GENE DISCOVERY IN SCHIZOPHRENIA THROUGH IDENTIFICATION OF COPY NUMBER VARIATIONS (CNVs) IN DISCORDANT MONOZYGOTIC (MZ) TWINS

Schizophrenia (MIM 181500) is a complex and devastating human disease that affects greater than 1% of the population (Huxley et al., 1964). The disease has a heritability of 80%, but a concordance rate in monozygotic (MZ) twins of only 48% (Gottesman, 1991). This suggests a role for both genetics and random/environmental factors in the etiology of this complex disorder (Singh et al., 2009). Copy Number Variation (CNV) is now a known candidate for disease associated variation in humans. CNVs have been identified as a common feature covering 12% of the genome in normal, healthy individuals (Feuk et al., 2006; Redon et al., 2006). Although little is known about the causes and consequences of this common genomic phenomenon, CNVs represent causal mutational events in any study of genetic causes of complex diseases. This study focuses on monozygotic twins who represent the best possible genetic match, but are discordant for the disease; this approach\u27 allows for a significant reduction of disease heterogeneity. A genome wide analysis of copy number variation on three pairs of monozygotic twins was performed using the Affymetrix Human Array 6.0. The results show that they differ significantly in variation profile and that the affected member of each twin pair had a significantly higher number of CNV when compared to their unaffected co-twin (p=0.01). In addition, a set of five de novo CNV’s were found to be shared in all three unrelated patients. These regions contain genes, many of which play a role in neurodevelopment and have the potential to cause schizophrenia pathogenesis. The results support a role for de novo CNVs in the etiology of schizophrenia

Investigating The Role Of the Cellular Prion Protein in Ca2+ Metabolism And Alzheimer's Disease

The cellular prion protein (PrPC) is a highly conserved cell-surface glycoprotein expressed in almost all mammalian tissues, in particular in the central nervous systems. The bad reputation acquired by PrPC originates from its capacity to convert into an aberrant conformer (PrPSc), which is the major component of the prion, the unconventional infectious particle causing fatal neurodegenerative disorders, known as prion diseases. Both the mechanism of prion related neurodegeneration and the physiologic role of PrPC are still unknown. However, use of animal and cell models has underscored a number of putative functions for PrPC, suggesting that it could serve in cell adhesion, migration, proliferation and differentiation, possibly by interacting with extracellular partners, and/or by taking part in multi-component signaling complexes at the cell surface. An intriguing hypothesis, based on increasing amounts of data that may explain the multifaceted behavior of PrPC, entails that the protein is involved in the regulation of Ca2+ homeostasis One major part of the present thesis deals with a close investigation of the alleged regulation of Ca2 homeostasis by PrPC. This study was carried out by monitoring local Ca2+ movements in primary cultures of cerebellar granule neurons (CGN) - obtained from PrP-knockout mice and transgenic PrPC-expressing mice - subjected to various stimuli. Measurements of Ca2+ fluxes in different cell domains were accomplished using the Ca2+- sensitive photo-protein aequorin genetically targeted to different cell domains (plasma membrane, cytosol, lumen of the endoplasmic reticulum and mitochondrial matrix). We found that, with respect to PrPC-expressing neurons, the absence of PrPC caused alterations of local Ca2+ movements, indicating that PrPC may be part of the cellular system(s) deputed to avoid toxic neuronal Ca2+ accumulation. As for the molecular mechanisms by which PrPC controls Ca2+ homeostasis, we found that this could be accomplished through the modulation of p59Fyn- and p42/p44-ERK-dependent signaling pathways. Another topic studied in this thesis stemmed from recent reports indicating that PrPC could acts as a high-affinity receptor for amyloid-beta (Abeta) peptides implicated in Alzheimer's disease. The possibility that PrPC-Abeta interactions may impair synaptic plasticity is, however, still highly debated. Thus, given that Ca2+ is intimately related to synaptic plasticity, we investigated whether Abeta peptides affected Ca2+ metabolism in a PrPC-dependent manner using the above-mentioned strategies and cell paradigms. The obtained results showed that interactions between PrPC and Abeta oligomers may cause Ca2+ accumulation following activation of store-operated Ca2+ entry, and that this may occur via a PrPC-dependent activation of p59Fyn kinase

Testing an interpersonal process model of intimacy using intimate discussions of committed romantic couples

