Biomarkers for experimental autoimmune encephalomyelitis and multiple sclerosis [Elektronische Ressource] : possible mechanisms mediating neurodegeneration / vorgelegt von Archana Mary Jastorff

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• Gesundheit

Informations

• Publié par: eberhard_karls_universitat_tubingen
• Publié le: 01 janvier 2007
• Nombre de lectures: 8
• Langue: English
• Poids de l'ouvrage: 29 Mo

Extrait

BiomarkersforExperimentalAutoimmune
EncephalomyelitisandMultipleSclerosis:
Possiblemechanismsmediating
neurodegeneration
Dissertation
zurErlangungdesGradeseinesDoktors
derNaturwissenschaften
derFakultat¨ fur¨ Biologie
und
derMedizinischenFakultat¨
derEberhard Karls Universit at¨ Tubingen¨
vorgelegt
von
ArchanaMaryJastorff
ausMavelikara,India
March 2007Tagdermundlichen¨ Prufung:¨ 14.03.07
DekanderFakultat¨ fur¨ Biologie: Prof. Dr. F.Schof¨ flderMedizinischenFakultat:¨ Prof. Dr. I.B.Autenrieth
1. Berichterstatter: Prof. Dr. C.W.Turck
2. Prof. Dr. Dr. R.Weissert
Prufungsk¨ ommission: Prof. Dr. C.W.Turck
Prof. Dr. Dr. R.Weissert
Prof. Dr. A.Melms
PDDr. B.Muller¨ Myhsok
PDDr. A.LuftTomylovingparentsAcknowledgments
Firstandforemost,mysincerethanksgotomysupervisorProf. ChirsTurck.
Chris, thank you very for the confidence you bestowed in me, and the freedom
you gave me in evolving my own ideas and focuses and for developing in me
a great sense of responsibility towards my work and my scientific carrier. I am
grateful to you for what I have learned and explored during the last four years.
I enjoyed all our conversations, be it scientific or casual. Thank you very much
foryourunderstandingandinterestinmyfamilialmattersbothduringhappyand
sadtimes.
IwouldliketoexpressmygratitudetoProf. MelmsandDr. Weissertfortheir
whole heartedwillingnessandinteresttobepartofmythesiscommitteeandfor
theirveryvaluablesuggestionsduringtheadvisoryboardmeeting.
ThisprojectwouldnothavebeenpossiblewithoutourcollaborationwithDr.
Tjalf Ziemssen. Thank you very much Tjalf, for the very successful interaction
betweenMunichandDresden. IwouldalsoliketothankTjalfandhiscolleagues,
Michaela and Steffi, for explaining to me the animal and cell culture studies
duringmytimeatDresden.
IwouldliketothankDr. FrankWeberforinitiatingthisprojectandorganizing
thecollaborationwithDresden.
My special thanks to Dr. Herbert for giving me the chance to submit my
thesis at the graduate school, for the warm welcome in Tuebingen and all his
timelyhelpduringmyMaster’sstudy.
Prof. Holsboer the director of our institute needs special mentioning. I thank
himformypositionattheinstitute and forthe excellentfacilities atthe institute
thatIhavemadeuseofforthethesiswork.
I would like to thank Katrin Haegler for her assistance. Most of all I would
liketothankyouforthegood spiritandcheerfulnessthatyouspread.
I am thinking of my parents and my sister for their love, prayers, and un
conditional support and for being a constant source of motivation from across
thousands of miles, and for all the values that they have nurtured in me. I thank
myuncleKarlearnstforhisgreathelpingettingstartedinGermany.
Heart feltthankstomyparentsin lawandeverymemberoftheJastorfffamily
fortheircontinuousinspirationandforteachingmetothinkpositiveandtolook
forward. Thanks, Bernd, for the immense interest you showed in my work and
fortheencouragementandsuggestions. Iwouldliketoremembermylatemother
in law,whowouldhavebeenoneofthehappiestpersonstoseemegraduate.During the last four years a lot of people grew close to my heart. Tons of
thanks to all the ”women”, Dale, Giuseppina, Eva, Katrin, Eser, Christiane,
Claudia, Jeeva, Maria, Laryssa, Isabel, Nicola and Sonya, and the ”boys”, Nic,
Christian, Victor, Daniel and Yaoyang, in our lab, for the very cheerful and
warm heartedambienceatworkandformakingourlabasecondhome. Thanks
Giuseppina, for all your help and for being a friend and a big sister. I thank you
Eva for your valuable help and for listening to my issues. Thanks Christiane
and Claudia, for the nice evenings we spent together. I thank every one of my
lab mates for your help and for making my time at AG Turck a very pleasant
experience.
IamgratefultomydearfriendsHelena,Mirjam,Melanie,LisaandWolfgang
for making my stay in Munich very enjoyable, the time they took to share my
allmyjoysandsmallconcernsandtheeffortsmadeinkeepingmefrommissing
myhusbandandparents.
I would like to thank my friend Rainer for giving me company during the
