Functional studies of GR and MR function by RNA interference [Elektronische Ressource] / vorgelegt von Hee-Young Lim

Lim Hee Yong

Sujets

• Biologie

Informations

• Publié par: julius-maximilians-universitat_wurzburg
• Publié le: 01 janvier 2007
• Nombre de lectures: 23
• Langue: English
• Poids de l'ouvrage: 7 Mo

Extrait

Functional studies of GR and MR function
by RNA interference









Dissertation zur Erlangung des
naturwissenschaftlichen Doktorgrades
der Bayerischen Julius-Maximilians-Universität Würzburg




vorgelegt von
Hee-Young Lim

aus
Seoul, Korea




Würzburg, 2007




















Eingereicht am:……………………………………


Mitglieder der Promotionskommission:

Prof. Dr. Martin J. Müller Vorsitzender : ……..…………………………………………………………….

Prof. Dr. Holger M. ReichardtGutachter : ………………………………………………………………………

Prof. Dr. Klaus SchellerGutachter : ………………………………………………………………………



Tag des Promotionskolloquiums:…………………………………………………



Doktorurkunde ausgehändigt am:…………………………………………………






Declaration


I hereby declare that the submitted dissertation was completed by myself and none other, and
I have not used any sources or materials other than those indicated.
Moreover, I declare this dissertation has not been submitted elsewhere in this form and has
not been used for obtaining any other equivalent qualification in any other organization.
Additionally, other than this degree I have not applied or will not attempt to apply for any
other degree, title or qualification in relation to this work.

















(Hee-Young Lim)
May 2007
Institute of Virology and Immunobiology
University of Würzburg, Germany





Acknowledgements


I am pleased to acknowledge and thank the many people who have helped and encouraged me
during my PhD time.
First of all I would like to grateful thank Prof. Dr. Holger M. Reichardt for supervising my
work and for giving me the opportunity to carry out my doctoral work in his lab, as well as for
his support and personal care. I am very thankful to Prof. Thomas Hünig for his generous help
and personal care.
I would like to thank Prof. K. Scheller for accepting to be my co-supervisor at the faculty of
biology and his support with all the formalities concerning the doctoral thesis.
I’m thanks to Dr. Jens van den Brandt for grateful help regarding my subjects and to Dr. Nora
Müller for kindly help for experiment. I am delighted to thank Dr. Marco Herold for his
helping me with kindness for discussion of experiment.
I’m thanks to Melanie, Kati, Denise, Dapeng and Christian for their help and the good times
we had together in the lab. I also thank my colleagues in other labs in the Institute.
Special thanks to Soon-Hwan and Shin-Young for many useful discussions and generous help,
and personal care, make me always delightful time in Germany.
I’m very grateful Prof. Yeo In-Kyu and Prof. Yoon Byoung-Su for his support and
encouragement during this time. I appreciate Pres. Lee Hyun-Jong for his support and
personal concerning.
I reserve the greatest thanks for my family, for their unlimited love and support.








Contents

I. Introduction..................................................................................................... 1
1.1 Steroid hormones; Glucocortioids.................................................................... 1
1.2 Steroid hormone receptors................................................................................ 2
1.2.1 Glucocorticoid receptor (GR)................................................................. 2
1.2.1.1 Structure of the glucocortcioid receptor............................................... 2
1.2.1.2 Mechanisms of glucocorticoid via the GR............................................ 3
1.2.2 Mineralocorticoid receptor (MR)........................................................... 6
1.2.2.1 Structure of the mineralocorticoid receptor.......................................... 6
1.2.2.2 The role of the MR................................................................................ 8
1.3 Immunomodulatory actions of glucocorticoids in macrophages...................... 10
1.3.1 Function of macrophages in innate immunity........................................ 11
1.3.2 Immunomodulatory actions of glucocorticoids...................................... 12
1.4 RNA interference (RNAi)................................................................................ 15

Aim of this study.................................................................................................... 20

II. Materials......................................................................................................... 22
2.1 Chemicals and general materials...................................................................... 22
2.2 Enzymes and inhibitors..................................................................................... 25
2.3 Reagent kits....................................................................................................... 25
2.4 Radioactive materials....................................................................................... 26
2.5 Size markers..................................................................................................... 26
2.6 Antibiotics........................................................................................................ 26
2.7 Antibodies......................................................................................................... 27
2.8 Consumables..................................................................................................... 27
2.9 Buffers and solutions........................................................................................ 28
2.9.1 Culture Medium...................................................................................... 28
2.9.1.1 Culture medium for Bacteria................................................................ 28
2.9.1.2 Culture medium for eukaryotic cells.................................................... 29
2.9.2 Molecular Biological Solutions.............................................................. 30
2.9.3 Cell Biological Solutions........................................................................ 32 2.10 Bacterial Hosts.................................................................................................. 34
2.11 Cell lines........................................................................................................... 35
2.12 Vectors.............................................................................................................. 35
2.13 Instruments....................................................................................................... 35
2.14 Primer pairs...................................................................................................... 37

III. Methods......................................................................................................... 40
3.1 Methods in Molecular biology.......................................................................... 40
3.1.1 DNA work............................................................................................... 40
3.1.1.1 Isolation of plasmid DNA..................................................................... 40
3.1.1.2 Isolation of genomic DNA.................................................................... 41
3.1.1.3 Electrophoresis...................................................................................... 42
3.1.1.3.1 Agarose gel electrophoresis............................................................. 42
3.1.1.3.2 TAE-Polyacrylamide gel electrophoresis........................................ 42
3.1.1.4 Restriction Reaction.............................................................................. 43
3.1.1.5 Elution of DNA..................................................................................... 44
3.1.1.5.1 Gel extraction kit.............................................................................. 44
3.1.1.5.2 Elution from polyacrylamide gels.................................................... 45
3.1.1.6 Recombinant DNA manipulation.......................................................... 45
3.1.1.6.1 Vector and Insert DNA preparation................................................. 45
3.1.1.6.2 Ligation............................................................................................ 46
3.1.1.6.3 Transformation to competent cells.................................................. 46
3.1.1.6.3.1 Heat-shock transformation.......................................................... 46
3.1.1.6.3.2 Electroporation............................................................................ 47
3.1.1.6.4 Screening of recombinant DNA....................................................... 47
3.1.1.6.4.1 Colony PCR for selection of positive recombinant DNA........... 47
3.1.1.6.4.2 Sequencing reaction..............................................................