Caratterizzazione mediante HPLC-MS di modifiche post-trasduzionali di proteine istoniche

Gli istoni sono proteine basiche che possono essere classificate in varie classi: H1, H2A, H2B, H3 e H4. Queste proteine formano l’ottamero proteico attorno al quale si avvolge il DNA per formare il nucleosoma che è l’unità fondamentale della cromatina. A livello delle code N-terminali, gli istoni possono essere soggetti a numerose modifiche posttraduzionali quali acetilazioni, metilazioni, fosforilazioni, ADP-ribosilazioni e ubiquitinazioni. Queste modifiche portano alla formazione di diversi siti di riconoscimento per diversi complessi enzimatici coinvolti in importanti processi come la riparazione e la replicazione del DNA e l’assemblaggio della cromatina. La più importante e la più studiata di queste modifiche è l’acetilazione che avviene a livello dei residui amminici della catena laterale dell’amminoacido lisina. I livelli corretti di acetilazione delle proteine istoniche sono mantenuti dall’attività combinata di due enzimi: istone acetil transferasi (HAT) e istone deacetilasi (HDAC). Gli enzimi appartenenti a questa famiglia possono essere suddivisi in varie classi a seconda delle loro diverse caratteristiche, quali la localizzazione cellulare, la dimensione, l’omologia strutturale e il meccanismo d’azione. Recentemente è stato osservato che livelli aberranti di HDAC sono coinvolti nella carcinogenesi; per questo motivo numerosi gruppi di ricerca sono interessati alla progettazione e alla sintesi di composti che siano in grado di inibire questa classe enzimatica. L’inibizione delle HDAC può infatti provocare arresto della crescita cellulare, apoptosi o morte cellulare. Per questo motivo la ricerca farmaceutica in campo antitumorale è mirata alla sintesi di inibitori selettivi verso le diverse classi di HDAC per sviluppare farmaci meno tossici e per cercare di comprendere con maggiore chiarezza il ruolo biologico di questi enzimi. Il potenziale antitumorale degli inibitori delle HDAC deriva infatti dalla loro capacità di interferire con diversi processi cellulari, generalmente non più controllati nelle cellule neoplastiche. Nella maggior parte dei casi l’attività antitumorale risiede nella capacità di attivare programmi di differenziamento, di inibire la progressione del ciclo cellulare e di indurre apoptosi. Inoltre sembra essere molto importante anche la capacità di attivare la risposta immunitaria e l’inibizione dell’angiogenesi. Gli inibitori delle HDAC possono essere a loro volta classificati in base alla struttura chimica, alla loro origine (naturale o sintetica), e alla loro capacità di inibire selettivamente le HDAC appartenenti a classi diverse. Non è ancora chiaro se la selettività di queste molecole verso una specifica classe di HDAC sia importante per ottenere un effetto antitumorale, ma sicuramente inibitori selettivi possono essere molto utili per investigare e chiarire il ruolo delle HDAC nei processi cellulari che portano all’insorgenza del tumore. Nel primo capitolo di questa tesi quindi è riportata un’introduzione sull’importanza delle proteine istoniche non solo da un punto di vista strutturale ma anche funzionale per il destino cellulare. Nel secondo capitolo è riportato lo stato dell’arte dell’analisi delle proteine istoniche che comprende sia i metodi tradizionali come il microsequenziamento e l’utilizzo di anticorpi, sia metodi più innovativi (RP-LC, HILIC, HPCE) ideati per poter essere accoppiati ad analisi mediante spettrometria di massa. Questa tecnica consente infatti di ottenere importanti e precise informazioni che possono aiutare sia a identificare gli istoni come proteine che a individuare i siti coinvolti nelle modifiche post-traduzionali. Nel capitolo 3 è riportata la prima parte del lavoro sperimentale di questa tesi volto alla caratterizzazione delle proteine istoniche mediante tecniche cromatografiche accoppiate alla spettrometria di massa. Nella prima fase del lavoro è stato messo a punto un nuovo metodo cromatografico HPLC che ha consentito di ottenere una buona separazione, alla linea di base, delle otto classi istoniche (H1-1, H1-2, H2A-1, H2A-2, H2B, H3-1, H3-2 e H4). La separazione HPLC delle proteine istoniche ha permesso di poter eseguire analisi accurate di spettrometria di massa mediante accoppiamento con un analizzatore a trappola ionica tramite la sorgente electrospray (ESI). E’ stato così possibile identificare e quantificare tutte le isoforme istoniche, che differiscono per il tipo e il numero di modifiche post-traduzionali alle quali sono soggette, previa estrazione da colture cellulari di HT29 (cancro del colon). Un’analisi così dettagliata delle isoforme non può essere ottenuta con i metodi