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Molecular characterization and expression of two new members of the SLC10 transporter family: SLC10A4 and SLC10A5 [Elektronische Ressource] / vorgelegt von Carla Freire Celedonio Fernandes

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Carla Freire Celedonio FernandesMolecular Characterization and Expression of Two New Members of the SLC10 Transporter Family: SLC10A4 and SLC10A5Dissertation zur Erlangung des Doktorgrades der Naturwissenchaften (Dr. rer. Nat.) édition scientifique dem Fachbereich Pharmazie der VVB LAUFERSWEILER VERLAGPhilipps-Universität MarburgVVB LAUFERSWEILER VERLAG ISBN 3-8359-5230-7 ST AU FEN BER G R I N G 1 5D - 3 5 3 9 6 G I E S S E NTel: 0641-5599888 Fax: -5599890redaktion@doktorverlag.dew w w . d o k t o r v e r l a g . d e 9 7 8 3 8 3 5 9 5 2 3 0 0édition scientifiqueVVB VVB LAUFERSWEILER VERLAGCARLA FREIRE CELEDONIO FERNANDES SLC10A4 AND SLC10A5Das Werk ist in allen seinen Teilen urheberrechtlich geschützt. Jede Verwertung ist ohne schriftliche Zustimmung des Autors oder des Verlages unzulässig. Das gilt insbesondere für Vervielfältigungen, Übersetzungen, Mikroverfilmungen und die Einspeicherung in und Verarbeitung durch elektronische Systeme.1. Auflage 2007All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of the Author or the Publishers.

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Published 01 January 2007
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Carla Freire Celedonio Fernandes
Molecular Characterization and
Expression of Two New Members
of the SLC10 Transporter Family:
SLC10A4 and SLC10A5
Dissertation zur Erlangung des
Doktorgrades der Naturwissenchaften
(Dr. rer. Nat.)
édition scientifique dem Fachbereich Pharmazie der
VVB LAUFERSWEILER VERLAG
Philipps-Universität Marburg
VVB LAUFERSWEILER VERLAG ISBN 3-8359-5230-7
ST AU FEN BER G R I N G 1 5
D - 3 5 3 9 6 G I E S S E N
Tel: 0641-5599888 Fax: -5599890
redaktion@doktorverlag.de
w w w . d o k t o r v e r l a g . d e 9 7 8 3 8 3 5 9 5 2 3 0 0
édition scientifique
VVB VVB LAUFERSWEILER VERLAG
CARLA FREIRE CELEDONIO FERNANDES SLC10A4 AND SLC10A5Das Werk ist in allen seinen Teilen urheberrechtlich geschützt.
Jede Verwertung ist ohne schriftliche Zustimmung des Autors
oder des Verlages unzulässig. Das gilt insbesondere für
Vervielfältigungen, Übersetzungen, Mikroverfilmungen
und die Einspeicherung in und Verarbeitung durch
elektronische Systeme.
1. Auflage 2007
All rights reserved. No part of this publication may be
reproduced, stored in a retrieval system, or transmitted,
in any form or by any means, electronic, mechanical,
photocopying, recording, or otherwise, without the prior
written permission of the Author or the Publishers.
st
1 Edition 2007
© 2007 by VVB LAUFERSWEILER VERLAG, Giessen
Printed in Germany
VVB LAUFERSWEILER VERLAG
édition scientifique
STAUFENBERGRING 15, D-35396 GIESSEN
Tel: 0641-5599888 Fax: 0641-5599890
email: redaktion@doktorverlag.de
www.doktorverlag.deAus dem Institut für Pharmakologie und Toxikologie
der Philipps-Universität Marburg

Betreuer: Prof. Dr. Dr. Joseph Krieglstein

und

dem Institut für Pharmakologie und Toxikologie
der Justus-Liebig-Universität Gießen

Betreuer: Prof. Dr. Ernst Petzinger


Molecular Characterization and Expression of
Two New Members of the SLC10 Transporter
Family: SLC10A4 and SLC10A5



Dissertation zur
Erlangung des Doktorgrades
der Naturwissenchaften
(Dr. rer. nat.)
dem Fachbereich Pharmazie
der Philipps-Universität Marburg




vorgelegt von





Carla Freire Celedonio Fernandes

Pharmazeutin aus Limoeiro do Norte,
Ceará, Brasilien




Marburg/Lahn 2007






















Vom Fachbereich Pharmazie der Philipps-Universität Marburg als Dissertation
am 02. November 2007 angenommen.

