dottorato yearbook2010_bioengineer


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Chair:

Prof. Maria Gabriella

Signorini

DOCTORAL PROGRAMIN BIOENGINEERING

The Doctoral Programme in Bioengineering trains graduatestudents through a strong interdisciplinary education on engineering,mathematics, medical and biological knowledge to develop highlevel engineering problem-solving abilities in life sciences insidea research group or in private or public industrial context.Students are involved in research works in elds currently ongoingat the Bioengineering Department of Politecnico di Milano whichorganizes the PhD track.PhD students in Bioengineering are about 15 per year, around 50in the three year course.Research themes include modelling and analysis of physiologicaldata, signals and systems; biomedical imaging processing andtechnologies; technologies and instrumentation for movementanalysis, rehabilitation, ergonomics and sports; therapeuticdevices and life support systems in cardiology, cardio/surgery

and pneumology; design and assessment of prostheses; computeraided surgery and surgery optimization through modelling;cardiovascular uid dynamics; molecular, cellular and tissueengineering for biomaterials and prostheses; neuro-engineeringand nanobiosystems; genomic and proteomic data analysis;bioinformatics. Stage periods in distinguished research institutesin Italy and abroad are an essential feature of the student training.

The educational offer includes ad hoc advanced courses specicallyprojected for the Ph.D. Among them, the school of the NationalBioengineering Group is held every year since 1981 for one weekin Bressanone(BZ). The content of the School is focused on themesof the bioengineering research and knowledge and it is organisedwith the support of national and international qualied teachers inthe specic eld coming both from academic and industrial research.The school is also a unique opportunity to put together students

from different Doctoral Programs coming from the entire country.This allows exchanging ideas and experiences also representinga very useful educational event.

Some themes of the recent past editions:2004 Advanced mthods of biomedical signal processing2005 Biomaterials: from protesic implants to regenerative medicine2006 Neuro-Robotics. Neuroscience e robotics

for the development of intelligent machines2007 Computational Genomics & Proteomics2008 Wearable Intelligent Devices for Human Health

and Protection2009 Bioengineering for Cognitive Neurosciences

Scientic and research PhD activities receive a strongsupport by Laboratories located inside and outside theDepartment in cooperation with other research bodiesand university hospitals:

Laboratory of 2D-3D analysis and modelling of neural

and sensory systems and bioelecromagnetism Biomaterials Laboratory Laboratory of biocompatibility and cell culture BioCell Laboratory of Biological Structure Mechanics LABS Laboratory of Computational Biomechanics The Luigi Divieti Postureand Movement Analysis Laboratory

Laboratory of micro and bio uid dynamics Biomedical Signal Processing Laboratory Medical Informatics Laboratory Biomedical Technologies Laboratories


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ADVISORY BOARD

Prof. Paolo Francescon (Direttore U. O. Fisica Sanitaria, Ospedale S. Bortolo, Dipartimento diNeuroscienze, Vicenza, Italy)

Dott. Emanuele Gatti (Fresenius Medical Care, Bad Homburg, Germany)

Prof. Ferdinando Grandori (Head Istituto Ingegneria Biomedica CNR, Milano, Italy)

Prof. Antonio Malgaroli, Universit Vita-Salute San Raffaele, Head of Molecular and CellularPhysiology Lab, Milano, Italy

Dott. Carlo Mambretti (ASSOBIOMEDICA, Milano)Dr. Ivan Martin (Head of Laboratory, University Hospital Basel, Institute for Surgical Researchand Hospital Management, Basel - Switzerland)

SCHOLARSHIP SPONSORS

Broncus Corp, Canada

Fondazione Giovanni e Annamaria Cottino, Torino

Fondazione MEDEA Bosisio Parini

IRCCS Fondazione Don Carlo Gnocchi, Milano

IRCSS Ospedale Pediatrico Bambino Ges, Roma

Istituto di Bioimmagini e Fisiologia Molecolare (IBFM) CNR- Segrate

Istituto di Ingegneria Biomedica ISIB e Istituto di Tecnologie Industriali e Automazione ITIA, CNR,

Milano

Istituto Ortopedico Galeazzi, Milano

Italian Institute of Technology, IIT Foundation

Marie Curie project EU

Sorin Group Italia S.p.A., Saluggia

Stazione Sperimentale per la Seta, Milano

The PhD in Bioengineering has an Advisory Board which has in charge all the student activities

The External Reference Committee is a fundamental link toward the industrial research,the clinical applications with an european and international perspective.

In 2010 new PhD positions as Executive PhDs have been created. They consist of a special PhDpath organized in 4 years and dedicated to PhD candidates that already work in a company/society.The Bioengineering PhD opened 3 positions in 2010.


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|2010STUDY Of THE NANOMECHANICAL PROPERTIES Of LUNG

EPITHELIAL CELLS AND THEIR ALTERATION IN PULMONARYfIBROSIS MEASURED BY ATOMIC fORCE MISCOSCOPY

The framework of this thesis hasbeen the scientic collaborationbetween the BioengineeringDepartment of the Politecnicodi Milano, and the Unitat deBiosica of the Universitat deBarcelona. This collaborationis still ingoing, and in fact thisthesis work is the starting pointfor future investigations of themechanical properties of lungcells in diseased conditionsby means of atomic forcemicroscopy (AFM) and other

nanotechnologies both at theUniversitat de Barcelona and atthe Politecnico di Milano.

The lung is a unique organsubjected to several complexmechanical perturbationsincluding breathing movements,pulmonary blood pressure andalveolar surface tension. Themain function of the lungs,i.e. the respiratory gas exchange,takes place at the respiratorybarrier which includes thealveolar epithelial cell layer andthe blood-containing micro-

capillaries underneath thislayer. The above mentionedmechanical stresses in the lungare intimately associated withthe normal process of breathing,and are ultimately transmittedfrom the tissue level to thecellular level. Thus, studyingthe mechanical response ofindividual lung cells is essentialto understand lung mechanicsand function both in normal and

Irene Acerbi

diseased conditions,as many pathologies areassociated with dramaticalterations in the mechanicsof the lung. Understanding themechanical response of lungcells is particularly relevant inlung brosis and lung cancer,since the mechanical integrityof the alveolar-capillaryinterphase is dramaticallycompromised in both diseases.However, the mechanicalproperties of lung cells in

general, and alveolar epithelialcells in particular, in normal andin diseased conditions remainpoorly understood, mainlydue to the lack of suitabletechniques.The recent developmentof nanotechnologies hasoffered novel technologicalopportunities to address thechallenges involved in thestudy of the mechanics of lungcells. Provided with adequatemodications, nanotechnologiesare able to mimic some of themechanical stimuli that affect

lung cells and to observe howcells respond to such stimuli,by applying deformations in therange of nanometers and forcesin the range of nanoNewtons.Among the differentnanotechniques, atomic forcemicroscopy (AFM) has emergedas the most versatile, as it allowsprobing mechanical propertiesof biological samples in nearlyphysiologic conditions including

single cells, extracellularmatrix gels, and tissue strips.The main goal of this thesiswas to provide new insightsin the mechanical behavior oflung alveolar epithelial cells inresponse to biomechanical andbiochemical signals that mimicdifferent aspects of normaland pathological conditions.In particular, the alterationsof the Youngs elastic modulusof lung epithelial cells underthe effect of either mechanical

stimuli (compression/distension)or biochemical signals (insolubleligands and pro-brotic solublefactors) were studied byAFM provided with suitablemodications. The main goalof the thesis was divided intotwo specic aims that aresummarized below.The rst specic aim was toinvestigate the local resistanceto deformation applied inopposing directions on livinghuman alveolar epithelial cells.The rationale underlying thisrst aim is the fact that alveolar

epithelial cells are permanentlysubjected to cyclic distensileand compressive forces in vivoduring breathing. However thisfundamental mechanical aspectof normal lung function hasnot been investigated in termsof mechanical responses at thecellular level yet. To addressthis aim, our experimentalapproach was to measure thecellular elasticity in response

mechanotransductionexperiments.The second specic aim wasto characterize the stiffeningof cultured lung epithelialcells in response to the potentpro-brotic soluble factortransforming growth factorbeta1 (TGF-1), which has

been directly associated withlung brogenesis as well aswith malignant transformationin the lung and in other organs.The rationale underlying thissecond aim is the fact thatabnormal tissue hardeningand destruction of the normalalveolar architecture arehallmarks of lung brosis, andthat the cell types responsiblefor such stiffening have notbeen clearly identied. BecauseTGF-1 is a potent pro-brotic factor, and it induces

epithelial-to-mesenchymaltransformation (EMT) in culture,and EMT has been observedin an animal model of lungbrosis, we hypothesized thatcells undergoing EMT maycontribute to the abnormalstiffening observed during lungbrosis. In addition, it shouldbe noted that EMT is a processby which epithelial cells loseepithelial characteristics, andgain mesenchymal propertiesincluding expression ofcytoskeletal proteins that mayconfer abnormal contractile

properties. To address this aim,I measured the elastic modulusof two different lung epithelialcell models (A549 and H441)untreated or treated withTGF-1 for 3 days, and checkedfor expression of EMT markersby immunouorescence. Thesetwo cell lines were selectedbased on previous studies thatrevealed that A549 cells mayundergo dramatic EMT upon

proper biochemical stimulation,whereas H441 do not. Ourresults revealed that TGF-1treatment induced an eight-and three-fold increase in theYoungs modulus of A549 andH441 cells, respectively. Thesendings strongly support thatTGF-1 may compromise the

normal mechanical propertiesof the lung microenvironmentby stiffening epithelial cells. Inaddition, these results reveala novel function of TGF-1,i.e. stiffening of lung epithelialcells, which may contribute tolung brogenesis by alteringthe normal balance of forces inthe lung microenvironment andultimately compromising lungfunction.In conclusion, this thesisprovides novel measurementprotocols based on advanced

nanobiotechnological toolsoptimized for lung cellmechanical measurements,and provides new insights inthe basic mechanical propertiesof the lung alveolar epithelialcells in response to mechanicaland biochemical signalsthat mimic different aspectsof normal and pathologicallung conditions.

