Dottorato in Biotecnologie

Università degli Studi di Perugia

The School is made by lectures and open discussions tailored for a broad audience of PhD students and youngresearchers from university, research centers and industries who are entering the fascinating, multi-disciplinaryfield of Biotechnology. This year, the School addresses emerging methods and technologies in the fieldof genomics and beyond with invited lectures, seminars and posters by the participants.

Pre-registration is mandatory, up to a maximum of 50 students

info: dottorato.biotecnologie@unipg.it

Invited Lectures (preliminary list):

FABIO BISCARINI, Life Sciences Dept.- University of Modena and Reggio EmiliaOrganic Bioelectronics: from fundamentals to applications to biosensors and implantable devices

PIERSANDRO COCCONCELLI, Università Cattolica del Sacro Cuore, PiacenzaMicrobial Metagenomics

ANNA MIGLIAZZA, Head, External Reseach Nerviano Medical SciencesNew drug R&D: How we translate biology into novel candidate therapies

VALERIO ORLANDO, Head of KAUST Environmental Epigenetics Res.Progr., King Abdullah Univ. of Sci. & Tech.Above the DNA: the role of the Epigenome in cell identity, plasticity and reprogramming

FRANCESCO PAVONE, UniFi and European Laboratory for Non-Linear Spectroscopy (LENS)Morpho-chemistry of tissues by optical imaging

DOMINIQUE SOLDATI-FAVRE, Dep. of Microbiology and Molecular Medicine, University of GenevaGenomic Manipulation in Apicomplexan Parasites

Scientific Committee: Fausto EliseiCarla EmilianiGiovanni GigliottiCarlo Riccardi

Organizing Committee: Stefano BruscoliGianluigi CardinaliDaniele Fioretto

Loredana LatteriniSabata MartinoEfisio Puxeddu

22-26 January 2018Teaching Center of the School of Medicine

WinterSchoolonBiotechnology2018

PROGRAMMAMonday,January22,2018Chair:SabataMartino9:00–9:30 9:30–11:00 Valerio

OrlandoAbovetheDNA:theroleoftheEpigenomeincellidentity,plasticityandreprogramming

11:00–11:30 COFFEEBREAK11:30–13:00 Francesco

PavoneMorpho-chemistryoftissuesbyopticalimaging

13:00–14:00 LUNCH14:00–15:30 FabioBiscarini OrganicBioelectronics:fromfundamentalstoapplicationsto

biosensorsandimplantabledevices15:30–16:00 Beatrice

GironiLowtemperaturebehaviorofmodellipidmembranes,hydrationandmore

16:00–16:30 FedericaPiro DissectionoftheTORCpathwayinToxoplasmagondii16:30–17:00 Chiara

AntoniniAnalysisofdynamicnetworksinproteomicsdataofNSCLCcelllinesusinganewpipelinebasedonmachinelearningtools

Tuesday,January23,2018Chair:StefanoBruscoli9:00–10:30 AnnaMigliazza NewdrugR&D:Howwetranslatebiologyintonovelcandidate

therapies10:30–11:00 COFFEEBREAK11:00–12:00 EfisioPuxeddu UseofNanoStringtechnologytodefinetheimmuneprofileof

thyroidcarcinoma12:00–13:00 OlgaBritanova Applicationofthemolecularbarcodinginimmunerepertoire

profiling 13:00–14:00 LUNCH14:00–15:00 GennaroDe

VitaFeaturesofantibodiesforlifescienceresearch.Applicationsinproteomicfield

15:00–15:30 SaraFlamini GlucocorticoidandBlymphocytesfunction:roleofglucocorticoid-inducedleucinezipper(GILZ)

15:30–16:00 AnnitaMontepeloso

Improvingtheabilityofhematopoieticstemandprogenitorcellstogeneratemicroglia-likeprogenyupontransplantation

16:00–16:30 SamueleSabbatini

EffectofprobioticonGardnellavaginalisaggregationandadhesiontoepithelialcells

!LESSONS!PRESENTATIONS

16:30–17:00 KriziaSagini Theeffectofdruginducedphospholipidosisonextracellularvesiclesrelease

Wednesday,January24,2018Chair:LoredanaLatterini9:00–10:30 MassimoVassalli Activemechanosensinginlivingcells:theemergingroleof

piezochannelsinhumanpatho-physiology10:30–11:00 COFFEEBREAK11:00–12:00 TamaraPosati Naturalpolymersasadvancedmaterialsforbio-medical

devices12:00–13:00 ErmannoFederici Metagenomicprofilingofmicrobialcommunitiesinextreme

environments13:00–14:00 LUNCH14:00–15:00 StefanoZancan ClinicalDevelopment:EnablingStepstoenterintotheClinic15:00–15:30 Francesco

