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Plant viruses: the many aspects of fascinating nano-biotechnological tool
Full text linkI capsidi virali sono strutture stabili e robuste composte da multiple copie di una o più tipi di subunità proteiche organizzate con simmetrie ordinate (icosaedriche o filmentose). I capsidi virali possono essere prodotti in sistemi vegetali in consistenti quantità sia tramite l’infezione delle piante sia tramite l’espressione delle subunità capsidiche. Data la semplicità e l’elevata stabilità, le Chimeric Virus Particles (CVPs) e le empty Virus Like Particles (eVLPs) hanno attirato l’attenzione della comunità scientifica per lo sviluppo di reagenti che possono trovare impiego nell’ambito delle nano-biotecnologie. In questo lavoro di tesi, le CVPs e le eVLPs sono state sfruttate per l’espressione di peptidi funzionali, al fine di stabilizzarli e di produrli a basso costo. In particolare, le piattaforme di espressione virali scelte si basano sui seguenti virus vegetali: Potato Virus X (PVX), Cowpea Mosaic Virus (CPMV), Tomato Bushy Stunt Virus (TBSV) e il Turnip Mosaic Virus (TuMV). La prima applicazione descritta riguarda il trattamento di due malattie autoimmuni che hanno un forte impatto sociale: il Diabete Melito di tipo 1 (T1D) e l’Artrite Reumatoide (AR). Attualmente, esistono trattamenti che sono solo in grado di arginare e gestire gli effetti di queste patologie senza però bloccarne definitivamente l’azione. In questo lavoro, virus vegetali che espongono peptidi associati al T1D e all’AR sono stati utilizzati per lo sviluppo rispettivamente di un trattamento preventivo e terapeutico. Le particelle virali sono state geneticamente modificate in modo da esporre peptidi autoantigenici associati al T1D e all’AR, espresse in pianta, purificate dal tessuto vegetale ed utilizzate per studi pre-clinici in animali modello. I risultati ottenuti mostrano che la struttura delle particelle virali è in grado di agire come adiuvante aumentando la capacità immunomodulante dei peptidi autoantigenici selezionati ed esposti sui capsidi virali. La seconda parte di questo lavoro di tesi, si concentra sull’utilizzo delle CVPs esprimenti peptidi associati alla Sindrome di Sjögren (SjS) e all’AR per lo sviluppo di innovativi kit diagnostici. Queste due malattie autoimmuni sono attualmente difficili da diagnosticare principalmente per la presenza di un sottogruppo di pazienti che risulta essere negativo alla presenza dei più comuni marcatori sierologici utilizzati per la diagnosi. Questo progetto si è focalizzato sull’espressione in pianta di CVPs di struttura filamentosa che, tramite l’espressione di specifici peptidi, hanno permesso di migliorare notevolmente le performance diagnostiche di un test ELISA sviluppato con tali particelle in confrontato con lo stesso sviluppato utilizzo i peptidi sintetici. Inoltre, sono state espresse in pianta ulteriori CVPs che espongono due peptidi associati all’AR. Tali CVPs saranno in futuro utilizzate per la messa a punto di un kit per la diagnosi dell’AR. L’ultima parte di questo lavoro, riguarda altre possibili applicazioni dei virus vegetali come strumenti nano-biotecnologici. Nello specifico, eVLPs sono state espresse in pianta al fine di esprimere un peptide antimicrobico (AMP) e un cell penetrating peptide (CPP) per ottenere rispettivamente un innovativo eco-pesticida e uno strumento biotecnologico per l’internalizzazione di peptidi funzionali o proteine di interesse nelle cellule.The capsids of most plant viruses are simple and robust structures consisting of multiple copies of one or few types of protein subunits arranged with either icosahedral or helical ordered symmetry. In many cases, capsids can be produced in large quantities either by the infection of plants or by the expression of the subunits. In view of their relative simplicity, stability and easy production, plant chimeric virus particles (CVPs) or empty virus-like particles (eVLPs) have attracted attention as potential reagents for applications in bionanotechnology. In this work CVPs and eVLPs have been exploited for the expression of functional peptides, in order to stabilize them and avoid peptide low intrinsic stability and susceptibility to degradation. In particular, the viral expression platforms chosen for the expression of target peptides are based on four plant viruses widely used as scaffold for peptide display: Potato Virus X (PVX), Cowpea Mosaic Virus (CPMV), Tomato Bushy Stunt Virus (TBSV) and Turnip Mosaic Virus (TuMV). The first application explored in this work regards the therapy of Type 1 diabetes (T1D) and Rheumatoid arthritis (RA), two autoimmune diseases that share a strong social impact. Currently, there are treatments able to manage and/or stem the effects of these disorders. In particular, plant viruses displaying peptides associated to T1D and RA have been used respectively for the development of a preventive and therapeutic drug. Virus particles displaying autoantigenic peptide specific for these diseases have been expressed and used for pre-clinical studies in T1D and RA animal models. The results observed suggest that the use of viral structure for peptide display works