to compression and distensionforces applied locally to thecell surface with AFM providedwith a specially nanofabricatednanocylindrical tip. The novelmethodological aspects that Iimplemented to address this rstaim include: (i) nanofabricatingcylindrical tips with the Focused

Ion Beam technique (FIB); (ii)coating the surface of thesenanocylinder tips with differentpeptides; (iii) designing a newmeasurement protocol toapply local deformation withdifferent directions (compressionand distension) on softsamples; and (iv) developingthe necessary software toolsto control the AFM and toperform the data processing.The measurement protocolwas validated on a linearelastic polyacrylamide (pAAm)

gel, and was subsequentlyapplied to cultured humanalveolar epithelial cells fromthe A549 cell line. The resultsshowed that, unlike pAAmgels, A549 cells were moredeformable to local distension,and less deformable to localcompression. Furthermorewe identied that the actincomponent of the cytoskeletonplayed a major role in theobserved differential response.In addition we observed thatcells exhibited quick stiffeningin presence of extracellular

peptide mediated deformations.This quick stiffening may beimportant as it may contributeto epithelial repair mechanismstriggered upon lung injury. Insummary, these data imply thatcell mechanosensing depends onthe direction of the deformationapplied, and thereby indicatesthat force directionalityshould be taken intoconsideration when interpreting


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NOVEL STRATEGY AND TOOLSfOR TRACHEA TISSUE ENGINEERINGfrom the bench to the bedside

End-stage organ failureor tissue loss is one of themost devastating and costlyproblems in medicine: over 8million surgical procedures areestimated to be performedevery year to treat thesedisorders in the US alone,incurring a tremendous healthcare cost of more than $400billion annually. Over the last50 years, tissue and organtransplantations, reconstructiveand replacement surgery have

signicantly improved patientoutcomes. Unfortunately, thesesolutions suffer from manylimitations, such as worseningdonor shortages, lifelongimmunosuppressive regimens,unwanted side effects and nitedurability. Therefore, increasinginterest has turned to the eldof tissue engineering (TE),which applies the principlesof engineering and lifesciences in an effort to reach afundamental understanding ofstructure-function relationshipsin normal and pathological

tissues and to develop in vitrobiological substitutes able torestore, maintain, or improvetissue and organ function. Thebasic principle is simple: cellscollected from a patient or adonor are either introducedwith or without modifyingtheir properties into a three-dimensional supporting materialand cultured under properenvironmental conditions (i.e.,

Maria Adelaide Asnaghi

in bioreactors); once the tissueis fully mature or the number ofcells adequate, engraftment maybe performed.Signicant advances havebeen achieved in the eld andexamples of successful clinicalimplementation of tissue-engineered products includeskin substitutes, restorationof corneal surfaces, andreconstruction of diseasedbladder, providing alternatives tothe shortage of suitable grafts

for replacement therapies ingeneral and for reconstructiveairway surgery in particular. Inthis eld, an unmet need fortracheal replacement exists inthe case of extensive defectscomprising more than 40-50%of the total length of the organ,since almost every attemptto provide an autologous,allogeneic or synthetic safeand reproducible trachealgraft has been disappointingso far because of stenosis,immunologic complication,and material failure. TE has

then emerged as the mostpromising technique to createa near-normal trachea, andencouraging early experimentalresults have been reported.In this context, the present studyfocussed on the developmentof an integrated approach onall relevant aspects of the TEprocess toward the denitionof an optimal strategy to bringtrachea substitutes to the

clinic. In particular, a detergent-enzymatic decellularisationmethod was applied to a non-immunogenic tracheal tubularmatrix of at least 6 cm in length.The protocol was effective,both in vitro and in vivo, inproviding matrices lacking anyMHC (major histocompatibilitycomplex) antigens whilemaintaining structural integrity,therefore a promising substratefor clinical use. To accomplishuniform and highly efcient

cell seeding on both the outerand the inner surface of the 3Dmatrix, co-culturing differentcell-types, a novel bioreactorwas developed (Fig.1). Thebioreactor provides two separatesterile compartments, each witha rotating air-medium interfaceto promote optimum masstransport between the culturemedium and the growing tissue;the rotation also provides thehydrodynamic shear stressnecessary to promote cellmetabolic activity and properdifferentiation. The relative

contribution of autologousepithelial cells and mesenchymalstem cells-derived chondrocyteswas then evaluated viaorthotopic implants ofbioreactor-recellularised trachealmatrices in pigs, collectingpromising results for a successfultranslation of the approach tothe clinic. Ultimate validation ofour strategy was provided by theapplication of this technology

in a 31-year old woman withend-stage bronchomalacia,with permission from allrelevant Committees. Therecipient had no complicationsfrom the operation and wasdischarged from hospital onthe tenth post operative day.The graft immediately provided

the recipient with a functionalairway (Fig.2) and improvedher quality of life, with noimmunosuppressive drugs.Cytological and histologicalstudies conrmed angiogenesisand the persistence of viablecells at two months post-surgery (Fig.3). The graft is stillfunctioning well and there is nosign of rejection at 18 monthspost-implantation.Our clinical experience showedthat autologous cells combinedwith a decellularised donor

matrix in a suitable bioreactorcould produce a functionaltissue-engineered airway, freefrom the risk of rejection. Oursuccess is highly encouraging,but also serves to highlightthe scientic, clinical andcommercial bottlenecks standingin the way of full integrationof this regenerative medicinetechnology into routine clinicalcare, translating our research-scale production modelinto a clinically applicablemanufacturing design that issafe, reproducible, cost-effective,

and compliant with the evolvingregulatory framework. In aneffort to move forward towardthis goal and to allow a moreefcient use of our bioreactor-based model system in bothscientic research and clinicallycompliant tissue manufacturing,we are developing an advancedbioreactor for simultaneousrotation and luminal perfusion oflong tubular scaffolds, and novel

tools to non-invasively monitorand control pH of the culturemedium. Whilst focussingon trachea engineering, thedeveloped approach and toolscould be efciently rolled out

for use in other tissues andwhole organs regenerationsettings.

1. Tracheal matrix engineering in the bioreactor and the nal graft beforesurgical implantation

2. Histology of engineered graft biopsiestwo months after implantation in the patient.Three-colour immunofuorescence histology: green, cytokeratins 5 and 8; red,collagen II; blue, DAPI (nuclei). A sheet o viable epithelial cells (green) is shownin (A), and many viable chondrocytes (red) are shown in (B). Haematoxylin andeosin image o deep biopsy (C) showing early revascularization

3. Volume-rendering CT before (A) and 1 month after engraftment (B)of tissue-engineered trachea to replace main bronchus.Terminal long segment narrowing is completely reversed ater surgery (arrows)


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POLYURETHANE fOAMS AND COMPOSITESfOR BONE TISSUE ENGINEERINGAND STEM CELLS DIffERENTIATION

Serena Bertoldi

Numerous efforts are ongoingto develop functional scaffoldsfor regeneration of tissuesand organs, particularly forbone tissue. Natural andbiodegradable syntheticpolymers have been proposedfor the fabrication of scaffolds.Among them, polyurethanes(PU) represent a very importantgroup, thanks to their versatilityallowing to achieve a wide rangeof physical and mechanicalproperties and making them

suitable for use in many areasof tissue engineering, eitherfor reconstruction of softtissue or for cartilage andbone regeneration.Considering the use ofbiodegradable materials,a good balance between invivo scaffold degradation andtissue regeneration is noteasily achievable. Furthermore,there are concerns about toxiceffects imputable to the localconcentration of degradationproducts, that can signicantlylower the local pH, inducing a

late inammatory response.Biointegration can be anappealing alternative tobiodegradation, and it can beachieved by use of polymericscaffolds with a very slowdegradation rate. In thisperspective, the rst part of thisPhD project was aimed at thesynthesis and characterizationof biointegrable cross-linked PUfoams with slow degradation rate.

The PU foams were synthesizedwith a one step bulkpolymerization in controlledvolume by reacting a polyether-polyol mixture with prepolymerMDI, using Fe-acetyl-acetonateas catalyst, and water asexpanding agent. The reactionbetween water and prepolymerMDI produces carbon dioxide,the porogen agent allowing toobtain the porous structure. Onthe whole, two families of foams(EC and EF) were developed

using two different polyether-polyol formulations, EF-basedfoams being more hydrophilicthan EC-based ones.Figure 1 shows the surfacemorphology of the obtainedfoams. Both the foam typeshave homogeneous morphologyand round-shaped pores. Formost of the obtained foams,a porosity higher than 50%and an average pore size inthe range 200-400 m weremeasured, with no differencesfor the two families. Consideringthe mechanical characterization,

all the PU foams show thetypical stress/strain behaviourof elasto-plastic foams, withan initial linear-elastic regimefollowed by a plateau of roughlyconstant stress correspondingto the pores collapse. In thedry state all the foams showhigher compressive propertiesthan in the wet state, andEF-based foams resulted moreexible than EC-based ones.

