Espressione e assemblaggio delle proteine di AAV: localizzazione ed incapsidazione del DNA in Saccharomyces cerevisiae.

Espressione e assemblaggio delle proteine di AAV: localizzazione ed incapsidazione del DNA in Saccharomyces cerevisiae. Il punto di partenza di questo progetto di tesi si basa sugli studi effettuati sul lievito S. cerevisiae come modello per la produzione di vettori derivati dal virus Adeno Associato (AAV) per la terapia genica. AAV è un virus difettivo, non patogeno della famiglia dei parvovirus che dipende dalla co-infezione di un virus Helper (come adenovirus o Herpes simplex) per una sua replicazione produttiva. Il genoma di AAV consiste di un DNA a singolo filamento contenente due open reading frames (ORF) fiancheggiate da due sequenze invertite ripetute dette ITR. Le due ORF codificano per le proteine Rep,Rep40, 52, 68 e 78, necessarie per la replicazione e le proteine Cap, VP1, 2 e 3, necessarie per la formazione del capside. In laboratorio, è stato dimostrato che il lievito è in grado sostenere la replicazione di vettori AAV ricombinanti (rAAV) e la formazione di capsidi. Lo scopo di questa tesi è pertanto quello di caratterizzare ulteriormente la formazione dei capsidi sia andando a studiare la localizzazione delle proteine del capside VP1, VP2 e VP3, sia sviluppando una nuova metodica di investigazione che riveli con più facilità la produzione di vettori virali in S.cerevisiae. Un altro aspetto che abbiamo studiato è stata l’incapsidazione del DNA. Il lavoro di tesi è stato eseguito sul ceppo di lievito aploide, RSY12, e sul ceppo diploide, BY4743. In laboratorio era stato costruito in precedenza il ceppo RSY12 contenente il gene che codifica per la proteina di replicazione di AAV REP68 integrato nel genoma, che è stato trasformato con il plasmide pESCVP2,3VP1KM, per l’espressione di VP1, VP2, VP3. Il ceppo diploide, ingegnerizzato allo stesso modo dell’aploide, è stato costruito durante questo lavoro di tesi al fine di avere un ceppo di lievito in grado di crescere più velocemente dell’aploide. Analisi mediante Western blot ha mostrato che l’espressione di VP1, 2 e 3 avviene nella giusta proporzione come avviene nell’aploide. Per determinare la localizzazione intracellulare di VP1, 2 e 3 nel ceppo RSY12, è stato messo a punto uno specifico protocollo di frazionamento che permette di caratterizzare la ripartizione delle proteine investigate nei vari compartimenti subcellulari. Analisi mediante Western blot delle proteine dei compartimenti subcellulari ha evidenziato una presenza omogenea di VP1, VP2, VP3 all’interno del lievito. Per verificare l’effettiva formazione di capsidi virali in vivo, smentendo ogni possibile dubbio sul fatto che questi si potessero formare durante le procedure di estrazione proteiche, é stato ideato e sviluppato un sistema Immuno blot che consente lo studio di una cinetica che mette in rapporto la produzione delle proteine virali con la formazione dei capsidi interi attraverso ibridazioni con anticorpi che riconoscono discriminatamente epitopi delle proteine libere ed epitopi delle proteine incapsidate. I risultati finora ottenuti su entrambi i ceppi indicano che con questo sistema è possibile capire se le cellule esprimono VP1,2 e 3 e se le tre proteine si assemblano correttamente. I due ceppi di lievito saranno trasformati con il plasmide pAAVpokURA contente il marker di selezione URA3 fiancheggiato dalle ITR del virus al fine di determinare se il ssDNA che viene formato viene incapsidato e se la localizzazione di VP1, VP2 e VP3 varia in presenza di DNA. A tal proposito sono in corso esperimenti di Southern blot per verificare la presenza del ssDNA e successivamente sarà determintata l’incapsidazione e localizzazione di VP1, 2 e 3 in presenza del DNA

Engineering responsive and biomimetic material based on elastin-like recombinamers for biomedical application