as an adjuvant by increasing peptide modulation capability. The second part of this work regards the use of plant viruses displaying peptide as reagents for the development of innovative kit for the Sjögren’s Syndrome (SjS) and RA diagnosis. These two autoimmune diseases are difficult to be diagnosed and either for SjS of RA there are subgroups of patient seronegative to the main diagnostic serological markers. In this work, the use of filamentous particles for the display of specific SjS peptide allowed to increase the diagnostic performances of an ELISA kit in comparison to the use of the peptide alone. Moreover, autoantigenic peptides associated to RA were successfully expressed in plants on the surface of viral particles that will be exploited in the future for the development of a kit for seronegative RA diagnosis. A third part of this PhD thesis regards another possible application of plant viruses as tools for peptide display. In particular, viral particles have been used for the expression of a antimicrobial peptide (AMP) that could be exploited as eco-friendly pesticide and for “nanoagriculture” application. Finally, the possibility of developing a biotechnological tool for peptide internalisation into the cells has been exploited by fusing on the surface of an icosahedral plant virus a cell-penetrating peptide (CPP) derive from HIV. Regarding this third part, CVPs and eVLPs displaying the selected peptides have been successfully expressed in plants; however, several drawbacks have been encountered in the purification process
Plant-Based Systems for Vaccine Production
No full text: Plant systems have been used as biofactories to produce recombinant proteins since 1983. The huge amount of data, collected so far in this framework, suggests that plants display several key advantages over existing traditional platforms when they are intended for therapeutic uses, including safety, scalability, and the speed in obtaining the final product.Here, we describe a method that could be applied for the expression and production of a candidate subunit vaccine in Nicotiana benthamiana plants by transient expression, defining all the protocols starting from plant cultivation to target recombinant protein purification
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe 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
Variations on the Author
Full text link“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
Transient Expression in Red Beet of a Biopharmaceutical Candidate Vaccine for Type-1 Diabetes
Full text linkPlant molecular farming is the use of plants to produce molecules of interest. In this perspective, plants may be used both as bioreactors for the production and subsequent purification of the final product and for the direct oral delivery of heterologous proteins when using edible plant species. In this work, we present the development of a candidate oral vaccine against Type 1 Diabetes (T1D) in edible plant systems using deconstructed plant virus-based recombinant DNA technology, delivered with vacuum infiltration. Our results show that a red beet is a suitable host for the transient expression of a human derived autoantigen associated to T1D, considered to be a promising candidate as a T1D vaccine. Leaves producing the autoantigen were thoroughly characterized for their resistance to gastric digestion, for the presence of residual bacterial charge and for their secondary metabolic profile, giving an overview of the process production for the potential use of plants for direct oral delivery of a heterologous protein. Our analysis showed almost complete degradation of the freeze-dried candidate oral vaccine following a simulated gastric digestion, suggesting that an encapsulation strategy in the manufacture of the plant-derived GAD vaccine is required
PVX a powerful tool for autoimmune disease diagnosis
No full textWe explored the use of Potato Virus X as a scaffold for the expression of an immunodominant peptide associated to Sjogren’s syndrome.
Sjögren's syndrome (SjS) is a chronic autoimmune disease in which the body's white cells destroy the exocrine glands, specifically the salivary and lacrimal glands, that produce saliva and tears, respectively.
Human lipocalin was recently identified as a primary autoantigen associated to the disease and the immunodominant peptide associated to the protein was discovered using the sera of SjS patients. Autoantibodies directed to the peptide allow to identify in the population patients whose sera was negative to anti nuclear antibodies and to the rheumatoid factor.
The lipocalin peptide was cloned into a PVX-based vector which mediates the peptide display on the virus coat protein, yielding Chimeric Virus Particles (CVPs). CVPs allow a high-density exposure of the peptide on the viral surface.
The CVPs were used for coating an ELISA plate giving an assay which reproducibility, stability and sensitive was compared to the use of the peptide alone
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe 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
Full text linkWe 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
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