1,721,143 research outputs found
Hindering NAT8L expression in hepatocellular carcinoma increases cytosolic aspartate delivery that fosters pentose phosphate pathway and purine biosynthesis promoting cell proliferation
Full text linkN-acetylaspartate (NAA) is synthesized by the mitochondrial enzyme NAT8L, which uses acetyl-CoA and aspartate as substrates. These metabolites are fundamental for bioenergetics and anabolic requirements of highly proliferating cells, thus, NAT8L modulation may impinge on the metabolic reprogramming of cancer cells. Specifically, aspartate represents a limiting amino acid for nucleotide synthesis in cancer. Here, the expression of the NAT8L enzyme was modulated to verify how it impacts the metabolic adaptations and proliferative capacity of hepatocellular carcinoma. We demonstrated that NAT8L downregulation is associated with increased proliferation of hepatocellular carcinoma cells and immortalized hepatocytes. The overexpression of NAT8L instead decreased cell growth. The pro-tumoral effect of NAT8L silencing depended on glutamine oxidation and the rewiring of glucose metabolism. Mechanistically, NAT8L downregulation triggers aspartate outflow from mitochondria via the exporter SLC25A13 to promote glucose flux into the pentose phosphate pathway, boosting purine biosynthesis. These results were corroborated by the analyses of human and mouse hepatocellular carcinoma samples revealing a decrease in NAT8L expression compared to adjacent non-tumoral tissues. Overall, this work demonstrates that NAT8L expression in liver cells limits the cytosolic availability of aspartate necessary for enhancing the pentose phosphate pathway and purine biosynthesis, counteracting cell proliferation
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
Role of mitophagy in kaempferol-induced neuroprotection in in vitro models of Parkinson’s Disease
No full textIl Morbo di Parkinson (PD) è una patologia degenerativa del Sistema Nervoso Centrale caratterizzata dalla progressiva perdita dei neuroni dopaminergici del pathway nigrostriatale. Sebbene finora siano stati condotti diversi studi riguardo il PD, la sua eziologia è ancora poco nota. Le forme familiari o ereditarie infatti, rappresentano soltanto il 5-10 % dei casi di PD, mentre il restante 95% è rappresentato dale forme idiopatiche o sporadiche,. Per tale ragione il PD viene considerata una malattia multifattoriale dove l’esposizione a fattori ambientali come pesticidi altri inquinanti gioca un ruolo fondamentale nell’insorgenza della malattia. E’ stato ormai ampiamente dimostrato che lo stress ossidativo e la disfunzione mitocondriale siano fortemente coinvolte nella morte neuronale nel PD. Inoltre, difetti nella rimozione delle proteine danneggiate e/o organelli, dovuto probabilmente a disfunzioni a carico del sistema ubiquitina-proteasoma e dell’autofagia, rappresentano un ulteriore evento coinvolto nell’eziopatogenesi del PD. In particolare, nei cervelli PD sono state osservate alterazioni del processo autofagico e in accordo con tali evidenze è stato recentemente dimostrato che il trattamento con la rapamicina, un noto induttore di autofagia, risulta protettivo contro tossine che causano il PD e che l’inibizione dell’autofagia basale è in grado di causare degenerazione nei topi. Diversi approcci terapeutici sono stati sviluppati finora per rallentare la sintomatologia del PD ma nessuno di questi trattamenti messi a punto è in grado di prevenire o curare la degenerazione neuronale. Negli ultimi anni l’attenzione di molti ricercatori si è focalizzata sullo studio di composti di derivazione naturale, come i polifenoli, in quanto il loro uso farmacologico potrebbe rappresentare un promettente approccio terapeutico/preventivo in patologie correlate allo stress ossidativo. Infatti, in aggiunta alla loro primaria attività antiossidante, è stato dimostrato come tali composti svolgano altre differenti attività biologiche, pertanto il loro utilizzo potrebbe risultare una strategia terapeutica vincente in patologie multifattoriali e complesse come il PD. Sulla base delle nostre conoscenze, questa tesi di Dottorato ha avuto lo scopo di caratterizzare gli effetti neuroprotettivi di un composto di derivazione naturale, il campferolo, in modelli in vitro di PD. Abbiamo dimostrato che il campferolo protegge le cellule SH-SY5Y e i neuroni corticali primari dalla tossicità del rotenone. Inoltre il campferolo è in grado di diminuire significativamente la produzione di specie reattive dell’ossigeno e l accumulo di proteine mitocondriali carbonilate, oltre a preservare la vitalità e la funzionalità della rete mitocondriale. Abbiamo dimostrato che l’evento principale reponsabile degli effetti antiapoptotici e antiossidanti del campferolo è l’induzione dell’aumento del turnover mitocondriale per mezzo dell’autofagia. Infatti analisi in microscopia elettronica e a fluorescenza hanno evidanziato un incremento del tasso di fissione mitocondriale e degli autofagosomi contenenti mitocondri. Inoltre la proteina LC3 (autophagosome-bound microtubule-associated protein light chain-3) aumenta durante il trattamento col campferolo e l’inibizione chimica o genetica del processo autofagico abolisce gli effetti protettivi di tale composto. La protezione mediata dal campferolo è inoltre specifica per altre tossine mitocondriali (1-metil-4-fenilpiridinio, paraquat) usate per