Higher values of compressiveparameters were related tohigher density values andlower values of open porespercentage.The repeatability of thesynthesis process was assessedsynthesizing three foamsat the same conditions oftemperature and humidityand using the same amountof reaction mixture (50g). Allthe results obtained by the

morphological characterizationby micro computed tomography(micro-CT) indicate the goodreproducibility of the synthesisprocess.Therefore, the syntheticprocedure proposed in thisproject turns out to be easyto apply, reproducible, andeffective in the production of PUfoam adequate for their use asscaffolds for tissue engineering.

1. Representative SEM imageof EF-based foams

Scaffold for bone tissueengineering should haveadequate mechanical propertiesto support the newly formingtissue. The mechanical propertiesand the osteoconductivity ofthe polymeric scaffolds canbe improved by the use ofcalcium phosphate (CaPs), in

particular hydroxyapatite (HA)and tricalcium phosphates(TCP) as ller or coating. Inthis perspective, the aim ofthe second part of the projectwas the production andcharacterization of compositesscaffolds using PU foams asmatrices and HA or -TCP asinorganic llers and/or coating.The composites were preparedwith a co-expansion process,in the presence of -TCP or HA,

adding to the same reagentsused for PU foams the properamount of powder, and/orby coating with CaPs the PUmatrices and composites byimmersion in a HA or -TCPsuspension.The results obtained by themorphological characterizationby SEM and micro-CT indicatesa homogeneous distributionof the CaPs particles within the

foam (Figure 2). Adding CaPsduring synthesis, in particularHA, allows to increase themechanical properties of thefoams, while the coating with-TCP can improve materialsosteoconduction. Therefore,PU-based composite with HAas llers and -TCP coating can

be considered the optimal choicefor bone tissue engineeringapplication.Besides scaffolds, also cells havekey role in the developmentof engineered constructs. Stemcells hold great promise, andhuman placenta represents anextremely innovative source ofstem cells. Aim of the third partof the work was the evaluationof proliferation and possibledifferentiation to osteoblastic

phenotype of amnion(AMCs) and chorion (CMCs)mesenchymal cells, isolatedfrom human placenta, culturedonto PU foams coated or notwith -TCP. By SEM, a goodcells colonization was observed,with cells well adherent withinthe foam pores, indicating theabsence of cells suffering (Figure3a). The Alizarin Red (Figure 3b)staining assesses the early cells

differentiation to theosteoblastic phenotype,thus conrming the basic roleof PU foams and compositesin supporting AMCs and CMCsdifferentiation.In conclusion, consideringall the obtained results, thedescribed PU-based materials

can be considered as appealingscaffolds for tissue engineeringapplication, in particularfor bone regeneration.Future developments willbe the evaluation of thein vivo biocompatibility andfunctionality of the PU-basedscaffolds in the animal bonemodel.

2. Representative 3-D reconstructionof EC-based composite with-TCP.The yellow part represents the

PU structure, while the blue onerepresents the-TCP particles

3. Representative SEM image of CMCs cultured onto coated PU foam (a)and histological image of Alizarin red staining of AMCs culturedonto PU foam (b)


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|2010DESIGN Of A DYNAMIC CULTURE SYSTEM

fOR THE IN VITRO MIMICRY Of THE BONEMARROW MICROENVIRONMENT, INTENDED fORTHE EXPANSION Of HAEMATOPOIETIC STEM CELLSMarco Cantini

Haematopoietic stem cells(HSCs) have been used routinelyin clinical practice for morethan 3 decades, particularlyin the form of bone marrowtransplantation, their mainindications being haematologicalmalignancies and rescue therapyafter high-dose chemotherapyor radiotherapy for non-haematological malignancies.Recent scientic advances haveproved that HSCs might be usedto treat several other pathologic

conditions, including aplasticanemia, thalassemia, sicklecell anemia, and autoimmunediseases.The difculty of harvestingHSCs and their scarcity hinderpresent and expected clinicalapplications, making thedevelopment of systems for theirin vitro expansion an appealingprospect for regenerativemedicine. The most promisingapproach for an efcientexpansion of undifferentiatedHSCs is the mimicry of theirphysiological microenvironment

(the bone marrow): 3D poroussubstrates should be properlystructured for the dynamic co-culture of two different cellularpopulations, HSCs and bonemarrow stromal cells (BMSCs);the latter cells and their 3Dorganization are expected toreproduce the in vivo physical,cellular and molecular signalingproperties of the bone marrow.Considering the cells that have

to be expanded (non-adherentHSCs), culture conditions andscaffold architecture have tobe optimized to allow a propermicroenvironment to develop,where non-physiological stimulileading to HSC differentiationare avoided while guaranteeingnutrient supply to all cells.In the present doctoral project,this issue is addressed throughthe integration of differentmethodologies, includingcomputational modeling ofthe hydrodynamic and mass

transport microenvironment,material processing forthe manufacture of micro-structured porous substratesfor cell culture, and design ofa bioreactor system able tohost the scaffolds and providethem with controlled cultureconditions.First of all, a model of scaffoldcharacterized by few, simple,effective and fabrication-

related geometric parametersis built, and uid-dynamic andnutrient transport phenomenawithin the entire scaffoldare modeled using a singlerepresentative periodic unit,with minor drawbacks withregard to the reliability ofthe computed results. Thesimulations recommend theaddition of properly spaced anddimensioned longitudinal micro-channels to an homogeneousporous scaffold as a meansto enhance nutrient delivery,

maintaining at the same timelow levels of drag forces andshear stresses within the porousbulk ( median ~ 0.08 mPa);moreover, the value of the owrate to apply to the scaffold isinferred (e.g., 0.02 ml/min fora 6-mm-diameter channeledscaffold, Fig. 1).Synthetic scaffolds respondingto the indications of thenumerical studies are produced

1. (a) Velocity eld, (b) wall shear stresses, and (c) oxygen partial pressureof a single periodic unit of channeled 85%-porous scaffold with 300-m-diameter pores and 1.1-mm-spaced, 450-m-diameter channels.

using a polymeric material(polycaprolactone, PCL) anda compression moulding/particulate leaching technique.A fairly reproduciblemanufacture process is setup and optimized, allowinggood control over thescaffold morphology. Using140220-m-diameterpolyethylmethacrylate beadsas leachable particles, porousscaffolds are obtained with wellinterconnected spherical poresand with a porosity that depends

on the initial porogen/polymerratio in a consistent fashion (Fig.2); longitudinal micro-channelsare added by drilling with anumerical control machine.The same optimized techniqueallows to manufacture bioactivePCL scaffolds containinghydroxyapatite nanoparticles,to promote adhesion andproliferation of the feeder cells.With regard to the dynamic

culture system, a connedperfusion strategy is chosen,and an easy to handle, versatile,compact, and functionalbioreactor is developed andoptimized. The idea is themanufacture of a dynamicmultiwell, that is a multi-chamber device able to holdseveral scaffolds at a time,allowing experiments to becarried out in parallel and, inperspective, allowing for a highnumber of expanded HSCs.The technological solution

consists of a modular perfusionchamber with integratedreservoir, hosting the scaffoldthrough an ad hoc designeddeformable scaffold holdercartridge (Fig. 3). The devicebenets from a closing systemsimilar to the one of commercialT-asks and from a bayonetcoupling between housingand reservoir, that make theassembling of the culture

2. SEM image of an 85%-porousPCL scaffold

3. CAD model of a single culturechamber of the bioreactor

chamber under laminar owhood straightforward; thedeformable scaffold holdercartridge ensures water-tightness, makes the handlingof the scaffold easier, and allowsto host scaffolds of differentsize or shape within the samehousing.Preliminary cell cultures arecarried out to prove the efcacyof the manufactured bioreactorat improving initial seeding offeeder cells within 85%-porousPCL scaffolds; an osteoblast-likecell line and primary humanskin brocytes (model of bonemarrow broblasts behavior)are used. These culturesrepresent the rst stepof a foreseen campaign of cellcultures, that include long termcultures for the repopulationof the scaffolds with feedercells and for their co-culturewith HSCs.


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NUMERICAL MODELING Of THE HEALTHYAND PATHOLOGICAL AORTIC ROOTApplication to the analysis o BAV-related alterations

Carlo A. Conti

Bicuspid aortic valve (BAV),the most frequent congenitalcardiac malformation, is presentin about 1-2% of live births.BAV is thought to result fromthe intrauterine fusion or non-separation of 2 underdevelopedcusps, the rst mechanism beingmore likely than the secondone: usually the right and leftcoronary leaet (R/L) or, lessfrequently, the right coronaryand non-coronary leaet (R/N)are fused. Most of the times,the fusion is characterized bya raphe, a region of brousthickening at the seambetween the two fused leaets.BAV is widely recognized as afrequent cause of aortic stenosisand/or aortic regurgitationand it is also a risk factorfor the development of aorticaneurysms: 50-70% of BAVpatients develop ascending aorticaneurysm. The still unidentiedgenetic defect causing BAVmalformation is thought to beresponsible for the occurrenceof these complications; however,

some authors have emphasizedthe role of altered biomechanicsand hemodynamics in the earlyfailure of valve function.The present work aims togaining insight into BAV functionand into its biomechanicalimplications on the surroundingstructures. At this purpose,numerical structural models ofthe aortic root were developedon the basis of human in vivo

morphometric data, acquiredthrough magnetic resonanceimaging (MRI) in collaborationwith the Cardiac SurgeryDivision of the Department ofCardiothoracic and RespiratorySciences, Second Universityof Naples, Italy.

