Reliability of short comparative genomic hybridization in fibroblasts and blastomeres for a comprehensive aneuploidy screening: first clinical application

BACKGROUND: Comparative genomic hybridization (CGH) is a valuable alternative to fluorescence in situ hybridization (FISH) for preimplantation genetic screening (PGS) because it allows full karyotype analysis. However, this approach requires the cryopreservation of biopsied embryos until results are available. The aim of this study is to reduce the hybridization period of CGH, in order to make this short-CGH technique suitable for PGS of Day-3 embryos, avoiding the cryopreservation step. METHODS: Thirty-two fibroblasts from six aneuploid cell lines (Coriell) and 48 blastomeres from 10 Day-4 embryos, discarded after PGS by FISH with 9 probes (9-chr-FISH), were analysed by short-CGH. A reanalysis by the standard 72 h-CGH and FISH using telomeric probes was performed when no concordant results between short-CGH and FISH diagnosis were observed. The short-CGH was subsequently applied in a clinical case of advanced maternal age. RESULTS: In 100% of the fibroblasts analysed, the characteristic aneuploidies of each cell line were detected by short-CGH. The results of the 48 blastomeres screened by short-CGH were supported by both 72 h-CGH results and FISH reanalysis. The chromosomes most frequently involved in aneuploidy were 22 and 16, but aneuploidies for the other chromosomes, excepting 1, 10 and 13, were also detected. Forty-one of the 94 aneuploid events observed (43.6%) corresponded to chromosomes which are not analysed by 9-chr-FISH. CONCLUSIONS: We have performed a preliminary validation of the short-CGH technique, including one clinical case, suggesting this approach may be applied to Day-3 aneuploidy analysis, thereby avoiding embryo cryopreservation and perhaps helping to improve implantation rate after PGS.

An integrative approach for studying the etiology of alcoholism and other addictions

Studies of alcoholism etiology often focus on genetic or psy-chosocial approaches, but not both. Greater understanding of the etiology of alcohol, tobacco and other addictions will come from integration of these research traditions. A research approach is outlined to test three models for the etiology of addictions — behavioral undercontrol, pharmacologic vulnerability, negative affect regulation — addressing key questions including (i) mediators of genetic effects, (ii) genotype-environment correlation effects, (iii) genotype x environment interaction effects, (iv) the developmental unfolding of genetic and environmental effects, (v) subtyping including identification of distinct trajectories of substance involvement, (vi) identification of individual genes that contribute to risk, and (vii) the consequences of excessive use. By using coordinated research designs, including prospective assessment of adolescent twins and their siblings and parents; of adult substance dependent and control twins and their MZ and DZ cotwins, the spouses of these pairs, and their adolescent offspring; and of regular families; by selecting for gene-mapping approaches sibships screened for extreme concordance or discordance on quantitative indices of substance use; and by using experimental (drug challenge) as well as survey approaches, a number of key questions concerning addiction etiology can be addressed. We discuss complementary strengths and weaknesses of different sampling strategies, as well as methods to implement such an integrated approach illustrated for the study of alcoholism etiology. A coordinated program of twin and family studies will allow a comprehensive dissection of the interplay of genetic and environmental risk-factors in the etiology of alcoholism and other addictions.

Host-symbiont interactions in the deep-sea vent mussel Bathymodiolus azoricus : a molecular approach

Tese de Doutoramento, Ciências do Mar, especialidade de Biologia Marinha, 19 de Dezembro de 2015, Universidade dos Açores.

Distributed computing for the factory-floor: a real-time approach using WorldFIP networks

Field communication systems (fieldbuses) are widely used as the communication support for distributed computer-controlled systems (DCCS) within all sort of process control and manufacturing applications. There are several advantages in the use of fieldbuses as a replacement for the traditional point-to-point links between sensors/actuators and computer-based control systems, within which the most relevant is the decentralisation and distribution of the processing power over the field. A widely used fieldbus is the WorldFIP, which is normalised as European standard EN 50170. Using WorldFIP to support DCCS, an important issue is “how to guarantee the timing requirements of the real-time traffic?” WorldFIP has very interesting mechanisms to schedule data transfers, since it explicitly distinguishes periodic and aperiodic traffic. In this paper, we describe how WorldFIP handles these two types of traffic, and more importantly, we provide a comprehensive analysis on how to guarantee the timing requirements of the real-time traffic.

The portuguese pharmaceutial market in the near future - A time series exploration approach

Dissertação apresentada como requisito parcial para obtenção do grau de Mestre em Estatística e Gestão de Informação

The Hybrid Approach for Palliation of Hypoplastic Left Heart Syndrome: Intermediate Results of a Single-Center Experience

INTRODUCTION: Hypoplastic left heart syndrome (HLHS) is a major cause of cardiac death during the first week of life. The hybrid approach is a reliable, reproducible treatment option for patients with HLHS. Herein we report our results using this approach, focusing on its efficacy, safety and late outcome. METHODS: We reviewed prospectively collected data on patients treated for HLHS using a hybrid approach between July 2007 and September 2014. RESULTS: Nine patients had a stage 1 hybrid procedure, with seven undergoing a comprehensive stage 2 procedure. One patient completed the Fontan procedure. Five patients underwent balloon atrial septostomy after the hybrid procedure; in three patients, a stent was placed across the atrial septum. There were three deaths: two early after the hybrid procedure and one early after stage two palliation. Overall survival was 66%. CONCLUSIONS: In our single-center series, the hybrid approach for HLHS yields intermediate results comparable to those of the Norwood strategy. The existence of dedicated teams for the diagnosis and management of these patients, preferably in high-volume centers, is of major importance in this condition.

