Identificação de Hotspots no gene MYH

Mestrado em Biologia Molecular e CelularA Polipose Associada ao MYH (MAP) é uma das síndromes de polipose cólica associadas a risco aumentado de cancro colorretal. Descoberta em 2002, a MAP é uma síndrome hereditária com transmissão autossómica recessiva. Causada por mutações bialélicas no gene MYH, a MAP é responsável por cerca de 0,5 - 1% dos carcinomas colorrectais. O gene MYH codifica a enzima MYH glicosilase que faz parte do sistema de reparação de DNA por excisão de bases (BER). O sistema BER desempenha um papel significativo na reparação de mutações causadas por espécies reativas de oxigénio (ROS). Este estudo teve como principal objetivo determinar as frequências alélicas das mutações patogénicas detetadas numa série de 115 amostras consecutivas estudada no GDPN, de modo a delinear um protocolo laboratorial de rastreio de alterações do gene MYH eficaz, rentável e adequado. Através das técnicas de DGGE e sequenciação direta, foram detetados 29 casos com variações de sequência (excluindo polimorfismos conhecidos), em que as mutações missense p.Tyr179Cys e p.Gly396Asp foram responsáveis por 72,2% dos alelos mutados. As outras mutações patogénicas encontradas foram a mutação frameshift c.1227_1228dupGG, já detetada na população portuguesa e a deleção extensa dos exões 4 ao 16 [c.349-?_(1650_?)del], com apenas dois relatos na literatura. A maioria das restantes variantes encontradas, embora classificadas como “provavelmente patogénicas” por programas computacionais preditivos do efeito das mutações na proteína, ainda carecem de estudos funcionais para que possam ser definitivamente “rotuladas” de patogénicas. Tendo em conta a variedade de mutações encontradas ao longo do gene e os casos de heterozigotia composta detetados, a melhor estratégia para o rastreio de alterações do gene MYH neste laboratório, deverá passar pelo estudo inicial dos exões 7 e 13 do gene, seguindo-se o estudo de toda a sequência codificante do gene em todos os casos negativos ou heterozigóticos para uma mutação patogénica. Por outro lado, nos pacientes portadores de mutação em aparente homozigotia ou heterozigotia deverá ser considerada a hipótese de uma análise adicional por MLPA no sentido de detetar/excluir a presença de grandes deleções/duplicações.MYH-associated polyposis (MAP) is one of the colon polyposis syndromes associated with a significant increased CCR risk. Discovered in 2002, MAP is a hereditary syndrome with autosomal recessive transmission. Caused by biallelic mutations in MYH gene, is responsible for about 0.5 – 1% of colorectal carcinomas. The MYH gene encodes the enzyme MYH glycosylase, which is part of the DNA repair by base excision (BER). The BER system plays a significant role in the repair of mutations caused by reactive oxygen species (ROS). This study aimed at determining the allele frequencies of MYH pathogenic mutations in order to devise an efficient, cost effective and suitable protocol for laboratory screening of MYH gene alterations. Using DGGE and direct sequencing 29 positive cases with MYH altered sequence (excluding known polymorphisms) were detected. Missense mutations p.Tyr179Cys and p.Gly396Asp accounted for 72,2% of mutated alleles. The other pathogenic mutations found were the frameshift mutation c.1227_1228dupGG, already detected in the Portuguese population and the extensive deletion of exons 4-16 [c.349-? _ (1650_?)del], with only two reports in the literature. Most of the remaining variants found, although classified as “likely pathogenic” by computational programs predicting the effect of mutations in the protein, still lack functional studies in order to be definitively “labelled” as pathogenic. Given the variety of mutations found throughout this gene and the detected cases of compound heterozygosity, the best strategy for the screening of the MYH gene in this laboratory should include the initial screening of exons 7 and 13, followed by the study of the entire coding sequence of the gene in all cases negative or heterozygous for a pathogenic mutation. Moreover, in patients who appear to be homozygous or heterozygous for a certain mutation further analysis by MLPA should be considered, in order to detect/exclude the presence of large deletions/duplications

Nanoparticles of titanium dioxide modulate the response to temperature by key enzymes involved in pyruvate availability in cytosol and mitochondria of Saccharomyces cerevisiae BY4741, CICTA 2013

