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Behaviour of endo-lysosomal and mitochondrial compartments in human NCL fibroblasts in vitro
Full text linkLe Ceroido Lipofuscinosi Neuronali (CLN) sono un gruppo di malattie degenerative dell’infanzia che colpiscono principalmente il sistema nervoso centrale (SNC). Le CLN appartengo al più vasto gruppo di patologie metaboliche da accumulo lisosomiale e sono caratterizzate dalla persistenza nel citoplasma di cellule neuronali, ma anche di altri tessuti periferici, di materiale di origine endo-lisosomiale che assume specifiche caratteristiche all’analisi ultrastrutturale. Le CLN possono interessare primariamente o secondariamente il compartimento lisosomiale, in quanto le proteine mutate nelle diverse forme di CLN sono localizzate non solo nei lisosomi, ma anche in altre strutture citoplasmatiche della cellula, quali ad esempio il reticolo endoplasmico.
Attualmente, attraverso l’utilizzo di criteri clinici, patologici e molecolari, si possono distinguere dieci diverse forme di CLN; inoltre, sono stati finora clonati otto geni-malattia, codificanti per proteine a diversa localizzazione cellulare, le cui funzioni non sono ancora del tutto ben caratterizzate.
Pertanto il termine CLN indica un gruppo eterogeneo di malattie neuro-degenerative, geneticamente determinate, che conducono prevalentemente ad un progressiva perdita di cellule neuronali. Un’importante tematica oggetto di studio nel campo delle CLN è la comprensione di quali meccanismi cellulari siano coinvolti in questo processo di morte neuronale, che nelle forme più gravi si manifesta nell’arco di pochi anni dall’insorgenza dei primi sintomi clinici. Lavori recenti hanno posto l’attenzione sugli effetti patogenetici che gli accumuli endo-lisosomiali possono avere sull’omeostasi e la sopravvivenza cellulare, dimostrando anche un coinvolgimento della via apototica caspasi-dipendente. Inoltre si sta cercando di comprendere meglio il ruolo dell’autofagia in questo processo di degenerazione, poiché esistono evidenze di un’attivazione di tale processo sia su campioni bioptici umani che in modelli sperimentali di malattia.
L’obiettivo di questo progetto di dottorato è stato quello di analizzare il comportamento cellulare in vitro di linee di fibroblasti umani, derivate da biopsie cutanee di pazienti affetti da forme geneticamente determinate di CLN, in particolare dalle forme CLN1, CLN3, CLN5, CLN6 e CLN7. Tali linee di fibroblasti umani sono state analizzate in un protocollo di ‘crescita protratta’ allo scopo di determinare quali risposte cellulari possano attivarsi per far fronte a questo processo di invecchiamento cellulare ‘forzato’ in vitro, condizione che stressa il compartimento endo-lisosomiale già coinvolto nella malattia.
Abbiamo posto l’attenzione principalmente sull’evoluzione del sistema endo-lisosomiale durante la crescita protratta in vitro; allo stesso tempo abbiamo analizzato la funzionalità mitocondriale e l’eventuale attivazione della via apoptotica. A tali scopi sono state impiegate sia metodiche morfologiche che biochimiche.
L’indagine morfologica del compartimento endo-lisosomiale ha rivelato che l’invecchiamento cellulare delle colture CLN era associato ad un progressivo accumulo di elementi lisosomiali (corpi lisosomiali, autolisosomi e vacuoli otticamente vuoti), in misura diversa rispetto alle cellule di controllo. Inoltre si è potuto osservare che le colture CLN1 mostravano un diverso comportamento nel processo di accumulo citoplasmatico di corpi densi e di vacuoli a singola membrana rispetto alle altre colture CLN. In alcuni casi di specifiche forme, quali CLN3 e CLN6, l’indagine ultrastrutturale ha permesso di rilevare strutture a doppia membrana, gli autofagosomi, e questa evidenza ci ha condotti ad analizzare anche il processo dell’autofagia, che è risultata essere attivata in diverse linee cellulari, indipendentemente dalla specifica forma di CLN o dalla gravità della mutazione.
Inoltre le alterazioni morfologiche del compartimento endo-lisosomiale, che avvengono nelle colture NCL, inducono una frammentazione del reticolo mitocondriale e una redistribuzione dei mitocondri polarizzati. Tuttavia, in condizioni basali non abbiamo mai osservato evidenze di attivazione della via apoptotica caspasi-dipendente in nessuna delle linee cellulari analizzate. Ciò nonostante, il trattamento con un agente chimico pro-apoptotico, la Staurosporina, ha rilevato una diversa attivazione della via apoptotica caspasi-3 mediata in almeno 2 linee CLN1 rispetto alle altre linee CLN e a quelle di controllo.
I risultati emersi da questo studio suggeriscono che il processo autofagico è attivato nei fibroblasti CLN, probabilmente come meccanismo cellulare di recupero per far fronte allo ‘stress’ del compartimento endo-lisosomiale, accentuato dalla crescita prolungata in vitro. Allo stesso tempo i mitocondri, organelli essenziali per la sopravvivenza cellulare, sono secondariamente coinvolti in questo modello in vitro, mentre le evidenze indirette di un coinvolgimento della via apoptotica solo nelle linee di CLN1 sembrano essere in accordo con recenti lavori scientifici, che hanno sottolineato il ruolo dell’apoptosi nella patogenesi di questo gruppo di malattie.Neuronal ceroid lipofuscinoses (NCL) are degenerative disorders of childhood, which affect the central nervous system (CNS). NCL belong to the wider group of lysosomal storage diseases and they are characterized by the accumulation of endo-lysosomal material with specific ultrastructural features, both in neuronal cells and other peripheral tissues. NCL arise from either primary or secondary involvement of the lysosomal compartment, since mutated NCL proteins reside not only in the lysosomes but also in other cellular structures, such as the endoplasmic reticulum.