Erstgutachter: Prof. Dr. Dr. Josef Krieglstein
Zweitgutachter: Prof. Dr. Ernst Petzinger

Tag der mündlichen Prüfung am 14. Dezember 2007































„DENN DER HIMMEL IST DER MENSCH
UND DER MENSCH IST DER HIMMEL
UND ALLE MENSCHEN EIN HIMMEL
UND DER HIMMEL NUR EIN MENSCH.“
PARACELSUS











This dissertation is dedicated to my parents Carlos & Ariza,
my siblings Carliza & Carlos Júnior, and my husband
Cléberson.

Com amor…





















ERKLÄRUNG



Ich versichere, dass ich meine Dissertation “ Molecular characterization and expression of two new
members of the SLC10 transporter family: SLC10A4 and SLC10A5 „ selbständig ohne unerlaubte
Hilfe angefertigt und mich dabei keiner anderen als der von mir ausdrücklich bezeichneten Quellen
bedient habe.

Die Dissertation wurde in der jetzigen oder einer ähnlichen Form noch bei keiner anderen
Hochschule eingereicht und hat noch keinen sonstigen Prüfungszwecken gedient.


Marburg, den 02 November 2007





Carla Freire Celedonio Fernandes



. CONTENTS
Contents
Figures and Tables .......................................................................................................................iv
Abbreviations ................................................................................................................................vi
1. Introduction ................................................................................................................................... 9
1.1. Review of Literature.................................................................................................................... 9
1.1.1. Principles of Membrane Transport ............................................................................................. 9
1.1.2. Membrane Transport Systems ................................................................................................. 10
1.1.3. The Solute Carrier Superfamily (SLC)...................................................................................... 10
1.1.4. The Solute Carrier 10 Family.................................................................................................... 12
1.1.4.1. NTCP...... 13
1.1.4.2. ASBT ........................................................................................................................ 14
1.1.4.3. SLC10A3 .................................................................................................................. 14
1.1.4.4. SLC10A4... 14
1.1.4.5. SLC10A5... 15
1.1.4.6. SOAT...... 15
1.1.4.7. SLC10A7... 15
1.1.5. Enterohepatic Circulation of Bile Acids .................................................................................... 16
1.1.6. The Role of CHT1, VAChT and ChAT in the Cholinergic System............................................ 17
1.2. Aim of the Work ....................................................................................................................... 20
2. Material ....................................................................................................................................... 21
2.1. Primers and Assays .............................................................................................................................. 21
2.1.1. Primers for sequencing............................................................................................................. 21
2.1.2. Primers for expression profile................................................................................................... 21
2.1.3. TaqMan gene expression assays for quantitative real time PCR (qPCR) ............................... 21
2.1.4. Primers for cloning.................................................................................................................... 22
2.1.5. Primers for sequence insertion of FLAG-epitope ..................................................................... 22
2.1.6. Primers for sequence insertion of HA-epitope.......................................................................... 22
2.1.7. Primers for subcloning in the vector pcDNA5/TO 23
2.1.8. Primers for control of the FLAG- and HA-insertions................................................................. 23
2.2. Agarose/Formaldehyde Gel Electrophoresis and Northern Blot........................................................... 23
2.2.1. Solutions and buffers................................................................................................................ 23
2.2.2. Gel electrophoresis.............. 24
2.2.3. Blotting...................................................................................................................................... 24
2.2.4. Other materials ......................................................................................................................... 25
2.3. Cloning, Expression Profiles, cRNA-Synthesis and Insertion of the FLAG and HA epitopes............... 25
2.3.1. Bacterial strains ........................................................................................................................ 25
2.3.2. Vectors...................................................................................................................................... 26
2.3.3. Media ........................................................................................................................................ 28
2.3.4. Agarose gel electrophoresis ..................................................................................................... 29
2.3.5. Enzymes.................. 29
2.3.6. Commercialized kits and material............................................................................................. 30
2.3.7. cDNA-Pannels and RNAs...... 30
2.4. Expression in Xenopus laevis Oocytes ................................................................................................. 31
2.4.1. Animals.................... 31
2.4.2. Solutions and buffers for the X.laevis oocytes.......................................................................... 31
2.4.3. Radiochemicals ........................................................................................................................ 31
2.4.4. Material ..................................................................................................................................... 31
2.5. Immunofluorescence in Xenopus laevis Oocytes ................................................................................. 32
2.5.1. Solution and buffers.................................................................................................................. 32
2.5.2. Antibodies ................................................................................................................................. 32
2.6. Cell Culture.............................. 33
