Human telomere disease due to disruption of the CCAAT box of the TERC promoter

Mutations in the coding region of telomerase complex genes can result in accelerated telomere attrition and human disease. Manifestations of telomere disease include the bone marrow failure syndromes dyskeratosis congenita and aplastic anemia, acute myeloid leukemia, liver cirrhosis, and pulmonary fibrosis. Here, we describe a mutation in the CCAAT box (GCAAT) of the TERC gene promoter in a family in which multiple members had typical features of telomeropathy. The genetic alteration in this critical regulatory sequence resulted in reduced reporter gene activity and absent binding of transcription factor NF-Y, likely responsible for reduced TERC levels, decreased telomerase activity, and short telomeres. This is the first description of a pathogenic mutation in the highly con-served CCAAT box and the first instance of a mutation in the promoter region of TERC producing a telomeropathy. We propose that current mutation-screening strategies should include gene promoter regions for the diagnosis of telomere diseases. This clinical trial was registered at www.clinicaltrials.gov as #NCT00071045. (Blood. 2012;119(13):3060-3063)

Duration of dual antiplatelet therapy and outcomes after coronary stenting with the genous (TM) bio-engineered R stent (TM) in patients from the e-healing registry

Objective: We investigated the relation between duration of dual antiplatelet therapy (DAPT) and clinical outcomes up to 12 months after Genous (TM) endothelial progenitor cell capturing R stent (TM) placement in patients from the e-HEALING registry. Background: Cessation of (DAPT) has been shown to be associated with the occurrence of stent thrombosis (ST). After Genous placement, 1 month of DAPT is recommended. Methods: Patients were analyzed according to continuation or discontinuation of DAPT at a 30-day and 6-month landmark, excluding patients with events before the landmark. Each landmark was a new baseline, and outcomes were followed up to 12 months after stenting. The main outcome for our current analysis was target vessel failure (TVF), defined as target vessel-related cardiac death or myocardial infarction and target vessel revascularization. Secondary outcomes included ST. (Un)adjusted hazard ratios (HR) for TVF were calculated with Cox regression. Results: No difference was observed in the incidence of TVF [HR: 1.03; 95% confidence intervals (CI): 0.651.65, P = 0.89] in patients continuing DAPT (n = 4,249) at 30 days versus patients stopped (n = 309), and HR: 0.82 (95% CI: 0.551.23, P = 0.34) in patients continuing DAPT (n = 2,654) at 6 months versus patients stopped [n = 1,408] DAPT). Furthermore, no differences were observed in ST. Even after addition of identified independent predictors for TVF, adjusted TVF hazards were comparable. Conclusions: In a post-hoc analysis of e-HEALING, duration of DAPT was not associated with the occurrence of the outcomes TVF or ST. The Genous stent may be an attractive treatment especially in patients at increased risk for (temporary) cessation of DAPT or bleeding. (C) 2011 Wiley Periodicals, Inc.

Simple heuristics for the assembly line worker assignment and balancing problem

We propose simple heuristics for the assembly line worker assignment and balancing problem. This problem typically occurs in assembly lines in sheltered work centers for the disabled. Different from the well-known simple assembly line balancing problem, the task execution times vary according to the assigned worker. We develop a constructive heuristic framework based on task and worker priority rules defining the order in which the tasks and workers should be assigned to the workstations. We present a number of such rules and compare their performance across three possible uses: as a stand-alone method, as an initial solution generator for meta-heuristics, and as a decoder for a hybrid genetic algorithm. Our results show that the heuristics are fast, they obtain good results as a stand-alone method and are efficient when used as a initial solution generator or as a solution decoder within more elaborate approaches.