The economic valuation of noise reduction projects: A cost-benefit analysis approach

The main purpose of this Work Project consists in performing a practical Cost-Benefit Analysis from a social perspective of two noise reduction projects in industrial sites that aim at complying with the existing regulation. By doing so, one may expect a more comprehensive view of the benefits and costs of both projects, as well as relevant insight to the way noise exposure regulation must be optimally defined in Portugal and within the EU area.

A novel and efficient approach to new N3-substituted-6,8-diaminopurines

[Excerpt] Purine nucleobases are fundamental biochemicals in living organisms. They have been a valuable inspiration for drug design once they play several key roles in the cell.1 To the best of our knowledge, reported routes to 8-aminopurines are still scarce due to the difficulty in introducing amino groups in this position of the purine ring. Here we report a novel, inexpensive and facile synthetic method to generate N3,N6-disubstituted-6,8-diaminopurines. In our research group, a number of substituted purines have been obtained from a common imidazole precursor, the 5-amino-4-cyanoformimidoyl imidazole 1. Recently, a comprehensive study on the reactivity of imidazoles 1 with nucleophiles under acidic conditions led us to develop experimental methods to incorporate primary amines into the cyanoformimidoyl group.2 (...)

A Computable general equilibrium approach to hypothetical extractions and missing links

Identifying key sectors or key locations in an interconnected economy is of paramount importance for improving policy planning and directing economic strategy. Hence the relevance of categorizing them and hence the corresponding need of evaluating their potential synergies in terms of their global economic thrust. We explain in this paper that standard measures based on gross outputs do not and cannot capture the relevant impact due to self- imposed modeling limitations. In fact, common gross output measures will be systematically downward biased. We argue that an economy wide Computable General Equilibrium (CGE) approach provides a modeling platform that overcomes these limitations since it provides (i) a more comprehensive measure of linkages and (ii) an alternate way of accounting for links' relevance that is in consonance with standard macromagnitudes in the National Income and Product Accounts.

Novel MLPA procedure using self-designed probes allows comprehensive analysis for CNVs of the genes involved in Hirschsprung disease.

Background: Hirschsprung disease is characterized by the absence of intramural ganglion cells in the enteric plexuses, due to a fail during enteric nervous system formation. Hirschsprung has a complex genetic aetiology and mutations in several genes have been related to the disease. There is a clear predominance of missense/nonsense mutations in these genes whereas copy number variations (CNVs) have been seldom described, probably due to the limitations of conventional techniques usually employed for mutational analysis. In this study, we have looked for CNVs in some of the genes related to Hirschsprung (EDNRB, GFRA1, NRTN and PHOX2B) using the Multiple Ligation-dependent Probe Amplification (MLPA) approach. Methods: CNVs screening was performed in 208 HSCR patients using a self-designed set of MLPA probes, covering the coding region of those genes. Results: A deletion comprising the first 4 exons in GFRA1 gene was detected in 2 sporadic HSCR patients and in silico approaches have shown that the critical translation initiation signal in the mutant gene was abolished. In this study, we have been able to validate the reliability of this technique for CNVs screening in HSCR. Conclusions: The implemented MLPA based technique presented here allows CNV analysis of genes involved in HSCR that have not been not previously evaluated. Our results indicate that CNVs could be implicated in the pathogenesis of HSCR, although they seem to be an uncommon molecular cause of HSCR.

II Comprehensive Mental Health Plan For Andalusia 2008-2012 : Andalusian Public Health System

A Comprehensive Plan is a medium-term planning instrument. Its development alone does not guarantee the achievement of the goals laid out in it, but by defining the goals, establishing priorities and setting out courses of action and concrete activities it will allow for an overall vision of the objectives being aimed towards and the tasks that will need to be carried out. The first Comprehensive Mental Health Plan for Andalusia 2003-2007 (I PISMA, Plan Integral de Salud Mental de Andalucía) was developed using this approach. Nine courses of action were covered in this Plan, which over its duration lead to noticeable progress in various fields.The assessment of the I PISMA and the experience gained from its development have channelled into this second Comprehensive Mental Health Plan for Andalusia 2008-2012 (II PISMA). The main principles for this second Plan are quality improvements, equality and efficiency of health services, aimed at public awareness of mental health in the Andalusian population, prevention of the illnesses and improvements to the care of patients and their families.

