The tectonically-confined Firenzuola turbidite system (Miocene, Marnoso-arenacea Formation, northern Apennines, Italy): an example of facies variations related to the basin morphology

The Firenzuola turbidite system formed during a paroxysmal phase of thrust propagation, involving the upper Serravallian deposits of the Marnoso-arenacea Formation (MAF). During this phase the coeval growth of two major tectonic structures, the M. Castellaccio thrust and the Verghereto high, played a key role, causing a closure of the inner basin and a coeval shift of the depocentre to the outer basin. This work focuses on this phase of fragmentation of the MAF basin; it is based on a new detailed high-resolution stratigraphic framework, which was used to determine the timing of growth of the involved structures and their direct influence on sediment dispersal and on the lateral and vertical turbidite facies distribution. The Firenzuola turbidite system stratigraphy is characterized by the occurrence of mass-transport complexes (MTCs) and thick sandstone accumulation in the depocentral area, which passes to finer drape over the structural highs; the differentiation between these two zones increases over time and ends with the deposition of marly units over the structural highs and the emplacement of the Visignano MTC. According to the stratigraphic pattern and turbidite facies characteristics, the Firenzuola System has been split into two phases, namely Firenzuola I and Firenzuola II: the former is quite similar to the underlying deposits, while the latter shows the main fragmentation phase, testifying the progressive isolation of the inner basin and a coeval shift of the depocentre to the outer basin. The final stratigraphic and sedimentological dataset has been used to create a quantitative high-resolution 3D facies distribution using the Petrel software platform. This model allows a detailed analysis of lateral and vertical facies variations that can be exported to several reservoirs settings in hydrocarbon exploration and exploitation areas, since facies distributions and geometries of the reservoir bodies of many sub-surface turbidite basins show a significant relationship to the syndepositional structural activity, but are beyond seismic resolution.

Liposome-mediated DNA immunisation via the subcutaneous route

Compared to naked DNA immunisation, entrapment of plasmid-based DNA vaccines into liposomes by the dehydration-rehydration method has shown to enhance both humoural and cell-mediated immune responses to encoded antigens administered by a variety of routes. In this paper, we have investigated the application of liposome-entrapped DNA and their cationic lipid composition on such potency after subcutaneous immunisation. Plasmid pI.18Sfi/NP containing the nucleoprotein (NP) gene of A/Sichuan/2/87 (H3N2) influenza virus in the pI.18 expression vector was incorporated by the dehydration-rehydration method into liposomes composed of 16 μmol egg phosphatidylcholine (PC), 8 μmoles dioleoyl phosphatidylethanolamine (DOPE) or cholesterol (Chol) and either the cationic lipid 1,2-diodeoyl-3-(trimethylammonium) propane (DOTAP) or cholesteryl 3-N-(dimethyl amino ethyl) carbamate (DC-Chol). This method, entailing mixing of small unilamellar vesicles (SUV) with DNA, followed by dehydration and rehydration, yielded incorporation values of 90-94% of the DNA used. Mixing or rehydration of preformed cationic liposomes with 100 μg plasmid DNA also led to similarly high complexation values (92-94%). In an attempt to establish differences in the nature of DNA association with these various liposome preparations their physico-chemical characteristics were investigated. Studies on vesicle size, zeta potential and gel electrophoresis in the presence of the anion sodium dodecyl sulphate (SDS) indicate that, under the conditions employed, formulation of liposomal DNA by the dehydration-rehydration generated submicron size liposomes incorporating most of the DNA in a manner that prevents DNA displacement through anion competition. The bilayer composition of these dehydration-rehydration vesicles (DRV(DNA)) can also further influence these physicochemical characteristics with the presence of DOPE within the liposome bilayer resulting in a reduced vesicle zeta potential. Subcutaneous liposome-mediated DNA immunisation employing two DRV(DNA) formulations as well as naked DNA revealed that humoural responses (immunoglobulin total IgG, and subclasses IgG1 and 1gG2a) engendered by the plasmid encoded NP were substantially higher after dosing twice, 28 days apart with 10 μg liposome-entrapped DNA compared to naked DNA. At all time points measured, mice immunised with naked DNA showed no greater immune response compared to the control, non-immunised group. In contrast, as early as day 49, responses were significantly higher in mice injected with DNA entrapped in DRV liposomes containing DOTAP compared to the control group and mice immunised with naked DNA. By day 56, all total IgG responses from mice immunised with both DRV formulations were significantly higher. Comparison between the DRV formulations revealed no significant difference in immune responses elicited except at day 114, where the humoural responses of the group injected with liposomal formulation containing DC-Chol dropped to significantly lower levels that those measured in mice which received the DOTAP formulation. Similar results were found when the IgG1 and IgG2a subclass responses were determined. These results suggest that, not only can DNA be effectively entrapped within liposomes using the DRV method but that such DRV liposomes containing DNA may be a useful system for subcutaneous delivery of DNA vaccines. © 2003 Taylor & Francis Ltd.

