105 research outputs found
Characterization of molecular bases of Myhre syndrome
No full textMyhre syndrome (MYHRS, MIM 139210) is a rare developmental disorder first described in 1981, for which about 50 cases are currently reported. Clinical features of MHYRS include typical facial gestalt (short palpebral fissures and philtrum, mid-face hypoplasia, prognathism, narrow mouth), thickened skin, joint limitation, muscular pseudohypertrophy, mild-to-moderate intellectual deficiency and hearing loss.
Our group (Caputo et al., 2012) and others (Le Goff et al., 2011a) identified SMAD4 (MIM 600993) as the gene mutated in MYHRS using Whole Exome Sequencing approach. Three different de novo missense changes involving Isoleucine 500 (p.Ile500Thr, p.Ile500Val and p.Ile500Met) within the evolutionary conserved MAD homology 2 domain of SMAD4 were detected in 19 patients.
SMAD4 plays a pivotal role in signal transduction of the transforming growth factor beta (TGFβ) superfamily cytokines, which exerts an important role from early embryogenesis to adulthood by mediating transcriptional activation of target genes involved in different cellular processes (such as cell division, differentiation, migration, and programmed cell death). SMAD4 has been established as a tumor suppressor gene, since loss-of-function mutations are known to cause two familiar cancer-prone diseases (juvenile polyposis syndrome (JPS, MIM 174900) and Juvenile polyposis/hereditary hemorrhagic telangiectasia (JPHT, MIM 175050), Gallione et al., 2004; Gallione et al., 2010), and occur in different types of carcinomas (pancreas, gastrointestinal tract and skin).
The main purpose of this thesis was the investigation of the molecular bases of Myhre syndrome through functional and in silico approaches. Molecular studies and assays on cell cultures were performed in order to characterize MYHRS causative variants.
Firstly, different cellular and biochemical assays were performed, in order to assess MYHRS Ile500 mutations functional impact on SMAD4 protein expression, localization, and on cell proliferation. Western blot analysis of MYHRS fibroblasts and HeLa SMAD4-mutated transfected cells, showed an increased endogenous and ectopic expression of the protein, respectively. Immunofluorescence analysis by confocal laser scanning microscopy of MYHRS fibroblasts disclosed an extra-nuclear accumulation of MYHRS-mutated SMAD4 in patient fibroblasts after 2.5 and 5 hours of TGFβ stimulation. Growth curve of MYHRS-mutated fibroblasts, as well as BrdU incorporation assay on MYHRS fibroblasts and 3T3 transfected cells, demonstrated a reduction in the proliferation levels of both patient and SMAD4-mutated overexpressing cells.
Moreover, we collected a cohort of cases with clinical features fitting MYHRS. Molecular screening of SMAD4 coding sequence in these patients identified a novel missense change affecting Arginine 496 (p.Arg496Cys) in three cases. In silico structural analysis, performed in collaboration with Tor Vergata University, suggested that conformational changes promoted by replacement of Arg496 impact the stability of the SMAD heterotrimer and/or proper SMAD4 ubiquitination (Caputo et al., 2014). Since the triplet coding for the Arginine at position 496 encompassed a CpG dinucleotide, we preliminarily investigated the methylation status of the cytosine at nucleotide c.1486, through digestion assays of genomic DNA from leukocytes of control subjects with Hpy99I methylation sensitive restriction enzyme which confirmed that c.1486 C>T mutation localized in a methylated CpG dinucleotide.
Finally, the investigation of the parental germline origin of MYHRS mutations was performed. Cloning of genomic fragments encompassing SMAD4 causative mutation and intronic polymorphic site for 11 informative MYHRS cases and segregation analysis demonstrated the paternal germline origin of Myhre pathogenic variants in all these patients, in line with the well-known gender bias in the origin of point mutations.
