Ultra-Deep Sequencing Reveals the microRNA Expression Pattern of the Human Stomach

Background: While microRNAs (miRNAs) play important roles in tissue differentiation and in maintaining basal physiology, little is known about the miRNA expression levels in stomach tissue. Alterations in the miRNA profile can lead to cell deregulation, which can induce neoplasia. Methodology/Principal Findings: A small RNA library of stomach tissue was sequenced using high-throughput SOLiD sequencing technology. We obtained 261,274 quality reads with perfect matches to the human miRnome, and 42% of known miRNAs were identified. Digital Gene Expression profiling (DGE) was performed based on read abundance and showed that fifteen miRNAs were highly expressed in gastric tissue. Subsequently, the expression of these miRNAs was validated in 10 healthy individuals by RT-PCR showed a significant correlation of 83.97% (P<0.05). Six miRNAs showed a low variable pattern of expression (miR-29b, miR-29c, miR-19b, miR-31, miR-148a, miR-451) and could be considered part of the expression pattern of the healthy gastric tissue. Conclusions/Significance: This study aimed to validate normal miRNA profiles of human gastric tissue to establish a reference profile for healthy individuals. Determining the regulatory processes acting in the stomach will be important in the fight against gastric cancer, which is the second-leading cause of cancer mortality worldwide.

Ultra deep blue: the ultimate chess player

Projecte d'adaptació del programa GNU Chess al sistema de grid computing 'Condor'. I amb això, es planteja un estudi sobre els algorismes de cerca i la seva aplicació en entorns distribuïts. Una sèrie de proves sobre unes mostres de una partida d'escacs contra el propi GNU Chess ens ajuden a posar de relleu els avantatges i inconvenients de cada un dels algorismes proposats.

Clinical pharmacogenomic testing of KRAS, BRAF and EGFR mutations by high resolution melting analysis and ultra-deep pyrosequencing

BACKGROUND: Epidermal growth factor receptor (EGFR) and its downstream factors KRAS and BRAF are mutated in several types of cancer, affecting the clinical response to EGFR inhibitors. Mutations in the EGFR kinase domain predict sensitivity to the tyrosine kinase inhibitors gefitinib and erlotinib in lung adenocarcinoma, while activating point mutations in KRAS and BRAF confer resistance to the anti-EGFR monoclonal antibody cetuximab in colorectal cancer. The development of new generation methods for systematic mutation screening of these genes will allow more appropriate therapeutic choices. METHODS: We describe a high resolution melting (HRM) assay for mutation detection in EGFR exons 19-21, KRAS codon 12/13 and BRAF V600 using formalin-fixed paraffin-embedded samples. Somatic variation of KRAS exon 2 was also analysed by massively parallel pyrosequencing of amplicons with the GS Junior 454 platform. RESULTS: We tested 120 routine diagnostic specimens from patients with colorectal or lung cancer. Mutations in KRAS, BRAF and EGFR were observed in 41.9%, 13.0% and 11.1% of the overall samples, respectively, being mutually exclusive. For KRAS, six types of substitutions were detected (17 G12D, 9 G13D, 7 G12C, 2 G12A, 2 G12V, 2 G12S), while V600E accounted for all the BRAF activating mutations. Regarding EGFR, two cases showed exon 19 deletions (delE746-A750 and delE746-T751insA) and another two substitutions in exon 21 (one showed L858R with the resistance mutation T590M in exon 20, and the other had P848L mutation). Consistent with earlier reports, our results show that KRAS and BRAF mutation frequencies in colorectal cancer were 44.3% and 13.0%, respectively, while EGFR mutations were detected in 11.1% of the lung cancer specimens. Ultra-deep amplicon pyrosequencing successfully validated the HRM results and allowed detection and quantitation of KRAS somatic mutations. CONCLUSIONS: HRM is a rapid and sensitive method for moderate-throughput cost-effective screening of oncogene mutations in clinical samples. Rather than Sanger sequence validation, next-generation sequencing technology results in more accurate quantitative results in somatic variation and can be achieved at a higher throughput scale.

Ultra-Deep Pyrosequencing Detects Conserved Genomic Sites and Quantifies Linkage of Drug-Resistant Amino Acid Changes in the Hepatitis B Virus Genome

Selection of amino acid substitutions associated with resistance to nucleos(t)ide-analog (NA) therapy in the hepatitis B virus (HBV) reverse transcriptase (RT) and their combination in a single viral genome complicates treatment of chronic HBV infection and may affect the overlapping surface coding region. In this study, the variability of an overlapping polymerase-surface region, critical for NA resistance, is investigated before treatment and under antiviral therapy, with assessment of NA-resistant amino acid changes simultaneously occurring in the same genome (linkage analysis) and their influence on the surface coding region.

