1,720,955 research outputs found
Kidney in a box: sviluppo di un modello renale in vitro per lo studio delle ciliopatie
No full textLe cellule epiteliali renali possiedono un'innata capacità di percepire e rispondere a segnali meccanici e chimici presenti nell'ambiente tubulare, una funzione essenziale per il mantenimento dell'omeostasi dei fluidi e degli elettroliti. Sebbene tradizionalmente attribuita a proteine di membrana plasmatica, come canali ionici e trasportatori, evidenze recenti evidenziano il ruolo fondamentale degli organelli intracellulari come centri di segnalazione in questo processo. Questa tesi indaga i meccanismi di segnalazione specifici degli organelli nelle cellule epiteliali renali, con particolare attenzione al segnale calcio-mediato dai lisosomi e alla segnalazione β-adrenergica mediata dalle ciglia primarie.
I nostri risultati mostrano che il rilascio di Ca2+ dai lisosomi tramite TRPML1 induce oscillazioni prolungate del Ca2+ citosolico attraverso i recettori inositolo 1,4,5-trifosfato (Ins3PRs) situati sul reticolo endoplasmatico. Questa cascata di segnalazione modula l'organizzazione del citoscheletro, attiva la segnalazione calcineurina/NFAT e promuove l’accumulo apicale del canale dell'acqua AQP2, essenziale per il riassorbimento dell'acqua nel dotto collettore. Inoltre, dimostriamo che le ciglia primarie fungono da hub di segnalazione β-adrenergica, con i recettori β2-adrenergici (β2ARs) localizzati sulla cilia che giocano un ruolo predominante nella regolazione dei livelli di cAMP citosolico. Tuttavia, i nostri risultati indicano che la segnalazione β-adrenergica ciliare dipende principalmente dal pool di cAMP citosolico, piuttosto che dalla generazione diretta di segnali all'interno della cilia stessa. Una delle principali difficoltà nello studio di questi complessi percorsi di segnalazione è l'assenza di modelli in vitro fisiologicamente rilevanti che riproducano il microambiente dinamico del rene. Per superare questo ostacolo, abbiamo utilizzato il bioreattore LiveBox2, in grado di simulare le condizioni di stress da flusso di fluidi che le cellule sperimentano in vivo. I nostri risultati dimostrano che la coltura dinamica all'interno di questo sistema migliora l'integrità epiteliale, modula la composizione delle proteine delle giunzioni strette e riorganizza il citoscheletro di actina, facilitando così il traffico di AQP2 verso la membrana apicale. Integrando tecniche avanzate di biologia molecolare con tecnologie innovative basate su bioreattori, questo lavoro fornisce nuove intuizioni sul ruolo degli organelli intracellulari nella segnalazione epiteliale renale. Questi risultati ampliano la nostra comprensione della fisiologia renale e aprono nuove prospettive per lo sviluppo di strategie terapeutiche mirate alle patologie renali.Kidney epithelial cells possess an intrinsic ability to sense and respond to mechanical and chemical cues within the tubular environment, a function essential for maintaining fluid and electrolyte homeostasis. While traditionally attributed to plasma membrane proteins such as ion channels and transporters, recent evidence highlights the pivotal role of intracellular organelles as key signaling hubs in this process. This thesis investigates organelle-specific signaling mechanisms in renal epithelial cells, with a focus on lysosomal calcium signaling and primary cilia-mediated β-adrenergic signaling.
Our findings reveal that lysosomal Ca2+ release via TRPML1 induces sustained cytosolic Ca2+ oscillations through inositol 1,4,5-trisphosphate receptors (Ins3PRs) on the endoplasmic reticulum. This signaling cascade modulates cytoskeletal organization, activates calcineurin/NFAT signaling, and promotes the apical accumulation of the water channel AQP2—critical for water reabsorption in the collecting duct. Additionally, we demonstrate that primary cilia serve as mechanosensors in β-adrenergic signaling, with β2-adrenergic receptors (β2ARs) localized to the cilium playing a predominant role in regulating cytosolic cAMP levels. However, our results indicate that ciliary β-adrenergic signaling is primarily dependent on cytosolic cAMP rather than direct signal generation within the cilium itself.
A key challenge in studying these intricate signaling pathways is the absence of physiologically relevant in vitro models that replicate the kidney’s dynamic microenvironment. To address this, we employed the LiveBox2 bioreactor, which simulates fluid shear stress conditions experienced in vivo. Our results show that dynamic culture within this system enhances epithelial integrity, modulates tight junction protein composition, and reorganizes the actin cytoskeleton, thereby facilitating AQP2 trafficking to the apical membrane.
