Development of a comprehensive on-line multidimensional high performance column liquid chromatography system for protein and peptide mapping with integrated size selective sample fractionation

Aufbau einer kontinuierlichen, mehrdimensionalen Hochleistungs-flüssigchromatographie-Anlage für die Trennung von Proteinen und Peptiden mit integrierter größenselektiver ProbenfraktionierungEs wurde eine mehrdimensionale HPLC-Trennmethode für Proteine und Peptide mit einem Molekulargewicht von <15 kDa entwickelt.Im ersten Schritt werden die Zielanalyte von höhermolekularen sowie nicht ionischen Bestandteilen mit Hilfe von 'Restricted Access Materialien' (RAM) mit Ionenaustauscher-Funktionalität getrennt. Anschließend werden die Proteine auf einer analytischen Ionenaustauscher-Säule sowie auf Reversed-Phase-Säulen getrennt. Zur Vermeidung von Probenverlusten wurde ein kontinuierlich arbeitendes, voll automatisiertes System auf Basis unterschiedlicher Trenngeschwindigkeiten und vier parallelen RP-Säulen aufgebaut.Es werden jeweils zwei RP-Säulen gleichzeitig, jedoch mit zeitlich versetztem Beginn eluiert, um durch flache Gradienten ausreichende Trennleistungen zu erhalten. Während die dritte Säule regeneriert wird, erfolgt das Beladen der vierte Säule durch Anreicherung der Proteine und Peptide am Säulenkopf. Während der Gesamtanalysenzeit von 96 Minuten werden in Intervallen von 4 Minuten Fraktionen aus der 1. Dimension auf die RP-Säulen überführt und innerhalb von 8 Minuten getrennt, wobei 24 RP-Chromatogramme resultieren.Als Testsubstanzen wurden u.a. Standardproteine, Proteine und Peptide aus humanem Hämofiltrat sowie aus Lungenfibroblast-Zellkulturüberständen eingesetzt. Weiterhin wurden Fraktionen gesammelt und mittels MALDI-TOF Massenspektrometrie untersucht. Bei einer Injektion wurden in den 24 RP-Chromatogrammen mehr als 1000 Peaks aufgelöst. Der theoretische Wert der Peakkapazität liegt bei ungefähr 3000.

Sintesi di immagini realistiche mediante l'uso del Photon differentials come estensione del Photon mapping

Questa tesi intende presentare una tecnica per la sintesi di immagini realistiche al calcolatore basata sul concetto di particle tracing. Il metodo proposto opera una stima sulla densità locale dei fotoni estendendo il concetto del photon differentials anche alla gestione delle riflessioni diffusive. Si è scelto di implementare il nuovo algoritmo di illuminazione globale all’interno di XCModel come estensione del photon mapping.

Sistemi Rh/CeO2-Al2O3 per l'ossidazione catalitica del metano

L’ossidazione catalitica parziale (CPO) del metano è un processo di elevato interesse scientifico ed industriale, permettendo la produzione su piccola scala di H2 o la produzione di syngas con un rapporto H2/CO = 2, utile per la produzione di metanolo o la sintesi di Fischer-Tropsch di idrocarburi. Inoltre, si possono raggiungere elevate conversioni del metano e selettività in syngas operando a bassi valori del tempo di contatto, riducendo così le dimensioni dei reattori. Tuttavia, gli elevati flussi e le temperature raggiunte nel letto possono condurre rapidamente alla disattivazione del catalizzatore; pertanto, è necessario lo sviluppo di materiali non soltanto attivi, ma anche stabili nelle condizioni di reazione. Lo scopo di questo lavoro è stato lo sviluppo di catalizzatori a base di Rh e ceria, utilizzati sia come pellets che supportati su una schiuma metallica. In particolare, il lavoro è stato focalizzato sulla sintesi, caratterizzazione ed attività catalitica di materiali Rh-CeO2-Al2O3. La presenza di CeO2 può modificare la dispersione del Rh metallico e la sua stabilità nei confronti della sinterizzazione e della formazione del carbone, mentre la stabilità termica è favorita dalla presenza di Al2O3. Poiché queste proprietà e, di conseguenza, le prestazione catalitiche dipendono dalla dimensioni delle particelle di CeO2 sono stati preparati catalizzatori con diverso contenuto di CeO2 (10 e 20 p/p %) ed utilizzando differenti metodi di preparazione per modularne le proprietà. Le sintesi sono effettuate per coprecipitazione e per sintesi con urea per trattamento micronde-idrotermale. Le prestazione dei catalizzatori in pellets sono state analizzate in un impianto di laboratorio operando a bassi valori del tempo di contatto e modificando la temperatura e la concentrazione della miscela gassosa, i.e. sia in condizioni lontane dall’equilibrio termodinamico che in condizioni prossime a quelle industriali. Un catalizzatore con lo stesso contenuto di Rh ed ottenuto da precursori tipo idrotalcite (HT) è stato utilizzato come riferimento. Per incrementare ulteriormente le prestazioni catalitiche, in particolare il trasferimento del calore lungo il letto catalitico, la migliore composizione individuata delle prove precedenti è stata depositata su pellets di una schiuma metallica (FeCrAlloy). E’ stato utilizzato il metodo dell’elettrosintesi per la deposizione di idrossidi di Rh, Ce e Al, ottenendo dopo calcinazione catalizzatori strutturati. Si è valutato l’effetto dei parametri di sintesi, potenziale applicato e tempo, sulle proprietà catalitiche. Anche in questo caso i risultati sono stati confrontati quelli ottenuti con un catalizzatori di riferimento ottenuti da un precursore HT preparati per elettrosintesi.