Regenerative Medicine is a well-established field of science that aims to replace, engineer and regenerate human cells, damaged tissues or organs to restore their normal function. This branch of translational research finds a deep interest in the Science of Biomaterials; indeed, the knowledge acquired in that field goes proportionally with the development of novel biomaterials. There is a great need in developing advanced biomaterials capable to fulfil the requirements of stability and bioactivity for their application in biomedicine. Moreover, considering the complexity of the human body, this system needs a certain rate of versatility in order to be tailored to a specific area of application. For all these reasons, recombinant proteins are an interesting approach, in which, elastin-like recombinamers (ELRs) represent one of the most promising biomaterials.ELRs are obtained through DNA recombinant technology, which allows the precise control at the genetic level, affording exquisite control over final protein functionality. ELRs are protein-based polypeptides that comprise repetitive units of the Val−Pro−Gly−X−Gly (VPGXG)n pentapeptide, in which X (guest residue) could be any amino acid except L-proline. In terms of biomaterial design, ELRs show several outstanding properties. ELRs are inspired by elastin, which is a component of natural extracellular matrix (ECM), showing excellent biocompatibility. One of the most important features of ELRs is that they exhibit thermo-responsiveness; this is due to the change of protein conformation above the so-called transition temperature (Tt), which depends on the amino acid composition of the polymer. Moreover, according to the ELRs design, they can be processed as several supramolecular structures, such as micelles, nanoparticles, films, and hydrogels. The large variety of ELRs, both in terms of structures and bioactivity, permits the application of these protein-based biomaterials to diverse biomedical applications. This Thesis represents a sort of journey towards the exploration of the evolution of ELRs as a powerful tool with great potential in the biomedical field.Departamento de Bioquímica y Biología Molecular y FisiologíaDoctorado en Investigación Biomédic

Cost-effectiveness profile, organizational implications and patient preferences on the use of exogenous TSH therapy (Thyrogen®) vs. THW in thyroid residue ablation in Italy

BACKGROUND: Radioiodine ablation is an adjuvant procedure used to treat patients with differentiated thyroid cancer. For ablation to be successful, patients must have elevated levels of thyroid stimulating hormone (TSH). This can be achieved by withholding thyroid hormone therapy (endogenous stimulation), or by administration of recombinant human thyroid stimulating hormone (rhTSH; Thyrogen®; exogenous stimulation) to patients in the euthyroid state.AIM: To compare the estimated health benefits, cost and cost-effectiveness of TSH stimulation with and without Thyrogen® in the Italian setting.METHODS: A cost-utility analysis was undertaken to assess the impact of exogenous vs. endogenous TSH stimulation before radioiodine remnant ablation of patients with newly diagnosed, well-differentiated papillary or follicular thyroid cancer who have undergone total or near-total thyroidectomy. A Markov model was developed to simulate treatment costs and health outcomes associated with exogenous and endogenous stimulation in four distinct health states: pre-ablation, ablation, post-ablation, and well/recovery. Treatment was stratified by patients who receive high- and low-activity (30-100 mCi, respectively) in the ablation state. The Italian National Health System perspective was adopted in the base case scenario while the impact of indirect costs was explored in a sensitivity analysis. Costs and quality-adjusted life years (QALY) specific to each health state were estimated, summarized and converted into a corresponding incremental cost-effectiveness ratio (ICER).RESULTS: We calculated a cost-effectiveness ratio of 18,357.18 €/QALY gained whereas the inclusion of indirect cost and accident cost produced reductions of the ICER to € 14,609.51 and € 15,515.26 per QALY, respectively. Finally, all results in the sensitivity analysis are below the lower bound of national and international cost- effective threshold.CONCLUSION: Thyrogen® represents a cost-effective option for patients with differentiated thyroid cancer who underwent total or near-total thyroidectomy in Italy. Our findings are consistent with other cost-utility analyses

The colour of stones and mosaics in Ravenna: new images of ancient monuments using the latest generation of unconventional photogrammetry

