A Leishmania ortholog of macrophage migration inhibitory factor modulates host macrophage responses [Elektronische Ressource] / vorgelegt von Daniela Kamir

Daniela Gallingani

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go A Leishmania Ortholog of Macrophage Migration Inhibitory Factor Modulates Host Macrophage Responses Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH Aachen University zur Erlangung des akademischen Grades einer Doktorin der Naturwissenschaften genehmigte Dissertation vorgelegt von Diplom-Biologin Daniela Kamir aus Aachen Berichter: Universitätsprofessor Dr. Jürgen Bernhagen Universitätsprofessor Dr. Fritz Kreuzaler Tag der mündlichen Prüfung: 17.07.08 Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar. Albert Einstein: “No amount of experimentation can ever prove me right; a single experiment can prove me wrong.” In memory of my grandfather Dr. med. Philipp Sachter who didn’t live long enough to see any of my graduations but whose memories will never be forgotten. Acknowledgments Completing a thesis is a challenge, thanking all those who contributed to it, is an even greater one. Due to the nature of my study, many people were involved in one or the other way. I want to thank all of them, including those not mentioned here by name. This PhD thesis was supervised by Prof. Dr. rer. nat. Jürgen Bernhagen, Department of Biochemistry and Molecular Cell Biology, Institute of Biochemistry, RWTH Aachen University, as well as by Prof.

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• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2008
• Nombre de lectures: 7
• Langue: English
• Poids de l'ouvrage: 1 Mo

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ALeishmaniaOrthologofMacrophageMigrationInhibitoryFactor

Modulates Host Macrophage Responses 

 

Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH

Aachen University zur Erlangung des akademischen

Naturwissenschaften genehmigte Dissertation









vorgelegt von

Diplom-Biologin  Daniela Kamir

aus Aachen

Berichter: Universitätsprofessor Dr. Jürgen Bernhagen

 Universitätsprofessor Dr. Fritz Kreuzaler

Tag der mündlichen Prüfung: 17.07.08

Grades

einer

Doktorin

der

Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar.

 







                                      Albert Einstein: No amount of experimentation can ever prove me right; a single

experiment can prove me wrong.

 

 

 

 

 

 

 

 

   In memory of my grandfather Dr. med. Philipp Sachter who didnt live long enough to see any of my graduations but whose memories will never be forgotten.             

Acknowledgments

Completing a thesis is a challenge, thanking all those who contributed to it, is an even

greater one. Due to the nature of my study, many people were involved in one or the

other way. I want to thank all of them, including those not mentioned here by name.

 This PhD thesis was supervised by Prof. Dr. rer. nat. Jürgen Bernhagen, Department of

Biochemistry and Molecular Cell Biology, Institute of Biochemistry, RWTH Aachen

University, as well as by Prof. Richard Bucala M.D./Ph.D., Department of Medicine and

Pathology, School of Medicine of the Yale University, New Haven, USA.



Im especially thankful to Prof. Bernhagen for the very interesting topic and for the

opportunity to carry out a major part of my thesis work at Yale University.

Thanks to Prof. Dr. rer. nat. F. Kreuzaler for taking time out from his busy schedule to

co-evaluate my thesis.



Id like to thank Prof. Bucala for giving me the opportunity to do my research in his

laboratory at Yale University. Im very grateful for his guidance, help in discussions, his

interest in supporting my thesis, his invaluable advice and constructive criticism.



Special thanks go to Dr. Lin Leng for his suggestions, useful advices and assistance

regarding numerous technical problems. Im very glad for his support during my PhD

work.



I would like to thank members of the Bucala lab, past and present, and those of the

Bernhagen lab for their support.



I am grateful to the Marianne und Dr. Fritz Walter Fischer-foundationfor generously

providing me a PhD scholarship.



Last but not least, I would like to thank my parents and my grandmother for their words

of encouragement and especially my father for his everlasting optimism and support

during all my academic studies.





Parts of this thesis have been published in a peer-reviewed international journal: Kamir, D., S. Zierow, L. Leng, Y. Cho, Y. Diaz, J. Griffith, C. McDonald, M. Merk, R. A. Mitchell, J. Trent, Y. Chen, Y. Kwong, H. Xiong, J., M. Cappello, D. McMahon-Pratt, J. Walker, J. Bernhagen, E. Lolis, R. Bucala. A Leishmania Ortholog of Macrophage Migration Inhibitory Factor Modulates Host Macrophage Responses. The Journal of Immunology, 2008, 180: 8250-8261. 

FBS

EtBr

HIV

hr(s)

EDTA

Ds

ERK

ELISA

DEPC

DDT

DNA

DMEM

C-terminal

BSA

COX2

CHMI

ABTS

AIDS

BMM

Bp

List of abbreviations: 

Å

Ab

Fig.