Applying the National Institute for Clinical Excellence criteria to patients treated with the Genous (TM) Bio-engineered R stent (TM): a sub-study of the e-HEALING (Healthy Endothelial Accelerated Lining Inhibits Neointimal Growth) worldwide registry

The National Institute for Clinical Excellence (NICE) guidelines recommend the use of bare-metal stents (BMS) in non-complex lesions with a low risk of restenosis (diameter a parts per thousand yen3 mm and lesion length a parts per thousand currency sign15 mm) and the use of drug-eluting stents (DES) in more complex lesions with a high risk of restenosis (diameter < 3.0 mm or lesion length > 15 mm). However, the guidelines were created based on studies evaluating BMS and DES only. We performed an analysis of patients undergoing non-urgent percutaneous coronary intervention with the novel endothelial cell capturing stent (ECS). The ECS is coated with CD34(+) antibodies that attract circulating endothelial progenitor cells to the stent surface, thereby accelerating the endothelialization of the stented area. We analyzed all patients enrolled in the worldwide e-HEALING registry that met the NICE criteria for either low-risk or high-risk lesions and were treated with a parts per thousand yen1 ECS. The main study outcome was target vessel failure (TVF) at 12-month follow-up, defined as the composite of cardiac death or MI and target vessel revascularization (TVR). A total of 4,241 patients were assessed in the current analysis. At 12-month follow-up, TVF occurred in 7.0% of the patients with low-risk lesions and in 8.8% of the patients with high-risk lesions (p = 0.045). When evaluating the diabetic patients versus the non-diabetic patients per risk group, no significant differences were found in TVF, MI or TVR in either risk group. The ECS shows good clinical outcomes in lesions carrying either a high or a low risk of restenosis according to the NICE guidelines with comparable rates of cardiac death, myocardial infarction, and stent thrombosis. The TVF rate with ECS was slightly higher in patients with high-risk lesions, driven by higher clinically driven TLR. The risk of restenosis with ECS in patients carrying high-risk lesions needs to be carefully considered relative to other risks associated with DES. Furthermore, the presence of diabetes mellitus did not influence the incidence of TVF in either risk group.

A comparison of non-HDL and LDL cholesterol goal attainment in a large, multinational patient population: The Lipid Treatment Assessment Project 2

Objective: This study evaluated the success in attaining non-HDL-cholesterol (non-HDL-C) goals in the multinational L-TAP 2 study. Methods: 9955 patients >= 20 years of age with dyslipidemia on stable lipid-lowering therapy were enrolled from nine countries. Results: Success rates for non-HDL-C goals were 86% in low, 70% in moderate, and 52% in high-risk patients (63% overall). In patients with triglycerides of >200 mg/dL success rates for non-HDL-C goals were 35% vs. 69% in those with <= 200 mg/dL (p < 0.0001). Among patients attaining their LDL-C goal, 18% did not attain their non-HDL-C goal. In those with coronary disease and at least two risk factors, only 34% and 30% attained respectively their non-HDL-C and LDL-C goals. Rates of failure in attaining both LDL-C and non-HDL-C goals were highest in Latin America. Conclusions: Non-HDL-C goal attainment lagged behind LDL-C goal attainment; this gap was greatest in higher-risk patients. (c) 2012 Elsevier Ireland Ltd. All rights reserved.

The presence of genes encoding for different virulence factors in clonally related Escherichia coli that produce CTX-Ms

Successful international clones have recently emerged among Escherichia coli that produce CTX-M beta-lactamases as important causes of community-onset urinary tract and bloodstream infections. One hundred and seven isolates that belong to sequence types (STs) ST38, ST131, ST405, ST648, and 38 nonrelated CTX-M producing E. coli from Canada and the Netherlands were assigned to phylogenetic groups and tested for the presence of genes encoding for virulence factors (VFs) using established multiplex polymerase chain reaction. The STs E. coli were significantly more resistant to antibiotics-ST38, ST405, and ST648 belonged to phylogenetic group D while ST131 belonged to B2. Secreted autotransporter toxin (sat), aerobactin receptor, and pathogenicity island marker were significantly more common among the STs; the heat-resistant agglutinin (hra) was present in ST38, sat, and uropathogenic-specific protein, and putative adhesin-siderophore receptor was more common in ST131, while outer membrane protease T was present in ST648. ST131 had a significantly higher VF score. In conclusion, the precise role of these VFs remains to be elucidated; however, we have identified certain putative VFs that possibly contribute to the fitness and success of certain sequence types. (C) 2012 Elsevier Inc. All rights reserved.