immunologici e permette di eseguire un’indagine molto accurata delle modifiche delle proteine istoniche correlandole ai diversi stadi della progressione del ciclo e alla morte cellulare. Il metodo messo a punto è stato convalidato mediante analisi comparative che prevedono la stessa separazione cromatografica ma accoppiata a uno spettrometro di massa avente sorgente ESI e analizzatore Q-TOF, dotato di maggiore sensibilità e risoluzione. Successivamente, per identificare quali sono gli specifici amminoacidi coinvolti nelle diverse modifiche post-traduzionali, l’istone H4 è stato sottoposto a digestione enzimatica e successiva analisi mediante tecniche MALDI-TOF e LC-ESI-MSMS. Queste analisi hanno permesso di identificare le specifiche lisine acetilate della coda N-terminale e la sequenza temporale di acetilazione delle lisine stesse. Nel quarto capitolo sono invece riportati gli studi di inibizione, mirati a caratterizzare le modifiche a carico delle proteine istoniche indotte da inibitori delle HDAC, dotati di diverso profilo di potenza e selettività. Dapprima Il metodo messo a punto per l’analisi delle proteine istoniche è stato applicato all’analisi di istoni estratti da cellule HT29 trattate con due noti inibitori delle HDAC, valproato e butirrato, somministrati alle cellule a dosi diverse, che corrispondono alle dosi con cui sono stati testati in vivo, per convalidare il metodo per studi di inibizione di composti incogniti. Successivamente, lo studio è proseguito con lo scopo di evidenziare effetti legati alla diversa potenza e selettività degli inibitori. Le cellule sono state trattate con due inibitori più potenti, SAHA e MS275, alla stessa concentrazione. In entrambi i casi il metodo messo a punto ha permesso di evidenziare l’aumento dei livelli di acetilazione indotto dal trattamento con gli inibitori; ha inoltre messo in luce differenti livelli di acetilazione. Ad esempio il SAHA, potente inibitore di tutte le classi di HDAC, ha prodotto un’estesa iperacetilazione di tutte le proteine istoniche, mentre MS275 selettivo per la classe I di HDAC, ha prodotto modifiche molto più blande. E’ stato quindi deciso di applicare questo metodo per studiare la dose e la tempo-dipendenza dell’effetto di quattro diversi inibitori delle HDAC (SAHA, MS275, MC1855 e MC1568) sulle modifiche post-traduzionali di istoni estratti da cellule HT29. Questi inibitori differiscono oltre che per la struttura chimica anche per il profilo di selettività nei confronti delle HDAC appartenenti alle diverse classi. Sono stati condotti quindi studi di dose-dipendenza che hanno consentito di ottenere i valori di IC50 (concentrazione capace di ridurre della metà la quantità relativa dell’istone meno acetilato) caratteristici per ogni inibitore nei confronti di tutte le classi istoniche. E’ stata inoltre calcolata la percentuale massima di inibizione per ogni inibitore. Infine sono stati eseguiti studi di tempo-dipendenza. I risultati ottenuti da questi studi hanno permesso di correlare i livelli di acetilazione delle varie classi istoniche con la selettività d’azione e la struttura chimica degli inibitori somministrati alle cellule. In particolare, SAHA e MC1855, inibitori delle HDAC di classi I e II a struttura idrossamica, hanno causato l’iperacetilazione di tutte le proteine istoniche, mentre MC1568 (inibitore selettivo per HDAC di classe II) ha prodotto l’iperacetilazione solo di H4. Inoltre la potenza e la selettività degli inibitori nel provocare un aumento dei livelli di acetilazione a livello delle distinte classi istoniche è stata correlata al destino biologico della cellula, tramite studi di vitalità cellulare. E’ stato osservato che il SAHA e MC1855, inibitori potenti e non selettivi, somministrati alla coltura HT29 a dose 50 μM producono morte cellulare, mentre MS275 alla stessa dose produce accumulo citostatico in G1/G0. MC1568, invece, non produce effetti significatici sul ciclo cellulare. Questo studio ha perciò dimostrato che l’analisi tramite HPLC-ESI-MS delle proteine istoniche permette di caratterizzare finemente la potenza e la selettività di nuovi composti inibitori delle HDAC, prevedendone l’effetto sul ciclo cellulare. In maggiore dettaglio è risultato che l’iperacetilazione di H4 non è in grado di provocare modifiche significative sul ciclo cellulare. Questo metodo, insieme alle analisi MALDI-TOF e LC-ESI-MSMS che permettono di individuare l’ordine di acetilazione delle lisine della coda N-terminale, potrà fornire importanti informazioni sugli inibitori delle HDAC e potrà essere applicato per delineare la potenza, la selettività e il meccanismo di azione di nuovi potenziali inibitori di questa classe enzimatica in colture cellulari tumorali.

