Produção e caracterização de quitinases ácidas de macieira em Pichia Pastoris

Tese de mestrado em Bioquímica (Bioquímica Médica), apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2011Este trabalho visou a clonagem e expressão de genes codificadores de quitinases ácidos de classe III de macieira no sistema heterólogo Pichia pastoris, nas estirpes GS115 e KM71H. Prepararam-se três construções com três genes já anteriormente identificados (C525, C209 e C203) e o sinal de secreção α-factor de S. cerevisiae, e duas construções com os genes C525 e C209 com o sinal de secreção nativo. Estas construções foram clonadas no vector pPICZαA e introduzidas na levedura. Obtiveram-se colónias positivas para todas as construções, excepto para a C209 com o sinal de secreção nativo, sugerindo uma análise de Southern a presença de apenas uma cópia da construção em cada recombinante. Após indução da expressão com metanol, mediu-se, em vários momentos, o crescimento e o teor total de proteína das culturas, bem como a actividade quitinolítica do secretoma das colónias recombinantes, usando glicolquitina como substrato. Seleccionaram-se as construções que apresentaram, pelo menos numa estirpe, actividade superior à dos controlos negativos correspondentes (estirpes WT e WT+pPICZαA), e os momentos que permitiram maior actividade enzimática para cada construção. Os melhores resultados foram obtidos para as isoformas C525 com o sinal de secreção nativo e C209 com o sinal de secreção α-factor, clonadas na estirpe GS115 (1,100U/mg de proteína às 24h e 0,233U/mg de proteína às 120h, respectivamente), e a isoforma C525 com o α-factor, clonada na estirpe KM71H (0,366U/mg de proteína às 139h). Destas três, a primeira foi a que expressou melhor e mais rapidamente a proteína. A análise de SDS-PAGE dos precipitados e dos secretomas respeitantes aos diferentes momentos não se revelou conclusiva, provavelmente devido à baixa concentração proteica das amostras. Propõe-se a produção em larga escala e purificação destas três proteínas recombinantes para caracterizar a sua actividade quitinolítica, para depois averiguar o seu potencial biofungicida contra fitopatogénios de macieira e pereira.This work focused on cloning and expressing three different genes encoding class III acidic chitinases from apple in Pichia pastoris heterologous expression system, using GS115 and KM71H strains. Three constructs were prepared with the genes C525, C209 e C203 and the α-factor secretion signal from S. cerevisiae, and two constructs with the genes C525 and C209 and the native secretion signal. The constructions were cloned in pPICZαA vector and introduced in the yeast. We obtained positive clones for all constructs, except for construct C209 with the native secretion signal, and a Southern analysis suggested the presence of only one copy of the construct in each recombinant. After inducing the expression with methanol, cultures‟ growth and total protein amount, at different moments, were evaluated, and the chitinolytic activity of the cell-free‟s medium was determined, using glycolchitin as substrate. The constructs that showed, at least in one strain, higher activity than the corresponding negative controls (WT strain and WT+pPICZαA) were chosen, as well as the moments with better enzymatic activity, for each construct. The best results derived from C525 gene with the native secretion signal and C209 gene with the α-factor signal, cloned in the GS115 strain (1,100U/mg protein at 24h and 0,233U/mg protein at 120h, respectively), and C525 gene with the α-factor signal cloned in the KM71H strain (0,366U/mg protein at 139h). Among these three, the first is the one that expressed the protein better and faster. A SDS-PAGE analysis of the pellets and supernatants from the different moments didn‟t show the expected results, probably due to the samples‟ low protein concentration. We propose to produce in large scale and purify these three recombinant proteins to characterize their chitinolytic activity and further analyze their potential as natural fungicide against phytopathogens that strongly affect apple and pear