Caspase-mediated cleavage of the exosome subunit PM/Scl-75 during apoptosis

Recent studies have implicated the dying cell as a potential reservoir of modified autoantigens that might initiate and drive systemic autoimmunity in susceptible hosts. A number of subunits of the exosome, a complex of 3'→5' exoribonucleases that functions in a variety of cellular processes, are recognized by the so-called anti-PM/Scl autoantibodies, found predominantly in patients suffering from an overlap syndrome of myositis and scleroderma. Here we show that one of these subunits, PM/Scl-75, is cleaved during apoptosis. PM/Scl-75 cleavage is inhibited by several different caspase inhibitors. The analysis of PM/Scl-75 cleavage by recombinant caspase proteins shows that PM/Scl-75 is efficiently cleaved by caspase-1, to a smaller extent by caspase-8, and relatively inefficiently by caspase-3 and caspase-7. Cleavage of the PM/Scl-75 protein occurs in the C-terminal part of the protein at Asp369 (IILD369↓G), and at least a fraction of the resulting N-terminal fragments of PM/Scl-75 remains associated with the exosome. Finally, the implications of PM/Scl-75 cleavage for exosome function and the generation of anti-PM/Scl-75 autoantibodies are discussed.

The double task of preventing malnutrition and overweight: a quasi-experimental community-based trial

Background: The Maternal-Child Pastoral is a volunteer-based community organization of the Dominican Republic that works with families to improve child survival and development. A program that promotes key practices of maternal and child care through meetings with pregnant women and home visits to promote child growth and development was designed and implemented. This study aims to evaluate the impact of the program on nutritional status indicators of children in the first two years of age. Methods: A quasi-experimental design was used, with groups paired according to a socioeconomic index, comparing eight geographical areas of intervention with eight control areas. The intervention was carried out by lay health volunteers. Mothers in the intervention areas received home visits each month and participated in a group activity held biweekly during pregnancy and monthly after birth. The primary outcomes were length and body mass index for age. Statistical analyses were based on linear and logistic regression models. Results: 196 children in the intervention group and 263 in the control group were evaluated. The intervention did not show statistically significant effects on length, but point estimates found were in the desired direction: mean difference 0.21 (95%CI −0.02; 0.44) for length-for-age Z-score and OR 0.50 (95%CI 0.22; 1.10) for stunting. Significant reductions of BMI-for-age Z-score (−0.31, 95%CI −0.49; -0.12) and of BMI-for-age > 85th percentile (0.43, 95%CI 0.23; 0.77) were observed. The intervention showed positive effects in some indicators of intermediary factors such as growth monitoring, health promotion activities, micronutrient supplementation, exclusive breastfeeding and complementary feeding. Conclusions: Despite finding effect measures pointing to effects in the desired direction related to malnutrition, we could only detect a reduction in the risk of overweight attributable to the intervention. The findings related to obesity prevention may be of interest in the context of the nutritional transition. Given the size of this study, the results are encouraging and we believe a larger study is warranted.

