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Publié par | humboldt-universitat_zu_berlin |
Publié le | 01 janvier 2008 |
Nombre de lectures | 44 |
Langue | English |
Poids de l'ouvrage | 5 Mo |
Extrait
NF-κB programme of
dendritic cell activation is affected
by vitamin D3
Dissertation
zur Erlangung des akademischen Grades
doctor rerum naturalium
(Dr. rer. nat.)
im Fach Biologie
eingereicht an der
Mathematisch-Naturwissenschaftlichen Fakultät I
der Humboldt-Universität zu Berlin
von Dipl. Biochemiker Mykola Goncharenko
geboren am 30.04.1978
in Kiew, Ukraine
Präsident der Humboldt-Universität zu Berlin
Prof. Dr. Christoph Markschies
Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I
Prof. Dr. Christian Limberg
Gutachter/innen: 1. Prof. Dr. Wolfgang Uckert
2. Prof. Dr. Claus Scheidereit
3. Prof. Dr. Martin Zenke
Tag der mündlichen Prüfung: 07.04.2008ACKNOWLEDGEMENTS
ACKNOWLEDGEMENTS
I would like to use this opportunity to express my deep gratitude to Prof. Martin Zenke, who
over the last number of years has guided me in every possible way with his abundant
knowledge and insightful ideas.
I appreciate the time invested by Prof. Wolfgang Lockau, Prof. Harald Saumweber, Prof.
Claus Scheidereit, Prof. Wolfgang Uckert and Prof. Martin Zenke for reading through the
numerous pages of this doctoral thesis manuscript and evaluating my work.
I would like to thank all members of the Institute of Biomedical Technology -Cell Biology- of
RWTH Aachen, especially Antonio, Piritta, David, Verena, Ivonne, Julia - thank you. I would
like to mention Thomas Hieronymus for his everyday support and enlightening discussions,
and Chris for making the lab go around. I deeply appreciated the help of Andrea who made
my daily life bearable, by taking care of every small crisis.
Many thanks to the members of the old “Zenke lab” of the MDC in Berlin: Petra Haink, Signe
Knespel, Christine Hacker and many others whom I have known only briefly. I would like to
say a special thank you to Gitta Blendinger for teaching me tissue culture techniques and to
Tatjana Gust for supervising my fist steps at the lab.
I cannot let this opportunity pass without extending my gratitude to Prof. Wolfgang Uckert
for hosting me for a year at his lab at the MDC after the move of the “Zenke lab” to Aachen,
as well as for his support and excellent judgement. Here I have to additionally thank Lisa,
Irmgard, Uta, Heike, Boris, Lilian, Cordelia and Matthias for providing everyday help and
support. Also special words of appreciation have to go to Prof. Thomas Blankenstein for his
constructive advice, and to all members of his team for their input and helpful scientific
opinions.
Additionally, I would like to thank Prof. Scheidereit and members of his group for some
revealing discussions on NF-κB signalling. I would like to extend a special word of gratitude
to Eva Kärgel for sharing the chromatin immunoprecipitation technique, for all joint
experiments, and for providing the moral support when it was needed so much.
I must regret that the limits of space prevent me having the satisfaction of acknowledging all
the generous assistance which I have received from many scientists during the years of my
Ph.D.
At last I would like to thank the members of my family, both past and present, and my dear
friends Thomas, Olga, Christian, Juri, Etienne, Adrian, Stefan, Matt and Anis for their
invaluable help, support and care.
IZUSAMMENFASSUNG
ZUSAMMENFASSUNG
Die Fähigkeit dendritischer Zellen (DC) Immunität zu erzeugen und Antigen-spezifische
Toleranz zu induzieren macht DC zu idealen Zielen pharmakologischer Interventionen zur
Beeinflussung von Immunreaktionen. NF-κB Faktoren sind eine Gruppe von
Transkriptionsregulatoren, die für die Entwicklung und Funktion von DC bedeutend sind.
