Role of the IKK complex during liver regeneration [Elektronische Ressource] : analysis of the differential role of IKKβ/IKK2 [IKKbeta/IKK2] and IKKγ/Nemo [IKKgamma/Nemo] during liver regeneration / vorgelegt von Yann Malato

Yann Robin

Sujets

• Gesundheit

Informations

• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2009
• Nombre de lectures: 10
• Langue: English
• Poids de l'ouvrage: 17 Mo

Extrait

Role of the IKK complex during liver
regeneration

Analysis of the differential role of IKKβ/IKK2 and IKKγ/Nemo during
liver regeneration



Von der Fakultät für Mathematik, Informatik und Naturwissenschaften
der RWTH Aachen University zur Erlangung des akademischen Grades
eines Doktors der Naturwissenschaften genehmigte Dissertation

vorgelegt von

Master of Science
Yann Malato
aus Bordeaux (Frankreich)


Berichter:
Universitätsprofessor Dr. med. Christian Trautwein
Universitätsprofessor Dipl. Ing. Dr. Werner Baumgartner

Tag der mündlichen Prüfung:
24. November 2009


Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar.
















"Check your heart, free your soul, never give up"
Mike Muir



Teile dieser Arbeit wurden bereits vorab veröffentlicht oder zur Publikation
eingereicht:

Y. Malato, N. Beraza, N. Gassler, M. Al-Masaoudi, C. Liedtke, C. Trautwein
activation of progenitor cells triggers the repopulation of conditional nemo KO livers
during liver regeneration in mice
Journal of Hepatology, April 2009 (Vol. 50, Page S310) Poster n°851
44th Annual Meeting of the European Association for the Study of the Liver (EASL),
Copenhagen, Denmark.


Y. Malato, L. E. Sander, C. Liedtke, M. Al-Masaoudi, F. Tacke, C. Trautwein, N.
Beraza
Hepatocyte-specific inhibitor-of-kappaB-kinase deletion triggers the innate immune
response and promotes earlier cell proliferation during liver regeneration
Hepatology. 2008 Jun;47(6):2036-50.


Y. Malato, N. Beraza, L.E. Sander, M. Almasaoudi, C. Liedtke, C. Trautwein
Hepatocyte specific IKK2 deletion causes imbalanced NF-κB activation after partial
hepatectomy and leads to an earlier resolution of liver damage
Journal of Hepatology, April 2008 (Vol. 48, Page S58) Oral Presentation n°135
43rd Annual Meeting of the European Association for the Study of the Liver (EASL),
Milan, Italy.


Y. Malato, N. Beraza, L.E. Sander, M. Almasaoudi, C. Liedtke, C. Trautwein
Enhanced innate immune response and accelerated liver regeneration in hepatocyte-
specific IKK2 deleted mice
Hepatology 46, November 2007. Oral presentation n°56. 58th Annual Meeting of the
American Association for the Study of Liver Diseases (AASLD), Boston, MA, USA.



Y. Malato, N. Beraza, M. Pasparakis, C. Trautwein
IKK2 hepatocyte specific deletion alters cell cycle progression after partial
hepatectomy
Journal of Hepatology, April 2007 (Vol. 46, Page S21) Oral presentation n°44
42nd Annual Meeting of the European Association for the Study of the Liver (EASL),
Barcelona, Spain.
Table of Contents I
Table of Contents

1. INTRODUCTION ....................................................................................... 1
1.1. PHYSIOLOGY AND CENTRAL FUNCTION OF THE LIVER .......................................... 1
1.2. LIVER REGENERATION ..................................................... 2
1.3. NF-ΚB, IKK2, NEMO AND LIVER REGENERATION ............... 3
1.4. THE CELL CYCLE ............................................................. 9
1.5. INVOLVEMENT OF THE INNATE IMMUNE RESPONSE DURING LIVER REGENERATION 11
1.6. REACTIVE OXYGEN SPECIES ......................................................................... 11
1.7. KNOCKOUT TECHNOLOGY AND TRANSGENIC MICE ............ 14
1.7.1. Generation of conditional IKK2 knockout mice .................................................................. 15
1.7.2. Generation of conditional Nemo knockout mice ................................................................ 16
1.8. AIM OF THE STUDY ........................................................................................ 18
2. MATERIAL AND METHODS ...................................... 19
2.1. CHEMICALS .................................................................. 19
2.1.1. Radiochemicals ...................................................................................................................... 21
2.2. INSTRUMENTS AND EQUIPMENT ...................................... 21
2.3. CONSUMABLES ............................................................................................. 22
2.3.1. General material .................................................................................................................... 22
2.3.2. Material for animal experimentation ............................................................. 22
2.3.3. Material for cell culture and flow cytometry ........................................................................ 23
2.3.4. ELISA and MACS kits ........................................................................................................... 23
2.3.5. Antibodies ............................................................................................................................... 24
2.3.5.1. Non-labeled primary antibodies ........................................................................................... 24
2.3.5.2. Labeled primary antibodies .......................................................................... 25
2.3.5.3. Secondary antibodies ............................................................................................................ 25
2.3.6. Enzymes.................................................................................................................................. 25
2.4. ANALYTICAL CHEMICALS, REAGENTS AND KITS ................................................. 26
2.5. ANIMALS ...................................................................... 27
2.5.1. Animal breeding ..................................................................................................................... 27
2.5.2. Partial hepatectomy ............................................................................................................... 27
2.5.3. Blood taking ............................................................................................................................ 27
2.5.4. Systemic IKK2 inhibition ....................................................................................................... 27
2.5.5. Oval cell expansion (DDC diet) ............................................................................................ 28 Table of Contents II
2.5.6. Antioxidant treatment (BHA diet) ......................................................................................... 28
2.5.7. Oxidative stress induction (PEITC injection)............................................... 28
2.5.8. Isolation of primary hepatocytes .......................................................................................... 28
2.5.9. Cell isolation and flow cytometry ................................................. 29
2.6. RNA ISOLATION ............................................................................................ 30
2.6.1. Isolation of RNA from liver tissue ........................................................................................ 30
2.6.2. Isolation of RNA from cell culture ....................... 30
2.6.3. Determination of RNA concentration .................................................................................. 31
2.6.4. Reverse Transcription (RT-PCR) ............................................. 31
2.6.5. Real-Time PCR ...................................................................................................................... 31
2.7. ISOLATION AND CHARACTERIZATION OF PROTEINS ............................................ 33
2.7.1. Protein extraction from liver tissue and primary hepatocytes ......................................... 33
2.7.2. Quantification of proteins by Bradford Assay .................................................................... 34
2.7.3. c-Jun-N-terminal-Kinase activity assay .............................................................................. 34
2.7.4. Zymography ........................................................................................................................... 35
2.8. GENOTYPING ................................................................................................ 35
2.8.1. Buffer ....................................................................................................................................... 35
2.8.2. PCR protocols and programs ............................................................................................... 36
2.9. PRIMERS LIST ............................................................................................... 38
2.9.1. Genotyping primers (5’ -> 3’) ............................................................................................... 38
2.9.2. Real-Time PCR primers ........................................................................................................ 38
2.10. GENERAL PROTOCOL FOR IMMUNOFLUORESCENCE .......................................... 40
2.10.1. BrdU and Histone H3 staining .............................................................................................. 41
2.10.2. CK19 staining .....