The activity of pH membrane disruptive pseudopeptides and their subcellular fate in mammalian cells cultured in-vitro

This thesis describes investigations upon pseudopeptides which were conducted to improve our understanding of the fate of synthetic macromolecules in cells and to develop approaches to influence that fate. The low uptake of molecules across the external cellular membrane is the principal barrier against effective delivery of therapeutic products to within the cell structure. In nature, disruption of this membrane by amphiphilic peptides plays a central role in the pathogenesis by bacterial and toxin infections. These amphiphilic peptides contain both hydrophobic and weakly charged hydrophilic amino acid residues and upon activation they become integrated into the lipid bilayers of the extracellular or endosomal membranes. The architectures of the pseudopeptides described here were designed to display similar pH dependent membrane rupturing activity to that of peptides derived from the influenza virus hemagglutinin HA-2. This HA protein promotes fusion of the influenza virus envelope with the cell endosome membrane due to a change in conformation in response to the acidic pH of the endosome lumen (pH 5.0-6.0). The pseudopeptides were obtained by the copolymerisation of L-lysine and L-lysine ethyl-ester with various dicarboxylic acid moieties. In this way a linear polyamide comprising of alternating pendant carboxylic acids and pendant hydrophobic moieties was made. At physiological pH (pH 7.4), electrostatic repulsion of pendant anionic carboxyl groups along the polymer backbone is sufficient to overcome the intramolecular association of the hydrophobic groups resulting in an extended conformation. At low pH (typically pH 4.8) loss of charge results in increased intramolecular hydrophobic association and the polymer chain collapses to a compact conformation, leading to precipitation of the polymer. Consequently, a conformation dependent functional property could be made to respond to small changes in the environmental pH. Pseudopepides were investigated for their cytoxicity towards a well known cell line, namely C26 (colorectal adenocarcinoma) and were shown through the use of a cell viability assay, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) to be well tolerated by C26 cells over a range of concentrations (2-500,μg/ml) at physiological pH (pH 7.4). A modified version of a shorter 30-minute coupled enzymatic assay, the LDH (lactate dehydrogenase) assay was used to evaluate the ability of the pseudopeptides to disrupt the membrane of two different cell lines (COS-1; African green monkey, kidney and A2780; human ovarian carcinoma) at low pH (pH 5.5). The cell membrane disruption property of the pseudopeptides was successfully demonstrated for COS-I and A2780 cell lines at this pH (pH 5.5). A variety of cell lines were chosen owing to limited availability and to compare the cytotoxic action of these pH responsive psudopeptides towards normal and tumorogenic cell lines. To investigate the intracellular delivery of one of the pseudopeptides, poly (L-lysine iso-phthalamide) and its subcellular location, a Cy3 bisamine fluorophore was conjugated into its backbone, at ratios of dye:lysine of 1:20, 1:30, 1:40, 1:60 and 1:80. Native polyacrylacrylamide gel electrophoresis (PAGE) and high voltage paper electrophoresis (HVPE) studies of the polydyes were conducted and provided evidence that that the Cy3 bisamine fluorophore was conjugated into the backbone of the polymer, poly (L-lysine iso-phthalamide). The subcellular fate of the fluorescentlylabelled "polydye" (hereafter PD20) was monitored by laser scanning confocal microscopy (LSCM) in CHO (Chinese hamster ovary) cells cultured in-vitro at various pH values (pH 7.4 and 5.0). LSCM images depicting time-dependent internalisation of PD20 indicated that PD20 traversed the extracellular membrane of CHO cells cultured in-vitro within ten minutes and migrated towards the endosomal regions where the pH is in the region of 5.0 to 6.0. Nuclear localisation of PD20 was demonstrated in a subpopulation of CHO cells. A further study was completed in CHO and HepG2 (hepatocellular carcinoma) cells cultured in-vitro using a lower molecular weight polymer to demonstrate that the molecular weight of "polydye" could be tailored to attain nuclear trafficking in cells. Prospective use of this technology encompasses a method of delivering a payload into a living cell based upon the hypercoiling nature of the pseudopeptides studied in this thesis and has led to a patent application (GB0228525.2; 20(2).