Figure

Antibody

Ångström

  

Bone-marrow macrophages

Acquired immune deficiency syndrome

sulfonate)

2,2-azino-di(3-ethylbenzthiazoline

Basepair

Bovine Serum Albumine

Carboxy-terminal

5-carboxymethyl-2-hydroxymuconate

isomerase

Cyclooxygenase 2

D-Dopachrome tautomerase

Diethyl pyrocarbonate

Dulbeccos modified Eagless medium

Desoxyribonucleic acid

Double-strand

Ethylendiamintetraacetat

Enzyme-linked immunosorbant assay

Extracellular signal-regulated kinase

Ethidium bromide

Fetal bovine serum

Human MIF

Hour(s) Human immunodeficiency virus

Interferon-γ

horseradish peroxidase

Inducible nitric oxide synthase

Interleukin

Isopropyl-β-D-thiogalactopyranosid

IPTG

iNOS

Abbreviations

HRP

hMIF

IL

IFN-γ 

Abbreviations

ISO-1

JNK Kb kDa LDS LmjF LmMIF LPS MAP Mb MIF mMIF MP Min N-terminal NO 4-OT PBS PCR PGE 2   PLA2 PMNs

PnP PnPP PVDF qPCR RNA Rpm

ROS sCD74 SDS-PAGE

(S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5- isoxazole acetic acid methyl ester Jun N-terminale kinase Kilo base pairs Kilo Dalton Lithium dodecyl sulphate sample buffer L. majorFriedlin L. majorMIF Lipopolysaccharid Mitogen-activated protein Mega base pairs Macrophage migration inhibitory factor Mouse MIF Microplate Minute(s) Amino-terminal Nitric oxide 4-oxalocrotonate tautomerase Phosphate-buffered saline Polymerase chain reaction Prostaglandin E2 Phospholipase A2 Polymorphonuclear neutrophil granulocytes p-Nitrophenole p-Nitrophenyl phosphate Polyvinylidene difluoride Quantitative PCR Ribonucleic acid Rotation per minute Reactive oxygen species Soluble CD74 sodium dodecyl sulfate polyacrylamide

Abbreviations

SNP sCD74 TAE Taq TE Th1 Th2 TLR4 Tm TNF-α TPOR Tris Trx TTBS U UV

gel electrophoresis Sodium nitroprusside Soluble CD74 Tris-Acetat-EDTA-Buffer Thermus aquaticus Tris-EDTA-buffer T-helper 1 lymphocytes T-helper 2 lymphocytes Toll-like receptor 4 Melting temperature Tumor necrosis factor-α Thiol-protein oxidoreductase Tris(hydroxymethyl)aminomethan Thioredoxin Tris-buffered salt solution with Tween 20 Specific enzyme activity Ultraviolet

Table of contents

Table of contents



1

2

1.1

1.2

Introduction....................................................................................................... 1-1

Host-parasite relationships between man and protozoa.................................... 1-1

Immunity........................................................................................................... 1-2

1.3 Aleppo Evil ....................................................................................................... 1-2

1.3.1

1.3.2

1.3.3

1.3.4

1.3.5

1.3.6

1.3.7

1.3.8

Leishmaniasis ........................................................................................... 1-2

Leishmania genome.................................................................................. 1-4

History ...................................................................................................... 1-5

Leishmaniasis: disease and parasitic life-cycle ........................................ 1-8

LeishmaniaRNA virus ........................................................................... 1-12 Leishmania/HIV co-infection................................................................. 1-12 The Trojan horse ofLeishmania.................................................. 1-13 .......... Th1 versus Th2 ....................................................................................... 1-14

1.4 The Cytokine MIF .......................................................................................... 1-15

1.4.1 Introduction ............................................................................................ 1-15

1.4.2 Structure and tautomerase/isomerase activity of MIF............................ 1-16

1.4.3

1.4.4

1.4.5

1.4.6

1.4.7

1.4.8

1.4.9

1.4.10

1.4.11

The role of TPOR activity in MIF function ........................................... 1-17

MIF polymorphisms and expression ...................................................... 1-19

MIF as a ligand....................................................................................... 1-20

MIF function........................................................................................... 1-21

MIF and ERK1/2 .................................................................................... 1-22

MIF and the tumor suppressor p53......................................................... 1-23

Role of MIF in infectious diseases ......................................................... 1-24

Mammalian MIF andL. majorinfection................................................ 1-25 Mouse models......................................................................................... 1-26

Specific Aims.................................................................................................. 2-28

3 Materials and Methods.................................................................................... 3-29

3.1 Materials ......................................................................................................... 3-29

3.1.1Mice........................................................................................................3-29

3.1.2 Material lists ........................................................................................... 3-29 Methods .................................................................................................................... 3-38 3.1.3 Sequence and phylogenetic analyses...................................................... 3-38