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Isolamento, espansione e caratterizzazione fenotipica delle cellule staminali masenchimali da emocomponenti, sangue cordonale e sangue periferico mobilizzato
No full textIn literature it has been described in the stromal component of the bone marrow the presence of a limited number of mesenchymal stem cells (MSCs) which posses multi-lineage potentiality and ability to differentiate into various tissues, such as, bone cartilage, fat, muscle, ligament, tendon and neural tissue. Plasticity of these cells could be shortly utilized in regenerative medicine for developing clinical trials in cell therapy. At the present, other human tissue are investigated as further alternative sources of MSCs.
This research project has these aims: development and optimization of isolation protocols specific for the different MSCs procurement sources, as well as cord blood (CB), cord tissue, bone marrow (BM) of patient affect by malignant empathy, mobilized peripheral blood (PB), peripheral blood from healthy donors, for example Buffy Coat (BC), or from patient affected by cutaneous sores (leuco-platelet concentrate CLP); expansion of isolated MSCs; comparison of phenotypical characterization between isolated MSCs and five different stromal cell lines HM1 SV40, HM2 SV40, HCB1 SV40, BAEP2 WILD, BAEP2 SV40.
Isolation of mononuclear cells by CB, BM, mobilized PB, BC and CLP samples was carried out by two different methods: isolation technique after separation by 1.077 g/ml density gradient centrifugation (Ficoll-Histoprep) according to Boyum technique and depletion of red blood cells with ammonium chloride. Cord tissue, was, instead, cut in small fragments and seeded in culture medium. Different primary culture parameters were evaluated: time between withdrawal and processing sample, culture medium, cell concentration seeding and adhesive characteristics of culture supports after different coating treatments. Primary culture were then incubated at 37° C in an atmosphere of 95 % air- 5% CO2 as long as they were semi-confluent; cells were, then, detached by a PBS solution with trypsin 0.25% and EDTA 0.02% and seeded again for expansion. Furthermore, for 10 BC samples separated on Ficoll-Histoprep (5 donors with age among 18 to 36 years old and 5 donors with age among 50 to 56 years old) limit dilution were set up on 96 wells plates to verify MSCs growth. Observation of the cultures were carried out to an inverse microscope after 96 hours and after 7 and 14 days from their setting.
Finally, phenotypic characterization of isolated cells from BC seeded on 96 wells plate were carried out after 24 hours, 48 hours, 7 and 14 days culture time by means of immunocytochemistry. A specific antibody panel was utilized to recognize specific MSCs surface antigens.
Number of attached cells for CB, mobilized PB and CLP cultures was limited after 14 days culture time; cells seem polymorph, spherical and rarely fibroblast-like. On the contrary, in BC cultures, in particular conditions, cells immediately adhere to the support and they assume stretched morphology. After 7 days culture time, cells of BM cultures from patient affected by malignant hematological pathology show a spherical, polymorph and essentially fibroblast-like shape and they get confluent after 14 days. With regard to processed cord, after about one month of culture time cells flow together and they organize themselves as a fibroblast-like monostrate.
In vitro expansion of isolated cells from different samples was difficult, because they tightly adhere to the substrate and with only 2 BC samples out of 32 it was reached the 4th serial passing. Limit dilution did not point out cell growth from 96 hours to 2 weeks culture time and there are not significant differences among BC samples of two different age range. Immunocytochemical investigations carried out on primary cultures of BC pointed out the following antibody specificities; EGF, EGF-R, FGF, FGF-R, ADM, RAMP2, ET-1, CD33, CD73, CD105, KDR and collagen. MSCs, instead, do not express the following surface markers: CD14, CD15, CD34, CD42b, CD71, HLA-DR, STRO-1, actin, desmin, fibronectin and laminin.
These results are comparable to those obtained with stromal cell lines HM1 SV40, HM2 SV40, HCB1 SV40, BAEP2 WILD, BAEP2 SV40 which can be considered as positive control. In particular, positivity for stromal mesenchymal markers CD73, CD105 besides KDR, demonstrate that isolated cells from BC are actually MSCs.
This preliminary experimentation shows some informations to optimize culture methods of MSCs isolated from sources alternative to bone marrow. As described in Literature, the results of the phonotypical characterization demonstrate that cell markers of cells isolated from BC are comparable to those of MSCs isolated from bone marrow. Therefore, BC could be an important source of adult stem cells immediately available for regenerative medicine, even the origin of these cells, their physiological-pathological meaning and their plastic potential are not yet cleared.In Letteratura è stata descritta la presenza nella componente stromale del midollo osseo di un ristretto numero di cellule staminali mesenchimali (MSCs) con potenzialità multi-linea, differenziabili in tessuto osseo, cartilagineo, adiposo, tendineo, muscolare e nervoso. La plasticità di tali cellule potrebbe essere sfruttata a breve termine nell’ambito della medicina rigenerativa per lo sviluppo di protocolli clinici applicativi di terapia cellulare. Attualmente, altri tessuti del corpo umano sono indagati come ulteriore fonti alternative di MSCs.
Il programma di ricerca si propone i seguenti obiettivi: sviluppo ed ottimizzazione di protocolli di isolamento specifici per le differenti fonti di approvvigionamento delle MSCs, quali sangue cordonale (CB), tessuto cordonale, midollo osseo (BM) di pazienti affetti da empatie maligne, sangue periferico (PB) mobilizzato, sangue periferico da donatori sani, come i Buffy Coat (BC), o da pazienti affetti da piaghe cutanee (CLP, concentrato leuco piastrinico); tentativo di espansione delle MSCs isolate; caratterizzazione fenotipica delle MSCs isolate rispetto a quella delle 5 linee stromali HM1 SV40, HM2 SV40, HCB1 SV40, BAEP2 WILD, BAEP2 SV40.
