1

AVIANO,OCTOBER6TH2011

1 TARGETEDNANO­DRUGDELIVERY 2

1.1 S.KROL:“NANODRUGDELIVERYWITHPOLYELECTROLYTEMULTIFUNCTIONALPARTICLES” 21.2 M.PRATO:"FUNCTIONALIZEDCARBONNANOTUBESAPPLICATIONSINMEDICINE" 31.3 M.COLOMBATTI:“ANTIBODY­TARGETEDNANOPARTICLESFORTUMORIMMUNODIAGNOSISANDIMMUNOTHERAPY" 41.4 E.FERRARI:"ASSEMBLYOFNEUROTOXINSBUILDINGBLOCKSFORTHETARGETEDSILENCINGOFNEUROTRANSMITTER

RELEASE" 5

2 ALLERGIES 6

2.1 M.DECARLI:“NANOTECHNOLOGYINHYPERSENSITIVITY” 62.2 G.GRI:“EXPLORINGAREGULATORYROLEFORMASTCELLSININVITROANDINVIVOMODELS” 72.3 F.SPINOZZI:“ANOPPORTUNITYFORRETHINKINGALLERGENSANDALLERGIES” 8

3 “NANOTECHNOLOGY:ANOPPORTUNITYFORPORDENONE’SAREADEVELOPMENT” 9

4 NANOTECHNOLOGYMEETSONCOLOGY 10

4.1 M.CONFALONIERI:“BIOMARKERSFORTHESCREENINGOFTHEBIGKILLER(LUNGCANCER)” 104.2 E.MINI:“PERSPECTIVESOFNANO­DRUGINONCOLOGY" 114.3 M.NAPOLI:"APIN1/MUTANTP53AXISPROMOTESAGGRESSIVENESSINBREASTCANCER" 124.4 D.CRIVELLARI:"NEWTHERAPEUTICOPTIONSINBREASTCANCER:THENAB­PACLITAXEL" 134.5 C.BELLUCO:“NANOTECHNOLOGYFORCLINICALPROTEOMICSINCOLORECTALCANCER” 144.6 M.TIRIBELLI:"METHODSOFDETECTIONOFLEUKEMICCELLSINAML:ADVANTAGESANDPITFALLS” 154.7 G.GASPARINI:“THEPARADIGMOFPERSONALIZEDTHERAPYINONCOLOGY” 16

UDINE,OCTOBER7TH2011

5 TOWARDSSINGLECELLPROTEOMICS 17

5.1 L.FRUK:“NANOINSPIREDTOOLSFORPROTEOMEINVESTIGATION" 175.2 L.CASALIS:"ATOMICFORCEMICROSCOPY­BASEDNANOSTRATEGIESFORSINGLECELLPROTEOMICS" 185.3 M.LAZZARINO:"MEMSBASEDMICROSCALESTRATEGIESFORSINGLECELLPROTEOMICS” 19

6 CARDIOLOGY 20

6.1 P.HUNZIKER:“ARTERIOSCLEROSIS–ALIFETHREATENINGINFLAMMATORYDISEASE” 206.2 C.LONG:“CARBONNANOTUBESFORTHERAPEUTICAPPLICATIONSINHEARTDISEASE” 216.3 A.P.BELTRAMI:“CELLULARSENESCENCEANDREGENERATIVEPARADIGMS" 226.4 M.GNECCHI:"STEMCELLSREPAIRMYOCARDIALDAMAGETHROUGHPARACRINEMECHANISMS" 236.5 C.FRESCO:“IHAVEADREAM” 24

7 UNCONVENTIONALBIOLOGY 25

7.1 N.ELVASSORE:“MICROSCALEMODELSFORSTUDYOFCELLFUNCTIONINDEVELOPMENTANDDISEASE” 257.2 F.VALLE:”MULTISCALEPATTERNINGFORLIFE­SCIENCEAPPLICATIONS” 267.3 S.PIANA­AGOSTINETTI:“THEQUESTFORTHEPERFECTFORCEFIELD” 277.4 F.SENO:“KNOWLEDGE­BASEDPOTENTIALS:APOSSIBLESTRATEGYTOASSISTTHEDESIGNOFPEPTIDESWITHHIGH

AFFINITYTOSPECIFICPROTEINS” 287.5 A.LAIO:“DESIGNINGSHORTPEPTIDESWITHHIGHAFFINITYFORORGANICMOLECULES:ACOMBINEDDOCKINGAND

MONTECARLOAPPROACH” 297.6 M.CASTRONOVO:DENSITY­DEPENDENTENZYMEBEHAVIOURINSIDELATERALLYCONFINEDDNAMONOLAYERS 30

UDINE,OCTOBER8TH2011

8 CLINICALNANOMEDICINE 31

8.1 B.LÖFFLER:THEEUROPEANFOUNDATIONFORCLINICALNANOMEDICINE(CLINAM):FACILITATORFORNANOMEDICINE:VISION,MISSION,ACHIEVEMENT) 31