D’AngeloFrancescoMartinoCarpi

Informaticssolutionforpersonalizedmedicineapproaches

15:30–16:00 DIATEC 16:00–16:30 ChiaraArgentati Stemcell-biomaterialinteractionsteersstemcellstowarda

selectedspecificationlineage16:30–17:00 EleonoraCalzoni Proteasesimmobilizationoninnovativematerialforthe

degradationofagribusinessbiomassesinwhitebiotechnologyapplications

Thursday,January25,2018Chair:EfisioPuxeddu9:00–10:30 Dominique

Soldati-FavreGenomicManipulationinApicomplexanParasites

10:30–11:00 COFFEEBREAK11:00–12:00 EdoardoPuglisi Understandingthestructureandfunctionsofmicrobial

communitiesinthebig-dataandpost-genomicera12:00–13:00 GiuseppeBellisola VibroSpectrOmics:justaStarTrecktechnology?13:00–14:00 LUNCH14:00–15:00 DuccioCavalieri RoleofthehumanMycobiomeinhealthanddisease15:00–15:30 Casagrande

PierantoniDeboraCandidabiofilm:amultidisciplinaryapproachtogaininsightonitsstructureandgrowth

15:30–16:00 NicoleNucci InvolvementoftheArylhydrocarbonreceptor(AhR)inthyroidcelltransformation

16:00–17:00 POSTERSESSION

Friday,January26,2018.Chair:GianluigiCardinali17:30–19:30SaladellaGalleriaNazionaledell’Umbria–C.soVannucciTavolarotondaapertaalpubblicosultema:BiotecnologieeSocietàLaRivoluzionedeiBigDataperloscienziatoepertuttinoiIntervengono:Prof.DuccioCavalieri− UniversitàdiFirenzeProf.saFlaviaMarcacci− UniversitàLateranenseMons.TomaszTrafnyPhD− SegretarioEsecutivoFondazioneSTOQPontificioConsigliodellaCulturaBiotecnologieoggiL’incontrodiorientamentoconstudentiesocietà,saràorganizzatoincollaborazioneconlaFondazioneITS–UmbriaedildottoratodiricercadiBiotecnologie.Ladatadell’incontrosaràcomunicatadurantelaWinterSchool2018.

INDEXOFPRESENTATIONS

OralPresentation Posters

O1BeatriceGironi P1BeatriceGironiO2FedericaPiro P2FedericaPiroO3ChiaraAntonini P3ChiaraAntoniniO4SaraFlamini P4SaraFlaminiO5AnnitaMontepeloso P5AnnitaMontepelosoO6SamueleSabbatini P6SamueleSabbatiniO7KriziaSagini P7KriziaSaginiO8ChiaraArgentati P8ChiaraArgentatiO9EleonoraCalzoni P9EleonoraCalzoniO10CasagrandePierantoniDebora P10CasagrandePierantoniDeboraO11NicoleNucci P11NicoleNucci P12MartinaAlunniCardinali P13MartaGambucci P14BenedettaRosiPetra P15DavideSala P16MarziaSichetti P17LorenzoTomassoni

Abstracts

O1/P1.Low-temperaturebehaviourofmodellipidmembranes:hydrationandmoreBeatriceGironia,MarcoPaolantonia,AssuntaMorresia,PaoloFoggia,b,PaolaSassia,caDipartimentodiChimica,BiologiaeBiotecnologie,UniversitàdiPerugia,ViaElcediSotto8,06123Perugia,ItalybEuropeanLaboratoryforNonLinearSpectroscopy(LENS),UniversitàdiFirenze,viaNelloCarrara1,50019SestoFiorentino,Florence,ItalycCentrodi Eccellenza suiMateriali InnovativiNanostrutturati (CEMIN),Università diPerugia,ViaElcedi Sotto8,06123Perugia,ItalyThepropertiesoflipidmembranesatlowtemperaturesareimportantforanumberofbiomedicalandbiotechnological applications and the success of these applications depends on understanding theeffects of temperature changes on intermolecular lipid-lipid and lipid-water interactions. FourierTransformInfraredSpectroscopy(FTIR)isapowerfulandlabelfreetechniquelargelyusedtoinvestigatethestructureoflipidmembranessincewecanobtaininformationaboutconformationanddynamicsofall membrane regions at the same time1. Here we use FTIR spectroscopy to study different lipidsuspensions in water/DMSO solutions in the -60÷30 °C range; the choice of DMSO as a cosolvent isjustifiedsinceitisoneofthemostusedcryoprotectiveagentsofcellularsystems.Inthepresentworkwe analyse the effects of the solvent composition on the structural and thermotropic properties ofliposomes composed of an unsaturated lipid and cholesterol since they are a suitable model ofplasmatic membrane2. To this extent, we compare the properties of lipid vesicles suspended inwater/DMSOsolutionat0.0and0.1DMSOmolefraction.1.R.N.A.H.Lewis,R.N.McElhaney,Biochim.etBiophys.Acta1828(2013)2347–23582.K.Simons,W.L.Vaz,Annu.Rev.Biophys.Biomol.Struct.,33(2004)269–295O2/P2.DissectionoftheTORCpathwayinToxoplasmagondiiManlioDiCristinaa,FedericaPiroaaUniversityofPerugia,DepartmentofChemistry,BiologyandBiotechnology,BuildingB,ViadelGiochetto,06122Perugia,Italy

