1,720,961 research outputs found
Counteracting intervertebral disc degeneration: a contribution from the development of Wharton's jelly-based scaffolds and the investigation of the purinergic P2X7 receptor
No full textIl lavoro di questa tesi ha avuto come scopo quello di fornire nuovi elementi per ottimizzare approcci terapeutici per contrastare la degenerazione del disco intervertebrale (IVDD). L'IVD, una giunzione fibrocartilaginea che collega due vertebre adiacenti, può andare incontro a degenerazione per diversi motivi (invecchiamento, traumi o infezioni), perdendo la sua funzionalità e causando mal di schiena, una malattia comune ancora senza cura. Durante il mio dottorato ho affrontato l'argomento seguendo due approcci: a. uno puramente molecolare riguardante l'identificazione di nuove molecole con ruoli critici nell'omeostasi dell'IVD, per sviluppare strategie terapeutiche mirate; b. l'altro legato all’ingegneria tissutale, finalizzato a sviluppare nuovi scaffold impiantabili basati sulla matrice di gelatina di Wharton decellularizzata (DWJM). In entrambi i casi il modello sperimentale utilizzato è rappresentato dalla coltura primaria di cellule IVD isolate da biopsie di pazienti con diverso grado di IVDD sottoposti a discectomia. L’approccio molecolare ha avuto lo scopo di studiare il recettore purinergico P2X7 (un canale ionico ATP-dipendente), dimostrando per la prima volta la sua localizzazione nel nucleo di cellule IVD, condrociti e osteoblasti e non solo sulla membrana plasmatica. Da qui l'ipotesi che P2X7R insieme a molecole partner da identificare possa agire come un collegamento tra l'esterno e l'interno, supportando sia l’IVDD sia svolgendo un ruolo nella meccanotrasduzione legata all'ATP extracellulare. I risultati principali sono stati: -l'espressione di P2X7R aumenta durante l’IVDD; -P2X7R è associato alla segnalazione di Ca++ attraverso l’interazione con il fattore di trascrizione NFATc1 in uno specifico complesso proteico; -NFATc1 è reclutato sul promotore del gene di P2X7R e ne aumenta l'espressione; -quando il fenotipo delle cellule IVD viene ripristinato dall'ipossia, l'espressione di P2X7R è diminuita da HIF-1α che viene reclutato sul promotore di P2RX7 spostando NFATc1; -P2X7R è associato alla lamina A/C, un componente principale della lamina nucleare, e a emerina, SUN2 e HDAC, tutti coinvolti nella regolazione della cromatina. Queste evidenze dimostrano per la prima volta che P2X7R e NFATc1 sono marcatori dell’IVDD. Inoltre, aprono la strada allo studio di ruoli di P2X7R non ancora descritti, avendo identificato proteine associate all'involucro nucleare come partners di P2X7R.
L’approccio di ingegneria tissutale ha avuto lo scopo di sviluppare un sistema in vitro simile al microambiente dell’IVD, fornendo sia una piattaforma adatta a monitorare il comportamento cellulare sia le condizioni per ottimizzare nuovi scaffold. Questo ha portato all'utilizzo della DWJM, i cui componenti biologici e strutturali sono molto simili alla matrice extracellulare dell’IVD. I risultati principali sono stati: -quando coltivate in combinazione con la DWJM, le cellule IVD degenerate recuperano il loro fenotipo simile ai condrociti, esprimendo marcatori persi durante l’IVDD (SOX9, TRPS1, FOXO3a). Questo effetto è potenziato dall’ipossia, con l’aumento di HIF-1α che viene reclutato sul promotore di SOX9 e FOXO3a, proteine cruciali per l'omeostasi e la riparazione dell’IVD;
-quando opportunamente combinata con alginato e gelatina, la DWJM forma millicilindri compositi (WJMs) più gestibili e geometricamente controllabili;
-i WJMs con la più alta percentuale di DWJM resistono alle forze meccaniche, supportano l'attività delle cellule progenitrici/notocordali residenti (aumento delle cellule CD24-positive) e influenzano positivamente la migrazione cellulare, la proliferazione e il ripristino del fenotipo IVD (aumento dell'espressione di collagene di tipo II, aggrecano, SOX9 e FOXO3a). In conclusione, questa tesi ha portato alla luce nuovi elementi (molecolari e biomaterialistici) per progettare in futuro nuove terapie mirate per l'IVDD.The work of this thesis was aimed at providing new elements to optimize therapeutic approaches to counteract intervertebral disc (IVD) degeneration. The IVD, a fibrocartilaginous junction that connects two adjacent vertebrae allowing limited movement, can undergo degeneration for different reasons (aging, trauma or infection), lose its functionality and cause back pain, a common disease that is still without a cure. During my PhD I addressed the topic following two main approaches: a. a more purely molecular one concerning the identification of new molecules that may play critical roles in IVD homeostasis, aimed to develop targeted therapeutic strategies; b. the other addressed to a tissue engineering field, aimed at developing new implantable scaffolds based on decellularized Wharton's jelly matrix (DWJM). In both cases the experimental model used were primary culture of IVD cells isolated from biopsies of patients undergoing discectomy with different grade of degeneration. The molecular field was aimed at studying the purinergic P2X7 receptor (an ATP-gated plasma membrane ion channel), that for the first time, we documented to be localized in the nucleus of IVD cells, chondrocytes, and osteoblasts and not only in the plasma membrane.