In conclusion, our functional data confirm increased expression of Ile500-mutated SMAD4 in MYHRS affected cells and point out a loss of function effect of MYHRS mutations on fibroblasts proliferation, a mechanism activated by TGFβ signaling. Furthermore, the identification of a new mutation causing this syndrome (c.1486 C>T; p.Arg496Cys), which encompass an amino acid close to Ile500 residue, suggests the impairment of ubiquitination and/or transcription complex stability as the probable outcome of this variant, highlighting the need of further investigations on possible different effect of MYHRS mutations on TGFβ signaling and gene transcription. Finally, the exclusive paternal origin of MYHRS variants in our 11 informative patients expand the number of point mutations causing dominant disorders which display a paternal bias in origin, and lead us to speculate on possibly other mechanisms which could produce a positive selection on MYHRS mutations in male testes
A generalized drift-diffusion model for rectifying Schottky contact simulation
No full textWe present a discussion on the modeling of Schottky barrier rectifying contacts (diodes) within the framework of partial-differential-equation-based physical simulations. We propose a physically consistent generalization of the drift-diffusion model to describe the boundary layer close to the Schottky barrier where thermionic emission leads to a non-Maxwellian carrier distribution, including a novel boundary condition at the contact. The modified drift-diffusion model is validated against Monte Carlo simulations of a GaAs device. The proposed model is in agreement with the Monte Carlo simulations not only in the current value but also in the spatial distributions of microscopic quantities like the electron velocity and concentratio
I discorsi sulla patria tra II e I secolo a.C.: il contributo dei populares
No full textThis paper focuses on the topic of patriotism in the rhetorical exempla dating from the Late Roman Republic. The aim is to demonstrate how the charge of military betrayal (proditio hostibus patriae) is connected to the conflicts between factiones after the fall of the Gracchi. The objective is achieved by analysing this matter from the perspective of the populares, reported by the author of the Rhetorica ad Herennium
Un discorso polemico sulla poena idonea nella Rhetorica ad Herennium
No full textThis essay aims to appreciate the Rhetorica ad Herennium in an historical perspective. The
analysis focuses on a declamatory speech set in the Social War, in which the author emphasises the
need of an appropriate matching between crime and punishment
Recetas para leer a Eliseo Verón
No full textEste articulo presenta cuatro claves de lectura a la obra de Eliseo Veron, colega y amigo del autor. Interrogándose sobre el paso del modelo binario, clásico de la epistemología estructuralista - que Verón contribuyo a instalar no solo en Argentina sino en América Latina- al modelo ternario de semiosis luego de la lectura del semiólogo y filósofo americano Charles S. Peirce, Traversa explora las formas en que Verón construye su propia epistemología.This article presents four keys to reading the work of Eliseo Verón, colleague and friend of the author. Interrogating the passage from the binary model, in a classic structuralist epistemology - which Verón contributed to install not only in Argentina but in Latin America - to the ternary model of semiosis after reading the American semiologist and philosopher Charles S. Peirce, Traversa explores the ways in which Verón builds his own epistemology
Exploring the Impact of European Union Law on Energy and Environmental Taxation
No full textIn this chapter, Alice Pirlot explores whether and how EU law has shaped the use of environmental tax measures – including environmental taxes and environmentally driven taxes and tax incentives – in the EU, drawing on existing literature on the topic. The author shows that EU law has shaped and continues to shape the development of environmental tax measures both at the level of the EU and at the level of the Member States. It is argued that at EU level, the institutional framework has not really helped in the enactment of environmentally driven taxes. The analysis of the historical development of EU provisions surrounding energy taxation illustrates this point. So far, the energy taxation directive remains largely disconnected from the EU’s climate policy, including the EU Emissions Trading Scheme. Second, EU substantive law has had an ambiguous impact on Member States’ environmental tax policy. On the one hand, EU substantive law has been interpreted by the Court of Justice in a way that encourages Member States to adopt tax measures that are environmentally driven. Indeed, the environmental purpose of Member States’ tax measures seems to play a positive role in the assessment of their compatibility with EU law, including State aid provisions, the fundamental freedoms and the energy taxation directive. On the other hand, in some instances, EU law strictly limits Member States’ ability to adopt environmentally driven tax measures. Moreover, it is shown that EU secondary law disregards the purpose of environmental taxes in order to classify them for statistical purposes
Absence of 6’Sialyllactose during lactation impairs cognitive capabilities and modulates gene expression
No full textAims: Human milk is the ideal source of nutrition for the new-born, promoting the development of cognitive capabilities. However, which components of maternal milk are involved in the proper development of executive functions has remained elusive. We hypothesized that the maturation of attention, cognitive flexibility, and memory depends on the neonatal bioavailability of a specific human milk oligosaccharide (HMOs), sialyl(alpha2,6)lactose (6’SL).