riprodurre le tipiche caratteristiche patogenetiche del PD, ma risulta completamente inefficiente contro altri stimuli che non danneggiano il mitocondrio (H2O2, 6-idrossidopamina, staurosporina). L’evidenza inoltre che il campferolo è in grado di preservare l’attività fisiologica dei neuroni striatali in fettine di cervello di ratto, ci suggerisce un protezione più generale di tale composto nel PD. Complessivamente tali evidenze forniscono ulteriori evidenze che il campferolo possa essere identificato come un composto in grado di incrementare il processo autofagico con nuove potenziali applicazioni terapeutiche.Parkinson Disease (PD) is a neurodegenerative disorder characterized by degenerative loss of neurons in the nigrostriatal dopaminergic pathway. So far, although several studies concerning PD have been performed, its aetiology remain still unclear. Familiar or hereditary forms account only for the 5 - 10% of the PD cases, while the remaining 95%, defined idiopathic or sporadic, rely on environmental factors without any certain genetic involvement. Therefore, PD can be considered a complex multifactorial pathology, where genetic background should be considered a predisposing factor towards environmental insults such as pesticides and chemicals. By now oxidative stress and mitochondrial injury have been extensively indicated to be involved in PD neuronal demise and are tightly related each other. Moreover the unsuccessful removal of damaged proteins and/or organelles, probably due to the impairment of either the ubiquitin-proteasome system or autophagy, is a further event associated with PD aetio-pathogenesis. In particular, autophagy has been observed to be deregulated in PD brains and in line with this evidence, it has been recently demonstrated that rapamycin, a well-known autophagic inducer, protects from PD toxins and that suppression of basal autophagy causes neurodegeneration in mice. Several therapeutical approaches to slow down PD symptoms have been developed but none of them is currently able to prevent neuronal degeneration. In the last few years the attention of many researchers has been focused on natural-occurring compounds, such as polyphenols, because their use has been suggested to be a promising therapeutic/preventing strategy in oxidative-related diseases. In fact, in addition to their primary antioxidant activity, they could display a wide variety of other biological functions and these properties could be helpful in a complex and multifactorial pathology such as PD.
On the basis of this knowledge, this PhD thesis is aimed to elucidate the processes underlying neuroprotection of a natural occurring compound kaempferol in in vitro models of PD. We demonstrate that kaempferol protects SH-SH5Y cells and primary cortical neurons from rotenone toxicity, as a reduction of caspases cleavage and apoptotic nuclei are observed. Reactive oxygen species levels and mitochondrial carbonyls decrease significantly. Mitochondrial network, transmembrane potential and oxygen consumption are also deeply preserved. We demonstrate that the main event responsible for the kaempferol-mediated antiapoptotic and antioxidant effects is the enhancement of mitochondrial turnover by autophagy. Indeed, fluorescence and electron microscopy analyses show an increase of the mitochondrial fission rate and mitochondria-containing autophagosomes. Moreover, the autophagosome-bound microtubule-associated protein light chain-3 increases during kaempferol treatment and chemical/genetic inhibitors of autophagy abolish kaempferol protective effects. Kaempferol induced-autophagy affords protection also toward other mitochondrial toxins (1-methyl-4-phenylpiridinium, paraquat) used to reproduce the typical features of PD, but is inefficient against apoptotic stimuli not directly affecting mitochondria (H2O2, 6-hydroxydopamine, staurosporine). Striatal glutamatergic response of rat brain slices is also preserved by kaempferol, suggesting a more general protection of kaempferol in PD. Overall, this data provide further evidence for kaempferol to be identified as an autophagic enancher with potential therapeutic capacity
Signalling pathways underlying metabolic adaptation of adipocytes to nutrient limitation
No full textFinding new molecular pathways and strategies modulating lipolysis in adipocytes is an attractive goal of the current research. In adipose tissue reduced blood vessel density occurring during aging, is related to hypoxia state, cell death and inflammation. Here it has been demonstrated that adipocytes of poorly vascularized enlarged visceral adipose tissue (i.e. adipose tissue of old mice) suffer from limited nutrient delivery. It has been showed that in adipocytes transcription factor FoxO1 is up-regulated by nutrient restriction (NR) via a ROS-dependent pathway involving the activation of mitochondrial proline oxidase (POX). Activated FoxO1 exerts the transcriptional control of lipid catabolism via the induction of lysosomal acid lipase (Lipa) and Adipose Triglyceride Lipase (ATGL). Liberated fatty acids (FAs) participate in signal transduction promoting the expression of genes related to mitochondrial oxidative metabolism (peroxisome proliferator-activated receptor-α, peroxisome proliferator-activated receptorγ coactivator-1α), thus setting a metabolic switch towards fat utilization. Furthermore, an increased