The XMM deep survey in the CDF-S. VI. Obscured AGN selected as infrared power-law galaxies

Context. Accretion onto supermassive black holes is believed to occur mostly in obscured active galactic nuclei (AGN). Such objects are proving rather elusive in surveys of distant galaxies, including those at X-ray energies. Aims. Our main goal is to determine whether the revised IRAC criteria of Donley et al. (2012, ApJ, 748, 142; objects with an infrared (IR) power-law spectral shape), are effective at selecting X-ray type-2 AGN (i.e., absorbed N_H > 10^22 cm^-2). Methods. We present the results from the X-ray spectral analysis of 147 AGN selected by cross-correlating the highest spectral quality ultra-deep XMM-Newton and the Spitzer/IRAC catalogues in the Chandra Deep Field South. Consequently it is biased towards sources with high S/N X-ray spectra. In order to measure the amount of intrinsic absorption in these sources, we adopt a simple X-ray spectral model that includes a power-law modified by intrinsic absorption at the redshift of each source and a possible soft X-ray component. Results. We find 21/147 sources to be heavily absorbed but the uncertainties in their obscuring column densities do not allow us to confirm their Compton-Thick nature without resorting to additional criteria. Although IR power-law galaxies are less numerous in our sample than IR non-power-law galaxies (60 versus 87 respectively), we find that the fraction of absorbed (N_H^intr > 10^22 cm^-2) AGN is significantly higher (at about 3 sigma level) for IR-power-law sources (similar to 2/3) than for those sources that do not meet this IR selection criteria (~1/2). This behaviour is particularly notable at low luminosities, but it appears to be present, although with a marginal significance, at all luminosities. Conclusions. We therefore conclude that the IR power-law method is efficient in finding X-ray-absorbed sources. We would then expect that the long-sought dominant population of absorbed AGN is abundant among IR power-law spectral shape sources not detected in X-rays.

Amarração de plataformas offshore flutuantes com cabos de poliéster

Trabalho Final de Mestrado para obtenção do grau de Mestre em Engenharia Civil na Área de Especialização de Estruturas

Rhinovirus Genome Variation during Chronic Upper and Lower Respiratory Tract Infections.

Routine screening of lung transplant recipients and hospital patients for respiratory virus infections allowed to identify human rhinovirus (HRV) in the upper and lower respiratory tracts, including immunocompromised hosts chronically infected with the same strain over weeks or months. Phylogenetic analysis of 144 HRV-positive samples showed no apparent correlation between a given viral genotype or species and their ability to invade the lower respiratory tract or lead to protracted infection. By contrast, protracted infections were found almost exclusively in immunocompromised patients, thus suggesting that host factors rather than the virus genotype modulate disease outcome, in particular the immune response. Complete genome sequencing of five chronic cases to study rhinovirus genome adaptation showed that the calculated mutation frequency was in the range observed during acute human infections. Analysis of mutation hot spot regions between specimens collected at different times or in different body sites revealed that non-synonymous changes were mostly concentrated in the viral capsid genes VP1, VP2 and VP3, independent of the HRV type. In an immunosuppressed lung transplant recipient infected with the same HRV strain for more than two years, both classical and ultra-deep sequencing of samples collected at different time points in the upper and lower respiratory tracts showed that these virus populations were phylogenetically indistinguishable over the course of infection, except for the last month. Specific signatures were found in the last two lower respiratory tract populations, including changes in the 5'UTR polypyrimidine tract and the VP2 immunogenic site 2. These results highlight for the first time the ability of a given rhinovirus to evolve in the course of a natural infection in immunocompromised patients and complement data obtained from previous experimental inoculation studies in immunocompetent volunteers.

