FE - Contribuições em Revistas Científicas / Contribution to Journals
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- Prediction of retention time of cutinases tagged with hydrophobic peptides in hydrophobic interaction chromatographyPublication . Lienqueo, M.E.; Salazar, O.; Henriquez, K.; Calado, C.R.C.; Fonseca, L.P.; Cabral, J.M.SHydrophobic interaction chromatography (HIC) is an important technique for protein purification, which exploits the separation of proteins based on hydrophobic interactions between the stationary phase ligands and hydrophobic regions on the protein surface. One way of enhancing the purification efficiency by HIC is the addition of short sequences of peptide tags to the target protein by genetic engineering, which could reduce the need for extra and expensive chromatographic steps. In the present work, a methodology for predicting retention times of cutinases tagged with hydrophobic peptides in HIC is presented. Cutinase from Fusarium solani pisi fused to tryptophan–proline (WP) tags, namely (WP)2 and (WP)4, and produced in Saccharomyces cerevisiae strains, were used as model proteins. From the simulations, the methodology based on tagged hydrophobic definition proposed by Simeonidis et al. (Φtagged), associated to a quadratic model for predicting dimensionless retention times, showed small differences (RMSE < 0.022) between observed and estimated retention times. The difference between observed and calculated retention times being lower than 2.0% (RMSE < 0.022) for the two tagged cutinases at three different stationary phases, except for the case of cut (wp)2 in octyl sepharose–2M ammonium sulphate. Therefore, we consider that the proposed strategy, based on tagged surface hydrophobicity, allows prediction of acceptable retention times of cutinases tagged with hydrophobic peptides in HIC.
- Geographic distribution of methyltransferases of Helicobacter pylori: evidence of human host population isolation and migrationPublication . Vale, Filipa F.; Mégraud, Francis; Vítor, Jorge M.Background. Helicobacter pylori colonizes the human stomach and is associated with gastritis, peptic ulcer, and gastric cancer. This ubiquitous association between H. pylori and humans is thought to be present since the origin of modern humans. The H. pylori genome encodes for an exceptional number of restriction and modifications (R-M) systems. To evaluate if R-M systems are an adequate tool to determine the geographic distribution of H. pylori strains, we typed 221 strains from Africa, America, Asia, and Europe, and evaluated the expression of different 29 methyltransferases. Results. Independence tests and logistic regression models revealed that ten R-M systems correlate with geographical localization. The distribution pattern of these methyltransferases may have been originated by co-divergence of regional H. pylori after its human host migrated out of Africa. The expression of specific methyltransferases in the H. pylori population may also reflect the genetic and cultural background of its human host. Methyltransferases common to all strains, M. HhaI and M. NaeI, are likely conserved in H. pylori, and may have been present in the bacteria genome since the human diaspora out of Africa. Conclusion. This study indicates that some methyltransferases are useful geomarkers, which allow discrimination of bacterial populations, and that can be added to our tools to investigate human migrations.
- Prediction of dynamic plasmid production by recombinant escherichia coli fed-batch cultivations with a generalized regression neural networkPublication . Silva, T.; Lima, P.; Roxo-Rosa, M.; Hageman, S.; Fonseca, L. P.; Calado, C. R. C.A generalized regression neural network with external feedback was used to predict plasmid production in a fed-batch cultivation of recombinant Escherichia coli. The neu ral network was built out of the experimental data obtained on a few cultivations, of which the general strategy was based on an initial batch phase followed by an exponen tial feeding phase. The different cultivation conditions used resulted in significant differ ences in bacterial growth and plasmid production. The obtained model allows estimation of the experimental outputs (biomass, glucose, acetate and plasmid) based on the bioreactor starting conditions and the following on-line inputs: feeding rate, dissolved oxygen concentration and bioreactor stirring speed. Therefore, the proposed methodol ogy presents a quick, simple and reliable way to perform on-line feedback prediction of the dynamic behaviour of the complex plasmid production process, based on simple on-line input data obtained directly from the bioreactor control unit and with few cultiva tion experiments for neural network learning.
