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FLOW CYTOMETRIC AND ARTIFICIAL INTELLIGENCE APPROACH TO DIAGNOSTIC MARKERS FOR B-CELL LYMPHOMAS
Full text linkB-cell Non-Hodgkin lymphomas (B-NHL) are a wide and highly heterogeneous group of malignancies, characterized by different morphology, phenotype, genotype, aggressiveness and response to therapy. Immunophenotypic characterization is a key element for their classification in order to guide to the correct therapeutic plan. Although the phenotypic study is routinely performed by immunohistochemistry (IHC), we verified that the use of flow cytometry (FC) could bring several advantages. Applied to samples from various sources derived from patients affected by the most common mature B-NHL, FC allows the quantitative expression of multiple markers to be evaluated by analyzing millions of cells simultaneously and easily defining clonal populations. This results in the ability to almost perfectly isolate each neoplastic subpopulation and study it in its uniqueness. This feature distinguishes FC analysis from IHC, which, by presenting the global appearance of the sample being examined, preserves the architecture of the tissue while revealing all mixed clones. In addition, like IHC, FC can provide information about intracellular antigens. In addition, FC can be applied to peripheral blood (PB) samples of leukemia stage lymphoma. In any case, FC provides faster and less biased results than IHC because the data are expressed quantitatively. However, the large amount of data generated by FC is very complex to analyze as a whole. In particular, it is difficult to correlate each marker with the precise diagnosis of a disease. Artificial intelligence (AI) can help by using sophisticated software and computing systems to compare and stratify all this data in a short time. In this way, AI provides more manageable data with a two-dimensional representation that is easier to interpret. In a previous study, we applied machine learning (ML) algorithms to a large dataset of B-NHL immunophenotypes to generate a robust and clinically applicable prediction system. This system would also allow us to overcome the time-consuming, optimize the use of antibodies and standardize a clinically applicable predictive system by establishing a panel of antibodies to be systematically used in a multiparametric immunophenotypic analysis of samples performed with a high-complexity flow cytometer.We then applied additional intracellular markers to a homogeneous case series of 615 tissue samples whose diagnoses, all confirmed by histologic analysis, were grouped into 8 major categories of B-NHL patients. The Predictive Power Score (ppscore) method allowed us to assess the impact of each marker in defining each lymphoma category. Considering that a ppscore greater than 0.22 (the baseline score) is statistically significant for discriminating diagnostic categories, we surprisingly noticed the discriminatory power of intracellular markers not commonly used in a multiparametric immunophenotypic approach to lymphoma diagnosis, such as IRF4 and Bcl6.The role of these markers was validated by combining each one with all the others in a classification tree, resulting in a structural relationship tree that separates the entire database into quasi-homogeneous groups of lymphomas. Finally, the UMAP dimensionality reduction technique, we observed that the 8 lymphoma categories were substantially grouped and separated in clusters. The results obtained demonstrate how the use of surface and intracellular markers allows us to define the major categories of B-NHL with a high degree of accuracy. 10 or less markers seem to be sufficient to achieve an adequate classification capability. Nevertheless, a greater number of markers, combining intracellular with unconventional markers (CD305, CD81), increases the ability of UMAP to separate different entities. In conclusion, it is conceivable that the implementation of AI applied to FC could contribute significantly to an optimal diagnostic process in B-NHL, where histopathological examination remains the gold standard
ADVANCED CHARACTERIZATION OF THE YEAST KEOPS/EKC COMPLEX
Full text linkDuring my PhD I have studied the properties of two yeast proteins, the protein kinase Bud32 and the putative protease Kae1, that take part in a nuclear complex named KEOPS/EKC. Actually, while Kae1 is associated uniquely to the proteins of the complex, Bud32 has many other partners in the cell; in fact, I have also studied its strong relationship with the Grx4 glutaredoxin.