autophagy and co-localization of lipid droplets (LDs) with lysosomes was observed implying lipophagy in NR-mediated LDs degradation. Interestingly, it has been found that metformin treatment, a biguanide drug commonly used to cure type-2 diabetes, recapitulates the metabolic adaptation to NR in adipose tissue. Actually, it is able to elicit FoxO1dependent ATGL and Lipa induction as well as LDs degradation through lipophagy. A crucial role of AMP-dependent protein kinase (AMPK) in sustaining FAs oxidation and up-regulating lipid oxidative genes in adipocytes has been also documented. In particular, it has been demonstrated that, besides promoting lipid signalling, FAs released by ATGL and Lipa are directed towards AMPK-mediated mitochondrial oxidation, thus maintaining energetic homeostasis, preventing adipocyte death and adipose tissue inflammation. In conclusion, our data show that cytoplasmic and lysosomalmediated lipid catabolism are activated by NR in adipocytes and give further support to the use of metformin as a NR mimetic to combat age-related diseases associated with altered lipid metabolism
Nitrosative stress due to GSNOR deficiency induces agingrelated mitochondrial impairment and enhances vulnerability to mitochondrial complex II-targeting drugs: a potential therapeutic approach for HCC
No full textThe denitrosylating enzyme GSNOR profoundly impact s on cellular signal transduction by controlling cellular concentrations of protein-SNOs. Excessive S-nitrosylation is a well-documented phenomenon known as nitrosative stress, which increases during age. In this phD thesis we provide evidence that GSNOR physiologically may undergo epigenetic silencing during aging probably due to DNA methylation of its promoter, carried out by TET proteins. It is plausible to speculate, therefore, that nitrosative stress occurring upon GSNOR deficiency, could compromise mitochondrial function, resembling, in such a way, aging-like conditions. By promoting mitochondrial defects, indeed, nitrosative stress has been associated with several features related to aging and to pathological states typical of advanced age, such as cancer. Coherently, GSNOR depletion has been recently demonstrated to contribute to development of hepatocellular carcinoma (HCC) and , being downregulated in almost the half of HCC cases, GSNOR deficiency characterizes a large subclass of this tumor. In this work we demostrate that hepatocarcinoma HepG2 cells stably downregulating GSNOR (shGSNOR HepG2) exhibit defective and fragmented mitochondria and that this condition is associated with the upregulation of succinate dehydrogenase (SDH) in order to sustain ATP production. Therefore, GSNOR deficiency, compromising mitochondrial function, prompts mitochondria to a higher vulnerability and this molecular adaptation enhances cell sensitivity of shGSNOR HepG2 cells to even low doses of different SDH-directed drugs (mitocans). In particular, we demonstrate that shGSNOR HepG2 cells undergo necroptosis via PARP1/RIP1 pathway. As a matter of fact, pharmacological inhibition of both proteins, as well as reverse genetics experiments completely rescue cell viability. We show that non-apoptotic cell death depends on caspase 3 Snitrosylation, which leads to its inactivation, and on the capability of SDHtargeting mitocans to produce ROS as side-effect of their mechanism of action. Indeed, pre-treatment with antioxidants fully abolishes cell demise. In addition, we revealed that SDH upregulation is associated with the cytosolic degradation of the mitochondrial chaperone TNF-associate protein 1 (TRAP1) via the ubiquitin-proteasome pathway. In particular, we provide the first evidence that TRAP1 is S-nitrosylated and that this modification is mandatory for its degradation. TRAP1 silencing by siRNA, indeed, recapitulates the molecular and cellular phenotype of shGSNOR HepG2 cells, and increases cell sensitivity to SDH-targeting mitocans. Overall, our results indicate that GSNOR is implicated in aging process, being epigenetically regulated during age and impacting on mitochondrial homeostasis, the first determinant of cell senescence/aging. Furthermore, excessive S-nitrosylation due to GSNOR downregulation elicits TRAP1 Snitrosylation and degradation, which in turn leads to increased SDH levels and enhanced sensitivity to SDH -targeting mitocans. Being GSNOR depletion distinctive of the 50% of HCC cases, our results argue for this class of molecules as new promising drugs to selectively eradicate HCC
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
Structural plasticity of the adult cerebellar climbing fibres: an in vivo study on the role of the growth-associated protein 43
Full text linkThe growth-associated protein GAP-43 has a pivotal role in axonal growth and guidance during development. Towards the end of postnatal development its expression dramatically declines in most brain regions with few exceptions, such as cerebellar cortex and inferior olive (IO). When experimentally over-expressed in murine CNS in vivo, it is able to induce axonal sprouting, moreover it is up-regulated in conditions known to induce a structural remodelling of neuronal connectivity, such as injuries to the CNS and neurodegenerative disorders. However the reason why high expression is maintained through adulthood only in some regions in the adult brain has never been clarified.
