ACOSTActionCA15214WG1-WG2-WG3-WG4NetworkingMeeting

Mechanobiologyofcellsandtissuesinhealthanddisease

8th-9thNovember2018,

Ljubljana,Slovenia

Host:

• MirjanaLiović,FacultyofMedicine,UniversityofLjubljana,Slovenia

ScientificCommittee:

• PavleAndjus,FacultyofBiology,UniversityofBelgrade,Serbia• AndrejaAmbriović-Ristov,RudjerBoškovićInstitute,Croatia• RuiTravasso,CenterforComputationalPhysics,UniversityofCoimbra,Portugal• NunoSaraiva,HealthSciencesandTechnologySchool,UniversidadeLusófona,

Lisbon,Portugal• MirjanaLiović,FacultyofMedicine,UniversityofLjubljana,Slovenia

Technicalhelp,organizationandlogistics:

• AdriaBiodoo,Postojna,Slovenia

Venue:

• CityHotel,Dalmatinovaulica15,SI-1000Ljubljana,Slovenia

Sponsor:

• CytoskeletonInc.,https://www.cytoskeleton.com/

• SlovenianBiochemicalSociety,http://www.sbd.si

• VWR,https://www.vwr.com/

• AdriaBiodoo,https://adriabio.com

MeetingProgram

• Thursday,8thNovember

BiophysicsofcellandtissuestructureI(allpresentationsare30minuteslongfollowedbya15minutediscussion,Q&A)

10:30am-10:45am–WelcomeIntroduction

10:45am–11:30am–RuiTravasso,UniversityofCoimbra,Portugal

”MATHEMATICALMODELINGOFCELLMOVEMENTANDANGIOGENESIS”

11:30am–12:15am–ŠpelaZemljič-Jokhadar,UniversityofLjubljana,Slovenia

”CYTOMECHANICSMEASUREDBYAFMANDOPTICALTWEEZERSAFTERF-ACTINDISRUPTION”

12:30pm–2pm-Buffetlunch

2pm-3pm-InvitedLecture,DuškoIlić,KingsCollegeLondon,UnitedKingdom

”FULLTHICKNESSSKINMODELSFROMHUMANPLURIPOTENTSTEMCELLSFORIDENTIFICATIONANDTESTINGEFFECTIVENESSOFPERSONALIZEDTHERAPIESINATOPICDERMATITIS”

BiophysicsofcellandtissuestructureII

3pm–3:45pm–JuanCarlosRodriguez-Manzaneque,UniversityofGranada,Spain

”ELUCIDATINGTHEACTIONSOFADAMTSEXTRACELLULARPROTEASES INHETEROGENEOUSEX-VIVOANDIN-VITROCULTUREMODELS“

3:45pm–4:30pm–MaraGrube,UniversityofLatvia,Latvia

”FTIRSPECTROSCOPYFOREVALUATIONOFTHECELLSMETABOLICSTRESS”

4:30pm–4:45pm–Coffeebreak

4:45pm-5:30pm–ReetKurg,UniversityofTartu,Estonia

”CANCER-TESTISANTIGENSMAGE-APROTEINSEXPRESSIONINEXTRACELLULARVESICLESRELEASEDBYCELLS”

5:30pm–6:15pm–PavleAndjus,UniveristyofBelgrade,Serbia

“THEEXTRACELLULARMATRIXGLYCOPROTEINTENASCIN-CANDMATRIXMETALLOPROTEINASESMODULATECEREBELLARSTRUCTURALANDFUNCTIONALPLASTICITY“

7pm–Networkingdinner

• Friday,9thNovember

Newmethodologiestostudymechanobiologyofcellsandtissues

9am–9:45am–NunoSaraiva,UniversityofLisbon,Portugal

”IMPACTOFAMANGANESE(III)PORPHYRININCANCERCELLMIGRATION”

9:45am–10:30am–RamunasValiokas,CenterforPhysicalSciencesandTechnology,Lithuania

”NANOPATTERNED HYDROGEL SURFACES FOR TISSUE FORMATION STUDIES ANDENGINEERING”

10:30am–10:45am–Coffeebreak

10:45am–11:30am–AndrejaAmbriović–Ristov,RudjerBoškovićInstitute,Croatia

”CHARACTERISATION OF THE INTEGRIN αV-DEPENDENT ADHESOME IN MDA-MB-435SMELANOMACELLS”

11:30am–12:15pm–PoloncaFerk,Universityofljubljana,Slovenia

”UV-INDUCEDGENTICANDEPIGENETICCHANGESINHUMANSKINCELLSINVITRO”

12:30–2pm-Buffetlunch

Mechanobiologicalprinciplesofrareandcommondiseases

2pm–2:45pm–BeataČunderlikova,ComeniusUniversity,Slovakia

”EXTRACELLULARMATRIXASANIMPORTANTCOMPONENTOFEXPERIMENTALINVITROMODELSOFDISEASES”

2:45pm–3:30pm–MelekÖzturk,IstanbulUniversity,Turkey

”OKADAICACID-INDUCEDTAUPATHYMODELANDPEPTIDYL-PROLYLCIS-TRANSISOMERASEEXPRESSIONINPRIMARYCORTICALNEURONS”

