Insight into oxidative stress mediated by nitric oxide synthase (NOS) isoforms in atherosclerosis [Elektronische Ressource] / by P. Padmapriya

Ponnuswamy_P

Sujets

• Biologie

Informations

• Publié par: julius-maximilians-universitat_wurzburg
• Publié le: 01 janvier 2008
• Nombre de lectures: 22
• Langue: Deutsch
• Poids de l'ouvrage: 6 Mo

Extrait

Insight into oxidative stress mediated by nitric
oxide synthase (NOS) isoforms in atherosclerosis

Thesis submitted towards fulfillment of the requirements for
doctoral degree to the Bayerischen Julius-Maximilians-
University, Würzburg, Germany,
By

P.Padmapriya
Coimbatore, TN, INDIA

Würzburg, GERMANY
September, 2008



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ndEingereicht am: 2 September 2008

Mitglieder der Promotionskommission:

Vorsitzender : Prof. Dr. Martin. J. Müller
Gutachter : PD. Dr. Peter. J. Kuhlencordt
Gutachter : Prof. Dr. Roland Benz

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

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









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Eidesstattliche Erklärungen



Hiermit erkläre ich ehrenwörtlich, dass die vorliegende Dissertation
“Insight into oxidative stress mediated by nitric oxide synthase
(NOS) isoforms in atherosclerosis” selbständig an der Medizinische
Klinik I der Universität Würzburg unter der Anleitung von Dr.Peter
Kuhlencordt angefertigt wurde und dass ich keine anderen als die
angegebenen Quellen und Hilfsmittel benutzt habe.
Weiterhin versichere ich, dass die vorliegende Dissertation weder in
gleicher oder ähnlicher Form noch nicht in einem anderen
Prüfungsverfahren vorgelegen hat und ich bisher noch keine akademischen
Grade erworben oder zu erwerben versucht habe.
Hiermit bewerbe ich mich erstmals um den Doktorgrad der
Naturwissenschaften der Bayerischen Julius-Maximilians-Universität
Würzburg.

(P.Padmapriya)
September, 2008
Medizinische Klinik I
Universität Würzburg
GERMANY


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I dedicate my doctoral thesis....
....to all the tiny little ones, whose lives have been
sacrificed for this work!





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v Acknowledgements


First and foremost I would like to thank Dr. Peter Kuhlencordt for his
scientific guidance and moral support. I am indebted to him for being there
for me at time of utmost need. He had been very patient and understanding
and had not only tolerated my failures but had also helped me overcome my
frustrations, by his pep talks. He had been the source of my inspiration and
motivation as a researcher and had also nurtured me into the researcher
that I am today. I sincerely thank him for giving me the liberty and
encouragement to make crucial decisions. I really appreciate his
constructive critical comments and the brainstorming discussions/”debates”
which eventually led to my progress. His amazing capability to quote
citations had been highly admirable. In addition to being my project
supervisor, he had also taken personal care and helped me acclimatise in
Germany. In addition to research, I had also learned a lot from him to deal
people diplomatically and suavely. Without his kindness, care and
understanding it would have been tough to have completed this work
against all the odds. I would always remember him for his smartness and
sweetness, which has made my doctoral thesis work an enjoyable
experience. In short, it had been a great pleasure and fun working with him.

I would like to thank Prof. Georg Ertl for giving me the opportunity to
perform my thesis in his reputed institute. I really appreciate his simplicity
and amicability. I also extend my sincere thanks to Prof. Roland Benz for
taking time to look into the progress of my project and his useful
suggestions during our discussions.

Many thanks to Dr.Fink (Noxygen Science Transfer & Diagnostics,
GmbH) for his ‘online’ guidance and helping me establish the ESR and
HPLC techniques in the lab. His generous help and expertise had aided me
in standardising these techniques. I would also like to thank Dr. Andreas
Kamlowski (Brukers Biospin GmbH) for his co-operation in establishing the
ESR technique in the lab and many thanks to IZKF (Interdisziplinären
Zentrum für Klinische Forschung) for the financial support.