Trotz ihrer zentralen Bedeutung für die DC Biologie ist die Identität von NF-κB regulierten
Genen in DC weitestgehend unbekannt. In der vorliegenden Studie wurde die Hemmung der
NF-κB Aktivierung durch den IκBα Super Repressor (IκBα-SR) und die Analyse der
Genexpression durch DNA Microarrays genutzt, um das Repertoire an NF-κB regulierten
Genen in DC zu ermitteln. Mit diesem Ansatz wurden unter anderem Connexin-43 und Fascin
als direkte NF-κB regulierte Gene identifiziert.
Die Beeinflussung des NF-κB Signalwegs wurde als möglicher Weg zur Modifizierung von
Immunantworten vorgeschlagen. Es wird vermutet, dass verschiedene
immunmoduloratorische Verbindungen wie z.B. Vitamin D3 (VD3) in NF-κB vermittelte
Immunmechanismen eingreifen. Um die Effekte von VD3 in der Aktivierung von DC zu
untersuchen, wurden DNA Microarray Analysen an DC von Mäusen mit mutiertem und
wildtyp Vitamin D3 Rezeptor (VDR) durchgeführt und die Beteiligung der VDR vermittelten
Repression von NF-κB regulierten Genen wie z.B. Connexin-43 untersucht. Die so
identifizierten Gene stellen potentielle Ansatzpunkte für die Entwicklung von spezifischeren
entzündungshemmenden Medikamenten für die klinische Anwendung dar.
IIABSTRACT
ABSTRACT
The ability of dendritic cells (DC) to initiate immunity and induce antigen-specific tolerance
makes DC ideal targets for pharmacological intervention into immune responses. NF-κB
factors are a family of transcriptional regulators important for DC development and function.
However, the identity of NF-κB target genes that are central to DC biology is largely
unknown. In the present study, inhibition of the NF-κB activation by the IκBα super repressor
(IκBα-SR) and DNA microarray analysis were used to determine the repertoire of NF-κB
responsive genes in DC. This approach identified, among others, connexin-43 and fascin as
direct NF-κB regulated genes.
The targeting of the NF-κB signalling pathway has been suggested as a useful means to
modify immune responses. A number of immunomodulatory compounds, such as vitamin D3
(VD3), are believed to affect NF-κB mediated immune mechanisms. Microarray analysis
employing vitamin D3 receptor (VDR) mutant and wild-type mice was used to survey the
effects of VD3 in DC. In this study, effects of VD3 on the activation of DC are evaluated, and
involvement of VDR mediated repression of NF-κB regulated genes, such as connexin-43, is
surveyed. Identified genes can be potentially useful targets for the development of more
specific anti-inflammatory agents for clinical applications.
IIITABLE OF CONTENTS
TABLE OF CONTENTS
Acknowledgements .....................................................................................................................I
ZUSAMMENFASSUNG .......................................................................................................... II
ABSTRACT .............................................................................................................................III
TABLE OF CONTENTS .........................................................................................................IV
ABBREVIATIONS................................................................................................................ VII
1. INTRODUCTION.......................................................................................................1
1.1 DC biology..................................................................................................................1
1.1.1 Immunity versus tolerance ..........................................................................................1
1.1.2 DC as potent initiators of immune responses ..............................................................2
1.1.3 Functional maturation of DC.......................................................................................2
1.2 Molecular regulation of DC maturation and function .................................................4
1.3 NF-κB signalling pathway...........................................................................................5
1.4 Immune effects of vitamin D3 on DC .........................................................................9
1.4.1 Vitamin D3 and intracellular signalling ....................................................................11
1.5 Gene transfer into DC................................................................................................14
1.5.1 Receptor-mediated gene delivery into DC with adenovirus as ligand ......................14
1.5.2 Adenoviral gene-delivery vectors..............................................................................14
1.5.3 CAR and CAR mice ..................................................................................................15
1.6 Aims and objectives16
2. MATERIALS AND METHODS17
2.1 Materials....................................................................................................................17
2.1.1 Appliances and computer software ...........................................................................17
2.1.2 Chemicals, solutions and buffers...............................................................................18
2.1.3 Kits ..........................................................................................................................20
2.1.4 Other materials.....................................