Learning for well being : Studies using the International Adult Literacy Survey

This thesis is a collection of five independent but closely related studies. The overall purpose is to approach the analysis of learning outcomes from a perspective that combines three major elements, namely lifelonglifewide learning, human capital, and the benefits of learning. The approach is based on an interdisciplinary perspective of the human capital paradigm. It considers the multiple learning contexts that are responsible for the development of embodied potential – including formal, nonformal and informal learning – and the multiple outcomes – including knowledge, skills, economic, social and others– that result from learning. The studies also seek to examine the extent and relative influence of learning in different contexts on the formation of embodied potential and how in turn that affects economic and social well being. The first study combines the three major elements, lifelonglifewide learning, human capital, and the benefits of learning into one common conceptual framework. This study forms a common basis for the four empirical studies that follow. All four empirical studies use data from the International Adult Literacy Survey (IALS) to investigate the relationships among the major elements of the conceptual framework presented in the first study. Study I. A conceptual framework for the analysis of learning outcomes This study brings together some key concepts and theories that are relevant for the analysis of learning outcomes. Many of the concepts and theories have emerged from varied disciplines including economics, educational psychology, cognitive science and sociology, to name only a few. Accordingly, some of the research questions inherent in the framework relate to different disciplinary perspectives. The primary purpose is to create a common basis for formulating and testing hypotheses as well as to interpret the findings in the empirical studies that follow. In particular, the framework facilitates the process of theorizing and hypothesizing on the relationships and processes concerning lifelong learning as well as their antecedents and consequences. Study II. Determinants of literacy proficiency: A lifelong-lifewide learning perspective This study investigates lifelong and lifewide processes of skill formation. In particular, it seeks to estimate the substitutability and complementarity effects of learning in multiple settings over the lifespan on literacy skill formation. This is done by investigating the predictive capacity of major determinants of literacy proficiency that are associated with a variety of learning contexts including school, home, work, community and leisure. An identical structural model based on previous research is fitted to the IALS data for 18 countries. The results show that even after accounting for all factors, education remains the most important predictor of literacy proficiency. In all countries, however, the total effect of education is significantly mediated through further learning occurring at work, at home and in the community. Therefore, the job and other literacy related factors complement education in predicting literacy proficiency. This result points to a virtual cycle of lifelong learning, particularly to how educational attainment influences other learning behaviours throughout life. In addition, results show that home background as measured by parents’ education is also a strong predictor of literacy proficiency, but in many countries this occurs only if a favourable home background is complemented with some post-secondary education. Study III. The effect of literacy proficiency on earnings: An aggregated occupational approach using the Canadian IALS data This study uses data from the Canadian Adult Literacy Survey to estimate the earnings return to literacy skills. The approach adapts a labour segmented view of the labour market by aggregating occupations into seven types, enabling the estimation of the variable impact of literacy proficiency on earnings, both within and between different types of occupations. This is done using Hierarchical Linear Modeling (HLM). The method used to construct the aggregated occupational classification is based on analysis that considers the role of cognitive and other skills in relation to the nature of occupational tasks. Substantial premiums are found to be associated with some occupational types even after adjusting for within occupational differences in individual characteristics such as schooling, literacy proficiency, labour force experience and gender. Average years of schooling and average levels of literacy proficiency at the between level account for over two-thirds of the premiums. Within occupations, there are significant returns to schooling but they vary depending on the type of occupations. In contrast, the within occupational return of literacy proficiency is not necessarily significant. The latter depends on the type of occupation. Study IV: Determinants of economic and social outcomes from a lifewide learning perspective in Canada In this study the relationship between learning in different contexts, which span the lifewide learning dimension, and individual earnings on the one hand and community participation on the other are examined in separate but comparable models. Data from the Canadian Adult Literacy Survey are used to estimate structural models, which correspond closely to the common conceptual framework outlined in Study I. The findings suggest that the relationship between formal education and economic and social outcomes is complex with confounding effects. The results indicate that learning occurring in different contexts and for different reasons leads to different kinds of benefits. The latter finding suggests a potential trade-off between realizing economic and social benefits through learning that are taken for either job-related or personal-interest related reasons. Study V: The effects of learning on economic and social well being: A comparative analysis Using the same structural model as in Study IV, hypotheses are comparatively examined using the International Adult Literacy Survey data for Canada, Denmark, the Netherlands, Norway, the United Kingdom, and the United States. The main finding from Study IV is confirmed for an additional five countries, namely that the effect of initial schooling on well being is more complex than a direct one and it is significantly mediated by subsequent learning. Additionally, findings suggest that people who devote more time to learning for job-related reasons than learning for personal-interest related reasons experience higher levels of economic well being. Moreover, devoting too much time to learning for personal-interest related reasons has a negative effect on earnings except in Denmark. But the more time people devote to learning for personal-interest related reasons tends to contribute to higher levels of social well being. These results again suggest a trade-off in learning for different reasons and in different contexts.