L’isolamento di cellule mononucleate da campioni di CB, BM, PB mobilizzato, BC e CLP è stato condotto mediante 2 metodiche: tecnica di separazione su gradiente Ficoll-Histoprep di densità 1.077 g/ml secondo la tecnica di Boyum e deplezione dei globuli rossi con ammonio cloruro. Il tessuto cordonale invece è stato tagliato in minuscoli frammenti e messo direttamente in coltura. Sono stati valutati vari parametri di coltura primaria, quali il tempo trascorso dalla raccolta del campione al suo processamento, il terreno di coltura, la concentrazione di semina e le caratteristiche adesive dei contenitori di coltura dopo vari trattamenti. Le colture primarie così allestite sono state incubate a 37°C e 5% di CO2 fino al raggiungimento della semi-confluenza; quindi le cellule sono state staccate del recipiente di coltura utilizzando una soluzione di Tripsina 0.25% ed EDTA 0.02% in PBS, e riseminate per l’espansione.
Inoltre su 10 campioni di BC separati su Ficoll-Histoprep (5 di donatori di età compresa tra 18 e 36 anni e 5 di età compresa tra 50 e 65 anni) sono state allestite 10 diluizioni limite su piastre da 96 pozzetti, per verificare la crescita delle MSCs. Le letture al microscopio ad inversione sono state eseguite dopo 96 ore, 7 giorni e 14 giorni dall’allestimento della coltura.
Infine è stata eseguita la caratterizzazione fenotipica delle cellule isolate da BC, seminate su piastre da 96 pozzetti, dopo 24 ore, 48 ore, 7 giorni e 14 giorni di coltura, mediante immunocitochimica, utilizzando un pannello di anticorpi per il riconoscimento di specifici antigeni di superficie per MSCs.
Nelle colture di CB, PB mobilizzato e CLP il numero di cellule adese dopo 14 giorni dalla semina è modesto; le cellule appaiono polimorfe, sferoidali e più raramente fibroblastoidi. Al contrario, nelle colture di BC, in particolari condizioni, le cellule aderiscono al recipiente quasi immediatamente ed assumono subito una morfologia fusata. Nelle colture di BM di pazienti affetti da emopatie maligne, le cellule presentano al 7° giorno di coltura forma sferica, polimorfa e prevalentemente fibroblastoide e vanno a confluenza al 14° giorno. Per quanto riguarda i cordoni processati, dopo circa un mese di coltura le cellule sono a confluenza e formano un monostrato di cellule fibroblastoidi.
L’espansione in vitro delle cellule isolate dai vari campioni è risultata difficoltosa, in quanto aderiscono fortemente al substrato e solo con 2 campioni di BC su 32 totali si è riusciti a raggiungere il 4° passaggio seriale. Le diluizioni limite non hanno evidenziato una crescita delle cellule dalle 96 ore alle 2 settimane di coltura e non si notano differenze significative tra i campioni di BC delle 2 diverse fasce di età.
Le indagini immunocitochimiche condotte sulle colture primarie di BC hanno evidenziato le seguenti specificità anticorpali: EGF, EGF-R, FGF, FGF-R, ADM, RAMP2, ET-1, CD33, CD73, CD105, KDR e collagene I. Invece le MSCs non esprimono i seguenti marcatori di superficie: CD14, CD15, CD34, CD42b, CD71, HLA-DR, STRO-1, actina, desmina, fibronectina e laminina. Tali risultati sono comparabili a quelli ottenuti con le linee stromali HM1 SV40, HM2 SV40, HCB1 SV40, BAEP2 WILD, BAEP2 SV40, che sono da considerare il controllo positivo. In particolare le positività per i marcatori stromali mesenchimali CD73, CD105, oltre che per KDR, dimostrano che le cellule isolate da BC sono effettivamente MSCs.
Questa sperimentazione preliminare fornisce alcune indicazioni per l’ottimizzazione dei metodi di coltura di MSCs isolate da fonti alternative al midollo osseo. Come descritto in Letteratura, i risultati della caratterizzazione fenotipica dimostrano che i marcatori cellulari delle cellule isolate da BC sono comparabili a quelli delle MSCs isolate da midollo osseo. Perciò i BC potrebbero essere un’importante fonte di cellule staminali adulte immediatamente sfruttabili in medicina rigenerativa, anche se sono ancora da chiarire l’origine di tali cellule, il loro significato fisio-patologico e il loro potenziale plastico
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
Urotensin-II and UII-receptor expression and function in the rat adrenal cortex
No full textUrotensin-II (UII) is a potent hypertensive peptide, which has been recognized as
an endogenous ligand of the G protein-coupled receptor (GPR)-14, now named UT-R.
Real-time PCR demonstrated the expression of UII and UT-R mRNAs in both dispersed
and in vitro cultured rat adrenocortical cells. UII concentration-dependently
decreased basal, but not ACTH-stimulated, corticosterone secretion from cultured
adrenocortical cells, and the effect was abolished by the UT-R antagonist
Palosuran. UII did not affect the proliferation rate of cultured cells. Taken
together, these findings suggest that UII may be included in the group of
peptides (adrenomedullin, atrial natriuretic peptide, neurotensin and beacon),
that, acting in an autocrine-paracrine manner, are involved in the inhibitory
tuning of adrenocortical secretion
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
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