[EN] This paper focuses on texture accuracy for a reliable simulation of construction materials present in some important historic monuments on the World Heritage List, dating back to Late Antiquity in Ravenna (Italy). A series of novel strategies will be explained in order to take advantage of automatic texturing solutions of 3D digital models created by Structure from Motion (SfM) applications. These monuments will be used as case studies toillustrate a shadow removal technique on Lambertian surfaces (stone, mortar, bricks, etc.) from apparent colour texture and a rapid approach for optic simulation of mosaic decoration. The latter consists of splitting a single apparent colour texture into a set of images whose purpose is to supply render engines more suitable shaders for representing coloured mosaic tiles in an optically reliable way[ES] El propósito de este trabajo es destacar el papel de la precisión de la textura para una representación fiable de los materiales presentes en algunos relevantes monumentos de Rávena (Italia), pertenecientes a la Lista del Patrimonio Mundial y que datan de la Antigüedad Tardía. Una serie de estrategias se explicarán con el fin de aprovechar las ventajas de las soluciones automáticas de texturizado de modelos 3D creados con aplicaciones Structure from Motion (SfM). Estos edificios serán los casos de estudios para ilustrar una técnica innovadora para la eliminación de las sombras de la textura aparente del color y un método rápido para la simulación de decoración taraceadas de mosaico. Este último problema consiste en dividir una única textura en un conjunto de imágenes cuya finalidad es ofrecer a los motores de renderizado los shaders más adecuados para la representación en una forma ópticamente fiable de las teselas coloreadas de mosaicoCipriani, L.; Fantini, F.; Bertacchi, S. (2015). El color en las piedras y en los mosaicos de Rávena: nuevas imágenes de los monumentos antiguos a través de la fotogrametría no convencional de última generación. EGA. Revista de Expresión Gráfica Arquitectónica. 20(26):190-201. doi:10.4995/ega.2015.4052SWORD1902012026Cipriani, L., Fantini, F., & Bertacchi, S. (2014). 3D models mapping optimization through an integrated parameterization approach: cases studies from Ravenna. ISPRS - International Archives of the Photogrammetry, Remote Sensing and Spatial Information Sciences, XL-5, 173-180. doi:10.5194/isprsarchives-xl-5-173-201

Abruzzo

In Abruzzo region 55,4% of women (aged 25-64) are screened regularly, meaning every 3 years. Considering regional tariffs and vaccine acquisition cost, the vaccination of 12-year-old girls with a 90% coverage could prevent 29 cases of cervicocarcinoma and 12 related deaths and thus results to be cost-effective (20.836 €/QALY). When the vaccination programme is extended to 16-year-old girls a further 28 cancer cases and 12 deaths could be prevented, with a very similar cost-effectiveness ratio. In Abruzzo region, the net cost for woman vaccinated is 199 € for the single cohort and 202 € for the multiple cohort

L'introduzione del vaccino anti-HPV bivalente adiuvato con AS04 nelle regioni italiane: impatto economico ed effetti sulla salute delle donne

Introduction: the impact of cervical cancer prevention, in particular through HPV female vaccination, has been published for many countries at the national level. However, to our knowledge no attempt has been made to address the impact at a regional level. Since the Italian health reforms of the early 1990s, introducing "managerialism", decentralization and quasi-market mechanisms, regional authorities have consequently been experimenting with different organizational and funding models to achieve an acceptable combination of equity, efficiency, freedom of choice and cost-containment. Methods: a Markov model, previously described and successfully adapted to the national scenario [La Torre, 2007], has been used to explore the impact of preventive cervical cancer vaccination with Cervarix™ at a regional level in Italy. Resource use was based on a standard therapeutic path applied to all regions. However we quantified the impact of the so-called "decentralization progress" by collecting regional data on: pap-test coverage, tariffs for treatments and cost of the vaccination course. We performed for each Italian region a cost-effectiveness analysis combined with a regional budget impact analysis. The regional analyses compared HPV vaccination, both of a single female cohort (12 years old) and a multiple female cohort (12+16 years old), plus screening to screening only. Results: 21 regional reports were produced presenting regional results on screening coverage, treatments costs, ICER and ICUR, net cost per subject vaccinated etc. Conclusions: national and regional analyses have two different aims: the former wants to address national regulatory agencies and needs to be representative of the national population whereas the latter deals with the real budget-holders, accountable in the eyes of patients. Furthermore in the Italian scenario, characterized by decentralization and local autonomy, a further level of detail is essential in order to describe the specific local settings and implications of a new health intervention

Puglia

In Apulia region 43,9% of women (aged 25-64) are screened regularly, meaning every 3 years. Considering regional tariffs and vaccine acquisition cost, the vaccination of 12-year-old girls with a 90% coverage could prevent 127 cases of cervicocarcinoma and 52 related deaths and thus results to be cost-effective (13.471€/QALY). When the vaccination programme is extended to 16-year-old girls a further 133 cancer cases and 54 deaths could be prevented, with a very similar cost-effectiveness ratio. In Apulia region, the net cost for woman vaccinated is 137€ for the single cohort and 141€ for the multiple cohort

Cerebellar Ataxia with Complete Clinical Recovery and Resolution of MRI Lesions Related to Central Pontine Myelinolysis: Case Report and Literature Review