Essays on the political economy of European integration

Recently, a rising interest in political and economic integration/disintegration issues has been developed in the political economy field. This growing strand of literature partly draws on traditional issues of fiscal federalism and optimum public good provision and focuses on a trade-off between the benefits of centralization, arising from economies of scale or externalities, and the costs of harmonizing policies as a consequence of the increased heterogeneity of individual preferences in an international union or in a country composed of at least two regions. This thesis stems from this strand of literature and aims to shed some light on two highly relevant aspects of the political economy of European integration. The first concerns the role of public opinion in the integration process; more precisely, how economic benefits and costs of integration shape citizens' support for European Union (EU) membership. The second is the allocation of policy competences among different levels of government: European, national and regional. Chapter 1 introduces the topics developed in this thesis by reviewing the main recent theoretical developments in the political economy analysis of integration processes. It is structured as follows. First, it briefly surveys a few relevant articles on economic theories of integration and disintegration processes (Alesina and Spolaore 1997, Bolton and Roland 1997, Alesina et al. 2000, Casella and Feinstein 2002) and discusses their relevance for the study of the impact of economic benefits and costs on public opinion attitude towards the EU. Subsequently, it explores the links existing between such political economy literature and theories of fiscal federalism, especially with regard to normative considerations concerning the optimal allocation of competences in a union. Chapter 2 firstly proposes a model of citizens’ support for membership of international unions, with explicit reference to the EU; subsequently it tests the model on a panel of EU countries. What are the factors that influence public opinion support for the European Union (EU)? In international relations theory, the idea that citizens' support for the EU depends on material benefits deriving from integration, i.e. whether European integration makes individuals economically better off (utilitarian support), has been common since the 1970s, but has never been the subject of a formal treatment (Hix 2005). A small number of studies in the 1990s have investigated econometrically the link between national economic performance and mass support for European integration (Eichenberg and Dalton 1993; Anderson and Kalthenthaler 1996), but only making informal assumptions. The main aim of Chapter 2 is thus to propose and test our model with a view to providing a more complete and theoretically grounded picture of public support for the EU. Following theories of utilitarian support, we assume that citizens are in favour of membership if they receive economic benefits from it. To develop this idea, we propose a simple political economic model drawing on the recent economic literature on integration and disintegration processes. The basic element is the existence of a trade-off between the benefits of centralisation and the costs of harmonising policies in presence of heterogeneous preferences among countries. The approach we follow is that of the recent literature on the political economy of international unions and the unification or break-up of nations (Bolton and Roland 1997, Alesina and Wacziarg 1999, Alesina et al. 2001, 2005a, to mention only the relevant). The general perspective is that unification provides returns to scale in the provision of public goods, but reduces each member state’s ability to determine its most favoured bundle of public goods. In the simple model presented in Chapter 2, support for membership of the union is increasing in the union’s average income and in the loss of efficiency stemming from being outside the union, and decreasing in a country’s average income, while increasing heterogeneity of preferences among countries points to a reduced scope of the union. Afterwards we empirically test the model with data on the EU; more precisely, we perform an econometric analysis employing a panel of member countries over time. The second part of Chapter 2 thus tries to answer the following question: does public opinion support for the EU really depend on economic factors? The findings are broadly consistent with our theoretical expectations: the conditions of the national economy, differences in income among member states and heterogeneity of preferences shape citizens’ attitude towards their country’s membership of the EU. Consequently, this analysis offers some interesting policy implications for the present debate about ratification of the European Constitution and, more generally, about how the EU could act in order to gain more support from the European public. Citizens in many member states are called to express their opinion in national referenda, which may well end up in rejection of the Constitution, as recently happened in France and the Netherlands, triggering a European-wide political crisis. These events show that nowadays understanding public attitude towards the EU is not only of academic interest, but has a strong relevance for policy-making too. Chapter 3 empirically investigates the link between European integration and regional autonomy in Italy. Over the last few decades, the double tendency towards supranationalism and regional autonomy, which has characterised some European States, has taken a very interesting form in this country, because Italy, besides being one of the founding members of the EU, also implemented a process of decentralisation during the 1970s, further strengthened by a constitutional reform in 2001. Moreover, the issue of the allocation of competences among the EU, the Member States and the regions is now especially topical. The process leading to the drafting of European Constitution (even if then it has not come into force) has attracted much attention from a constitutional political economy perspective both on a normative and positive point of view (Breuss and Eller 2004, Mueller 2005). The Italian parliament has recently passed a new thorough constitutional reform, still to be approved by citizens in a referendum, which includes, among other things, the so called “devolution”, i.e. granting the regions exclusive competence in public health care, education and local police. Following and extending the methodology proposed in a recent influential article by Alesina et al. (2005b), which only concentrated on the EU activity (treaties, legislation, and European Court of Justice’s rulings), we develop a set of quantitative indicators measuring the intensity of the legislative activity of the Italian State, the EU and the Italian regions from 1973 to 2005 in a large number of policy categories. By doing so, we seek to answer the following broad questions. Are European and regional legislations substitutes for state laws? To what extent are the competences attributed by the European treaties or the Italian Constitution actually exerted in the various policy areas? Is their exertion consistent with the normative recommendations from the economic literature about their optimum allocation among different levels of government? The main results show that, first, there seems to be a certain substitutability between EU and national legislations (even if not a very strong one), but not between regional and national ones. Second, the EU concentrates its legislative activity mainly in international trade and agriculture, whilst social policy is where the regions and the State (which is also the main actor in foreign policy) are more active. Third, at least two levels of government (in some cases all of them) are significantly involved in the legislative activity in many sectors, even where the rationale for that is, at best, very questionable, indicating that they actually share a larger number of policy tasks than that suggested by the economic theory. It appears therefore that an excessive number of competences are actually shared among different levels of government. From an economic perspective, it may well be recommended that some competences be shared, but only when the balance between scale or spillover effects and heterogeneity of preferences suggests so. When, on the contrary, too many levels of government are involved in a certain policy area, the distinction between their different responsibilities easily becomes unnecessarily blurred. This may not only leads to a slower and inefficient policy-making process, but also risks to make it too complicate to understand for citizens, who, on the contrary, should be able to know who is really responsible for a certain policy when they vote in national,local or European elections or in referenda on national or European constitutional issues.

