Cadmium uptake and induction of metallothionein synthesis in a renal epithelial cell line (LLC-PK1).

LLC-PK1 cells, an established cell line from pig kidney with proximal tubule properties, were cultivated in vitro at confluence on plastic dishes. They were then exposed (apical side) to inorganic cadmium (CdCl2, 5 microM) for periods ranging between 1 to 24 h. Analysis of the cell supernatant after homogenisation and ultracentrifugation indicated that Cd taken up in the first 3 h was bound to cytosolic high molecular weight proteins, but was redistributed to low molecular weight proteins at later stages. Induction of Cd-metallothionein (Cd-Mt) synthesis, as judged from Cd-Mt binding to a specific anti-Cd-Mt antibody and from the rate of 35S-cys incorporation into a specific protein fraction, was apparent 3-6 h after the addition of Cd to the incubation medium.

Optimized exosome isolation protocol for cell culture supernatant and human plasma.

Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of disease. However, there is currently limited information elucidating the most efficient methods for obtaining high yields of pure exosomes, a subset of extracellular vesicles, from cell culture supernatant and complex biological fluids such as plasma. To this end, we comprehensively characterize a variety of exosome isolation protocols for their efficiency, yield and purity of isolated exosomes. Repeated ultracentrifugation steps can reduce the quality of exosome preparations leading to lower exosome yield. We show that concentration of cell culture conditioned media using ultrafiltration devices results in increased vesicle isolation when compared to traditional ultracentrifugation protocols. However, our data on using conditioned media isolated from the Non-Small-Cell Lung Cancer (NSCLC) SK-MES-1 cell line demonstrates that the choice of concentrating device can greatly impact the yield of isolated exosomes. We find that centrifuge-based concentrating methods are more appropriate than pressure-driven concentrating devices and allow the rapid isolation of exosomes from both NSCLC cell culture conditioned media and complex biological fluids. In fact to date, no protocol detailing exosome isolation utilizing current commercial methods from both cells and patient samples has been described. Utilizing tunable resistive pulse sensing and protein analysis, we provide a comparative analysis of 4 exosome isolation techniques, indicating their efficacy and preparation purity. Our results demonstrate that current precipitation protocols for the isolation of exosomes from cell culture conditioned media and plasma provide the least pure preparations of exosomes, whereas size exclusion isolation is comparable to density gradient purification of exosomes. We have identified current shortcomings in common extracellular vesicle isolation methods and provide a potential standardized method that is effective, reproducible and can be utilized for various starting materials. We believe this method will have extensive application in the growing field of extracellular vesicle research.

Fast and Rigorous Computation of Gene and Pathway Scores from SNP-Based Summary Statistics.

Integrating single nucleotide polymorphism (SNP) p-values from genome-wide association studies (GWAS) across genes and pathways is a strategy to improve statistical power and gain biological insight. Here, we present Pascal (Pathway scoring algorithm), a powerful tool for computing gene and pathway scores from SNP-phenotype association summary statistics. For gene score computation, we implemented analytic and efficient numerical solutions to calculate test statistics. We examined in particular the sum and the maximum of chi-squared statistics, which measure the strongest and the average association signals per gene, respectively. For pathway scoring, we use a modified Fisher method, which offers not only significant power improvement over more traditional enrichment strategies, but also eliminates the problem of arbitrary threshold selection inherent in any binary membership based pathway enrichment approach. We demonstrate the marked increase in power by analyzing summary statistics from dozens of large meta-studies for various traits. Our extensive testing indicates that our method not only excels in rigorous type I error control, but also results in more biologically meaningful discoveries.

Trasporti e sviluppo territoriale negli spazi intermedi : l'esempio della regione transfrontaliera Insubrica