Over last 30 year ten NCL forms have been identified according to clinical, pathological and molecular criteria; moreover, eight NCL genes have been cloned, coding for proteins of specific and different functions. Therefore, today, NCL represents the acronym of a group of genetically different disorders of the CNS, leading to progressive shrinkage of the brain.
The main issue in the NCL pathology is the comprehension of which cellular mechanisms are involved in neuronal cell death in these conditions. Recently the attention has been addressed mainly to the pathogenetic role that the abnormal storage may exert on the cellular homeostasis and survival, even through apoptotic cell death. Moreover, a role for autophagy is also under scrutiny, since morphological and molecular evidences suggest the activation of this cellular mechanism both in humans and animal models of disease.
The aim of this doctorate project was to analyze in vitro the cellular behaviour of human genetically determined NCL fibroblasts derived from skin biopsies of patients affected with NCL forms, harbouring mutations on CLN1, CLN3, CLN5, CLN6 and CLN7 genes. Specifically, fibroblasts cell lines were analyzed in a ‘prolonged culture paradigm’ in order to assess which cellular responses could be activated by NCL cells, carrying a pre-existing endo-lysosomal dysfunction, to cope with the aging process in vitro.
We focused our attention principally on the evolution of the endo-lysosomal system during the prolonged growth; at the same time, we checked the mitochondria functionality and the activation of the apoptotic pathway. Both morphological and biochemical tools were used.
The morphological investigation of the endo-lysosomal system revealed that the aging of NCL cultures was associated with a progressive accumulation of lysosomal elements (lysosomal bodies, autolysosomes and empty vacuoles) in different manner as compared to controls. Furthermore, a different behaviour in the accumulation process of dense bodies and single membrane vacuoles was seen between CLN1 and the other forms. The detection of autophagosomes led to investigate the autophagic pathway, that was found to be activated in several cell lines, regardless the NCL forms and the severity of mutation. Moreover, the morphological alterations of the endo-lysosomal compartment occurring in all NCL cell lines affected the mitochondrial network and the distribution of polarized mitochondria as well. No evidences of apoptotic activation were seen under basal conditions during the prolonged growth in all the cell lines analyzed. However, the treatment with the apoptotic chemical inducer Staurosporine revealed a different activation of the caspase-3 dependent apoptosis in two CLN1 lines as compared to the other NCL forms and controls.
These results suggested that autophagy is activated in NCL fibroblasts, probably as a rescue cellular program to cope with the endo-lysosomal stress, intensified by the prolonged growth in vitro. At the same time, mitochondria, a vital compartment for the cell survival, were secondary affected in this in vitro model, whereas the indirect evidence of involved apoptotic pathway in CLN1 only seems to be consistent with recent scientific reports
Neuronal ceroid lipofuscinosis: the increasing spectrum of an old disease.
No full textNeuronal Ceroid Lipofuscinoses (NCL) are genetically heterogeneous heritable neurodegenerative disorders with worldwide distribution. They are considered as childhood diseases; however rare adult onset forms are known. NCL have a progressive course, affecting visual, motor and cognitive functions, and are associated with myoclonic epilepsy; behavioural problems can be observed at the onset. The outcome is invariably fatal, mostly during the second or third decade. The denomination is based on pathological criteria, i.e. the presence of intralysosomal storage of autofluorescent lipopigment of glycoprotein origin with characteristic ultrastructural features. The NCL are autosomal recessive diseases (but a rare autosomal dominant form of adult onset). Thirteen NCL associated genes have been identified so far, which allow a definite diagnosis to be reached and provide genetic counselling to the families. Still unidentified NCL genes are foreseen. Allelic heterogeneity is observed in some mutated genes; likewise phenotypic heterogeneity is seen in several NCL. The gene products are either soluble proteins (such as lysosomal enzymes) or membrane proteins related to lysosomes, endoplasmic reticulum, synaptic vesicles. Little is known about pathogenetic mechanisms, leading to storage formation and cell death. Current research is focusing on intracellular trafficking, neurotransmission and storage removal. No cure is available for any form. Innovative treatments led to some results in mouse models related to lysosome hydrolase defects. Evidences that autophagy, oxidative stress, excitotoxicity play roles in NCL cell pathology raise the possibility that selected steps of these processes might become target of treatments, and therefore modify the disease course
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
Neuronal ceroid lipofuscinoses : many players, and more to come
No full textThe neuronal ceroid lipofuscinoses (NCL) are the most common group of progressive neurodegenerative diseases of childhood. The overall clinical features are highly similar regardless of the age at disease manifestation, the extent and shape of abnormally stored cytosomes and the severity of clinical course, and are generally characterized by failure and regression of psychomotor development, impaired vision, seizures and fatal outcome. The expanding array of genetic etiologies and disease-associated mutations in NCL provide the basis for the heterogeneity of these clinical conditions and are the focus of this review. Less understood are the pathogenic mechanisms, but common themes and molecular pathways are now emerging and new players are expected to come into the scene of NCL
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