The effect of cross-linker on endogenous enzymatic activity within radicular dentin and bond strength between intraradicular post and radicular dentin

The primary aim was to evaluate the effect of 1-ethyl-3-(3-dimethylamino-propyl) carbodiimide (EDC) on endogenous enzymatic activity within radicular dentin and push-out bond strength of adhesively luted fiber posts, at baseline and after artificial aging. Additionally, the effect of different cementation strategies on endogenous enzymatic activity and fiber post retention was evaluated. The experiment was carried out on extracted human teeth, following endodontic treatment and fiber post cementation. Three cementation strategies were performed: resin cement in combination with etch-and-rinse (EAR) adhesive system, with self-etch (SE) system and self-adhesive (SE) cement. Each of the mentioned strategies had a control and experimental (EDC) group in which root canal was irrigated with 0.3M EDC for 1 minute. The push-out bond strength test was performed 24h after cementation and after 40.000 thermocycles. In order to investigate the effect of EDC and different cementation strategies, in situ zymography analyses of the resin-dentin interfaces were conducted. Statistical analyses were conducted with the software Stata 12.0 (Stata Corp, College Station, Texas, USA) and the significance was set for p<0.05. The results of statistical analysis (ANOVA) showed that the variables “EDC”, “root region” and “artificial aging” significantly influenced fiber posts’ retention to root canal (p<0.05). The highest values were observed in coronal third. The mean values observed after artificial aging were lower when compared to baseline, however EDC was effective in preserving bond strength. The level of enzymatic activity varied between the groups and EDC had a beneficial effect on silencing the enzymatic activity. Within the limitations of the study, it was concluded that the choice of cementation strategy did not influence posts’ retention, while EDC contributed to the preservation of bond strength after artificial aging and reduced enzymatic activity within radicular dentin. In vivo trials are necessary to confirm the results of this in vitro study.

Influence of application mode of universal adhesives on bond strength performances and enzymatic activity of coronal and radicular dentin

Objective: The aims of this thesis were to analyze the application mode of the universal adhesives (UA) and to give instructions for clinical procedures. The etching mode of UA on the bond strength to dentin and on the risk of retention, marginal discoloration, marginal adaptation and post-operative sensitivity (POS) was analyzed by two systematic reviews. Three in vitro studies were conducted: 1) evaporation mode of a UA on coronal dentin; 2) cementation approach on radicular dentin; 3) adhesion of metal brackets to enamel. Materials and methods: Two systematic review were conducted firstly, then in vitro study to investigate the evaporation mode in presence or not of pulpal pressure by means of μTBS, and the enzymatic activity using in situ zymography, at T0 and T6. The cementation of a fiber into radicular dentin with different resin-cements was studied, by push-out bond strength evaluation. Orthodontic brackets were cemented according to 4 adhesive protocols and shear bond strength test was conducted. Two adhesive removal techniques were evaluated, and spectrophotometry was used. Results: The probability of POS occurrence was less in SE. SEE approach seems to perform better than SE. Air-drying resulted in higher μTBS. Suction-evaporation, aging and ER mode increased MMPs activity. Differences in NL expression were present at T0 for fiber post study, and the aging produced an increase in marginal infiltration. Brackets cemented with new universal cement with previous etchant application showed good μTBS values. Conclusion: SEE performed better than SE and TE with UA in terms of uTBS. Evaporating with air-drying is better for UA in terms of uTBS and enzymatic activity. Aging and choice of resin cement for cementation of fiber posts influenced the PBS. Brackets cementation with a new resin- cement seems to offer the highest bond strength and leaves more cement remnants after the bracket removal.