Physiological aortic rootnite element (FE) modelThe geometry of the model isbased on MRI data obtainedfrom 10 healthy subjects. Thefollowing aortic root maingeometric parameters weremeasured: annulus diameter,commissural positions, widthof the Valsalva sinuses, andascending aorta orientation.As far as tissues mechanicalproperties are concerned, leaetstissue was modeledvia a transversely isotropichyperelastic model, while theValsalva sinuses and ascendingaorta tissue were modeledas linear elastic and isotropic.A dynamic FE analysis wasperformed.

The results show that anatomicaldifferences between leaet-sinus units cause differencesin stress and strain patterns.These are notably higher forthe leaets and smaller forthe sinuses. For the maximumtransvalvular pressure value,maximum principal stresses onthe leaets are equal to 759,613 and 603 kPa on the noncoronary, right and left leaet,

respectively. Although liable offurther improvements, the modelseems to reliably reproduce thebehaviour of the real aortic root:the models leaet stretches,leaet coaptation lengths andcommissure motions, as wellas the timings of the aorticleaet closures and openings,all matched with theexperimental ndings reported inthe literature.

FE modelling of the BAVThe geometry of the modelwas based on measurementsfrom MR images acquiredin 8 normotensive andotherwise healthy subjects withechocardiographically normalfunction of their BAV. The sametissues mechanical propertiesimplemented for the TAV modelwas adopted for the BAV one.Numerical results were comparedwith those obtained from ourprevious model representingthe normal root with a TAV.During systole, the bicuspid valveopened asymmetrically compared

to normal, resulting in anelliptical shape of its orice.During diastole, the conjointcusp occluded a larger proportionof the valve orice and leaetsbending was altered, althoughcompetence was preserved. TheBAV model presented higherstresses with respect to the TAVone, particularlyin the central basal regionof the conjoint cusp: the

presence of a raphe partiallyreduced stresses in this region,but increased stresses in theother cusp. As regards theproximal part of the ascendingaorta, circumferential andlongitudinal stresses are increasedrespectively by 8% and 20%at the intrados and by 17%

and 36% at the extrados, whencompared to the TAV model.The results show that the aorticvalve function is altered also inclinically normally functioningBAVs. Bicuspid geometry perse entails abnormal leaetstresses: their location suggeststhat they may play a role intissue remodeling at the rapheregion. Morphological anomaliesassociated to BAV lead toincreased stresses not onlyin the leaets, but also in allof the surrounding structures.In particular, longitudinal stresseswere increased, as compared tothe TAV model, in the extradosof the ascending aorta: this

stress location is consistentwith the region where dilatationassociated to BAV is commonlymore pronounced. This region isalso the most frequent site whereaortic dissection occurs. Thus, themere BAV anatomy, regardless ofthe possible coexistence of someinherited aortic wall weakness,affectsthe ascending aorta with alteredstress amount and distribution,

which may play a role in theonset and development of thequite unique form of aortopathyfrequently developing with BAV.

Effects of anisotropy andnon-linearity on aortic rootand ascending aorta wallmodelling

Different thickness and collagenber orientations were assigned

to each arterial layer (intima,media, adventitia) of theaortic wall. Layer-specicmechanical properties weredened using an anisotropicnon-linear hyperelastic modelable to represent collagen bresdispersion. The coefcientsof the strain energy function

were obtained fromexperimental data reportedin literature. The mechanicalresponse of the leaet tissue wasmodelled with the same strategypreviously adopted.The comparison betweencircumferential and longitudinalstresses in the three layersunderlined the anisotropicbehaviour of the aortic wall,due to the different orientationof dispersed collagen bers.An overstressed intimal layer,compared to media andadventitia, was highlighted bothin TAV and BAV. In particular,the intimal layer showed levelsof circumferential stressesalmost two orders of magnitudehigher than the middle and theadventitial layer and one orderof magnitude higher for thelongitudinal stresses.

Comparative nite volumeanalysis of ascending aortaow in BAV and TAV

To analyze the uiddynamics implications ofBAV, computational uid

dynamics (CFD) simulationswere performed. The systemconsidered for CFD analysesconsisted of the aortic root,the ascending aorta, the aorticarch, the supra-aortic vesselsand the proximal tract of thedescending aorta. In the TAVmodel, a symmetrical velocityeld was computed for thetwo more proximal consideredsections, coherently with the

morphological symmetry of theaortic valve. When the section 2cm downstream of the STJ wasconsidered, a slight asymmetrywas observed: values of velocitymagnitude were higher, with apeak value of 1.02 m/s, towardsthe concavity of the aortic arch.On the contrary, the velocity

eld computed for the BAVmodel, was symmetrical at theValsalva sinus level, but washighly asymmetrical at boththe STJ level and mid-ascendingaorta level. In particular, atthe mid ascending aorta theow was skewed towardsthe convexity of the aortaand a peak value of velocitymagnitude equal to 1.39 m/swas observed.The study offers preliminaryevidence that even normallyfunctioning BAV may generatealtered ow patterns. Thecomparison between TAVand BAV models showsan asymmetrical and highervelocity in the bicuspid one.The asymmetry is morepronounced at the aortic level,known to be more exposedto the early formation ofaneurisms in bicuspid patients.This supports the hypothesisthat haemodynamic factorsmay contribute to ascendingaorta pathology in this subsetof patients.


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METHODOLOGICAL DEVELOPMENTSOf THE APPLICATION Of INDEPENDENTCOMPONENT ANALYSIS IN EEG-fMRI

Tiziana Franchin

In the last decade,simultaneous acquisitions ofelectroencephalographic (EEG)recordings during functionalmagnetic resonance imaging(fMRI) sessions allowed thepossibility to greatly enrich thestudy of the brain functionalspecialization and integration.The advantages of this multi-modal approach are mainlyrelated to the opportunity ofmaking use of both temporaland spatial resolutions.Established standards ofprocessing and analysis of thissort of data are not denedyet because of several problemsstill not solved. Independentcomponent analysis (ICA) isan exploratory data analysisapproach successfully appliedin biomedical data processing,such as the analysis of EEGsignals and functional magneticresonance images. It consistsof searching for a lineartransformation that minimizesthe statistical dependencebetween its components. ICA

consents to analyze the datawithout a-priori model and theonly condition to be warrantedis the statistical independenceassumption, which is plausiblyaccepted in the eld ofbiomedical signal processing.The aim of this workis to contribute to themethodological advancementof using the independentcomponent analysis in the

biomedical signal processing.An optimized ICA algorithmwas implemented to suggest analternative and more performingsolution to the difcultiesrelated to the computationalcomplexity and to the accuracyof the blind extraction ofindependent sources. Thepresented algorithm was usedfor: removing ballistocardiogramartefacts from EEG recordingsin MR eld, by means ofthe extraction of the signalcomponents more correlatedwith an artefact template;analyzing fMRI data by means ofa spatial ICA method to separatemore signicant components forthe identication of functionalareas.In blind extraction ofindependent sources, differentalgorithms were introducedto solve the problem of a correctrecovering of a set ofunobservable independentsignals from observedmixtures of sources. A possibleclassication can be feasible

considering the problem ofthe convergence speed andthe condition for the separabilityof linearly mixed source signals.The convergence in the solutionrecovering is easily attainablewith block implementations,which are not necessarily morecostly than adaptive (recursive,on-line, sample-by-sample andneural) algorithms: moreover,they are able to use more

effectively the informationcontained in the observed signalblock. The separability of linearlymixed source signals is relatedto the quantitative measureof non-Gaussianity of the datawhich is possible introducing anappropriate contrast function.Contrast function is a functionalin the distribution of theseparator output quantifyinga measurable property ofthe signals and related to thedegree of separation. Kurtosisis a well-established measureof non-Gaussianity. Two typesof this cost functions existin this setting. The rst onerests on a direct minimisation/maximisation of the kurtosis,which is meaningful only ifthe variance of the extractedsource signal is bounded. Theother type of cost functionis based on the normalisedkurtosis, the advantage ofwhich is that we do not needto perform the otherwiserequired pre-whitening andweight normalisation operations.