Nanotechnology can be used to obtain materials at nanoscale (<100 nm) with new physicochemical and structural properties which depend on particle size and, probably, may trigger new biological effects. As Saccharomyces cerevisiae is an excellent model for study molecular and cell biology responses is growingly used in the toxicological evaluation of chemicals, such as heavy metals or nanoparticles of metal oxide due increasing use of these materials in consumables as cosmetics and textiles. The malate dehydrogenase (EC 1.1.1.37, MDH2) and malic enzyme (EC 1.1.1.38/39, ME1) of S. cerevisiae catalyze the oxidative decarboxylation of L-malate to pyruvate and CO2 coupled to reduction of NAD(P)+ in NAD(P)H of cytoplasm and mitochondria. These enzymes are part of metabolic crossroads that are implicated in regeneration of pyruvate, thereby contributing to the functionality of the citric acid cycle and generation of reducing equivalents as NADPH or NADH, required for de novo fatty acid biosynthesis and antioxidant response or respiratory chain. Hence, the main purpose of this work was to evaluate how nanoparticles of titanium dioxide modulate the effects of temperature on pyruvate availability in S. cerevisiae BY4741, a EUROCAST strain. Yeast (106 cells mL-1) at mid-exponential phase were inoculated in YEPD medium with 2% (w/v) glucose and allowed grown in a water bath, with orbital stirring at 25, 28, 30 or 40ºC, during 200 min in absence or presence of 0.1 or 1.0 µg/mL TiO2-NP. Samples from each treatment, suspended in 10 mM phosphate buffer, pH 7.0 were lysed by sonication and centrifuged at 12,000 g during 20 min, at 4ºC. Aliquots of supernatant and pellet were stored at -20ºC for later use. Protein contents in the cell fractions as well as enzyme activities MDH2, G6PD and ME1 were determined in the post-12,000 g supernatant or pellet by spectrophotometry. ROS and MDA contents were estimated in the post-12,000 g supernatant by fluorimetry. Nanoparticles of titanium dioxide (<100 nm) were purchased from Sigma-Aldrich. Statistical analysis (five independent experiments) included ANOVA I and Duncan test. The results showed that the enzymes MDH2, ME1 and G6PD of S. cerevisiae BY4741 exhibited an optimal of activity at 28ºC. Secondly, it was observed a significant increase in the ROS and MDA levels when temperature range from 25 to 30°C, countered by a significant drop at 40 °C. Thus, the increase of temperature in the range from 25 to 30°C may have blocked the renewal of cytoplasmic and mitochondrial pyruvate, slowing down the carbon flux via citric acid cycle and de novo fatty acids biosynthesis, assisted by G6PD. The decrease of MDH2, ME1 and G6PD activities detected at 40°C may be interpreted as cell death, confirmed by the increase in the MDA levels. The exposure to TiO2-NPs triggered an increase in the MDH2 activity in any realized assays, effect that was more pronounced at 28°C. On the other hand, the ME1 activity which decreased in yeast grown at 25°C and 28°C, exposed to TiO2-NPs, underwent an increase in yeasts grown at 30°C or 40°C. Although the ROS levels have increased with the presence of TiO2-NP in any realized assays, it was only detected an increase of cell damages in cell grown at 25, 28 and 40°C. Thus, it can be inferred that exposure of S. cerevisiae BY4741 to TiO2-NP can counteract the adaptation to temperature of their energy metabolism, reversing the cytoplasmic and mitochondrial pyruvate availability, that in the latter case only occurred at 30 and 40ºC

How does heat-shock affect the influence of titanium dioxide nanoparticles in growth and antioxidant power of Saccharomyces cerevisiae BY4741?