- Unconstrained mining of transcript data reveals increased alternative splicing complexity in the human transcriptomePublication . Mollet, I. G.; Ben-Dov, Claudia; Felício-Silva, Daniel; Grosso, A. R.; Eleutério, Pedro; Alves, Ruben; Staller, Ray; Silva, Tito Santos; Carmo-Fonseca, MariaMining massive amounts of transcript data for alternative splicing information is paramount to help understand how the maturation of RNA regulates gene expression. We developed an algorithm to cluster transcript data to annotated genes to detect unannotated splice variants. A higher number of alternatively spliced genes and isoforms were found compared to other alternative splicing databases. Comparison of human and mouse data revealed a marked increase, in human, of splice variants incorporating novel exons and retained introns. Previously unannotated exons were validated by tiling array expression data and shown to correspond preferentially to novel first exons. Retained introns were validated by tiling array and deep sequencing data. The majority of retained introns were shorter than 500 nt and had weak polypyrimidine tracts. A subset of retained introns matching small RNAs and displaying a high GC content suggests a possible coordination between splicing regulation and production of noncoding RNAs. Conservation of unannotated exons and retained introns was higher in horse, dog and cow than in rodents, and 64% of exon sequences were only found in primates. This analysis highlights previously bypassed alternative splice variants, which may be crucial to deciphering more complex pathways of gene regulation in human.
- Optimization of the culture medium composition using response surface methodology for new recombinant cyprosin B production in bioreactor for cheese productionPublication . Sampaio, Pedro N.; Calado, Cecília R. C.; Sousa, Lisete; Bressler, David C.; Pais, Maria Salomé; Fonseca, Luís PinaThe optimization of culture medium compositionwas carried out for improvement the recombinantcyprosin B production, an enzyme with high milk-clottingactivity. Response surface methodology (RSM) wasapplied to evaluate the effect of variables namely glucose,yeast extract (YE) and bactopeptone present in the culturemedium, used for recombinant cyprosin B production bytransformed Saccharomyces cerevisiae BJ1991 strain inshake-flask and bioreactor culture conditions. The centracomposite experimental design (CCD) was adopted toderive a statistical model for optimizing the composition ofthe fermentation medium. The optimal concentration estimatedfor each variable related to a theoretical maximumof cyprosin B activity (488 U mL-1) was 30 g L-1 glucose,15 g L-1 YE and 27 g L-1 bactopeptone. The optimizedmedium composition, based on empirical model, ledto a cyprosin B activity of 519 U mL-1, which correspondsto an increase of 46%. The fermentation using optimizedculture medium in a 5-L bioreactor allowed a significantincrease in biomass (82%) and recombinant cyprosin Bproduction (139%). The improvement in the recombinantcyprosin B production after optimization process can beconsidered adequate for large-scale applications, and theclotting activity of cyprosin B account for their use inindustrial cheese making.
- Robot skills and cognitive performance of preschool childrenPublication . Poletz, Linda; Encarnação, Pedro; Adams, Kim; Cook, AlSeveral studies have demonstrated the potential of robots as assistive tools for play activities. Through the use of robots, children with motor impairments may be able to manipulate objects and engage in play activities as their typically developing peers, thus having the same opportunities to learn cognitive, social, motor and linguistic skills. Robot use can also provide a proxy measure of disabled children's cognitive abilities by comparing their performance with that of typically developing children. This paper reports a study with eighteen typically developing children aged three, four and five years to assess at which ages the cognitive concepts of causality, negation, binary logic, and sequencing are demonstrated during Lego robot use.
- The effects of cryogenic cooling on surface integrity in hard machiningPublication . Umbrello, D.; Pu, Z.; Caruso, S.; Outeiro, J. C.; Jayal, A. D.; Dillon, O. W.; Jawahir, I. S.This paper presents the results of an experimental investigation to determine the effects of cryogenic coolant on surface integrity in orthogonal machining of hardened AISI 52100 bearing steel. Experiments were performed under dry and cryogenic conditions using chamfered CBN tool inserts. Several experimental techniques were used in the analyzing of the machined surface and subsurface: optical and scanning electron microscopes (SEM) were utilized for the surface topography characterization; chemical characterization (phase study) was carried out by means of Energydispersive spectroscopy (EDS) techniques; and X-ray diffraction (XRD) technique was used to determine residual stresses and phase changes induced by dry and cryogenic machining. The results show the benefits and the future potential of cryogenic cooling for surface integrity enhancement to achieve improved product's functional performance in hard machining.