The possibility of sparing thispre-processing step makes thecomponents identication fasterand more reliable.The proposed algorithm is anoptimization of a deection-mode block-implemented xedstep size algorithm in whichthe cost function is a normalizedkurtosis which considers theeffect of noise directly within it.A set of simulation experiments,

differing from the samplesize of the synthetic data andthe intensity of additive noise,was conducted in order toinvestigate the performanceof the proposed non-parametricmethod. The convergence speedin terms of the computationalcost and the quality of

component extraction usingthis optimized algorithm werecompared with the resultsobtained by the standard versionof RobustICA algorithm andby FastICA algorithm, one ofthe most popular and used ICAalgorithm in the bioengineeringeld. Results showed that theproposed method is truly blindto the particular distribution ofthe original sources and doesnot require the selection ofoptimal working parameters,or suitable non-linearitiesto act as contrast functions.The optimized ICA algorithmhas been successfully usedfor a better denition ofballistocardiogram (BCG)artefactual components ofEEG recordings during fMRIsessions. Auditory and visualERPs recorded during fMRIsessions were acquired in 7healthy subjects and used forassessing the method. Thesource components related toBCG artefacts were separatedusing three ICA algorithm(FastICA, RobustICA and the

optimized algorithm). Theywere selected computing thelinear correlation between thesources and a template createdfrom the ECG recorded signaland an averaged BCG signal.Such selected componentswere subtracted by rawsignals, taking into account therespective estimated weights.For validating the proposedalgorithm, a comparing of

the quality of BCG artefactremoval obtained by fourremoval method was carriedon by means of: ERP scalp mapreconstruction analysis; BCGartefact attenuation percentage;comparison of EEG powerspectral density. Despite of thelimited available number of

subjects, which is a weak pointin the validation of the proposedmethod, the temporal andspectral trend analysis conrmedthe quality of the cleanedEEG recordings, showing a goodreproducibility and comparisonof P100 and P200 peaks in andout the MR eld.An open challenge in applicationof spatial IndependentComponent Analysis for fMRIanalysis is the correct separationof components which reectinteresting spatiotemporalpatterns of stimulus-inducedor spontaneous brain activityfrom other ones which reectsignal artefacts or noise.An alternative proposal ofclassication of signicantindependent components basedon the analysis of the spatialfeatures of the signals obtainedby fMRI images is proposed.The aim is to implementan easier method for blindidentication of cerebralfunctional areas investigatedin single-subject andmultiple subject studies. It is

characterized by using theabove-written optimizedalgorithm, which consenta good separation of thesignicant components relatedto signal information respectto noisy contributions, whosestatistical properties are simplyidentiable. For this reason,the proposed methodwas tested on an fMRI groupdataset obtained during

a cognitive processes study andon two single cases of differenttype of epilepsy. Separatingcomponents were classiedconsidering the correlation ofthe independent sources withthe spatial template of greyand white matter of thesubjects. This rst step of

classication was followedby the analysis for thekurtosis value of each singlecomponent. Components witha high amplitude during a littlefrequent period and close tozero amplitude for the rest ofthe time usually own a super-Gaussian random distributions:otherwise, small continuoustemporal trends have a quasi-Gaussian distributions. Thesestatistical features are easilydistinguished using the kurtosisvalue. The last step was to maskthe signicant areas obtainedby the over-described sortingcomponents. An automatedanatomical labelling offunctional activation areaswas performed starting bya pre-existing anatomicalparcellation atlas: for eachspatial IC map identied bythe above-written steps,activated areas correspondedto the parcellated onesindividuated. Activated areasindividuated by spatial IC mapsshow similar results obtainablewith well-established processing

method, proving the goodnessof the method.


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HOME MONITORING Of RESPIRATORY MECHANICSIN PATIENTS WITH CHRONIC OBSTRUCTIVELUNG DISEASES

Alessandro Gobbi

Asthma and chronic obstructivepulmonary disease (COPD)are the most diffused chronicrespiratory disorders affectingmillions of children and adultsworldwide. Common featuresof these diseases are thepresence of a state of chronicinammation of the respiratorytract associated to airwayobstruction and the periodiconset of acute exacerbations,one of the main causes ofpatient suffering.In clinical settings, the diagnosisand staging of asthma andCOPD still rely on a onetime punctual assessment oflung function by spirometry.However, recent ndings havesuggested that, given theabnormal temporal uctuationsof many respiratory parametersassociated with the disease,a proper evaluation of thepatients condition at any pointin time would require frequent,at least daily, repetitions of thetests. Up to now, this has beenpossible mainly with qualitative

solutions (diaries, nurseryassistance, etc.) while the onlyquantitative attempt used peakexpiratory ow (PEF) metersthat, however, provide data withlow accuracy and reproducibility.Indeed, even under controlledsituations, elderly patientsand young children may havedifculty completing spirometryand the supervision of trainedtechnicians is often needed to

obtain results with thedesired quality standards.In this work we investigateda different approach for thediagnosis of chronic respiratorydiseases, for the association ofclinical symptoms to quantitativeparameters and for the earlydetection of acute events. Thisapproach was based on themeasurement of the respiratorymechanics (Zrs) by forcedoscillation technique (FOT) andon the evaluation of temporalvariability of the associatedrespiratory parameters.Accordingly, we designed a newcompact FOT device suitablefor the home monitoring of Zrs.This device is able to manageautomatically several types ofmeasurement protocols and toguide the patient while makingan unsupervised assessmentof Zrs. An automatic algorithmis able to compute respiratoryparameters in real-time and toidentify and discard breathswith artifacts. The results ofeach test are then encrypted

and transmitted over theInternet to a central dataserver using whichever type ofconnection available at patientshouse (g. 1). We also designeda database, in which the lessent from each FOT deviceare memorized and organized.These data can be retrieved fromauthorized physicians usinga web browser from anycomputer connected to the

Internet.The accuracy, stability andrepeatability of unsupervisedself-measurements of Zrswere conrmed using in-vitrovalidation studies.The device was also testedfor a long-term period bymonitoring Zrs of one healthysubject and one COPD patientat home for 36 consecutivedays. The results showed verylimited variations in the healthysubject and a greater variabilityin the COPD, suggesting that

while the respiratory indicesare highly stable in the normal,the uctuations observed in theCOPD might reect a periodicchange in airway obstruction.These positive ndings and thegood results obtained fromreliability tests made on thenetwork architecture and onthe automatic algorithm for thecomputation of Zrs allowed todesign a more extensive pilot

1. Home monitoringnetwork architecture

clinical study, involvinga greater number of subjectsand recording their Zrs at homefor a longer period.Therefore, two age-matchedpopulations of 10 mildasthmatics and 10 controls wereincluded progressively in thisstudy and they were requiredto make the test daily in themorning and in the eveningfor at least 6 months. Theirmeasurements were used tobuild the time series of Zrs.A subgroup of 3 asthmaticsand one control were givena portable PEF-meter and theyrecorded their morning andevening PEF for at least onemonth. The time series of thePEF were compared to thetime series recorded by FOT.The results were unclear, sincethe correlation between PEFand the mechanical respiratoryparameters was signicantin some cases but weak orabsent in others. These resultsstrengthened our belief thatZrs data might provide a valid

alternative to PEF to look atchronic respiratory diseasesfrom several new angles, fora better understanding of themechanisms of their variability.We compared the globalstatistical properties (mean,standard deviation and inter-quartile range) of the FOT timeseries with and without missingdata, nding no signicantdifferences between them.

Neither the comparison betweenthe controls and the asthmaticsshowed signicant differencesin these global indices,suggesting to analyze at adeeper level the notion ofvariability, considering differenttime scales of observation.The variability of Zrs at thesmaller time scales, up toseveral minutes, did not differsignicantly between theasthmatic and control group,while signicant differencesbetween them were observedstarting from the time scale ofone day, indicating that twodaily tests is the minimumrequired to separate thepopulations.Consequently, we calculatedthe probability of beingasthmatic given a relative dailychange in Zrs. At time scalesgreater than one day we wereable to correlate the gravityof asthma to the observedvariability: the greater theabsolute value of variabilityand its rate of increase with

time the more severe theclinical symptoms of thepathology. These results providea very useful diagnostic tool insituations where the standardspirometric tests are not reliable.Additionally, in asthmaticsubjects already under therapy,the home monitoring basedon FOT can be convenientlyused for a better managementof their pathology and for

the evaluation of the effectsof the medications over time.From the same time serieswe also derived the predictorof the likelihood of futureexacerbations, given thevariability of Zrs measuredat a given time scale.Additionally, in a parallel clinicalstudy we found that obesityis associated to an excessiveairway responsiveness andvariability in otherwise healthyhumans. We studied 41healthy subjects with bodymass index (BMI) ranging from20 to 56. The main resultsof this study indicated thatairway responsiveness wassignicantly correlated withBMI in nonasthmatic subjectsand that this increase wassignicantly related to lungvolume restriction and to anincrease of breathing frequency.These ndings indicate thatobesity is an important clinicalfactor to take into considerationwhen analyzing the long-term variability of respiratory

parameters in subjects withoutchronic respiratory disorders.In conclusion, this work showedthat a home daily monitoring ofrespiratory mechanics is feasibleand it may contribute to improvethe clinical management ofpatients with chronic obstructivelung diseases.


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lesion damages in individual

tractographic reconstruction was

developed and validated on corpus

callosum (CC) tracts in a set of 35

multiple sclerosis (MS) patients.

Four Atlas types were constructed

from 18 healthy volunteers (HV):

A1) linear registration (LR) of

diffusion weighted images (DWIs)

prior to DTI computation; A2)

non-linear registration (NLR) of

DWIs; A3) LR of subjects DTIs;

A4) NLR of subjects DTIs. The

simplest atlas, A1, was sufcient

for the CC, while A3 was

necessary for the reconstruction

of peripheral bundles (inferior-

fronto-occipital-fasciculus: IFOF;

inferior-longitudinal-fasciculus:

ILF; Arcuate fasciculus: AcF)

(Figure 1). The computationally

expensive NLR of HV did not

introduce any improvement in

atlas reconstruction. Conversely,

NLR for the normalization of

individual patients to atlas, proved

to be necessary both for CC and

the other bundles.