A combined approach of VNTR and MLST analysis: improving molecular typing of Argentinean isolates of Leptospira interrogans

Leptospirosis is an emerging infectious disease that has been identified as both a human and animal health problem worldwide. Regular outbreaks associated with specific risk factors have been reported in Argentina. However, there are no available data concerning the genetic population level for this pathogen. Therefore, the aim of this work was to describe the genetic diversity of Leptospira interrogans through the application of two molecular typing strategies: variable number of tandem repeats (VNTR) and multilocus sequence typing (MLST). For this purpose, seven reference strains and 18 non-epidemiologically related isolates from diverse hosts and Argentinean regions were analysed. Among them, nine genotypes and seven sequence types (STs), including three unreported STs, were described using VNTR and MLST, respectively. eBURST analysis demonstrated that ST37 was the most frequent and founder genotype of a clonal complex (CCs) containing STN1 and STN3, suggesting the importance of studying the serovars belonging to this CC in Argentina. The data from maximum parsimony analysis, which combined both techniques, achieved intra-serovar discrimination, surmounted microscopic agglutination test discrepancies and increased the discriminatory power of each technique applied separately. This study is the first to combine both strategies for L. interrogans typing to generate a more comprehensive molecular genotyping of isolates from Argentina in a global context.

Ab initio absorption spectrum of NiO combining molecular dynamics with the embedded cluster approach in a discrete reaction field

We developed a procedure that combines three complementary computational methodologies to improve the theoretical description of the electronic structure of nickel oxide. The starting point is a Car-Parrinello molecular dynamics simulation to incorporate vibrorotational degrees of freedom into the material model. By means ofcomplete active space self-consistent field second-order perturbation theory (CASPT2) calculations on embedded clusters extracted from the resulting trajectory, we describe localized spectroscopic phenomena on NiO with an efficient treatment of electron correlation. The inclusion of thermal motion into the theoretical description allowsus to study electronic transitions that, otherwise, would be dipole forbidden in the ideal structure and results in a natural reproduction of the band broadening. Moreover, we improved the embedded cluster model by incorporating self-consistently at the complete active space self-consistent field (CASSCF) level a discrete (or direct) reaction field (DRF) in the cluster surroundings. The DRF approach offers an efficient treatment ofelectric response effects of the crystalline embedding to the electronic transitions localized in the cluster. We offer accurate theoretical estimates of the absorption spectrum and the density of states around the Fermi level of NiO, and a comprehensive explanation of the source of the broadening and the relaxation of the charge transferstates due to the adaptation of the environment

Generic approach for the sensitive absolute quantification of large undigested peptides in plasma using a particular liquid chromatography-mass spectrometry setup.

A generic LC-MS approach for the absolute quantification of undigested peptides in plasma at mid-picomolar levels is described. Nine human peptides namely, brain natriuretic peptide (BNP), substance P (SubP), parathyroid hormone 1-34 (PTH), C-peptide, orexines A and B (Orex-A and -B), oxytocin (Oxy), gonadoliberin-1 (gonadothropin releasing-hormone or luteinizing hormone-releasing hormone, LHRH) and α-melanotropin (α-MSH) were targeted. Plasma samples were extracted via a 2-step procedure: protein precipitation using 1vol of acetonitrile followed by ultrafiltration of supernatants on membranes with a MW cut-off of 30 kDa. By applying a specific LC-MS setup, large volumes of filtrates (e.g., 2×750 μL) were injected and the peptides were trapped on a 1mm i.d.×10 mm length C8 column using a 10× on-line dilution. Then, the peptides were back-flushed and a second on-line dilution (2×) was applied during the transfer step. The refocalized peptides were resolved on a 0.3mm i.d. C18 analytical column. Extraction recovery, matrix effect and limits of detection were evaluated. Our comprehensive protocol demonstrates a simple and efficient sample preparation procedure followed by the analysis of peptides with limits of detection in the mid-picomolar range. This generic approach can be applied for the determination of most therapeutic peptides and possibly for endogenous peptides with latest state-of-the-art instruments.

Comprehensive evaluation of the IEEE 802.15.4 MAC layer performance with retransmissions

Supported by IEEE 802.15.4 standardization activities, embedded networks have been gaining popularity in recent years. The focus of this paper is to quantify the behavior of key networking metrics of IEEE 802.15.4 beacon-enabled nodes under typical operating conditions, with the inclusion of packet retransmissions. We corrected and extended previous analyses by scrutinizing the assumptions on which the prevalent Markovian modeling is generally based. By means of a comparative study, we singled out which of the assumptions impact each of the performance metrics (throughput, delay, power consumption, collision probability, and packet-discard probability). In particular, we showed that - unlike what is usually assumed - the probability that a node senses the channel busy is not constant for all the stages of the backoff procedure and that these differences have a noticeable impact on backoff delay, packet-discard probability, and power consumption. Similarly, we showed that - again contrary to common assumption - the probability of obtaining transmission access to the channel depends on the number of nodes that is simultaneously sensing it. We evidenced that ignoring this dependence has a significant impact on the calculated values of throughput and collision probability. Circumventing these and other assumptions, we rigorously characterize, through a semianalytical approach, the key metrics in a beacon-enabled IEEE 802.15.4 system with retransmissions.