Sviluppo di metodologie di localizzazione nel contesto del Comprehensive Nuclear Test-Ban Treaty

Since the first underground nuclear explosion, carried out in 1958, the analysis of seismic signals generated by these sources has allowed seismologists to refine the travel times of seismic waves through the Earth and to verify the accuracy of the location algorithms (the ground truth for these sources was often known). Long international negotiates have been devoted to limit the proliferation and testing of nuclear weapons. In particular the Treaty for the comprehensive nuclear test ban (CTBT), was opened to signatures in 1996, though, even if it has been signed by 178 States, has not yet entered into force, The Treaty underlines the fundamental role of the seismological observations to verify its compliance, by detecting and locating seismic events, and identifying the nature of their sources. A precise definition of the hypocentral parameters represents the first step to discriminate whether a given seismic event is natural or not. In case that a specific event is retained suspicious by the majority of the State Parties, the Treaty contains provisions for conducting an on-site inspection (OSI) in the area surrounding the epicenter of the event, located through the International Monitoring System (IMS) of the CTBT Organization. An OSI is supposed to include the use of passive seismic techniques in the area of the suspected clandestine underground nuclear test. In fact, high quality seismological systems are thought to be capable to detect and locate very weak aftershocks triggered by underground nuclear explosions in the first days or weeks following the test. This PhD thesis deals with the development of two different seismic location techniques: the first one, known as the double difference joint hypocenter determination (DDJHD) technique, is aimed at locating closely spaced events at a global scale. The locations obtained by this method are characterized by a high relative accuracy, although the absolute location of the whole cluster remains uncertain. We eliminate this problem introducing a priori information: the known location of a selected event. The second technique concerns the reliable estimates of back azimuth and apparent velocity of seismic waves from local events of very low magnitude recorded by a trypartite array at a very local scale. For the two above-mentioned techniques, we have used the crosscorrelation technique among digital waveforms in order to minimize the errors linked with incorrect phase picking. The cross-correlation method relies on the similarity between waveforms of a pair of events at the same station, at the global scale, and on the similarity between waveforms of the same event at two different sensors of the try-partite array, at the local scale. After preliminary tests on the reliability of our location techniques based on simulations, we have applied both methodologies to real seismic events. The DDJHD technique has been applied to a seismic sequence occurred in the Turkey-Iran border region, using the data recorded by the IMS. At the beginning, the algorithm was applied to the differences among the original arrival times of the P phases, so the cross-correlation was not used. We have obtained that the relevant geometrical spreading, noticeable in the standard locations (namely the locations produced by the analysts of the International Data Center (IDC) of the CTBT Organization, assumed as our reference), has been considerably reduced by the application of our technique. This is what we expected, since the methodology has been applied to a sequence of events for which we can suppose a real closeness among the hypocenters, belonging to the same seismic structure. Our results point out the main advantage of this methodology: the systematic errors affecting the arrival times have been removed or at least reduced. The introduction of the cross-correlation has not brought evident improvements to our results: the two sets of locations (without and with the application of the cross-correlation technique) are very similar to each other. This can be commented saying that the use of the crosscorrelation has not substantially improved the precision of the manual pickings. Probably the pickings reported by the IDC are good enough to make the random picking error less important than the systematic error on travel times. As a further justification for the scarce quality of the results given by the cross-correlation, it should be remarked that the events included in our data set don’t have generally a good signal to noise ratio (SNR): the selected sequence is composed of weak events ( magnitude 4 or smaller) and the signals are strongly attenuated because of the large distance between the stations and the hypocentral area. In the local scale, in addition to the cross-correlation, we have performed a signal interpolation in order to improve the time resolution. The algorithm so developed has been applied to the data collected during an experiment carried out in Israel between 1998 and 1999. The results pointed out the following relevant conclusions: a) it is necessary to correlate waveform segments corresponding to the same seismic phases; b) it is not essential to select the exact first arrivals; and c) relevant information can be also obtained from the maximum amplitude wavelet of the waveforms (particularly in bad SNR conditions). Another remarkable point of our procedure is that its application doesn’t demand a long time to process the data, and therefore the user can immediately check the results. During a field survey, such feature will make possible a quasi real-time check allowing the immediate optimization of the array geometry, if so suggested by the results at an early stage.