Studio dei meccanismi molecolari coinvolti nel determinismo sintomatologico di piante infette da virus e virus-satelliti

The aim of our work was to study the molecular mechanisms involved in symptoms appearance of plants inoculated either with a virus or with a virus-satellite complex. In the first case, we tried to set up a reliable method for an early identification of PVYNTN strains present in Italy and causing potato tuber necrosis. This, to prevent their spread in the field and to avoid severe yield losses, especially in seed potato production. We tried to localize the particular genomic region responsible for tuber necrosis. To this purpose, we carried out RT-PCR experiments using various primer combinations, covering PVY genomic regions larger than those previously used by other authors. As the previous researchers, though, we were not able to differentiate all NTN from others PVY strains. This probably because of the frequent virus variability, due to both genomic mutations and possible recombination events among different strains. In the second case, we studied the influence of Y-sat (CaRNA5 satellite) on symptoms of CMV (Cucumber mosaic virus) in Nicotiana benthamiana plants: strong yellowing appearance instead of simple mosaic. Wang et al (2004), inoculating the same infectious complex on tobacco plants transformed with a viral suppressor of plant silencing (HC-PRO), did not experience the occurrence of yellowing anymore and, therefore, hypotesized that changes in symptoms were due to plant post transcriptional gene silencing (PTGS) mechanism. In our case, inoculation of N. benthamiana plants transformed with another PTGS viral suppressor (p19), and other plants defective for RNA polymerase 6 (involved in systemic silencing), still resulted in yellowing appearance. This, to our opinion, suggests that in our system another possible mechanism is involved.

Costruzione di uno scaffold vascolarizzato per la chirurgia ortopedica

Specific aims The aim is to improve the treatment of the bone losses at the metacarpal bones level (both diaphysis and epiphysis) combining microsurgery, tissue engineering and biomaterials, so to minimize the donor side morbidity and optimize healing and outcomes. Methods Pre-operative controlateral X-ray or 3-D CT to allow custom-made HA scaffolds. Cement as temporary spacer in acute lesion and monitoring of infective risks. Treatment of the bone loss recurring to pre-fabricated or custom-made HA scaffolds, adding platelet gel or growth factor OP1. Stable synthesis. Control group with auto/omografts. Outcome indices: % of bone-union; finger TAM, Kapandji, DASH score; NMR and Scintigraphy at 180 days for revascularisation and bio-substitution of the scaffold. Preliminary results The authors just treated 6 patients, 4 males and 2 females, with an average age of 38.5 yrs, affected by segmental bone losses at the hand and wrist, recurring to pre-fabricated not vascularised scaffolds. In all cases the synthesis was performed with angular stability plates and a stable synthesis achieved. All patients have been controlled at a mean follow-up of 10.5 months (from 2 to 16 ). In all case but one the bone-scaffold osteo-integration was achieved at an average of 38 days at the hand, and 46 days at the wrist. The outcome studies, according to the DASH score, finger TAM, and Kapandji, were good and excellent in 5 cases, poor in one.

Analisi della rete di trasporto di Manduria: implementazione di un modello di simulazione

Il sistema viabile rappresenta una parte fondamentale nella dinamica dei trasporti al servizio della mobilità di trasporti e merci. La mobilità, ha fatto registrare ritmi di crescita sostenuti nel corso degli anni. Le trasformazioni sociali, del mercato del lavoro, lo sviluppo produttivo e commerciale, la posizione baricentrica tra le province jonca salentina e brindisina fanno di Manduria, una realtà dove è necessario prestare attenzione al sistema dei trasporti. Tuttavia la rete statale di vecchio stampo che risulta utilizzata, soprattutto da quote di mobilità interprovinciale per questioni di economicità del percorso e dal momento che manca una rete viaria ordinaria funzionale e scorrevole che colleghi in modo capillare il territorio fanno si che la Strada Statale 7ter risulti congestionata in maniera diffusa. La Superstrada di nuova costruzione sarebbe potuta essere un importante arteria stradale allacciandosi alla SS Taranto Brindisi Lecce collegando il sud della provincia Jonica in modo capillare ai comuni della provincia Taranto e Lecce, la quale non è stata ultimata ed avrebbe rappresentato una valida alternativa alla statale esistenti. Oltre a favorire i collegamenti tra il territorio jonico, brindisino e quello salentino, tale intervento sarebbe stato necessario per ridurre la componente di traffico di attraversamento di una serie di centri urbani, tra cui il Comune di Manduria. In questa tesi, sulla base dei dati raccolti dall’ultimo censimento nell’ambito provinciale e del Comune di Manduria, verrà implementato il funzionamento del Piano del Traffico stradale del Comune di Manduria e verranno condotte delle simulazioni, con l’ausilio del software di pianificazione di sistemi di trasporto stradale Omnitrans, versione demo, scaricabile gratuitamente dal sito www.omnitrans-international.com. Si ricercheranno stime sul funzionamento della rete, valutando la congestione del traffico ed eventualmente in termini di aumento della sicurezza e riduzione dell’inquinamento atmosferico.