The 'Healthy Passport' intervention with older people in an English urban environment:effects of incentives and peer-group organisers in promoting healthy living

This paper reports the evaluation of the effectiveness of incentives (viz. points and prizes) and of peer-group organisers ('older people's champions') in the outcomes of a health-improvement programme for people aged 50 + years in a multi-ethnic district of the West Midlands, England. Health promotion activities Were provided, and adherence, outcome variables and barriers to adherence were assessed over six months, using a `passport' format. Those aged in the fifties and of Asian origin Were under represented, but people of Afro-Caribbean origin were well represented and proportionately most likely to stay in the project. Those of greater age and With more illness were most likely to drop out. There were significant improvements in exercise, diet and the uptake of influenza vaccines and eyesight tests, but slighter improvements in wellbeing. Positive outcomes related to the incentives and to liking the format. The number of reported barriers was associated with lower involvement and lack of change, as was finding activities too difficult, the level of understanding, and transport and mobility problems, but when these were controlled, age did not predict involvement. Enjoying the scheme was related to positive changes, and this was associated with support from the older people's champions.

Surfactant vesicle-mediated delivery of DNA vaccines via the subcutaneous route

Compared to naked DNA immunisation, entrapment of plasmid-based DNA vaccines into liposomes by the dehydration-rehydration method has shown to enhance both humoural and cell-mediated immune responses to encoded antigens administered by a variety of routes. In this paper we have compared the potency of lipid-based and non-ionic surfactant based vesicle carrier systems for DNA vaccines after subcutaneous immunisation. Plasmid pI.18Sfi/NP containing the nucleoprotein (NP) gene of A/Sichuan/2/87 (H3N2) influenza virus in the pI.18 expression vector was incorporated by the dehydration-rehydration method into various vesicle formulations. The DRV method, entailing mixing of small unilamellar vesicles (SUV) with DNA, followed by dehydration and rehydration, yielded high DNA vaccine incorporation values (85-97% of the DNA used) in all formulations. Studies on vesicle size revealed lipid-based systems formed cationic submicron size vesicles whilst constructs containing a non-ionic surfactant had significantly large z-average diameters (>1500 nm). Subcutaneous vesicle-mediated DNA immunisation employing two DRV(DNA) formulations as well as naked DNA revealed that humoural responses (immunoglobulin total IgG, and subclasses IgG 1 and 1gG 2a) engendered by the plasmid encoded nucleoprotein were substantially higher after dosing twice, 28 days apart with 10 μg DRV-entrapped DNA compared to naked DNA. Comparison between the lipid and non-ionic based vesicle formulations revealed no significant difference in stimulated antibody production. These results suggest that, not only can DNA be effectively entrapped within a range of lipid and non-ionic based vesicle formulations using the DRV method but that such DRV vesicles containing DNA may be a useful system for subcutaneous delivery of DNA vaccines. © 2004 Elsevier B.V. All rights reserved.

Characterization and optimization of bilosomes for oral vaccine delivery

Oral vaccines offer significant benefits due to the ease of administration, better patient compliance and non-invasive, needle-free administration. However, this route is marred by the harsh gastro intestinal environment which is detrimental to many vaccine formats. To address this, a range of delivery systems have been considered including bilosomes; these are bilayer vesicles constructed from non-ionic surfactants combined with the inclusion of bile salts which can stabilize the vesicles in the gastro intestinal tract by preventing membrane destabilization. The aim of this study was to investigate the effect of formulation parameters on bilosome carriers using Design of Experiments to select an appropriate formulation to assess in vivo. Bilosomes were constructed from monopalmitoylglycerol, cholesterol, dicetyl phosphate and sodium deoxycholate at different blends ratios. The optimized bilosome formulation was identified and the potential of this formulation as an oral vaccine delivery system were assessed in biodistribution and vaccine efficacy studies. Results showed that the larger bilosomes vesicles (~6 µm versus 2 µm in diameter) increased uptake within the Peyer's patches and were able to reduce median temperature differential change and promote a reduction in viral cell load in an influenza challenge study. © 2013 Informa UK, Ltd.

