Functional epithelial cell proteomics under conditions of chronic intestinal inflammation [Elektronische Ressource] / Anna Shkoda

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• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2007
• Nombre de lectures: 25
• Langue: Deutsch
• Poids de l'ouvrage: 5 Mo

Extrait

Fachgebiet für Experimentelle Ernährungsmedizin der
Technischen Universität München






Functional epithelial cell proteomics under conditions of

chronic intestinal inflammation


Anna Shkoda



Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan
für Ernährung, Landnutzung und Umwelt der Technischen Universität München
zur Erlangung des akademischen Grades eines
Doktors der Naturwissenschaften (Dr. rer. nat.)
genehmigten Dissertation.


Vorsitzender: Univ.-Prof. Dr. H. Daniel

Prüfer der Dissertation: 1. Univ.-Prof. Dr. D. Haller
2. apl.Prof. Dr. A. Görg




Die Dissertation wurde am 30.08.2006 bei der Technischen Universität München eingereicht und
durch die Fakultät Wissenschaftzentrum Weihenstephan für Ernährung, Landnutzung und Umwelt
am 14.11.2006 angenommen. Publications

1. Ruiz PA, Shkoda A, Kim SC, Sartor RB, Haller D. IL-10 gene-deficient mice lack TGF-
beta/Smad signaling and fail to inhibit proinflammatory gene expression in intestinal
epithelial cells after the colonization with colitogenic Enterococcus faecalis. J Immunol
2005; 174(5): 2990-9.

2. Shkoda AP, Ruiz PA, Daniel H, Kim SC, Rogler G, Sartor RB, Haller D. Interleukin 10
blocked endoplasmatic reticulum stress in the intestinal epithelium: impact on chronic
inflammation. Gastroenterology 2007; 132: 190-207.

3. Shkoda A, Werner T, Daniel H, Gunckel M, Rogler G, Haller D. Differential protein
expression profile in intestinal epithelium from patients with inflammatory bowel disease. J.
Proteome Res. 2007 (in press)





Poster presentations

1. Shkoda A, Haller D, Sartor RB. Charakterisierung von Signaltransduktion und
Proteinexpressionsprofilen in nativen Darmepithelzellen unter Bedingungen chronischer
Entzündung: Patienten mit chronisch entzündlichen Darmerkrankungen im Vergleich zum
IL-10 gene-defizienten Mausmodell. 42. Wissenschaftlicher Kongress der DGE Kiel 17-18
March, 2005. First poster prize.

2. Shkoda A, Ruiz PA, Kim SC, Sartor RB, Daniel H, Haller D. Identification of dynamic
proteome changes in primary intestinal epithelial cells after the colonization of germ-free
wild type and IL-10 gene deficient mice with colitogenic Enterococcus faecalis.
Inflammatory Bowel Disease: Research Drives Clinics Münster 2-3 September, 2005.

3. Shkoda A, Ruiz PA, Kim SC, Sartor RB, Daniel H, Haller D. Identification of dynamic
proteome changes in primary intestinal epithelial cells after the colonization of germ-free ficient mice with colitogenic Enterococcus faecalis. Innate
Immunity and IBD Stuttgart 4-5 November, 2005.

4. Shkoda A, Sartor RB, Daniel H, Haller D. Charakterisierung der physiologischen Relevanz
und molekularen Funktion von grp-78, kreatin kinase und Galectin-3 in der Regulation von
Entzündungsprozessen in Darmepithelzellen. 43. Wissenschaftlicher Kongress der DGE
Hohenheim 9-10 March 2006.

5. Shkoda A, Sartor RB, Daniel H, Haller D. Functional epithelial cell proteomics identified
novel disease targets for the colitogenic mechanisms of Enerococcus faecalis in IL-10 gene
deficient mice. Crohn and Colitis Foundation of America (CCFA) Microbial-Host
Interaction Workshop St. Petersburg, Florida USA 16-19 March, 2006. Travel award and
poster presentation.
2Acknowledgements


There are many people who directly or indirectly contributed to my scientific life, to the supervision
of this thesis and generally, to my successful adaptation in Germany during the last 3 years. In
particular, I would like to express my special thanks to the following persons:

• To Prof. Dr. Dirk Haller for creation of the outstanding research group and well-equipped
laboratory; for his scientific competence, supervision and personal time investment; for the
constant generation of new scientific ideas and for the competitive spirit in the lab with
elements of “positive pressure” , “brainstorming” and humour
• To Prof. Dr. Hannelore Daniel, Prof. Dr. Angelika Görg and Prof. Dr. Michael Schemann
who kindly agreed to revise this manuscript
• To Prof. Balfour Sartor and his colleagues from University of North Caroline, USA for the
participation in joint research projects in gnotobiotic facilities
• To Angelika Herzog, Dagmar Fuchs, Isabel Winkelmann and to all people from Molecular
Nutrition Unit for the comprehensive help, advice and patience during my first steps in
mastering the proteomic technique
• To Pedro Ruiz for the scientific partnership, flexibility and for his 24-hours availability for
all possible working questions
• To our laboratory members Micha, Anja, Theresa, Eva and Gabi for the constant assistance,
collaboration and nice time spent also outside the lab
• To Tanja Werner for the help in conduction of clinical proteomic studies
• To Anja Osterhues, Nico Gebhardt and our former building-neighbours for the pleasant
sharing of equipment and time
• To my parents, grandmother and relatives, supporting me so much despite being so far
• To Clemens for his great optimism, moral support and simply for the presence in my life


THANK YOU VERY MUCH!!!
3Content
List of abbreviations.................................................................................................... 8
1 Introduction ........................................................................................................ 11
1.1 Immune response in the gut ............................................................................................11
1.1.1 Commensal microbiota of the gut..................................................................................12
1.1.2 Role of the intestinal epithelium in the host protection.................................................13
1.1.3 Role of the gut-associated lymphatic tissue in mucosal homeostasis............................15
1.2 Innate immune recognition .............................................................................................16
1.2.1 Toll receptor signalling..................................................................................................17
1.3 Signalling to NF-κB: importance for innate immunity and inflammation.................19
1.4 NF-κB activation and apoptosis......................................................................................22
1.5 Animal models of chronic intestinal inflammation.......................................................23
1.6 Human Inflammatory Bowel Disease.............................................................................26
1.7 Proteomics: identification of novel targets and molecular mechanisms.....................29
1.7.1 Proteome analysis: principles and main strategy...........................................................29
1.7.2 Proteomics for disease: importance of clinical studies..................................................32
1.7.3 Epithelial protein expression profiling ..........................................................................33
2 Aims of the present work................................................................................... 35
3 Materials and Methods ...................................................................................... 36
3.1 Reagents and Chemicals..................................................................................................36
3.2 Buffers and Solutions.......................................................................................................40
3.3 Technical equipment........................................................................................................43
3.4 Methods and techniques45
3.4.1 Isolation of primary intestinal epithelial cells from IBD patients .................................45
3.4.2 Animals and bacterial monoassociation ........................................................................46
3.4.3 Isolation of primary mouse intestinal epithelial cells....................................................46
3.4.4 Cell culture.....................................................................................................................47
3.4.5 Bacterial infection and cell stimulation .........................................................................47
3.4.6 Adenoviral infection......................................................................................................48
3.4.7 Small interference RNA cell transfections ....................................................................48
3.4.8 Real-Time PCR (Light Cycler)......................................................................................49
43.4.8.1 Isolation of total RNA........................................................................................49
3.4.8.2 cDNA generation...............................................................................................49
3.4.8.3 Primer design and Real-Time PCR.................................................