The effects of hypocalcemia on spatial alternans and ventricular fibrillation studied with the optical mapping technique

The heart is a wonderful but complex organ: it uses electrochemical mechanisms in order to produce mechanical energy to pump the blood throughout the body and allow the life of humans and animals. This organ can be subject to several diseases and sudden cardiac death (SCD) is the most catastrophic manifestation of these diseases, responsible for the death of a large number of people throughout the world. It is estimated that 325000 Americans annually die for SCD. SCD most commonly occurs as a result of reentrant tachyarrhythmias (ventricular tachycardia (VT) and ventricular fibrillation (VF)) and the identification of those patients at higher risk for the development of SCD has been a difficult clinical challenge. Nowadays, a particular electrocardiogram (ECG) abnormality, “T-wave alternans” (TWA), is considered a precursor of lethal cardiac arrhythmias and sudden death, a sensitive indicator of risk for SCD. TWA is defined as a beat-to-beat alternation in the shape, amplitude, or timing of the T-wave on the ECG, indicative of the underlying repolarization of cardiac cells [5]. In other words TWA is the macroscopic effect of subcellular and celluar mechanisms involving ionic kinetics and the consequent depolarization and repolarization of the myocytes. Experimental activities have shown that TWA on the ECG is a manifestation of an underlying alternation of long and short action potential durations (APDs), the so called APD-alternans, of cardiac myocytes in the myocardium. Understanding the mechanism of APDs-alternans is the first step for preventing them to occur. In order to investigate these mechanisms it’s very important to understand that the biological systems are complex systems and their macroscopic properties arise from the nonlinear interactions among the parts. The whole is greater than the sum of the parts, and it cannot be understood only by studying the single parts. In this sense the heart is a complex nonlinear system and its way of working follows nonlinear dynamics; alternans also, they are a manifestation of a phenomenon typical in nonlinear dynamical systems, called “period-dubling bifurcation”. Over the past decade, it has been demonstrated that electrical alternans in cardiac tissue is an important marker for the development of ventricular fibrillation and a significant predictor for mortality. It has been observed that acute exposure to low concentration of calcium does not decrease the magnitude of alternans and sustained ventricular Fibrillation (VF) is still easily induced under these condition. However with prolonged exposure to low concentration of calcium, alternans disappears, but VF is still inducible. This work is based on this observation and tries to make it clearer. The aim of this thesis is investigate the effect of hypocalcemia spatial alternans and VF doing experiments with canine hearts and perfusing them with a solution with physiological ionic concentration and with a solution with low calcium concentration (hypocalcemia); in order to investigate the so called memory effect, the experimental activity was modified during the way. The experiments were performed with the optical mapping technique, using voltage-sensitive dye, and a custom made Java code was used in post-processing. Finding the Nolasco and Dahlen’s criterion [8] inadequate for the prediction of alternans, and takin into account the experimental results, another criterion, which consider the memory effect, has been implemented. The implementation of this criterion could be the first step in the creation of a method, AP-based, discriminating who is at risk if developing VF. This work is divided into four chapters: the first is a brief presentation of the physiology of the heart; the second is a review of the major theories and discovers in the study of cardiac dynamics; the third chapter presents an overview on the experimental activity and the optical mapping technique; the forth chapter contains the presentation of the results and the conclusions.

