Structural analysis of reconstituted collagen type I-heparin cofibrils [Elektronische Ressource] / von Dimitar Stamov

Structural analysis of reconstituted collagen type I-heparin cofibrils [Elektronische Ressource] / von Dimitar Stamov

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derStructuralorsitzenderAnalysisProf.Dr.phil.habil.ofamReconstitutedVCollagenProf.Dr.rer.nat.Tvyparna,e24.11.2009.I1.-2.HeparinProf.Dr.rer.nat.habil.CobrilsStzurgebErlangungimdesTakhademiagscg:henhGradesanielDoktoringenieurh(Dr.-Ing.)WernerVBleyonPromotionskderamoF(M.Sc.)akulorent02.10.1979.?tVMascBulgarienhinenagwEinreicesenung:derTTderecerteidigunhnisc15.03.2010.henGutacUnivter:erDsiM?llertGutac?tter:DresdenCarstenangenomennegez:DissertationThomasvVonderDimitommissionarEinsteinNaturupe(1858cdgeompuposesansomeimpofatiheroloveliestwithpamoawemsImaginationforlethed.micrtheoscropgreisandMaxthe1947)telescooptoe.eTheodoremyRmoroszakthan(1933Know-li)magiAencirscienticAlbertruth-doatiesnnotowstriumphthatbyfamiliarcit.onvincingPlanckits-oppIonentsenoughandfmakingartistthemdrsefreelytheonlight,imagination.butisreatherortantbknowedge.cleauseisitsmiteoppIonentsneventualonlyclesdieworld.andta(1879new1955)gene.Con.ten.ts.Con.ten.ts.iv.1.MaterialsIn.tro.duction.1PGs2..Theoreticalhanicalbac.kgroundnanomec5.2.1..The3.1.2.extracellular.matrix.as.a.biological.scaold..23...as.2.4.1....to...proteins..............5.2.1.1..CollagensSoftand.glycoproteins........of.......y.......morphologi...op.....'Non-in.analysis...29.t..6ds2.1.2..Glycosaminoglycans.and.proteoglyca.n35s................Devices................8412.1.3..Therap.eutic.necessit.y.for.xenogeneic2.3.5.scaoldsGs.............In.other........10.2.2.2.4.Collagenol'tandypharacterize.Iprinciple.mo.........26.asiv.for........

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derStructuralorsitzenderAnalysisProf.Dr.phil.habil.ofamReconstitutedVCollagenProf.Dr.rer.nat.Tvyparna,e24.11.2009.I1.-2.HeparinProf.Dr.rer.nat.habil.CobrilsStzurgebErlangungimdesTakhademiagscg:henhGradesanielDoktoringenieurh(Dr.-Ing.)WernerVBleyonPromotionskderamoF(M.Sc.)akulorent02.10.1979.?tVMascBulgarienhinenagwEinreicesenung:derTTderecerteidigunhnisc15.03.2010.henGutacUnivter:erDsiM?llertGutac?tter:DresdenCarstenangenomennegez:DissertationThomasvVonderDimitommissionarEinsteinNaturupe(1858cdgeompuposesansomeimpofatiheroloveliestwithpamoawemsImaginationforlethed.micrtheoscropgreisandMaxthe1947)telescooptoe.eTheodoremyRmoroszakthan(1933Know-li)magiAencirscienticAlbertruth-doatiesnnotowstriumphthatbyfamiliarcit.onvincingPlanckits-oppIonentsenoughandfmakingartistthemdrsefreelytheonlight,imagination.butisreatherortantbknowedge.cleauseisitsmiteoppIonentsneventualonlyclesdieworld.andta(1879new1955)gene.Con.ten.ts.Con.ten.ts.iv.1.MaterialsIn.tro.duction.1PGs2..Theoreticalhanicalbac.kgroundnanomec5.2.1..The3.1.2.extracellular.matrix.as.a.biological.scaold..23...as.2.4.1....to...proteins..............5.2.1.1..CollagensSoftand.glycoproteins........of.......y.......morphologi...op.....'Non-in.analysis...29.t..6ds2.1.2..Glycosaminoglycans.and.proteoglyca.n35s................Devices................8412.1.3..Therap.eutic.necessit.y.for.xenogeneic2.3.5.scaoldsGs.............In.other........10.2.2.2.4.Collagenol'tandypharacterize.Iprinciple.mo.........26.asiv.for.........Application.c.rization...31.d.3.1....................Chemicals........11.2.2.1..St.ructure.of.trop.o.c.ollagen........38.................39.are.................tration11.2.2.2..Molecular.pac.king.of.collagen.t.yp.e.I.in.brils22.Impact.GA.and..................13.2.2.3..St.ructural2.3.6.pterplaolymorphismwith.collagens.....................25.SFM.'nanoto.for.cal.mec.c.ation...26.Basic.and.erating17di2.3..F.actors.aecting.collagen.b.ril.formation.and.structure2.4.2.invvitre'ool.structural..........19.2.3.1..T.emp2.4.3.eraturein.hanical.harac.e.of.......3..an.Metho.35.Materials.....................................3.1.1.20.2.3.2..pH.v.alue........................35.Materials.................................3.1.3.......21.2.3.3..Pr.e.sence/absence.of.telo.p.eptides............3.1.4..w........................21.2.3.4..E.e.ct.ofconcen.vi.Con.tenbandingts.3.1.5..Microscoppes..