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ASPECTS OF CELLULAR IRON HOMEOSTASIS: NRAMP TRANSPORTER FUNCTION AND ERYPTOSIS
Full text linkThe present study focus attention on “Aspects of cellular iron homeostasis: NRAMP transporter function and eryptosis” combining aspects of both basic (first chapter: “Iron transporters NRAMP1 and NRAMP2 from Dictyostelium discoideum as a model of cellular iron homeostasis”) and applied physiology (second chapter: “Effects of xenobiotics on the suicidal death of erythrocytes”). Iron plays a central role in a large number of essential cellular functions but it is also potentially toxic being able to generate reactive oxygen species (ROS). SLC11 and SLC40 families are involved in iron transport and play an important role in the maintenance of iron homeostasis. The SLC11 family is comprised of two members, SLC11A1 and SLC11A2. SLC11A1 is expressed in the phagolysosome of macrophages and in the tertiary granules of neutrophils and it contributes to the innate resistance against bacterial infection. SLC11A2 (also known as DMT1) is expressed in the proximal duodenum, immature erythroid cells, brain, placenta and kidney and is a key player in iron metabolism. Intestinal iron absorption is indeed mediated by SLC11A2 at the apical membrane of enterocytes and is followed by basolateral exit via SLC40A1. D. discoideum represents a model for the study of cellular iron homeostasis, showing subcellular localization of iron transporters resembling that of macrophages. The Dictyostelium genome shares with mammals many genes regulating iron homeostasis; in particular, D. discoideum expresses the ortholog of SLC11A1 transporter in phagolysosomes and that of SLC11A2 in the contractile vacuole. To better understand the function of Dictyostelium NRAMP proteins, they were expressed in Xenopus laevis oocytes by cRNA injection and functionally tested by radiochemical techniques and by a novel assay based on metal-induced changes in calcein fluorescence. Radiochemical assays showed that NRAMP1 induced iron transport is proton-dependent and it is inhibited by Mn2+, Cd2+, Co2+, Ni2+, Cu2+ and to a lesser extent by Zn2+. In calcein-injected oocytes expressing NRAMP1 and analyzed using confocal microscopy, Fe2+, Mn2+ and but not Fe3+ or Cu2+ led to fluorescence quenching due to their transport and accumulation into the cytoplasm of the oocytes. Therefore Dictyostelium NRAMP1 is an electrogenic proton-dependent divalent metal ion transporter with a cation selectivity comparable to that of rat DMT1. NRAMP1 colocalizes with V-ATPase in the membrane of phagolysosomes. Thus, it exploits the proton gradient maintained by the V-ATPase to mediate the efflux of iron from the phagolysosomes to the cytosol after bacterial engulfment. Preliminary studies showed that D. discoideum NRAMP2 can transport ferrous iron at neutral pH and it appears independent from proton gradient but dependent on Na+, nevertheless its transport activity is strongly reduced compared with that observed for NRAMP1. The second topic of this PhD thesis is eryptosis, the suicidal death of erythrocytes. Mutations that reduce DMT1 activity in human are associated with a severe defect in erythroid iron utilization and are correlated with several diseases. DMT1 deficiency causes an impaired erythroid differentiation hallmarked by accumulation of immature forms of erythroblast, accelerated death of erythroid precursors and a decreased survival in the erythroid progenitors. Iron deficiency is associated with shortened life span of erythrocytes. The accelerated clearance of