There are several reports of central pontine myelinolysis (CPM) in a setting of malnutrition, alcoholism, and chronic debilitating illness associated with electrolyte abnormalities, especially hyponatremia. The cause of myelinolysis is still under debate, and, although osmotic effects are thought to be responsible in most cases, alternative pathological factors should be considered [King et al.: Am J Med Sci 2010;339:561–567]. We report a case of CPM in a patient with recent chemotherapy for colon cancer without electrolyte unbalance and otherwise unexplained causes. Moreover, the present case is an example of the unusual clinical ataxic variant, followed by complete recovery without any specific treatment. The diagnosis was confirmed by MRI, which showed a characteristic hyperintense signal abnormality in the central part of the pons with an unaffected outer rim. One month later, we observed complete resolution of clinical and radiological symptoms

Modeling the Cost-Effectiveness for Cement-Less and Hybrid Prosthesis in Total Hip Replacement in Emilia Romagna, Italy

Background. The aim of the present study was to assess the cost-effectiveness of cement-less versus hybrid prostheses in total hip replacement (THR) in patients diagnosed with primary osteoarthritis. Methods. Effectiveness data were obtained from the Emilia-Romagna Regional Registry on Orthopaedic Prosthesis (RIPO), which collects information on all orthopaedic intervention performed in Emilia-Romagna (41,199 total hip replacements performed from 2000 to 2007), and from which we obtained survival curves and transition probabilities for the cement-less and hybrid prostheses, respectively. Conversely, costs were derived from regional databases through a specific procedure, which allowed us to register individual component's costs for both primary and subsequent revision interventions. A specific Markov transition model was constructed in order to consider the 3 types of revisions that an implant could possibly undergo through its life-span: total, cup or stem, head insert or neck. The cost-effectiveness was expressed in terms of cost per "revision-free" life year. Results and conclusions. Considering a 70-y old patient undergoing THR, the cementless strategy resulted more effective but more costly than the hybrid solution, with an incremental cost effectiveness ratio of 2401.63 (sic) per revision-free life year. Following a deterministic sensitivity analysis, hybrid and cementless fixation showed, respectively, a dominance profile for patients older than 83 y and younger than 43 y, whereas for all ages in between, we report a progressive increase in the ICER of cementless prostheses. Our results proved to be robust, as underlined by the probabilistic sensitivity analysis performed using cost distributions. (C) 2011 Elsevier Inc. All rights reserved

Cartilage Regeneration in Preannealed Silk Elastin-Like Co-Recombinamers Injectable Hydrogel Embedded with Mature Chondrocytes in an Ex Vivo Culture Platform

Producción CientíficaTissue engineering for cartilage repair requires biomaterials that show rapid gelation and adequate mechanical properties. Although the use of hydrogel is the most promising biomaterial, it often lacks in rigidity and anchorage of cells when they are surrounded by synovial fluid while they are subjected to heavy loads. We developed and produced the Silk Elastin-Like co-Recombinamer (SELR), which contains both the physical interaction from elastin motifs and from silk motifs. In the first part of this work, we set up and optimized a preannealing treatment based on the evolution of silk motifs into β-sheet structures in order to fulfill the required mechanical properties of hydrogels for cartilage repair. The new preannealed SELRs (pA(EIS)2-(I5R)6) were characterized with the combination of several experimental techniques (CD, TEM, SEM, and rheology) to provide a deep insight into the material features. Finally, the regeneration properties of the pA(EIS)2-(I5R)6 hydrogel embedded with chondrocytes were evaluated. After 4 weeks of culturing in a standardized and representative ex vivo model, the biochemical and histological analysis revealed the production of glycosaminglycans and collagen. Moreover, the immunohistochemistry showed the absence of fibro-cartilage and the presence of hyaline cartilage. Hence, we conclude that the pA(EIS)2-(I5R)6 hydrogel presents improved mechanical properties while conserving the injectability, which leads to successful regeneration of hyaline cartilage in an ex vivo model.2019-10-202019-10-20Ministerio de Economía, Industria y Competitividad (Proyects PCIN-2015-010, MAT2015-68901-R, MAT2016-78903-R,MAT2016-79435-RJunta de Castilla y León (programa de apoyo a proyectos de investigación – Ref. Project VA015U16)European Union’sHorizon 2020 research and innovation programme under theMarie Sklodowska-Curie grant agreement No. 6426