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Mass spectrometry as an emerging tool for the detection of proteins in complex matrices: from untargeted to targeted analysis
No full textMy PhD was performed in Mérieux NutriSciences, a company which provides analytical services. During my PhD I worked on three projects, whose common determinant was the application of mass spectrometry (MS) to the analysis of proteins. The main study deals with MS as a new tool for the analysis of food allergens. Food allergy is an important health problem involving immunological reactions that arise following exposure to protein allergens. In the absence of a cure, patients need to avoid the offending food to prevent allergic reactions. In the European Union, 14 allergens must be indicated in food labels when intentionally added. However, one of the main causes triggering allergic reactions is represented by undesired contamination of food by allergens in production facilities. Even tiny amounts of allergens can trigger severe manifestations; thus, to protect consumers sensitive analytical methods are required. Recently, methods based on MS have received increasing attention for the quantification of food allergens in complex matrices. In the present study, the development of a method based on MS for the simultaneous detection of egg, milk, tree nuts and peanuts allergens into bakery products is described. The method is based on the detection of specific peptides generated from the enzymatic hydrolysis of the target allergens and employs a technique called Multiple Reaction Monitoring, in which the mass spectrometer is operated to selectively acquire signals deriving from specific couples of m/z values, corresponding to a peptide ion and to one of its fragments. The method developed allows to detect target allergens in a specific way and with acceptable sensitivities and can be considered as a valuable alternative to other common analytical techniques, such as ELISA and PCR. A second topic of this thesis is bovine beta-casein, a polymorphic protein for which 12 genetic variants have been identified, the most common being A1 and A2. Some reports suggested a possible association between the consumption of A1 beta-casein and the etiology of some human diseases, including ischemic heart disease and diabetes. At the basis of the effects caused by A1 beta-casein there would be a bioactive peptide with opioid-like activity, released by proteolytic enzymes upon gastrointestinal digestion. This peptide, called beta-casomorphin-7, was shown to be produced only from certain beta-casein isoforms, having a histidine in position 67, including the A1 variant. On the other hand, variants possessing a proline in position 67, such as the A2 variant, would not be able to generate beta-casomorphin-7. Based on these assumptions, some companies now sell “A2 milk”, a milk containing only A2 beta-casein. In this project, a LC-MS analytical method was developed to discriminate between A2 milk and commercial milk, which typically contains a mixture of A1 and A2 beta-casein. The final purpose is to offer milk producers an analytical tool to certify that a milk labeled as “A2 milk” is really as such, eventually capable to identify possible frauds or contaminations. Finally, in this thesis a minor project is described, having as object the enzyme transglutaminase (TGase) from microbial origin. TGase catalyses the formation of isopeptide bonds between carboxamides of glutamine residues and amine groups of lysine, resulting in protein cross-linking. The action of TGase can determine significant changes in the physico-chemical properties of proteins, leading to changes in viscosity, thermal stability and elasticity. For these reasons, TGase finds application as an additive in the food industry. In this study a TGase from an unknown microbial source has been characterized and identified by applying a bottom-up proteomic approach. The identified enzyme is produced from a bacterial strain different from the one most commonly used in food industrial applications, S. mobaraense. Moreover, a method for the measurement of TGase enzymatic activity by the hydroxamate assay has been set up and has now become a service offered by the Mérieux NutriSciences.Il mio dottorato di ricerca si è svolto in Mérieux NutriSciences, un’ azienda che fornisce servizi analitici. Durante il mio periodo di dottorato ho lavorato su tre progetti, il cui determinante comune era l'applicazione della spettrometria di massa (SM) all'analisi delle proteine. Lo studio principale riguarda la SM applicata all'analisi degli allergeni alimentari. L'allergia alimentare è una patologia importante, dovuta a reazioni immunologiche che insorgono a seguito dell'esposizione di