erythrocytes can be attributed to excessive hemolysis or induction of programmed cell death of erythrocytes, called eryptosis. Eryptosis is fostered by an increase in cytosolic calcium. Iron deficient erythrocytes when exposed to stress conditions has been demonstrated to activate Ca2+-permeable cation channel allowing Ca2+ entry. Ca2+ entry through this channels leads to activation of a scramblase with subsequent phosphatidylserine exposure and to activation of the Gardos channels leading to KCl loss and cell shrinkage. This study was conducted in order to investigate prospective antitumoral products which are employed against tumor growth in humans, in particular CA4P or Pazopanib, or compounds used in vitro, such as Nocodazole, Terfenadine, Piceatannol, Ceranib-2 and Sclareol, in order to unveil the effects on erythrocytes survival and to clarify the mechanisms and signalling involved in their action. Human erythrocytes drawn from healthy individual were incubated in vitro at a hematocrit of 0.4% in Ringer solution. Where indicated, RBCs were exposed for 48 hours to the drugs at the indicated concentrations. The main hallmarks of eryptosis were investigated by flow cytometry. Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, reactive oxygen species (ROS) formation from DCF-dependent fluorescence, GSH levels by CMF-dependent fluorescence and ceramide abundance utilizing specific antibodies. Hemoglobin concentration in the supernatant was taken as measure of hemolysis. ATP levels following CA4P treatment were measured using luciferin-luciferase assay kit. For studying the effect of Nocodazole on tubulin in human erythrocytes, TubulinTrackerTM Green reagent was used. A 48 hours exposure of human erythrocytes to CA4P, Pazopanib, Nocodazole, Terfenadine, Piceatannol, Ceranib-2 or Sclareol treatment increased the percentage of annexin-V-binding cells. Furthermore, the effect of Sclareol on annexin-V-binding was significantly blunted in the presence of p38 kinase inhibitor skepinone and in the presence of casein kinase 1α inhibitor D4476. CA4P and Piceatannol significantly decreased forward scatter. CA4P, Nocodazole, Terfenadine, Ceranib-2 and Sclareol treatment significantly increased Fluo3-fluorescence. CA4P significantly decreased GSH abundance and ATP levels. In addition, Pazopanib, Terfenadine, Ceranib-2 and Sclareol further resulted in significant hemolysis. Pazopanib, Nocodazole and Ceranib-2 significantly increased DCF-fluorescence and ceramide abundance. Nocodazole treatment reduced total tubulin abundance. In conclusion, these xenobiotics trigger eryptosis through different mechanisms
Funzionalizzazione di Nanoparticelle con mAbSp17 per la cura del carcinoma ovarico
Full text linkNonostante l’avvento di nuovi agenti chemioterapici nella terapia contro il carcinoma ovarico, la mortalità causata da questo tumore maligno rimane invariata. Il problema maggiore è che il tumore viene diagnosticato in fase tardiva e la sopravvivenza, a 5 anni dalla diagnosi, è del 27% contro il 45,6% se la diagnosi è precoce. Sperm Protein 17 (SP17) è una proteina altamente conservata nei mammiferi ed è implicata nel legame dello spermatozoo alla zona pellucida. SP17 fa parte della famiglia dei cancer testis antigen ed è considerato un possibile target per l’immunoterapia. La presenza di questa proteina è stata osservata nelle linee cellulari di OC umano, considerandolo come un possibile biomarker per questo tumore. Studi pre-clinici, in modello murino con OC, hanno dimostrato che la proteina SP17 funziona come vaccino prevenendo la formazione del tumore.