3:30pm–3:45pm–Coffeebreak

3:45pm–4:30pm–SerapArbak,AcibademUniversity,Turkey

”MICROSCOPICAL INVESTIGATIONS ON THE PUTATIVE EFFECTS OF NANOPARTICLES ONTESTISTISSUE”

4:30pm–5:15pm–MirjanaLiović,UniversityofLjubljana,Slovenia

”IPSC CELLS DERIVED FROM EPIDERMOLYSIS BULLOSA PATIENTS AS DISEASE MODELSYSTEMS”

Structuralanalysisofbiomoleculesinvolvedinmechanobiology

5:15pm–6:00pm–SergeiStrelkov,UniversityofLeuven,Belgium

”KERATINIFSTRUCTURE,REVISITED”

6:00pm–8pm-Meetingwrap-upandnetworkingdinner

• Saturday,10thNovember

Breakfastanddeparture

ABSTRACTS

INVITEDLECTURE

FULL THICKNESS SKIN MODELS FROM HUMAN PLURIPOTENT STEM CELLS FORIDENTIFICATION AND TESTING EFFECTIVENESS OF PERSONALIZED THERAPIES IN ATOPICDERMATITIS(WORKINPROGRESS)DuškoIlićKingsCollegeLondon,LondonUKdusko.ilic@kcl.ac.ukI will present a current project that we are working on, which is supported by the LEOFoundationinDenmark.Theprojectaimstoutilizethelatestadvancesinstemcellscience,gene-editing and tissue engineering to develop and fully validate innovative 3D in vitromodelsofskin,similartothenativeskininatopicdermatitis(eczema)patients.Dusko Ilic received his MD degree and BSci in Molecular Biology at the University ofBelgrade, Serbia. After obtaining a PhD degree at Tokyo University, Japan, he didpostdoctoralwork at theUniversityofCalifornia in San Francisco.Heheld thepositionofAdjunctAssociateProfessorattheUniversityofCaliforniaSanFrancisco,ConsultantattheVeteran Affairs Medical Center, San Francisco, and Director of R&D in StemLifeLine, aCalifornia-licensed,state-of-the-artcellculturefacilitythatfollowedcGTPforderivationandthelong-termcryopreservationofhumanembryonicstemcelllines(hESC).Since2009,heisattheAssistedConceptionUnitatGuy’sHospital,King’sCollegeLondon,wherehederivedthe first clinical grade hESC under xeno-free conditions. Focus of hiswork is translationalstemcellresearch.

CHARACTERISATION OF THE INTEGRIN αV-DEPENDENT ADHESOME IN MDA-MB-435SMELANOMACELLSMladen Paradžik1, Jonathan D. Humphries2, Davor Nestić1, Dragomira Majhen1, AnaDekanić1, Nikolina Stojanović1, Delphine Sedda1, Igor Weber3, Martin J. Humphries2,AndrejaAmbriović-Ristov1

1Laboratory for Cell Biology and Signalling, Division ofMolecular Biology, Ruđer BoškovićInstitute,Zagreb,Croatia2Wellcome Trust Centre for Cell-Matrix Research, Faculty of Life Sciences, University ofManchester,ManchesterM139PT,England,UK3Laboratory for Cell Biophysics, Division of Molecular Biology, Ruđer Bošković Institute,Zagreb,CroatiaAndreja.Ambriovic.Ristov@irb.hrIntegrinsareheterodimericglycoproteinsconsistedofαandβsubunits thatbindscells toextracellular matrix proteins. Upon integrin clustering, multimolecular integrin adhesioncomplexes (IACs) form that facilitate the linkage between integrins and the actincytoskeletonandpermitbidirectionalsignalling.TheαVintegrinisexpressedinmosttumourcellswhereitregulatesadiversearrayofcellularfunctionsandplaysaroleinanti-tumourdrug resistance. The aim of our work was to assess αV-dependent changes in IACcomposition inMDA-MB-435Smelanomacells inordertobetterunderstandthe increasedsensitivity to paclitaxel and vincristine upon integrin αV knockdown. Integrin αV-specificshRNA was cloned into pSUPER.puro, transfected into MDA-MB-435S cells usingLipofectamine, and cell clones were selected using puromycin. The sensitivity of cells toantitumordrugswasdeterminedusinganMTTassay.CellmigrationwasmonitoredusingaTranswellassay. IACswere isolated followingcrosslinkingand theirmolecularcompositionanalysed usingmass spectrometry (MS)–based proteomics. Flow cytometry,western blot,confocal microscopy and Interference Reflection Microscopy (IRM) were done usingstandardprotocols. IntwoMDA-MB-435S-derivedcellcloneswithdecreasedexpressionofintegrin αV, expressing 15% (2αV) or 5% (3αV) of the control cells amount, increasedsensitivity to paclitaxel and vincristine, decreased sensitivity to cisplatin, and decreasedmigration were observed in line with previous results obtained following transienttransfectionwith integrinαV siRNA. In cell clones 2αV and3αV,whichwere smaller thancontrol cells and lacked stress fibres, the number of focal adhesions was shown to besignificantly lower as observed by IRM and immunofluorescence detection of phospho-paxillin,phosphoFAKandphospho-Src.MSanalysisof isolatedIACsfromcontrolMDA-MB-435S,2αVand3αVcellsidentified282proteins,including36outof60consensusadhesomeproteins. As expected, in clones 2αV and 3αV, integrins αV, β3 and β5were detected atmuch lower levels comparedwith control cells. In addition, lower levels of talin-1 and 2,vinculin,α-actinin-4, tensin-3, filamin-Aand -B, liprinβ1andplectinweredetected.Thesedatawill enable follow-up analyses of themechanisms of signalling by integrins αVβ3/β5and therefore, represent a valuable resource to improve our understanding of themechanisms involved in adhesion control of cell sensitivity to antitumor drugs andmetastaticpotential.