weekends that I was alone in Munich. I am especially grateful to him for the
longhoursthathespenddayafterdaytomakemythesislookgood.
Finally special thanks go to my loving husband, Jan, for the million ways
throughwhichheshowedhisgreatinterestandsupportineachandeverystepof
mythesis. Heconstantlyworkedonbuildingupself confidenceinmeespecially,
whenthingsdidnotlookupandlenthisattentiontoallthedetailsandcomplaints
about work. I thank him for making every weekend a pleasant experience, for
giving me company in the middle of the night when occasion demanded and i
had to be at the lab (during the short weekends that we met the last 4 years) and
forgoingthrougheverysingleslideofminebeforeapresentation.Contents
1 Introduction 11
1.1 Objectiveandrationale . . . . . . . . . . . . . . . . . . . . . . 11
1.2 MultipleSclerosis . . . . . . . . . . . . . . . . . . . . . . . . 14
1.2.1 PathogenesisofMS . . . . . . . . . . . . . . . . . . . . 14
1.2.2 ClinicaldiagnosisofMS . . . . . . . . . . . . . . . . . 18
1.2.3 TreatmentofMS . . . . . . . . . . . . . . . . . . . . . 19
1.3 EAE:AnimalmodelforMS . . . . . . . . . . . . . . . . . . . 20
1.4 Proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
1.4.1 Twodimensionalpolyacrylamidegeleletrophoresis . . . 28
1.4.2 Massspectrometry . . . . . . . . . . . . . . . . . . . . 33
1.4.3 Proteinidentificationbymassspectrometry . . . . . . . 40
1.5 BiomarkersandproteomicsforMS . . . . . . . . . . . . . . . . 43
2 Materialsandmethods 45
2.1 EAEinduction . . . . . . . . . . . . . . . . . . . . . . . . . . 45
2.1.1 ImmunizationofmiceandevaluationofEAE . . . . . . 45
2.1.2 PassiveEAEbytransferofencephalitogenicT cells . . . 46
2.2 HumanMSspecimen . . . . . . . . . . . . . . . . . . . . . . . 46
2.3 Twodimensionalgelelectrophoresis . . . . . . . . . . . . . . . 46
2.3.1 Samplepreparationoftissuesamples . . . . . . . . . . 46
2.3.2 Firstandseconddimensiongelelectrophoresis . . . . . 48
2.3.3 Proteinstaining . . . . . . . . . . . . . . . . . . . . . . 50
78 CONTENTS
2.4 Imageanalysis . . . . . . . . . . . . . . . . . . . . . . . . . . 51
2.5 Gelspotprocessingformassspectrometry . . . . . . . . . . . . 54
2.6 Modifiedprotocolformass . . . . . . . . . . . . . 55
2.7 Massspectrometryandproteinidentification . . . . . . . . . . . 55
2.7.1 MALDI TOF TOFanalysis . . . . . . . . . . . . . . . 55
2.7.2 LC MS/MSanalysis . . . . . . . . . . . . . . . . . . . 57
2.8 Membraneproteinanalysis . . . . . . . . . . . . . . . . . . . . 58
2.9 Westernblotanalysis . . . . . . . . . . . . . . . . . . . . . . . 59
3 Results 63
3.1 Experimentalautoimmuneencephalomyelitis(EAE) . . . . . . 64
3.1.1 ProteinmarkersinadvancedEAE . . . . . . . . . . . . 65
3.1.2 ProteinmarkersindifferentstagesofEAE . . . . . . . 72
3.1.3 Devic’sEAE . . . . . . . . . . . . . . . . . . . . . . . 78
3.1.4 Membraneproteinanalysis . . . . . . . . . . . . . . . . 80
3.1.5 Differentialregulationinthebrainandspinalcord . . . 82
3.1.6 CommonproteinmarkersinEAE . . . . . . . . . . . . 82
3.1.7 Summary: EAEresults . . . . . . . . . . . . . . . . . . 86
3.2 Humanspecimen . . . . . . . . . . . . . . . . . . . . . . . . . 87
3.2.1 Comparisonsandproteinmarkers . . . . . . . . . . . . 87
3.2.2 Summary: humanspecimenresults . . . . . . . . . . . 89
3.3 ProteinmarkerscommontoEAEandMS . . . . . . . . . . . . 92
3.4 Selectedproteinmarkersandtheirvalidation . . . . . . . . . . . 92
3.4.1 Summary: selectedproteinmarkers . . . . . . . . . . . 98
3.5 Functionalclassificationandpathwayanalysis . . . . . . . . . . 99
4 Discussion 103
4.1 GeneralDiscussion . . . . . . . . . . . . . . . . . . . . . . . . 103
4.2 Selectedproteinmarkers . . . . . . . . . . . . . . . . . . . . . 109
4.2.1 Neurotoxicity . . . . . . . . . . . . . . . . . . . . . . . 109
4.2.2 Sirtuins . . . . . . . . . . . . . . . . . . . . . . . . . . 112CONTENTS 9
4.2.3 CreatineKinase . . . . . . . . . . . . . . . . . . . . . . 116
4.2.4 Ferritinheavychain . . . . . . . . . . . . . . . . . . . 118
4.2.5 Proteinsinvolvedinaxonalguidance . . . . . . . . . . 121
4.2.6 Stathmin . . . . . . . . . . . . . . . . . . . . . . . . . 124
4.3 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
4.4 Futureperspectives . . . . . . . . . . . . . . . . . . . . . . . . 132
5 References 135
A Results: MOGactiveEAE 163
B Abbreviations 171
C T cellsamplepreparation 175
D PDQuestimageanalysis 177
E CurriculumVitae 18510 CONTENTS