Urinary proteomics and the role of orosomucoid (ORM) in vascularization of bladder cancer [Elektronische Ressource] / vorgelegt von Ster Irmak

Ster

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Urinary proteomics and the role of orosomucoid (ORM) in vascularization of bladder cancer Inaugural-Dissertation Zur Erlangung des Doktorgrads der Naturwissenschaften (Dr. rer. nat.) am Fachbereich Chemie an der Universität Duisburg-Essen vorgelegt von Ster Irmak aus Mardin Essen 2007 Die der vorliegenden Arbeit zugrunde liegenden Experimente wurden am Institut für Anatomie der Universität Duisburg-Essen und des Universitätsklinikums Hamburg-Eppendorf durchgeführt. 1. Gutachter: Prof. Dr. R. Sustmann 2. Gutachter: Prof. Dr. Dr. H. de Groot 3. Gutachter: Prof. Dr. S. Ergün Vorsitzender des Prüfungsausschusses: Prof. Dr. A. Schönbucher Tag der mündlichen Prüfung: 03.07.2007 To my Family and nephew Yusuf Heja Ever tried, ever failed. Try again, fail again. Fail better… Samuel Beckett Acknowledgement Acknowledgements The present study has been carried out between March 2003 - August 2006 at the Department of Anatomy, University Hospital Hamburg-Eppendorf, Germany and between August 2006 – March 2007 at the Department of Anatomy, University Hospital Essen, Germany. I wish to express my sincere gratitude to everyone who aided, supported and inspired me, in one way to another, throughout this study.

Sujets

• Gesundheit

Informations

• Publié par: universitat_duisburg-essen
• Publié le: 01 janvier 2007
• Nombre de lectures: 144
• Langue: English
• Poids de l'ouvrage: 13 Mo

Extrait

Urinary proteomics and the role of orosomucoid

(ORM) in vascularization of bladder cancer

Dissertation Inaugural-

Zur Erlangung des

aften hDoktorgrads der Naturwissensc

(Dr. rer. nat.)

am Fachbereich Chemie

ersität Duisburg-Essenan der Univ

vorgelegt von

Ster Irmak

aus Mardin

Essen 2007

Die der vorliegenden Arbei

t zugrunde liegenden Experiment

e wurden am Institut

-Essen und des Universitätsklinikum Duisburgfür Anatomie der Universität

Hamburg-Eppendorf durchgeführt.

Gutachter: Prof. Dr. R. Sustmann 1.

Gutachter: Prof. Dr. Dr2. t o. H. de Gro

Gutachter: Prof. Dr. S. Ergün 3.

r chönbuchehusses: Prof. Dr. A. SVorsitzender des Prüfungsaussc

hen PrTag der mündlicüfung: 03.07.2007

s

To my Family and

nephew Yusuf Heja

Ever tried, ever failed. Try again, fail again. Fail better Samuel Beckett

Acknowledgement

Acknowledgements

tween March 2003 - August 2006 at the The present study has been carried out be

spital Hamburg-Eppendorf, Germany and Department of Anatomy, University Ho

Anatomy, University 2007 at the Department ofbetween August 2006  March

Hospital Essen, Germany.

ted andto everyone who aided, suppor my sincere gratitude I wish to express

, in one way to another, throughout this study. einspired m

thank my supervisor Professor Dr. Süleyman I would especially like to First of all,

ting field of research, for his constant Ergün for introducing me to work in an exci

e study and for the friendly atmosphere interest and support in the progress of th

within the department.

Hamburg-Eppendorf, I would ogy, University Hospital From the Department of Urol

support and the opportunity to work in the like to thank Professor Dr. Huland for his

e to thank PD Dr. Martin y likiallogical Department. I would speclaboratory of the Urol

Friedrich for his encouragement, optimism and for giving the clinicians point of view. I

laboratory of the Urologyformer and present, at the thank all the lab members,

Hospital Hamburg -Eppendorf. Department at the University

ra-e to thank Dr. Leticia OliveiI would likerrer for her help and scientific and F

in the first months of my life in sometimes non-scientific support especially

uschild for her support, always having a ssica HaeGermany. I would like to thank J

breakfasts and coffe breaks. smile and the happy times, daily

I thank Dr. Nerbil Kilic, Dr. Ergin Kilic and Dr. Derya Tilki for their warm support and

fellowship. I would like to thank Kiersten Miete, Elvin Zengin, Ege Erenler, Biranda

ly atmosphere in lab.doviding a frienKocaoglu, Mehmet Varol, Deniz Tilki for pr

al Essen, I would firstly like to omy, University HospitWithin the Department of Anat

-Yasar for her help anthank Dr. Ferya Banazd support during the writing of my thesis.