artigo 154º-A do Código Penal

On 5th September 2015, the 38th amendment to the Criminal Code was introduced by Law n. 83/2015, of August 5th. In line with the provisions of the Istanbul Convention, this amendment added three new crimes to the Criminal Code, including Stalking. Stalking is a form of harassment that corresponds to a set of repeated behaviors by one individual towards another, through various ways of nonconsensual or unwanted contact, harmful to his sphere of privacy, which may cause psycho-emotional and/or physical damages and is restrictive of his freedom of action and decision. With the consecration of this legal type, dignity and legal-criminal valuation were attributed to the social type of Stalking. Having this been a reality dealt with closely during an internship at APAV, Stalking and its recent criminalization are matters of undeniable interest and actuality. Aware of it, this Internship Report, with valuable support on real cases, aims to prove the necessity of the crime of Stalking, to characterize the legal type and to evaluate its effectiveness regarding the victims’ protection. After a brief introduction to APAV and the work developed, attempts to demonstrate the need of the crime of Stalking. As a recent crime, its characterization is essential to understand its scope. Considering the legal type, questions may arise related with the principle of legality, the requirement of reiteration of conduct, the possibility of homogeneous competition and the clause of subsidiarity expressed in Article 154-A (1) of the Criminal Code. Regarding the effectiveness of Article 154-A of the Criminal Code, the configuration of the Portuguese legal system doesn’t allow the responsible entities to apply adequate measures and to respond quickly to all forms of violence, not offering adequate and immediate protection to the victims of the crime of Stalking if non-aggravated. This conclusion is possible by assessing which coercive and protective measures and the accessory penalties that can be applied to this crime. Furthermore, the victim may adopt security measures and it is important to know what to do if the agent cannot be held responsible due to psychic anomaly, since these are frequent situations when this crime occurs. This Report aims to not only inspire the investigation and development of studies on the new crime of Stalking, but also alert the community to the need to protect all victims of crime.A 5 de setembro de 2015 entrou em vigor a 38ª alteração ao CP, introduzida pela Lei n.º 83/2015, de 5 de agosto. Cumprindo o disposto na Convenção de Istambul, esta alteração aditou três novos crimes ao Código Penal, entre os quais o de perseguição. A perseguição é uma forma de assédio correspondente a um conjunto de condutas reiteradas de um indivíduo contra outro, através de diversos modos de contacto não consentido ou indesejado, lesivo da sua esfera de privacidade, que pode causar danos psicoemocionais e/ou físicos e que é ainda restritivo da sua liberdade de ação e decisão. Atribui-se, com a consagração deste tipo legal, dignidade e valoração jurídico-penal ao tipo social do Stalking. Sendo uma realidade com a qual se contactou de perto durante a realização de estágio na APAV, o Stalking e a sua recente criminalização são assuntos de inegável interesse e atualidade. Ciente disso, este Relatório de Estágio, com valioso suporte em casos reais, tem como objetivos comprovar a necessidade do crime de perseguição, caracterizar o tipo legal e avaliar a sua eficácia no que respeita à proteção das vítimas. Após uma breve introdução à APAV e ao trabalho desenvolvido, procura demonstrar-se a necessidade do crime de perseguição. Sendo um crime recente, a sua caracterização é essencial para compreender o seu alcance. Atendendo ao tipo legal, surgem questões relacionadas com o princípio da legalidade, a exigência de reiteração de condutas, a possibilidade ou não de haver concurso homogéneo e com a cláusula de subsidiariedade expressa no artigo 154º-A n.º1 do Código Penal. Quanto à eficácia do artigo 154º-A do Código Penal, a configuração do tipo no ordenamento jurídico português não permite às entidades responsáveis aplicar medidas adequadas e de resposta rápida a todas as formas de violência, não oferecendo proteção adequada e imediata às vítimas do crime de perseguição não agravado. Tal conclusão é possível avaliando-se quais as medidas de coação e proteção e as penas acessórias que que podem ser aplicadas a este crime. Para além disso, a vítima pode adotar medidas de proteção e importa saber o que fazer se o agente for inimputável por anomalia psíquica, uma vez que são situações frequentes aquando da prática deste crime. Este Relatório ambiciona não só inspirar a investigação e o desenvolvimento de estudos sobre o novo crime de perseguição, mas também alertar a comunidade para a necessidade de proteger todas as vítimas de crime

TECNOLOGIA E INOVAÇÃO AO SERVIÇO DO EXERCÍCIO E SAÚDE. Exercício. Pandemia COVID-19. Tecnologia Vs. Isolamento Social

info:eu-repo/semantics/publishedVersio

NEOTROPICAL ALIEN MAMMALS: a data set of occurrence and abundance of alien mammals in the Neotropics