Development of a cross-European virtual sample repository and HIV resistance database

BACKGROUND: Several European HIV observational data bases have, over the last decade, accumulated a substantial number of resistance test results and developed large sample repositories, There is a need to link these efforts together, We here describe the development of such a novel tool that allows to bind these data bases together in a distributed fashion for which the control and data remains with the cohorts rather than classic data mergers.METHODS: As proof-of-concept we entered two basic queries into the tool: available resistance tests and available samples. We asked for patients still alive after 1998-01-01, and between 180 and 195 cm of height, and how many samples or resistance tests there would be available for these patients, The queries were uploaded with the tool to a central web server from which each participating cohort downloaded the queries with the tool and ran them against their database, The numbers gathered were then submitted back to the server and we could accumulate the number of available samples and resistance tests.RESULTS: We obtained the following results from the cohorts on available samples/resistance test: EuResist: not availableI11,194; EuroSIDA: 20,71611,992; ICONA: 3,751/500; Rega: 302/302; SHCS: 53,78311,485, In total, 78,552 samples and 15,473 resistance tests were available amongst these five cohorts. Once these data items have been identified, it is trivial to generate lists of relevant samples that would be usefuI for ultra deep sequencing in addition to the already available resistance tests, Saon the tool will include small analysis packages that allow each cohort to pull a report on their cohort profile and also survey emerging resistance trends in their own cohort,CONCLUSIONS: We plan on providing this tool to all cohorts within the Collaborative HIV and Anti-HIV Drug Resistance Network (CHAIN) and will provide the tool free of charge to others for any non-commercial use, The potential of this tool is to ease collaborations, that is, in projects requiring data to speed up identification of novel resistance mutations by increasing the number of observations across multiple cohorts instead of awaiting single cohorts or studies to reach the critical number needed to address such issues.

Cinemática de partículas em fluidos de viscosidade variável com o tempo e sua aplicação na construção de poços de petróleo: avaliação durante paradas operacionais

The new oil reservoirs discoveries in onshore and ultra deep water offshore fields and complex trajectories require the optimization of procedures to reduce the stops operation during the well drilling, especially because the platforms and equipment high cost, and risks which are inherent to the operation. Among the most important aspects stands out the drilling fluids project and their behavior against different situations that may occur during the process. By means of sedimentation experiments, a correlation has been validated to determe the sedimentation particles velocity in variable viscosity fluids over time, applying the correction due to effective viscosity that is a shear rate and time function. The viscosity evolution over time was obtained by carrying out rheologic tests using a fixed shear rate, small enough to not interfere in the fluid gelling process. With the sedimentation particles velocity and the fluid viscosity over time equations an iterative procedure was proposed to determine the particles displacement over time. These equations were implemented in a case study to simulate the cuttings sedimentation generated in the oil well drilling during stops operation, especially in the connections and tripping, allowing the drilling fluid project in order to maintain the cuttings in suspension, avoiding risks, such as stuck pipe and in more drastic conditions, the loss of the well

Novo método para estimativa do gradiente de fratura para poços de petróleo

The development of oil wells drilling requires additional cares mainly if the drilling is in offshore ultra deep water with low overburden pressure gradients which cause low fracture gradients and, consequently, difficult the well drilling by the reduction of the operational window. To minimize, in the well planning phases, the difficulties faced by the drilling in those sceneries, indirect models are used to estimate fracture gradient that foresees approximate values for leakoff tests. These models generate curves of geopressures that allow detailed analysis of the pressure behavior for the whole well. Most of these models are based on the Terzaghi equation, just differentiating in the determination of the values of rock tension coefficient. This work proposes an alternative method for prediction of fracture pressure gradient based on a geometric correlation that relates the pressure gradients proportionally for a given depth and extrapolates it for the whole well depth, meaning that theses parameters vary in a fixed proportion. The model is based on the application of analytical proportion segments corresponding to the differential pressure related to the rock tension. The study shows that the proposed analytical proportion segments reaches values of fracture gradient with good agreement with those available for leakoff tests in the field area. The obtained results were compared with twelve different indirect models for fracture pressure gradient prediction based on the compacting effect. For this, a software was developed using Matlab language. The comparison was also made varying the water depth from zero (onshore wellbores) to 1500 meters. The leakoff tests are also used to compare the different methods including the one proposed in this work. The presented work gives good results for error analysis compared to other methods and, due to its simplicity, justify its possible application

A tutela constitucional da exploração de petróleo em águas internacionais em face do princípio do desenvolvimento sustentável: a possibilidade de responsabilização civil do dano futuro