Editorial overview:new protein production tools for structural biology

Full text: The idea of producing proteins from recombinant DNA hatched almost half a century ago. In his PhD thesis, Peter Lobban foresaw the prospect of inserting foreign DNA (from any source, including mammalian cells) into the genome of a λ phage in order to detect and recover protein products from Escherichia coli [ 1 and 2]. Only a few years later, in 1977, Herbert Boyer and his colleagues succeeded in the first ever expression of a peptide-coding gene in E. coli — they produced recombinant somatostatin [ 3] followed shortly after by human insulin. The field has advanced enormously since those early days and today recombinant proteins have become indispensable in advancing research and development in all fields of the life sciences. Structural biology, in particular, has benefitted tremendously from recombinant protein biotechnology, and an overwhelming proportion of the entries in the Protein Data Bank (PDB) are based on heterologously expressed proteins. Nonetheless, synthesizing, purifying and stabilizing recombinant proteins can still be thoroughly challenging. For example, the soluble proteome is organized to a large part into multicomponent complexes (in humans often comprising ten or more subunits), posing critical challenges for recombinant production. A third of all proteins in cells are located in the membrane, and pose special challenges that require a more bespoke approach. Recent advances may now mean that even these most recalcitrant of proteins could become tenable structural biology targets on a more routine basis. In this special issue, we examine progress in key areas that suggests this is indeed the case. Our first contribution examines the importance of understanding quality control in the host cell during recombinant protein production, and pays particular attention to the synthesis of recombinant membrane proteins. A major challenge faced by any host cell factory is the balance it must strike between its own requirements for growth and the fact that its cellular machinery has essentially been hijacked by an expression construct. In this context, Bill and von der Haar examine emerging insights into the role of the dependent pathways of translation and protein folding in defining high-yielding recombinant membrane protein production experiments for the common prokaryotic and eukaryotic expression hosts. Rather than acting as isolated entities, many membrane proteins form complexes to carry out their functions. To understand their biological mechanisms, it is essential to study the molecular structure of the intact membrane protein assemblies. Recombinant production of membrane protein complexes is still a formidable, at times insurmountable, challenge. In these cases, extraction from natural sources is the only option to prepare samples for structural and functional studies. Zorman and co-workers, in our second contribution, provide an overview of recent advances in the production of multi-subunit membrane protein complexes and highlight recent achievements in membrane protein structural research brought about by state-of-the-art near-atomic resolution cryo-electron microscopy techniques. E. coli has been the dominant host cell for recombinant protein production. Nonetheless, eukaryotic expression systems, including yeasts, insect cells and mammalian cells, are increasingly gaining prominence in the field. The yeast species Pichia pastoris, is a well-established recombinant expression system for a number of applications, including the production of a range of different membrane proteins. Byrne reviews high-resolution structures that have been determined using this methylotroph as an expression host. Although it is not yet clear why P. pastoris is suited to producing such a wide range of membrane proteins, its ease of use and the availability of diverse tools that can be readily implemented in standard bioscience laboratories mean that it is likely to become an increasingly popular option in structural biology pipelines. The contribution by Columbus concludes the membrane protein section of this volume. In her overview of post-expression strategies, Columbus surveys the four most common biochemical approaches for the structural investigation of membrane proteins. Limited proteolysis has successfully aided structure determination of membrane proteins in many cases. Deglycosylation of membrane proteins following production and purification analysis has also facilitated membrane protein structure analysis. Moreover, chemical modifications, such as lysine methylation and cysteine alkylation, have proven their worth to facilitate crystallization of membrane proteins, as well as NMR investigations of membrane protein conformational sampling. Together these approaches have greatly facilitated the structure determination of more than 40 membrane proteins to