Nanomaterials include all substances that contain nanoscale structures sized between 1 and 100 nm. At this size, the characteristics of materials change: their strength, conductivity, and reactivity, which differ substantially from macro- or micron- sized materials, shifting the rules of physics and chemistry to the sidelines. Although, the geological origin and the ubiquitous occurrence of nanoparticles in the earth crust can lead to suppose a good phylogenetic adaptation of living beings to such substances, the unique charateristics of nanoparticles (NPs) bring a new dimension to environmental effects testing. The industrial development coupled with vast new applications of nanomaterials, have contributed to raise their environmental levels, reason because, concern over the environmental pressure of the nanoparticles in certain regions of the world as well as its effects on the biosphere has grown in recent years, since its reactivity with biomolecules mainly depends on the surface area/molecular size ratio and physicochemical factors such as pH and temperature. Thus, the main objective of this study was to evaluate how heat-shock affects cell survival and antioxidant response of S. cerevisiae BY4741, a Eurocast strain, exposed to titanium dioxide nanoparticles (TiO2-NPs). Cells in exponential phase were inoculated in liquid YEPD medium 2 % (w/v) glucose at 28 °C are exposed at 0.1 or 1.0 μg/mL NP-TiO2 prepared by sonication, during 200 min at 40 °C. Samples from each treatment were used to obtain the post-12000 g fractions, which were used for protein content, DPPH, glutathione antioxidant capacity and, ALP, catalase and LOX activities determinations. The results show that the presence of TiO2-NPs in the culture medium induced cell death, response evidenced by a decrease of proliferative capacity detected by the alkaline phosphatase (ALP, EC 3.1.3.1) activity, loss of redox buffer capacity mediated by glutathione, evidenced by a decrease of GSH+GSSG contents and GSH/GSSG ratio. On the other hand, cell death also appears depend on the loss of ability to scavenge free radicals, estimated by DPPH method. We also observed an increase of lipoxygenase (LOX, EC 1.13.11.12) activity, a marker of lipid peroxidation, which may be related with a loss of antioxidant power mediated by peroxisomal catalase (CAT A, EC 1.11.1.6), probably due a slowdown of β-oxidation. Finally it was observed an increase of the antioxidant cytoplasmic catalase (CAT T, EC 1.11.1.6) in cells exposed to concentrations of 0.1 mg/mL, but a significantly decrease of this enzyme activity in cells exposed to 1 mg/mL TiO2-NPs. This apparently bimodal response indicates a loss of proliferative capacity by an active process when the level exposure was 0.1 mg/mL. However, for 1 mg/mL TiO2-NPs level, appears to occur a transition for necrosis

Estratégias da regeneração urbana através da criação de novos espaços públicos

Dissertação de Mestrado Integrado em Arquitetura, com a especialização em Arquitetura, apresentada na Faculdade de Arquitetura da Universidade de Lisboa para obtenção do grau de Mestre.N/

O ensino do vocabulário, em particular dos falsos amigos, em ELE

Mestrado em Ensino de Português no 3º ciclo do Ensino Básico e no Secundário e de Língua Estrangeira [Espanhol] nos Ensino Básico e SecundárioApós uma breve viagem pelos diferentes métodos de ensino de línguas estrangeiras - em particular de ELE e EL2 - procurando as suas contribuições em matéria de léxico e de vocabulário, propomo-nos a demonstrar o interesse que suscitam os falsos amigos, em duas línguas tão próximas como o Português e o Espanhol. Verificámos que os aprendizes reagiam de forma positiva perante o contraste ou choque que provoca este aspeto linguístico em particular, concluindo que os falsos amigos podem funcionar como recurso de grande motivação para a aprendizagem e aquisição de vocabulário nas aulas de ELE, com alunos do 7º ano do Ensino Básico. Elaborámos um estudo de caso, com características de investigação-ação, no qual se utilizaram instrumentos muito diversificados, inserindo-se todos eles dentro das práticas comunicativas que se exercem atualmente no ensino de Espanhol como língua estrangeira.Tras un breve recorrido por los diferentes métodos de enseñanza de lenguas extranjeras -en particular de ELE y EL2-, recabando sus aportaciones en materia de léxico y vocabulario, nos proponemos demostrar el interés que suscitan los falsos amigos en las dos lenguas próximas portugués y español. Hemos comprobado que los aprendices reaccionaban de manera positiva ante el contraste o choque que provoca este particular aspecto lingüístico, llegando a concluir que los falsos amigos pueden funcionar como recurso de gran motivación para el aprendizaje y adquisición de vocabulario en las aulas de ELE, con alumnos de 7º ano do Ensino Básico. Hemos llevado a cabo un estudio de caso, con características de investigación-acción, en el que se han utilizado instrumentos muy diversos; todos ellos, dentro de las prácticas comunicativas que se ejercitan actualmente en la enseñanza del español como lengua extranjera.After a journey through the different methods of foreign language teaching – particularly Spanish Foreign Language and Spanish Second Language – searching the contributions for teaching lexicon and vocabulary, we have tried to demonstrate the importance that false friends assume in two languages as close as Portuguese and Spanish. We have found that learners reacted positively to the contrast or shock caused by this particular aspect of linguistic, coming to the conclusion that false friends can serve as a resource of great motivation for vocabulary acquisition in the Spanish Foreign Language classroom, with students of 7º ano do Ensino Básico . Finished the book revision, a case study with characteristics of research-action was built, having been used in the classroom diversified instruments inherent to the communicative approach that currently applies in the teaching of Spanish as a foreign language