Taking advantage of lentiviral vectors and shRNAs to induce an efficient and stable gene silencing specifically in the IO, we chose to investigate GAP-43 role in axonal remodelling of the adult CFs, since they originate from one of the nuclei which retain GAP-43 high expression levels, they are conveniently arranged for morphological studies and they are endowed with profound plastic and regenerative potentials. Here we show in vivo, in adult rats, that silencing of GAP-43 causes an atrophy of olivocerebellar terminal axons and significant modification of its presynaptic boutons.
These data suggest that GAP-43 plays a pivotal role in the maintenance of axonal structure of CF and of the organization of presynaptic plasma membrane in physiological conditions and it plays a complex role in CF lesion-induced sprouting.
Finally, by means of two-photon microscopy and laser nanosurgery, we describe here for the first time in vivo a lesion-induced axonal sprouting in the mammalian CNS
Superossido dismutasi a Cu,Zn nella funzionalità mitocondriale e citoscheletrica: importanza nel mantenimento dell'omeostasi in cellule di origine neuronale
Full text linkTra le varie isoforme di SOD quella contenente Cu e Zn è la proteina maggiormente studiata e meglio caratterizzata dal punto di vista strutturale e funzionale. Nonostante ciò, in questi ultimi anni stanno emergendo nuovi aspetti riguardanti il ruolo fisiologico di questo enzima e riguardo gli effetti della sua carenza. In particolare, si è sviluppato un grosso interesse della comunità scientifica internazionale nello studio del ruolo della Cu,ZnSOD nell’insorgenza di disordini del mitocondrio e del citoscheletro correlati alle malattie neurodegenerative e l’invecchiamento.
Tramite l’uso dell’RNA interference, è stato possibile studiare adeguatamente una finestra temporale nella quale si evidenziano i principali effetti intracellulari della carenza della Cu,ZnSOD in cellule di neuroblastoma umano. Gli studi sulla down-regolazione della Cu,ZnSOD ci hanno mostrato come una piccola variazione della sua concentrazione induca comunque un effetto rapido e rilevante sullo stato redox intracellulare. e come questo abbia una sua influenza sull'omeostasi mitocondriale e sull'equilibrio del citoscheletro essenzialmente in cellule di origine neuronale. Questi dati offrono una nuova chiave di lettura per comprendere la reale importanza della Cu,ZnSOD nel sistema nervoso ed in particolare al fine di chiarire il significato della elevata concentrazione di questo enzima in questo distretto.Among Superoxide dismutases, the copper,zinc containing form is the most studied and well caracterized. The pathological effects of its disfunction linked to mutations on its gene have been extensively studied in the occurrence of neurodegenerative diseases as ALS. Although, many emerging data link not only its disfunction but also its decrease to disorders of cytoskeleton and mitochondria as ageing. By the use of RNA interference, we studied the effects of Cu,ZnSOD down-regulation in neuroblastoma cell lines in a short time-window. We demonstrated that a little decrease of Cu,ZnSOD concentration can lead to a significant change in redox homeostasis which in turn is responsible for the impairment of mitochondrial function and the transient disruption of cytoskeletal network only in neuronal deriving cells. These data give effort to the importance of Cu,ZnSOD in neuronal cells and to the real significance of its high concentration in these cells
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