THE EXTRACELLULAR MATRIX GLYCOPROTEIN TENASCIN-C AND MATRIXMETALLOPROTEINASES MODULATE CEREBELLAR STRUCTURAL AND FUNCTIONALPLASTICITYStamenkovićV1,JakovcevskiI2,WilczynskiGM3,KaczmarekL3,SchachnerM2,AndjusPR1

1Centerforlasermicroscopy,FacultyofBiologyUniversityofBelgrade,Belgrade,Serbia2Center for Molecular Neurobiology, University Medical Center Hamburg-Eppendorf,HamburgGermany3LaboratoryofNeurobiology,NenckiInstituteofExperimentalBiology,Warsaw,Polandpandjus@bio.bg.ac.rsThestudyfocusesontheroleofTnCinthemodulationoftheenrichhedenvironment(EE)-induced structural plasticity in the cerebellum by applying imaging to monitor thedistribution of perineuronal nets (PNN), changes in the presynaptic terminals, and theactivitiesofthemajorenzymesthatdegradeECM.Forthispurpose,micewithadeficiencyofTnC(TnC-/-)orMMP-9(MMP-9-/-)aged3weekswereexposedtostandardbreedingconditions(SC)orEE4or8weeks.We showed that the exposure to EE for 8 weeks leads to a decrease in the intensity ofstainingofPNN,aswellasadecreaseinthesizeofGABAergicandanincreaseinthenumberand size of glutamatergic synaptic terminals in control mice. In contrast, TnC - / - miceshowed a reduced intensity of PNN staining comparedwith control animals grown in SC,whiletheirexposuretotheEEdidnotleadtoadecrease,buttoaslightincreaseinintensityofPNN.Inaddition,theEEdidnotaffectthedensityofthetwotypesofsynapticterminalsinTnC - / -mice,while the sizeof inhibitory,albeitnotexcitatory, synaptic terminalswasincreased. Itwas also shown thatMMP-9 affects the rearrangement of PNNand synapticplasticity in thecerebellumbetween4thand8thweekofEEexposure.These findingsarefurtherconfirmedbytheresultsobtainedonMMP-9-/-mice.Thestruturalplasticityfindingswereconfirmedonthebehaivoural levelon TnC-/-micethat show spontaneous nocturnal hyperactivity, as well as poor sensomotor coordinationand low swimming speed. These changes were partly abrogated after exposure to EE.Coversely, lackofTnCdiminished thepositiveeffectsof theEEon theability to learnandmemorize,anditstheanxiolyticeffects.Thus, the interactionsbetweenTnCandMMP-9arecrucial for theregulationofstructuralplasticityinthecerebellum.Theeffectofthegeneticbasisonthebehavioralresponsescanbealteredbytheexposureofanimalstoahighlystimulatingenvironment.

MICROSCOPICAL INVESTIGATIONS ON THE PUTATIVE EFFECTS OF NANOPARTICLES ONTESTISTISSUESerkan Deveci1, Merve Acikel Elmas2, Deniz Yucel2, M. Selcuk Keskin3, Sibel DemirciDelipinar4,IsmailSeckin4,AhmetSahin,3SerapArbak21ReyapHospital,DepartmentofUrology,Corlu,Turkey2 Acıbadem University, School of Medicine, Department of Histology and Embryology,Istanbul,Turkey3AcıbademUniversity,SchoolofMedicine,DepartmentofUrology,Istanbul,Turkey4 Istanbul University, Cerrahpasa Faculty of Medicine, Department of Histology andEmbryology,Istanbul,Turkeyarbaks@yahoo.comThe use of nanobiotechnology in human health has been increased in recent years. Drugcarrier nanoparticles with their wide range of uses and advantages are promisingapproachesforthetreatmentofmanydiseases.Theaimofthisstudy isto investigatetheeffectsoforalandlocaltreatmentofthesilicabasednanoparticlesontestisstructuretobeusedclinicallyinthefieldofurology.Nanoparticles(NanoXactTMSilica,fromNanoComposix,Inc)withadiameterof80nmwereusedinthisstudy.Solutionofnanoparticles(10mg/mL,inMilli-Qwater)wassonicatedfor20mininwaterbathsonicatorbeforeapplication.Inoraladministrationgroup,nanoparticleswereappliedtoanimalsviagavageas20mg/kg.Ontheotherhand,nanoparticleswereappliedtopicallytothepenisincoconutoilas2mg/cm2forlocalapplication.Both localandoralapplicationsweredoneevery threedays for35days,and the animals were sacrificed on day 35. Testes samples were fixed with 10% neutralbufferedformalinsolution.Afterfixation,testestissueswerepreparedforparaffinsections.Sections were stained with Haematoxylin–Eosin (H&E) and Periodic acid–Schiff (PAS)reactionforlightmicroscopicalexamination.H&EandPAS-stainedsectionswereevaluatedforhistopathological scoring.Histopathological scoringwasperformedby themodificationof Hess’s data and number of normal, regressive, degenerative or atrophic tubules weredetermined(Hess,Linder,Strader,&Perreault,1988).Inordertodeterminethelocalizationand expression of occludin, an integral tight junctionmembrane protein, in seminiferousepithelium, samples were examined using semiquantitative and statistical analysisof occludin immunreactivity. For transmissionelectronmicroscopical examination, testestissuesampleswerefixedin2.5%glutaraldehydeandprocessedforepoxyresinembedding.Normalmorphologyofseminiferoustubuleswereobservedincontrolgroup.Inbothgavageandlocalgroups,thenumberofnormaltubulesdecreasedwhilethenumberofregressivetubuleshasbeenincreased.Itwasobservedthatthenumberofdegenerativeandatrophictubuleswas higher in oral administration group compared to local application group. Theimmunohistochemical results showed that occluding immunoreactivitywas higher in localgroup comparing to the oral administration group. Ultrastructural examinationsdemonstratedgerminalepithelialdamage,whichwas less in localgroupcompared tooraladministration group. The results revealed that toxicity of silica nanoparticle on themalereproductivesystemis lowinbothapplications,particularly in localapplication.Therefore,thesesilicananoparticleswouldbeusedasadrugdeliverysysteminthetreatmentofmalereproductivesystemdisordersforthefurtherstudies.