A clinical study for individual-

and atlas-based techniques

performances comparison wasperformed with the examination

of 35 MS patients (14 Relapsing-

Remitting, RR, and 21 Secondary-

progressive, SP) and 18 HV.

The DT-derived metrics (i.e.

fractional anisotropy, FA, and

mean diffusivity, MD) of the CC

WM bers were computed using

the optimized individual-based

method for ber tracking and two

atlas-based methods with different

kinds of subjects registration

(LT or NLR) to the atlas A1.

All the methods were able to

distinguish between MS patients

and HV, any statistically signicant

difference was found between

measures derived with individual

method and those with atlas A1

and NLR, but using the individual-

based tracking method with

customized termination criteria,

stronger relationships (the highest

Spearmans correlation

coefcients, Table 1) were found

between CC DT-derived metrics

and the subjects clinical condition.

Indeed, the Friedman test

demonstrated that the magnitude

of the observed correlations was

greater when using individual-

based than when using atlas-

based LR (p=0.003 and 0.002) and

atlas-based NLR (p=0.04 for both

comparisons) methods. However,

the individual-based approach is

not without limitations. First of all,

MS patients CC reconstruction

was feasible in vast majority (94%)

of examined patients (Figure 2

A), but tractography terminated

near lesions in 2 out of 35patients (Figure 2 B). Beside that,

while the atlas-based methods

are completely automated, the

individual-based one can be

time-consuming for the operator,

due to the manual positioning of

ROIs, which is about 15 minutes

for an experienced operator.

Since the individual-based

method is operator-dependent,

the intra-rater and inter-rater

QUANTIfICATION Of MICROSCOPIC TISSUEDAMAGES IN NEUROLOGICAL DISORDERSDiusion Tensor Imaging and fiber Tracking

Maria Marcella Lagan

Clinical application of diffusion

tensor imaging (DTI) and

tractography poses challenging

problems as to the diversity of

brain morphologies to be analyzed

in the presence of lesions,

diffused damages, or atrophy

and also to the deviations of DTI

parameters driving tractographic

reconstruction. The development

and validation of specic protocols

were based on a thorough

methodological analysis of the

single steps and on extended

clinical trials on brain and spine

structures. Specic aspects dealt

with were: the unfavourable SNR

of DTI; the limited acquisition

times permitted in clinical studies;

optimization of registration

and averaging procedures;

the interference of lesions with

tractographic reconstruction; the

dependence of reconstructed bre

bundles (tracts) from seeding ROIs;

the segmentation of tract volume

for the evaluation of micro-

structural diffusion by means of

individual or atlas based methods.

Brain DTI sequence selection,

based on SNR and tr actographicreconstruction optimization

was performed among 26

combinations of scanning

parameters evaluated by means

of 7 methods for SNR estimate

on phantom and human data.

Segmentation of bundles was

performed by individual and

atlas based methods. A procedure

for stopping criteria threshold

adaptation to microscopic

2. A) Successful individual-based tractography reconstruction of the CC froma 43-year old female SPMS patient. The patient has a brain T2 lesion load(LL) of 34.99 ml. T2-visible MS lesions (green) are superimposed to thereconstructed CC. B) Incomplete, individual-based tractography reconstructionof the CC from a 38- old female SPMS patient. The patient has a brain T2 LLof 13.52 ml. T2-visible MS lesions (in green) are superimposed to the partiallyreconstructed CC and white arrows indicate the premature ending of anteriorbody bers.

1. Arcuate fasciculus (red), inferior longitudinal fasciculus (yellow),inferior fronto-occipital fasciculus (green) reconstruction on Atlas 3.

Tab 1. Spearmans rank correlation coefcients between DT-derived measurescomputed in CC and the subjects condition (HV v s. RRMS vs. SPMS)

measurement dependency was

tested with a reproducibility study

in a subgroup of 12 MS patients,

where the main rater repeated

the measurements twice, while

a second rater performed the

same measurements without

being aware of the other raters

results. This analysis demonstrated

a very high intra- and inter-ratermeasurement reproducibility

for the average CC DT-derived

measures.

As about the performance of

peripheral bundle tractography,

it was preliminarily tested on

3 stroke patients with large

hemispheric lesions and 3

patients with dementia. In stroke

the individual analysis was not

appropriate and NLR on A3

best performed. In dementia

the individual approach was

used to evaluate white matter

degeneration accompanying

gray matter damage.

Preliminary studies were

performed for the optimization

of DTI in the spine and validated

on 11 HV. Nineteen DTI (8 sagittal

and 11 axial) sequences were

compared for SNR and resolution.

A method for DT-derived measures

analysis in reconstructed tracts

inside, more caudal or more

cranial to the lesion level was

performed and validated on 3

MS patients with at least one

cervical cord lesion: tractography

demonstrated to produce indices

correlated to pathology orphysiology more than the simple

and commonly used ROI analysis.

In conclusion, this study

demonstrates the feasibility of an

extension of DTI and tractographic

procedures to clinical neurology,

through the optimization of DWI

sequence and image processing

methods, which were applied and

validated in specic clinical studies.

AVERAGE CC fA

Individual-basedtractography

Atlas-based LTtractography

Atlas-based NLTtractography

Subjectscondition(HV vs RRMSvs SPMS)

-0.83** -0.65** -0.72**

AVERAGE CC MD

Individual-basedtractography

Atlas-based LTtractography

Atlas-based NLTtractography

Subjectscondition(HV vs RRMSvs SPMS)

0.81** 0.60** 0.69**

** p


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DEVELOPMENT AND APPLICATION OfCOMPUTATIONAL METHODS fOR THE ANALYSISOf TWO-DIMENSIONAL GEL ELECROPHORESIS MAPS

Saveria Mazzara

The last decade in life scienceswas deeply inuenced by thedevelopment of the Omicstechnologies (genomics,transcriptomics, proteomicsand metabolomics), which aimfor a global view on biologicalsystems. Correlating proteomicwith genomic information is notan easy task: not only expressionlevels of proteins vary widelywithin a cell, but also theircorrelation with mRNA levelis rather poor. In addition,especially in higher organisms,

post-translational modicationsand differential splicing cansignicantly hide the informationgained at the DNA level. Itis thus that proteomics isbecoming an emerging area ofresearch in the postgenomic erathat deals with the complexityof protein expression via acombination of techniquesfor resolving, identifying,quantitating, and characterizingproteins. The drive to analyzeproteins at a genomic scalehas led to the development

of a number of parallelapproaches. Among them,two-dimensional polyacrylamidegel electrophoresis (2D-PAGE)still plays a central rolefor analyzing the proteincomposition of a given cell typeand for monitoring changesin gene activity through thequantitative and qualitativeanalysis of the thousands ofproteins that orchestrate various

cellular functions. 2DE providesa proteome mapping of thesample via orthogonal mass/charge analysis. The sample(e.g. tissue, serum) is solubilised,and the proteins are denaturedinto their polypeptide subunits.This mixture is then separatedby isoelectric focusing; on theapplication of a current, thecharged polypeptide subunitsmigrate in a polyacrylamidegel strip that contains animmobilized pH gradient untilthey reach the pH at which

their overall charge is neutral(isoelectric point orpI), henceproducing a gel strip containingdiscrete protein bands alongits length. This gel strip is thenapplied to the edge of arectangular slab polyacrylamidegel containing sodium dodecylsulphate, and the focusedpolypeptides migrate in anelectric current into the secondgel and are separated on thebasis of molecular size. Theproteins are represented byspots stained with special

dyes. However, convertingthe large amount of noisydata obtained with 2-DE intoreliable and interpretablebiological information is still atime consuming process, thus,there is an increasing need offully automated approaches.Objective of the project hasbeen to develop complementaryapproaches aiming at anautomatic extraction of the

essential information emergingfrom separation imagesintended as ngerprints ofthe correspondent biologicalconditions able to identifypotential discriminant patternsbetween different clinical/biological conditions. What wewould like to propose is a newdiagnosis criteria that overcomethe diagnostic methods basedon single or few marker/s,using a statisticalmultiparametric analysis.To this aim, image descriptors

are derived, on the basis ofthe extracted quantitativeparameters to be used in thesuccessive exploratory analysis.In this way, each gel image,represented as a vector ofquantitative features, canbe investigated by means ofappropriate analytical tools.The development andapplication of computationalmethods to the data generatedby two-dimensionalelectrophoresis representsan exciting way to explore

the pathophysiologicalprocesses at the molecularlevel; multivariate methods likeprincipal component analysis(PCA), partial least squares(PLS) and local tangent spacealignment (LTSA) have beenused to analyze several two-dimensional gel electrophoresisdatasets. In the presentproject it is demonstrated howinformation can be extracted

by multivariate data analysisto efciently analyze theamount of data generatedby 2DE; these techniques areapplied on the quanticationparameters, obtained throughthe commercial software,at the aim of exploring thereliability of the features

extracted from the experimentalsubregions of the separationimages and providing the basesfor a protocol of automaticclassication. The processingof the descriptors correspondingto different regions of theseparation area can providethe identication of portionof image, and thus subset ofspots, that are most responsiblein the observing the existenceof clusters of samples. In thisway it is possible to go back tothe ranges of pI and MW thatare most informative and that