Il contributo dell’Unione europea all’affermazione dello stato di diritto nella comunità internazionale

This thesis is aimed at analysing EU external relations from the perspective of the promotion of the rule of law in order to evaluate the effectiveness and consistency of its action within the international community. The research starts with an examination of the notion of the rule of law from a theoretical point of view. The first chapter initially describes the historical-political evolution of the establishment of the notion of the rule of law. Some of the most significant national experiences (France, the UK, Germany and Austria) are discussed. Then, the focus is put on the need to propose interpretations which explain the grounds of the rule of law, by highlighting the different formal and substantive interpretations. This philosophical-historical analysis is complemented by a reconstruction of how the notion of the rule of law was developed by the international community, with a view to searching a common notion at the international level by comparing theory and practice within the main international organisations such as the UN, OECD and the Council of Europe. Specific mention is made of the EU experience, whose configuration as a Community based on the rule of law is often debated, starting from the case law of the European Court of Justice. The second chapter deals with the conditionality policy and focuses on the development and scope of democratic conditionality according to the dominant approach of the doctrine. First, the birth of conditionality is analysed from an economic point of view, especially within international financial organisations and the different types of conditionality recreated in the scientific sector. Then an analysis is provided about the birth of democratic conditionality in the EC – in relation to its external relations – firstly as a mere political exercise to be then turned into a standardised system of clauses. Specific reference is made to the main scope of conditionality, that is to say enlargement policy and the development of the Copenhagen criteria. The third chapter provides further details about the legal questions connected to the use of democratic clauses: on the one hand, the power of the EC to include human rights clauses in international agreements, on the other, the variety and overlapping in the use of the legal basis. The chapter ends with an analysis of the measures of suspension of agreements with third countries in those rare but significant cases in which the suspension clause, included in the Lomè Convention first and in the Cotonou Agreement then, is applied. The last chapter is devoted to the analysis of democratic clauses in unilateral acts adopted by the European Union which affect third countries. The examination of this practice and the comparison with the approach analysed in the previous chapter entails a major theoretical question. It is the clear-cut distinction between conditionality and international sanction. This distinction is to be taken into account when considering the premises and consequences, in terms of legal relations, which are generated when democratic clauses are not complied with. The chapter ends with a brief analysis of what, according to the reconstruction suggested, can be rightly labelled as real democratic conditionality, that is to say the system of incentives, positive measures developed within the community GSP. The dissertation ends with a few general considerations about the difficulties experienced by the EU in promoting the rule of law. The contradictory aspects of the EU external actions are manifold, as well as its difficulties in choosing the most appropriate measures to be taken which, however, reflect all the repercussions and tension resulting from the balance of power within the international community. The thesis argues that it is difficult to grant full credibility to an entity like the EU which, although it proclaims itself as the guardian and promoter of the rule of law, in practice, is too often biased in managing its relations with third countries. However, she adds, we must acknowledge that the EU is committed and constantly strives towards identifying new spaces and strategies of action.

Progettazione acustica per il recupero polifunzionale di un ambiente a pianta ottagonale

L’obiettivo di questo lavoro è illustrare l’analisi dello stato di fatto e la progettazione degli interventi di correzione acustica della torre dell’ ex Carico dell’Acqua di Budrio, nella prospettiva di renderla utilizzabile come sala polifunzionale. La norma tecnica ISO 3382-1 [8] delinea criteri generali per l’analisi qualitativa degli ambienti di ascolto. Il capitolo 1 presenta un panorama sintetico sull’acustica architettonica, con particolare riferimento ai descrittori acustici riportati nella ISO 3382 e riporta cenni teorici sulla simulazione numerica. Nel capitolo 2 sono descritte in dettaglio le misurazioni all’interno dell’ambiente. Sono indicate inoltre motivazioni tecniche e teoriche riguardo i metodi di analisi. I valori misurati sono successivamente confrontati con i valori ricavati mediante simulazione numerica realizzata con un software previsionale. La realizzazione e la successiva taratura del modello numerico chiudono il capitolo 2. Il capitolo 3 relaziona in merito agli interventi proposti di correzione acustica. Attraverso il modello tridimensionale tarato si eseguono simulazioni numeriche di diversi interventi di correzione acustica fino a definire una rosa di possibili soluzioni che realizzino gli obiettivi qualitativi riportati nella ISO 3382. Materiali e geometrie sono riportati in dettaglio, insieme ad una esauriente mappatura acustica dell’ambiente, dallo stato di fatto alla progressiva ottimizzazione acustica.

Viola-Jones based detectors: How much affects the training set?

[EN]This paper presents a study on the facial feature detection performance achieved using the Viola-Jones framework. A set of classi- ers using two di erent focuses to gather the training samples is created and tested on four di erent datasets covering a wide range of possibili- ties. The results achieved should serve researchers to choose the classi er that better ts their demands.