Sustainability of treatment technologies for industrial biowastes effluents

Despite the huge efforts to develop efficient technologies for the treatment of recalcitrant biowastes and other emerging pollutants, selecting the most sustainable method among the possible alternatives is still a formidable task. This is mainly because of the integration of technical, economic, environmental, and social criteria in decision-making process. Traditionally, various multi-criteria decision-making approaches have been adopted to integrate innumerable criteria for environmental applications. In this study, we have examined the fuzzy-Delphi approach to evaluate seventeen parameters for integrating technical, economic, environmental and social criteria in order to rank the nine treatment technologies divided in two categories (physico-chemical and biological processes). The results of this study indicated that although efficiency of treatment methods is the most important criterion, but contribution of other sustainability criteria should also be considered because they are of high importance for the selection of sustainable wastewater treatment methods. As per our proposed framework on membrane technologies (among the many other physico-chemical methods) and anaerobic sludge blanket technology (among the biological treatment methods) are the most promising approaches for the treatment of highly polluted emerging industrial pollutants. The findings of this study are fully supported by the consensus achieved by a group of fifty experts from nineteen different countries. Opportunities for the improvement of the methods as per data generated are discussed.publishe

Green synthesis of nanomaterials - a scientometric assessment

The green synthesis of engineered nanomaterials (NMs) has deserved an enormous academic interest and huge financial investments during the last decades. However, this prominent position has not been followed by the rapid commercialization of NMs for real applications thus rendering their practical usefulness very doubtful and the appropriateness of novel investments in the field highly questionable. The present manuscript presents the first scientometric study on the green synthesis of NMs aiming to survey the scientific progress in this particular field and identify its main gaps while providing applicable suggestions to facilitate the knowledge transfer from laboratories to real full scale production and applications. The research on green synthesis of nanomaterials published in Web of Science during the period 1991–2019 is here carefully analyzed. Overall, 9 scientometric indicators are employed to interpret the results retrieved from the 8761 documents collected. It is found that 107 countries and nearly 22,400 authors have contributed to this subject, hence highlighting the relevance of this topic. The keywords spectrum is dominated by the term “nanoparticle” which full adoption takes place at the beginning of the 21st century. Some few years later, a batch of words like “silver nanoparticle”, “gold nanoparticle” and “nanocomposite” reaches a significant impact reflecting the emergence of commercial applications for these nanomaterials. It is only in 2009 that the keyword “green synthesis” gains strength, followed then by “biosynthesis” in 2010, making it evident a trend towards environmentally friendly reagents. The number of publications on green synthesis of nanomaterials displays up to now a sigmoidal like growth pattern, which points actually to a decrease on new arrivals, thus suggesting a possible forthcoming decline in this field. However, the analysis carried out in the present work allows identifying various gaps related to sustainability, which, if appropriately addressed, may contribute to a resurgence of the research on nanomaterials synthesis while fostering more frugal approaches on material synthesis tendencies.publishe

Infecções do tracto urinário no Litoral Alentejano : etiologia e susceptibilidade aos antimicrobianos

