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Induzione della fosfatasi MKP-1 ad opera di NOTCH3: ruolo di questa proteina nell'apoptosi in cellule di leucemia T
Full text linkT cell acute lymphoblastic leukaemia (T-ALL) is a thymocyte's derived malignant disease characterized by an abnormal lymphoblast numbers that represents 10-15% of all leukaemias in childhood and 25% in adulthood. The Notch pathway has been involved in the pathogenesis of this disease. This is an evolutionary conserved pathway involved not only in leukaemia biology but also in different physiologic processes, including T cell differentiation and angiogenesis. New blood vessels development is mandatory to foster tumor growth, because insufficient perfusion can induce the establishment of a state of dormancy, a condition characterized by sustained tumor latency.
The present thesis work tries to expand our knowledge on the mechanisms involved in regulation of tumor dormancy by exploiting a T-ALL model where escape from dormancy is due to activation of Notch3 signalling induced by angiogenesis. We investigated the mechanisms downstream to this activation. Moreover, we tried to extend our findings to other T-ALL cells in order to draw some therapeutic implications.
This study demonstrated that escape from tumor dormancy is connected with low activation of MAPKs ERK1/2 and p38, in part confirming literature data describing the involvement of this signalling pathway in dormancy. In parallel to MAPKs silencing we found an increase in MKP-1 expression, a phosphatase known to target ERK1/2 and p38 by inhibiting their activity. We demonstrated that MKP-1 upregulation is induced by Notch3 activation through a mechanism based on reduction of its ubiquitination and protein degradation.
We further investigated the role of MKP-1 in our model, demonstrating that its overexpression is not sufficient to prevent tumor dormancy, but yet MKP-1 expression is important in early phases of tumor growth. This role seems to be linked to MKP-1 anti-apoptotic properties following challenge of T-ALL cells with serum starvation and some chemotherapeutic drugs.
In conclusion, our findings describe on MKP-1 phosphatase as one key effector of the anti-apoptotic activities of Notch3 that, in our model, regulates escape from dormancy. We also demonstrated that Notch3 control of MKP-1 levels is mediated by protein stabilization, an uncommon mechanism for this class of receptors usually involved in gene expression regulation.
Finally, we confirmed the strong anti-apoptotic properties of MKP-1 in T-ALL cells, a finding which could have therapeutic implications.La leucemia linfoblastica acuta a cellule T (T-ALL) è un'emopatia maligna dei timociti caratterizzata da un alto numero di linfoblasti che rappresenta il 10-15% dei casi di leucemia in età pediatrica ed il 25% dei casi in età adulta. Tra le vie di signalling maggiormente coinvolte in questo tipo di neoplasia figura quello governato dai recettori Notch. Questa pathway evolutivamente conservata, oltre ad essere alterata in queste leucemie, ricopre un ruolo molto importante anche in diversi processi fisiologici, tra cui ricordiamo il differenziamento dei linfociti T e l'angiogenesi. Lo sviluppo di nuovi vasi sanguigni è un evento centrale nello sviluppo tumorale in quanto un'insufficiente perfusione può determinare, tra l'altro, l'instaurarsi di uno stato di dormienza, una condizione caratterizzata dalla stasi prolungata della crescita tumorale.
Il presente lavoro propone di ampliare le conoscenze sul fenomeno della dormienza tumorale impiegando un modello in cui cellule di T-ALL sono in grado di sfuggire dallo stato di dormienza grazie all'attivazione del signalling di Notch3, e di indagare più in dettaglio quelli che sono i meccanismi a valle di questa attivazione. Inoltre, si è cercato di estendere le evidenze ottenute in questo modello anche ad altre cellule di T-ALL traendone anche alcune implicazioni terapeutiche.
Lo studio ha dimostrato che la condizione di uscita dalla dormienza è associata ad una diminuzione dell'attivazione delle MAP chinasi ERK1/2 e p38, confermando in parte dati di letteratura che descrivono il coinvolgimento di queste vie di signalling nel fenomeno della dormienza tumorale. Parallelamente a questo spegnimento delle MAP chinasi è stato rilevato un aumento dei livelli della fosfatasi MKP-1. Abbiamo quindi dimostrato che l'incremento di questa ultima è indotto dall'attivazione di Notch3, attraverso un meccanismo basato sulla riduzione dell'ubiquitinazione di MKP-1 e quindi sulla conseguente stabilizzazione della proteina.
E' stato successivamente indagato il ruolo di questa fosfatasi nel nostro modello, dimostrando che la sua overespressione da sola non è sufficiente a interrompere lo stato di dormienza, ma che, nonostante questo, risulta essere comunque importante nelle prime fasi di crescita tumorale. Questo suo ruolo sembra essere legato alla capacità di MKP-1 di proteggere le cellule di T-ALL dall'apoptosi, sia in condizioni di stress cellulare che a seguito di trattamento con alcuni chemioterapici.