Technical innovations for the diagnosis and the rehabilitation of motor and perceptive impairments of the child with Cerebral Palsy

The treatment of the Cerebral Palsy (CP) is considered as the “core problem” for the whole field of the pediatric rehabilitation. The reason why this pathology has such a primary role, can be ascribed to two main aspects. First of all CP is the form of disability most frequent in childhood (one new case per 500 birth alive, (1)), secondarily the functional recovery of the “spastic” child is, historically, the clinical field in which the majority of the therapeutic methods and techniques (physiotherapy, orthotic, pharmacologic, orthopedic-surgical, neurosurgical) were first applied and tested. The currently accepted definition of CP – Group of disorders of the development of movement and posture causing activity limitation (2) – is the result of a recent update by the World Health Organization to the language of the International Classification of Functioning Disability and Health, from the original proposal of Ingram – A persistent but not unchangeable disorder of posture and movement – dated 1955 (3). This definition considers CP as a permanent ailment, i.e. a “fixed” condition, that however can be modified both functionally and structurally by means of child spontaneous evolution and treatments carried out during childhood. The lesion that causes the palsy, happens in a structurally immature brain in the pre-, peri- or post-birth period (but only during the firsts months of life). The most frequent causes of CP are: prematurity, insufficient cerebral perfusion, arterial haemorrhage, venous infarction, hypoxia caused by various origin (for example from the ingestion of amniotic liquid), malnutrition, infection and maternal or fetal poisoning. In addition to these causes, traumas and malformations have to be included. The lesion, whether focused or spread over the nervous system, impairs the whole functioning of the Central Nervous System (CNS). As a consequence, they affect the construction of the adaptive functions (4), first of all posture control, locomotion and manipulation. The palsy itself does not vary over time, however it assumes an unavoidable “evolutionary” feature when during growth the child is requested to meet new and different needs through the construction of new and different functions. It is essential to consider that clinically CP is not only a direct expression of structural impairment, that is of etiology, pathogenesis and lesion timing, but it is mainly the manifestation of the path followed by the CNS to “re”-construct the adaptive functions “despite” the presence of the damage. “Palsy” is “the form of the function that is implemented by an individual whose CNS has been damaged in order to satisfy the demands coming from the environment” (4). Therefore it is only possible to establish general relations between lesion site, nature and size, and palsy and recovery processes. It is quite common to observe that children with very similar neuroimaging can have very different clinical manifestations of CP and, on the other hand, children with very similar motor behaviors can have completely different lesion histories. A very clear example of this is represented by hemiplegic forms, which show bilateral hemispheric lesions in a high percentage of cases. The first section of this thesis is aimed at guiding the interpretation of CP. First of all the issue of the detection of the palsy is treated from historical viewpoint. Consequently, an extended analysis of the current definition of CP, as internationally accepted, is provided. The definition is then outlined in terms of a space dimension and then of a time dimension, hence it is highlighted where this definition is unacceptably lacking. The last part of the first section further stresses the importance of shifting from the traditional concept of CP as a palsy of development (defect analysis) towards the notion of development of palsy, i.e., as the product of the relationship that the individual however tries to dynamically build with the surrounding environment (resource semeiotics) starting and growing from a different availability of resources, needs, dreams, rights and duties (4). In the scientific and clinic community no common classification system of CP has so far been universally accepted. Besides, no standard operative method or technique have been acknowledged to effectively assess the different disabilities and impairments exhibited by children with CP. CP is still “an artificial concept, comprising several causes and clinical syndromes that have been grouped together for a convenience of management” (5). The lack of standard and common protocols able to effectively diagnose the palsy, and as a consequence to establish specific treatments and prognosis, is mainly because of the difficulty to elevate this field to a level based on scientific evidence. A solution aimed at overcoming the current incomplete treatment of CP children is represented by the clinical systematic adoption of objective tools able to measure motor defects and movement impairments. A widespread application of reliable instruments and techniques able to objectively evaluate both the form of the palsy (diagnosis) and the efficacy of the treatments provided (prognosis), constitutes a valuable method able to validate care protocols, establish the efficacy of classification systems and assess the validity of definitions. Since the ‘80s, instruments specifically oriented to the analysis of the human movement have been advantageously designed and applied in the context of CP with the aim of measuring motor deficits and, especially, gait deviations. The gait analysis (GA) technique has been increasingly used over the years to assess, analyze, classify, and support the process of clinical decisions making, allowing for a complete investigation of gait with an increased temporal and spatial resolution. GA has provided a basis for improving the outcome of surgical and nonsurgical treatments and for introducing a new modus operandi in the identification of defects and functional adaptations to the musculoskeletal disorders. Historically, the first laboratories set up for gait analysis developed their own protocol (set of procedures for data collection and for data reduction) independently, according to performances of the technologies available at that time. In particular, the stereophotogrammetric systems mainly based on optoelectronic technology, soon became a gold-standard for motion analysis. They have