Lo scopo di questo lavoro è quello di legare l’anticorpo monoclonale contro SP17 (mAbSP17) a nanoparticelle di ossido di ferro (Fe3O4NP), iniettare il sistema in un modello murino con carcinoma ovarico umano, per valutare se l’anticorpo legato direzioni le NP verso il tumore. In parallelo si vuole anche valutare la biodistribuzione delle Fe3O4NP. A tale scopo, le Fe3O4NP sono state rivestite con amminopropiltrietossisilano (APTES) e coniugate all'anticorpo, usando EDC (1-Etil-3-(3-dimetilamminopropil)-carbodiimmide cloridrato) e NHS (N-idrossisulfosuccinimide). Il legame covalente dell’APTES con le NP è stato confermato mediante spettroscopia FT-IR, mentre il legame dell’anticorpo alle NP è stata determinata mediante saggio ELISA. La capacità di internalizzazione del sistema [email protected] è stata valutata, mediante microscopio confocale, trattando la linea cellulare di carcinoma ovarico umano (SKOV3). I sistemi, NP@APTES-mAbSP17 e [email protected], sono stati testati in vivo su modello murino con carcinoma ovarico indotto da SKOV3. NP@APTES-mAbSP17 e [email protected] sono stati iniettati, per via intraperitoneale, dopo 7 giorni dall’induzione del tumore. Nelle immagini acquisite, utilizzando lo strumento IVIS-LUMINA (Caliper, LifeSciences), si osserva una forte fluorescenza nella zona tumorale, che indica che le [email protected] sono localizzate nel tumore. La nostra ipotesi ancora da confermare è che le NP siano state internalizzate dalle cellule tumorali, come già dimostrato in vitro. Per quanto riguarda il sistemaNP@APTES-mAbSP17, il lavoro è ancora in corso. Ad oggi si ha solo una valutazione macroscopica del fenomeno, ma le NP non sembrano aver causato risposta immunitaria o tossicità. Ovaio, polmone e fegato verranno valutati sia, per via immunoistochimica, per verificare la presenza di SP17 e quindi del tumore, che analizzati al TEM per osservare l’eventuale presenza delle NP
Stimulating Effect of Sclareol on Suicidal Death of Human Erythrocytes
Full text linkBackground/Aims: The diterpene alcohol Sclareol has been proposed for the treatment of malignancy. In analogy to apoptosis of nucleated cells, erythrocytes may enter eryptosis, a suicidal cell death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Cellular mechanisms involved in the triggering of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, ceramide, p38 kinase and casein kinase 1α. The present study explored, whether Sclareol induces eryptosis and, if so, shed light on the mechanisms involved. Methods: Phosphatidylserine abundance at the erythrocyte surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, abundance of reactive oxygen species (ROS) from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA)-dependent fluorescence, and ceramide abundance at the erythrocyte surface utilizing specific antibodies. Hemolysis was estimated from haemoglobin concentration in the supernatant. Results: A 48 hours exposure of human erythrocytes to Sclareol (≥ 50 µM) significantly increased the percentage of annexin-V-binding cells without significantly modifying the average forward scatter, DCF-fluorescence or ceramide abundance. Sclareol (≥ 50 µM) further triggered hemolysis. Sclareol (100 µM) significantly increased Fluo3-fluorescence, but the effect of Sclareol on annexin-V-binding was not significantly blunted by removal of extracellular Ca2+. Instead, the effect of Sclareol on annexin-V-binding was significantly blunted in the presence of p38 kinase inhibitor skepinone (2 µM) and in the presence of casein kinase 1α inhibitor D4476 (10 µM). Conclusions: Sclareol triggers phospholipid scrambling of the erythrocyte cell membrane, an effect in part due to activation of p38 kinase and casein kinase 1α
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
Stimulation of Eryptosis, the Suicidal Erythrocyte Death by Piceatannol
Full text linkBackground/Aims: Piceatannol, an analog and metabolite of resveratrol, is effective against various disorders including malignancy. It is in part effective by triggering suicidal death or apoptosis of tumor cells. Cellular mechanisms mediating the proapoptotic effect of Piceatannol include mitochondrial depolarization and cytochrome c release. Erythrocytes lack mitochondria but may nevertheless enter suicidal death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Cellular mechanisms involved in the triggering of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress and ceramide formation. The present study explored, whether Piceatannol induces eryptosis and, if so, to shed some light on the cellular mechanisms involved. Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, reactive oxygen species (ROS) formation from 2',7'-dichlorodihydrofluorescein (DCF) diacetate-dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemoglobin concentration in the supernatant was taken as measure of hemolysis. Results: A 48 hours exposure of human erythrocytes to Piceatannol (10 - 20 µM) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter, significantly increased DCFDA-fluorescence, significantly increased ceramide abundance, but did not significantly increase Fluo3-fluorescence. Removal of extracellular Ca2+ slightly blunted but did not abolish the effect of Piceatannol on annexin-V-binding and forward scatter. Piceatannol (20 µM) significantly augmented the increase of annexin-V-binding, but significantly blunted the decrease of forward scatter following treatment with the Ca2+ ionophore ionomycin. Conclusions: Piceatannol triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part downstream of Ca2+ and involving oxidative stress and ceramide formation
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
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