Modulation of Na and acetate transport by heat shock proteins (Hsp) across the rumen epithelium of sheep [Elektronische Ressource] / vorgelegt von Imtiaz Rabbani

Rabbani.Imtiaz

Informations

• Publié par: freie_universitat_berlin
• Publié le: 01 janvier 2010
• Nombre de lectures: 16
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Institute of Veterinary Physiology
Faculty of Veterinary Medicine
Free University of Berlin




Modulation of Na and acetate transport by heat shock proteins (Hsp)
across the rumen epithelium of sheep




Thesis submitted for the partial fulfillment of a doctoral degree in
Veterinary Medicine (Dr. med. vet.) at the Free University of Berlin



submitted by
Imtiaz Rabbani
D.V.M, M.Sc. (Hons)
Veterinarian from Lahore/Pakistan
Berlin 2010
Journal Nr.: 3429 Aus dem Institut für Veterinär-Physiologie
des Fachbereichs Veterinärmedizin
der Freien Universität Berlin








Modulation of Na and acetate transport by heat shock proteins (Hsp)
across the rumen epithelium of sheep





Inaugural-Dissertation zur Erlangung des Grades eines
Doktors der Veterinärmedizin (Dr. med. vet.)
an der Freien Universität Berlin



vorgelegt von
Imtiaz Rabbani
Tierarzt aus Lahore/Pakistan
Berlin 2010
Journal Nr.: 3429
Gedruckt mit Genehmigung des Fachbereichs Veterinärmedizin
der Freien Universität Berlin


Dekan: Univ.-Prof. Dr. L. Brunnberg
Erster Gutachter: Univ.-Prof. Dr. H. Martens
Zweiter Gutachter: Univ.-Prof. Dr. A. Richter
Dritter Gutachter: Univ.-Prof. Dr. K. E. Müller


Deskriptoren (nach CAB-Thesaurus):
rumen, epithelium, ion transport, sodium, heat shock proteins, sheep


Tag der Promotion: 05.11.2010





Bibliografische Information der Deutschen Nationalbibliothek

Die Deutsche Nationalbibliothek verzeichnet diese Publikation in der Deutschen
Nationalbibliografie; detaillierte bibliografische Daten sind im Internet über
<http://dnb.ddb.de> abrufbar.

ISBN: 978-3-86664-861-6
Zugl.: Berlin, Freie Univ., Diss., 2010
Dissertation, Freie Universität Berlin
D 188

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Dedicated to
My parents for their continuous support
and unselfish love
Contents:
1 General Introduction and literature review .............................................................. 2
1.1 Introduction ................................................................................................................ 3
1.2 Review of literature .................................................................................................... 5
1.2.1 Anatomy of the rumen ................................................................................................ 5
1.2.2 Rumen fermentation ................................................................................................... 6
1.2.3 Transport mechanisms across the rumen ................................................................... 7
1.2.4 Consequences of improper fermentation and absorption ......................................... 11
1.2.5 Ruminal adaptation .................................................................................................. 16
1.2.6 Stress recovery mechanisms and pH regulation ...................................................... 18 i
1.2.7 Heat shock proteins (Hsp) ........................................................................................ 24
1.2.8 Hsp70 and acidosis 28
2 Material and Methods ................................................................................................ 33
2.1 Experimental animals ............................................................................................... 35
2.1.1 Isolation, preparation, and handling of epithelial tissues/cells .................................
2.2 Ussing chamber technique ....................................................................................... 37
2.2.1 Electrical measurements ........................................................................................... 38
2.3 Calculation of ion flux rates ..................................................................................... 40
2.3.1 Measurement of Na and acetate fluxes .................................................................... 41
2.4 Estimation of heat shock proteins (Hsp70) .............................................................. 41
2.4.1 Incubation and collection of tissues/cells ................................................................. 41
2.4.2 Western blot ............................................................................................................. 42
2.4.3 Reverse transcription with quantitative polymerase chain reaction (RT-qPCR) ..... 43
2.5 Buffer solutions ........................................................................................................ 43
2.6 Inhibitors .................................................................................................................. 44
2.6.1 Amiloride ................................................................................................................. 44
2.6.2 Cariporide (HOE 642) .............................................................................................. 44
2.6.3 S3226 ........................................................................................................................ 45
2.6.4 Cycloheximide ......................................................................................................... 45
2.6.5 Triptolide .................................................................................................................. 46
2.7 Data analysis ............................................................................................................ 46
3 Results ......................................................................................................................... 47
3.1 In vitro Induction of Hsp70 ...................................................................................... 49
i 3.1.1 Effect of temperature ................................................................................................ 49
3.1.2 Effect of pH .............................................................................................................. 60
3.1.3 Effect of SCFA concentration and mucosal pH ....................................................... 63
3.1.4 Effect of mucosal osmolarity ................................................................................... 65
3.1.5 Effect of inhibitors ................................................................................................... 66
3.2 Measurement of Na flux rates .................................................................................. 69
3.2.1 Introduction .. 69
3.2.2 Effect of NHE inhibitors .......................................................................................... 69
3.2.3 Effect of Hsp70 inhibitor 72
3.3 Measurement of acetate fluxes ................................................................................. 75
3.3.1 Introduction .............................................................................................................. 75
3.4 In vivo induction of Hsp70 ....................................................................................... 77
3.4.1 Introduction .. 77
3.4.2 Measurement of Hsp70 expression .......................................................................... 79
3.4.3 ent of Na and acetate fluxes .................................................................... 80
4 General Discussion and Conclusion 83
4.1 Induction of Hsp70 ................................................................................................... 86
4.2 Modulation of Hsp70 expression by inhibitors ........................................................ 91
4.3 Hsp and transport physiology ................................................................................... 93
4.4 Na and acetate transport ........................................................................................... 94
5 Summary ..................................................................................................................... 99
6 Zusammenfassung .................................................................................................... 103
7 References ..........................