Analisi molecolare e funzionale della variabilità del gene della succinico semialdeide deidrogenasi (SSADH) umana

L’SSADH è un enzima mitocondriale NAD+-dipendente e svolge il suo ruolo principale nel catabolismo del GABA, il principale neurotrasmettitore inibitore del Sistema Nervoso Centrale (SNC), attraverso l’ossidazione del substrato succinico semialdeide (SSA) a succinato, il quale a sua volta entra nel ciclo di Krebs; l’SSA può essere anche convertito nell’acido 4-idrossibutirrico (GHB) da SSA reduttasi tessuto-specifiche. La deficienza completa di SSADH risulta nella 4-idrossibutirrico aciduria (4-HBA), una rara malattia autosomica recessiva del metabolismo umano. I malati di 4-HBA manifestano un’ampia variabilità di fenotipi clinici, che vanno dal ritardo psicomotorio a gravi difetti neurologici, dovuti agli effetti neurotossici dell’anormale accumulo di GABA e GHB nei tessuti e nei liquidi fisiologici. Studi recenti hanno rivelato un secondo ruolo dell’SSADH: essa è una delle due aldeidi deidrogenasi mitocondriali responsabili della detossificazione del 4-idrossi-2-nonenale (HNE). L’HNE è un’aldeide reattiva prodotta fisiologicamente in quantità relativamente elevate in seguito a perossiadazione lipidica. Livelli elevati di HNE nel Sistema Nervoso Centrale sono implicati nella patogenesi di numerose malattie neurodegenerative quali la malattia di Parkinson e di Alzheimer. Dallo studio di più di 50 famiglie deficienti di SSADH, abbiamo identificato numerose varianti patologiche. Abbiamo anche identificato varianti non patologiche nella regione codificante. Alcune di queste sono rare e altre mostrano frequenze polimorfiche come ad esempio la trasversione da G a C in posizione 106 e la transizione da C a T nelle posizioni 538 e 545. Tramite il sequenziamento del DNA di primati non umani, abbiamo identificato gli alleli ancestrali e derivati per ciascun tipo polimorfico e abbiamo identificato quelli che si trovano in modo specifico nella linea umana. Abbiamo osservato che per le posizioni c.106 e c.545 è presente sia l’allele ancestrale che quello derivato in quasi tutte le popolazioni umane e che l’allele ancestrale è quello più frequente; invece per la posizione c.538 l’allele derivato ha aumentato la propria frequenza e ora è quello più comune nella popolazione umana. Dal sequenziamento di famiglie e singoli individui abbiamo ottenuto 3 differenti aplotipi per i tre polimorfismi più comuni (c.106 G>C, c.538 C>T and c.545 C>T): l’aplotipo 1 (GCC), l’aplotipo 4 (GTC) e l’aplotipo 5 (CTT). Abbiamo effettuato trasfezioni transienti con i cDNA corrispondenti a questi tre aplotipi nella linea cellulare HEK293. L’attività enzimatica associata ai tre aplotipi ha mostrato la stessa efficienza sia con il substrato SSA che HNE. Esprimendo l’attività SSADH degli aplotipi 4 e 5 come percentuale dell’aplotipo 1 (100%), questi hanno prodotto un’attività pari rispettivamente al 62-72% e 23-25%, usando l’SSA e l’HNE come substrati. Per caratterizzare la regione del promotore del gene SSADH abbiamo analizzato circa 100 individui di differente origine geografica identificando così nove posizioni varianti e ricostruendo gli aplotipi e le loro frequenze. Gli aplotipi sono stati subclonati in un vettore d’espressione che avesse la luciferasi come gene reporter e sono stati chiamati BK31, BK45 e BK70; abbiamo ottenuto i seguenti risultati: il clone BK31 ha mostrato l’attività più alta (100%) mentre gli altri,BK45 and 70, hanno mostrato circa il 50% dell’attività rispetto a BK31. Altro scopo di questo studio è stato quello di confermare il secondo ruolo dell’SSADH verificando se conferisce protezione contro l’HNE e altri agenti ossidanti. Abbiamo ottenuto linee cellulari trasfettate stabilmente che overesprimessero l’aplotipo 1 della regione codificante del gene SSADH con differente dosaggio genico. Per ciascuna linea stabile abbiamo misurato l’espressione dell’RNA e l’attività enzimatica utilizzando sia il substrato SSA che l’HNE. Per ciascuna linea cellulare abbiamo osservato che la differenza nei livelli di espressione dell’RNA è in accordo con la differenza nell’attività enzimatica. Usando il saggio MTT come misura della capacità di sopravvivenza cellulare, abbiamo osservato che le linee cellulari con la più alta attività enzimatica mostravano anche una maggiore resistenza al trattamento con differenti dosaggi di HNE and H2O2, confermando che le cellule che overesprimono SSADH sono anche le più effficienti nella detossificazione. Abbiamo quindi concluso che sia la regione codificante che