Harmful algae and their potential impacts on the coastal ecosystem: growth and toxin production dynamics

The main goal of the present thesis was to study some harmful algal species which cause blooms in Italian coastal waters, leading to consequences for human health, coastal ecosystem, fishery and tourism. In particular, in the first part of this thesis the toxicity of Adriatic strains of the raphidophyte Fibrocapsa japonica was investigated. Despite several hypotheses have been proposed for the toxic mechanism of the raphidophytes, especially for the species Chattonella antiqua and C. marina, which have been studied more extensively, just a few studies on the toxic effects of these species for different organisms were reported. Moreover, a careful reading of the literature evidenced as any ichthyotoxic events reported worldwide can be linked to F. japonica blooms. Although recently several studies were performed on F. japonica strains from the USA, Japan, Australia, New Zealand, the Netherlands, Germany, and France in order to characterize their growth and toxicity features, the work reported in this thesis results one of the first investigation on the toxic effects of F. japonica for different organisms, such as bacteria, crustaceans and fish. Mortality effects, together with haemolysis of fish erythrocytes, probably due to the relatively high amount of PUFAs produced by this species, were observed. Mortality for fish, however, was reported only at a high cell density and after a long exposition period (9-10 days); moreover a significant increase of H2O2 obtained in the tanks where sea basses were exposed to F. japonica was also relevant. This result may justify the absence of ichthyotoxic events in the Italian coasts, despite F. japonica blooms detected in these areas were characterized by high cell densities. This work reports also a first complete characterization of the fatty acids produced and extracellularly released by the Adriatic F. japonica, and results were also compared with the fatty acid profile of other strains. The absence of known brevetoxins in F. japonica algal extracts was also highlighted, leading to the hypothesis that the toxicity of F. japonica may be due to a synergic effect of PUFAs and ROS. Another microalgae that was studied in this thesis is the benthic dinoflagellate Ostreopsis cf. ovata. This species was investigated with the aim to investigate the effect of environmental parameters on its growth and toxicity. O. cf. ovata, in fact, shows different blooming periods along the Italian coasts and even the reported toxic effects are variable. The results of this work confirmed the high variability in the growth dynamic and toxin content of several Italian strains which were isolated in recent years along the Adriatic and Tyrrhenian Seas. Moreover, the effects of temperature and salinity on the behaviour of the different isolates are in good agreement with the results obtained from field surveys, which evidence as the environmental parameters are important factors modulating O. cf. ovata proliferation. Another relevant result that was highlighted is the anomaly in the production of palytoxin-like compounds reported by one of the studied isolate, in particular the one isolated in 2008 in Ancona (Adriatic Sea). Only this strain reported the absence of two (ovatoxin-b and –c) of the five ovatoxins so far known in the toxin profile and a different relative abundance of the other toxins. The last aspect that was studied in this thesis regards the toxin biosythesis. In fact, toxins produced (palytoxin-like compounds) or supposed to be produced (brevetoxin-like compounds) by O. cf. ovata and F. japonica, respectively, are polyketides, which are highly oxygenated compounds synthesized by complex enzymes known as polyketide synthase (PKS) enzymes. These enzymes are multi-domain complexes that structurally and functionally resemble the fatty acid synthases (FASs). This work reports the first study of PKS proteins in the dinoflagellates O. cf. ovata, C. monotis and in the raphidophyte F. japonica. For the first time some PKSs were identified in these species, confirming the presence of PKS proteins predicted by the in silico translation of the transcripts found in K. brevis also in other species. The identification of O. cf. ovata PKSs and the localization of the palytoxin-like compounds produced by this dinoflagellate in a similar location (chloroplast) as that observed for other dinoflagellate and cyanobacterial toxins provides some indication that these proteins may be involved in polyketide biosynthesis. However, their potential function as fatty acid synthases cannot be ruled out, as plant fatty acid synthesis also occurs within chloroplasts. This last hypothesis is also supported by the fact that in all the investigated species, and in particular in F. japonica, PKS proteins were present. Therefore, these results provide an important contribution to the study of the polyketides and of the involvement of PKS proteins in the toxin biosynthesis.