un soggetto ad allergeni proteici. Poiché ad oggi non esiste cura, per prevenire le reazioni allergiche i pazienti devono evitare di assumere alimenti contenenti allergeni. Nell'Unione Europea, 14 allergeni devono essere indicati sulle etichette degli alimenti se aggiunti intenzionalmente. Tuttavia, una delle principali cause di reazione allergica è rappresentata dalla contaminazione indesiderata degli alimenti con allergeni all'interno degli impianti di produzione. Anche piccole quantità di allergene possono scatenare gravi reazioni; dunque, per proteggere i consumatori sono necessari metodi analitici sensibili. Recentemente, i metodi basati sulla SM hanno ricevuto crescente attenzione per la quantificazione degli allergeni alimentari in matrici complesse. Nel presente studio viene descritto lo sviluppo di un metodo basato sulla SM per il rilevamento simultaneo di allergeni da uova, latte, arachidi e frutta secca in prodotti da forno. Il metodo si basa sull'identificazione di specifici peptidi generati dall'idrolisi enzimatica degli allergeni target ed impiega una tecnica chiamata Multiple Reaction Monitoring, in cui lo spettrometro di massa è utilizzato per acquisire selettivamente segnali derivanti da coppie di specifici valori m/z, corrispondenti a uno ione peptidico e ad uno dei suoi frammenti. Il metodo sviluppato consente di rilevare gli allergeni target in modo specifico e con sensibilità accettabile e può essere considerato una valida alternativa ad altre comuni tecniche analitiche, come l’ELISA e la PCR. Un secondo argomento trattato in questa tesi riguarda la beta-caseina bovina, una proteina polimorfica per la quale sono state identificate 12 varianti genetiche, fra cui le più comuni sono la A1 e la A2. Alcuni studi hanno suggerito una possibile associazione fra il consumo di beta-caseina A1 e l'eziologia di alcune malattie, tra cui l’ischemia cardiaca e il diabete. Alla base degli effetti causati dalla beta-caseina A1 ci sarebbe un peptide bioattivo con attività simil-oppiode, rilasciato da specifici enzimi proteolitici durante la digestione. Questo peptide, chiamato beta-casomorphin-7, viene generato solo a partire da alcune isoforme di beta-caseina, contenenti un'istidina in posizione 67, inclusa la variante A1. Al contrario, le varianti che possiedono una prolina in posizione 67, come la variante A2, non sarebbero in grado di generare il peptide beta-casomorphin-7. Sulla base di queste ipotesi, alcune aziende vendono ora il cosiddetto "latte A2", un tipo di latte contenente solo beta-caseina A2. In questo progetto di dottorato è stato sviluppato un metodo analitico LC-MS per discriminare il latte A2 dal latte commerciale, che tipicamente contiene una miscela di beta-caseina A1 e A2. Lo scopo finale è offrire ai produttori di latte uno strumento analitico per certificare che un latte etichettato come "latte A2" sia realmente tale, ed eventualmente in grado di identificare possibili frodi o contaminazioni. Infine, in questa tesi viene descritto un progetto che ha come oggetto l'enzima transglutaminasi (TGasi) di origine microbica. La TGasi catalizza la formazione di legami isopeptidici tra residui di glutammina e di lisina, determinando la formazione di cross-linking fra proteine. L'azione della TGasi può determinare cambiamenti significativi nelle proprietà fisico-chimiche delle proteine, portando a modifiche nella viscosità, nella stabilità termica e nella elasticità. Per questi motivi, la TGasi trova applicazione come additivo nell'industria alimentare. In questo studio, una specie di TGasi di origine microbica è stata caratterizzata e identificata applicando un approccio proteomico “bottom-up”. L'enzima identificato viene prodotto da un ceppo batterico diverso da quello più comunemente utilizzato nelle applicazioni industriali alimentari, denominato S. mobaraense. Infine, è stato sviluppato un metodo per la misurazione dell'attività enzimatica della TGasi mediante il saggio dell'idrossammato, che è ora diventato un servizio analitico offerto da Mérieux NutriSciences
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Caratterizzazione di nuovi sistemi nanoparticellari per la somministrazione di farmaci anti-tumorali
No full textIn questo lavoro di tesi sono stati caratterizzati nuovi sistemi di nanocapsule per il trasporto di farmaci anti-tumorali. Il bersaglio di questi nanocarrier sono le MDSC, una popolazione di cellule mieloidi che ha la capacità di inibire lo sviluppo di una risposta immunitaria T cellulare contro il tumore. La biocompatibilità e la capacità di effettuare drug-delivery di questi sistemi è stata testata mediante studi sia in vivo che in vitr
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
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