The demands brought by a society doomed to the constant production of global risks, which whose effects are not immediately noticed effects are not perceived immediately, claim from the Law a new Theory about the Risk, that would offer a broad environmental protection, at the same time it would still be compatible with the idea of economic efficiency, required by the Modern Industry. The expansion of the methods and technologies regarding the exploitation and production of oil causes the constant expansion of the exploitable boundaries, especially in ultra-deep waters with the Pre-salt layer, in Brazil, or the still incipient research about the polymetallic nodules and other mineral sources in international waters, like the Atlântico Sudoeste, by the Programme on Ocean Science in Relation do Non Living Resources (OSNLR), a global study performed in partnership with the Intergovernmental Oceanographic Commission, from UNESCO (IOC UNESCO) and also with the Division of Ocean Affairs and Law of the Sea (UNDOALOS). Thus, we aim to analyze the correlation, and possible collisions between the right to a balanced environment and the free exercise of economic activity and the occurrence of environmental damages from the perspective of the exploitation activities of oil and other natural resources in international waters, specifically in the Area, from the constitutional principle of sustainable development and its legitimacy by the environmental international protection. Therefore, this study also aims to evaluate the legal framework for exploration and production of oil in international waters, particularly in the Area, and appraise how the constitutional instruments and mechanisms for environmental protection can impact on the international environmental protection system in order to ensure the present and future generations an ecologically balanced environment, laid down in Article 225 of the Brazilian Constitution, even with so many risks posed by the activities of exploitation and production of oil in international waters. In the meantime, we intend to also intend to investigate the possibility of future liability for environmental damage in order to ensure that constitutional principle and, consequently, and try to define the concept of environmental damage and its implications on the constitutional principle of environmental protection. Given all that was in summary, this work aims to contribute to the evolution of the new Theory of Environmental Risk, turning the law into something more than a punitive or corrective element in this society, but into a legal risk management, that may be triggered even before the consolidation of the damage

Caracterização tectono-estratigráfica da sequência transicional na sub-bacia de Sergipe

This thesis deals with the tectonic-stratigraphic evolution of the Transitional Sequence in the Sergipe Sub-basin (the southern segment of the Sergipe-Alagoas Basin, Northeast Brazil), deposited in the time interval of the upper Alagoas/Aptian stage. Sequence boundaries and higher order internal sequences were identified, as well as the structures that affect or control its deposition. This integrated approach aimed to characterize the geodynamic setting and processes active during deposition of the Transitional Sequence, and its relations with the evolutionary tectonic stages recognized in the East Brazilian Margin basins. This subject addresses more general questions discussed in the literature, regarding the evolution from the Rift to the Drift stages, the expression and significance of the breakup unconformity, the relationships between sedimentation and tectonics at extensional settings, as well as the control on subsidence processes during this time interval. The tectonic-stratigraphic analysis of the Transitional Sequence was based on seismic sections and well logs, distributed along the Sergipe Sub-basin (SBSE). Geoseismic sections and seismic facies analysis, stratigraphic profiles and sections, were compiled through the main structural blocks of this sub-basin. These products support the depositional and tectonic-stratigraphic evolutionary models built for this sequence. The structural analysis highlighted similarities in deformation styles and kinematics during deposition of the Rift and Transitional sequences, pointing to continuing lithospheric extensional processes along a NW trend (X strain axis) until the end of deposition of the latter sequence was finished by the end of late Aptian. The late stage of extension/rifting was marked by (i) continuous (or as pulses) fault activity along the basin, controling subsidence and creation of depositional space, thereby characterizing upper crustal thinning and (ii) sagstyle deposition of the Transitional Sequence at a larger scale, reflecting the ductile stretching and thinnning of lower and sub crustal layers combined with an increasing importance of the thermal subsidence regime. Besides the late increments of rift tectonics, the Transitional Sequence is also affected by reactivation of the border faults of SBSE, during and after deposition of the Riachuelo Formation (lower section of the Transgressive Marine Sequence, of Albian age). It is possible that this reactivation reflects (through stress propagation along the newlycreated continental margin) the rifting processes still active further north, between the Alagoas Sub-basin and the Pernambuco-Paraíba Basin. The evaporitic beds of the Transitional Sequence contributed to the development of post-rift structures related to halokinesis and the continental margin collapse, affecting strata of the overlying marine sequences during the Middle Albian to the Maastrichtian, or even the Paleogene time interval. The stratigraphic analysis evidenced 5 depositional sequences of higher order, whose vertical succession indicates an upward increase of the base level, marked by deposition of continental siliciclastic systems overlain by lagunar-evaporitic and restricted marine systems, indicating that the Transitional Sequence was deposited during relative increase of the eustatic sea level. At a 2nd order cycle, the Transitional Sequence may represent the initial deposition of a Transgressive Systems Tract, whose passage to a Marine Transgressive Sequence would also be marked by the drowning of the depositional systems. At a 3rd order cycle, the sequence boundary corresponds to a local unconformity that laterally grades to a widespread correlative conformity. This boundary surface corresponds to a breakup unconformity , being equivalent to the Pre-Albian Unconformity at the SBSE and contrasting with the outstanding Pre-upper Alagoas Unconformity at the base of the Transitional Sequence; the latter is alternatively referred, in the literature, as the breakup unconformity. This Thesis supports the Pre-Albian Unconformity as marker of a major change in the (Rift-Drift) depositional and tectonic setting at SBSE, with equivalent but also diachronous boundary surfaces in other basins of the Atlantic margin. The Pre-upper Alagoas Unconformity developed due to astenosphere uplift (heating under high lithospheric extension rates) and post-dates the last major fault pulse and subsequent extensive block erosion. Later on, the number and net slip of active faults significantly decrease. At deep to ultra deep water basin segments, seaward-dipping reflectors (SDRs) are unconformably overlain by the seismic horizons correlated to the Transitional Sequence. The SDRs volcanic rocks overly (at least in part) continental crust and are tentatively ascribed to melting by adiabatic decompression of the rising astenospheric mantle. Even though being a major feature of SBSE (and possibly of other basins), the Pre-upper Alagoas Unconformity do not correspond to the end of lithospheric extension processes and beginning of seafloor spreading, as shown by the crustal-scale extensional structures that post-date the Transitional Sequence. Based on this whole context, deposition of the Transitional Sequence is better placed at a late interval of the Rift Stage, with the advance of an epicontinental sea over a crustal segment still undergoing extension. Along this segment, sedimentation was controled by a combination of thermal and mechanical subsidence. In continuation, the creation of oceanic lithosphere led to a decline in the mechanical subsidence component, extension was transferred to the mesoceanic ridge and the newly-formed continental margin (and the corresponding Marine Sequence) began to be controlled exclusively by the thermal subsidence component. Classical concepts, multidisciplinary data and new architectural and evolutionary crustal models can be reconciled and better understood under these lines