8.2 R.ELLISBEHNKE:NANOMEDICINE:THEINTERSECTIONOFNANOTECHNOLOGYANDHEALTHCARE 31

2

AVIANO,OCTOBER6TH2011

1 TARGETEDNANO‐DRUGDELIVERYChair:DanielaCESSELLI(UniversityofUdine)

1.1 “Nanodrugdeliverywithpolyelectrolytemultifunctionalparticles”Silke KROL (I.R.C.C.S. Neurological Institute “Carlo Besta”, Milan, Italy, email: silke.krol@ifom‐ieo‐campus.it)Nanotechnologyorbetternanoparticlesfoundtheirwayintomedicalapplicationsespeciallyasdrugdelivery system but also for diagnosis funding a new discipline in science, the nanomedicine.Nowadays nanomaterialsmade fromdifferentmaterials such as polymers, noblemetals or oxidesareexploredfortheirpropertieslikebiocompatibility,toxicityofthemetabolitesandbiodistribution.Another important feature is how the particle size and surface charge and functional groups areinfluencingtheirbiodistributioninthebody[1]andthepharmacokineticsofthebounddrugs.In thepresentedwork twodifferentmultifunctionalnanoparticleswill be introduced. The first aregoldnanoparticleswhichwerefunctionalizedtoinhibittheaggregationofprionproteinaggregation[2], theoriginofneurodegeneration in“madcowdisease”,scrapie insheep,andCreutzfeldt‐Jakobdiseaseinhumans.ThedistributioninmicewasdeterminedbyNIR‐TD(nearinfrared‐timedomain)imaging in a livingmouse at different time points after intravenous injection using a fluorescencelabelonthemultilayershell.Thenthemiceweresacrificedandexvivoa3Dreconstructionofx‐raymicrotomographydetectingtheelectron‐densegoldwasperformedofthebrain.Thisspecialinterestin the brain and the brain distribution arises from the fact that treatments of neurodegenerativedisease as well as brain tumours usually fail because drugs are not able to pass the blood brainbarrier (BBB) in sufficient quantities. The BBB consists of endothelial cells (blood vessels) whichbecome impermeable by tight junctions stitching them together. Only hydrophobic molecules ormolecules with a transporter‐mediated up‐take can cross. But by fluorescence microscopy and aselectiveco‐stainingtheparticlesweredetectedinthebraintissue[3].Aquantitativebiodistributionwasperformedbyradioactivegoldnanoparticles.Themultilayersservedinthepresentedstudyfordiagnosis,andasdrug.Thesecondexampleforamultifunctionalnanoparticlebuildupbyamultilayerapproachintroduceanovelgenetherapeuticcancerdrug.HerethecompletelyorganicconstructwasbuilduptodeliversiRNAtoinhibitcelldivision,targetagainstcancercells,providedeliverytothenucleusandreleaseand express a wild‐type DNA for a gene which often is mutated or deleted in tumour cells. Theparticles were able to selectively kill cancer cells by delivering the gene therapy with nearly viralefficacy[4].[1]ChoiH.S.,AshitateY.,LeeJ.H.,KimS.H.,MatsuiA.,InsinN.,BawendiM.G,Semmler‐BehnkeM.,FrangioniJ.V,TsudaA.2010Rapidtranslocationofnanoparticlesfromthelungairspacestothebody.Naturebiotechnology28,1300‐1304.[2]SousaF.,TranH.N.A.,ModaF.,MandalS.,ChananaM.,VimercatiC.,MorbinM.,KrolS.,TagliaviniF.,LegnameG.(2010)Anovelclassofpotentialpriondrugs:preliminaryinvitroandinvivodataformultilayercoatedgoldnanoparticles.Nanoscale2,2724‐2732.[3] Sousa F.,Mandal S., Garrovo C., AlfonsoA., BonifacioA., LatawiecD.,MenkR.H., Arfelli F., LegnameG.,GallaH.‐J., Krol S. (2010)FunctionalizedGoldNanoparticles:DetailedInVivoMultimodalMicroscopicBrainDistributionStudy.Nanoscale,2(12),2826‐2834.[4]MandalS.,RossoN.,TiribelliC.,ScolesG.,KrolS.(2011)TargetedMulticomponentPolysomesforHighEfficiency,SimultaneousAnti‐senseandGeneDelivery.SoftMatter,2011,7(19),9424–9434

Notes:

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1.2 "FunctionalizedCarbonNanotubesapplicationsinMedicine"Maurizio PRATO (Dipartimento di Scienze Farmaceutiche, Università di Trieste, Piazzale Europa 1,34127Trieste,Italye‐mail:prato@units.it)Carbonnanotubes(CNT)havegeneratedgreatexpectationsduetotheirelectronicandmechanicalproperties.However, highmolecularweights and stronghydrophobic forces keepCNT together inbundles,making theirmanipulation, characterizationandanalytical investigationverydifficult. Theorganicfunctionalizationoffersthegreatadvantageofproducingsolubleandeasy‐to‐handleCNT.1As a consequence, compatibility of CNT with other materials, such as polymers, is expected toimprove. In addition, once properly functionalized, CNT become soluble inmany solvents, so thattheir solution properties can be studied. Many functionalized carbon nanotubes may find usefulapplications in the field of materials science and technology, including photovoltaics.2 Also innanomedicine carbon nanotubes are set to play an important role.3 Their use as drug deliveryscaffolds and substrates for vaccines has already been demonstrated. CNT functionalized withbioactivemoieties areparticularly suited for targeteddrugdelivery. In fact, notonly theybecomelesstoxicthanpristinenanotubesbutalsoexhibitahighpropensitytocrosscellmembranes.4Towardthefullintegrationofcarbonnanotubesintobiologicalsystems,severalstrategieshavebeendevised. Among these, themost successful is the covalent functionalization of sp2 carbons at thesidewallswithorganicpendantgroups.Within this contribution,wewill reviewourmost recent achievements in the field of synthesis offunctionalizedcarbonnanotubesand theirapplications inbiomedicine.NotonlyCNTappear tobeidealsubstratesforneuronalgrowth,buttheyarealsoexcellentcarriersfordrugdelivery.References:1.Tasis,D.;Tagmatarchis,N.;Bianco,A.;Prato,M.Chem.Rev.,2006,106,1105.2.Guldi,D.M.;Rahman,G.M.A.;Zerbetto,F.;Prato,M.Acc.Chem.Res.2005,38,871.3.(a)Bianco,A.;Kostarelos,K.;Prato,M.Curr.Opin.Chem.Biol.2005,9,674.(b)Prato,M.;Kostarelos,K.;Bianco,A.Acc.Chem.Res.2008,41,60.4.Pantarotto,D.;etal.Angew.Chem.,Int.Ed.2004,43,5242.

Notes:

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1.3 “Antibody‐targetednanoparticlesfortumorimmunodiagnosisandimmunotherapy"MarcoCOLOMBATTI(UniversityofVerona;e‐mail:marco.colombatti@univr.it)

Inthelasttenyearsmonoclonalantibodieshavebeenappliedclinicallyintumorswithconsiderableresults,bothforimmunodiagnosisandforimmunotherapy;inprostatecarcinomaspecificmoleculartargetshavebeenidentifiedsuchasPSMA,PSCA,STEAPandtheantibodiesrecognizingthesetumor‐associatedantigensareunderinvestigationinphaseIandIIclinicaltrials.Inthiscontextnewimmuno‐imagingapproachesbasedontheuniquetumorcellrecognitionabilitiesofantibodiesareneededto improvediagnosisandtreatmentofprimaryandmetastatictumorstohelpphysiciansindiagnosis,stagingandevaluatingtherapeutictreatmentefficacy.We are therefore investigating the use of antibodies as vehicle moieties to specifically drivenanostructurestothetumorsites;becausenanoparticlescanaccumulateinthetumormassduetotheEPR(enhancedpermeabilityandretention)effect,antibodiescouldplayacriticalroletoimprovethenanomaterialaccumulationwithintumorsitestherebyreachingclinicallyrelevantconcentrationsoftrackingand/ortherapeuticmoleculesandreducingnon‐specificdeliverytohealthytissues.Wehaveconjugatedantibodiestogoldnanoparticles (AuNPs) loadedwithSERS(SurfaceEnhancedRamanSpectroscopy)trackers;thesuperiorabilityofRamanspectroscopytodetectantibody‐guidedAuNPswas exploited and the potential of this technologywas evaluated in vitro.Due to the verynarrowpeakofRamansignalsitwasalsopossibletoperformmultiplexedimagingwithasinglelaseremission.Notes:

5

1.4 "Assemblyofneurotoxinsbuildingblocksforthetargetedsilencingofneurotransmitterrelease"EnricoFERRARI(MRCLaboratoryofMolecularBiology,NeurobiologyDivision,Cambridge,UK;e‐mail:eferrari@mrc‐lmb.cam.ac.uk)

The assembled binary neurotoxins are protein‐protein conjugates synthesized by a non chemical,although irreversible, linkingmethod,basedon twodistinct polypeptide linkers that canbeeitherexpressedorconjugatedtoeachbuildingblock.Thetwo linkershavetheuniquecapability toself‐assembleonly inpresenceofaspecific,chemicallysynthesizedpeptidestaple.This linkingstrategyallows the assembly of toxic recombinant proteins starting from safe sub‐units using a straight‐forward modular approach. Moreover, the enzymatic part of the neurotoxins can be easily re‐targetedbysimplyswappingthereceptorbindingdomainbuildingblock.Notes:

6

2 ALLERGIESChair:CarlaEMILIANI(UniversityofPerugia)

2.1 “Nanotechnologyinhypersensitivity”Marco DE CARLI (Ambulatorio di Immunologia, SOCMedicina 2, AziendaOspedaliero‐UniversitariaSantaMariadellaMisericordiadiUdine;e‐mail:decarli.marco@aoud.sanita.fvg.it)

Engineered particles at nanometer size have the potential to provide new approaches, thusimproving current therapeutic and diagnostic tools in the field of immunopathology, too. Newmaterials interacting with different cells and molecules of the immune system can influence theoutcome of the immune response to antigens. Our presentation will be focused on the possibleapplicationsofnanotechnologiesinthediagnosisandtherapyofhypersensitivityreactions,abroadspectrum of immunological disorders originated from the activation of T cell subset specific forinnocuousantigensthatareusuallytoleratedbymostpeople.Notes:

7

2.2 “Exploringaregulatoryroleformastcellsininvitroandinvivomodels”Giorgia GRI (Section of Immunology, Department ofMedical and Biological Sciences, University ofUdine.Email:giorgia.gri@uniud.it)Regulatory cells have the aptitude to suppress immune system effector mechanisms and are theessentialplayerslimitingtheextentoftheimmuneresponse,thuspreventingitspotentiallyharmfuloutcome.ThesefunctionsareclassicallyascribedtodifferentsubsetsofTcells.Recently,thefocusofresearch on the composition of normal‐tolerogenic and inflamed‐tissuemicroenvironments led totheidentificationofaplethoraofcellswithdifferentregulatoryskills.Inthislight,anemergingroleofmastcells isdelineatingasmastcells regulatephysiologicalandpathological immuneresponsesacting at the interface between the innate and adaptive branches of the immune system, butassumingdifferentfunctionandbehavioratdiscretestageoftheimmuneresponse.Aim of the presentation is to review the latest discoveries in the field ofmast cell biology, withparticularemphasisonnanobiotechnologyapplications.Notes:

8

2.3 “Anopportunityforrethinkingallergensandallergies”FrancescoSPINOZZI(LaboratoryofExperimentalImmunologyandAllergy,PerugiaUniversity;e‐mail:SPINOZZI@UNIPG.IT)

Recent advances in allergy research mostly focused on two major headings: the amelioration ofproteinallergenpurification,which isaimed toabetter characterizationof IgE‐andT cell‐reactiveepitopes,andthepotentialnewroleforunconventionalinnateandregulatoryTcellsindeterminingand controlling inflammation. These advancements could appear in conflict to each other, sinceinnate T cells have a poorly‐defined antigen specificity, that is often directed toward non‐proteinsubstances,suchaslipids.Ontheotherhand,thefundamentalmechanismofpollengraincapturebydendriticcellsandrecognitionbyTcellsatmucosalsurfacesisnotwellunderstood.Moreover,micemodels cannot take intoaccount thewaybywhich lifelongenvironmental stimuli, actingover thegeneticbackground,mayinteractwithhostmucosalimmunesysteminthefinaldevelopmentofthedisease.In the aim to reconcilemany of these contrasting findings, we propose to consider themodel ofcypress pollinosis as paradigmatic for the study of allergic diseases in adults. The biochemicalcharacterizationofmajornativeproteinallergensfromundenaturedpollengrainhasdemonstratedthat the most relevant substance with IgE‐binding activity is represented by a glycohydrolaseenzyme,similartothosefoundinotherpollensandfruits,whicheasilydenaturize instoredgrains.Moreover, lipids from the pollen membrane are implicated in early pollen grain capture andrecognitionbyCD1+dendriticcellsandCD1‐restrictedTlymphocytes,whichincludeunconventionalmucosal TCRgd+, aswell as TCRab+ but less frequently TCRVa24+ invariantNKT cells. Such T cellsdisplay the functional activities of Th0/Th2 lymphocytes, in some cases are able to produceregulatorycytokines,suchasIL‐10andTGF‐b,butcanalsoelicitanIgE‐mediatedimmuneresponsetophospholipids.SinceCD1+immaturedendriticcellsexpandinthemucosaofallergicsubjectsandhavethepropertytoprocessbothproteinandlipidsintheirlysosomalcompartments,afinalscenariomaysuggestasarule that expansion and functional activation of CD1+ dendritic cells is a key step formounting aTh0/Th2‐deviated immune response, and that such innate response does not confer long‐lastingprotectiveimmunity.Thisfactmayuncovernewperplexitiesindesigntheactualimmunotherapyforallergicdiseases.