been successfully applied especially for scientific purposes. Nowadays the optoelectronic systems have significantly improved their performances in term of spatial and temporal resolution, however many laboratories continue to use the protocols designed on the technology available in the ‘70s and now out-of-date. Furthermore, these protocols are not coherent both for the biomechanical models and for the adopted collection procedures. In spite of these differences, GA data are shared, exchanged and interpreted irrespectively to the adopted protocol without a full awareness to what extent these protocols are compatible and comparable with each other. Following the extraordinary advances in computer science and electronics, new systems for GA no longer based on optoelectronic technology, are now becoming available. They are the Inertial and Magnetic Measurement Systems (IMMSs), based on miniature MEMS (Microelectromechanical systems) inertial sensor technology. These systems are cost effective, wearable and fully portable motion analysis systems, these features gives IMMSs the potential to be used both outside specialized laboratories and to consecutive collect series of tens of gait cycles. The recognition and selection of the most representative gait cycle is then easier and more reliable especially in CP children, considering their relevant gait cycle variability. The second section of this thesis is focused on GA. In particular, it is firstly aimed at examining the differences among five most representative GA protocols in order to assess the state of the art with respect to the inter-protocol variability. The design of a new protocol is then proposed and presented with the aim of achieving gait analysis on CP children by means of IMMS. The protocol, named ‘Outwalk’, contains original and innovative solutions oriented at obtaining joint kinematic with calibration procedures extremely comfortable for the patients. The results of a first in-vivo validation of Outwalk on healthy subjects are then provided. In particular, this study was carried out by comparing Outwalk used in combination with an IMMS with respect to a reference protocol and an optoelectronic system. In order to set a more accurate and precise comparison of the systems and the protocols, ad hoc methods were designed and an original formulation of the statistical parameter coefficient of multiple correlation was developed and effectively applied. On the basis of the experimental design proposed for the validation on healthy subjects, a first assessment of Outwalk, together with an IMMS, was also carried out on CP children. The third section of this thesis is dedicated to the treatment of walking in CP children. Commonly prescribed treatments in addressing gait abnormalities in CP children include physical therapy, surgery (orthopedic and rhizotomy), and orthoses. The orthotic approach is conservative, being reversible, and widespread in many therapeutic regimes. Orthoses are used to improve the gait of children with CP, by preventing deformities, controlling joint position, and offering an effective lever for the ankle joint. Orthoses are prescribed for the additional aims of increasing walking speed, improving stability, preventing stumbling, and decreasing muscular fatigue. The ankle-foot orthosis (AFO), with a rigid ankle, are primarily designed to prevent equinus and other foot deformities with a positive effect also on more proximal joints. However, AFOs prevent the natural excursion of the tibio-tarsic joint during the second rocker, hence hampering the natural leaning progression of the whole body under the effect of the inertia (6). A new modular (submalleolar) astragalus-calcanear orthosis, named OMAC, has recently been proposed with the intention of substituting the prescription of AFOs in those CP children exhibiting a flat and valgus-pronated foot. The aim of this section is thus to present the mechanical and technical features of the OMAC by means of an accurate description of the device. In particular, the integral document of the deposited Italian patent, is provided. A preliminary validation of OMAC with respect to AFO is also reported as resulted from an experimental campaign on diplegic CP children, during a three month period, aimed at quantitatively assessing the benefit provided by the two orthoses on walking and at qualitatively evaluating the changes in the quality of life and motor abilities. As already stated, CP is universally considered as a persistent but not unchangeable disorder of posture and movement. Conversely to this definition, some clinicians (4) have recently pointed out that movement disorders may be primarily caused by the presence of perceptive disorders, where perception is not merely the acquisition of sensory information, but an active process aimed at guiding the execution of movements through the integration of sensory information properly representing the state of one’s body and of the environment. Children with perceptive impairments show an overall fear of moving and the onset of strongly unnatural walking schemes directly caused by the presence of perceptive system disorders. The fourth section of the thesis thus deals with accurately defining the perceptive impairment exhibited by diplegic CP children. A detailed description of the clinical signs revealing the presence of the perceptive impairment, and a classification scheme of the clinical aspects of perceptual disorders is provided. In the end, a functional reaching test is proposed as an instrumental test able to disclosure the perceptive impairment. References 1. Prevalence and characteristics of children with cerebral palsy in Europe. Dev Med Child Neurol. 2002 Set;44(9):633-640. 2. Bax M, Goldstein M, Rosenbaum P, Leviton A, Paneth N, Dan B, et al. Proposed definition and classification of cerebral palsy, April 2005. Dev Med Child Neurol. 2005 Ago;47(8):571-576. 3. Ingram TT. A study of cerebral palsy in the childhood population of Edinburgh. Arch. Dis. Child. 1955 Apr;30(150):85-98. 4. Ferrari A, Cioni G. The spastic forms of cerebral palsy : a guide to the assessment of adaptive functions. Milan: Springer; 2009. 5. Olney SJ, Wright MJ. Cerebral Palsy. Campbell S et al. Physical Therapy for Children. 2nd Ed. Philadelphia: Saunders. 2000;:533-570. 6. Desloovere K, Molenaers G, Van Gestel L, Huenaerts C, Van Campenhout A, Callewaert B, et al. How can push-off be preserved during use of an ankle foot orthosis in children with hemiplegia? A prospective controlled study. Gait Posture. 2006 Ott;24(2):142-151.