This study attempts to better understand relationship processes that promote or enhance a coupleÂs experience of emotional intimacy in their relationship. An overarching goal of the research is to test Reis and ShaverÂs (1988) interpersonal process model of intimacy with a sample of committed, romantic couples. The interpersonal process model asserts that discussions involving self-disclosure and empathic responding will result in subjective feelings of emotional intimacy. Reis and ShaverÂs model suggests that more vulnerable self-disclosure will promote deeper levels of emotional understanding and concern, subsequently resulting in greater subjective intimacy. Analyses tested the interpersonal process model of intimacy by examining self- and partner-reports of self-disclosure, empathic responding, and emotional intimacy. In this study, data were collected on 108 committed romantic couples from the community. Couples completed a packet of questionnaires individually and then engaged in videotaped interactions in which they discussed times when (a) someone other than their partner hurt their feelings (low-risk), and (b) their partner hurt their feelings (high-risk). The discussion topics were aimed at eliciting vulnerable self-disclosure and empathic responding. Results support the interpersonal process model, showing that self-disclosure and empathy are positively related to greater reports of post-interaction intimacy. Empathy proved to have a stronger impact on intimacy in high-risk discussions than low-risk discussions. The impact of self-disclosure and empathy on intimacy did not differ for men and women, suggesting that similar processes are at work for both genders. Methodological and clinical implications are discussed, along with suggestions for future research

The prion protein regulates glutamate-mediated Ca2+ entry and mitochondrial Ca2+ accumulation in neurons

The cellular prion protein (PrPC) whose conformational misfolding leads to the production of deadly prions, has a still-unclarified cellular function despite decades of intensive research. Following our recent finding that PrPC limits Ca2+ entry via store-operated Ca2+ channels in neurons, we investigated whether the protein could also control the activity of ionotropic glutamate receptors (iGluRs). To this end, we compared local Ca2+ movements in primary cerebellar granule neurons and cortical neurons transduced with genetically encoded Ca2+ probes and expressing, or not expressing, PrPC. Our investigation demonstrated that PrPC downregulates Ca2+ entry through each specific agonist-stimulated iGluR and after stimulation by glutamate. We found that, although PrP-knockout (KO) mitochondria were displaced from the plasma membrane, glutamate addition resulted in a higher mitochondrial Ca2+ uptake in PrP-KO neurons than in their PrPC-expressing counterpart. This was because the increased Ca2+ entry through iGluRs in PrP-KO neurons led to a parallel increase in Ca2+-induced Ca2+ release via ryanodine receptor channels. These data thus suggest that PrPC takes part in the cell apparatus controlling Ca2+ homeostasis, and that PrPC is involved in protecting neurons from toxic Ca2+ overloads

Impact of Lentiviral Vector-Mediated Transduction on the Tightness of a Polarized Model of Airway Epithelium and Effect of Cationic Polymer Polyethylenimine

Lentiviral (LV) vectors are promising agents for efficient and long-lasting gene transfer into the lung and for gene therapy of genetically determined pulmonary diseases, such as cystic fibrosis, however, they have not been evaluated for cytotoxicity and impact on the tightness of the airway epithelium. In this study, we evaluated the transduction efficiency of a last-generation LV vector bearing Green Fluorescent Protein (GFP) gene as well as cytotoxicity and tight junction (TJ) integrity in a polarized model of airway epithelial cells. High multiplicities of infection (MOI) showed to be cytotoxic, as assessed by increase in propidium iodide staining and decrease in cell viability, and harmful for the epithelial tightness, as demonstrated by the decrease of transepithelial resistance (TER) and delocalization of occludin from the TJs. To increase LV efficiency at low LV:cell ratio, we employed noncovalent association with the polycation branched 25 kDa polyethylenimine (PEI). Transduction of cells with PEI/LV particles resulted in 2.5–3.6-fold increase of percentage of GFP-positive cells only at the highest PEI:LV ratios (1×107 PEI molecules/transducing units with 50 MOI LV) as compared to plain LV. At this dose PEI/LV transduction resulted in 6.5 ± 2.4% of propidium iodide-positive cells. On the other hand, PEI/LV particles did not determine any alteration of TER and occludin localization. We conclude that PEI may be useful for improving the efficiency of gene transfer mediated by LV vectors in airway epithelial cells, in the absence of high acute cytotoxicity and alteration in epithelial tightness