Notes:

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3 ROUNDTABLE:“NANOTECHNOLOGY:ANOPPORTUNITYFORPORDENONE’SAREADEVELOPMENT”

WelcomefromthePresidentoftheCRO,DrP.CappellettiG.Scoles(UniUD/CRO):“Untrenoincorsadanonperdere!”G.Toffoli(CRO,Aviano):“Dallaricercaclinicaall’impresaperavvicinarsial<<lettodelpaziente>>”M.Agrusti(PoloTecnologicoPordenone):“Riportiamocompetenzeeccellentinelterritorio”F.Scolari(PoloTecnologicoPordenone):“Nanotechnology:abridgeforclinicaldevelopment”Notes:

10

4 NANOTECHNOLOGYMEETSONCOLOGYChairmen:GiuseppeTOFFOLI(CRO,Aviano)andMarcoCONFALONIERI(CattinaraHospital,Trieste)

4.1 “Biomarkersforthescreeningofthebigkiller(LungCancer)”MarcoCONFALONIERI(CattinaraHospital,Trieste;e‐mail:marco.confalonieri@aots.sanita.fvg.it)Notes:

11

4.2 “Perspectivesofnano‐druginOncology"Enrico MINI (Department of Pharmacology, University of Florence, Florence, Italy; e‐mail:enrico.mini@unifi.it)

The application of nanotechnologies to diagnosticsand therapeuticsmay offer particularlyinterestingperspectivesinoncology. These newtechnologiesare basedon the engineeringofnanometric‐size particles that areabletocross biological barriers,including theblood‐brain barrier,transit out blood vessel wallsorcellmembranesbydifferenttransportmechanisms. Thus,nanoparticlesmayinteractwithabroadspectrumofbiologicalentities(fromproteinstocells).Theyalsohave tuneablephysical, chemicalorbiologicalpropertiesdifferent from thatof the samematerialsinlargerscaleforms,becauseofmodifiedquantummechanicalproperties. In relationto the kind of application, nanoparticles may havevariable shapeand be madeofdifferent materials. Thesefeaturesprovidenanoparticleswith a high capability of interactionandtransport,makingthemattractivetoolsforthedesignoftherapeuticcarriers. Researchersare very interested in this function for therapeutic purposessinceavailable anticancerdrugsarestillmainlymoleculeswithcharacteristicsthatlimittheirclinicaluse:e.g.poorsolubilityinwater,non‐specificbiodistributionand targetingand low therapeutic index.Furthermore, the rapiddevelopmentoftumordrugresistancereducestheefficacyofconventionalandtargetedanticancerdrugs.Theselimitationscouldbeovercomebyvehiculationofanticancerdrugsbynanoparticles.Therapeuticnanoparticleshavebeenpreparedusingseveralmaterials.Liposomesandpolymersareexamples ofnanocarriersalready widelystudied and used successfully for several years inclinicaloncology. Currently, liposomal formulationsof anthracyclinesare used for the treatmentofvarioussolidtumors. Afurtheradvancementwithrespecttothesefirst‐generationnanodrugsisrepresentedbytargetednanodrugsthatbindtospecifictumorreceptorsortoendothelialtumorcells.Examplesofthesenanodrugsalreadyinclinicaluseareproteinssuchasalbumin,orantibodieslinkedtoanticancerdrugs.Manyothertypesofnanoparticles(micelles,dendrimers,viral‐likenanoparticles)arenowtestedaspotentialvectorsofanticancerdrugs.Theobjectiveofmultidisciplinaryresearchteamsinvolved in thisfield is tovalidatesafeandactivenanodrugsandnanocarriersforuseinclinicaloncologywithintheframeworkofthenext10years. Notes:

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4.3 "APin1/mutantp53axispromotesaggressivenessinbreastcancer"M.NAPOLI1,2,#,J.E.Girardini1,2,#,S.Piazza1,A.Rustighi1,2,C.Marotta1,2,V.Capaci1,2,A.Thompson3,A.Rosato4,T.Crook3,A.R.Means5,G.Lozano6andG.DelSal1,2.1LaboratorioNazionaleCIB(LNCIB),AreaSciencePark,34149Trieste,Italy2DipartimentodiScienzedellaVita,UniversitàdegliStudidiTrieste,34127Trieste,Italy3DundeeCancerCentre,UniversityofDundee,DD19SY,UK4DipartimentodiScienzeOncologicheeChirurgiche,UniversitàdegliStudidiPadova,eIstitutoOncologicoVenetoIRCCS,35128Padova,Italy5DepartmentofPharmacologyandCancerBiology,DukeUniversity,Durham,NC27710,USA6DepartmentofCancerGenetics,M.D.AndersonCancerCenter,Houston,Texas77030,USA#Theseauthorscontributedequallytotheworke‐mail:MARCO.NAPOLI@LNCIB.ITTP53missensemutationsdramaticallyinfluencetumorprogression,howevertheirmechanismofactionisstillpoorlyunderstood.HerewedemonstratethefundamentalroleoftheprolylisomerasePin1inmutantp53oncogenicfunctions.Pin1enhancestumorigenesisinaLi‐Fraumenimousemodelandcooperateswithmutantp53inRasdependenttransformation.Inbreastcancercells,Pin1promotesmutantp53dependentinhibitionoftheanti‐metastaticfactorp63andinductionofamutantp53transcriptionalprogramtoincreaseaggressiveness.Furthermore,weidentifiedatranscriptionalsignatureassociatedwithpoorprognosisinbreastcancerand,inacohortofpatients,Pin1overexpressioninfluencedtheprognosticvalueofp53mutation.TheseresultsdefineaPin1/mutantp53axisthatconveysoncogenicsignalstopromoteaggressivenessinhumancancers.Notes:

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4.4 "Newtherapeuticoptionsinbreastcancer:thenab‐paclitaxel"DianaCRIVELLARI(CRO,Aviano;e‐mail:dcrivellari@cro.it)Thetaxanes(paclitaxelanddocetaxel)areamongthemostactivedrugs inthetreatmentofbreastcancerparticularlyinthemetastaticsetting.Theyareactiveeitherinmonotherapyorincombinationwithothercytotoxicdrugs.Despitetheirwidespreaduse,bothdrugsareassociatedwithsignificanttoxicitiesasmyelosuppression,neuropathyandhypersensitivityreactions.Nab‐paclitaxel isanovelpaclitaxel formulation inwhichpaclitaxel isboundtonanoparticlesof thenaturallyoccurringvehicleforhydrophobicmolecules:albumin.The nab platform exploits the properties of albumin to falicitate uptake of drug directly into thetumor cells via the gp60 receptor‐mediatedpathway, resulting in increasedantitumor activity andreducedtoxicitycomparedwithsolvent‐basedpaclitaxel.After preclinical, animal, and phase I studies had been performed, phase II studies of this newcompoundrevealedaninterestingclinicalactivity inpatientswithmetastaticbreastcancerwithanencouragingresponserateof48%whichincreasedto64%inthesubgroupofpatientswhoreceivedthis drug as first‐line therapy. Perhaps more important was the fact that myelosuppression andperipheralneuropathywerelessfrequentandlessseverethanwouldhavebeenexpectedwiththeparent compounds. Another important advantage was the almost complete absence ofhypersensitivityreactionsdespitethedrugbeinggivenwithoutpremedication.