quella del promotore del gene SSADH presentano una variabilità molto elevata nella popolazione umana e che alcune varianti aplotipiche riducono l’attività enzimatica fino al 50% rispetto a quella dell’aplotipo più comune. Abbiamo inoltre confermato il secondo ruolo dell’SSADH nel proteggere le cellule dall’HNE e altri agenti ossidanti.SSADH is a mitochondrial NAD+-dependent enzyme and serves its major role in the catabolism of the GABA, the most important inhibitory neurotransmitter of the Central Nervous System (CNS), by oxidation of the substrate succinic semialdehyde (SSA) to succinate which enters the Krebs cycle; SSA can be also converted into 4-hydroxybutyric acid (GHB) by tissue specific SSA reductases. Complete SSADH deficiency results in 4-hydroxybutyric aciduria, a rare recessive autosomal disorder of human metabolism. 4-HBA patients manifest a considerable variability of the clinical phenotype, ranging from mild psychomotor retardation to severe neurological defects, due to the neurotoxic effects of the abnormal accumulation of GABA and GHB in tissues and physiologic fluids. Recent studies reveal a second role of SSADH: it is one of the two mitochondrial aldehyde dehydrogenases responsible of detoxification of 4-hydroxy-2-nonenal (HNE). HNE is a reactive aldehyde physiologically produced in relatively large amounts from lipid peroxidation. Elevated levels of HNE in CNS are implicated in the pathogenesis of numerous neurodegenerative disorders such as Parkinson’s and Alzheimer’s diseases. By studying more than 50 SSADH deficient families, we identified several pathological variants. We also identified not pathological variants in the coding region. Some of the polymorphic variants are rare and some reach polymorphic frequencies: the G to C transversion in position c.106 and C to T transitions in positions c.538 and c.545. Resequencing non human primates DNA, we identified the ancestral and derived alleles for each polymorphic site and we identified those that occurred in the human specific lineage. We observed that both ancestral and derived alleles for the positions c.106 and c.545 are present in almost all human populations and that the ancestral allele is more frequent; whereas the derived allele at the position c.538 has increased its frequency and now represent the majority in human populations. Resequencing families and single subjects, we obtained 3 different haplotypes for the most common polymorphisms (c.106 G>C, c.538 C>T and c.545 C>T): haplotype 1 (GCC), haplotype 4 (GTC) and haplotype 5 (CTT). We performed transient transfections with cDNAs corresponding to these three haplotypes into HEK293 cell line. The enzyme activity associated with the three haplotypes showed the same efficiency when the substrate was SSA or HNE. When expressing SSADH activity of haplotypes 4 and 5 as percentage of haplotype 1 (100%), they produced 62-72% and 23% of the activity, respectively, using SSA and HNE as substrates. In order to characterized the SSADH promoter region we analysed about 100 subjects of different geographic origins; we identified nine variant positions and obtained the reconstruction of haplotypes and their frequencies. The haplotypes were subcloned in an expression vector with luciferase as reporter gene and named BK31, BK45 and BK70; we obtained the following results: clone BK31 showed the highest activity (100%) while the others, BK45 and 70, showed about 50% of the activity with respect to BK31. Another aim of this study was to confirm the second role of SSADH verifying whether it confers protection against HNE and other oxidants. We obtained stable transfected cell lines overexpressing haplotype 1 of SSADH coding region with different gene-dosage. For each stable cell line we measured RNA expression and enzyme activity, on both SSA and HNE as substrates. We observed that the difference in RNA expression levels parallels the difference in enzyme activities. Using MTT assay as a measure of cell viability, we observed that the stable transfected cell lines, showing the highest enzyme activity, showed also a significant increase of cell viability after treatment with different concentrations of HNE and H2O2, confirming a very high efficacy of SSADH overexpressing cells in detoxification. We concluded that both coding and promoter regions show a very high variability in the human population and some haplotype variant reduce the enzyme activity up to 50% of the most common ones. We confirmed the second role of ALDH5A in the protection against HNE and other oxidants