EXTRACELLULARMATRIX AS ANIMPORTANT COMPONENT OF EXPERIMENTAL IN VITROMODELSOFDISEASESB. Čunderlíková1,2, C. Jayachandran3, D. Haško2, B. Filová1, K. Kajo4, M. Vallová4, F.Rehfeldt3,A.Mateašík21InstituteofMedicalPhysics,Biophysics, InformaticsandTelemedicine,FacultyofMedicineComeniusUniversity,Bratislava,Slovakia2InternationalLaserCentre,Bratislava,Slovakia3ThirdInstituteofPhysics-Biophysics,Georg-August-Universität,Göttingen,Germany4DepartmentofPathology,OncologicInstitueofst.Elisabeth,Bratislava,Slovakiabeata.cunderlikova@fmed.uniba.skThe proper interaction between the cells and surrounding extracellular matrix (ECM) iscrucialinphysiologyandisdisruptedinmanypathologies.InordertoidentifyeffectsofECMon cellular processes that are active under clinically relevant conditions, in vitro modelsenabling studies under defined experimental conditionswith possibility tomodify cellularand noncellular composition and increase complexity are helpful. Three-dimensional (3D)cellculturesbasedonECMofferthispossibility.Applicationof3DculturesbasedonECMcomponentsrelevanttocarcinogenesishasshownthat ECM affects different aspects of cell behaviour frommorphogenesis, differentiation,production of extracellular vesicles, metabolic activity as well as response to treatmentinterventionintheformofphotodynamicinactivation,incell-typeandECM-typedependentmanner.VimentinexpressingcelllinewasinfluencedbysurroundingECMatmost,especiallywithregardtomorphogenesis,differentiation,andresponsetophotodynamic inactivation.BiophysicalpropertiesoftheECMsurroundingthecells,namelycollagentypeIfiberdensity,have shown the most considerable modifications in the course of multicellular clusterformation in the case of vimentin expressing cell line. Mechanobiological measurementshavesuggestedmoredistinctaccompanyingchangesinthestiffnessofcollagentypeImatrixaround clustersof vimentinexpressing cells thanaround clustersof cellsnegative for thisdifferentiationmarker.Results of our research indicate that presence of the proper ECM composition inexperimental in vitro models is crucial for understanding mechanisms behindpathophysiologyofcancerandotherdiseases.Acknowledgement: This work was supported by VEGA 1/0070/16 and STSM Grant fromCOSTActionCA15214.

UV-INDUCEDGENTICANDEPIGENETICCHANGESINHUMANSKINCELLSINVITRO1PoloncaFerk,2TanjaPrunkZdravković,1AndrejKastrin,1BraneLeskošek,3ChristineG.Lian,3GeorgeF.Murphy.1FacultyofMedicine,UniversityofLjubljana,Slovenia,andELIXIR-SI2CeljeGeneralHospital,Slovenia3Brigham&Women'sHospital,HarvardMedicalSchool,Boston,USApolonca.ferk@guest.arnes.siSun exposure with sunburn is established as a major environmental risk factor formelanoma,themaincauseofdeathfromskincancers.OurstudiesonUV-inducedprimaryhumanmelanocytes showed that crucial eventson transcriptomic level (nanostringmRNAanalyses) happen mainly in MAPK signalling. The key switch in PanCancer® signallingpathways was observed after UVA irradiation. The investigated transcriptomic changesshowedwavelengthanddosedependency.Inadditiontogeneticchangesthatcontributetothe initiation and propagation of melanoma, epigenetic alterations, i.e. lower expressionlevelsof5-hydroxymethylcytosine(5-hmC)havebeenreported.Ourresultsshowthe5-hmCloss might be UV-driven in a dose-dependent manner. These findings need furtherevaluationfortheirbiologicalsignificanceindeterminingpotentialmelanomaprecursors.

