Sviluppo di un set-up di misura innovativo per lo studio del fenomeno del dielectric charging in dispositivi RF-MEMS

Il lavoro contenuto in questa tesi riguarda lo studio del fenomeno del dielectric charging in dispositivi RF-MEMS, in particolare in switch dielectric-less. Viene in oltre presentato un set-up di misura innovativo per l’attuazione dei dispositivi suddetti, indipendentemente dalla tensione elettrostaticaopenEmbargo per motivi di priorità nella ricerca (previo accordo con terze parti

Histone Hyperacetylation in Mitosis Prevents Sister Chromatid Separation and Produces Chromosome Segregation Defects

Posttranslational modifications of core histones contribute to driving changes in chromatin conformation and compaction. Herein, we investigated the role of histone deacetylation on the mitotic process by inhibiting histone deacetylases shortly before mitosis in human primary fibroblasts. Cells entering mitosis with hyperacetylated histones displayed altered chromatin conformation associated with decreased reactivity to the anti-Ser 10 phospho H3 antibody, increased recruitment of protein phosphatase 1-δ on mitotic chromosomes, and depletion of heterochromatin protein 1 from the centromeric heterochromatin. Inhibition of histone deacetylation before mitosis produced defective chromosome condensation and impaired mitotic progression in living cells, suggesting that improper chromosome condensation may induce mitotic checkpoint activation. In situ hybridization analysis on anaphase cells demonstrated the presence of chromatin bridges, which were caused by persisting cohesion along sister chromatid arms after centromere separation. Thus, the presence of hyperacetylated chromatin during mitosis impairs proper chromosome condensation during the pre-anaphase stages, resulting in poor sister chromatid resolution. Lagging chromosomes consisting of single or paired sisters were also induced by the presence of hyperacetylated histones, indicating that the less constrained centromeric organization associated with heterochromatin protein 1 depletion may promote the attachment of kinetochores to microtubules coming from both poles

Sviluppo di un set-up di misura innovativo per lo studio del fenomeno del dielectric charging in dispositivi RF-MEMS

Il lavoro contenuto in questa tesi riguarda lo studio del fenomeno del dielectric charging in dispositivi RF-MEMS, in particolare in switch dielectric-less. Viene in oltre presentato un set-up di misura innovativo per l’attuazione dei dispositivi suddetti, indipendentemente dalla tensione elettrostatic

Abnormal kinetochore-generated pulling forces from expressing a N-terminally modified Hec1.

BACKGROUND: Highly Expressed in Cancer protein 1 (Hec1) is a constituent of the Ndc80 complex, a kinetochore component that has been shown to have a fundamental role in stable kinetochore-microtubule attachment, chromosome alignment and spindle checkpoint activation at mitosis. HEC1 RNA is found up-regulated in several cancer cells, suggesting a role for HEC1 deregulation in cancer. In light of this, we have investigated the consequences of experimentally-driven Hec1 expression on mitosis and chromosome segregation in an inducible expression system from human cells. METHODOLOGY/PRINCIPAL FINDINGS: Overexpression of Hec1 could never be obtained in HeLa clones inducibly expressing C-terminally tagged Hec1 or untagged Hec1, suggesting that Hec1 cellular levels are tightly controlled. On the contrary, a chimeric protein with an EGFP tag fused to the Hec1 N-terminus accumulated in cells and disrupted mitotic division. EGFP- Hec1 cells underwent altered chromosome segregation within multipolar spindles that originated from centriole splitting. We found that EGFP-Hec1 assembled a mutant Ndc80 complex that was unable to rescue the mitotic phenotypes of Hec1 depletion. Kinetochores harboring EGFP-Hec1 formed persisting lateral microtubule-kinetochore interactions that recruited the plus-end depolymerase MCAK and the microtubule stabilizing protein HURP on K-fibers. In these conditions the plus-end kinesin CENP-E was preferentially retained at kinetochores. RNAi-mediated CENP-E depletion further demonstrated that CENP-E function was required for multipolar spindle formation in EGFP-Hec1 expressing cells. CONCLUSIONS/SIGNIFICANCE: Our study suggests that modifications on Hec1 N-terminal tail can alter kinetochore-microtubule attachment stability and influence Ndc80 complex function independently from the intracellular levels of the protein. N-terminally modified Hec1 promotes spindle pole fragmentation by CENP-E-mediated plus-end directed kinetochore pulling forces that disrupt the fine balance of kinetochore- and centrosome-associated forces regulating spindle bipolarity. Overall, our findings support a model in which centrosome integrity is influenced by the pathways regulating kinetochore-microtubule attachment stability

Combined isobutyryl‐CoA and multiple acyl‐CoA dehydrogenase deficiency in a boy with altered riboflavin homeostasis

In this report, we describe the case of an 11-year-old boy, who came to ourattention for myalgia and muscle weakness, associated with inappetence andvomiting. Hypertransaminasemia was also noted, with ultrasound evidence ofhepatomegaly. Biochemical investigations revealed acylcarnitine and organicacid profiles resembling those seen in MADD, that is, multiple acyl-CoA dehy-drogenase deficiencies (OMIM #231680) a rare inherited disorder of fatty acids,amino acids, and choline metabolism. The patient carried a single pathoge-netic variant in theETFDHgene (c.524G>A, p.Arg175His) and no pathoge-netic variant in the riboflavin (Rf) homeostasis related genes (SLC52A1,SLC52A2,SLC52A3,SLC25A32,FLAD1). Instead, compound heterozygosity wasfound in theACAD8gene (c.512C>G, p.Ser171Cys; c.822C>A, p.Asn274Lys),coding for isobutyryl-CoA dehydrogenase (IBD), whose pathogenic variantsare associated to IBD deficiency (OMIM #611283), a rare autosomal recessivedisorder of valine catabolism. The c.822C>A was never previously describedin a patient. Subsequent further analyses of Rf homeostasis showed reducedlevels of flavins in plasma and altered FAD-dependent enzymatic activities inerythrocytes, as well as a significant reduction in the level of the plasma mem-brane Rf transporter 2 in erythrocytes. The observed Rf/flavin scarcity in thispatient, possibly associated with a decreased ETF:QO efficiency might beresponsible for the observed MADD-like phenotype. The patient's clinical pic-ture improved after supplementation of Rf,L-carnitine, Coenzyme Q10, andalso 3OH-butyrate. This report demonstrates that, even in the absence ofgenetic defects in genes involved in Rf homeostasis, further targeted molecu-lar analysis may reveal secondary and possibly treatable biochemical alter-ations in this pattern