Valutazione delle politiche di sviluppo locale: una comparazione internazionale

Lo sviluppo locale rappresenta, non solo per gli economisti, un tema di analisi sempre più rilevante sia al livello istituzionale che al livello scientifico. La complessità degli aspetti inerenti lo sviluppo locale richiede il coinvolgimento di diverse discipline, in ambito economico, politico, sociale e ambientale e di tutti i livelli istituzionali. Parallelamente è cresciuta l’esigenza di processi valutativi coerenti e sistematici, basati su di un numero sempre maggiore di strumenti e metodologie di valutazione. Dall’orientamento della Commissione Europea emerge del resto con sempre maggiore evidenza il binomio fra politica di sviluppo locale e valutazione, che coinvolge i diversi livelli di governo. Il presente lavoro realizza un quadro delle politiche di sviluppo locale, partendo dal livello europeo fino ad arrivare al livello locale, ed una successiva analisi di metodologie e strumenti di valutazione consolidati e di frontiera. La considerazione della valutazione come strumento strategico per le politiche di sviluppo locale trova applicazione nella realizzazione di una analisi comparativa di due aree di montagna. Tali aree, identificate nell’Appennino Bolognese e nell’area montana della Contea di Brasov in Romania, pur collocate in paesi a diverso livello di sviluppo, risultano confrontabili, in termini di similitudini e criticità, al fine di trarre considerazioni di policy inerenti il disegno di adeguate politiche di riqualificazione, mettendo in luce l’importanza del processo valutativo e la necessità di contribuire a diffondere una vera e propria cultura della valutazione.

Materiali a memoria di forma: caratterizzazione e applicazioni nel campo dei beni culturali

Questo lavoro di tesi nasce da un progetto di ricerca promosso nel 2001 dal Prof. Leonardo Seccia (Seconda Facoltà di Ingegneria, sede di Forlì, e C.I.R.A.M., Università di Bologna), dal Prof. Nicola Santopuoli (Facoltà di Architettura Valle Giulia, Sapienza Università di Roma), dal Prof. Ingo Muller e dal Dott. André Musolff (Technical University Berlin, Facultat III, Thermodynamics). Tale progetto ha avuto come obiettivo lo studio, la progettazione e la realizzazione di un dispositivo di ancoraggio in lega a memoria di forma per il restauro di affreschi e mosaici parietali, che presentino distacchi più o meno evidenti fra gli strati di intonaco di supporto, proponendosi come mezzo efficace per la salvaguardia strutturale di tali zone variamente ammalorate. In particolare, è stata programmata una serie di prove di laboratorio per caratterizzare in modo preciso il comportamento del materiale prescelto, al fine di realizzare un prototipo rispondente alle caratteristiche di progetto ed anche per implementare un modello numerico sufficientemente realistico. A questo proposito, è stato anche approfondito il problema della scelta del modello costitutivo più adeguato. Successivamente, i risultati ottenuti sono stati impiegati nella progettazione e realizzazione di nuovi dispositivi in lega a memoria di forma da impiegare nel campo dei beni culturali, fra cui sistemi reversibili per il ricongiungimento di parti fratturate e sistemi di movimentazione intelligenti sia per lastre di protezione di superfici affrescate, sia per finestre da inserire in contesti museali per il controllo del microclima.

Study of apoptotic deletion mediated by Bifidobacterium longum with construction of recombinant strains for Serpin encoding gene and phenotypes comparison in a pig cell model

