Influence of selenium on pancreatic carcinogenesis and the role of the selenoproteins cytosolic and mitochondrial thioredoxin reductase in the pancreas [Elektronische Ressource] / vorgelegt von Michaela Yvonne Aichler
175 pages
Deutsch
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Influence of selenium on pancreatic carcinogenesis and the role of the selenoproteins cytosolic and mitochondrial thioredoxin reductase in the pancreas [Elektronische Ressource] / vorgelegt von Michaela Yvonne Aichler

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175 pages
Deutsch

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Influence of selenium on pancreatic carcinogenesis and the role of the selenoproteins cytosolic and mitochondrial thioredoxin reductase in the pancreas Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) Fakultät Naturwissenschaften Universität Hohenheim Institut für Biologische Chemie und Ernährungswissenschaften und Abteilung für Vergleichende Medizin, Helmholtz Zentrum München – Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH) vorgelegt von Michaela Yvonne Aichler aus Backnang 2007 Dekan: Prof. Dr. rer. nat. H. Breer 1. berichtende Person: Prof. Dr. rer. nat. L. Graeve 2. berichtende Person: Dr. vet. med. M. Brielmeier Eingereicht am: 21.12.2007 Mündliche Prüfung am: 08.05.2008 Die vorliegende Arbeit wurde am 25.03.2008 von der Fakultät Naturwissenschaften der Universität Hohenheim als „Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften― angenommen. To my parents. Preliminary remarks The present work has in part be published or presented as follows: Publications in peer-reviewed journals Aichler, M., Algül, H., Behne, D., Hölzlwimmer, G., Michalke, B., Quintanilla-Martinez, L., Schmidt, J., Schmid, R.M., Brielmeier, M.

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Publié par
Publié le 01 janvier 2008
Nombre de lectures 49
Langue Deutsch
Poids de l'ouvrage 7 Mo

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Influence of selenium on pancreatic carcinogenesis
and the role of the selenoproteins
cytosolic and mitochondrial thioredoxin reductase
in the pancreas


Dissertation zur Erlangung des Doktorgrades
der Naturwissenschaften (Dr. rer. nat.)

Fakultät Naturwissenschaften
Universität Hohenheim

Institut für Biologische Chemie und Ernährungswissenschaften
und
Abteilung für Vergleichende Medizin,
Helmholtz Zentrum München –
Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH)

vorgelegt von
Michaela Yvonne Aichler
aus Backnang
2007

Dekan: Prof. Dr. rer. nat. H. Breer
1. berichtende Person: Prof. Dr. rer. nat. L. Graeve
2. berichtende Person: Dr. vet. med. M. Brielmeier
Eingereicht am: 21.12.2007
Mündliche Prüfung am: 08.05.2008




Die vorliegende Arbeit wurde am 25.03.2008 von der Fakultät Naturwissenschaften der
Universität Hohenheim als „Dissertation zur Erlangung des Doktorgrades der
Naturwissenschaften― angenommen.















































To my parents.




































Preliminary remarks
The present work has in part be published or presented as follows:

Publications in peer-reviewed journals
Aichler, M., Algül, H., Behne, D., Hölzlwimmer, G., Michalke, B., Quintanilla-Martinez, L.,
Schmidt, J., Schmid, R.M., Brielmeier, M.
Selenium status alters tumour differentiation but not incidence or latency of pancreatic
+/-adenocarcinomas in Ela-TGF-α p53 mice.
Carcinogenesis, 28 (9), 2002-2007, 2007.

Talks
Aichler, M., Algül, H., Behne, D., Hölzlwimmer, G., Michalke, B., Quintanilla-Martinez, L.,
Schmidt, J., Schmid, R.M., Brielmeier, M.
Does the selenium status influence the incidence of pancreatic carcinomas and
hematopoietic tumours in tumour prone p53-hemizygous mice?
th th(Danish –German Symposium, June 27 – 28 , 2006, Environmental Research Station
Schneefernerhaus – Zugspitze, Garmisch)
Aichler, M., Algül, H., Behne, D., Hölzlwimmer, G., Michalke, B., Quintanilla-Martinez, L.,
Schmidt, J., Schmid, R.M., Brielmeier, M.
Effect of selenium on incidence, latency and tumour differentiation in pancreatic cancer.
th th th(8 International Symposium on Selenium in Biology and Medicine, July 25 – 30 , 2006,
University of Wisconsin, Madison WI, USA)