Mapping the elastic response of epithelial apical cell membranes suspended across porous array

As the elastic response of cell membranes to mechanical stimuli plays a key role in various cellular processes, novel biophysical strategies to quantify the elasticity of native membranes under physiological conditions at a nanometer scale are gaining interest. In order to investigate the elastic response of apical membranes, elasticity maps of native membrane sheets, isolated from MDCK II (Madine Darby Canine kidney strain II) epithelial cells, were recorded by local indentation with an Atomic Force Microscope (AFM). To exclude the underlying substrate effect on membrane indentation, a highly ordered gold coated porous array with a pore diameter of 1.2 μm was used to support apical membranes. Overlays of fluorescence and AFM images show that intact apical membrane sheets are attached to poly-D-lysine coated porous substrate. Force indentation measurements reveal an extremely soft elastic membrane response if it is indented at the center of the pore in comparison to a hard repulsion on the adjacent rim used to define the exact contact point. A linear dependency of force versus indentation (-dF/dh) up to 100 nm penetration depth enabled us to define an apparent membrane spring constant (kapp) as the slope of a linear fit with a stiffness value of for native apical membrane in PBS. A correlation between fluorescence intensity and kapp is also reported. Time dependent hysteresis observed with native membranes is explained by a viscoelastic solid model of a spring connected to a Kelvin-Voight solid with a time constant of 0.04 s. No hysteresis was reported with chemically fixated membranes. A combined linear and non linear elastic response is suggested to relate the experimental data of force indentation curves to the elastic modulus and the membrane thickness. Membrane bending is the dominant contributor to linear elastic indentation at low loads, whereas stretching is the dominant contributor for non linear elastic response at higher loads. The membrane elastic response was controlled either by stiffening with chemical fixatives or by softening with F-actin disrupters. Overall, the presented setup is ideally suitable to study the interactions of the apical membrane with the underlying cytoskeleton by means of force indentation elasticity maps combined with fluorescence imaging.

Association mapping of stem rust resistance in durum wheat at the seedling and adult plant stages

In wheat, stem rust is known to rapidly evolve new virulence to resistance genes. While more than 50 stem rust resistance (Sr) loci have been identified in wheat, only a few remain effective, particularly against the highly virulent race Ug99 (TTKSK race) and a mixture of durum-specific races. An association mapping (AM) study based on 183 durum wheat accessions was utilized to identify resistance loci for stem rust response in Ethiopia over four seasons and artificial inoculation with Ug99 (TTKSK race) and a mixture of durum-specific races under field conditions as well as in greenhouse test at seedling stage under controlled conditions for resistance to four highly virulent stem rust races: TRTTF, TTTTF, (TTKSK (Ug99) and JRCQC. The panel was profiled with 1,253 SSR and DArT markers. Twelve QTL-tagging markers were significant (P < 0.05) across three to four seasons. The role of Sr13, Sr9, Sr14, Sr17, and Sr28 was confirmed. Thirteen significant markers were in regions with no Sr genes/QTLs. The results under controlled conditions showed that 15, 20, 19 and 19 chromosome regions harbored markers that showed significant effects for races TRTTF, TTTTF, TTKSK and JRCQC, respectively. These genomic regions showed marker R2 values ranging from 1.13 to 8.34, 1.92 to 17.64, 1.75 to 23.12 and 1.51 to 15.33% for races TRTTF, TTTTF, TTKSK and JRCQC, respectively. The study demonstrates that stem rust resistance in durum wheat is governed in part by shared loci and in part by race-specific ones. The QTLs identified in this study through AM will be useful in the marker-assisted development of durum wheat cultivars with durable stem rust resistance.