The first part of the research project of the Co-Advisorship Ph.D Thesis was aimed to select the best Bifidobacterium longum strains suitable to set the basis of our study. We were looking for strains with the abilities to colonize the intestinal mucosa and with good adhesion capacities, so that we can test these strains to investigate their ability to induce apoptosis in “damaged” intestinal cells. Adhesion and apoptosis are the two process that we want to study to better understand the role of an adhesion protein that we have previously identified and that have top scores homologies with the recent serpin encoding gene identified in B. longum by Nestlè researchers. Bifidobacterium longum is a probiotic, known for its beneficial effects to the human gut and even for its immunomodulatory and antitumor activities. Recently, many studies have stressed out the intimate relation between probiotic bacteria and the GIT mucosa and their influence on human cellular homeostasis. We focused on the apoptotic deletion of cancer cells induced by B. longum. This has been valued in vitro, performing the incubation of three B.longum strains with enterocyte-like Caco- 2 cells, to evidence DNA fragmentation, a cornerstone of apoptosis. The three strains tested were defined for their adhesion properties using adhesion and autoaggregation assays. These features are considered necessary to select a probiotic strain. The three strains named B12, B18 and B2990 resulted respectively: “strong adherent”, “adherent” and “non adherent”. Then, bacteria were incubated with Caco-2 cells to investigate apoptotic deletion. Cocultures of Caco-2 cells with B. longum resulted positive in DNA fragmentation test, only when adherent strains were used (B12 and B18). These results indicate that the interaction with adherent B. longum can induce apoptotic deletion of Caco-2 cells, suggesting a role in cellular homeostasis of the gastrointestinal tract and in restoring the ecology of damaged colon tissues. These results were used to keep on researching and the strains tested were used as recipient of recombinant techniques aimed to originate new B.longum strains with enhanced capacity of apoptotic induction in “damaged” intestinal cells. To achieve this new goal it was decided to clone the serpin encoding gene of B. longum, so that we can understand its role in adhesion and apoptosis induction. Bifidobacterium longum has immunostimulant activity that in vitro can lead to apoptotic response of Caco-2 cell line. It secretes a hypothetical eukaryotic type serpin protein, which could be involved in this kind of deletion of damaged cells. We had previously characterised a protein that has homologies with the hypothetical serpin of B. longum (DD087853). In order to create Bifidobacterium serpin transformants, a B. longum cosmid library was screened with a PCR protocol using specific primers for serpin gene. After fragment extraction, the insert named S1 was sub-cloned into pRM2, an Escherichia coli - Bifidobacterium shuttle vector, to construct pRM3. Several protocols for B. longum transformation were performed and the best efficiency was obtained using MRS medium and raffinose. Finally bacterial cell supernatants were tested in a dotblot assay to detect antigens presence against anti-antitrypsin polyclonal antibody. The best signal was produced by one starin that has been renamed B. longum BLKS 7. Our research study was aimed to generate transformants able to over express serpin encoding gene, so that we can have the tools for a further study on bacterial apoptotic induction of Caco-2 cell line. After that we have originated new trasformants the next step to do was to test transformants abilities when exposed to an intestinal cell model. In fact, this part of the project was achieved in the Department of Biochemistry of the Medical Faculty of the University of Maribor, guest of the abroad supervisor of the Co-Advisorship Doctoral Thesis: Prof. Avrelija Cencic. In this study we examined the probiotic ability of some bacterial strains using intestinal cells from a 6 years old pig. The use of intestinal mammalian cells is essential to study this symbiosis and a functional cell model mimics a polarised epithelium in which enterocytes are separated by tight junctions. In this list of strains we have included the Bifidobacterium longum BKS7 transformant strain that we have previously originated; in order to compare its abilities. B. longum B12 wild type and B. longum BKS7 transformant and eight Lactobacillus strains of different sources were co-cultured with porcine small intestine epithelial cells (PSI C1) and porcine blood monocytes (PoM2) in Transwell filter inserts. The strains, including Lb. gasseri, Lb. fermentum, Lb. reuterii, Lb. plantarum and unidentified Lactobacillus from kenyan maasai milk and tanzanian coffee, were assayed for activation of cell lines, measuring nitric oxide by Griess reaction, H202 by tetramethylbenzidine reaction and O2 - by cytochrome C reduction. Cytotoxic effect by crystal violet staining and induction on metabolic activity by MTT cell proliferation assay were tested too. Transepithelial electrical resistance (TER) of polarised PSI C1 was measured during 48 hours co-culture. TER, used to observe epithelium permeability, decrease during pathogenesis and tissue becomes permeable to ion passive flow lowering epithelial barrier function. Probiotics can prevent or restore increased permeability. Lastly, dot-blot was achieved against Interleukin-6 of treated cells supernatants. The metabolic activity of PoM2 and PSI C1 increased slightly after co-culture not affecting mitochondrial functions. No strain was cytotoxic over PSI C1 and PoM2 and no cell activation was observed, as measured by the release of NO2, H202 and O2 - by PoM2 and PSI C1. During coculture TER of polarised PSI C1 was two-fold higher comparing with constant TER (~3000 ) of untreated cells. TER raise generated by bacteria maintains a low permeability of the epithelium. During treatment Interleukin-6 was detected in cell supernatants at several time points, confirming immunostimulant activity. All results were obtained using Lactobacillus paracasei Shirota e Carnobacterium divergens as controls. In conclusion we can state that both the list of putative probiotic bacteria and our new transformant strain of B. longum are not harmful when exposed to intestinal cells and could be selected as probiotics, because can strengthen epithelial barrier function and stimulate nonspecific immunity of intestinal cells on a pig cell model. Indeed, we have found out that none of the strains tested that have good adhesion abilities presents citotoxicity to the intestinal cells and that non of the strains tested can induce cell lines to produce high level of ROS, neither NO2. Moreover we have assayed even the capacity of producing certain citokynes that are correlated with immune response. The detection of Interleukin-6 was assayed in all our samples, including B.longum transformant BKS 7 strain, this result indicates that these bacteria can induce a non specific immune response in the intestinal cells. In fact, when we assayed the presence of Interferon-gamma in cells supernatant after bacterial exposure, we have no positive signals, that means that there is no activation of a specific immune response, thus confirming that these bacteria are not recognize as pathogen by the intestinal cells and are certainly not harmful for intestinal cells. The most important result is the measure of Trans Epithelial Electric Resistance that have shown how the intestinal barrier function get strengthen when cells are exposed to bacteria, due to a reduction of the epithelium permeability. We have now a new strain of B. longum that will be used for further studies above the mechanism of apoptotic induction to “damaged cells” and above the process of “restoring ecology”. This strain will be the basis to originate new transformant strains for Serpin encoding gene that must have better performance and shall be used one day even in clinical cases as in “gene therapy” for cancer treatment and prevention.

Analisi del comportamento a caldo dell'acciaio AISI - H11 per la stima della vita utile di matrici per estrusione

The effect of process parameters on the creep-fatigue behavior of a hot-work tool steel for aluminum extrusion die was investigated through a technological test in which the specimen geometry resembled the mandrel of a hollow extrusion die. Tests were performed on a Gleeble thermomechanical simulator by heating the specimen using joule’s effect and by applying cyclic loading up to 6.30 h or till specimen failure. Displacements during the tests at 380, 490, 540 and 580°C and under the average stresses of 400, 600 and 800 MPa were determined. In the first set of test a dwell time of 3 min was introduced during each of the tests to understand the creep behavior. The results showed that the test could indeed physically simulate the cyclic loading on the hollow die during extrusion and reveal all the mechanisms of creep-fatigue interaction. In the second set a pure fatigue laod were induced and in the third set a static creep load were induced in the specimens. Furher type of tests, finite element and microstructural analysis were presented.