date. It may be an advantage to produce a target protein in mammalian cells, especially if authentic post-translational modifications such as glycosylation are required for proper activity. Chinese Hamster Ovary (CHO) cells and Human Embryonic Kidney (HEK) 293 cell lines have emerged as excellent hosts for heterologous production. The generation of stable cell-lines is often an aspiration for synthesizing proteins expressed in mammalian cells, in particular if high volumetric yields are to be achieved. In his report, Buessow surveys recent structures of proteins produced using stable mammalian cells and summarizes both well-established and novel approaches to facilitate stable cell-line generation for structural biology applications. The ambition of many biologists is to observe a protein's structure in the native environment of the cell itself. Until recently, this seemed to be more of a dream than a reality. Advances in nuclear magnetic resonance (NMR) spectroscopy techniques, however, have now made possible the observation of mechanistic events at the molecular level of protein structure. Smith and colleagues, in an exciting contribution, review emerging ‘in-cell NMR’ techniques that demonstrate the potential to monitor biological activities by NMR in real time in native physiological environments. A current drawback of NMR as a structure determination tool derives from size limitations of the molecule under investigation and the structures of large proteins and their complexes are therefore typically intractable by NMR. A solution to this challenge is the use of selective isotope labeling of the target protein, which results in a marked reduction of the complexity of NMR spectra and allows dynamic processes even in very large proteins and even ribosomes to be investigated. Kerfah and co-workers introduce methyl-specific isotopic labeling as a molecular tool-box, and review its applications to the solution NMR analysis of large proteins. Tyagi and Lemke next examine single-molecule FRET and crosslinking following the co-translational incorporation of non-canonical amino acids (ncAAs); the goal here is to move beyond static snap-shots of proteins and their complexes and to observe them as dynamic entities. The encoding of ncAAs through codon-suppression technology allows biomolecules to be investigated with diverse structural biology methods. In their article, Tyagi and Lemke discuss these approaches and speculate on the design of improved host organisms for ‘integrative structural biology research’. Our volume concludes with two contributions that resolve particular bottlenecks in the protein structure determination pipeline. The contribution by Crepin and co-workers introduces the concept of polyproteins in contemporary structural biology. Polyproteins are widespread in nature. They represent long polypeptide chains in which individual smaller proteins with different biological function are covalently linked together. Highly specific proteases then tailor the polyprotein into its constituent proteins. Many viruses use polyproteins as a means of organizing their proteome. The concept of polyproteins has now been exploited successfully to produce hitherto inaccessible recombinant protein complexes. For instance, by means of a self-processing synthetic polyprotein, the influenza polymerase, a high-value drug target that had remained elusive for decades, has been produced, and its high-resolution structure determined. In the contribution by Desmyter and co-workers, a further, often imposing, bottleneck in high-resolution protein structure determination is addressed: The requirement to form stable three-dimensional crystal lattices that diffract incident X-ray radiation to high resolution. Nanobodies have proven to be uniquely useful as crystallization chaperones, to coax challenging targets into suitable crystal lattices. Desmyter and co-workers review the generation of nanobodies by immunization, and highlight the application of this powerful technology to the crystallography of important protein specimens including G protein-coupled receptors (GPCRs). Recombinant protein production has come a long way since Peter Lobban's hypothesis in the late 1960s, with recombinant proteins now a dominant force in structural biology. The contributions in this volume showcase an impressive array of inventive approaches that are being developed and implemented, ever increasing the scope of recombinant technology to facilitate the determination of elusive protein structures. Powerful new methods from synthetic biology are further accelerating progress. Structure determination is now reaching into the living cell with the ultimate goal of observing functional molecular architectures in action in their native physiological environment. We anticipate that even the most challenging protein assemblies will be tackled by recombinant technology in the near future.