From here we pursued the hypothesis that P2X7R together with partner molecules to be identified could act as a link between the outside and the inside both supporting the degeneration of the IVD and playing a role in the mechanotransduction that relies on extracellular ATP. The main results were: -P2X7R expression increased during the IVD degeneration; -P2X7R is associated with Ca++ signaling through a direct binding to the Ca++-sensing transcription factor NFATc1 in a specific protein complex; -NFATc1 is recruited at the P2RX7 gene promoter and increased P2X7R expression; -when the degenerated phenotype of IVD cells was reversed by hypoxia, P2X7R expression decreased by HIF-1α which is recruited at the P2RX7 promoter displacing NFATc1; -P2X7R is closely associated with lamin A/C, a major component of the nuclear lamina, emerin, SUN2 and HDAC, all involved in a chromatin regulatory network. With this evidence we identified for the first time that P2X7R and NFATc1 are markers of IVD degeneration. Moreover, we have opened the way to reasoning about roles of P2X7R not yet described, such as scaffold protein or regulator of gene expression, having identified specific protein associated with the nuclear envelope as partners of P2X7R. The field of tissue engineering has been to develop an in vitro system that mimics the IVD microenvironment, providing both a suitable platform to monitor cellular behavior and conditions to optimize new scaffolds. This led to the use of DWJM, whose biological and structural components are very similar to the IVD extracellular matrix. The main results were: -when cultured in combination with DWJM, degenerated IVD cells responded positively by recovering their chondrocyte-like phenotype, expressing markers lost during degeneration (SOX9, TRPS1, FOXO3a). This effect was enhanced when, after exposure to hypoxia, HIF-1α increased in the expression level and was recruited to the promoter of SOX9 and FOXO3a, which are crucial proteins for IVD homeostasis and repair; -when appropriately combined with alginate and gelatin, DWJM gave rise to more manageable and geometrically controllable composite millicylinders (WJMs); -WJMs with the highest percentage of DWJM were prone to resist mechanical forces, supported the activity of resident progenitor/notochordal cells (as shown by an increase in CD24-positive cells), positively affected cell migration, proliferation, and restoration of the IVD phenotype (as shown by an increase of collagen type II, aggrecan, SOX9, and FOXO3a expression). We can conclude that this thesis has brought to light new elements (molecular and biomaterialistic) to design in the future new targeted therapies for the regeneration of the degenerated IVD
A 3D in vitro model of biphasic calcium phosphate (BCP) scaffold combined with human osteoblasts, osteoclasts, and endothelial cells as a platform to mimic the oral microenvironment for tissue regeneration
Full text linkObjectives: This study aimed to develop an innovative 3D in vitro model based on the biphasic calcium phosphate (BCP) scaffold combined with human osteoblasts (hOBs), osteoclasts (hOCs), and endothelial cells to evaluate its effects on bone and vascular cells behavior.
Methods: To this end, an optimized mixture of hydroxyapatite (HA) and β-tricalcium phosphate (TCP) with a weight ratio of 30/70 was employed to develop a BCP scaffold using the computer-aided design (CAD) approach. The BCP scaffold was combined with primary cultures of hOBs, hOCs and human umbilical vein endothelial cells (HUVECs).
Results: Morphometric analyses using scanning electron microscopy (SEM) and X-ray micro-computed tomography, along with biomechanical testing, revealed that BCP scaffold exhibited a regular 3D structure with large interconnected internal pores (700 μm) and high mechanical strength. In terms of biological behavior, after 14 days of tri-culture with hOBs, hMCs and HUVECs, SEM, immunofluorescence, and histological analyses showed that all cell types were viable and adhered well to the entire surface of the scaffold. Interestingly, SEM and energy-dispersive X-ray spectroscopy analyses also revealed on the BCP scaffold the presence of mineralized matrix crystals of Ca, P, O and C within a tissue-like cell layer produced by the interaction of the three cell types.
Conclusions: Data confirmed the high performance of the BCP scaffold through biomechanical studies. Notably, for the first time, this study demonstrated the feasibility of combining BCP scaffold with hOBs, hOCs, and HUVEC, which remained viable and maintained their native phenotypes, creating also tissue-like cell layer.
Clinical significance: Although further investigation is needed, these results underscore the potential to develop a 3D in vitro model that mimics the oral microenvironment, which could be valuable for BTE approaches in in vivo studies
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
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
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
Author-wise bibliometric analysis based on entropy.
No full textAuthor-wise bibliometric analysis based on entropy.</p
- …
