demonstrated that zinc is the only cofactor have revealed that rhomboid proteases contain a catalytic serine required for proteolysis, and further work using this system is recessed into the plane of the membrane, within a hydrophilic likely to reveal how intramembrane proteolysis is catalyzed by cavity that opens to the extracellular face, but protected laterally this class of enzymes. from membrane lipids by a ring of transmembrane segments. This Rhomboid proteins are intramembrane serine proteases that architecture poses questions about how substrates enter the in- are among the most widely conserved membrane proteins known ternal active site laterally from membrane lipid. Because structures (12, 13). Despite significant sequence divergence, many rhom- are static glimpses of a dynamic enzyme, we have taken a boid enzymes from both prokaryotes and eukaryotes share structure–function approach analyzing >40 engineered variants to biochemical characteristics (14, 15).
    [Show full text]
  • Molecular Mechanisms and Effect of Acute and Psychosocial Stress on the Intestinal Barrier Function
    ADVERTIMENT. Lʼaccés als continguts dʼaquesta tesi queda condicionat a lʼacceptació de les condicions dʼús establertes per la següent llicència Creative Commons: http://cat.creativecommons.org/?page_id=184 ADVERTENCIA. El acceso a los contenidos de esta tesis queda condicionado a la aceptación de las condiciones de uso establecidas por la siguiente licencia Creative Commons: http://es.creativecommons.org/blog/licencias/ WARNING. The access to the contents of this doctoral thesis it is limited to the acceptance of the use conditions set by the following Creative Commons license: https://creativecommons.org/licenses/?lang=en DOCTORAL THESIS MOLECULAR MECHANISMS AND EFFECT OF ACUTE AND PSYCHOSOCIAL STRESS ON THE INTESTINAL BARRIER FUNCTION. IMPLICATIONS ON THE IRRITABLE BOWEL SYNDROME. Presented by MARC PIGRAU PASTOR Directed by Francisco Javier Santos Vicente, María Vicario Pérez & Carmen Alonso Cotoner Tutor Fernando Azpiroz Vidaur Thesis submitted to obtain the degree of Doctor PhD program in Medicine Department of Medicine, Medical School, Universitat Autònoma de Barcelona Barcelona, September 2018 FRANCISCO JAVIER SANTOS VICENTE, Doctor en Medicina, MARÍA VICARIO PÉREZ, Doctora en Farmacia y CARMEN ALONSO COTONER, Doctora en Medicina HACEN CONSTAR Que la tesis titulada “Molecular mechanisms and effect of acute and psychosocial stress on the intestinal barrier function. Implications on the irritable bowel syndrome.” presentada por Marc Pigrau Pastor para optar al grado de Doctor, se ha realizado bajo su dirección, y al considerarla concluida, autorizan su presentación para ser juzgada por el tribunal correspondiente. Y para que conste a los efectos firman la presente. Barcelona, Septiembre de 2018 Dr. Francisco Javier Dra. María Dra. Carmen Santos Vicente Vicario Pérez Alonso Cotoner (Directores de la tesis) Dr.
    [Show full text]
  • Tesis Doctoral Alejandro Piñeiro Ugalde Oviedo 2011
    Universidad de Oviedo Departamento de Bioquímica y Biología Molecular Identificación de nuevos componentes del degradoma y estudio de la regulación biológica por miRNAs en un modelo animal deficiente en la metaloproteasa Face-1 Tesis Doctoral Alejandro Piñeiro Ugalde Oviedo 2011 Director de la Tesis Carlos López-Otín ABREVIATURAS, SIGLAS Y SÍMBOLOS ADAM metaloproteasa y desintegrina ADAMTS ADAM con dominios trombospondina cDNA DNA copia Ci curio dCTP desoxicitidina trifosfato DNA ácido desoxirribonucleico dNTPs desoxinucleósidos trifosfato GH hormona de crecimiento HGPS síndrome de Hutchinson-Gilford IGF-1 factor de crecimiento similar a insulina 1 kb kilobase MBS sitio de unión de microRNAs MMP metaloproteasa de matriz miRNA microRNA mRNA RNA mensajero pb pares de bases PCR reacción en cadena de la polimerasa RNA ácido ribonucleico ROS especie reactiva del oxígeno RISC complejo de silenciamiento inducido por RNA SSC tampón citrato sódico TAE tampón Tris/acetato con EDTA TBE tampón Tris/borato con EDTA TE tampón Tris con EDTA TIMP inhibidor tisular de metaloproteasa Tris tris(hidroximetil)-aminometano ÍNDICE Introducción ................................................................................................. 1 Los sistemas proteolíticos .................................................................................................. 4 Metaloproteasas ................................................................................................. 6 Aminopeptidasas ...............................................................................................
    [Show full text]