- Surface integrity in dry and cryogenic machining of AZ31B Mg alloy with varying cutting edge radius toolsPublication . Pu, Z.; Outeiro, J. C.; Batista, A. C.; Dillon, O. W.; Puleo, D. A.; Jawahir, I. S.Surface integrity of machined products has a critical impact on their functional performance. Magnesium alloys are lightweight materials for transportation industry and are also emerging as a potential material for temporary biomedical implants. However, their unsatisfactory corrosion resistance limits their application to a great extent. Surface integrity factors, such as grain size, crystallographic orientation and residual stresses, were reported to have significant influence on corrosion resistance of AZ31 Mg alloys. In this study, AZ31B Mg discs were orthogonally turned using cutting tools with two edge radii under both dry and cryogenic conditions. The influence of cutting edge radius and cooling method on surface integrity was investigated. Cryogenic machining using a large edge radius tool led to a thicker grain refinement layer, larger compressive residual stresses and stronger intensity of basal texture, which may remarkably enhance the corrosion performance of magnesium alloys.
- An experimental investigation of residual stresses in hard machining of AISI 52100 steelPublication . Caruso, S.; Umbrello, D.; Outeiro, J. C.; Filice, L.; Micari, F.In this paper an experimental investigation was conducted to determine the effects of the tool cutting-edge geometry, workpiece hardness, cutting speed, and microstructural changes (white and dark layers) on the residual stresses in dry orthogonal hard machining of AISI 52100 steel. X-ray diffraction technique was used to obtain in-depth residual stresses profiles in both axial and circumferential directions. The results show that tool geometry, workpiece hardness and cutting parameters significantly affect the surface residual stress, maximum compressive residual stress below the machined surface and its location. Moreover, microstructural analysis shows that thermally-induced phase transformations have a significant impact on the magnitude and location of this maximum compressive residual stress peak.
- Ulcerogenic helicobacter pylori strains isolated from children: a contribution to get insight into the virulence of the bacteriaPublication . Vitoriano, Inês; Saraiva-Pava, Kathy D.; Rocha-Gonçalves, Alexandra; Santo, Andrea; Lopes, Ana I.; Oleastro, Mónica; Roxo-Rosa, MónicaInfection with Helicobacter pylori is the major cause for the development of peptic ulcer disease (PUD). In children, with no other etiology for the disease, this rare event occurs shortly after infection. In these young patients, habits of smoking, diet, consumption of alcohol and non-steroid anti-inflammatory drugs and stress, in addition to the genetic susceptibility of the patient, represent a minor influence. Accordingly, the virulence of the implicated H. pylori strain should play a crucial role in the development of PUD. Corroborating this, our in vitro infection assays comparing a pool of five H. pylori strains isolated from children with PUD to a pool of five other pediatric clinical isolates associated with non-ulcer dyspepsia (NUD) showed the greater ability of PUD strains to induce a marked decrease in the viability of gastric cells and to cause severe damage in the cells cytoskeleton as well as an impairment in the production/secretion of mucins. To uncover virulence features, we compared the proteome of these two groups of H. pylori strains. Two-dimensional gel electrophoresis followed by mass-spectrometry allowed us to detect 27 differentially expressed proteins between them. In addition to the presence of genes encoding well established virulence factors, namely cagA, vacAs1, oipA "on" status, homB and jhp562 genes, the pediatric ulcerogenic strains shared a proteome profile characterized by changes in the abundance of: motility-associated proteins, accounting for higher motility; antioxidant proteins, which may confer increased resistance to inflammation; and enzymes involved in key steps in the metabolism of glucose, amino acids and urea, which may be advantageous to face fluctuations of nutrients. In conclusion, the enhanced virulence of the pediatric ulcerogenic H. pylori strains may result from a synergy between their natural ability to better adapt to the hostile human stomach and the expression of the established virulence factors.