Functionalized carbon nanotube nanoelectrodes for biomolecular recognition

The objective of this research is to develop nanoscale ultrasensitive transducers for detection of biological species at molecular level using carbon nanotubes as nanoelectrodes. Rapid detection of ultra low concentration or even single DNA molecules are essential for medical diagnosis and treatment, pharmaceutical applications, gene sequencing as well as forensic analysis. Here the use of functionalized single walled carbon nanotubes (SWNT) as nanoscale detection platform for rapid detection of single DNA molecules is demonstrated. The detection principle is based on obtaining electrical signal from a single amine terminated DNA molecule which is covalently bridged between two ends of an SWNT separated by a nanoscale gap. The synthesis, fabrication, chemical functionalization of nanoelectrodes and DNA attachment were optimized to perform reliable electrical characterization these molecules. Using this detection system fundamental study on charge transport in DNA molecule of both genomic and non genomic sequences is performed. We measured an electrical signal of about 30 pA through a hybridized DNA molecule of 80 base pair in length which encodes a portion of sequence of H5N1 gene of avian Influenza A virus. Due the dynamic nature of the DNA molecules the local environment such as ion concentration, pH and temperature significantly influence its physical properties. We observed a decrease in DNA conductance of about 33% in high vacuum conditions. The counterion variation was analyzed by changing the buffer from sodium acetate to tris(hydroxymethyl) aminomethane, which resulted in a two orders of magnitude increase in the conductivity of the DNA. The fabrication of large array of identical SWNT nanoelectrodes was achieved by using ultralong SWNTs. Using these nanoelectrode array we have investigated the sequence dependent charge transport in DNA. A systematic study performed on PolyG - PolyC sequence with varying number of intervening PolyA - PolyT pairs showed a decrease in electrical signal from 180 pA (PolyG - PolyC) to 30 pA with increasing number of the PolyA - PolyT pairs. This work also led to the development of ultrasensitive nanoelectrodes based on enzyme functionalized vertically aligned high density multiwalled CNTs for electrochemical detection of cholesterol. The nanoelectrodes exhibited selectively detection of cholesterol in the presence of common interferents found in human blood.

Definizione e validazione di uno strumento per la misurazione dell'impatto delle variabili redazionali di un item matematico sulla performance degli studenti

Questa tesi descrive la ricerca condotta tra l'autunno e l'inverno di quest'anno da un gruppo di ricercatori in didattica della matematica relativamente all'influenza che le variazioni redazionali di un quesito matematico hanno sulle performance degli studenti. Lo scopo della ricerca è quella di strutturare e validare una metodologia e uno strumento che permettano di individuare e quantificare l'influenza delle variazioni del testo sulle prestazioni dello studente. Si è sentita l'esigenza di condurre uno studio di questo tipo poichè è sempre più evidente il profondo legame tra il linguaggio e l'apprendimento della matematica. La messa a punto di questo strumento aprirebbe le porte a una serie di ricerche più approfondite sulle varie tipologie di variazioni numeriche e/o linguistiche finora individuate. Nel primo capitolo è presentato il quadro teorico di riferimento relativo agli studi condotti fino ad ora nell'ambito della didattica della matematica, dai quali emerge la grossa influenza che la componente linguistica ha sulla comprensione e la trasmissione della matematica. Si farà quindi riferimento alle ricerche passate volte all'individuazione e alla schematizzazione delle variazioni redazionali dei Word Problems. Nel secondo capitolo, invece si passerà alla descrizione teorica relativa allo strumento statistico utilizzato. Si tratta del modello di Rasch appartenente alla famiglia dei modelli statistici dell'Item Response Theory, particolarmente utilizzato nella ricerca in didattica. Il terzo capitolo sarà dedicato alla descrizione dettagliata della sperimentazione svolta. Il quarto capitolo sarà il cuore di questa tesi; in esso infatti verrà descritta e validata la nuova metodologia utilizzata. Nel quinto sarà eseguita un analisi puntuale di come lo strumento ha messo in evidenza le differenze per ogni item variato. Infine verranno tratte le conclusioni complessive dello studio condotto.

Simulazione della bradicinesia in soggetti parkinsoniani attraverso modello neurale