Automated Analysis Methods for the Assessment of Bicycle Infrastructure and Demand

A first phase of the research activity has been related to the study of the state of art of the infrastructures for cycling, bicycle use and methods for evaluation. In this part, the candidate has studied the "bicycle system" in countries with high bicycle use and in particular in the Netherlands. Has been carried out an evaluation of the questionnaires of the survey conducted within the European project BICY on mobility in general in 13 cities of the participating countries. The questionnaire was designed, tested and implemented, and was later validated by a test in Bologna. The results were corrected with information on demographic situation and compared with official data. The cycling infrastructure analysis was conducted on the basis of information from the OpenStreetMap database. The activity consisted in programming algorithms in Python that allow to extract data from the database infrastructure for a region, to sort and filter cycling infrastructure calculating some attributes, such as the length of the arcs paths. The results obtained were compared with official data where available. The structure of the thesis is as follows: 1. Introduction: description of the state of cycling in several advanced countries, description of methods of analysis and their importance to implement appropriate policies for cycling. Supply and demand of bicycle infrastructures. 2. Survey on mobility: it gives details of the investigation developed and the method of evaluation. The results obtained are presented and compared with official data. 3. Analysis cycling infrastructure based on information from the database of OpenStreetMap: describes the methods and algorithms developed during the PhD. The results obtained by the algorithms are compared with official data. 4. Discussion: The above results are discussed and compared. In particular the cycle demand is compared with the length of cycle networks within a city. 5. Conclusions

Novel isolates of Cydia pomonella granulovirus (CpGV): deciphering the molecular mechanism for overcoming CpGV resistance in codling moth (Cydia pomonella)