Sviluppo di un tomografo multi-energy per lo studio pre-clinico di nuove metodiche diagnostiche finalizzate al riconoscimento precoce della patologia tumorale

A new multi-energy CT for small animals is being developed at the Physics Department of the University of Bologna, Italy. The system makes use of a set of quasi-monochromatic X-ray beams, with energy tunable in a range from 26 KeV to 72 KeV. These beams are produced by Bragg diffraction on a Highly Oriented Pyrolytic Graphite crystal. With quasi-monochromatic sources it is possible to perform multi-energy investigation in a more effective way, as compared with conventional X-ray tubes. Multi-energy techniques allow extracting physical information from the materials, such as effective atomic number, mass-thickness, density, that can be used to distinguish and quantitatively characterize the irradiated tissues. The aim of the system is the investigation and the development of new pre-clinic methods for the early detection of the tumors in small animals. An innovative technique, the Triple-Energy Radiography with Contrast Medium (TER), has been successfully implemented on our system. TER consist in combining a set of three quasi-monochromatic images of an object, in order to obtain a corresponding set of three single-tissue images, which are the mass-thickness map of three reference materials. TER can be applied to the quantitative mass-thickness-map reconstruction of a contrast medium, because it is able to remove completely the signal due to other tissues (i.e. the structural background noise). The technique is very sensitive to the contrast medium and is insensitive to the superposition of different materials. The method is a good candidate to the early detection of the tumor angiogenesis in mice. In this work we describe the tomographic system, with a particular focus on the quasi-monochromatic source. Moreover the TER method is presented with some preliminary results about small animal imaging.

New approaches to open problems in gene expression microarray data

In the past decade, the advent of efficient genome sequencing tools and high-throughput experimental biotechnology has lead to enormous progress in the life science. Among the most important innovations is the microarray tecnology. It allows to quantify the expression for thousands of genes simultaneously by measurin the hybridization from a tissue of interest to probes on a small glass or plastic slide. The characteristics of these data include a fair amount of random noise, a predictor dimension in the thousand, and a sample noise in the dozens. One of the most exciting areas to which microarray technology has been applied is the challenge of deciphering complex disease such as cancer. In these studies, samples are taken from two or more groups of individuals with heterogeneous phenotypes, pathologies, or clinical outcomes. these samples are hybridized to microarrays in an effort to find a small number of genes which are strongly correlated with the group of individuals. Eventhough today methods to analyse the data are welle developed and close to reach a standard organization (through the effort of preposed International project like Microarray Gene Expression Data -MGED- Society [1]) it is not unfrequant to stumble in a clinician's question that do not have a compelling statistical method that could permit to answer it.The contribution of this dissertation in deciphering disease regards the development of new approaches aiming at handle open problems posed by clinicians in handle specific experimental designs. In Chapter 1 starting from a biological necessary introduction, we revise the microarray tecnologies and all the important steps that involve an experiment from the production of the array, to the quality controls ending with preprocessing steps that will be used into the data analysis in the rest of the dissertation. While in Chapter 2 a critical review of standard analysis methods are provided stressing most of problems that In Chapter 3 is introduced a method to adress the issue of unbalanced design of miacroarray experiments. In microarray experiments, experimental design is a crucial starting-point for obtaining reasonable results. In a two-class problem, an equal or similar number of samples it should be collected between the two classes. However in some cases, e.g. rare pathologies, the approach to be taken is less evident. We propose to address this issue by applying a modified version of SAM [2]. MultiSAM consists in a reiterated application of a SAM analysis, comparing the less populated class (LPC) with 1,000 random samplings of the same size from the more populated class (MPC) A list of the differentially expressed genes is generated for each SAM application. After 1,000 reiterations, each single probe given a "score" ranging from 0 to 1,000 based on its recurrence in the 1,000 lists as differentially expressed. The performance of MultiSAM was compared to the performance of SAM and LIMMA [3] over two simulated data sets via beta and exponential distribution. The results of all three algorithms over low- noise data sets seems acceptable However, on a real unbalanced two-channel data set reagardin Chronic Lymphocitic Leukemia, LIMMA finds no significant probe, SAM finds 23 significantly changed probes but cannot separate the two classes, while MultiSAM finds 122 probes with score >300 and separates the data into two clusters by hierarchical clustering. We also report extra-assay validation in terms of differentially expressed genes Although standard algorithms perform well over low-noise simulated data sets, multi-SAM seems to be the only one able to reveal subtle differences in gene expression profiles on real unbalanced data. In Chapter 4 a method to adress similarities evaluation in a three-class prblem by means of Relevance Vector Machine [4] is described. In fact, looking at microarray data in a prognostic and diagnostic clinical framework, not only differences could have a crucial role. In some cases similarities can give useful and, sometimes even more, important information. The goal, given three classes, could be to establish, with a certain level of confidence, if the third one is similar to the first or the second one. In this work we show that Relevance Vector Machine (RVM) [2] could be a possible solutions to the limitation of standard supervised classification. In fact, RVM offers many advantages compared, for example, with his well-known precursor (Support Vector Machine - SVM [3]). Among these advantages, the estimate of posterior probability of class membership represents a key feature to address the similarity issue. This is a highly important, but often overlooked, option of any practical pattern recognition system. We focused on Tumor-Grade-three-class problem, so we have 67 samples of grade I (G1), 54 samples of grade 3 (G3) and 100 samples of grade 2 (G2). The goal is to find a model able to separate G1 from G3, then evaluate the third class G2 as test-set to obtain the probability for samples of G2 to be member of class G1 or class G3. The analysis showed that breast cancer samples of grade II have a molecular profile more similar to breast cancer samples of grade I. Looking at the literature this result have been guessed, but no measure of significance was gived before.