most contribute to the analyticrepresentation that shows gelscorresponding to differentclinical conditions as positionsconned in different zones ofthe new space of observation.In this sense, the developed toolmay represent a further supportto the identicationof potential pathologicalmarkers through the selectionof critical patterns of image gel.The outlined strategy was testedon three datasets: samplesfrom prostatic cancer tissues,

samples of human cerebrospinaluid (CSF) acquired withina framework of a study ofperipheral neuropathy andsamples of human CSF acquiredin a study on amyotrophiclateral sclerosis. Cancerresearch is a highly suitable areafor the application of such anapproach. This is because inmany cases it is possible to makea direct comparison of proteins

expressed in controland diseased tissues.The comparison of 2DEseparation of such tissuescan highlight proteins thatare present in greater or lesserquantities and new proteinswhich may only be expressedin cancerous samples. Such

approach appears particularlytoward for the characterizationof the brain proteome undernormal and disease condition;biomarkers of brain disordersare urgently needed to aiddiagnosis, monitor diseaseprogression, and, as newmedicines are introduced,detect the patients response totreatment. In conclusion, it isworth noting that the samplesof the data set are not technicalreplicates, i.e. gels obtained fromfractions of the same biologicalsample. In this work, biological

replicates were processed, i.e.each gel image is representativeof a different human subject,so the gels are characterized bylow homogeneity; this bringsthe tackled problem to a muchhigher level of variability andcomplexity. Moreover, there exista difculty of the reperibility ofsamples, in particular for controlsubjects. Notwithstanding thesecritical aspects, the strategydeveloped, based on techniquesof multivariate analysisapplied on the quantication

parameters obtained throughthe commercial software,gave promising and reliableresults. The use of 2D gelelectrophoresis coupled tomultivariate data analysis seemsto be promising as a fast, reliableway of transforming many gelsinto spot quantities. The methoddeveloped can be a usefulcomplement in the routineof a proteomics laboratory,

because it is highly repeatableand does not need any apriori information. It mayprovide an effective visualizationtool and lead to the denitionof a protocol of automaticclassication, that is able toshow discriminating patternswithin a pool of gels from a

clinical/biological study in whichquality and reproducibilityare challenged by the presenceof samples not obtained adhoc, capturing the ngerprintleaved by the sample on the twodimensional separation image.Moreover, this kind of analysismay provide an useful supportin the identication of themost informative areas (spotsor groups of spots) that havethe major discriminant powerbetween different clinicalconditions towardsthe denition of new potential

biomarkers. Finally, nobodyexpert eye is able from a visualinspection of 2D electrophoreticgels to discriminate physiologicalfrom pathological conditions;thus, the information extractionin the processing of 2DEimages is an important topic incomputational biology and theproposed strategy may providean interesting and fruitful pointof view capturing the essentialinformation from gel images.


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DEVELOPMENT Of AN EXPERT SYSTEM BASED ONARTIfICIAL NEURAL NETWOR fOR BIOMEDICALEQUIPMENTS RIS ASSESSMENT

Matteo Ritrovato

In todays health care servicesbiomedical technologies playan exceptionally relevant role:ubiquitous and essential tomodern clinical procedures,are one of the main keys forhealth practices improvement,not only because of the betterdiagnostic and therapeuticcapabilities, but also due tothe increased health accessibility,and therefore for theimprovement of the outcome,as well means by which wehave developed research and

knowledge in medical sciences.However, there is a downside:the state of functioning andconservation of these devicesaffects the outcome and safetyof procedures; the pace oftechnological innovation hasincreased the complexity of useand management, as well asthe magnitude of associatedcosts. Moreover, the complexityand the intrinsic riskiness of eachunit are capable to interact inthose contexts that require, forthe specic clinical procedure,

the convergence and integrationof different devices, thusincreasing the criticalities relatedto patients and operators safety.The assessment of riskassociated with medical devicesin general and in particular withbiomedical equipments, hasthus become an increasinglyrecognized activity by end-usersand managers, both for theimplications on patient safety

and for those associated withthe management of medicaldevices.The risk assessment methodhere developed is based ontraditional methods of riskcomputation: the value ofrisk associated with a medicalequipment or biomedicaltechnology is the productof two factors: the factorProbability (estimatedoccurrence frequency ofpotentially damaging event)and the factor Damage (type

and extent of damage associatedwith each event). Thus, thecalculated risk value is mappedon a risk matrix and associatedto an operational meaningcorresponding to an Acceptable,Tolerable and I ntolerable risk.To separately computeProbability and Damage factors,we referred to a linear model,previously developed bya working group of the ItalianAssociation of Clinical Engineersand here updated with thecontribution of the Clinical

Engineering Service at PediatricHospital Bambino Ges andfellow experts, that identiesand quanties the variablesthat separately contributeto those factors.The main objective of this workwas to overcome the obviouslimitations of the initial model,limitations due to its linearity,thus solving the recognizedunderestimation of the value of

risk associated withmany specic combinationsof input variables. This goalwas approached byimplementing, as computationalmethod, two neural networks,one for the calculationof Probability, one for thecalculation of the Damage.Therefore, in accordance withthe classical method of riskestimation two separate neuralnetworks were implemented,each consisting of one inputlayer, one hidden layer and

one output layer. As transferfunction of neurons constitutingthe input and hidden layerswe used a sigmoidal function(whose output is limited tothe range -1+1), whereas forthe output layer, consisting ofa single neuron, the functionis linear, since the output ofeach network has to vary in therange 0 4. The neural networkdedicated to the estimation ofthe probability of occurrenceshas no. 16 inputs, while thenetwork that computes the

value of Damage processesno 6 input variables.The data used for the trainingphase of the two neuralnetworks are the result ofsimulations performed on thebasis of linear mathematicalmodel from which we started.The data thus obtained wereadded to those attributed toparticular congurations of theinput variables that, although

the linear model is not ableto indicate as highly risky,has been associated with a highdegree of risk on the basis ofthe opinion of experts. In thisway we introduced the non-linearity useful to overcomethe limitations of classicationof the linear model.

The training of the neuralnetwork is based on acombination of two algorithms:the rst, chronologically, isa genetic algorithm whoseobjective is to identify an initialconguration of weights ofthe network sufciently closeto an absolute minimum oferror function dened for thenetwork. The second algorithm,given the number of layersand the differentiability ofthe error function, is a classicbackpropagation algorithm.Using a genetic algorithm

as a rst step in the processof network training, it hasbecome necessary due to thegreat dependence on initial(random) parameters given asinput to the training algorithmbased on backpropagation andconsequent instability shownby the trained network.At the end of the geneticalgorithm, i.e. when the averagevalue of the tness functiongiven by the entire populationof strings converge to theminimum value of the same

function given by an element ofthe population itself (called bestindividual), it is identied the setof parameters to be passed asinitial conguration of theneural network to the nextphase of training, phasein which backpropagationalgorithm is used.The results indicate that theuse of a genetic algorithm hassuccessfully guaranteed, in all

the performed tests, a correctand stable training of thenetwork.Backpropagation algorithmimplemented uses theLevenberg-Marquardt algorithmfor updating the synapsesweights, and as error functionthe mean square error. For

better generalization, besideslimiting the number of neuronsforming the network (to avoidthe overtting problem), anearly stopping mechanism wasused (to avoid the problemof memorization). Thesimulation of synthetic data,the neural networks, and thetraining algorithms have beenimplemented in MatLab 7.0.The expert system was testedon a set of about 180equipment used at the PediatricHospital Bambino Ges.The average value of risk

identied was equal to 3.4,with a variance equal to 2.6.Specically, we obtained anaverage value for the damageof 3.2 and a variance of 1.2,while the probability averagevalue was equal to 1.06 andvariance 0.12, with only 1devices with risk value >8(limit of risk intolerable)and 4 devices with risk value>4 (limit of risk tolerable).These 5 devices were subjectedto the necessary repairs andimprovements so that the

recalculated risk returnedback within the range 14(acceptable risk).Moreover,such results were also takeninto account in the preparationof the technology replacementand renovation plans.From the monitoring workconducted for 24 monthsfollowing the initial acquisitionof data, with the aim ofassessing the ability of

classication of the expertsystem, there was a minimum(depending on how the riskclassication tolerable havebeen considered) specicityequal to 0.97 and a NegativePredicative Value of 1, whilenothing can be said aboutsensitivity, because no adverse

events associated with theexamined equipments havebeen occurred.Marginally with respect to suchaspects and since during thephase of data acquisition onthe set of equipment underinvestigation there was a clear

judgment variability, amongoperators who have tested themethod, while associating valuesto the variables required bythe model, it was investigatedthe possibility of exploit thepotential of fuzzy logic andsystems in order to reduce

the inherent subjectivityof the expert system.It was therefore implementeda fuzzy system that acts asa layer of data preprocessing.In particular, the data beingprocessed by the fuzzy layerare those relating to subjectiveassessments of factors notinstrumentally measured,required by the neural networkthat calculates the probability ofoccurrence, for a total of no. 7input variables. Therefore it hasbeen dened no. 3 membership

functions for each data input, aset of fuzzy rules and operatorsfor the composition of fuzzyset and, nally, the operatorof output defuzzication.