I am thankful to my colleagues Gabriele Riehl and Alla Ganscher,
who had helped me in many ways and for their kindness and care. They had
taken personal care of me and had always wished for my well-being. I am
grateful to Gabriele for having taken immense responsibility in taking care
of all my animals. I greatly appreciate Alla for her patience in helping me
learn German and for having tolerated my poor German!!

I sincerely thank my other lab mates, Johannes Schödle, Sebastian
Rützel, Eva Ostermeier, Angelika Schröttle, Elisabeth Bendel, Nadja Miller
and Carolin Knoll and institute members especially Lisa Bauer and Helga
Wagner for all their timely help and for creating a good working
atmosphere. I am thankful to Elisabeth and Nadja for their help in German
vi translations. Special thanks to my lab mate Eva Ostermeier for her great
experimental assistance, team spirit and co-operation in completing the
project.

I am very thankful to all of my Indian friends who had helped me in
all possible ways and for creating a home away from home ambience. My
special thanks to Reena, Shruti and Tripat for their friendship and for being
patient listeners of all my stories.

And of course, last but not the least, I would like to thank my family
for their constant motivation. I am indebted to my dad- my best friend,
philosopher and guide for his encouragement through out my childhood, till
date and forever and for believing more in me than what I believe in myself.
I hope to live up to his dreams! Many thanks to my mom, whose prayers
have always been with me and for her moral support. My mom and dad had
been the best parents a kid could ever hope for! Tons of thanks to my
sisters- Thara and Sasi, for their motherly care, priceless love, affection and
encouragement. I also thank my nephews, Sriram and Sanath for being my
future. I have no words to express my gratitude to my family. Though miles
away, their warmth, love and care had always kindled my happiness. Most
of all, I thank my God father for being with me always, in thoughts.











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“Research is to see what everybody else has seen and to
think what nobody else has thought”
-Albert Szent-Gyorgyi
Hungarian Biochemist,
1937 Nobel Prize for Medicine








viii Contents
Chapter 1 1
1.0 Introduction 2
1.1 Pathogenesis of atherosclerosis 2
1.2 Risk factors 6
1.2.1 Factors with a strong genetic component 6
1.2.2 Environmental factors 8
1.3 Oxidative stress 10
1.3.1 Sources of oxidants in the vasculature 11
1.3.2 Functional role of superoxide in atherosclerosis 12
1.3.3 Functional role of nitric oxide in atherosclerosis 13
1.4 The nitric oxide pathway in atherosclerosis 16
1.4.1 Overview of NOS family 17
1.4.2 Unique features of NOS isoforms 19
1.4.2.1 Endothelial nitric oxide synthase (eNOS) 20
1.4.2.2 Neuronal nitric oxide synthase (nNOS) 22
1.4.2.3 Inducible nitric oxide synthase (iNOS) 25
1.5 The apoE ko model of atherosclerosis 27
1.6 Role of NOS isoforms in cardiovascular diseases 28
1.6.1 Role of eNOS in cardiovascular diseases 28
1.6.2 Role of nNOS in cardiovascular diseases 30
1.6.3 Role of iNOS in cardiovascular diseases 32
1.7 Aim of the study 35


ix Chapter 2 37
2.0 Materials and Methods 38
2.1 Materials 38
2.1.1 Mice 38
2.1.2 Chemicals and reagents 38
2.1.3 Preparation of reagents 43
2.2 Methods 44
2.2.1 Detection of free radicals in the vasculature 44
2.2.1.1 Electron spin resonance (ESR) 45
2.2.1.2 Principle of ESR 46
2.2.2 Measurement of vascular nitric oxide production by
electron paramagnetic spin trapping 48
2.2.3 Measurement of vascular oxygen radical production
by ESR 49
2.2.3.1 Sample preparation for ESR measurements 51
2.2.4 Measurement of nitric oxide bioavailability in the
bloodstream 51
2.2.5 Measurement of intracellular superoxide production
by HPLC detection of oxyethidium 52
2.2.6 Tissue preparation and immunohistochemistry 53
2.2.7 Histomorphometry 54
2.2.8 Protein Estimation 54
2.2.9 Statistical Analyses 54


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