1,720,963 research outputs found
Integrated analysis of novel signal transduction pathways in red cells from patients with neuroacanthocytosis
Full text linkIl termine neuroacantocitosi (NA) raggruppa diverse malattie genetiche rare che condividono manifestazioni neurologiche simili e la presenza di globuli rossi stellati nella circolazione periferica, gli acantociti. Le due principali malattie classificate come NA sono Corea acantocitosi (ChAc) e sindrome di McLeod (MLS).
Poiché la presenza di acantociti è una caratteristica comune di questi disordini, lo studio dei meccanismi alla base della loro formazione può aiutare a comprendere la patogenesi delle NA. In questa tesi viene presentata una serie di studi sui meccanismi di signaling e sulle modificazioni strutturali di globuli rossi di pazienti di ChAc e MLS.
Nel primo studio abbiamo analizzato con tecniche di proteomica la fosforilazione in tirosina di globuli rossi di pazienti affetti da ChAc .
Nei globuli rossi di questi pazienti abbiamo riscontrato un aumento della fosforilazione in tirosina su diverse proteine di membrana e legate alla membrana tra cui banda 3, β-spectrina e adducina. In particolare, la fosforilazione sul residuo Tyr-904 della banda 3, target della chinasi Lyn, era molto elevata, mentre sul residuo Tyr-8 della stessa proteina, target della chinasi Syk, non abbiamo riscontrato un aumento della fosforilazione. Nei pazienti di ChAc, la fosforilazione della banda 3 da parte di Lyn è indipendente dal meccanismo canonico di fosforilazione sequenziale mediato da Syk.
Le alterazioni dell’organizzazione delle proteine di membrana correlate con ChAc sembrano quindi essere il risultato di un aumento della fosforilazione in tirosina che porta a cambiamenti nel legame della banda 3 con i ponti multiproteici tra la membrana e il citoscheletro. Proponiamo quindi quest’alterazione nell’associazione tra membrana e citoscheletro mediata da fosforilazione in tirosina come un nuovo meccanismo che porta alla formazione di acantociti in ChAc.
Nel secondo studio abbiamo combinato i nostri set di dati fosfo-proteomici su eritrociti di pazienti di ChAc e MLS con l’analisi topologica di network proteici per predirre quali sub-network della trasduzione del segnale possano essere coinvolti nella formazione degli acantociti.
Abbiamo identificato tutte le interazioni che legano le due proteine mutate nelle due patologie in esame (rispettivamente coreina e XK) con le proteine differenzialmente fosforilate dei nostri dati sperimentali. Quindi, abbiamo analizzato nello specifico solo cluster di proteine coinvolte nella trasduzione del segnale che interagiscono molto strettamente tra loro e che possono essere coinvolte nella formazione di acantociti in entrambe le patologie. Abbiamo identificato un cluster di 14 chinasi che possono essere coinvolte in tale processo e meritano ulteriori approfondimenti.
Come studio preliminare nel contesto di una collaborazione internazionale abbiamo analizzato globuli rossi da pazienti affetti da Neurodegenerazione con Accumulo di Ferro nel Cervello (NBIA) e di loro parenti di primo grado. Il nostro scopo era quello di determinare se fossero presenti acantociti nei pazienti e nei soggetti correlati ma privi di sintomi clinici e di studiare le caratteristi che strutturali dei loro eritrociti.
Nell’ultimo studio abbiamo validato con l’applicazione a tecniche di analisi proteomica un nuovo copolimero basato su acrilamide e polivinil alcool modificato con gruppi olefinici. Questo nuovo idrogel è semplice da maneggiare anche a basse concentrazioni e la sua macroporosità lo rende particolarmente adatto alla separazione di proteine ad alto peso molecolare quale la coreina.Neuroacanthocytosis (NA) is a group of rare genetic disorders that share similar neurological clinical manifestations and the presence of thorny red cells in the peripheral circulation, the acanthocytes. The two core NA diseases are Chorea-Acanthocytosis (ChAc) and McLeod Syndrome (MLS). Since acanthocytes are an hallmark of NA, studying the mechanisms underlying the generation of acanthocytes might shed light on the pathogenesis of NA syndromes. Here, we present a set of studies on the signaling mechanisms and structural changes in red cells from ChAc and MLS patients.