FTIRSPECTROSCOPYFOREVALUATIONOFTHECELLSMETABOLICSTRESSMaraGrubeInstituteofMicrobiologyandBiotechnology,UniversityofLatvia,Latviagrube@lanet.lv

Fourier transform infrared (FTIR) spectroscopy ismoreandmore frequently appliedas anapprovedtechniqueinnaturalandlifesciences.Aninfraredspectrumprovidesspecificdataforidentificationandevaluationoftheoverallmolecularcomposition,chemicalbondingandstructure,alsoenablingqualitativeandquantitativeanalysesofsamplesincl.microbialcells,mesenchymalstemcells,cancercells,etc.Minimalrequirementsforsamplepretreatment,significantly lower sample amounts to compare with biochemical or chromatographymethods, and the fact that all analyzed components are represented together in a singlespectrum, are just some of FTIR advantages. Therefore, FTIR spectroscopy methods arefrequentlyusedinbiomedicalscienceasalabel-freemethodprovidingobjectiveandreliablediagnostic information ina relativelynon-invasivemanner.FTIRspectroscopy isapplied toevaluate, characterize, classify, or distinguish the cell composition based on the dataanalysesofspecificabsorptionbands.TheresponseofcellstovariousgrowthfactorsviathebiochemicalcompositioniswellknownandhasbeenstudiedbyFT-IRspectroscopy.Anovelmethod for FTIR-microscopy of small quantities of biosamples as a hydrated film on adiamondanvilcellwillbepresented.ThismethodallowstospeeduptheFTIRspectroscopicanalysis of various biosamples, particularly in those cases,where the available amount ofbiomaterialisalimitingfactor.Toourexperienceit isappropriatetouseFTIRspectroscopytoestimatethecellmetabolicresponses inducedbyvariousgrowthenvironment-inducedstressfactors. Incl.,mechanicalandthermalstressduringbioprinting.We suggest that FTIR spectroscopy analyses along with other modern cell biology andbiochemistrymethods could contribute tomultidisciplinary collaboration studies applyingfor appropriate Co-ERANET or ERANET grant programs or other relevantnational/internationalfunding’s.

CANCER-TESTIS ANTIGENSMAGE-A PROTEINS EXPRESSION IN EXTRACELLULAR VESICLESRELEASEDBYCELLSReetKurgInstituteofTechnology,UniversityofTartu,Tartu,Estoniareet.kurg@ut.eeMelanoma antigens (MAGE-A) represent a unique class of tumor antigens which areexpressed inawidevarietyofmalignant tumors,while theirexpression inhealthynormaltissues is restricted to germ cells of testis, fetal ovary and placenta. Their restrictedexpression and immunogenicity make them ideal targets for immunotherapy in humancancers. MAGE-A expression is observed mainly in cancers that have acquired malignantphenotypes, invasivenessormetastasis,andtheexpressionofMAGE-Afamilyproteinshasbeenlinkedtoapoorprognosisincancerpatients.We have previously shown that MAGEA4 and MAGEA10 proteins are expressed on thesurface of retrovirus virus-like particles (VLP-s) induced by over-expression of MLV Gag-protein. In the current study, we have analyzed the expression of MAGE-A proteins innaturallyoccurringextracellularvesicles (EVs) releasedbymammaliancells.Weshowthatectopically expressed MAGE-A proteins are incorporated into extracellular vesicles usingdifferentmammalian cell lines.MAGE-Aproteinsareexpressedon the surfaceofEVsandareresistanttothetreatmentwithsaltandnon-ionicdetergents.MAGE-Aproteinscanalsobe used to guide recombinant proteins, e.g. EGFP and Cherry, onto the surface of EVsallowing to follow the behavior of EVs in real time. Our study shows that someMAGE-Aproteins,locatingbothinthenucleusandcytoplasmofthecell,aredirectedtothesurfaceofEVsreleasedbycells.However,themechanismofthisphenomenaanditsbiological

IPSC CELLS DERIVED FROM EPIDERMOLYSIS BULLOSA PATIENTS AS DISEASE MODELSYSTEMSNikolaKolundzic1,PreetiKhurana1,MarigaRogar2,DuskoIlic1,MirjanaLiovic2

1ACUUnit,KingsCollegeLondon,LondonUK2MedicalCenterforMolecularBiology,FacultyofMedicine,UniversityofLjubljana,Sloveniamirjana.liovic@mf.uni-lj.siWehaveengineerediPSCcellsfromprimarykeratinocytesderivedfromskinbiopsiesofanepidermolysisbullosasimplexDowling-Mearaphenotypepatientwithaknownmutationinkeratin 5 (K5 E475G), and a control sample obtained from a healthy donor. Thereprogramming process included non-integrating Sendai virus vector cell reprogramming,selection, cell line expansion, cell line characterisation (CGH, STR, differentiationmarkers,teratomaassay)andskinequivalentdevelopment.ThetworesultingiPSCcelllineshavenowbeen also registered with the Pluripotent Stem Cell Registry (https://hpscreg.eu/), asMLi002-A(iEBS)andMLi003-A(iWT).Wehavesetupprotocols fordifferentiationof iPSCscellsintodifferentepithelialcelltypes,keratinocytesandfibroblasts,whichwillbethenusedtoassembleanew3D full skinequivalent for studyingandtestingwoundhealingand theeffectsofdrugrepurposing(thispartofworkisincludedintheEUproject4D-HEALING).InthiscontextwearealsogoingtoprepareiPScellsand3DskinequivalentsforDystrophicEB,a very severe type of EB that is due to mutations in vimentin. We are also working onprotocolsforcelldifferentiationintoothercelltypestoincreasethecomplexityofourfuture3Dmodelsystems.