Autophagyisamechanismabletoproducenutrientswhenacellisinstarvationinordertomaintainitviable. Toxoplasma gondii exploits this mechanism in its life cycle and we want to understand howautophagyisimportantinthesurvivalofthisparasite.Tobetterunderstand,wedecidedtostudywhicharethemostimportantregulatorproteinsinT.gondiithatareinvolvedintheregulationofautophagy.Thanks to bioinformatics analysis we selected some proteins that may play key roles in autophagyregulation, i.e. TGME49_227430, TGME49_227570 e TGME49_227580. These proteins have beenpredictedaslysosomalaminoacidtransportersandsensorssimilarlytoSLC38A9,Rag-aRag-candLST8proteinsthathavearoleinregulationofTORC1complexinhumans1.Ouraim is tocharacterize theseproteins in termsof roleand localization indifferentstagesof theT.gondiilifecycleandidentifypossibletherapeutictargetstoeradicatethisparasiteinfection2.1.RebsamenM.,Superti-FurgaG. (2016).SLC38A9:a lysosomalaminoacidtransporteratthecoreoftheaminoacid-sensingmachinerythatcontrolsmTORC1.Autophagy12(6):1061-1062.2. Yao Y., Jones E., Inoki K. (2017). Lysosomal Regulation of mTORC1 by Amino Acids in Mammalian Cells.Biomolecules.7(3):51.

O3/P3. Analysis of dynamic networks in proteomics data of NSCLC cell lines using a new pipelinebasedonmachinelearningtools.Chiara Antonini a, Lorenzo Tomassoni a, Elisa Baldelli b, Vienna Ludovini c, Sara Baglivo c,MariaelenaPierobonb,EmanuelFPetricoinb,LucioCrinòd,PaoloValigia,FortunatoBianconieaUniversityofPerugia,Perugia,Italy;bGeorgeMasonUniversity,Manassas,VA;cAziendaOspedalieradiPerugia,Perugia, Italy; d Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST), Meldola, Italy; eIndependentresearcher,Montefalco,Italy.Non-smallcell lungcancer (NSCLC) is themostcommonsubtypeof lungcancer.Theaimofthisstudywas todevelopa systemsbiologyapproach tounderstandcomplex interactions inNSCLCandpredictresponse to target treatments.We combined proteomic datawithmachine learning to analyze eightNSCLC cell lines (CL) (5 KRAS MUT and 3 KRAS WT). CL were subjected to reverse phase proteinmicroarray(RPPA)tomeasure183proteinstreatedwithSelumetinib(SE),at6timepoints.WeappliedSupport Vector Machine (SVM) to discover the most divergent proteins both between control andtreated samples and between KRASMUT andWT CLs. To build the interaction networks from thesesubsets,weusedtwoCytoscapeplugins.ThealgorithmrevealedthatSEwasmainlyeffectiveinashorttime,sincethenetworkat1hourcontained25proteinsand76 interactions,whileat24hoursonly4proteins.Theoutputnetwork includednotonlytheSEtarget(MEK),butalsootherpathways,suchasNGFR,mTORandPI3K-Akt.Moreover,inthefirsthour,MEKandERKwerethemainrelevantproteinsinWT samples, while in MUT samples a complex network of 70 proteins and 278 interactions wasactivated.O4/P4. Glucocorticoid and B Lymphocytes Function: Role Of Glucocorticoid-Induced Leucine Zipper(GILZ)Sara Flamini 1, Stefano Bruscoli 1, Francesco Adamo 1, AndreaGagliardi 1, Oxana Bereshchenko 1 andCarloRiccardi11DepartmentofMedicine,SectionofPharmacology,UniversityofPerugia,Perugia,Italy.Glucocorticoid-induced leucine zipper (GILZ) is rapidly and invariably inducedbyGlucocorticoids (GC),whicharethemostcommonlyuseddrugsfortreatmentofautoimmuneandinflammatorydiseases.Ithas been previously shown that GILZmediatesmany GC actions including anti-inflammatory effects.Recently, we have demonstrated that GILZ is as an important regulator of B cell survival andhomeostasis.Bcellactivityhasbeenlinkedtomodulationoftheinflammatoryresponsesbyantibody-dependentandindependentmechanisms.Antibody-independentfunctionsofBcellsincludeproductionofthepro-andanti-inflammatorycytokines.HereweshowthatlackofGILZinBcellsleadstoanincreasedproductionofINFγproductionnotonlyingilzcKOBcells,butalso inco-culturedWTCD4+Tcells,thussuggestingthatGILZdeficiency inBcellsdrivesWTTcellstowardaTh1-likephenotype.ThetranscriptionalactivationofINFγpromoterisdue,atleastinpart,toanenhancedtranscriptionalactivityofAP1inGILZcKOBcells.Moreover,wefoundthatGILZ deficiency drives to an enhanced susceptibility to experimental colitis in mice and signs of thedisease are more severe in gilz cKO B cells compared to WT. Interestingly, we have observed anincreasedproductionof INFγ in infiltratingBandTcells in laminapropriaofcKOmice, indicatingthatGILZcontributestotheregulationofBandTcellfunctionsininflammatoryprocessesduringcolitis.1. Lack of Glucocorticoid-induced leucine zipper (GILZ) deregulates B cell survival and results in B celllymphocytosis in mice. Stefano Bruscoli, Michele Biagioli, Daniele Sorcini, Tiziana Frammartino, MonicaCimino,PaoloSportoletti,EmanuelaMazzon,OxanaBereshchenkoandCarloRiccardi.Blood-2015-03-631580PrepublishedonlineAugust14,2015;2.Glucocorticoid-inducedleucinezipper(GILZ):anewimportantmediatorofglucocorticoidaction.AyroldiE,RiccardiC.FASEBJ.2009Nov;23(11):3649-58.doi:10.1096/fj.09-134684.Epub2009Jun30;