Wordline approach to higher spin fields

The main object of this thesis is the analysis and the quantization of spinning particle models which employ extended ”one dimensional supergravity” on the worldline, and their relation to the theory of higher spin fields (HS). In the first part of this work we have described the classical theory of massless spinning particles with an SO(N) extended supergravity multiplet on the worldline, in flat and more generally in maximally symmetric backgrounds. These (non)linear sigma models describe, upon quantization, the dynamics of particles with spin N/2. Then we have analyzed carefully the quantization of spinning particles with SO(N) extended supergravity on the worldline, for every N and in every dimension D. The physical sector of the Hilbert space reveals an interesting geometrical structure: the generalized higher spin curvature (HSC). We have shown, in particular, that these models of spinning particles describe a subclass of HS fields whose equations of motions are conformally invariant at the free level; in D = 4 this subclass describes all massless representations of the Poincar´e group. In the third part of this work we have considered the one-loop quantization of SO(N) spinning particle models by studying the corresponding partition function on the circle. After the gauge fixing of the supergravity multiplet, the partition function reduces to an integral over the corresponding moduli space which have been computed by using orthogonal polynomial techniques. Finally we have extend our canonical analysis, described previously for flat space, to maximally symmetric target spaces (i.e. (A)dS background). The quantization of these models produce (A)dS HSC as the physical states of the Hilbert space; we have used an iterative procedure and Pochhammer functions to solve the differential Bianchi identity in maximally symmetric spaces. Motivated by the correspondence between SO(N) spinning particle models and HS gauge theory, and by the notorious difficulty one finds in constructing an interacting theory for fields with spin greater than two, we have used these one dimensional supergravity models to study and extract informations on HS. In the last part of this work we have constructed spinning particle models with sp(2) R symmetry, coupled to Hyper K¨ahler and Quaternionic-K¨ahler (QK) backgrounds.

Computational methods for genome screening

Motivation An actual issue of great interest, both under a theoretical and an applicative perspective, is the analysis of biological sequences for disclosing the information that they encode. The development of new technologies for genome sequencing in the last years, opened new fundamental problems since huge amounts of biological data still deserve an interpretation. Indeed, the sequencing is only the first step of the genome annotation process that consists in the assignment of biological information to each sequence. Hence given the large amount of available data, in silico methods became useful and necessary in order to extract relevant information from sequences. The availability of data from Genome Projects gave rise to new strategies for tackling the basic problems of computational biology such as the determination of the tridimensional structures of proteins, their biological function and their reciprocal interactions. Results The aim of this work has been the implementation of predictive methods that allow the extraction of information on the properties of genomes and proteins starting from the nucleotide and aminoacidic sequences, by taking advantage of the information provided by the comparison of the genome sequences from different species. In the first part of the work a comprehensive large scale genome comparison of 599 organisms is described. 2,6 million of sequences coming from 551 prokaryotic and 48 eukaryotic genomes were aligned and clustered on the basis of their sequence identity. This procedure led to the identification of classes of proteins that are peculiar to the different groups of organisms. Moreover the adopted similarity threshold produced clusters that are homogeneous on the structural point of view and that can be used for structural annotation of uncharacterized sequences. The second part of the work focuses on the characterization of thermostable proteins and on the development of tools able to predict the thermostability of a protein starting from its sequence. By means of Principal Component Analysis the codon composition of a non redundant database comprising 116 prokaryotic genomes has been analyzed and it has been showed that a cross genomic approach can allow the extraction of common determinants of thermostability at the genome level, leading to an overall accuracy in discriminating thermophilic coding sequences equal to 95%. This result outperform those obtained in previous studies. Moreover, we investigated the effect of multiple mutations on protein thermostability. This issue is of great importance in the field of protein engineering, since thermostable proteins are generally more suitable than their mesostable counterparts in technological applications. A Support Vector Machine based method has been trained to predict if a set of mutations can enhance the thermostability of a given protein sequence. The developed predictor achieves 88% accuracy.