The yeast KEOPS/EKC complex has been isolated in 2006 by two different groups and has been shown to be involved in both telomere homeostasis and transcription regulation. The complex is evolutionarily conserved and is composed of five proteins: the protein kinase Bud32, the hypothetical metallo-protease Kae1, the still uncharacterized Cgi121, and two other proteins of small size, Pcc1 and Pcc2/Gon7. For many years, our attention has been focused on the atypical protein kinase Bud32, which interacts with many other yeast proteins, suggesting that it may play several roles in the cell. Among these Bud32 partners, we demonstrated that the glutaredoxin Grx4 is a substrate of the protein kinase, being readily phosphorylated by Bud32 mainly at Ser 134. Also, this modification is upregulated by the previous phosphorylation of Bud32 at its Ser258 residue by the Sch9 protein kinase (the yeast homologue of mammalian Akt/PKB). During the first part of my PhD I deepened the study of the physiological significance of this new phosphorylation cascade. By the phenotypic analysis of yeast strains expressing mutagenized forms of Grx4, I demonstrated that the phosphorylation of Grx4 by Bud32 is important for Grx4 functionality in vivo. However I could not identify a specific effect of the Bud32-mediated phosphorylation of Grx4 on the known activities of the glutaredoxin, wich is involved in iron cellular homeostasis and in the survival under oxidative stress conditions. This result suggests that the Bud32-mediated phosphorylation of Grx4 play a role in different, uncharacterized functions of the glutaredoxin. I also checked if the phosphorylation of Bud32 by Sch9 could modulate the activity of the whole KEOPS complex, but the analysis of telomeres length and of the activation rate of the galactose-inducible GAL1 gene (one of the main transcriptional targets of KEOPS) showed that these functions are unaffected in a Bud32 unphosphorylatable mutant (S258A). These results suggest that the phosphorylation of Bud32 at Ser258 is unrelated to its function within the KEOPS complex.
I then addressed my attention to the Kae1 subunit of the complex. By using two strains expressing mutagenized forms of Kae1, I could demonstrate that the activity of this protein is essential for the complex, both at the telomere and at the transcriptional level. The biochemical function of Kae1 is however still unknown. It was initially classified as a protease, and, in effect, in 2006 an endopeptidase activity was indirectly demonstrated for the human homologue of Kae1, OSGEP. On the contrary, in 2007 Hecker et al. demonstrated that an archaeal orthologue of Kae1 is an AP-endonuclease . During my PhD I tried to define the activity of yeast Kae1, but the results obtained are not sufficient to clarify this point.
Finally, I decided to verify the hypothesis, coming from a recent work that describes the atomic structure of an archaeal-derived KEOPS complex, that Kae1 could be a substrate of Bud32. Using the yeast Bud32 and Kae1 proteins, co-expressed and purified from E.coli, I observed that the also the recombinant proteins are tightly associated, forming a kind of catalytic KEOPS subcomplex. Using several mutagenized forms of these proteins I demonstrated, by in vitro phosphotranspherase assays, that Bud32 is able to phosporylate Kae1 and that the binding of Kae1 has an inhibitory effect on the catalytic activity of the kinase. An important confirmation comes from the MS analysis of phosphorylated Kae1, that identified Ser 367 as a target of Bud32. However this might not be the only phosphorylated residue.
Altogether these results indicate that, at least in vitro, a regulatory relationship exists between Bud32 and Kae1. This is interesting as the two proteins are liable to carefully modulate the functions of the entire KEOPS complex.Durante il Dottorato di ricerca, mi sono occupata dello studio di due proteine di lievito, la proteinchinasi Bud32 e l’ipotetica proteasi Kae1, che fanno parte di un complesso nucleare denominato KEOPS/EKC. Mentre Kae1 è associata unicamente alle proteine del complesso, Bud32 ha molti altri partner all’interno della cellula; oggetto del mio studio è stata infatti anche la sua forte relazione con la glutaredoxina Grx4.