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REGIONAL ANALYSIS Of LUNG DENSITY fORPLANNING AND EVALUATION Of ENDOBRONCHIALINTERVENTIONS IN SEVERE EMPHYSEMA

Caterina Salito

Emphysema is a worldwideleading cause of morbidityand mortality. As denedby the American ThoracicSociety, emphysema isa condition of the lungcharacterized by permanent,abnormal enlargement ofairspaces distal to the terminalbronchiole, accompanied bythe destruction of their walls.Emphysema is characterizedon computed tomography (CT)by the presence of areas ofabnormally low attenuation.

CT, in particular, volumetricacquired high-resolution CT(0.6 to 2 mm collimation)is excellent in detecting thepresence and in demonstratingthe pattern, distribution, andextent of emphysema. HRCTscans of emphysema patientscan be and have been used toidentify regions of trapped gas.Denitive therapy for severeemphysema is limited to invasiveand expensive surgical measures:lung transplantation and lungvolume reduction surgery.

Recently a new minimallyinvasive surgical intervention,airway bypass, has beenintroduced. This interventionis constituted by the placementof bronchopulmonary conduits,stents, between conductingairways and emphysematouslung parenchyma for expiration.The success of the techniquestrongly depends on thepossibility to identify regions of

trapped gas for interventionaltarget areas. In this Thesis aset of new methodologies andtechniques specically developedin order to identify and quantifyregional trapped gas on a lungregional basis is described. Thesame methods can be usefullyused also for surgical planning,namely the choice of thetarget sites for airway bypassintervention. Here below, a listof more detailed specic aimsof this Thesis is reported.

Specifc Aim 1- To identifyand quantify regional trappedgas in human lung with severeemphysema via HRCT atdisparate lung volumes.Air trapping reects theretention of excess gas in all orpart of the lung and is detectedas decreased attenuation onexpiratory CT as compared withthe corresponding inspiratoryimages. The methods proposedis addressed to measure thechanges in specic gas volume,i.e. the volume of gas per

gram of tissue (SVg, ml/g), aslung volume decreases from ahigh volume close to total lungcapacity (TLC) to a lower volumeclose to residual volume (RV) byusing HRCT. Lobar and overallSV

gwere measured by HRCT

scanning at total lung capacity(TLC) and at residual volume(RV) in a group of patients withsevere emphysema and healthycontrol subjects. The study

allowed to determine regionaldistribution of specic gasvolume, which can be eventuallytaken to identify target regionsfor fenestrations. We foundthat interlobar heterogeneityin lung emptying was highlyvariable between patients withsevere emphysema. Thus thereare patients with large inter-and intralobar regions of gastrapping that act like bullae.Mean SV

gwas 3-fold higher

than normal. Gas trappingpredominates in upper lobes

with less decrease in SV/unitdecrease in lung volumecompared to lower lobes.

Specifc Aim 2- To verify,on an ex-vivo controlled modelof airway obstruction, if specicgas volume measured fromCT images, can be a reliableestimate of regional gastrapping.In order to utilize high resolutionCT scanning and 3He MRI andapply them to the regionalquantication of trapped gas

in an unambiguous way to testthe techniques, we developeda model of airway obstructionand trapped gas in explantedporcine lungs, which lackcollateral ventilation betweenlung lobules. Dynamic expiratorythin-section CT and 3He MRIwere performed during passivedeation from total lungcapacity after obstructions werecreated with inverted, one-way

endobronchial exit valves insegmental or lobar bronchi,to produce identiable regionsof trapped gas in four porcinelungs removed after sacricefor unrelated cardiacexperiments. Changes in SV

g

were assessed from CT datafor dened regions of interest

(ROI) within and outside ofthe obstructed segments andfor entire lobes. 3He data wereanalyzed by the correspondingregional signal reduction duringexpiration, compared to thetotal magnetic moment at eachtime point. The obtained resultsdemonstrate that both CT-determined SV

gand 3He MRI

can identify and quantify theextent of regional trapped gasin explanted porcine lungs.

Specifc Aim 3- To determinethe effects of extremely low

attenuation values on thecalculation of specic gasvolume for the assessment ofemphysema and gas trapping.Being SV

gdened as the inverse

of the lung density (minus aconstant which is assumed tobe the specic volume of lungtissue), it is highly sensitiveto very low attenuation values.Very low attenuation valuesfrequently occur in the caseof severe emphysema, wherethe density of the lungs is muchlower than that of water or

bone and falls in a range wherenot all manufacturers haveoptimized their scanners.In the present Thesis wetherefore investigated, foremphysematous and nonemphysematous lungs, in-vivoand ex-vivo, the inuence of theextremely low attenuation pixelvalues on lung densitometry,more specically on specicgas volume calculation (which

is extremely useful for theassessment of emphysemaand gas trapping). Differentsets of in-vivo and ex-vivo CTlung images were analyzedat two different volume,high and low. In order toevaluate the incidence ofvery low attenuation pixels

on the calculation of thespecic volume of gas andreduce the disproportionateweighting of these very lowattenuation pixels in specicgas volume assessment, wecalculated average values ofSV

gby reassigning all pixels

below a given Hounseldvalue a threshold value. Thisthreshold varied between -1023and -1000. We found that asignicant number of pixelswith very low attenuation valuesare present in the emphysemapatient both at TLC and RV. This

is not the case in the healthysubject histograms. As expected,SV

gvalues were signicantly

higher in patients withemphysema than in the healthysubjects, at both TLC and RV. Inemphysema patients, absoluteSV

gvalues were signicantly

inuenced by the thresholdapplied, on the contrary forhealthy subjects, SV

gwasnot signicantly inuencedby the threshold. The differencebetween the ex-vivo and in-vivoSV

gvalues was remarkably small.

Our results show that,in emphysematous but notin healthy lungs, SV

gis

signicantly affected by verylow attenuation values andthis effect is emphasized whensmaller regions of interestare considered.

Specifc Aim 4 - To developa system for 3D airway treereconstruction by HRCT images

specically addressed to theregistration of images atdifferent lung volumesin severe emphysema.The work presented in this partof the Thesis is divided in twoparts: airway segmentationand quantitative measurements.The rst step was to develop

a set of new algorithms basedon three-dimensional region-growing methods, able toreconstruct the airway treefrom the CT datasets. Thebranch-based region growingalgorithm recognizes branchpoints during the regiongrowing process, and providesas output a binary tree structure.After airway tree segmentationobtained at two different lungvolumes, RV and TLC, thesoftware allowed to choosecorresponding branches onthe two airway trees, particularly

those corresponding to allthe segmental bronchi whichare suitable for airway bypassprocedure. For each site a ROIat both lung volumes was thenanalyzed in terms of HU and SV

g

density histograms and meanSV

gand SV

g. The developed

algorithm allowed to reconstructin a successful way the airwaytrees in both healthy and severeemphysematous lungs, alwaysreaching at least the 3rd/4thbronchial generation. The resultsobtained in the different ROIs

conrmed the general behaviorof the lung in terms of HU andSV

gvariations.


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NEW METHODS fOR THE OPTIMIzATIONOf MECHANICAL VENTILATION IN RESPIRATORYDISTRESS SYNDROME (RDS)

Emanuela Zannin

Acute Respiratory DistressSyndrome (ARDS) is a specicform of respiratory failurecharacterized by diffusealveolar damage, associatedto inammation and increasedpermeability of the alveolar-capillary membrane. InfantRespiratory Distress Syndrome(IRDS) is another causeof respiratory failure dueto surfactant deciencyand structural immaturityof the lungs.Although mechanical ventilation

is crucial for survival in bothadults and infants affectedby RDS, if it is not administeredadequately it may cause asecondary ventilation inducedlung injury (VILI).Thus the goal of any ventilationstrategy should be not onlyto support but also protectpulmonary function andstructure. Any ventilatorystrategy addressed to RDSpatients should open the lungand maintain alveolar volume,avoiding overdistension and

preventing cyclic shear-stressassociated with intra-tidalphenomena. However howto achieve this goal remainscontroversial.The assessment of lung functionhas been largely studied forthe optimization of ventilatorysettings. However all thecurrently available methodsfor lung function testing havesome problems that limit their

use in the clinical managementof ventilated patients. Thusa proper tool able to guidethe clinician in tailoring theventilatory strategy on theindividual patient is still missing.The aim of the present workwas to develop new methodsable to accurately monitor lungfunction in ventilated patientsand to guide the physicianin the optimization of theventilatory strategy bothin adults and infants withrespiratory distress.

Forced oscillation technique(FOT) and optoelectronicplethysmography (OEP) havebeen used and adapted to suitspecic clinical applicationsrelated to the management ofmechanically ventilated patients.FOT is a simple non-invasivetechnique for the assessmentof respiratory mechanics.It provides a measurementof the respiratory systemimpedance (Zrs), which is acomplex number that can besplit into a real part called

resistance (Rrs) and an imaginarypart, called reactance (Xrs), thatdescribes the compliance andinertance of the system.At rst we evaluated the abilityof Xrs measured by FOT at 5Hz to identify lung volumerecruitment/derecruitment.A very strong correlationwas found between Xrs andthe amount of non-aeratedtissue measured by CT scans,

indicating that Xrs is ableto quantify changes inlung volume recruitment/derecruitment associated todifferent interventions affectinglung volume.Then we tested the abilityof Xrs to identify the optimalPositive End-ExpiratoryPressure (PEEP). Xrs was ableto identify PEEP-inducedrecruitment/derecruitmentand overdistension and tidalhyperinatio

dottorato yearbook2010_bioengineer

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