Poster presentations
Brielmeier, M., Aichler, M., Behne, D., Schmid, R., Schmidt, J.
The selenium platform: selenium deficiency as pathogenic factor.
th th(Gemeinsame Herbsttagung, 24 -25 November, 2005, Hahn-Meitner-Institut, Berlin,
Germany)
Aichler, M., Algül, H., Behne, D., Hölzlwimmer, G., Michalke, B., Quintanilla-Martinez, L.,
Schmidt, J., Schmid, R.M., Brielmeier, M.
Effect of selenium on incidence, latency and differentiation in pancreatic cancer prone
mice.
th th(Gemeinsame Herbsttagung ―Metallobiolomics‖, 6 – 7 December, 2006, Hahn-Meitner-
Institut, Berlin, Germany)
Aichler, M., Algül, H., Behne, D., Hölzlwimmer, G., Michalke, B., Quintanilla-Martinez, L.,
Schmidt, J., Schmid, R.M., Brielmeier, M.
Selenium supplementation in mice influences the malignancy of pancreatic
adenocarcinomas.
th th(FELASA-ICLAS Joint Meeting, 11 – 14 June, 2007, Villa Erba, Cernobbioa (CO), Italy)
Aichler, M., Algül, H., Behne, D., Hölzlwimmer, G., Michalke, B., Quintanilla-Martinez, L.,
Schmidt, J., Schmid, R.M., Brielmeier, M.
Selenium supplementation in mice influences the tumour differentiation of pancreatic
adenocarinomas.
st th(1 Life-Science PhD Symposium, 7 December, 2007, MPI of Neurobiology/Biochemistry,
Munich, Germany)



































Index
1 Introduction............................................................................................................ 1


1.1 Selenium...................................................................................................... 1
1.1.1 Selenium metabolism......................................................................... 2
1.1.2 Selenium in cancer prevention........................................................... 4
1.1.3 Selenoproteins................................................................................... 6
1.1.4 Thioredoxin reductases...................................................................... 7
1.1.5 The thioredoxin/thioredoxin reductase system in
carcinogenesis and tumour progression............................................ 10

1.2 Pancreas...................................................................................................... 12
1.2.1 Pancreatic organogenesis.................................................................. 12
1.2.2 Ptf1a-p48 as a promoter for Cre-expression...................................... 14
1.2.3 Pancreas anatomy, morphology and physiology................................ 14
1.2.4 Pancreatic disorders........................................................................... 17
Acute and chronic pancreatitis........................................................... 17
Pancreatic ductal adenocarcinoma.................................................... 23
1.2.5 Thioredoxin reductases in the pancreas............................................. 24



1.3 Aim of this thesis........................................................................................ 24


2 Materials and methods.......................................................................................... 26

2.1 Materials....................................................................................................... 26
2.1.1 Chemicals........................................................................................... 26
2.1.2 Desoxyoligoribonucleotides................................................................ 28
2.1.3 Enzymes............................................................................................. 29
2.1.4 Kits...................................................................................................... 29
2.1.5 Antibodies for immunohistochemistry................................................. 30

2.2 Transgenic mice.......................................................................................... 31
31 2.2.1 EL-TGF -hGH....................................................................................
+/- 2.2.2 P53 .................................................................................................. 31
2.2.3 Floxed cytosolic thioredoxin reductase mouse................................... 32
2.2.4 Floxed mitochondrial thioredoxin reductase mouse........................... 32
2.2.5 ROSA26 Cre reporter......................................................................... 33
ex1 2.2.6 Ptf1a-Cre ........................................................................................ 33

2.3 Experimental groups and breeding........................................................... 34
2.3.1 Pancreatic cancer mouse model........................................................ 34
2.3.2 Pancreas-specific Txnrd1 knockout mouse strain.............................. 35
2.3.3 Pancreas-specific Txnrd2 knockout mouse strain.............................. 36
2.3.4 Pancreas-specific knockout mouse strains with Cre reporter............. 36
2.3.5 Genotyping......................................................................................... 37

2.4 Animal husbandry....................................................................................... 37

2.5 Molecular biological methods.................................................................... 38
2.5.1 Preparation of genomic DNA.............................................................. 38
2.5.2 Preparation of RNA............................................................................ 38
2.5.3 Polymerase chain reaction (PCR)...................................................... 39
2.5.4 Semiquantitative reverse transcription-polymerase
chain reaction (RT-PCR).................................................................... 41
2.5.5 Gel electrophoresis and detection...................................................... 42
2.5.6 Preparation of cytosolic and mitochondrial protein fractions.............. 42
2.5.7 Protein quantification according to Bradford...................................... 43
2.5.8 Thioredoxin reductase activity assay.................................................. 43

2.6 Histological methods.................................................................................. 43
2.6.1 Preparation and fixation of organs for paraffin sections..................... 43
2.6.2 Preparation and fixation of organs for cryosections........................... 44
2.6.3 Paraffin sections................................................................................. 44
2.6.4 Cryosections....................................................................................... 45
2.6.5 Hematoxylin and Eosin (H&E) staining............................................... 45
2.6.6 Immunohistochemistry....................................................................... 45
2.6.7 Masson-Trichrom staining.................................................................. 47
2.6.8 Periodic Acid Schiff (PAS) staining..................................................... 48
2.6.9 X-Gal detection of β-galactosidase..................................................... 48
2.6.10 Transmission electron microscopy..................................................... 49

2.7 Physiological and metabolically screens................................................. 49
2.7.1 Body weight........................................................................................ 49
2.7.2 Blood glucose..................................................................................... 49
2.7.3 Blood serum analysis for amylase and lipase.................................... 49
2.7.4 Lipid absorption test........................................................................... 50
2.7.5 Intraperitoneal glucose tolerance test (IP-GTT)................................ 50