3.IdentificationofB-cellsubsets:anexpositionof11-color(Hi-D)FACSmethods.TungJW1,ParksDR,MooreWA,HerzenbergLA,HerzenbergLA.MethodsMolBiol.2004;271:37-58.O5/P5. Improving the ability of hematopoietic stem and progenitor cells to generatemicroglia-likeprogenyupontransplantationA.Montepeloso1,F.J.Molina-Estevez1,C.Baricordi1,L.Biasco1,4,M.Peviani1,A.Biffi1,2,31 Gene Therapy Program, Dana-Farber/Boston Children's Cancer and Blood Disorders Center, Harvard MedicalSchool,Boston,MA,USA2GeneTherapyProgram,DepartmentofMedicine,BostonChildren'sHospital,Boston,MA,USA3SanRaffaeleTelethonInstituteforGeneTherapy,DivisionofRegenerativeMedicine,StemCellandGeneTherapy,SanRaffaeleScientificInstitute,Milano,Italy4UCLGreatOrmondStreetInstituteofChildHealth,London,UKRecent findings indicate that hematopoietic stem and progenitor cells (HSPCs) can contribute to theturnover of resident brain myeloid cell populations, upon administration of a proper conditioningregimen. In the context of metabolic and neurological diseases, microglia-like cells replaced by theprogenyofHSPCscanactasvehiclesfortherapeuticstothebrainofaffectedpatientsaswellascriticalmodulatorsoftheinflammatoryenvironment.However,theimpactofthisapproachisaffectedbyi)thelack of information concerning the nature and turnover of a functionally defined brain microgliaprecursorpopulation(μP)andii)theslowpaceofreconstitutionbyHSPCsprogeny,ascomparedwiththerapidprogressionoftheneurologicaldisease.With the goal of designing advanced HSPCs transplantation protocols, we provide a functionalcharacterizationof abona fideµP,endowedwith clonogenicpotential, susceptible toBusulfan-basedconditioningregimenandcapableofengraftingupontransplant inhematopoietic tissuesandbrainofmyeloablatedrecipients.Thiswillbeinstrumentalfortargetingthesecellsspecifically.O6/P6.EffectofprobioticsonGardnerellavaginalisaggregationandadhesiontoepithelialcellsSabbatiniS.a,MonariC.aandAnnaVecchiarelliaaDepartmentofMedicine,MicrobiologySection,UniversityofPerugia,Perugia,ItalyBacterial vaginosis (BV) is a disease that contributes over 60% to all vulvovaginal infections. It’sassociatedwithvariousdisordersofthereproductiveapparatusincludinginfertility,manyinflammatorydiseasesand,ifpresentatearlystagesofpregnancy,pretermbirthorspontaneousabortion.Recently,ithasbeenshownthatBVincreasessusceptibilitytoinfectionssuchasHIV,HPV,ChlamydiaandNeisseria.BV ischaracterizedbydysbiosisofthehealthyvaginal floraresulting inanovergrowthofbacteria likeGardnerellavaginalis.Because of the high rates of recurrences of BV, patients undergo to repeated antibiotic therapy andalternativeapproachestopreventortoeradicatetheinfectionarenecessary.Inthisstudytheeffectofthree probiotic strains (Saccharomyces cerevisiae, Lactobacillus rhamnosus and Lactobacillusplantarum),onG.vaginalisaggregationandadhesioncapacityonvaginalepithelialcellsinan“invitro”experimentalmodelwasevaluated.The results obtained showed that some probiotics usedwere able to promote the aggregation of G.vaginalis.Furthermore,aninhibitionofG.vaginalisadhesiontoepithelialcellsandadisplacementofG.vaginaliswhenalreadyadheredtoepithelialcellswasobserved.Inaddition,preliminaryresultsfrominvivoexperimentsshowedatherapeuticeffectofprobioticsduringG.vaginalisinfection.