Avec cette thèse de doctorat nous proposons une réflexion transversale concernant les relations entre infrastructures de transport et développement territorial dans des espaces dits « intermédiaires ». Le concept d'espace intermédiaire, relativement nouveau en géographie, est conçu en fonction d'une double approche : celle des infrastructures où les espaces intermédiaires constituent des zones de transit obligées entre des pôles urbains hiérarchiquement supérieurs (par rapport une échelle horizontale) et celle des frontières où les espaces intermédiaires constituent des territoires de coopération entre différents niveaux politico-institutionnels (par rapport à une échelle verticale). Cette problématique de recherche est traitée aussi bien du point de vue théorique qu'avec des études de cas portant sur les effets des nouvelles infrastructures de transports dans la région transfrontalière insubrique (entre le Canton du Tessin et la Lombardie). L'objectif visé est de défendre un scénario d'organisation spatiale polycentrique à plusieurs niveaux comme solution pour le développement durable et cohérent de ces espaces intermédiaires. Ainsi, pour le « niveau macro », nous proposons une analyse des changements d'accessibilité spatiale et des potentiels de développement territorial pour les agglomérations concernées par la mise en service du nouveau tunnel ferroviaire de base du Monte Ceneri (TBC) et de la nouvelle ligne Lugano/Como-Mendrisio-Varese-Malpensa (FMV) à l'horizon 2020. Pour le « niveau meso », nous analysons les effets de la nouvelle ligne FMV en termes de potentiel de densification polycentrique autours des gares ferroviaires. Pour le « niveau micro », nous proposons une analyse sur les comportements de mobilité ainsi que des améliorations ciblées du système de transport pour la ville de Mendrisio visant à promouvoir le développement polycentrique de cette commune. De plus, un système d'analyse permettant de mettre en lien les divers facteurs explicatifs dans l'analyse des relations entre les nouvelles infrastructures de transport et les effets sur la mobilité et le développement territorial est également élaboré et testé dans notre recherche. -- With this Ph.D. thesis we investigate the relationship between transport infrastructures and territory development inside the so called "in-between spaces". The idea of "in-between space", relatively novel in geography, is the formal outcome of a double approach: the one of the infrastructures, saying that these spaces can be described as areas of constrained transit between urban centres of superior hierarchical level (on a horizontal scale), and the one of the borders, stating that in-between spaces are areas of cooperation between various political-institutional levels. The above mentioned research issues are deepened both at theoretical and empirical level, being the latter based on field studies of the cross-boundary Western-Lombard area (between the Swiss canton of Ticino and the Italian region of Lombardy). This research pursues the goal of defending the argument that a multi-level polycentric spatial scenario can be a possible solution fora sustainable development of the above described in- between areas. From a "macro" perspective, what we submit here is an analysis on the expected changes in spatial accessibility and on the potential territorial development for the built-up areas influenced by the construction of the new train tunnel of the Monte Ceneri (TBC) and of the new railway line Lugano/Como-Mendrisio-Varese-Malpensa (FMV). At a "meso" level we analyse the effects exerted by the new FMV line taking into account the potential densification of the areas surrounding the railway stations. Finally, at a "micro" level, we analyse the mobility behaviours in the town of Mendrisio and we propose some possible improvements to the local public transport system, with the scope to promote a polycentric development of this municipality. Moreover, we developed and tested an analytic system able to define the existing links between the various explaining factors characterizing the relationship between new transport infrastructures and effects on mobility. -- With this Ph.D. thesis we investigate the relationship between transport infrastructures and territory development inside the so called "in-between spaces". The idea of "in-between space", relatively novel in geography, is the formal outcome of a double approach: the one of the infrastructures, saying that these spaces can be described as areas of constrained transit between urban centres of superior hierarchical level (on a horizontal scale), and the one of the borders, stating that in-between spaces are areas of cooperation between various political-institutional levels. The above mentioned research issues are deepened both at theoretical and empirical level, being the latter based on field studies of the cross-boundary Western-Lombard area (between the Swiss canton of Ticino and the Italian region of Lombardy). This research pursues the goal of defending the argument that a multi-level polycentric spatial scenario can be a possible solution for a sustainable development of the above described in- between areas. From a "macro" perspective, what we submit here is an analysis on the expected changes in spatial accessibility and on the potential territorial development for the built-up areas influenced by the construction of the new train tunnel of the Monte Ceneri (TBC) and of the new railway line Lugano/Como-Mendrisio-Varese-Malpensa (FMV). At a "meso" level we analyse the effects exerted by the new FMV line taking into account the potential densification of the areas surrounding the railway stations. Finally, at a "micro" level, we analyse the mobility behaviours in the town of Mendrisio and we propose some possible improvements to the local public transport system, with the scope to promote a polycentric development of this municipality. Moreover, we developed and tested an analytic system able to define the existing links between the various explaining factors characterizing the relationship between new transport infrastructures and effects on mobility.

Changes in cannabis and tobacco use trajectories from age 17 to age 23

Much like the first volume published in 2011, the second volume of this anthology series gathers a selection of analyses which are empirically based on the data of the Swiss panel study TREE (Transitions from Education to Employment). The contributions of this volume carry on the investigation of the critical transitions during youth and young adulthood, drawing on sociological, economic, psychological and pedagogical research questions and thus highlighting the analytic and pluri-disciplinary research potential of the TREE data. One of the topical foci is the long-term influence of social origin on education and labour market pathways, particularly with regard to access to higher education.