I Gangli della Base svolgono un ruolo molto importante nel movimento volontario, ovvero nel meccanismo di azione-selezione, e la loro influenza è evidente soprattutto in alcune patologie che ancora ad oggi sono in fase di studio: una di queste è il Morbo di Parkinson. I Gangli della Base comprendono quattro formazioni nervose: lo striato, il globus pallidus, la substantia nigra e il nucleo subtalamico: essi ricevono le principali afferenze dalla corteccia cerebrale ed inviano le principali efferenze al tronco dell’encefalo, e, per mezzo del talamo, alle corteccia prefrontale, premotoria e motrice. A differenza della maggior parte delle altre componenti dei sistemi motori, i Gangli della Base non stabiliscono direttamente né connessioni afferenti, né efferenti con il midollo spinale. Il compito principale svolto dai Gangli dDella Base è la selezione di un’azione: esso permette ad un’azione di essere selezionata rispetto ad un’altra, che in questo modo viene inibita. La descrizione dell’anatomia, dei meccanismi fisiologici e del Morbo di Parkinson è trattata nel Capitolo 1. In questo elaborato è utilizzato il modello computazionale di Mauro Ursino e Chiara Baston, che sarà illustrato dettagliatamente nel Capitolo 2, riguardante il meccanismo di azione-selezione svolto dai Gangli della Base. E’ descritto un sistema di valutazione di un paziente parkinsoniano, il tapping test: esso consiste in un movimento alternato del dito e ad oggi risulta essere uno dei metodi più semplici per ottenere informazioni sulla gravità della bradicinesia. L’obiettivo di questo lavoro è quello di comprendere, tramite l’analisi di simulazioni effettuate per mezzo del modello computazionale di Mauro Ursino e Chiara Baston, come la frequenza di tapping dipenda dal variare di alcuni parametri delle equazioni del modello: gli effetti dovuti alla variazione di un singolo parametro o più di uno, saranno mostrati nel Capitolo 3.

Analisi modellistica di un intenso ciclone di tipo tropicale nel Mediterraneo (7-8 novembre 2014): predicibilità, caratteristiche termodinamiche e confronto con osservazioni da satellite

Il lavoro di tesi riguarda lo studio dettagliato di un ciclone di tipo tropicale (tropical like cyclone, TLC) verificatosi nel Canale di Sicilia nel novembre 2014, realizzato attraverso un'analisi modellistica effettuata con i modelli BOLAM e MOLOCH (sviluppati presso il CNR-ISAC di Bologna) e il confronto con osservazioni. Nel primo capitolo è fornita una descrizione generale dei cicloni tropicali e dei TLC, indicando come la formazione di questi ultimi sia spesso il risultato dell'evoluzione di cicloni extratropicali baroclini nel Mediterraneo; sono identificate le aree geografiche con i periodi dell'anno maggiormente soggetti all'influenza di questi fenomeni, riportando un elenco dei principali TLC verificatisi nel Mediterraneo negli utlimi tre decenni e lo stato dell'arte sullo studio di questi eventi. Nel secondo capitolo sono descritte le modalità di implementazione delle simulazioni effettuate per il caso di studio e presentati i principali prodotti dell'analisi modellistica e osservazioni da satellite. Il terzo capitolo si apre con la descrizione della situazione sinottica e l'analisi osservativa con immagini Meteosat e rilevazioni radar che hanno permesso di ricostruire la traiettoria osservata del TLC. In seguito, viene dapprima fornito l'elenco completo delle simulazioni numeriche, quindi sono presentati alcuni dei più importanti risultati ottenuti, dai quali emerge che la previsione della traiettoria e intensità del TLC differisce notevolmente dalle osservazioni. Tenendo conto della bassa predicibilità che ha caratterizzato l'evento, nel quarto capitolo è descritto il metodo usato per ricostruire in maniera ottimale la traiettoria, utilizzando spezzoni da varie simulazioni, che ha permesso un confronto più realistico con i dati osservati e un'analisi dei processi fisici. Nel quinto capitolo sono riportati i principali risultati di alcuni test mirati a valutare l'impatto di aspetti legati all'implementazione delle simulazioni e ad altre forzanti fisiche.

Studio di contenitori in PET per riempimento a caldo nitro-hot-fill

Lo scopo di questa attività è approfondire le conoscenze sul processo di riempimento a caldo noto come nitro-hot-fill (NHF) utilizzato per contenitori in PET. Il nostro obiettivo è quello di simulare su scala di laboratorio il processo industriale al fine di ottimizzarne i parametri e aumentare la stabilità dei contenitori anche attraverso l’utilizzo di materie prime con caratteristiche migliorate utilizzando formulazioni adatte ai trattamenti a caldo. Il processo consiste nel riempimento della bottiglia ad una temperatura tra gli 80°/85°C, successivo al quale vi è l’iniezione di azoto al fine di evitare l’implosione durante il raffreddamento fino a temperatura ambiente. Questo settore del mercato è in forte crescita, molte bevande infatti hanno la necessità di un contenitore asettico; il processo di NHF ha il vantaggio di utilizzare il calore del prodotto stesso al fine di rendere la bottiglia sterile. Da qui nascono le criticità legate al processo, occorre prendere diversi accorgimenti al fine di rendere processabile in questo modo una bottiglia, infatti l’aumento di pressione interna dovuto all’iniezione di azoto si accompagna una temperatura vicina alla temperatura di transizione vetrosa. La nostra attività di ricerca ha focalizzato la propria attenzione sul design della bottiglia, sul processo di stiro-soffiaggio, sull’influenza dell’umidità assorbita nel PET, sul materiale utilizzato e su altri parametri di processo al fine di produrre contenitori in grado di resistere al riempimento NHF.

