82. Cftr Gene Targeting in Murine ES Cells Mediated by the SFHR Technique

Small Fragment Homologous Recombination (SFHR)-mediated targeting is a gene therapy strategy where a specific genomic locus is modified through a target exchange between a small DNA fragment (SDF) and genomic DNA. Here we demonstrate that SFHR can stably introduce a 3-bp deletion (corresponding to |[Delta]|F508) within Cftr (Cystic Fibrosis Transmembrane Conductance Regulator) locus in the genome of mouse embryonic stem (ES) cells. SDFs (about 6.4|[times]|105 molecules per cell) carrying the |[Delta]|F508 mutation were transfected by nucleofection protocol. About 12% of transcript corresponding to deleted allele was detected and about 60% of the electroporated cells no longer had measurable CFTR-dependent chloride efflux. The CFTR activity was also analyzed by measuring the chloride efflux by the fluorescence microscopy-coupled digital video imaging system in each ES cell colony, previously loaded with MQAE, a chloride sensitive dye. An average of 4-6 regions for each cell colony was analysed to verify the genotypic homogeneity of each colony. In fact all regions examined in each colony showed a similar significant chloride efflux after PKA activation. Moreover on twelve electroporated ES colonies analysed, eight were successfully mutated (Cl- efflux not significantly different from zero) while four colonies showed Cl-efflux CFTR-dependent not significantly different from the untreated ones

Electron and ion spectroscopy of Azobenzene in the valence and core shells

Azobenzene is a prototype and building block of a class of molecules of extreme technological interest as molecularphoto-switches. We present a joint experimental and theoretical study of its response to irradiation with light across theUV to X-ray spectrum. The study of valence and inner shell photo-ionization and excitation processes, combined withmeasurement of valence photoelectron-photoion coincidence (PEPICO) and of mass spectra across the core thresholdsprovides a detailed insight onto the site- and state-selected photo-induced processes. Photo-ionization and excita-tion measurements are interpreted via the multi-configurational restricted active space self-consistent field (RASSCF)method corrected by second order perturbation theory (RASPT2). Using static modelling, we demonstrate that thecarbon and nitrogen K edges of Azobenzene are suitable candidates for exploring its photoinduced dynamics thanks tothe transient signals appearing in background-free regions of the NEXAFS and XP

Radiation Damage Mechanisms of Chemotherapeutically Active Nitroimidazole Derived Compounds

Photoionization mass spectrometry, photoelectron-photoion coincidence spectroscopic technique, and computational methods have been combined to investigate the fragmentation of two nitroimidazole derived compounds: the metronidazole and misonidazole. These molecules are used in radiotherapy thanks to their capability to sensitize hypoxic tumor cells to radiation by “mimicking” the effects of the presence of oxygen as a damaging agent. Previous investigations of the fragmentation patterns of the nitroimidazole isomers (Bolognesi et al., 2016; Cartoni et al., 2018) have shown their capacity to produce reactive molecular species such as nitric oxide, carbon monoxide or hydrogen cyanide, and their potential impact on the biological system. The results of the present work suggest that different mechanisms are active for the more complex metronidazole and misonidazole molecules. The release of nitric oxide is hampered by the efficient formation of nitrous acid or nitrogen dioxide. Although both metronidazole and misonidazole contain imidazole ring in the backbone, the side branches of these molecules lead to very different bonding mechanisms and properties

The Firm as an Institution : Recent Evolution in the Contractual Perspective

The aim of this paper is to clarify the connection between types of firms and types of assumptions about the information structure. Building on the role of information structure, notions of the firm as an organisation have been put forward which suggest that the firm can assume an alternative (to the market) coordinating role in conditions of contractual incompleteness. A comparison (and opposition) between two recent approaches to the theory of the firm as an organisation seems to suggest that different notions of incomplete contracts imply substantial differences in terms of the meaning of a Pareto-efficiency improvement, of the role to assign to vertical integration, and of the determinants of the dimension of the firm.

Effect of ElectroSpark Process Parameters on the WE43 Magnesium Alloy Deposition Quality

This research aims to investigate the effects of process parameters on the quality of WE43 coatings deposited on homologue substrate by ElectroSpark Deposition (ESD) technology. ESD is new technology used to apply coatings or for the restoration and refurbishment of worn or damaged high valued parts. The depositions were processed using five different levels of Energy input (Es, Spark Energy). The microstructure of both the base material and deposits cross-section were characterized by optical and scanning electron microscopies. Also, X-ray diffraction technique was used. In addition, stereological studies of the through-thickness heterogeneities of the deposits (e.g., voids) were performed. The mechanical properties were evaluated by Vickers micro-hardness. The results show that the deposits exhibited a fine grained microstructure due to the rapid solidification. The average micro-hardness values of the deposits are lower than that of the substrate and distributed in a small range (49-60 HV). The lower hardness of the deposits respect to the base material is due to the presence of defectiveness such as spherical, laminar and random shaped voids. The defects area percentage inside the deposits remains well below than 11%. All the deposits were mainly affected by laminar morphology defects. The results indicate that the deposits defectiveness decreases as the energy input increases

Indicators and Targets in a Multilevel Government Setting with Increasing Budget Responsibilities. Lessons from Targeting Essential Services in the South of Italy

The debate on progress in Italy is affected by the backwardness of the Mezzogiorno. The recent fiscal federalism reform hence raises new questions on how to attain more comparable living conditions for residents in regions that currently differ in levels of development and access to basic services. The on-going experience of a performance-based mechanism directed to the eight regions of the Mezzogiorno suggests interesting routes for setting services standards, supporting the achievement of objectives and enhancing accountability. Far from being the outcome of merely technical activities, the chosen options might greatly impact the overall progress of the country and its internal inequalities.federalism, indicators and targets

Technical Progres and Wage Dispersion in Italy: Evidence from Firm's Data

In this paper we test for the effect of technological progress on the labour demand and relative earnings by skill in Italy from 1986 to 1990. We consider a large panel of firms, about 36,000: the data allow us to construct a proxy of the use of new technology by each firm. Our main conclusions are that: (i) wage dispersion in Italy did not increase by the same extent as in the US and UK; (ii) technological progress led to a remarkable increase in the employment of skilled (captured in our data by white-collars) vis-à-vis blue-collar workers. However relative earnings did not experience a similar rise. We suggest that both a shift in the supply of skills and the features of the Italian wage bargaining system counteracted the rise in earnings dispersion.