On-disc observations of flux rope formation prior to its eruption

Coronal mass ejections (CMEs) are one of the primary manifestations of solar activity and can drive severe space weather effects. Therefore, it is vital to work towards being able to predict their occurrence. However, many aspects of CME formation and eruption remain unclear, including whether magnetic flux ropes are present before the onset of eruption and the key mechanisms that cause CMEs to occur. In this work, the pre-eruptive coronal configuration of an active region that produced an interplanetary CME with a clear magnetic flux rope structure at 1 AU is studied. A forward-S sigmoid appears in extreme-ultraviolet (EUV) data two hours before the onset of the eruption (SOL2012-06-14), which is interpreted as a signature of a right-handed flux rope that formed prior to the eruption. Flare ribbons and EUV dimmings are used to infer the locations of the flux rope footpoints. These locations, together with observations of the global magnetic flux distribution, indicate that an interaction between newly emerged magnetic flux and pre-existing sunspot field in the days prior to the eruption may have enabled the coronal flux rope to form via tether-cutting-like reconnection. Composition analysis suggests that the flux rope had a coronal plasma composition, supporting our interpretation that the flux rope formed via magnetic reconnection in the corona. Once formed, the flux rope remained stable for two hours before erupting as a CME

Accuracy barriers in mesh adaptation

The convergence rate of the approximation of a non-smooth, typically discontinuous function can be made higher than usual when an adaptative mesh refinement strategy is applied (in contrast to the uniform mesh refinemen- t). But, for isotropic mesh refinement, this order can be limited to d/p(d-1) for the L^p norm and a space dimension d

Restauro e materiali innovativi: come il recupero architettonico si concilia con la funzionalità e la fruizione di spazi antichi in tempi moderni.

Il restauro è un campo in cui l'Italia può considerarsi all'avanguardia. Su questo tema alcuni esperti, docenti e architetti si confrontano per dare risposta alle problematiche connesse al rapporto tra conservazione e fruizione di spazi antichi in tempi moderni, con uno sguardo alle nuove tecnologie

Self-adaptive F.E.M.Algorithms for the Euler equations

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Mesh adaptation for compressible flows

We describe here some recent developments in mesh adaption for compressible viscous inviscid flow simulation. One of the most challenging task is to capture efficiently thin layers such as boundary layers, mixing layers. Two important issues, the coupling between mesh and flow, and the use of flat elements for stretching are considered in the case of deformation mesh systems

A consistent ale-rezoned mesh adaption algorithm for compressible flow finite-element calculations

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The light and shadow of senescence and inflammation in cardiovascular pathology and regenerative medicine

Recent epidemiologic studies evidence a dramatic increase of cardiovascular diseases, especially associated with the aging of the world population. During aging, the progressive impairment of the cardiovascular functions results from the compromised tissue abilities to protect the heart against stress. At the molecular level, in fact, a gradual weakening of the cellular processes regulating cardiovascular homeostasis occurs in aging cells. Atherosclerosis and heart failure are particularly correlated with aging-related cardiovascular senescence, that is, the inability of cells to progress in the mitotic program until completion of cytokinesis. In this review, we explore the intrinsic and extrinsic causes of cellular senescence and their role in the onset of these cardiovascular pathologies. Additionally, we dissect the effects of aging on the cardiac endogenous and exogenous reservoirs of stem cells. Finally, we offer an overview on the strategies of regenerative medicine that have been advanced in the quest for heart rejuvenation

Fine structures in coronal mass ejection-driven sheath regions

Coronal Mass Ejections (CMEs) often travel in the interplanetary space faster than the ambient solar wind. When their relative velocities exceed the local magnetosonic speed, a shock wave forms. The region between the shock front and the leading edge is known as a sheath region. Sheaths are compressed regions characterized by turbulent magnetic field and plasma properties and they can cause significant space weather disturbances. Within the sheath region, it is possible to find fine structures such as planar magnetic structures (PMSs). The magnetic field vectors in a PMS are characterized by abrupt changes in direction and magnitude, but they all remain for a time interval of several hours nearly parallel to a single plane that includes the interplanetary magnetic field (IMF) spiral direction. PMSs have been associated to several regions and phenomena in the heliosphere, but many of them occur in CME sheath regions. This suggests that CMEs play a central role in the formation of PMSs, probably by provoking the amplification and the alignment of pre-existing discontinuities by compression of the solar wind at the CME-driven shock or because of the draping of the magnetic field lines around the CME ejecta. The presence of PMSs in sheath regions, moreover, suggests that PMSs themselves can be related to space weather effects at Earth, therefore a comprehensive study of PMS formation and structure might lead to a better knowledge of the geoeffectiveness of CMEs. This work presents the study of PMSs in the sheath region of CMEs with a magnetic cloud (MC) structure for a sample of events observed in situ by the ACE and WIND spacecraft between 1997 and 2013. The presence of fine structures is evaluated through the minimum variance analysis (MVA) method, needed for determining the normal vector to the PMS-plane. Then, the position of each PMS within its corresponding sheath region is determined and the encountered cases are divided into different groups. Eventually, a number of shock, sheath and MC properties is evaluated for each group, aiming to perform a statistical analysis. The conclusions are that PMSs are observed in 80% of the studied sheath events and their average duration is ∼5 hours. PMSs tend to form in certain locations within the sheath: they are generally observed close to the CME-driven shock, close to the MC leading edge or they span the whole sheath. PMSs observed near the shock can be associated to strong shocks, while PMSs located near the MC leading edge can be related to high density regions and, therefore, to compression