Qualifica a Vibrazioni di Componenti Meccanici: Studio e Verifica di una Procedura di Test Tailoring.

I test di qualifica a vibrazioni vengono usati in fase di progettazione di un componente per verificarne la resistenza meccanica alle sollecitazioni dinamiche (di natura vibratoria) applicate durante la sua vita utile. La durata delle vibrazioni applicate al componente durante la sua vita utile (migliaia di ore) deve essere ridotta al fine di realizzare test fattibili in laboratorio, condotti in genere utilizzando uno shaker elettrodinamico. L’idea è quella di aumentare l’intensità delle vibrazioni riducendone la durata. Esistono diverse procedure di Test Tailoring che tramite un metodo di sintesi definiscono un profilo vibratorio da applicare in laboratorio a partire dalle reali vibrazioni applicate al componente: una delle metodologie più comuni si basa sull’equivalenza del danno a fatica prodotto dalle reali vibrazioni e dalle vibrazioni sintetizzate. Questo approccio è piuttosto diffuso tuttavia all’autore non risulta presente nessun riferimento in letteratura che ne certifichi la validità tramite evidenza sperimentalmente. L’obiettivo dell’attività di ricerca è stato di verificare la validità del metodo tramite una campagna sperimentale condotta su opportuni provini. Il metodo viene inizialmente usato per sintetizzare un profilo vibratorio (random stazionario) avente la stessa durata di un profilo vibratorio non stazionario acquisito in condizioni reali. Il danno a fatica prodotto dalla vibrazione sintetizzata è stato confrontato con quello della vibrazione reale in termini di tempo di rottura dei provini. I risultati mostrano che il danno prodotto dalla vibrazione sintetizzata è sovrastimato, quindi l’equivalenza non è rispettata. Sono stati individuati alcuni punti critici e sono state proposte alcune modifiche al metodo per rendere la teoria più robusta. Il metodo è stato verificato con altri test e i risultati confermano la validità del metodo a condizione che i punti critici individuati siano correttamente analizzati.