Methods: To test this hypothesis, we evaluated the aforementioned cognitive capabilities in adult mice that received maternal milk containing different concentrations of 6’SL. To modulate the availability of 6’SL during lactation, we leveraged a genetically engineered mouse (C57BL/6-St6gal1tm1.1Jxm/J, KO), that provides milk without 6‘SL. Specifically, we performed a cross-fostering study in which wild-type (WT) mice were reared to either KO or WT dams. During lactation, maternal behaviour was analysed for potential effects of maternal care on offspring. Adult subjects were tested for spatial memory, working memory and sensorimotor gating. To understand the underpinning molecular mechanisms of potential effect, we performed an RNA-seq analysis on PFC and hippocampal samples. Furthermore, we addressed ex vivo long-term potentiation (LTP, an electrophysiological correlate of memory performance) in hippocampal slices.
Results: Mice that received 6’SL deficient milk showed an impoverishment of spatial reference memory, working memory and attentional capabilities compared to control. Furthermore, this group showed an altered regulation in the expression of genes involved in PFC development and an alteration in LTP.
Conclusions: These findings show that absence of 6’SL in maternal milk impairs cognitive functions, such as memory and attention
Exploring non-coding genetic variability in ACE2: Functional annotation and in vitro validation of regulatory variants
No full textThe surge in human whole-genome sequencing data has facilitated the study of non-coding region variations, yet understanding their biological significance remains a challenge. We used a computational workflow to assess the regulatory potential of non-coding variants, with a particular focus on the Angiotensin Converting Enzyme 2 (ACE2) gene. This gene is crucial in physiological processes and serves as the entry point for severe acute respiratory syndrome coronavirus 2 (SARSCoV-2), the virus causing coronavirus disease 19 (COVID-19). In our analysis, using data from the gnomAD population database and functional annotation, we identified 17 significant Single Nucleotide Variants (SNVs) in ACE2, particularly in its enhancers, promoters, and 3’ untranslated regions (UTRs). We found preliminary evidence supporting the regulatory impact of some of these variants on ACE2 expression. Our detailed examination of two SNVs, rs147718775 and rs140394675, in the ACE2 promoter revealed that these co-occurring SNVs, when mutated, significantly enhance promoter activity, suggesting a possible increase in specific ACE2 isoform expression. This method proves effective in identifying and interpreting impactful non-coding variants, aiding in further studies and enhancing understanding of molecular bases of monogenic and complex traits
Molecular Approaches in Fetal Malformations, Dynamic Anomalies and Soft Markers: Diagnostic Rates and Challenges—Systematic Review of the Literature and Meta-Analysis
No full textFetal malformations occur in 2–3% of pregnancies. They require invasive procedures for cytogenetics and molecular testing. “Structural anomalies” include non-transient anatomic alterations. “Soft markers” are often transient minor ultrasound findings. Anomalies not fitting these definitions are categorized as “dynamic”. This meta-analysis aims to evaluate the diagnostic yield and the rates of variants of uncertain significance (VUSs) in fetuses undergoing molecular testing (chromosomal microarray (CMA), exome sequencing (ES), genome sequencing (WGS)) due to ultrasound findings. The CMA diagnostic yield was 2.15% in single soft markers (vs. 0.79% baseline risk), 3.44% in multiple soft markers, 3.66% in single structural anomalies and 8.57% in multiple structural anomalies. Rates for specific subcategories vary significantly. ES showed a diagnostic rate of 19.47%, reaching 27.47% in multiple structural anomalies. WGS data did not allow meta-analysis. In fetal structural anomalies, CMA is a first-tier test, but should be integrated with karyotype and parental segregations. In this class of fetuses, ES presents a very high incremental yield, with a significant VUSs burden, so we encourage its use in selected cases. Soft markers present heterogeneous CMA results from each other, some of them with risks comparable to structural anomalies, and would benefit from molecular analysis. The diagnostic rate of multiple soft markers poses a solid indication to CMA