Biological invasion is one of the main threats to native biodiversity. For a species to become invasive, it must be voluntarily or involuntarily introduced by humans into a nonnative habitat. Mammals were among first taxa to be introduced worldwide for game, meat, and labor, yet the number of species introduced in the Neotropics remains unknown. In this data set, we make available occurrence and abundance data on mammal species that (1) transposed a geographical barrier and (2) were voluntarily or involuntarily introduced by humans into the Neotropics. Our data set is composed of 73,738 historical and current georeferenced records on alien mammal species of which around 96% correspond to occurrence data on 77 species belonging to eight orders and 26 families. Data cover 26 continental countries in the Neotropics, ranging from Mexico and its frontier regions (southern Florida and coastal-central Florida in the southeast United States) to Argentina, Paraguay, Chile, and Uruguay, and the 13 countries of Caribbean islands. Our data set also includes neotropical species (e.g., Callithrix sp., Myocastor coypus, Nasua nasua) considered alien in particular areas of Neotropics. The most numerous species in terms of records are from Bos sp. (n = 37,782), Sus scrofa (n = 6,730), and Canis familiaris (n = 10,084); 17 species were represented by only one record (e.g., Syncerus caffer, Cervus timorensis, Cervus unicolor, Canis latrans). Primates have the highest number of species in the data set (n = 20 species), partly because of uncertainties regarding taxonomic identification of the genera Callithrix, which includes the species Callithrix aurita, Callithrix flaviceps, Callithrix geoffroyi, Callithrix jacchus, Callithrix kuhlii, Callithrix penicillata, and their hybrids. This unique data set will be a valuable source of information on invasion risk assessments, biodiversity redistribution and conservation-related research. There are no copyright restrictions. Please cite this data paper when using the data in publications. We also request that researchers and teachers inform us on how they are using the data

Health-status outcomes with invasive or conservative care in coronary disease

BACKGROUND In the ISCHEMIA trial, an invasive strategy with angiographic assessment and revascularization did not reduce clinical events among patients with stable ischemic heart disease and moderate or severe ischemia. A secondary objective of the trial was to assess angina-related health status among these patients. METHODS We assessed angina-related symptoms, function, and quality of life with the Seattle Angina Questionnaire (SAQ) at randomization, at months 1.5, 3, and 6, and every 6 months thereafter in participants who had been randomly assigned to an invasive treatment strategy (2295 participants) or a conservative strategy (2322). Mixed-effects cumulative probability models within a Bayesian framework were used to estimate differences between the treatment groups. The primary outcome of this health-status analysis was the SAQ summary score (scores range from 0 to 100, with higher scores indicating better health status). All analyses were performed in the overall population and according to baseline angina frequency. RESULTS At baseline, 35% of patients reported having no angina in the previous month. SAQ summary scores increased in both treatment groups, with increases at 3, 12, and 36 months that were 4.1 points (95% credible interval, 3.2 to 5.0), 4.2 points (95% credible interval, 3.3 to 5.1), and 2.9 points (95% credible interval, 2.2 to 3.7) higher with the invasive strategy than with the conservative strategy. Differences were larger among participants who had more frequent angina at baseline (8.5 vs. 0.1 points at 3 months and 5.3 vs. 1.2 points at 36 months among participants with daily or weekly angina as compared with no angina). CONCLUSIONS In the overall trial population with moderate or severe ischemia, which included 35% of participants without angina at baseline, patients randomly assigned to the invasive strategy had greater improvement in angina-related health status than those assigned to the conservative strategy. The modest mean differences favoring the invasive strategy in the overall group reflected minimal differences among asymptomatic patients and larger differences among patients who had had angina at baseline

Initial invasive or conservative strategy for stable coronary disease

BACKGROUND Among patients with stable coronary disease and moderate or severe ischemia, whether clinical outcomes are better in those who receive an invasive intervention plus medical therapy than in those who receive medical therapy alone is uncertain. METHODS We randomly assigned 5179 patients with moderate or severe ischemia to an initial invasive strategy (angiography and revascularization when feasible) and medical therapy or to an initial conservative strategy of medical therapy alone and angiography if medical therapy failed. The primary outcome was a composite of death from cardiovascular causes, myocardial infarction, or hospitalization for unstable angina, heart failure, or resuscitated cardiac arrest. A key secondary outcome was death from cardiovascular causes or myocardial infarction. RESULTS Over a median of 3.2 years, 318 primary outcome events occurred in the invasive-strategy group and 352 occurred in the conservative-strategy group. At 6 months, the cumulative event rate was 5.3% in the invasive-strategy group and 3.4% in the conservative-strategy group (difference, 1.9 percentage points; 95% confidence interval [CI], 0.8 to 3.0); at 5 years, the cumulative event rate was 16.4% and 18.2%, respectively (difference, 121.8 percentage points; 95% CI, 124.7 to 1.0). Results were similar with respect to the key secondary outcome. The incidence of the primary outcome was sensitive to the definition of myocardial infarction; a secondary analysis yielded more procedural myocardial infarctions of uncertain clinical importance. There were 145 deaths in the invasive-strategy group and 144 deaths in the conservative-strategy group (hazard ratio, 1.05; 95% CI, 0.83 to 1.32). CONCLUSIONS Among patients with stable coronary disease and moderate or severe ischemia, we did not find evidence that an initial invasive strategy, as compared with an initial conservative strategy, reduced the risk of ischemic cardiovascular events or death from any cause over a median of 3.2 years. The trial findings were sensitive to the definition of myocardial infarction that was used