During the last twenty years, Cydia pomonolla granulovirus (CpGV, Baculoviridae) has become the most important biological control agent for the codling moth (CM) in organic and integrated apple production. All registered products in Europe are based on the isolate CpGV-M, which was discovered 1964 in Mexico. A serious threat to future application of CpGV is the occurrence of CM field populations resistant to CpGV. Since 2003, populations with up to 10,000-fold reduced susceptibility were reported from orchards in Germany, France, Italy, Switzerland, Austria and the Netherlands. A putative alternative to CpGV-M are novel CpGV isolates which are able to overcome CM resistance. This thesis focuses on the identification and characterisation of resistance overcoming CpGV isolates and the analysis of their molecular difference to CpGV-M.rnSixteen CpGV isolates were tested against CM lab strains in bioassays. Hereby, five isolates were identified which were able to completely overcome resistance. The genomes of these isolates were compared to CpGV-M by restriction fragment length polymorphism (RFLP) analysis. To identify the molecular factor responsible for improved virulence of some CpGV isolates, major genomic differences were sequenced and analysed. A 0.7 kb insertion was found in CpGV-I01, -I12 and -E2, but not in other resistance overcoming isolates. Analysis of the insertions sequence revealed that it might be due to a transposition event, but not involved in overcoming resistance. rnFor unequivocal identification of CpGV isolates, a new method based on molecular analysis was established. Partial sequencing of the conserved polyhedrin/granulin (polh/gran), late expression factor-8 (lef-8) and late expression factor-9 (lef-9) genes revealed single nucleotide polymorphisms (SNPs). SNP analysis correlated with the grouping obtained by RFLP analysis. A phylogenetic classification due to different genome types A-E is proposed. Phylogenetic analysis suggested that CpGV-M was the phylogenetically youngest of the tested CpGV isolates.rnWhole genome sequencing of two resistance overcoming isolates CpGV-I12 (type D genome) and -S (type E genome) and CpGV-M (type A genome) was performed. Comparison of the three genomes revealed a high sequence identity. Several insertions and deletions ranging from 1-700 nucleotides (nt) were found. Comparison on open reading frame (ORF) level revealed that CpGV-I12 and -S shared only one protein alteration when compared to CpGV-M: a stretch of 24 nt present in ORF cp24 was not found in any of the resistance overcoming isolates. Cp24 codes for the early gene pe38. Combined with the results of phylogenetic analysis, it is proposed that these 24 nt are a recent insertion into the CpGV-M genome. The role of pe38 in overcoming resistance was investigated by knocking out pe38 of a CpGV-M based bacmid and swapping of CpGV-I12 pe38 of into the k.o. bacmid. When pe38 of CpGV-I12 was inserted into the k.o. bacmid, the infectivity could not be rescued, suggesting that the genomic portion of pe38 might play a role in its function.rnIt can be concluded that the recently observed CpGV resistance in CM is only related to type A genomes. RFLP and SNP analysis provide tools for identifying and characterising different CpGV isolates reliably, a pre-condition for a future registration of CpGV products based on novel CpGV isolates.rnrnrn

Relationship between the logistic EuroSCORE and the Society of Thoracic Surgeons Predicted Risk of Mortality score in patients implanted with the CoreValve ReValving system--a Bern-Rotterdam Study

Background Surgical risk scores, such as the logistic EuroSCORE (LES) and Society of Thoracic Surgeons Predicted Risk of Mortality (STS) score, are commonly used to identify high-risk or “inoperable” patients for transcatheter aortic valve implantation (TAVI). In Europe, the LES plays an important role in selecting patients for implantation with the Medtronic CoreValve System. What is less clear, however, is the role of the STS score of these patients and the relationship between the LES and STS. Objective The purpose of this study is to examine the correlation between LES and STS scores and their performance characteristics in high-risk surgical patients implanted with the Medtronic CoreValve System. Methods All consecutive patients (n = 168) in whom a CoreValve bioprosthesis was implanted between November 2005 and June 2009 at 2 centers (Bern University Hospital, Bern, Switzerland, and Erasmus Medical Center, Rotterdam, The Netherlands) were included for analysis. Patient demographics were recorded in a prospective database. Logistic EuroSCORE and STS scores were calculated on a prospective and retrospective basis, respectively. Results Observed mortality was 11.1%. The mean LES was 3 times higher than the mean STS score (LES 20.2% ± 13.9% vs STS 6.7% ± 5.8%). Based on the various LES and STS cutoff values used in previous and ongoing TAVI trials, 53% of patients had an LES ≥15%, 16% had an STS ≥10%, and 40% had an LES ≥20% or STS ≥10%. Pearson correlation coefficient revealed a reasonable (moderate) linear relationship between the LES and STS scores, r = 0.58, P < .001. Although the STS score outperformed the LES, both models had suboptimal discriminatory power (c-statistic, 0.49 for LES and 0.69 for STS) and calibration. Conclusions Clinical judgment and the Heart Team concept should play a key role in selecting patients for TAVI, whereas currently available surgical risk score algorithms should be used to guide clinical decision making.