Valutazione dell’impiego dei test per la genotipizzazione di HPV e l’espressione degli oncogeni virali nel follow-up di donne conizzate per lesioni cervicali di alto grado nello screening del cervico-carcinoma della Regione Emilia-Romagna

Obiettivi: Valutare la prevalenza dei diversi genotipi di HPV in pazienti con diagnosi di CIN2/3 nella Regione Emilia-Romagna, la persistenza genotipo-specifica di HPV e l’espressione degli oncogeni virali E6/E7 nel follow-up post-trattamento come fattori di rischio di recidiva/persistenza o progressione di malattia; verificare l’applicabilità di nuovi test diagnostici biomolecolari nello screening del cervicocarcinoma. Metodi: Sono state incluse pazienti con citologia di screening anormale, sottoposte a trattamento escissionale (T0) per diagnosi di CIN2/3 su biopsia mirata. Al T0 e durante il follow-up a 6, 12, 18 e 24 mesi, oltre al Pap test e alla colposcopia, sono state effettuate la ricerca e la genotipizzazione dell'HPV DNA di 28 genotipi. In caso di positività al DNA dei 5 genotipi 16, 18, 31, 33 e/o 45, si è proceduto alla ricerca dell'HPV mRNA di E6/E7. Risultati preliminari: Il 95.8% delle 168 pazienti selezionate è risultato HPV DNA positivo al T0. Nel 60.9% dei casi le infezioni erano singole (prevalentemente da HPV 16 e 31), nel 39.1% erano multiple. L'HPV 16 è stato il genotipo maggiormente rilevato (57%). Il 94.3% (117/124) delle pazienti positive per i 5 genotipi di HPV DNA sono risultate mRNA positive. Abbiamo avuto un drop-out di 38/168 pazienti. A 18 mesi (95% delle pazienti) la persistenza dell'HPV DNA di qualsiasi genotipo era del 46%, quella dell'HPV DNA dei 5 genotipi era del 39%, con espressione di mRNA nel 21%. Abbiamo avuto recidiva di malattia (CIN2+) nel 10.8% (14/130) a 18 mesi. Il pap test era negativo in 4/14 casi, l'HPV DNA test era positivo in tutti i casi, l'mRNA test in 11/12 casi. Conclusioni: L'HR-HPV DNA test è più sensibile della citologia, l'mRNA test è più specifico nell'individuare una recidiva. I dati definitivi saranno disponibili al termine del follow-up programmato.

Valutazione della mobilita' di contaminanti inorganici tramite test di cessione su matrici ambientali contaminate