-.....e.......p...heparin...brillar.4.3.2........99...brils.4.2.1..........p...dieren.king.....eptides42.3.1.6..Canbrilstilevhanicalers..duli...in...sion.......t.....al.....Cobrils.....IV.o...4.2.4........co.77.on.of......42.3.2.ofMethotdsAXS).......t...........Bending.brils...Discussion.telop.assem.......9.I.........4.2..of.e.........c.els.erio......43683.2.1.asymmetricBio.c.hemical.metho.ds4.2.3..with.r.y.......origin.forms.........4.2.5..ransition.of.structures.Impact.tercalati.trabrillar.mec.I..........43.3.2.2..Chemical.mo4.3.1.dicationsand.coll.e.trastructure...............v.of.......4.3.3..ending...........84.shear.col45.3.2.3..Microscopical.metho86dsComp.site.and.n.............5..6..Bibliograph.of.I.................64.Structural.olymorphism49collagen4.ypResultsIand.Discussion.53.4.1..Morphological.c.haracterization.of68collagenStrutturypleveandIp-dicitiesG.A.G.assem.blies......4.2.2..with53D-banding4.1.1..Impact.of.GA.G.t.yp.e.on70theFLSmorpholognanobrilsysymmetricofetheicdicitob.ri.ls........73.Nucleic.of.olymorphic54.4.1.2..Impact.of.conce.n.tration.on.the.morphology74ofDiscussioncollagenTIand-existenceLtheMWtH.cobrils4.3..of.in.on.in.pac.and.hanics.collagen.brils.................................80.Impact.heparin.telop.on.agen.yp.I56ul-4.1.3.(WKinetics.of.PSC.-.LMWH.cobrillogenesi.s..............80.Co.alen.immobilization.freestanding......57.4.1.4..In.tercal83ationMicromecofbheparin.within.the.cobrils..................4.3.4..and.mo.of.lagen..59.4.1.5..Impact.of.degree.of.sulfation,4.3.5.m-olecularetitivwbindingeighofteptidesandheparinpHcollagevbrilaluebly........62.4.1.6..Discussion..........88.Conclu.93.Summary.7.y.List.Figures.I...Con.ten.ts.vii.List.of.T.ables.V.Nomenclature.VI.A..Appcendices.A.-1.A.1..Impact.of.PSC.concen.trationofon.the.kinetics.of.bril.formatio.n....B-5..........A-1.A.2..Impact.of.pH.on.the.kineticsB.2.of.PSC.brillogenesis............B.3..wledgmen............A-2.A.3..ImpactDeclarationof.HS.concen.tration.on.the.size.distribution.ofB-6PSC.-.HS.brils.....A.-2.A.4..R.outine.for.tting.of.adheren.tB-1brilListcross-sectionspublicationsvia.mo.died.in.v.erse.p.oly-.nomial.function............B-4.A.kno.ts.................................B.4...............A-3.B..Other.s.B-1.B.1..Statemen.ts.........imp1.leInthetroECMductionmolecules,Onecofcollagenstheemenmostypcotalhallengingbtasksbinationof(3).tissuelyengineeringbrillarnoadditionworadatainyshisrequirementonoexploitlaterthevideprincib-thplesyofbuildrationalthedesignorkofascaoldsglycosaminoglycansandctowielicitelemenappropriateforsignalsintohcells.ECM,Sucfurthermorehnativscaoldofcwhicharacteristicsdaremostexptheticectedscaoldtoinpromoteresistbiologicalandpronetcesses,text,thtusIleadingstructurestomassprotductionconsideredoflemennewproteins,tissuesoforirepairproteoglycansofndamagedECM,ones.isInbioadhesivthisreconsense,text,predestinedthetonature'stegritideal5biologicallitatingscaf-supramolecularfoldas-isthedeneextracellulartmatrixfor(ECM),matrixcomesaasducingaelkiseyuregulatortissueofIncelltalbfunction,ehashouldvior,struc-proinvidingtheindividualuid,cellsablewithnarcthehitecturalecueshofork)timethisandmaspstraproteinsce,ypmoIV)dulatinghanicallybioatvthanailabilitortionysystem.oflevsolublebgronetwthtensileandconsistingdierenamorphoustiationtinousfactors,wandeorganizingconsitheofmG)ulticellular(6).tissuemimicdevtheel-ofopmenvtt(1).parallelySincehthepropcomplexcalled3-dimensionaloforganizInationlagenofIthtissueeabilitEonlyCMihasbutnotwithbdiereneenfacifullysucunderstoaohierarcdyandthecitharacterized,pivittoiandsfulllpivheotaltstoandemeansontowexploreytheproinateractionvbbioscafolde-httoweeensedierenfortengineeringstructuralon.andtheirfunctionalelemenmolecules,biomeandhanicalthsynusECMsproprovideaaturaltothatolcomfowithrsurroundingconterstitialtrollingshouldcellebtoehateviorsiand(viatissuebrils)mcompressivor(viaphogeenesisydratedupwonstressesconIntactconwtheijoritthoftheseucmicrouralen(mostlyvironmenttse(2).andAlthoughcannaturallymecderivrobustedwithECMsypicalhalessv1%epropprotovwholeedAeectivoneeel,incanmaneyabasicwandofcleinicalts,appli-ofcations,andthestructuralneedbiconforwithcustom-madenetmatricesorkforcompressivtissue-spel-ecicts,cellstbiologicalnginamorphousv(GAestigationsanddriv(PG)esInreconstitutiontoofkitheirgkstructuraleyharacterctheharacteristicsainerysynortantheticsteportobiohcomplyybridtmaterialsthe(3),eesperties,eciallyelseinmolecularthecognitionlighitstts.ofthatproblemscolwtiethisimmforunologicengineeringrespitsonsesy(4,not5).mainIntissuethenabsenceyofalsonature'steractsupnearlyerior0buildingtmechanismsalso2pastCHAPTERgro1.edInECMtrohighductionasincludingfoalmostvallandnaturalorkPGs,factorandbthematricesmaysjorit(17).ytoofbECMisproteinst(7).highOthertmoleculesmicroscopwithbreciognizedlsadhesivofemanfunctionsorkincludewthbronectin,delivlaminin,hanismscollagencase,IV.cellularBecauseapproacofinthetissuetalenitoghsubsttpureconnectioncbstructurallyetts,wUtilizingeentotherialscytoskoeletonsomandcesstheinECMbthroughicellvsurface(1416).receptors,cessescellsasensesignalingandthanrespe,ondhemestoultiplethebiologicalmecfactorhanicaldpropfactorertiesreloflotheirmatrixenmimicsvitheseronotsmventriguingttbtheytheconThvvertinginmeceryhanicalcomsignalshargeinaimtoncmatrixhemicaleonese(8,harged9).viaThewviscogroelasticfrombvehapresenvior,erbiomecearshanicalipropsuerties,vandrepabilmoitregenerationyandtoellsuppductiortascularization,cellpattachh-erthelessmencellulartvthrmorphogenesisonetusevghwcollagen,usuallybronectinsingleandtlamininrepligandsonaresequeninsucienoftwthaloneusetokexplaingrotheeryconstructiveeneInremogrodelingbevmatrixenuptsthroughthatproteolyticarefromobserv20);edsubstanfollomecwingwhicinarevivofromimplanthetationyofcellsECMAnscaoldsu(2).whatIscoisbindingerecognition,ssenytialwththattheirsynrefore,theticearsmatricesseenarendesignedininfactorsuconhinafromweaGAy(21).thatthetheytoalsoyzeservharacterizeebasedasoreservcompoirtforandgroelywthGfactorsstructuralandrangecytokinestecandshedthengineeruswthalloreleasewingbiomate-thehaexibiliteyeentotedmovdulatethetissueydynamicsand(3,e10).nIntialnaturceha,etheseeenbioinductivortedeanimalpropdeertiesforareofensuredonebskinywtheaspresencenofonnegativvelyexploitingcarioushargedolymerGAandGsydrogelsandNevPGsaswhicyhproactinasolvmandatoryincoreceptorsrequireforcomplexnwumofberalerpath-ofagandrmoreoawthgrofactorsfactorasypbroblastothergroortswthcusedfactorssc(FforGF),tialhepatoerycytemgrogrowthfactorsfactorsThe(HGF),ofvfeedbacascularmecendothelialingrowthwthdelivfactorshas(VEGF),betcexplore(1113).(18).Bythisianwthteractingiseoundlec-thetrostaticallyandwitheasedtheseonmoleculesdemandthecell-mediatedgrocalizedwthcleafactorsagemathey(19,bthisecomehsequesteredtiallyfromthetheirhanismsignalingyreceptors,htheyfactorsmareleasedyvivobdepeincomenaturalactivbatedin(viaadingproteolyticinprorepair.cessing),inandcontheyndrummatoyextenbtheevprteaectssenstructuraltedfoldingtoabilittheofcellsgroinfactorsaelicitmannereect.signicanetlyrecenalteringytheirhabioactiviteya(10).aAstialgroterestwththefactorswtharedelivrequiredbasedintheonlyelectrostaticsmallteractionamouningtsthetonegativelicitcaofbiologicalGresphainsonse,Thetheofmainwfoiscusainaldeandsigningcsynatheticbuild-upmatricesonforwgronativwthECMfactoronenpresencollagentationypisItotheconnegativtrolctheirGAloheparin.calizedadelivanalysiserya.ofSevyeralhniquesstreatesomegies