Infrastrutture basate su Edge Computing per Supporto a Servizi Mobili in Ambienti Ostili

Questa tesi è incentrata sulla revisione del classico modello di infrastruttura Cloud. Le motivazioni sono da ricercare nelle condizioni operative reali della maggior parte dei dispositivi connessi alla rete attualmente. Si parla di ambiente ostile riferendosi a network popolate da molti dispositivi dalle limitate caratteristiche tecniche e spesso collegati con canali radio, molto più instabili delle connessioni cablate. Allo scenario va ad aggiungersi la necessità crescente di mobilità che limita ulteriormente i vantaggi derivanti dall'utilizzo dell’infrastruttura Cloud originale. La trattazione propone il modello Edge come estensione del Cloud. Esso ne amplia il ventaglio di utilizzo, favorendo aree di applicazione che stanno acquisendo maggiore influenza negli ultimi periodi e che richiedono una revisione delle vecchie infrastrutture Cloud, dettata dalle caratteristiche stringenti che necessitano per un'operatività soddisfacente.

D4.3 Prototype Software (Documentation)

The agent-based social simulation component of the TELL ME project (WP4) developed prototype software to assist communications planners to understand the complex relationships between communication, personal protective behaviour and epidemic spread. Using the simulation, planners can enter different potential communications plans, and see their simulated effect on attitudes, behaviour and the consequent effect on an influenza epidemic.

The model and the software to run the model are both freely available (see section 2.2.1 for instructions on how to obtain the relevant files). This report provides the documentation for the prototype software. The major component is the user guide (Section 2). This provides instructions on how to set up the software, some training scenarios to become familiar with the model operation and use, and details about the model controls and output.

The model contains many parameters. Default values and their source are described at Section 3. These are unlikely to be suitable for all countries, and may also need to be changed as new research is conducted. Instructions for how to customise these values are also included (see section 3.5).

The final technical reference contains two parts. The first is a guide for advanced users who wish to run multiple simulations and analyse the results (section 4.1). The second is to orient programmers who wish to adapt or extend the simulation model (section 4.2). This material is not suitable for general users.

Respiratory virus-associated severe acute respiratory illness (SARI) and viral clustering in Malawian children in a setting with a high prevalence of HIV, malaria and malnutrition

BACKGROUND:  We used four years of paediatric severe acute respiratory illness (SARI) sentinel surveillance in Blantyre, Malawi to identify factors associated with clinical severity and co-viral clustering.

METHODS:  From January 2011 to December 2014, 2363 children aged 3 months to 14 years presenting to hospital with SARI were enrolled. Nasopharyngeal aspirates were tested for influenza and other respiratory viruses. We assessed risk factors for clinical severity and conducted clustering analysis to identify viral clusters in children with co-viral detection.

RESULTS:  Hospital-attended influenza-positive SARI incidence was 2.0 cases per 10,000 children annually; it was highest children aged under 1 year (6.3 cases per 10,000), and HIV-infected children aged 5 to 9 years (6.0 cases per 10,000). 605 (26.8%) SARI cases had warning signs, which were positively associated with HIV infection (adjusted risk ratio [aRR]: 2.4, 95% CI: 1.4, 3.9), RSV infection (aRR: 1.9, 95% CI: 1.3, 3.0) and rainy season (aRR: 2.4, 95% CI: 1.6, 3.8). We identified six co-viral clusters; one cluster was associated with SARI with warning signs.

CONCLUSIONS:  Influenza vaccination may benefit young children and HIV infected children in this setting. Viral clustering may be associated with SARI severity; its assessment should be included in routine SARI surveillance.