OKADAIC ACID-INDUCED TAUPATHY MODEL AND PEPTIDYL-PROLYL CIS-TRANSISOMERASEEXPRESSIONINPRIMARYCORTICALNEURONSMelekÖztürkIstanbulUniversity-Cerrahpaşa Faculty ofMedicine,Medical BiologyDepartment, Istanbul,TURKEYmozturk@istanbul.edu.trThe microtubule associated protein tau plays important roles in microtubule stability,neuritedevelopmentandaxonaltransport.Theaccumulationofintracellularneurofibrillarytangles(NFTs)arethemajorpathologicalhallmarksoftheneurodegenerationinAlzheimer’sdisease (AD). NFTs are mainly composed of hyperphosphorylated tau. In pathologicalconditions,tauhyperphosphorylationinhibitsthebindingoftautomicrotubules,socausingdestabilization of microtubules and leading to neurodegeneration. Tau proteinhyperphosphorylation is also associated with the abnormal activities of kinases andphosphatasesthatareregulatedbyPin1protein.Pin-1(peptidyl-prolylcis-transisomerase)plays important roles in various cellular processes such as cancer, aging andneurodegenerativediseases. Pin-1hasaneuroprotectiveroleinAD,andco-localizeswithNFTs, mediates tau dephosphorylation by PP2A. Pin1 regulates the phosphorylation ofSer/Thrsitesoftauprotein,andpromotesmicrotubuleassembly.Theactivationandactionmechanisms of Pin1 are still unclear in AD. It is suggested that Pin-1 acts on tauconformationand function.We focusedonconsequencesof tauhyperphosphorylationonPin-1 expression and its subcellular localization in hippocampal pimary cortical neurons.GiventherelationbetweenPin1expressionandtauhyperphosphorylationwehypothesizedthatthetaupathologymayalsoinvolvePin1dysregulation.Okadaicacid(OKA)thatisusedforexperimentalmodelfortauopathyandADstudiesinhibitsPP2Aactivity.OKAsuppressesPP2A and causes tau hyperphosphorylation. 25 nMOKA at 8h is an efficient dose for tauhyperphosphorylation in cultured neurons. In our studies we observed the high levels ofcytotoxicity in the OKA-treated groups. It might be indicate that OKA-dependent neurondamagecouldalsoimplicatetauhyperphosphorylation.However,wereported25nMOKAtreatment induced neuronal damage but not significant cell loss.We observed that OKA-induced tau hyperphosphorylation resulted in impairment and retraction of neurites. Ourresultsindicatedthattauopathiesmightbeassociatedwiththedefectsinneuritestructure.Boththeobservedneuronaldamageandhyperphosphorylationoftauwereconsistentwiththe use of OKA treatment as a suggested model of neurodegenerative tauopathy. Wedemonstrate that OKA induced tau hyperphosphorylation and OKA induced Pin1downregulationmightbetwodifferentmechanismswherethealterationinPin1expressionisexpected to result in further tauphosphorylation.With further studies, the relationshipbetween PP2A and Pin1-mediated neuronal death through the inhibition of tauhyperphosphorylation will help to explain the molecular mechanisms underlying ADpathologyandpossibletherapeuticuseofPin1inADinthefuture.

ELUCIDATINGTHEACTIONSOFADAMTSEXTRACELLULARPROTEASESINHETEROGENEOUSEX-VIVOANDIN-VITROCULTUREMODELSJuanCarlosRodríguez-ManzanequeGENYO. Centre for Genomics and Oncological Research: Pfizer, Universidad de Granada,JuntadeAndalucía,Granada,Spainjuancarlos.rodriguez@genyo.esRecent studies on tumor heterogeneity are including the dynamism of extracellularcomponentsduetoproteolyticremodeling.Thehighnumberofproteasesandthespecificcharacteristics of each biological setting make necessary a deeper knowledge of theseprocesses.InourlaboratorywearestudyingtheextracellularproteaseADAMTS1formanyyears. This molecule was first reported as an anti-angiogenic factor, and likewise morerecent works supported this activity. However, both pro-tumorigenic and pro-metastaticproperties have also been described in distinct tumor models, highlighting itsmicroenvironment-dependentactions.WhileourworksupportsaprotumorigeniccontributionofstromaADAMTS1inasyngeneictumormodelwithB16F1murinemelanomacells,weobservedadysfunctionalvasculaturecorrelatingwithhypoxiaandanalteredinfiltrationofmacrophageandimmune-relatedcells.Inanattempttounderstandthesephenotypicchanges,weapproachedex-vivoaorticassaysthat confirmed a deficient sprouting activity in the absence of the protease.We are alsoinvestigatingthepropertiesofendothelialandtumourcellswhentheyareco-culturedundervariousconditions.Importantly,weobservedthatthecharacteristiccapillary-likephenotypeofendothelial cellsandsomeplastic tumorcellswasclearlycompromisedbyother tumorcells that do not express ADAMTS1, and we are interested to evaluate effects on thepolarizationofmacrophages.Inlinewithongoingwork,wewouldpursueadeepanalysisoftheconstituentsoftheECM,includingADAMTSssubstrates,tounveilthesignificanceoftheextracellularmicroenvironment.