O7/P7.TheEffectsofDrugInducedPhospholipidosisonExtracellularVesiclesReleaseLorenaUrbanelli1,SandraBuratta1,KriziaSagini1,andCarlaEmiliani1,21DepartmentofChemistry,BiologyandBiotechnology,UniversityofPerugia,Perugia,Italy2CEMIN-CenterofExcellenceforInnovativeNanostructuredMaterial,Perugia,ItalyDrug-induced phospholipidosis (PLD), a transient intracellular accumulation of phospholipids intomultilamellarinclusionbodieswithinlateendosomal/lysosomalcompartments,representsamajorside-effect for many drugs. Amiodarone (AM) is a cationic amphiphilic drug (CAD) widely used as anti-arrhythmicagentandawell-knownPLDinducer;mostCADsinducePLDpresumablybyinterferingwiththe late endosomal compartments. Membrane invagination of late endosome is known to originateMultivesicular Bodies (MVBs) containing up to hundreds Intraluminal Vesicles (ILVs), which can bereleased outside cells upon exocytosis (“exosomes”). Exosomes have been implicated in cell-to-cellcommunication inphysiologicalandpathologicalcondition.PLDcouldeventually influencethereleaseofexosomesand,thus,modulatethetransmissionofvesicle-mediatedsignalstotargetcells.Tounravelthe connectionbetweenPLDandexosome release,wedevelopedcellmodels that stablyexpress thefluorescent fusionmembrane proteins EGFP-CD63 andmCherry-CD63, resulting in the production offluorescentvesicles.We,then,investigatedAM-inducedexosomereleaseandshowedthatfluorescencecould be directly assessed in cell culturemedium. Isolation of exosomes by serial ultracentrifugationclearly indicated that AM treatment interferes with exosome release, suggesting an impairment ofMVBs secretion. Instead of undergoing exocytosis, MVBs could collapse and contribute to thegenerationofPLD-associatedmultilamellarinclusionbodies.O8/P8.Stemcell-BiomaterialinteractionsteersstemcellstowardaselectedspecificationlineageArgentati,C.a;Morena,F.a;BazzucchiM.a;Montanucci,P.b;Armentano,I.c;Emiliani,C.a,d;Martino,S.aa Department of Chemistry, Biology and Biotechnologies, University of Perugia; b Department of Medicine,UniversityofPerugia;cCivilandEnvironmentalEngineeringDepartment,UdRINSTM,UniversityofPerugia,Terni;d

CEMIN-CenterofExcellenceforInnovativeNanostructuredMaterial,Perugia,ItalyThetissueengineeringstrategiesarebasedonthecombinationofaselectedstemcelltype,whichhasthe ability to differentiate toward committed cell lineages, and a biomaterial, that, due to owncharacteristics,couldserveasactivescaffoldtogenerateabio-hybridtissue/organ1.Withinthisissue,wearestudyingthemolecularmechanismsunderlyingthestemcell-biomaterialinterface.Inthisworkwepresentdatademonstratinghowtheactiveinteractionbetweenhumanumbilicalcordmatrixstemcellsandpoly(l-lactide)(PLLA)andPLLA/MultiWalledCarbonNanotubes(MWCNT)films,ensurestheformationof three-dimensionalspheroidalstructuresandaffects the fate transitionofstemcells.Thespheroidsresponddirectlytoatunablesurfaceandthebulkproperties(electric,dielectricandthermal)of PLLA and nanocomposite PLLA/MWCNTs films by triggering a mechanotransduction axis. Thisphenomenonbeginsbybinding theFocalAdhesionproteinsof thecells to thesurfacesofboth films,andbytheAdherensJunctionsbetweenthecells.Thesecomplexestransmittheforcestocytoskeletonproteins (to F-Actin stress fibres that link Filamin-A andMyosin-IIA proteins), generating a biologicalscaffold,withincreasingstiffeningconformationfromPLLAtoPLLAnanocompositefilm,thatmodulatethe nucloeskeleton proteins to boost chromatin reprogramming processes. Here, the oppositeexpressionofNANOGandGATA6transcriptionfactors,togetherwithotherlineagespecificationrelatedproteins,steerspheroidstowardanEpiblast-likelineagecommitmentonPLLAfilms,andtoaPrimitiveEndoderm2.1.Morena,F.,Argentati,C.,Calzoni,E.,Cordellini,M.etal.Ex-VivoTissuesEngineeringModelingforReconstructiveSurgeryUsingHumanAdultAdiposeStemCellsandPolymericNanostructuredMatrix,Nanomaterials,2016,6,4.