Reduced availability of a selective human milk oligosaccharide during lactation impairs post-weaning executive functions
No full textMaternal milk is considered the ideal source of early life nutrition. It contains nutritional, immunological and signalling factors that are essential for the correct development of the new-born, including development of cognitive capabilities, gastrointestinal tract, and immune functions. Thus, breast-feeding has been associated with improved cognitive capabilities and increased resistance to infection. Specifically, there is an association between duration of breast feeding and verbal IQ. Human milk oligosaccharides (HMOs) represent the third most abundant component of breast milk; many of them are composed by sialylated molecules that represent the principal source of sialic acid (Sia) for the infant. Sia is an essential component of glycolipids (gangliosides) and glycoproteins (polySia-NCAM), two highly represented molecules in the brain. Since endogenous synthesis of Sia is insufficient to meet the nutritional needs of the neonate, its dietary provision is fundamental. Among the components of the breast-milk, sialylated-HMOs have been indicated as possible mediators of its beneficial effects on neurodevelopment and brain function. This study investigated the effects of one specific HMO, sialyl(alpha2,3)lactose (3’SL), on cognitive development. Importantly, 3’SL is abundant in maternal milk, and absent in infant formula. We hypothesised that 3’SL during lactation has permanent effects on executive functions in adulthood and that variations in this HMO during lactation related to a differential development of learning, memory, attention and locomotion. Methods: To modulate the availability of 3’SL during lactation, we performed a cross-fostering study in which wild-type mice were reared to knock-out mice constitutively deficient of 3’SL. Specifically, the study involves C57BL/6J wild-type and C57BL/6-St3gal4tm1.1Jxm/J transgenic male mice. The deletion of the ST3GAL4 gene (B6-129 St3gal4-/-) resulted in an 80% decrease of 3’SL in milk. The cross-fostering design resulted in the following groups: WT mice reared to WT dams (WT to WT, N=11), WT mice reared to KO dams (WT to KO, N=12), KO mice reared to WT dams (KO to WT, N=12) and KO mice reared to KO dams (KO to KO, N=12). Adult subjects were tested for spatial memory (Barnes maze), recognition memory (Novel object recognition task), attention (Attentional set shifting task, ASST), impulsivity (T-maze), sensorimotor gating (Prepulse inhibition) and anxiety (Elevated plus maze). Data was analysed using a factorial ANOVA using 2 between subject factors (dams genotype and pups genotype). Results: compared to control subjects, all experimental groups exhibited a reduction of retention memory in the Barnes maze one day after training, but not in the novel object recognition task, indicating that early life presence of 3’SL may support spatial memory. When evaluating attentional capabilities, mice that received 3’SL deficient-milk exhibited poor performance in several stages of the ASST. Furthermore, the same experimental group showed increased impulsivity. The experimental groups did not differ in anxiety-like behaviour, evaluated through the elevated plus maze. Conclusion: reduced availability of 3’SL during lactation caused long-lasting deficits in cognitive abilities, thus confirming the importance of the presence of this breast-milk component during the crucial period of lactation. Since this study was the first to evaluate this deficiency, further studies will be needed to confirm these findings and deepen the mechanism underlying the effect of 3’SL
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