i CONTENTS
2.6.1. Eukariotic cell line HEK293....................................................................................................... 33
2.6.2. Eukaryotic cell line PC12.......................................................................................................... 33
2.6.3. Material for cell culture............... 33
2.6.4. Cell culture medium and supplements ..................................................................................... 33
2.6.5. Transient transfection using HEK293 cells............................................................................... 33
2.7. Immunocytochemistry ........................................................................................................................... 34
2.7.1. Primary and secondary antibodies ........................................................................................... 34
2.7.2. Reagents .................................................................................................................................. 34
2.7.3. Buffer and solutions.................................................................................................................. 34
2.8. Western Blot.......................................................................................................................................... 34
2.8.1. HEK293 cells ............................................................................................................................ 35
2.8.2. Solutions and buffers................................................................................................................ 35
2.8.3. Membranes............................................................................................................................... 35
2.8.4. Other materials and reagents ................................................................................................... 36
2.9. In situ Hybridization............................................................................................................................... 36
2.9.1. Animals.................... 36
2.9.2. Solutions and buffers............. 36
2.9.3. Probe preparation ..................................................................................................................... 38
2.9.4. Other materials and reagents....... 38
2.10. Immunohistochemistry ........................................................................................................................ 38
2.10.1. Animals ................................................................................................................ 38
2.10.2. Solutions and buffers.............................................................................................................. 38
2.10.3. Antibodies ............................................................................................................................... 39
2.10.4. Other materials and reagents ................................................................................................. 39
2.11. Reagents............................ 40
2.12. Equipments ......................................................................................................................................... 41
2.13. Bioinformatic............................................................................................................ 42
3. Methods ...................................................................................................................................... 43
3.1. General Molecular Biology Methods ..................................................................................................... 43
3.1.1. PCR Product purification .......................................................................................................... 43
3.1.2. Concentration and purity determination of nucleic acid preparations....................................... 43
3.1.3. Agarose-gel electrophoresis..................................................................................................... 43
3.1.4. Restriction enzyme digestion.................................................................................................... 44
3.1.5. Plasmid purification................................................................................................................... 44
3.1.6. Preparation of electrocompetent E.coli cells ............................................................................ 44
3.2. RNA Extraction...................................................................................................................................... 46
3.2.1. Total RNA isolation and RNA quality........................................................................................ 46
+3.2.2. Purification of Poly (A) RNA from total RNA ........................................................................... 46
3.3. Northern Blot ......................................................................................................................................... 47
3.3.1. Preparation of radiolabeled cDNA probes................................................................................ 47
3.3.2. Blotting and hybridization.......................................................................................................... 48
3.4. cDNA Synthesis........................ 48
3.5. PCR – Polymerase Chain Reaction ...................................................................................................... 48
3.5.1. RT-PCR .................................................................................................................................... 48
3.5.2. Real time quantitative PCR......... 49
3.5.3. Site-directed mutagenesis by PCR to insert FLAG and HA epitopes ...................................... 50
3.6. Cloning .................................................................................................................................................. 50
3.7. Heterologous Expression in Xenopus laevis Oocytes for Transport..................................................... 52
3.7.1. Linearization of plasmid DNA ................................................................................................... 52
3.7.2. cRNA synthesis ........................................................................................................................ 53
3.7.3. Frog surgery and preparation of oocytes.................................................................................. 53
3.7.4. Microinjection.................. 54
3.8. Functional Characterization................................................................................................................... 54
ii

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