2.Morena, F., Armentano, I.,Montanucci, P., Argentati, C. et al.Design of a nanocomposite substrate inducingadult stem cell assembly and progression toward an Epiblast-like or Primitive Endoderm-like phenotype viamechanotransduction,Biomaterials.2017,144,211-229.O9/P9. Proteases immobilization on innovative material for the degradation of agribusinessbiomassesinWhiteBiotechnologyApplicationsEleonora Calzoni a, Alessio Cesaretti a, Alessandro di Michele b, Silvia Tacchi c, Silvia Caponi c, IlariaArmentanod,DanieleFiorettobandCarlaEmiliania,eaDepartmentofChemistry,BiologyandBiotechnologyandCEMIN,UniversityofPerugia;bDepartmentofPhysicsand Geology, University of Perugia; c Istituto Officina dei Materiali del CNR (CNR-IOM) - Unità di Perugia, c/oDepartment of Physics and Geology, University of Perugia; d Department of Ecological and Biological Sciences,TusciaUniversity,Italy;eCEMIN-CenterofExcellenceforInnovativeNanostructuredMaterial,Perugia,ItalyProteasesrepresentafamilyofenzymesabletocatalysehydrolysisofthepeptidebondsbetweentheamineandcarboxylicacidgroupofproteins. It isthelargestgroupofenzymesusedinbio-industrybyvirtue of their high catalytic activity in a broad range of temperature and pHs, enabling industrialprocesses toshift toamoresustainableapproach. In thisworkwehave immobilizedproteasesonaninnovative bio-material due to the growing interest in the use of immobilized enzymes,more stablethansolubleenzymes,whichcanbeusedincontinuousproductionprocesses.Inparticular,immobilizedproteases do not undergo autolysis or denaturing processes1. We used proteases from AspergillusoryzaethathavebeencovalentlyimmobilizedonPoly-L-LacticAcidbyamethodthatusesanamineandglutaraldehydetoactivatethesupportandmakepossiblethebindingofenzymaticmolecules.Wehavecarry out a biochemical characterization of the immobilized proteases, by evaluating the stability totemperatureandpHvariations,andphysical characterizationbyScanningElectronMicroscopy (SEM),AtomicForceMicroscopy(AFM)andRamanSpectroscopy.Moreover,weevaluatedthestabilityoftheenzymaticactivityovertimeandtheabilityofhydrolysingbiomassesfromwasteagribusinessproducts.1ZaborskyOR.Immobilizedenzymes.Cleveland,Ott:CRCPress,1974O10/P10.Candidabiofilm:amultidisciplinaryapproachtogaininsightonitsstructureandgrowthDeboraCasagrandePierantoni1,MartinaAlunniCardinali2,DanieleFioretto2,GianluigiCardinali11DipartimentodiScienzeFarmaceutiche,UniversitàdiPerugia2DipartimentodiFisicaeGeologia,UniversitàdiPerugiaTheabilityofmanyyeastspeciestoformbiofilmisathreattothehumanhealthandaprobleminthefoodindustrywherespoilerspeciescanresistdisinfectioneffortsbyformingthisstructure.Biofilmsarenotorious for forming on different types of solid surfaces including implanted medical devices,catheters, pacemakers, heart valves, but alsoplasticmaterials and stainless steel, i.e. onmost of thematerialsusedtoproduceindustrialmachinesandworkingsurfaces.Candidaalbicans,forexample,isanaturalcomponentofthehumanmicrobiome1,butitisalsothemajorfungalpathogenofhumansforthepersistenceoftheorganismsonthedevicesonthebiofilmformandmakesthemhighlyresistanttodrugsandstresses.WeuseddifferentapproachestoanalysethebiofilmformingabilityandthebiofilmstructureofCandidaalbicansandnon-CandidaalbicansCandidaspecies (NCAC)strains.Thanks to thenew joint Brillouin-Raman microspectroscopy (BMRS) technique we had an insight of the biofilmstructure. Furthermore, a quantitative analysis hasbeen carriedout consideringdifferentparameterssuchastemperature,adhesiontimeandopticaldensity,toelucidatethemodalityofbiofilmgrowth.1.S.Silvaetal.,TrendsMicrobiol2011,19,241-247.

O11/P11.InvolvementoftheArylhydrocarbonreceptor(AhR)inthyroidcelltransformationMenicaliE.1,NucciN.1,MorelliS.1,ColellaR.2,MorettiS.1,PuxedduE.11Dep.ofMedicine,UniversityofPerugia2Dep.ofExperimentalMedicine,UniversityofPerugiaTheArylhydrocarbonreceptor(AhR)isoverexpressedbymanytumorsandinvolvedinthetumorigenicprocess.KynurenineproducedIDO1inthetumormicroenvironment,contributestoimmunetolerance,motilityandgrowthofcancercellsbybindingAhRexpressedbyT lymphocytesandtransformedcells.Veryrecently,wedemonstratedthatIDOisoverexpressedbythyroidcarcinomas.AimofourworkwastoevaluateexpressionandfunctionofAhRinthyroidcarcinomas.AhR expression and function was evaluated in thyroid carcinoma samples and in human thyroidcarcinoma-derivedcelllines.AhRresultedoverexpressedinallthyroidcarcinomahistologiescomparedtonormalthyroid,eitherasmRNAorprotein.AsignificantassociationbetweenAhRanditstranscriptionaltargetCYP1B1couldbedetected,indicatingAhRfunctionalactivationinthetumorsamples.TostudyAhRfunctioninthyroidcancerweselectedFTC133celllinethatshowedthecoexistenceofIDOandAhRoverexpressionandaspontaneousKynurenineoverproductionandwetreatedthemwithtwoagonists of the receptor, kynurenine and ITE. Both treatments resulted in a general activation of thereceptor.Atvariance,kynurenireupregualtedstemnessmarkers,EMTmarkers,IDOandAHRexpressionandcellularmotilitywhileITEdownregulatedthem.P12. Candida spp. identification by Raman microspectroscopy: the effect of rapid heating on theidentificationprocess.MartinaAlunniCardinalia;DeboraCasagrandePierantonibaDipartimentodiFisicaeGeologia,UniversitàdiPerugia,ViaPascoli,I-06123Perugia,ItalybDepartmentofPharmaceuticalSciences-Microbiology,UniversityofPerugia,Borgo20Giugno74,06121Perugia,ItalyTherapid identificationofpathogenicyeasts,asCandida, is foundamental in theraphybothtoreducethe mortality rate due to nosocomial infections, both to fight the increasing problem of antifungalresistance. In recent years, Raman microspectroscopy has been used successfully for microbialidentification,sinceitallowsthedirectanalysisofearlymicrocoloniesgrownonasolidmediumculture.Howeverthisapproachrequiredboththesubtractionofthemediumcontributionasadditionalstepinthedataelaborationprocessboththeuseofanhighcofocalset-upwhichis,unfortunally,muchmoresensitive to sample heterogeneity. In thisworkwe propose another approach to sample preparationthatpermitsustosolvebothproblems.FourdifferentCandidaspeciesweregrownat37°Cfor24hours,then they were washed twice from medium, centrifuged, pelleted and finally dried following twodifferentprocedures:arapidheatingat42°Cfor20minutesandanair-dryingatroomtemperatureforabout5hours.Results suggestnotonly that thedifferentpellets canbedistinguishedby speciesbutalso that air-drying procedure provides better results since a rapid heating of the samples causes anincreasingon fluorescencebackground, that strongly affect the spectral quality and the identificationprocess.P13.EffectsofHydrophobicSilverNanoparticlesonGramicidinaPeptideConformationMartaGambuccia,PaolaSassia,LoredanaLatteriniaaDipartimentodiChimica,BiologiaeBiotecnologie,UniversitàdiPerugia-ViaElcediSotto,8,06123Perugia,Italy