Further phase III trials confirmed and validated the drug in themetastatic setting.We willreview the clinicaldata that support the idea that thisdrug isnot simplyanother taxanebut is atargetedchemotherapythatrepresentsanewtreatmentadvanceforpatientsaffectedbymetastaticbreastcancer.Notes:

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4.5 “Nanotechnologyforclinicalproteomicsincolorectalcancer”ClaudioBELLUCO(CRO,Aviano;e‐mail:cbelluco@cro.it)ColorectalcanceristhesecondmostcommoncauseofcancerrelateddeathinmenandwomeninWesternCountries. The strategy for reducing colorectal cancer incidenceandmortality focusesontwomainobjects:- Identificationandremovalofpremalignantlesions(polyps),andearlydiagnosis;- Personalizedtreatmentoflocallyadvancedandmetastatictumors.Clinicalproteomicsrepresentsapromisingtranslationalresearchapproachinthisareasinceitcanbeusedforidentifying:- Serummarkersforearlydiagnosis;- Prognosticandpredictivemolecularfactors,andpotentialtargetsfortreatment.Nanotechnology devices offer a great opportunity for exploiting clinical proteomics by serumpeptidome harvesting using engineerized nanoparticles, and by high‐troughput proteinmicroarrayanalysis.Thesetwoapproacheswillbepresentedanddiscussed.Notes:

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4.6 “MethodsofdetectionofleukemiccellsinAML:advantagesandpitfalls”MarioTIRIBELLI(DivisionofHematologyandBoneMarrowTransplantation,AOUUdine,Udine,Italy;mario.tiribelli@uniud.it)The recent discoveries in the molecular biology of leukemia have changed the approaches todiagnosisandtreatmentofmostofthehematologicalmalignancies.Inparticular,thefocushasbeenpointed,ononeside,tothetargetingofspecificabnormalitiesintheneoplasticcells(targettherapy)and, on the other side, to the detection of leukemic cells at diagnosis and after therapy (minimalresidualdisease).Insomeneoplasms,wherethemolecularpathogenesisisclear(suchasPhiladelphia‐positivechronicmyeloid leukemia), this approach has led to astonishing clinical results, with the design of highlyactivedrugsandastandardizedmethodofdiseasequantificationanddefinitionofresponse.But,inmanyotherhematologicaldiseases,suchasacutemyeloidleukemia,thelackof“universal”sensitiveand specificmarkers, has a negative impact on evaluation and characterization of neoplastic cellsand, therefore, on design of therapeutic strategies. Inmore of 50%of AMLpatients, detection ofblast still relies substantially only on “conventional” methods as microscopic evaluation or flowcytometry, that are either subjective and/or scarcely sensitive. Recently, some molecularabnormalities (such asWT‐1 gene overexpression or FLT3 and NPM1 genes mutations) has beenstudiedaspotentialmarkersfordetectionofAMLcellsatdiagnosisandaftertherapy,andtargetofspecifictherapy.Thetopicsofthistalkwillbetherevisionofactuallyavailablemethodsofdetectionofleukemiccells,with particular focus on AML, and the possibilities that new technologies, such as nanomedicine,couldopeninthefieldofhematology.Notes:

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4.7 “Theparadigmofpersonalizedtherapyinoncology”GiampietroGASPARINI(StrutturaComplessadiOncologia‐AziendaOspedalieraSanFilippoNeri,Roma;gasparini.oncology@tiscalinet.it)

Currently,anticancertherapyismainlydirectedtotumortypeandbygivingthesametreatmenttopresumed homogeneous patients. The switch from histologic‐driven therapy to molecular clinicaloncologyisallowedbyanimprovedunderstandingofthe“moleculartaxonomy”oftumorsthatcanprovide us the targets for specific drugs. Therefore, cancer therapy is moving irreversibly towardpersonalized therapy that benefits selected patients. Once potential therapeutic targets areidentified, the availability of predictive biomarkers is the key element and their prospectiveevaluationshouldbeaparallelcomponentofclinicalevaluationofanewtargeteddrugs.Thestateofartof clinical resultsofpersonalized therapywillbepresentedanddiscussed.However,inpatientswithadvanceddisease, a limitednumberof targetedagents improveoverall survival, themajorityhavingan impactonlyon response rateor time to tumorprogressionwithefficacy limited in timeduetoacquiredresistance.Themechanismsleadingtoresistancearerelatedtotumorcellheterogeneityandinpartexplainedbythecancerstemcellmodelandgeneticinstability.Thestepstowardtheoptimizationoftailoredtherapy need validated predictive biomarkers, pharmacogenetic analysis and a close collaborationbetweenbenchandbedside.Notes:

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UDINE,OCTOBER7TH2011

5 TOWARDSSINGLECELLPROTEOMICSChairman:F.Romanati(IOM‐CNR,Trieste)

5.1 “NanoInspiredToolsforProteomeInvestigation"Ljiljana FRUK (DFG‐Centre for FunctionalNanostructures, Karlsruhe Institute of Technology; E‐mail:ljiljana.fruk@kit.edu)Aswestarteddecipheringgeneticcode,thecomplexityoftheproteomeemergedasoneofthekeyissuestoaddressedtoenablethefurtheradvanceinmedicineandpersonaliseddrugdesign.Toeasethe understanding of numerous processes involved in the control of structural and functionalproperties of different protein classes as well as protein‐ protein interactions, interdisciplinaryresearchpullingtogethertoolsfromdifferentscientificsubbranchesisneeded.Nanotechnologyofferssomepotentialsolutionstoenablemonitoringoftheproteinfunctionaswellastheinvestigationoftheircomplexinteractionsonananometerscalewithinandoutofthecell.Anintroductiontosomeoftherecentapplicationsofnanotechnologyinthefieldofproteomicswillbegiven and a design of protein biosensor based on DNA directed immobilisation presented. Inaddition, design of the nano tools will be discussed with the particular emphasis on the use ofnanoparticlesandtheirpropertiestoeasesinglecellstudies.Notes:

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5.2 "AtomicForceMicroscopy‐basednanostrategiesforsinglecellproteomics"LoredanaCASALIS(ELETTRA,Trieste;e‐mail:loredana.casalis@elettra.trieste.it)Currentin‐vitrotechniqueshaveproblemswhenusedforaccuratelyidentifyingthesmalldifferencesinprotein content, functionand interactions,which are characteristic of disease.Oneof themainreasonsforlackofreliabilityinproteinanalysisfordiseasediagnosticsisalackoftestsensitivity.Thisis because, for many tests, to be reliable, they need to be performed on a homogeneous, andtherefore very small, sample.Ananotechnologyapproachmayovercome the current limits in lowabundantproteindetection.Weproposeheretousenovelnano‐immunoassaystrategiesbasedonAtomicForceMicroscopynanografting,forcapturingproteinsinnLvolumesandconcentrationuptotensofpM.Suchnanoscaledevicesallowfortheregulationofthedensity,accessibility,andlateralhomogeneity of active biorecognition sites. When combined with microwells, fabricated by soft‐lithographyandimplementedonacell‐sortingset‐up,canbeusedfortrappinghistologicallydefinedcells,fortheindependentproteinprofiling/secretomeanalysisineachwell.Notes:

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5.3 "MEMSBasedMicroscaleStrategiesforSingleCellProteomics”Marco LAZZARINO (IOM‐CNR, Area Science Park‐Basovizza, 34149 Trieste ITALY; e‐mail: lazzarino@tasc.infm.it)

Biological sensing in the mechanical domain providesunique opportunities to measure forces, displacementsandmasschangesfromcellularandsubcellularprocesses.Nanomechanical systems are naturally well matched insize with molecular interactions, and, because of theirpotential single‐molecule sensitivity, they represent theidealcandidatesforinvestigatebiologicalprocessesatthesingle cell levels. Applications of micro andnanomechanical systems range from basic biological

investigationstorealtimeinsitudiagnostics.Severalstrategieshavebeenproposedtoimplementmicro‐andnano‐mechanicalsystemsassensingtoolsinbiologyandmedicine.Biosensingrelyonachemicalinteractionsbetweenthesensorandtheanalyte: inaffinity‐basedassayshighlyselectivetargetidentificationandcaptureisachievedbytheemploying high specificity (that is, affinity) between the target and the ‘functionalization’ at thedevicesurface.Infingerprintassaysamultiplicityofless‐selectivefunctionalizationlayerstoidentifyatargetthroughcharacteristicbindingaffinitiestoanensembleofsensors.Insinglecellproteomics,wherehundredsofproteinshighlydiluitedneedtobe identifiedatthesametimeonlythesecondapproachcanbeadopted.Mechanicaldetectioncanberealizedusingseveraldifferentapproaches:the most common are: surface‐stress mechanical biosensors; dynamic‐mode cantilevers andsuspended bridges operated in vacuo; suspended microchannels; optical microring resonators;quartz crystalmicrobalances; opticalmicroring resonators; force detecting sensors and nanowiressensors. For each of those approach a tradeoff between advantage and disadvantage should bereached,withaparticular attention inbalancing sensitivity versus response time.A secondaspectrely on the possibility of operating those sensors in wet environment to perform real timeexperiments.Arecentdevelopmentofmechanicalsensorsbasedonmicropillarshasbeenproposedbyuswhichhasthepotentialityofcombiningsinglemoleculesensitivity,wetoperation,fastresponsetimeandmultiplexingforfingerprintassaying.Theperformancesofmicropillarsensorswillbediscussedintheframework of diffusion limited molecular reaction and we will show that microsizing the sensosrsurfacewillenhancedetectionrateorconcentrationsensitivitybyabout3ordersofmagnitude.Notes:

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6 CARDIOLOGYChair:UgolinoLIVI

6.1 “Arteriosclerosis–ALifeThreateningInflammatoryDisease”PatrickHUNZIKER(PresidentoftheEuropeanSocietyforNanomedicine;DeputyHeadoftheIntensiveCareClinicoftheUniversityofBasel,CH;e‐mail:hunziker@clinam.org)Notes:

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6.2 “Carbonnanotubesfortherapeuticapplicationsinheartdisease”CarlinLONG1,2(1DenverHealthMedicalCenter,2UniversityofColoradoHealthSciencesCenter,USA;e‐mail:Carlin.Long@dhha.org)Carbon nanotube scaffolds have been found to promote growth, differentiation and survival ofneurons and tomodify their electrophysiological properties.We hypothesized that similar effectsmight be seen in cardiacmyocytes,which share several characteristics (terminal differentiation aswell as molecular and electrophysiological properties) with neurons For our investigations, wecombinedmicroscopy,biologicalandsingle‐cellelectrophysiologicalmethodologies toexamine thephenotypeofneonatalratventricularmyocytes(NRVM)culturedonsubstratesofmultiwallcarbonnanotubes(MWCNTs)orongelatin(control).Withtransmissionelectronmicroscopy,wefoundthatNRVMmembranesinteractwithcarbonnanotubesbyformingtightcontacts.Comparedtocontrol,NRVM grown on carbon nanotube platforms showed both increased viability and proliferation.Furthermore, we observed changes in the electrophysiological properties of NRVM on carbonnanotubes, suggesting that the MWCNTs may promote cardiac myocyte maturation. Preliminaryinvestigations into the gene expression patterns of these cells support such an effect on cellularmaturation.Wefeelstronglythattheseresultsholdthepotentialforthedevelopmentofinnovativeclinical, pharmacological and tissueengineeringapplications for carbonnanotube‐baseddevices incardiacdisease.Notes:

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6.3 “CellularSenescenceandRegenerativeParadigms"Antonio P. BELTRAMI (Department ofMedical and Biological Sciences, University of Udine; e‐mail:Antonio.beltrami@uniud.it)The term cellular senescence identifies a cellular response to several stressors that results in: anirreversiblegrowtharrest,alterationsofthegeneexpressionprofile,epigeneticmodificationsandanalteredsecretome,thateventuallyimpairthereparativepropertiesofprimitivecells,addingalayerofcomplexity to theregenerativemedicine field.Over the lastyears,cellularsenescencehasbeendescribed to occur in the settings of several pathological alterations of the cardiovascular system,ranging from metabolic syndrome and diabetes, atherosclerosis, cardiovascular aging and heartfailure. Several molecular pathways have been either associated with the development of cellsenescence or, on the contrary, with organism longevity. Interestingly, the main components ofthese two are common and their manipulation may be explored both to ameliorate stem cellregenerativeapproachesandtopreventstemcelldysfunctionandorganpathology.

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6.4 StemcellsrepairmyocardialdamagethroughparacrinemechanismsMassimilianoGNECCHI(UniversityofPavia;e‐mail:massimiliano.gnecchi@unipv.it)Transdifferentiation of transplanted stem cells into cardiomyocytes and into vascular lineage cellshas been originally proposed as principal mechanism underlying ASC’s therapeutic action. Morerecently,other investigatorshave failed todetectpermanentengraftmentand transdifferentiationof transplanted ASCs. Furthermore, cell fusion of BM‐derived donor cells with recipientcardiomyocyteshasbeenreported.Finally,sofar ithasnotbeenpossibletoreproducibly induceafunctionalcardiacphenotypeinBM‐derivedASCsinvitrousingphysiologicalgrowthfactorsornon‐toxic chemical compounds. These negative results have questioned the plasticity of bothendogenousandtransplantedBM‐derivedstemcells.Regardless of whether stem cells transdifferentiate via a fusion‐dependent or ‐independentmechanism,ithasbeenshownthatinmanycasesthenumberofnewlygeneratedcardiomyocytesistoo low to justify functional improvements. Therefore, it has been proposed that the functionalbenefits observed after stem‐cell transfer in animal models of cardiac injury might be related tosecretion of soluble factors that, acting in a paracrine fashion, protect the heart. The mostcompellingdatapertaintoBM‐derivedmesenchymalstemcells(MSCs).Themechanismsmediatedby paracrine factors are many. Cytokines and growth factors may favor neovascularization,cytoprotectionandendogenouscardiacregeneration.Furthermore,thepostinfarctioninflammatoryandfibrogenicprocesses,cardiaccontractility,andcardiacmetabolismmayalsobe influenced inaparacrine fashion. The demonstration that stem cells may secrete therapeutic factors provides apotential breakthrough in that, rather than administering cells, one may be able to administerspecific proteins produced by these cells for cardiac therapy. Here, we will review the potentialparacrinemechanismsinvolvedinadultstemcellsignalingandtherapy.Notes:

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6.5 “Ihaveadream”ClaudioFRESCO(CardiologyDepartment,AziendaOspedalieroUniversitariadiUdine;e‐mail: fresco.claudio@aoud.sanita.fvg.it)Despitesignificantadvancesmadeinthelast25years,cardiovasculardiseasesremainthenumber1killerinwesterncountries.Furthermore,indevelopingcountriestheimpactofischemicheartdiseaseisdramaticallyincreasing.Nanotechnologymayofferuniqueopportunitiestorethinkourapproachtocardiovasculardiseases,fromphysiopathologytodiagnosisandtotreatment.Unmetneedswillbediscussedfromthepointofviewofapracticingcardiologist,workingeverydayintherealworld.Notes:

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7 UNCONVENTIONALBIOLOGYChairmen:AlfonsoCOLOMBATTI(CRO,Aviano)andCarloA.BELTRAMI(UniversityofUdine)

7.1 “Microscalemodelsforstudyofcellfunctionindevelopmentanddisease”NicolaELVASSORE1,2(1Dept.ofChemicalEngineering,UniversityofPadova,viaMarzolo,9,Padova,Italy;2VenetianInstituteofMolecularMedicine,viaOrus2,Padova,Italy;e‐mail:nelvas@MIT.EDU)Recently, engineered tissueshave foundapromising application as in vitromodels; they couldbeused to support the human tissue physiology and pathophysiology study. For instance, thedevelopmentofnewdrugsandtherapiesfordiseasessuchasmyocardiumfailureisgreatlyslowedandhinderedbythelackofadequateinvitromodels,whichshouldbefunctional,representativeofhuman tissue,easy touseandeconomic.Therefore the requirementsare theuseofahumancellsource and the microscale design, in order to reproduce with high precision the cellmicroenvironment and to guide cell differentiation correctly. The microscale gives the additionalpossibilityofdevelopingmicrometricarrayofcells,whichcanbecoupledtomicrofluidicplatformsforhighthroughputexperiments,whicharefundamentalformultifactorialdiseases.Inthisscenario,theaimofthisworkistheobtainmentofhumanfunctionalcardiactissues,throughtheapplicationof innovative microscale techniques to standard cell culture devices. The strategy employed isbiomimeticandmultidisciplinary:thecellculturemicroenvironmenthasbeenengineeredinordertoreproduceinvitrothemajorstimulithatguidecelldifferentiationinvivo.Thecouplingoftoolsandmethodologiesofthetissueengineeringwithmicroscaletechnologieshasledtoaprecisecontrolofthecellmicroenvironmentinspaceandtime.Notes:

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7.2 ”Multiscalepatterningforlife‐scienceapplications”FrancescoVALLE1,BeatriceChelli1,MicheleBianchi1,EvaBystrenova1,MariannaBarbalinardo3,ArianShehu1,TobiasCramer1,MauroMurgia1,GiuliaFoschi1,PierpaoloGreco2,MassimilianoCavallini1,FabioBiscarini1.1ISMN‐CNR,viaGobetti101,I‐40129Bologna,Italy;2ScribaNanotecnologie,Bologna,Italy;3Nano4bios.r.l.VialeG.Fanin48,40127Bologna,Italy.

e‐mail: f.valle@bo.ismn.cnr.it

The combination of unconventional fabrication technology andbiomaterials allows both to realizestate‐of‐the‐art deviceswith highly controlled lateral features and performances and to study themainpropertiesof thebiomolecules themselvesbyoperatingata scale level comparablewith theonecrucialfortheiractivity.

Soft lithographyandmicrofluidicdevicesoffer a tool‐boxboth to studybiomoleculesunderhighlyconfinedenvironmentsandtofabricateinaneasywaytopographicfeatureswithlocallycontrolledmechanical and chemical surface properties, thus leading to a finer control of the interplay ofmechanicsandchemistry.

Iwillpresentanapplicationofthistechnologytothecontrolofcellfatethatisbecomingakeyissuein regenerative medicine in the perspective of generating novel artificial tissues. Patterns ofExtracellularMatrix(ECM)proteinshavebeenfabricated,byamodifiedLithographicallyControlledWetting (LCW), on the highly antifouling surface of Teflon‐AF to guide the adhesion, growth anddifferentiationofneuralcells(SHSY5Y,1321N1,NE‐4C)achievinganextremelyaccurateguidanc.

Localsurfacetopographyisalsoknowntoinfluencethecellfate,thus,integratingthisparamenterinthe substrate fabrication could increase the complexity of the signals supplied to the cells. In thisperspective we have developed a novel fabrication technique, named Lithographically controlledEtching (LCE), allowing, in one step, to engrave and to functionalize the substrate surface overdifferentlengthscalesandwithdifferentfunctionalities.

Iwillconcludeshowinghowwehavebeendevelopingultra‐thinfilmorganicfieldeffecttransistors(OFETs) as label‐free biological transducers and sensors of biological systems. OFETs are low‐dimensional devices where ordered conjugated molecules act as charge transport material.UnconventionalpatterningtechniquesandmicrofluidicshavebeenadaptedtoproteinsandnucleicacidstodosethemoleculesontheOFETchannelwithahighcontroloftheconcentration.Inanotherset of experiments, we have also been addressing the signalling from neural cells and networksgrownonpentaceneultra‐thinfilmtransistosr.