By integrating advanced molecular biology techniques with innovative bioreactor technology, this work provides new insights into the role of intracellular organelles in renal epithelial signaling. These findings advance our understanding of kidney function and open new avenues for therapeutic strategies targeting kidney disease
Targeting unfolded protein response reverts ER stress and ER Ca2+ homeostasis in cardiomyocytes expressing the pathogenic variant of Lamin A/C R321X
Get PDFBackground: We previously demonstrated that an Italian family affected by a severe dilated cardiomyopathy (DCM) with history of sudden deaths at young age, carried a mutation in the Lmna gene encoding for a truncated variant of the Lamin A/C protein (LMNA), R321X. When expressed in heterologous systems, such variant accumulates into the endoplasmic reticulum (ER), inducing the activation of the PERK-CHOP pathway of the unfolded protein response (UPR), ER dysfunction and increased rate of apoptosis. The aim of this work was to analyze whether targeting the UPR can be used to revert the ER dysfunction associated with LMNA R321X expression in HL-1 cardiac cells. Methods: HL-1 cardiomyocytes stably expressing LMNA R321X were used to assess the ability of 3 different drugs targeting the UPR, salubrinal, guanabenz and empagliflozin to rescue ER stress and dysfunction. In these cells, the state of activation of both the UPR and the pro-apoptotic pathway were analyzed monitoring the expression levels of phospho-PERK, phospho-eIF2α, ATF4, CHOP and PARP-CL. In addition, we measured ER-dependent intracellular Ca2+ dynamics as indicator of proper ER functionality. Results: We found that salubrinal and guanabenz increased the expression levels of phospho-eIF2α and downregulated the apoptosis markers CHOP and PARP-CL in LMNA R321X-cardiomyocytes, maintaining the so-called adaptive UPR. These drugs also restored ER ability to handle Ca2+ in these cardiomyocytes. Interestingly, we found that empagliflozin downregulated the apoptosis markers CHOP and PARP-CL shutting down the UPR itself through the inhibition of PERK phosphorylation in LMNA R321X-cardiomyocytes. Furthermore, upon empagliflozin treatment, ER homeostasis, in terms of ER ability to store and release intracellular Ca2+ was also restored in these cardiomyocytes. Conclusions: We provided evidence that the different drugs, although interfering with different steps of the UPR, were able to counteract pro-apoptotic processes and to preserve the ER homeostasis in R321X LMNA-cardiomyocytes. Of note, two of the tested drugs, guanabenz and empagliflozin, are already used in the clinical practice, thus providing preclinical evidence for ready-to-use therapies in patients affected by the LMNA R321X associated cardiomyocytes
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe 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
Get PDF“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
Get PDFWe 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
Get PDFWe 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
TRPML1-Induced Lysosomal Ca2+ Signals Activate AQP2 Translocation and Water Flux in Renal Collecting Duct Cells
Get PDFLysosomes are acidic Ca2+ storage organelles that actively generate local Ca2+ signaling events to regulate a plethora of cell functions. Here, we characterized lysosomal Ca2+ signals in mouse renal collecting duct (CD) cells and we assessed their putative role in aquaporin 2 (AQP2)-dependent water reabsorption. Bafilomycin A1 and ML-SA1 triggered similar Ca2+ oscillations, in the absence of extracellular Ca2+, by alkalizing the acidic lysosomal pH or activating the lysosomal cation channel mucolipin 1 (TRPML1), respectively. TRPML1-dependent Ca2+ signals were blocked either pharmacologically or by lysosomes’ osmotic permeabilization, thus indicating these organelles as primary sources of Ca2+ release. Lysosome-induced Ca2+ oscillations were sustained by endoplasmic reticulum (ER) Ca2+ content, while bafilomycin A1 and ML-SA1 did not directly interfere with ER Ca2+ homeostasis per se. TRPML1 activation strongly increased AQP2 apical expression and depolymerized the actin cytoskeleton, thereby boosting water flux in response to an hypoosmotic stimulus. These effects were strictly dependent on the activation of the Ca2+/calcineurin pathway. Conversely, bafilomycin A1 led to perinuclear accumulation of AQP2 vesicles without affecting water permeability. Overall, lysosomal Ca2+ signaling events can be differently decoded to modulate Ca2+-dependent cellular functions related to the dock/fusion of AQP2-transporting vesicles in principal cells of the CD
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
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