IMPACTOFAMANGANESE(III)PORPHYRININCANCERCELLMIGRATIONNunoSaraiva1,Ana Flórido1,2, JoãoCosta1,MaddyParsons3, InesBatinic-Haberle4, JoanaPaivaMiranda2,MatildeCastro2,NunoGuerreiroOliveira2,AnaSofiaFernandes1

1CBIOS, Universidade Lusófona Research Center for Biosciences & Health Technologies,Lisboa,Portugal;2Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade deLisboa,Av.ProfessorGamaPinto,1649-003Lisboa,Portugal3RandallDivisionofCellandMolecularBiophysics,King`sCollegeLondon,LondonW21PG,England,UK4DepartmentofRadiationOncology,DukeUniversityMedicalSchool,Durham,NC,USAp4757@ulusofona.pt

Manganese(III)porphyrins(MnPs)mimicsuperoxidedismutase,scavengedifferentreactivespecies (RS) andmodulate redox signaling.MnPs are currently in clinical trials in patientssubmitted to chemo- or radiotherapy, due to their ability to boost anticancer treatmentswhile protecting off-target tissues. Although RS are implicated in themetastatic process,only scarce studies have addressed the impact of MnPs in metastases. Herein wecharacterized the impact of non-cytotoxic levels of an MnP (MnTnHex-2-PyP5+) inmetastases-related processes. In renal cancer cells 786-O, MnP (0.25 µM) decreasedchemotaxis.ThisMnP(5μM)wasalsostudiedinMCF7andMDA-MB-231breastcancercellsaloneandincombinationwithdoxorubicin(dox;0.1μM).Theco-treatmentdecreasedthecollectivemotilityofMCF7,thechemotacticmigrationofbothcelllines,andtheproteolyticinvasionofMDA-MB-231cells.MnPalsocounteractedtheincreaseinrandomMDA-MB-231cell migration induced by dox. To explore the underlying mechanisms, the effects in cellspread/area, focal adhesions, intracellular RS levels, andNF-kB activitywere studied. OurresultssuggestthatMnPmayhaveabeneficialimpactinreducingcancercellsmigrationandwarrantfurtherstudiesregardingMnP-basedanticancerapproaches.

KeratinIFstructure,revisited

SergeiStrelkov

DepartmentofPharmaceuticalandPharmacologicalSciences,KULeuven,Belgium

sergei.strelkov@kuleuven.be

Historicallykeratinswereamongthefirst intermediate filament (IF)proteinstobestudiedstructurally. Such studies are clearly indispensable towards the understanding of normalkeratinIFfunctioningaswellasthepathologicalmechanismofkeratinmutationsleadingtodisease.Morerecentlytherehasbeenasubstantialprogress,throughbothcrystallographyandinsilicostructureprediction,towardselucidatingtheatomicstructureoftheelementaryIFdimerandthesolubletetramer.However,experimentaldataonhigher-orderarchitectureof keratin IFs are still lacking sufficient detail. Application of modern methods, such ascryoelectron microscopy and chemical crosslinking coupled to MS analysis, are a key tofurtherprogressinthekeratinstructurefield.

MATHEMATICALMODELINGOFCELLMOVEMENTANDANGIOGENESIS

RuiTravasso1,M.Moreira-Soares1,MarcosGouveia1,JoãoCarvalho1

1CFisUC,DepartmentofPhysics,UniversityofCoimbra,RLarga,3004-516Coimbra,Portugal

ruit@uc.ptBiochemicalprocessesareoftentightlycoupledwithphysicalmechanisms.Forexample,theway cells organize in a tissue is dependent on their epigenetic states but also on thepropertiesoftheadhesionforcesbetweenthecellsandwiththeextracellularmatrix.Vesselformation and remodeling depend on blood flow, vessel mechanics, tissue mechanics,growthfactordiffusion,andmatrixmechanicalpropertiesanddegradation.InthistalkIwillpresent severalexamplesof computationalmodels that simulatecellmigrationandvesselformation taking into account the mechanical interplay between the cells and theirmicroenvironment.Iwillfocusontheabilityforthesemodelstosuggesttestablehypothesisandtoprovidenewinsightsintothemechanismsthatdrivethedynamicsinthesebiologicalsystems.

REFERENCES

[1] Santos-Oliveira, P., Correia, A., Rodrigues, T.,Ribeiro-Rodrigues, T.M., Matafome, P.,Rodríguez-Manzaneque, J. C., ... Travasso,R.D. (2015). The forceat the tip-modellingtensionandproliferationinsproutingangiogenesis.PLoScomputationalbiology,11(8),e1004436.

[2] M.Moreira-Soares, R. Coimbra, L. Rebelo, J. Carvalho, R. D.M. Travasso, Angiogenicfactors produced by hypoxic cells drive anastomoses in sprouting angiogenesis – acomputationalstudy.(2018).ScientificReports,8,8726

[3] Ramos, J. R., Travasso, R., Carvalho, J. (2018). Capillary network formation fromdispersed endothelial cells: Influence of cell traction, cell adhesion, and extracellularmatrixrigidity.PhysicalReviewE,97(1),012408.