In the first study, we evaluated tyrosine phosphorylation of red cells from ChAc patients by proteomics analysis. Increased Tyr-phosphorylation state of several membrane proteins including band 3, β-spectrin and adducin was found in ChAc RBCs. In particular, band 3 was highly phosphorylated on the Tyr-904 residue, a functional target of Lyn, but not on Tyr-8, a functional target of Syk. In ChAc RBCs band 3 Tyr-phosphorylation by Lyn was independent of the canonical Syk mediated pathway. The ChAc-associated alterations in RBC membrane-protein organization appear to be the result of increased Tyr-phosphorylation leading to altered linkage of band 3 to the junctional complexes involved in anchoring the membrane to the cytoskeleton. We propose this altered association between cytoskeleton and membrane proteins as a novel mechanism in the generation of acanthocytes in ChAc.
In the second study, we combined phosphoproteomics datasets on ChAc and MLS with network topology analysis to predict signaling sub-networks involved in acanthocyte generation. We identified all the interactomic shortest paths linking the two proteins mutated in NA syndromes, respectively chorein and XK, to the differentially phosphorylated proteins in our proteomics data. Then, we refined the analysis considering only restricted clusters of highly interacting signaling proteins which can be involved in acanthocyte formation in both diseases. We identified a cluster of 14 kinases that might be related to red cell shape alterations and deserve further investigation.
As preliminary study in the context of an international collaboration we analyzed red cells from Neurodegeneration with Brain Iron Accumulation (NBIA) patients and their first degree relatives. Our aim was to assess the presence of acanthocytes in these subjects and to study their structural characteristics.
In the last study, we validated a new co-polymer based on acrylamide and polyvinyl alcohol bearing olefinic moieties in proteomic analysis of red cells. This new hydrogel is easy to handle and its macroporosity makes it suitable for the separation of high molecular weight proteins such as chorein
Microplate spectrophotometry for the high-throughput screening of cytotoxic molecules
No full textObjectives: High-throughput chemical and biochemi- cal technologies are now exploited by modern phar- macology and toxicology to synthesize a multitude of new molecules with bioactive potential, or to isolate them from living matter. Testing molecules in cell sys- tems on a large scale, however, is a rate-limiting step in drug discovery or in toxicity assessment. In this study, we developed a low-cost high-throughput method for first-level screening of cytotoxic molecules.
Materials and methods: We used microplate spectro- photometry to measure growth kinetics of human tumour cells that grow in suspension (Molt3) or adherent to the plastic surface of culture wells (HeLa) in standard RPMI medium. Cells were trea- ted with colchicin, idarubicin or paclitaxel under var- ious treatment schedules. The effects were quantified and compared with those measured by optical microscopy using the trypan blue dye exclusion method to reveal dead cells.
Results: Proliferation kinetics of tumour cells can be quantified by measuring variations in optical densi- ties of cell samples at 410 and 560 nm wavelengths. For cells that grow in suspension, one single reading at 730 nm may be sufficient to reconstruct growth curves that parallel those obtained by direct cell counting. Effects of the cytotoxic treatments could also be quantified and results compared very favour- ably with those obtained using standard techniques. Conclusions: Microplate spectrophotometry is a robust and sensitive method to monitor growth of animal cell populations both in the absence and in
the presence of cytotoxic drugs. This method imple- ments existing technologies and can be fully auto- mated
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
Performance of a novel sieving matrix of Poly(vinyl alcohol)ACRYLAMIDE copolymer in elecrophoretic separations of high molecular weight proteins from red cell membrane.
Full text linkThe analysis of high molecular weight (HMW) proteins from complex mixtures is still a
challenge in proteomics. This work introduces a novel hydrogel obtained by the copolymerization
of an allyl-PVA derivative with acrylamide and bisacrylamide and applies this
matrix to the electrophoretic separation of HMW proteins. By inducing gelation of polyacrylamide
in the presence of variable amounts of allyl-PVA, it is possible to control and
vary the average gel porosity. This gel is easy to produce and handle and offers the advantage
of being highly mechanically resistant and macroporous. The new matrix was
tested in mono-dimensional separations of complex protein mixtures extracted from red
cell membranes with different detergents. The improved performance of this macroporous
matrix allowed to identify new proteins by MS and immunoblot analysis using specific
antibodies. In particular, the resolution of proteins ranging in size between 97 and
279 kDa was greatly improved here compared to standard polyacrylamide gels, suggesting
that this matrix can be a useful tool in routine analysis of HMW proteins in cell biology
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
- …