Prof. Dr. Ergün. Among them, I speciallyI thank all members of the research group of

thank Dorothee Schünke for her help.

for his help in editing of figures. I would like to thank Oguzhan A. Polat

support encouragement and their continuousFinally, I want to thank my parents for

during all the years. Without them this work would not have been possible. I would

en for her deep friendship and motivation. like to thank my best friend Elmas Yurtsev

Index

I

Index 1 Introduction.......................................................................................................1
1.2 1.1 ProteomicsTechnology of proteomics: 2-Dim...................................................................................................ensional Gelelectrophoresis (2-DE).........3 2
1.2.2 Protein 1.2.1 Sample extractionpreparation.................................................................................................................................................................4 4
1.2.2.2 De1.2.2.1 Chaottergentsropic agent........................................................................................................................................................................5 5
6 ................................................................................gentsing a1.2.2.3 Reduc1.2.3 1.2.4 Second First Dimension: Isoeldimension: SDectric focuS-PAGEsing (IEF)......................................................................................................10 7
1.2.5 Equi1.2.6 Identificatiolibrationn of proteins..............................................................................................................................................................11 11
1.3.1 1.3 Urinary proteomicsFormation of urine........................................................................................12 12
1.4 Bladder1.3.2 Sources cancerof urinary proteins............................................................................................................................................................15 12
1.4.2 Cellular 1.4.1 Staging of classifibladder cationcancer..............................................................................................................................................16 15
1.5 Clinical 1.6 Urinary proteombiomarkersics and biomarkers.................................................................................................................................................18 18
19 ...................................................................................isangiogenes1.7 Tumor 1.8 1.9 The One of urinplasminogen ary proteiactivatins: Orosomon systemucoid (ORM).......................................................................................................2220

2 The aim of study..............................................................................................26
3 Material and Methods......................................................................................27
3.1 Mate3.1.1 Chemical rialsand Consumables....................................................................................................................................................................27 27
28 ......................................................................................................Kits3.1.2 3.1.3 Stock Solutions and buffers................................................................29
3.1.5 Anti3.1.4 Equipment bodiesand applications...........................................................................................................................................................31 32
3.1.6 Cell lines and medium for cultivation of cell lines................................32
3.1.8 Primers3.1.7 Bacterial strains...................................................................................................................................................................................33 32
34 .....................................................................................................thods3.2 Me3.2.1 Protei3.2.1.1 Determinatn analysesion of total protein..........................................................................................................................................34 34
3.2.1.3 3.2.1.2 Preparation of Two dimensional polyacrylurine Samples for 2-DEamide gel electrophoresis (2D-PAGE)...........................................35 35
3.2.1.4 3.2.1.5 Rehydration First dimensional separation: Isoelectric ...................................................................................focusing (IEF)................36 36
3.2.1.7 3.2.1.6 E Second dimquilibrationensional separation........................................................................................................................................37 37
3.2.1.9 3.2.1.8 Silv Proter staiein detecningtion............................................................................................................................................................38 38
3.2.1.10 Coomassie staining.......................................................................39

Index

II

39 ...............................................................analysisage and data 3.2.1.11 Im3.2.1.13 SDS-polyacry3.2.1.12 Mass spectrometrylamide gelele and bioictrophornformaticsesis.................................................................................41 39
3.2.1.14 We3.2.2 Molecularbiostern bllogical ottingmethods...........................................................................................................................................42 41
42 ..................................................E. coli Cultivation and storage of 3.2.2.1 3.2.2.3 3.2.2.2 Bacter Preparatiial transfoon of comprmaettionent cells...................................................