[4] Gandica, Y., Schwarz, T., Oliveira, O., Travasso, R. D. (2014). Hypoxia in vascularnetworks:acomplexsystemapproachtounravelthediabeticparadox.PloSone,9(11),e113165.

NANOPATTERNED HYDROGEL SURFACES FOR TISSUE FORMATION STUDIES ANDENGINEERINGV. CėplaX, R. Eimont&, A. MazėtytėX, A. Vailionytė&, G. Garbenčiūtė&, V. NavikasX, I.MasilionisX,R.ValiokasX

XDepartment of Nanoengineering, Center for Physical Sciences and Technology, Savanorių231,LT02300Vilnius,Lithuania&UABFerentis,Savanorių235,LT02300Vilnius,Lithuaniavaliokas@ftmc.ltWe present applications of unconventional nanofabrication techniques for studyingfunctional modifications of biocompatible hydrogels. Chemically crosslinked collagenhydrogels have been successfully tested as materials for the next generation of tissuescaffolds and implants. However, little is known about the influence of their surfacecompositionandtopographyoninteractionswithadherentcells. Forexample, Islametal.observed a surface pattern-dependent enhancement of the proliferation rate onrecombinant human collagen type III hydrogels, however the origin of this effect has notbeenanalyzed (1). Theexcellent elastomechanical properties (Young’smodulus close to1MPa) and optical transparency of these hydrogels make them suitable for studying therelatedmechanismson themolecular level. Therefore,wehavedevelopedaplatform forprobingthe influenceofnanoscopicECMproteinpatternsonthecytoskeletalorganizationofsinglecells,e.g.humanforeskindermalfibroblasts(HDFs).Also,wehavestructuredthesurfaceof crosslinkedporcinecollagen (PC)and2-methacryloyloxyethylphosphorylcholine(MPC) hydrogel by creating regular patterns of nanoscopic topographies: nanoholes andnanospikes. We have found that the surface nanostructures promote adhesion of HDFs,despite the studied material itself displays strong interaction with this type of cells.Interestingly, the cells conformed to the topographies on the hydrogel surface, preciselyrepeating the surface nanostructures in their intra-cellular organization. Morphometricalanalysis showed that the axial ratio (AR) of the cells, which were growing on flat vs.nanostructured PC-MPC hydrogel, decreased and they becamemoremultipolar.We alsoobservedvinculinconglomerationsincellscultivatedontothenanoholetopograhpies.Thus,wehavedemonstratedthatthedevelopedtoolboxissuitableforprecisecontroloftheinnerarchitecture on the single-cell level as well as for systematic dissection and studies ofdifferentmechanismsrelatedtocell-ECMinteractionsandtissueformation.References:

1. IslamM.M.,CėplaV.,HeC.,EdinJ.,KobuchK.,RuželėŽ.,JacksonW.B.,RakickasT.,RafatM., LohmannC.P.,ValiokasR.,GriffithM.,Post-FabricationModifications toRecombinantHumanCollagen-PhosphorylcholineHydrogelsforRegenerativeMedicineApplications,ActaBiomaterialia,12,70–80,2015.

CYTOMECHANICSMEASUREDBYAFMANDOPTICALTWEEZERSAFTERF-ACTINDISRUPTIONŠpelaZemljičJokhadar1,JureDerganc1,JoseLuisToca-Herrera2,JagobaIturri2

1InstituteofBiophysics,FacultyofMedicine,UniversityofLjubljana,Slovenia2InstituteofBiophysics,DepartmentofNanobiotechnology,UniversityofNaturalResourcesandLifeScienceVienna,Austriaspela.zemljic-jokhadar@mf.uni-lj.siThe sub-membrane actin cortex in animal cells undergoes a continuous turnover andenablesaquickmechanical responsetostimuli.Themajorityof thecorticalF-actin (whichrepresentsroughly50%oftotalcellularF-actin)isgeneratedonlybytwonucleators:forminDiaph1andArp2/3complexes.ToanalyzethepotentialinfluenceofArp2/3onthecellularmechanicswemeasured cell stiffness of endothelial cells before and after the treatmentwithCK-869,which is a known suppressorofArp2/3activity.Complementarily, cellswerealso treated with F-actin disrupting drugs Cytochalasin-D (an inhibitor of actinpolymerization) and Jasplakinolide (an actin polymerization promoter) for referencepurposes.Thecellularstiffnesswasmeasuredinparallelbymeansoftwodifferenttoolsthatoperateindifferentforceregimesandwithprobesofdifferentgeometricalshapes.Atomicforcemicroscopy (AFM) induces largedeformationswithapyramidal typandprobesbulkcellular stiffness, and optical tweezers (OT), which induce shallower deformations with amicro-sphere.Inagreementwithpreviousstudies,theresultswithbothmethodsshowedareduction incell stiffness forCytochalasin-D treatedcells,andnosignificantdifference forJasplakinolide. Importantly, the CK-869 treatment caused a significant reduction in cellstiffnesswhenprobedwithbothmethods.Hence, thecombinedemploymentofAFMandOTtechniquesappearsasanexcellenttoolforamorecomplete,levelwise,characterizationofcellstiffness.