Adeeperknowledgeoftheeffectsofnanomaterialsonproteinconformationandfoldingisfundamentalfor the development of new therapeutic and diagnostic devices.Gramicidin A (GramA) is a

pentadecapeptidewhich can form ion channels inside phospholipidmembranes, thus is an excellentmodelformembraneproteinsandionchannels1.Inthiswork,theeffectofsilvernanoparticles(AgNPs)onGramAconformationintoPOPCliposomesispresented.Dodecanethiol-stabilizedsphericalAgNPs(D-AgNPs)2arepreparedtohavedimensions(5nm)andanhydrophobicnaturecompatiblewiththePOPClipidbilayer.TryptophanfluorescenceandRamansignalswereusedtoprobethepositionofthepeptideinsidethebilayerduetotheirsensitivitytothelocalenvironment3,4.AmideIandIIvibrationalbandsinATR-FTIR spectrumwereemployed to studyGramAconformation 5.Our results suggest thatD-AgNPsmayaffectthepeptidepositioninthebilayerandtheformationoftheionchannel.

1.D.A.Kelkar,A.Chattopadhyay,BBA-Biomembranes,2007,1768(9),2011-2025.2.S.Y.Kang,K.Kim,Langmuir,1998,14(1),226-230.3.J.R.Lakowicz,PrinciplesofFluorescenceSpectroscopy,2006,Springer,NewYork.4.P.Sassietal.J.RamanSpectrosc.,2012,43(2),273-279.5.A.Barth,BBA-Bioenergetics,2007,1767(9),1073-1101.P14.Studyofsolvationinpoly(N-isopropylacrylamide)thermoresponsivemicrogelparticlesSilviaCorezzi,BenedettaPetraRosi,LuciaComezMany biologically relevant phenomena result from a competition between hydrophobicity andhydrogenbonding[1,2].Inmixedaqueoussolutionscosolventsinfluencebothtypesofinteractionandpromotebiologicallyrelevantactions.Tominimizethecomplexity,thechoiceofamodelamphiphiletomimic biological processesmay be of help in the study of cosolvent-induced effects. One promisingsystem consists of poly(N-isopropylacrylamide) (PNIPAM)microgel particles, nanometre-size polymernetworks which respond to changes of temperature by undergoing a transition from a swollen to acollapsed state at a critical temperature (LCST) that can be regarded as an analogue of the colddenaturation in proteins [3].UV-Raman scatteringmeasurements have been used to getmicroscopicdynamical information to be correlated tomacroscopic structural information obtained by PCS. As apreliminaryresultwehavebeenabletodetectvibrationalsignaturesoftherearrangementsoccurringinthemicrogelstructureduringthevolumephasetransition,inparticularthefrequencyshiftofthepeaksintheC-Hbendingandtheamideregionthatareextremelyinterestingastheycorrelatewiththesizechangeoftheparticles.[1]T.Wyttenbach,M.T.Bowers,ChemicalPhysicsLetters480,1(2009)[2]I.M.Klotz,ProteinScience2,1992-1999(1993)[3]Microgelsuspensions:FundamentalandApplications,editedbyA.Fernandez-Nievesetal.,Wiley-VCH(2011)P15.Novelcombinedgene/celltherapystrategiestoprovidefullrescueoftheSandhoffpathologicalphenotypeDavide Sala 1,2, Francesca Ornaghi 1, Fabiana Tedeschi 1, Francesco Morena 2, Valeria Alberizzi 3,AlessandraBolino3,SabataMartino2andAngelaGritti11IRCCSOspedaleSanRaffaele,SR-TIGET,Milano,Italy2UniversityofPerugia,Perugia,Italy3IRCCSOspedaleSanRaffaele,DivisionediNeuroscienze,Milano,ItalySandhoffdisease isa rareneurodegenerative lysosomal storagedisordercausedbygeneticdefects inthe b-N acetylhexosaminidase (Hex) activity, which leads to lysosomal accumulation ofglycosphingolipids, glycoproteins and glycosaminoglycans, with devastating effects on the centralnervoussystem(CNS)(Sandhoffetal.,1968).Notreatmentsarecurrentlyavailable(Regieretal.,2016).Resultsfrompre-clinicalstudiesandclinicalevidencestressedtheimportanceofearlyintervention,high