Elettrosintesi di catalizzatori contenenti Rh e Pt su supporti metallici

La sintesi di catalizzatori Rh-Al2O3 e Pt-Al2O3 è stata condotta per via elettrochimica su dei supporti con diverse geometrie (lastrine, schiume) e diversa composizione (Pt e FeCrAlY). Per l’elettrosintesi dei precursori dei catalizzatori sono state utilizzate due diversi tipi di celle elettrochimiche (a singolo comparto, nella quale il moto degli elettroliti segue un regime di tipo diffusivo, e cella in flusso a comparti separati). Per il catalizzatore Rh-Al2O3 sono stati sintetizzati i precursori composti da idrossidi di Al3+ e Rh3+. Per il catalizzatore Pt-Al2O3 sono state condotte sia delle sintesi mono-stadio di nanoparticelle di Pt sul supporto ricoperto di Al2O3 che delle sintesi cercando di inglobare le nanoparticelle di Pt in un film di idrossido di alluminio. La caratterizzazione morfologica e composizionale della superficie del supporto metallico, ha permesso di studiare l’influenza delle varie condizioni di sintesi sulla qualità del ricoprimento ottenuto tramite elettrosintesi. I migliori risultati in termini di ricoprimento del supporto e omogeneità dello strato catalitico depositato, sono stati ottenuti tramite l’utilizzo della cella in flusso e confrontando i campioni ottenuti con delle schiume ricoperte di allumina tramite washcoating, sono state osservate le principali differenze tra i due metodi. L’elettrosintesi dei precursori del catalizzatore Rh-Al2O3 ha portato ad uno scarso ricoprimento ma la fattibilità del processo è stata dimostrata tramite l’utilizzo di un elettrodo di Pt. Per il catalizzatore Pt-Al2O3, lo scarso controllo nella grandezza e distribuzione delle nanoparticelle di Pt elettrosintetizzate sulle schiume ricoperte di Al2O3 ha dimostrato l’esigenza di un metodo alternativo di sintesi. Questo prevede l’incorporazione delle nanoparticelle di Pt durante l’elettrosintesi del film di idrossido di alluminio. Utilizzando un elettrodo di Pt la fattibilità del processo è stata dimostrata, tuttavia il metodo richiede dei miglioramenti, in termini di ricoprimento e distribuzione delle particelle se condotto su schiume.

Mapping di Access Point WiFi su Android: architettura

Sviluppo di un'applicazione Android che effettua scansioni di reti Wi-Fi in una determinata area al fine di mappare tutti gli Access Point rilevabili.

Mapping di access point wifi su android: valutazione

Sviluppo di un'applicazione Android che effettua scansioni di reti Wi-Fi in una determinata area al fine di mappare tutti gli Access Point rilevabili.

Stream sediments analysis for geochemical mapping of Romagna Apennines (Northern Italy): monitoring and management tool of environmental resources at various scales

Geochemical mapping is a valuable tool for the control of territory that can be used not only in the identification of mineral resources and geological, agricultural and forestry studies but also in the monitoring of natural resources by giving solutions to environmental and economic problems. Stream sediments are widely used in the sampling campaigns carried out by the world's governments and research groups for their characteristics of broad representativeness of rocks and soils, for ease of sampling and for the possibility to conduct very detailed sampling In this context, the environmental role of stream sediments provides a good basis for the implementation of environmental management measures, in fact the composition of river sediments is an important factor in understanding the complex dynamics that develop within catchment basins therefore they represent a critical environmental compartment: they can persistently incorporate pollutants after a process of contamination and release into the biosphere if the environmental conditions change. It is essential to determine whether the concentrations of certain elements, in particular heavy metals, can be the result of natural erosion of rocks containing high concentrations of specific elements or are generated as residues of human activities related to a certain study area. This PhD thesis aims to extract from an extensive database on stream sediments of the Romagna rivers the widest spectrum of informations. The study involved low and high order stream in the mountain and hilly area, but also the sediments of the floodplain area, where intensive agriculture is active. The geochemical signals recorded by the stream sediments will be interpreted in order to reconstruct the natural variability related to bedrock and soil contribution, the effects of the river dynamics, the anomalous sites, and with the calculation of background values be able to evaluate their level of degradation and predict the environmental risk.

Analysis of the introduction of a business process mapping system in Rosetti Marino spa

After a first theoric introduction about Business Process Re-engineering (BPR), are considered in particular the possible options found in literature regarding the following three macro-elements: the methodologies, the modelling notations and the tools employed for process mapping. The theoric section is the base for the analysis of the same elements into the specific case of Rosetti Marino S.p.A., an EPC contractor, operating in the Oil&Gas industry. Rosetti Marino implemented a tool developped internally in order to satisfy its needs in the most suitable way possible and buit a Map of all business processes,navigable on the Company Intranet. Moreover it adopted a methodology based upon participation, interfunctional communication and sharing. The GIGA introduction is analysed from a structural, human resources, political and symbolic point of view.