In conclusione, le nostre ricerche hanno individuato nella fosfatasi MKP-1 uno degli effettori dell'attività anti-apoptotica e di Notch3 che, nel nostro modello, sembra essere coinvolta nell'uscita dalla condizione di dormienza tumorale. Abbiamo inoltre dimostrato che l'azione di Notch3 è mediata da un meccanismo atipico per questa classe di recettori, molto più comunemente coinvolti nella regolazione della trascrizione genica. In ultimo, abbiamo dimostrato le rilevanti capacità anti-apoptotiche di MKP-1, formulando quindi l'ipotesi che l'espressione di questa proteina possa avere anche un significato traslazionale per la terapia delle T-ALL
Fluorescence polarization immunoassay for cyclosporine A determination in whole blood.
No full textA fluorescence polarization immunoassay (FPIA) for cyclosporine A (CsA) determination in blood was evaluated. The method demonstrated a good reproducibility both in within-run and between-run assays. The linearity range was 35-990 micrograms/L. Comparison of measurements in patients' whole blood with radioimmunoassay results showed higher estimates by the FPIA despite the good correlation coefficient. Finally, a poor agreement was observed between the evaluated method and the high-performance liquid chromatographic technique for CsA determinatio
CA 19-9 and CA 125 determination by immunoluminometric assay.
Full text linkWe evaluated a new immunoluminometric technique (ILMA) for the measurement of the cancer antigens, CA 19-9 and CA 125 in serum. A satisfactory reproducibility was found, coefficients of variation ranging from 4.2 to 7.3% in within-run and between-run assays. The linearity of tests was maintained over a wide concentration range. Mean analytical recovery was 94% for CA 19-9 and 98% for CA 125. A significant agreement between results obtained by immunoradiometric assays and evaluated methods was found both for CA 125 (ILMA = 0.917 IRMA + 1.048; r = 0.966; n = 98) and CA 19-9 (ILMA = 1.156 IRMA + 0.996; r = 0.995; n = 100)
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
Eicosanoid release from human bronchial epithelial cells upon exposure to toluene diisocyanate in vitro.
No full textEpithelial injury and inflammation are involved in airway hyperresponsiveness and asthma induced by toluene diisocyanate. In that isocyanates are insoluble and highly reactive compounds, bronchial epithelial cells may represent the most important target cells of their toxic effect. We hypothesized that damage to airway epithelium by toluene diisocyanate may result in the release of metabolites of arachidonic acid, which are known to promote inflammation and to alter epithelial cell function and airway smooth muscle responsiveness. To test this hypothesis we examined eicosanoid products in the culture media of bronchial epithelial cells exposed in vitro to 8 and 18 ppb toluene diisocyanate. Epithelial cells derived from human bronchi obtained at surgery were cultured to confluency on collagen-coated microporous membranes. Those cells, which expressed differentiated characteristics of epithelial cells (they showed keratin-containing filaments and had a cobblestone appearance), were alternatively exposed to toluene diisocyanate or air for 30 min in a specially designed in vitro chamber. The production of metabolites of arachidonic acid was assessed by measuring the release of immunoreactive products into the cell medium at the end of the exposure and during a 2 hr period after exposure. This method revealed a predominant isocyanate-induced release of immunoreactive 15-hydroxyeicosatetraenoic acid. Release rate of this compound tended to be dose-related and was associated with cell damage as assessed by the release of lactate dehydrogenase in the medium
Leukotriene B4 and late asthmatic reactions induced by toluene diisocyanate.
No full textWe investigated whether leukotriene B4 (LTB4) is released from the lungs of sensitized subjects during asthmatic reactions induced by toluene diisocyanate (TDI). We examined three groups of TDI-sensitized subjects, one after no exposure to TDI, the second 8 h after an exposure to TDI that caused an early asthmatic reaction, and the third 8 h after an exposure to TDI that caused a late asthmatic reaction. We analyzed bronchoalveolar lavage (BAL) fluid by reverse-phase high-performance liquid chromatography and by specific radioimmunoassay. The mean concentration of LTB4 was higher [0.31 +/- 0.09 (SE) ng/ml, range 0.15-0.51] in BAL fluid of sensitized subjects who developed a late asthmatic reaction than in BAL fluid of subjects who developed an early asthmatic reaction (0.05 +/- 0.04 ng/ml, range 0-0.224), and no LTB4 was detectable in the control subjects. We also performed BAL 8 h after TDI exposure on four TDI-sensitized late-dual reactors who were on steroid treatment. In this group of subjects no LTB4 was detectable. These results suggest that LTB4 may be involved in late asthmatic reactions induced by TDI
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