levels of enzymatic reconstitution in the CNS and global targeting of affected tissues as fundamentalrequirementstoobtainsubstantialtherapeuticbenefit.Herewewillassesswhetheraclinicallyrelevantcombinatorialstrategybasedonneuralstemcell transplantation(NSCT)or intracerebralgenetherapy(ICGT)coupledtobonemarrowtransplantation (BMT)cansupply timelyandtherapeutically relevantlevels of functional Hex enzyme in the CNS and periphery of Sandhoff mouse model, thuspreventing/delaying disease onset and progression, prolonging lifespan and correcting pathologicalhallmarks. Animals treatedwith single or combined approacheswill be analysed to evaluate disease-associated hallmarks, their progression and extent of correction. Results of this studywill assess therelativecontributionoftreatmentstothetherapeuticoutcomeinCNSandperipheryofaffectedmice,withimportantimplicationsforclinicaltranslationoftheseinnovativegene/celltherapystrategies.P16.TheleadingroleoftheepithelialpermeabilityinguthomeostasisMarziaSichettia,DonatellaPietrellaa,GiovannaTrainaaaDepartmentofPharmaceuticalSciences,UniversityofPerugia,ITALYTranswellmodeliswidelyexploitedtorecreateinvitroafunctioningepithelialbarrier.Westudiedthebehaviourofdifferentkindofepithelialcelllinesfromgastro-intestinaltractstimulatedwithethanolorlipopolysaccharide (LPS)knownto induce intestinalbarrierdisruption.Theeffectsof this stimuliwereevaluatedmeasuringthetransepithelialelectricalresistance(TEER)usingamillicell-ERSvolt-ohmmeter(Millipore).Comparedtonon-treatedcontrols,TEERofthecellmonolayerstreatedwithethanolorLPSinduceasignificantdecreaseafter6hwithamaximumofefficacyafter12h.Subsequentlyweusedthistranswell model to investigate the effects of a probiotic formulation (Serobioma) to modulate theinflammatoryresponseofexvivomonocyte-derivedmacrophagesthroughtheepithelialbarrier.Humanintestinal epithelial cell line HT-29 was cultured for 5 days on tranwell membranes to achieve fullymonolayers and stimulated with LPS. After 24h of treatment with LPS, we notice an increase in theproduction of the anti-inflammatory cytokine (IL-10) and a down-regulation of the pro-inflammatorycytokine (IL-1β). Serobiomawas able tomodulate the inflammatory response ofmacrophages acrosstheepithelialbarrier.Thesedatashednewlightsonthehopefullyuseofprobioticsasacoadjutant intheanti-inflammatorytreatmentofthegutepithelialbarrier.P17.ParameterestimationandidentifiabilityanalysisinCancerSystemsBiology:robustcalibrationofhighdimensionnonlineardynamicalmodelsthroughomicsdata.LorenzoTomassoni1,ChiaraAntonini1,PaoloValigi1andFortunatoBianconi2.1 Department of Engineering,University of Perugia, G. Duranti 95, 06132 Perugia; 2 Independent Researcher,Belvedere 44, 06036 Montefalco, Perugia, Italy e-mail: fortunato.bianconi@gmail.com (seehttp://www.fortunatobianconi.eu/).Incomputationalmathematicalmodelling,mostmodelparametersareunknownbecausetheycannotbe directly measured. Therefore, key issues in System Identification of nonlinear systems aremodelcalibrationand identifiability analysis. Existingmethodologies forparameterestimationaredivided intwoclasses:frequentistandBayesianmethods.Thefirstonesoptimizeacostfunctionwhilethesecondonesestimatetheposteriordistributionofparameters.Thesemethodologiessufferfromanincreasingcomputational costwith high dimensionalmodels. Here,we present an innovative Bayesianmethod,ConditionalRobustCalibration(CRC),formodelcalibrationandidentifiabilityanalysis.Thealgorithmisan iterativeprocedurebasedonuniformandjointperturbationoftheparameterspace.Ateachstep,CRC returns the probability density functions of parameters that progressively shrink toward specificpointsintheparameterspace.Thesedistributionsareestimatedonparametersamplesthatguaranteeacertainlevelofagreementbetweenobservablesandinsilicomeasures.WeapplyCRCtoanonlinear

high-dimensional Ordinary Differential Equations model representing the pathway of p38MAPK inmultiple myeloma. The dataset consists of proteomic cancerous data.We test CRC performances incomparison with profile-likelihood and a Bayesian algorithm. We obtain a more precise and robustsolutionwithareducedcomputationalcost.

Dottorato in BiotecnologieUniversità degli Studi di Perugia

Scientific Committee: Fausto EliseiCarla EmilianiGiovanni GigliottiCarlo Riccardi

Organizing Committee: Stefano BruscoliGianluigi CardinaliDaniele Fioretto

Loredana LatteriniSabata MartinoEfisio Puxeddu

26 January 2018 ore 17:30Open day

Biotecnologie e Società Sala della Galleria Nazionale dell’Umbria – C.so Vannucci

La Rivoluzione dei Big Data per lo scienziato e per tutti noi

Intervengono

Prof. Duccio Cavalieri Università di Firenze

Prof.sa Flavia MarcacciUniversità Lateranense

Mons. Tomasz Trafny PhDSegretario Esecutivo Fondazione STOQ

Pontificio Consiglio della Cultura

ModeratoreProf. Gianluigi Cardinali

Dottorato di Ricerca in BiotecnologieUniversità degli Studi di Perugia

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