Il complesso di lievito KEOPS/EKC, isolato nel 2006 da due diversi gruppi di ricerca, è coinvolto nell’omeostasi telomerica e nella regolazione della trascrizione. Il complesso è evolutivamente conservato ed è composto da cinque proteine: la chinasi Bud32, l’ipotetica proteasi Kae1, Cgi121, proteina non ancora caratterizzata, e altre due piccole subunità, Pcc1 e Pcc2/Gon7. Per molti anni la nostra attenzione è stata rivolta alla proteinchinasi atipica Bud32, che interagisce con molte altre proteine di lievito, suggerendo come essa possa avere altri ruoli all’interno della cellula. Tra i vari partner di Bud32, abbiamo dimostrato che la glutaredoxina Grx4 risulta essere un substrato della chinasi sia in vivo che in vitro, ed è infatti fosforilata da Bud32 principalmente nella Ser 134. Questa relazione è inoltre a sua volta modulata dalla precedente fosforilazione di Bud32 nella Ser258 da parte della chinasi Sch9 (l’omologa in lievito della chinasi di mammifero Akt/PKB). Durante la prima parte del mio dottorato mi sono concentrata sulla ricerca di un possibile significato fisiologico di questa nuova cascata di fosforilazioni. Analizzando ceppi di lievito che esprimono forme mutagenizzate di Grx4, ho dimostrato come la fosforilazione di Grx4 da parte di Bud32 sia importante per la funzionalità della proteina in vivo. Sfortunatamente, non ho potuto identificare un effetto di questa fosforilazione sulle attività della glutaredoxina, coinvolta nella regolazione dell’omeostasi cellulare del ferro e nella sopravvivenza in condizioni di stress ossidativo. Questi risultati portano all’ipotesi che la fosforilazione di Grx4 da parte di Bud32 giochi un ruolo in qualche funzione diversa e ancora non caratterizzata della glutaredoxina.
Ho inoltre verificato se la fosforilazione nella Serina 258 di Bud32 da parte di Sch9 potesse modulare l’attività dell’intero complesso KEOPS, ma l’analisi della lunghezza dei telomeri e del livello di attivazione del gene inducibile GAL1 (uno dei maggiori target trascrizionali di KEOPS) ha rivelato che queste funzioni non erano colpite nel mutante BUDS258A. Questi risultati suggeriscono che la fosforilazione della serina 258 di Bud32 non sia collegata alle funzioni della chinasi nel complesso KEOPS.
Ho successivamente indirizzato la mia attenzione alla proteina Kae1. Attraverso l’utilizzo di due ceppi, esprimenti forme mutagenizzate di Kae1, ho potuto dimostrare come l’attività di questa proteina sia essenziale per l’intero complesso, sia a livello dei telomeri che della trascrizione. La funzione biochimica di Kae1 è tuttavia ancora sconosciuta. Inizialmente la proteina è stata classificata come una proteasi, e in effetti nel 2006 è stata indirettamente dimostrata un'attività endopeptidasica per l'omologa umana di Kae1, OSGEP. Al contrario, nel 2007 Hecker et al. hanno dimostrato che l’omologa di Kae1 in Archea è un’AP-endonucleasi. Durante il mio dottorato ho provato a definire l’attività della proteina Kae1 di lievito, ma i risultati ottenuti non sono stati sufficienti per chiarire questo punto.
Infine, ho deciso di verificare l’ipotesi, derivante da un recente lavoro in cui viene descritta la struttura atomica del complesso KEOPS negli Archaea, che Kae1 possa essere un substrato di Bud32. Utilizzando le proteine di lievito Bud32 e Kae1, espresse in E.coli, ho osservato che, come accade nelle cellule di lievito, le proteine ricombinanti sono strettamente associate e formano quindi una sorta di sub-complesso catalitico di KEOPS.
Utilizzando diverse forme mutagenizzate delle due proteine, in test chinasici in vitro, ho dimostrato che Bud32 è in grado di fosforilare Kae1 e che il legame di Kae1 ha un effetto inibitorio sull’attività catalitica della chinasi. Un’importante conferma è derivata dall’analisi di spettrometria di massa su Kae1 fosforilata, in cui la Ser 367 è stata identificata come target di Bud32. Tuttavia questo potrebbe non essere l’unico residuo fosforilato.
Nel complesso, questi dati indicano che, perlomeno in vitro Bud32 e Kae1 vengono reciprocamente regolate. Questo dato è interessante, dal momento che le due proteine potrebbero modulare le funzioni dell’intero complesso KEOPS
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
Scham und Haare. Zur Verflochtenheit zweier Motivstränge bei Paul Celan und Elfriede Jelinek
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