Tese de mestrado, Microbiologia Clínica, Faculdade de Medicina, Universidade de Lisboa, 2010RESUMO A infecção do tracto urinário (ITU) é considerada a segunda infecção mais comum na comunidade e a mais frequente entre as infecções associadas aos cuidados de saúde. A terapêutica empírica é a abordagem escolhida na maioria das situações, e, portanto, é importante o conhecimento da etiologia e perfis de susceptibilidade, de forma a instituir-se uma política de antimicrobianos a utilizar para uma dada região geográfica. O tratamento antimicrobiano inadequado destas infecções, devido à instituição de um protocolo terapêutico inadequado à população de uma dada região geográfica e/ou pela falta de dados relativamente a essa mesma região poderá levar ao aparecimento e disseminação de resistências, dificultando o seu tratamento e levando a um aumento significativo nas taxas de mortalidade, morbilidade, custos de tratamento e período de internamento. Com este trabalho pretendeu-se desenvolver um estudo que permitisse conhecer os agentes patogénicos implicados na etiologia das ITU numa área geográfica abrangida por uma unidade hospitalar recente (Hospital do Litoral Alentejano), procedendo a um levantamento e análise de dados etiológicos e de susceptibilidade aos antimicrobianos. Das 594 amostras processadas, 232 foram consideradas positivas. Destas, 115 foram classificadas como ITU associadas aos cuidados de saúde (ITUACS) e 117 como ITU da comunidade (ITUC). Ambas as populações são caracterizadas por uma idade superior a 50 anos, tendo a população entre 0 e 19 anos constituído um grupo à parte. Para todos os grupos de amostras, o agente etiológico mais frequentemente isolado foi E. coli. No entanto, para as ITUACS, outros agentes para além de E. coli foram mais frequentes e, para este grupo e para ITUC, predominaram no sexo masculino. Os valores de taxas de resistência aos antimicrobianos mais utilizados no tratamento deste tipo de infecções (nomeadamente fluoroquinolonas, betalactâmicos e trimetoprim-sulfametoxazol), são preocupantes e contrariam os indicados por outros estudos, urgindo a sua correcção e vigilância.ABSTRACT Considered the second most common infection in the community the urinary tract infection (UTI) is also the most frequent infection among those associated to health care. Empirical therapy is the approach chosen for the majority of the cases, thus it is important to know the aetiology and susceptibility profiles to implement an antimicrobial policy to a given geographical region. An inadequate antimicrobial treatment of these infections can lead to the emergence and spread of resistances, impeding the treatment and leading to a significant raise of the death rate, morbidity, treatment costs and hospitalization period as a result of the employment of an inadequate therapeutic protocol to the inhabitants of a given geographical region and/or due to the lack of data as regards of that same region. The scope of this work was to develop a study that would provide knowledge of the pathological agents involved in the UTI’s aetiology in a geographical area covered by a recent hospital unit (Hospital do Litoral Alentejano) assessing the aetiological and susceptibility data regarding the antimicrobial profile that would allow the comparison of the obtained results concerning the protocols recommended in the antimicrobial therapy of UTI’s. 232 of 594 samples processed were positive in which 115 were considered to be UTI’s associated to health care procedures (UTIAHCP) and 117 were considered to be community acquired UTI’s (CUTI). Both groups are characterised as being above 50 years old. The age group of 0-19 is set apart. For all the sample groups, E. coli was the most frequently isolated agent. However, for UTIAHCP, non-E. coli agents were more frequent and for this group and CUTI they predominate in the male gender. Resistance rate numbers to the most used antimicrobials in the treatment of UTI’s, amely fluoroquinolones, beta-lactams and, trimetoprimsulfamethoxazole, are already concerning and oppose those indicated in other studies, requiring close watch and urgency in its correction

Sustainability considerations in membrane-based technologies for industrial effluents treatment

Treatment of industrial effluents (EFs) from the polluted wastewater sources using membrane technologies is an effective and attractive alternative to overcome the weaknesses of some of the conventional wastewater treatment processes, especially when dealing with EFs loaded with recalcitrant organic pollutants and toxic substances. The application of various polymeric and inorganic membrane based technologies to be used for the treatment of industrial EFs has attracted a considerable attention in the past decades. In this regard, a critical discussion on the sustainability of various aspects of membrane technologies would promote the commercialization of these technologies. In this review, various sustainability criteria in technical, economic, environmental, and social categories have been considered for a critical discussion on the current status and improvement opportunities of membrane technologies for the treatment of industrial EFs. While the application of polymeric membranes has been restricted by some bottlenecks to deal with some industrial effluents, metal oxides fabricated ceramic membranes, and especially those fabricated with nanostructured materials such as nano-zeolites, those made of metal organic frameworks as well as carbon-based fabricated membranes have shown a promising performance in the rejection of recalcitrant organic pollutants. In addition, the combinations of inorganic membrane technologies with other novel methods such as advanced oxidation processes (e.g., using engineered nanomaterials) can be considered among the best options to deal with such highly polluted effluents.publishe