Dimensionamento e análise numérica de cascas de concreto armado para aplicações em plataformas offshore

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Pirossequenciamento de alta cobertura da região hipervariável 1 do Vírus da Hepatite C

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Next-Generation Sequencing-Based Mutations Scanning Strategy of the BCR-ABL Kinase Domain in Patients with PhiladelPhia-Chromosome Positive Leukemias Treated with Tyrosine Kinase Inhibitors

In chronic myeloid leukemia and Philadelphia-positive acute lymphoblastic leukemia patients resistant to tyrosine kinase inhibitors (TKIs), BCR-ABL kinase domain mutation status is an essential component of the therapeutic decision algorithm. The recent development of Ultra-Deep Sequencing approach (UDS) has opened the way to a more accurate characterization of the mutant clones surviving TKIs conjugating assay sensitivity and throughput. We decided to set-up and validated an UDS-based for BCR-ABL KD mutation screening in order to i) resolve qualitatively and quantitatively the complexity and the clonal structure of mutated populations surviving TKIs, ii) study the dynamic of expansion of mutated clones in relation to TKIs therapy, iii) assess whether UDS may allow more sensitive detection of emerging clones, harboring critical 2GTKIs-resistant mutations predicting for an impending relapse, earlier than SS. UDS was performed on a Roche GS Junior instrument, according to an amplicon sequencing design and protocol set up and validated in the framework of the IRON-II (Interlaboratory Robustness of Next-Generation Sequencing) International consortium.Samples from CML and Ph+ ALL patients who had developed resistance to one or multiple TKIs and collected at regular time-points during treatment were selected for this study. Our results indicate the technical feasibility, accuracy and robustness of our UDS-based BCR-ABL KD mutation screening approach. UDS was found to provide a more accurate picture of BCR-ABL KD mutation status, both in terms of presence/absence of mutations and in terms of clonal complexity and showed that BCR-ABL KD mutations detected by SS are only the “tip of iceberg”. In addition UDS may reliably pick 2GTKIs-resistant mutations earlier than SS in a significantly greater proportion of patients.The enhanced sensitivity as well as the possibility to identify low level mutations point the UDS-based approach as an ideal alternative to conventional sequencing for BCR-ABL KD mutation screening in TKIs-resistant Ph+ leukemia patients