Developmentally-regulated localization and possible functions of HRP-3 in the murine nervous tissue [Elektronische Ressource] / vorgelegt von Heba Mahmoud Ahmed Eltahir

Heba Mahmoud Ahmed El-Tahir

Sujets

• Biologie

Informations

• Publié par: rheinische_friedrich-wilhelms-universitat_bonn
• Publié le: 01 janvier 2009
• Nombre de lectures: 21
• Langue: English
• Poids de l'ouvrage: 5 Mo

Extrait

Developmentally-regulated localization and
possible functions of HRP-3 in the murine
nervous tissue
Dissertation
zur
Erlangung des Doktorgrades (Dr. rer. Nat.)
der
Mathematische Naturwissenschaftlichen Fakultät
der
Rheinischen Friedrich-Wilhelms Universität Bonn
vorgelegt von
Heba Mahmoud Ahmed Eltahir
Aus
Kena, Egypt
Bonn, Oktober 2009 Angefertigt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät
der Rheinischen Friedrich-Wilhelms-Universität Bonn





















1. Gutachter: Prof. Dr. med. Volkmar Gieselmann
2. Gutachter: Prof. Dr. Klaus Mohr


Tag der Promotion: 10. März 2010
Erscheinungsjahr: 2010



For
My small family,
& for the soul of my
parents. Acknowledgement
First, I would thank God for helping me to finish this work in the best I can way
then the people who helped me in every step of this work.
I would like to thank
Prof. Dr. med. Volkmar Gieselmann for giving me the generous chance to do my
doctoral thesis in his lab. His interesting ideas and constructive discussion have
been of major importance for this work.
Prof. Dr. Klaus Mohr for taking the responsibility of being my second referee
I would also like to deeply thank my co-supervisor Dr. Sebastian Franken for his
excellent supervision of my work. He was always able to invent new ideas that
gave my work much better dimensions. His constructive criticism, great support,
help and cooperation were of great importance for my work and always gave me a
push forward. He was always there answering my questions and had made a
great effort correcting and revising until this work had come out in its final shape.
The group of Dr. Franken; Rainer Gallitzendörffer, Katharina Klein, Stephanie
Bremer, Robert and specially Angela sedlmeier for the precise reading of the last
manuscript of this work.
The members of the anatomy department for their great help and instructions in
preparing the Vibratome sections. And also I would like to thank them for the
facilities they provided in using the confocal laser microscope.
The group of Prof. Dr. Magin for the microscope facilities they provided.
Dr. Kappler, Dr. Eckhardt and Dr. Matzner for their help and cooperation.

All co-workers for their cooperation and for providing a friendly work atmosphere.
Mrs. Simonis for her great help in general DNA techniques, Mrs. Buttkau and Mr.
Rösel for their great help in technical work.
Mr. Pflüger for his great and brilliant help in the use of computer programs.
My husband, Mekky, for his incredible help and endless support. He sacrificed
many important things to provide me with the suitable atmosphere to do my work.
Without his help, love and patience, I would never be able to do this work.
My children, Ahmed and Radwa who pushed me forward with their love and
smiles.
My brother and sister and my big family in Egypt, they provided me with support
and encouragement and they have done too much for me especially my mother.
Finally, I wanted to thank my mother for all what she had done for me, for being
always behind me and for all her words, advices and love even when she is not
there any more to read these words. Table of content
Table of content
Table of content ........................................................................... I
List of figures ............................................................................... V
List of tables ................................................................................. VIII
Abbreviations ............................................................................... IX
1. Introduction .............................................................................. 1
1.1 Development of the murine nervous system ................................ 1
1.2 Extracellular matrix ....................................................................... 5
1.3 Growth factors, cytokines and neurotrophic factors ........... 7
1.3.1 Nomenclature ................................................................................. 7
1.3.2 Roles of growth factors .................................................................. 9
1.3.2.1 General roles ...................................................................... 9
1.3.2.2 Neuron-related roles ........................................................... 9
1.3.3 Hepatoma-derived growth factor and its related proteins .............. 11
1.3.3.1 Family-member overview .................................................... 11
1.3.3.2 Hepatoma-derived growth factor related protein -3 ............ 13
1.4 Aim of work ................................................................................... 13
2. Materials .................................................................................... 15
2.1 Instruments ................................................................................... 15
2.2 Equipments .............. 16
2.3 Reagents ...................................................................................... 16
2.3.1 Chemicals ...................................................................................... 16
2.3.2 Standard solutions and buffers ...................................................... 19
2.3.3 Media ............................................................................................. 20
2.3.4 Antibodies ...................................................................................... 20
2.3.5 Restriction enzymes ....................................................................... 21
2.3.6 Bacteria, cell lines, and animals ..................................................... 21
2.3.7 Primers ........................................................................................... 22
3. Methods .................................................................................... 23
3.1 Cell culture protocols .................................................................... 23
3.1.1 Primary mouse cortical neurons culture ......................................... 23
3.1.2 Protein coating of tissue culture vessels ........................................ 23
3.1.3 Determination of neurite length ...................................................... 24
3.1.4 Neuron rescue by the addition of purified proteins ......................... 24
® 3.1.5 Quantitative determination of cell survival using Alamar blue ...... 25
3.1.5.1 Principle .............................................................................. 25
3.1.5.2 Protocol ............................................................................... 25
3.1.6 Antibody treatment of neurons ....................................................... 26
3.1.7 Gene silencing ............................................................................... 27
3.1.7.1 Definition and types ............................................................ 27
3.1.7.2 RNAi approach ................................................................... 27
ITable of content
3.1.7.3 Transfection of neurons with siRNA oligos using
®Lipofectamin 2000 ............................................................. 29
3.1.7.4 Transfection of neurons with vector-based siRNA using
®
MATra ............................................................................... 29
3.1.7.4.1 Principle ................................................................ 29
3.1.7.4.2 Protocol ................................................................. 30
3.1.8 Primary mouse sensory neuron culture ......................................... 30
3.1.9 B35 neuroblastoma cells ................................................................ 31
3.1.9.1 B35 cells in culture .............................................................. 31
3.1.9.2 Transient transfection of B35 cells with HRP-3 constructs . 32
3.1.10 Daoy medulloblastoma cells ........................................................ 32
3.1.10.1 Transient transfection of Daoy cells using calcium
phosphate ........................................................................ 32
3.1.10.2 Stable transfection and selection of Daoy cells ................ 33
3.1.10.3 Proliferation assay of Daoy cells using MTS .................... 34
3.1.10.3.1 Principle .............................................................. 34
3.1.10.3.2 Protocol ............................................................... 34
3.1.11 Freezing of cells ........................................................................... 34
3.1.12 Thawing of cells ........................................................................... 35
3.2 Immunolocalization protocols ............................................................ 35
3.2.1 Immunolocalization of interest proteins in mouse tissues .............. 35
3.2.2 Immunolocalization of interest proteins in neuron cultures ............ 36
3.3 DNA protocols .............................................................................. 37
3.3.1 Cloning of HRP-3 variants .............................................................. 37
3.3.2 Construction