A multiparental cross population for mapping QTL for relevant agronomic traits in durum wheat (Triticum durum Desf.)

Multiparental cross designs for mapping quantitative trait loci (QTL) in crops are efficient alternatives to conventional biparental experimental populations because they exploit a broader genetic basis and higher mapping resolution. We describe the development and deployment of a multiparental recombinant inbred line (RIL) population in durum wheat (Triticum durum Desf.) obtained by crossing four elite cultivars characterized by different traits of agronomic value. A linkage map spanning 2,663 cM and including 7,594 single nucleotide polymorphisms (SNPs) was produced by genotyping 338 RILs with a wheat-dedicated 90k SNP chip. A cluster file was developed for correct allele calling in the framework of the tetraploid durum wheat genome. Based on phenotypic data collected over four field experiments, a multi-trait quantitative trait loci (QTL) analysis was carried out for 18 traits of agronomic relevance (including yield, yield-components, morpho-physiological and seed quality traits). Across environments, a total of 63 QTL were identified and characterized in terms of the four founder haplotypes. We mapped two QTL for grain yield across environments and 23 QTL for grain yield components. A novel major QTL for number of grain per spikelet/ear was mapped on chr 2A and shown to control up to 39% of phenotypic variance in this cross. Functionally different QTL alleles, in terms of direction and size of genetic effect, were distributed among the four parents. Based on the occurrence of QTL-clusters, we characterized the breeding values (in terms of effects on yield) of most of QTL for heading and maturity as well as yield component and quality QTL. This multiparental RIL population provides the wheat community with a highly informative QTL mapping resource enabling the dissection of the genetic architecture of multiple agronomic relevant traits in durum wheat.

Un sistema per il Mapping di Access Point Wi-Fi in ambito Android

Un sistema per il mapping di access point wi-fi utilizzando un client scritto in android ed un server scritto in php. Il client si occuperà di scansionare le reti disponibili ed inviarle al server che con tali informazioni potrà inferire sulle posizioni effettive degli access point e fornire ad altri client informazioni più dettagliate per potersi connettere.

Fine Mapping of qroot-yield-1.06, a QTL for Root, Plant Vigor and Yield in Maize

Root-yield-1.06 is a major QTL affecting root system architecture (RSA) and other agronomic traits in maize. The effect of this QTL has been evaluated with the development of near isogenic lines (NILs) differing at the QTL position. The objective of this study was to fine map qroot-yield-1.06 by marker-assisted searching for chromosome recombinants in the QTL interval and concurrent root phenotyping in both controlled and field conditions, through successive generations. Complementary approaches such as QTL meta-analysis and RNA-seq were deployed in order to help prioritizing candidate genes within the QTL target region. Using a selected group of genotypes, field based root analysis by ‘shovelomics’ enabled to accurately collect RSA information of adult maize plants. Shovelomics combined with software-assisted root imaging analysis proved to be an informative and relatively highly automated phenotyping protocol. A QTL interval mapping was conducted using a segregating population at the seedling stage grown in controlled environment. Results enabled to narrow down the QTL interval and to identify new polymorphic markers for MAS in field experiments. A collection of homozygous recombinant NILs was developed by screening segregating populations with markers flanking qroot-yield-1.06. A first set of lines from this collection was phenotyped based on the adapted shovelomics protocol. QTL analysis based on these data highlighted an interval of 1.3 Mb as completely linked with the target QTL but, a larger safer interval of 4.1 Mb was selected for further investigations. QTL meta-analysis allows to synthetize information on root QTLs and two mQTLs were identified in the qroot-yield-1.06 interval. Trascriptomics analysis based on RNA-seq data of the two contrasting QTL-NILs, confirmed alternative haplotypes at chromosome bin 1.06. qroot-yield-1.06 has now been delimited to a 4.1-Mb interval, and thanks to the availability of additional untested homozygous recombinant NILs, the potentially achievable mapping resolution at qroot-yield-1.06 is c. 50 kb.