La caratterizzazione di sedimenti contaminati è un problema complesso, in questo lavoro ci si è occupati di individuare una metodologia di caratterizzazione che tenesse conto sia delle caratteristiche della contaminazione, con analisi volte a determinare il contenuto totale di contaminanti, sia della mobilità degli inquinanti stessi. Una adeguata strategia di caratterizzazione può essere applicata per la valutazione di trattamenti di bonifica, a questo scopo si è valutato il trattamento di soil washing, andando ad indagare le caratteristiche dei sedimenti dragati e del materiale in uscita dal processo, sabbie e frazione fine, andando inoltre a confrontare le caratteristiche della sabbia in uscita con quelle delle sabbie comunemente usate per diverse applicazioni. Si è ritenuto necessario indagare la compatibilità dal punto di vista chimico, granulometrico e morfologico. Per indagare la mobilità si è scelto di applicare i test di cessione definiti sia a livello internazionale che italiano (UNI) e quindi si sono sviluppate le tecnologie necessarie alla effettuazione di test di cessione in modo efficace, automatizzando la gestione del test a pHstat UNI CEN 14997. Questo si è reso necessario a causa della difficoltà di gestire il test manualmente, per via delle tempistiche difficilmente attuabili da parte di un operatore. Le condizioni redox influenzano la mobilità degli inquinanti, in particolare l’invecchiamento all’aria di sedimenti anossici provoca variazioni sensibili nello stato d’ossidazione di alcune componenti, incrementandone la mobilità, si tratta quindi di un aspetto da considerare quando si individuano le adeguate condizioni di stoccaggio-smaltimento, si è eseguita a questo scopo una campagna sperimentale.

Sviluppo di un framework per l'automazione dei test al banco e la prototipazione di algoritmi di controllo di motori a combustione interna

Negli ultimi anni, i limiti sempre più stringenti sulle emissioni inquinanti dei gas di scarico, hanno portato ad un notevole aumento della complessità dei motori a combustione interna. Questa complicazione determina un aumento esponenziale del numero di test da effettuare nella sala prova. I metodi tipici di gestione dei test non possono più essere utilizzati, ma è essenziale creare un sistema che ottimizzi le prove. Per ridurre drasticamente il tempo di esecuzione, è necessario implementare un'architettura in grado di facilitare lo scambio di dati tra i sistemi presenti nella sala prova, e, in aggiunta, definire le strategie di automazione dei test. L'approccio a taluni metodi si presenta ancora complicato in molti gruppi di sviluppo di strategie di controllo motore, anche se, una volta sviluppati, portano e a grandi benefici durante la fase di test. Il lavoro illustra i metodi implementati per la gestione di queste strategie. Prima si descrive l'approccio utilizzato nella calibrazione di anticipo di accensione per mantenere livelli accettabili di detonazione durante il processo di calibrazione. Successivamente è mostrato il sistema di automazione dei test che consente il pieno controllo del punto di funzionamento del motore, la gestione dell'acquisizione e la verifica della stabilità delle condizioni ottenute. L'ultima parte mostra sistemi di prototipazione rapida per la gestione di componenti innovatici del motore.

Development of 3D Cancer Cell Models to Test Metabolic Interventions as an Anticancer Strategy

In recent years, it has become evident that the role of mitochondria in the metabolic rewiring is essential for cancer development and progression. The metabolic profile during tumorigenesis has been performed mainly in traditional 2D cell models, including cell lines of various lineages and phenotypes. Although useful in many ways, their relevance can be often debatable, as they lack the interactions between different cells of the tumour microenvironment and/or interaction with the extracellular matrix 1,2. Improved models are now being developed using 3D cell culture technology, contributing with increased physiological relevance 3,4. In this work, we improved a method for the generation of 3D models from healthy and tumour colon tissue, based on organoid technology, and performed their molecular and biochemical characterization and validation. Further, in-plate cryopreservation was applied to these models, and optimal results were obtained in terms of cell viability and functionality of the cryopreserved models. We also cryopreserved colon fibroblasts with the aim to introduce them in a co-culture cryopreserved model with organoids. This technology allows the conversion of cell models into “plug and play” formats. Therefore, cryopreservation in-plate facilitates the accessibility of specialized cell models to cell-based research and application, in cases where otherwise such specialized models would be out of reach. Finally, we briefly explored the field of bioprinting, by testing a new matrix to support the growth of colon tumour organoids, which revealed promising preliminary results. To facilitate the reader, we organized this thesis into chapters, divided by the main points of work which include development, characterization and validation of the model, commercial output, and associated applications. Each chapter has a brief introduction, followed by results and discussion and a final conclusion. The thesis has also a general discussion and conclusion section in the end, which covers the main results obtained during this work.