Three new bricks in the wall: Berkeley 23, Berkeley 31, and King 8

A comprehensive census of Galactic open cluster properties places unique constraints on the Galactic disc structure and evolution. In this framework we investigate the evolutionary status of three poorly-studied open clusters, Berkeley 31, Berkeley 23 and King 8, all located in the Galactic anti-centre direction. To this aim, we make use of deep LBT observations, reaching more than 6 mag below the main sequence Turn- Off. To determine the cluster parameters, namely age, metallicity, distance, reddening and binary fraction, we compare the observational colour-magnitude diagrams (CMDs) with a library of synthetic CMDs generated with different evolutionary sets (Padova, FRANEC and FST) and metallicities. We find that Berkeley 31 is relatively old, with an age between 2.3 and 2.9 Gyr, and rather high above the Galactic plane, at about 700 pc. Berkeley 23 and King 8 are younger, with best fitting ages in the range 1.1-1.3 Gyr and 0.8-1.3 Gyr, respectively. The position above the Galactic plane is about 500- 600 pc for the former, and 200 pc for the latter. Although a spectroscopic confirmation is needed, our analysis suggests a sub-solar metallicity for all three clusters.Comment: 17 Pages, Accepted for publication in MNRA

Preparation of drug-loaded small unilamellar liposomes and evaluation of their potential for the treatment of chronic respiratory diseases

The aim of the present investigation was to evaluate the influence of liposome formulation on the ability of vesicles to penetrate a pathological mucus model obtained from COPD affected patients in order to assess the potential of such vesicles for the treatment of chronic respiratory diseases by inhalation. Therefore, Small Unilamellar Liposomes (PLAIN-LIPOSOMEs), Pluronic® F127- surface modified liposomes (PF-LIPOSOMEs) and PEG 2000PE-surface modified liposomes (PEG-LIPOSOMEs) were prepared using the micelle-to-vesicle transition (MVT) method and beclomethasone dipropionate (BDP) as model drug. The obtained liposomes showed diameters in the range of 40-65 nm, PDI values between 0.25-0.30 and surface electric charge essentially close to zero. The encapsulation efficiency was found to be dependent on the BDP/lipid ratio used and, furthermore, BDP-loaded liposomes were stable in size both at 37°C and at 4°C. All liposomes were not cytotoxic on H441 cell line as assessed by the MTT assay. The liposome uptake was evaluated through a cytofluorimetric assay that showed a non-significant reduction in the internalization of PEG-LIPOSOMEs as compared with PLAIN-LIPOSOMEs. The penetration studies of mucus from COPD patients showed that the PEG-LIPOSOMEs were the most mucuspenetrating vesicles after 27 hours. In addition, PEG- and PF-LIPOSOMEs did not cause any effect on bronchoalveolar lavage fluid proteins after aerosol administration in the mouse. The results highlight that PEG-LIPOSOMEs show the most interesting features in terms of penetration through the pathologic sputum, uptake by airway epithelial cells and safety profile

Correctors of mutant CFTR enhance subcortical cAMP-PKA signaling through modulating ezrin phosphorylation and cytoskeleton organization

The most common mutation of the cystic fibrosis transmembrane regulator (CFTR) gene, F508del, produces a misfolded protein resulting in its defective trafficking to the cell surface and an impaired chloride secretion. Pharmacological treatments partially rescue F508del CFTR activity either directly by interacting with the mutant protein and/or indirectly by altering the cellular protein homeostasis. Here, we show that the phosphorylation of ezrin together with its binding to phosphatidylinositol-4,5-bisphosphate (PIP2) tethers the F508del CFTR to the actin cytoskeleton, stabilizing it on the apical membrane and rescuing the sub-membrane compartmentalization of cAMP and activated PKA. Both the small molecules trimethylangelicin (TMA) and VX-809, which act as 'correctors' for F508del CFTR by rescuing F508del-CFTR-dependent chloride secretion, also restore the apical expression of phosphorylated ezrin and actin organization and increase cAMP and activated PKA submembrane compartmentalization in both primary and secondary cystic fibrosis airway cells. Latrunculin B treatment or expression of the inactive ezrin mutant T567A reverse the TMA and VX-809-induced effects highlighting the role of corrector-dependent ezrin activation and actin re-organization in creating the conditions to generate a sub-cortical cAMP pool of adequate amplitude to activate the F508del-CFTR-dependent chloride secretion