Notes:

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7.3 “Thequestfortheperfectforcefield”Stefano PIANA‐AGOSTINETTI (D.E. Shaw Reserach, New York; e‐mail: Stefano.Piana‐

Agostinetti@DEShawResearch.com)

Advances in hardware and software have enabled increasingly long atomisticmolecular dynamicssimulations of biomolecules, allowing the exploration of processes occurring on timescales ofhundredsofmicroseconds to a fewmilliseconds. Increasing the lengthof simulations beyond themicrosecondtimescalehasexposedanumberoflimitationsintheaccuracyofcommonlyemployedforce fields. Such limitationsbecomemore severeas the sizeof the systems investigatedand thelength of the simulations increase. Here I will describe the force field problems that we haveencounteredinourstudies,howweidentifiedandaddressedthem,andwhatwehavelearnedintheprocessabout thebiophysicsof the systemsweare investigating.While thequest fora “perfect”forcefieldisnotover(andmayneverbe),ourworkhasgreatlyimprovedtheaccuracyandrangeofapplicabilityof simplephysics‐based force fields, to thepoint that reliablepredictions cannowbeobtainedfrommillisecond‐timescalesimulationsofbiomolecules.Notes:

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7.4 “Knowledge‐basedpotentials:apossiblestrategytoassistthedesignofpeptideswithhighaffinitytospecificproteins”FlavioSENO(UniversityofPadua;e‐mail: flavio.seno@unipd.it)

Thedesignofpeptides capableof selectivelybinding toa specificprotein is a formidable task: itssolutioncouldhaveanenormous impactformedicalandpharmaceuticalapplications .Oneofthekeyaspectsoftheproblemistheimprovementofthereliabilityofthescorefunctionsthatareusedto estimate the affinity of a peptide towards its target protein or target drug. In this talkwewillillustrate how knowledge based potentials, e.g. potentials that are derived directly by a statisticalanalysisofdatabankstructures,canplayacentralroleinthisdirection.Inparticularwewilldiscussan approach (BACH) that we have recently implemented in the context of protein structurerecognition.BACHachievesanunprecedentedcapabilityof recognizing the foldedstate inadecoyset,namelyasetofalternative,highlycompetitive,conformations.Thisispossiblebyemployinganelegantstatisticalanalysisthatallowsforasimultaneousandhomogeneousanalysisofpairwiseandsolvationpotentialswithouttherequirementofanyadhocoptimization.Becauseofitsaccuracyandcomputationalefficiency,BACHmightbethe ideal tool tostudyprotein‐proteinandprotein‐ligandinteractions,aspreliminaryresultsarealreadyshowing.Notes:

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7.5 “Designingshortpeptideswithhighaffinityfororganicmolecules:acombineddockingandMonteCarloapproach”AlessandroLAIO,RolandoHong&FedericoBerti(SISSA,Trieste;e‐mail:Laio@sissa.it)Wepresentanovelcomputationaltoolfordesigningartificialreceptorscapableofbindingwithhighaffinitytoatargetorganicmolecule,forexampleadrug.Thedesignedreceptorisashortpeptideof15 amino acids or less, whose primary sequence is optimizedmaximizing its binding affinity. Thealgorithmbuildsonacombinationofmoleculardynamics,semi‐flexibledockingandreplicaexchangeMonteCarlo, andperformsa simultaneous sampling in sequenceand structure spaces taking intoaccount the flexibility of the peptide. The approach is used to design a decapeptide capable ofbindinganEfavirenzmolecule.Asignificantbindingaffinityofthedesignedpeptide(64nmol/L)wasafterwardsconfirmedexperimentallybyfluorescencemeasurements.NMRspectroscopyconfirmedtheinteractionsbetweenthepeptideandtheEfavirenzmoleculepredictedbythealgorithm.Notes:

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7.6 Density‐DependentEnzymeBehaviourInsideLaterallyConfinedDNAMonolayersDianneChoi1,ShivK.Redhu1,2,VincentInverso2,NehabenPatel1,GiacintoScoles1,3,AllenNicholson1,2,FlavioRizzolio1,4&MatteoCASTRONOVO1,4

(1) Temple University, College of Science & Technology, Department of Biology, 1900 N. 12th Street, 19122,Philadelphia,PA,USA;(2)TempleUniversity,CollegeofScience&Technology,DepartmentofChemistry,1901N. 13th Street, 19122, Philadelphia, PA, USA; (3) Temple University, College of Science & Technology,DepartmentofPhysics,1900N.13thStreet,19122,Philadelphia,PA,USA;(4)CentrodiRiferimentoOncologico‐CRO,FarmacologiaSperimentaleeClinica,ViaF.Gallini2,33081,Aviano,Italy.

e‐mail:mcastro@temple.edu

Standard in vitro techniques do not allow studying biomolecular functions in highly concentratedenvironment,whilecellularfunctionsemergefrombiomolecularreactionsinrathercrowdedcellularcompartments.Nanotechnologyallowsconfiningbiomoleculesintopackedsystemswherethelevelof crowding is close to the cellular condition, and, furthermore, provides analytical methods toinvestigatetheeffectofcrowdingonbiomolecularreactions.

Inourexperimentalwork,wehavestudiedthemechanismbywhichrestrictionenzymesworkinsidepatches of monolayers of short double stranded (ds)DNA molecules, confined on ultra‐flat goldsurfacesbyusingNanografting,anAtomicForceMicroscopy(AFM)basednanolithographymethod.dsDNAmoleculeshavearestrictionsiteathalfheightand,andinturnsuccessfulrestrictionreactionsleadtoa50%decreaseofthepatchheightwithrespecttothesurroundingsurface,thatwemeasurebyAFM.ByvaryingthedsDNAdensitywehaveunequivocallyshownthatwhenthedsDNAdensityissufficiently high, the restriction reaction can successfully occur also on a partial‐consensus dsDNAsite.

OurfindingsdemonstratethattheDNApackinghasaquantifiableeffectonthereactionmechanismof restrictionenzymes, suggesting that in crowdedsystems,enzymesworkverydifferently than insolution. These findings may have broad implications on the development of in vitro approachesuseful, for instance, to improve the understanding of biomolecular functions in living cells orgenerateinnovativebiologicalsensorsbasedonmasstransfer.

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UDINE,OCTOBER8TH2011

8 CLINICALNANOMEDICINE

8.1 PRESENTATIONOFTHEEUROPEANFOUNDATIONFORCLINICALNANOMEDICINE(CLINAM:FacilitatorforNanomedicine:Vision,Mission,Achievement)BeatLÖFFLER(MACEOoftheEuropeanFoundationforClinicalNanomedicine,Basel,CH;e‐mail:loeffler@clinam.org)Notes:

8.2 NANOMEDICINE:THEINTERSECTIONOFNANOTECHNOLOGYANDHEALTHCARERutledgeG.ELLIS‐BEHNKE(DepartmentofBrainandCognitiveSciences,MassachusettsInstituteofTechnology,Cambridge,MA,USA.rutledg@mit.edu)Notes:

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