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The present study aimed to test, in vitro, the antimicrobial activity against Candida albicans and Streptococcus mutans and the surface roughness of a 3D-printed polymethylmethacrylate dental resin enhanced with graphene. A 3D-printed polymethylmethacrylate dental resin was reinforced with four different concentrations of graphene: 0.01, 0.1, 0.25 and 0.5 wt%. Neat resin was used as a control. The specimens were printed in a liquid crystal display printer. Disc specimens were used in antimicrobial evaluation, and bar-shaped specimens were used to measure surface roughness. The study of antimicrobial activity included the inhibition of the growth of C. albicans and S. mutans and their adhesion to the resin’s surface. Surface roughness increased with the increase in the graphene concentration. The growth inhibition of C. albicans was observed in the different concentrations of graphene after 24 h, with no recovery after 48 h. The specimens doped with graphene were capable of inactivating S. mutans after 48 h. The surface-adhesion studies showed that the density of microbial biofilms decreases in the case of specimens doped with graphene. Graphene, despite increasing the resin’s surface roughness, was effective in inhibiting the growth and the adhesion to the resin’s surface of the main inducers of prosthetic stomatitis.

Corroles possess key photophysical and photochemical properties to be exploited as thera-peutic agents in antimicrobial photodynamic therapy (aPDT). Herein, we present for the first time the antimicrobial efficiency of three corrole dimers and of the corresponding precursor against the Gram(+) bacterium Staphylococcus aureus. Additionally, to explore future clinical applications, the cytotoxicity of the most promising derivatives towards Vero cells was evaluated. The aPDT assays performed under white light irradiation (50 mW/cm2; light dose 450 J/cm2) and at a corrole concen-tration of 15 µM showed that some dimers were able to reduce 99.9999% of S. aureus strain (decrease of 5 log10 CFU/mL) and their photodynamic efficiency was dependent on position, type of linkage, and aggregation behavior. Under the same light conditions, the corrole precursor 1 demonstrated notable photodynamic efficiency, achieving total photoinactivation (>8.0 log10 CFU/mL reduction) after the same period of irradiation (light dose 450 J/cm2). No cytotoxicity was observed when Vero cells were exposed to corrole 1 and dimer 3 for 24 h according to ISO guidelines (ISO 10993-5) for in vitro cytotoxicity of medical devices. The results show that corrole dimers, dependent on their structures, can be considered good photosensitizers to kill Staphylococcus aureus.

Botryosphaeriaceae fungi are phytopathogens and human opportunists. The influence of temperature on the phytotoxicity and cytotoxicity of culture filtrates of five Botryosphaeriaceae species was investigated. All culture filtrates of fungi grown at 25 °C were phytotoxic: symptoms were evaluated based on visual inspection of necrosis areas and on the maximum quantum yield of photosystem II, Fv/Fm. Diplodia corticola and Neofusicoccum kwambonambiense were the most phytotoxic, followed by Neofusicoccum parvum CAA704 and Botryosphaeria dothidea. Phytotoxicity dramatically decreased when strains were grown at 37 °C, except for B. dothidea. All strains, except N. parvum CAA366 and Neofusicoccum eucalyptorum, grown either at 25 °C or 37 °C, were toxic to mammalian cells; at 25 °C and at 37°C, D. corticola and B. dothidea were the most cytotoxic, respectively. Although the toxicity of B. dothidea to both cell lines and of N. kwambonambiense to Vero cells increased with temperature, the opposite was found for the other species tested. Our results suggest that temperature modulates the expression of toxic compounds that, in a scenario of a global increase of temperature, may contribute to new plant infections but also human infections, especially in the case of B. dothidea.

Dissertação de mestrado em Direito das Crianças, Família e Sucessões, O objetivo da presente dissertação é a realização de um estudo crítico sobre o crime de violação da obrigação de alimentos, previsto no artigo 250.º do Código Penal. Procedemos à caracterização da obrigação de alimentos enquanto prestação pecuniária atribuída pelo progenitor que não convive habitualmente com o menor, com o objetivo de satisfazer todas as necessidades fundamentais deste. Acontece que, infelizmente, é comum os obrigados a prestar alimentos incumprirem essa mesma obrigação. Por este motivo, cumpre referir que este incumprimento se encontra perfeitamente tutelado pelo direito civil e é este que resolve a maior parte dos litígios. O legislador, no entanto, considerou ser, ainda, necessária a tutela penal. Como tal, analisamos, em concreto, o crime previsto no artigo 250.º do Código Penal, tendo como finalidade responder às principais questões relativas a este crime, isto é, tentamos compreender se esta tutela é suficiente, eficaz e adequada, desde logo, para satisfazer as necessidades do menor. Concluímos que este artigo deverá ser alvo de uma revisão e, assim sendo, propusemos os termos em que essa mesma revisão deve acontecer e quais os pontos que podem e devem ser revistos., The purpose of this dissertation is to conduct a critical study of the crime of violation of the maintenance obligation, provided in article 250.º of the Penal Code. We proceed with the characterization of the maintenance obligation as a cash benefit attributed by the parent who does not usually live with the child, in order to satisfy all the fundamental needs of them. It turns out that, unfortunately, it is common for those obliged to provide to breach that same obligation. For this reason, it should be noted that this non-compliance is perfectly protected by civil law and it is this, that resolves most of the disputes. However, the legislator considered that criminal protection is still necessary. As such, we analyze, in particular, the crime provided for in article 250.º of the Penal Code, with the purpose of answering the main questions related to this crime, in other words, we try to understand if this protection is sufficient, effective and adequate to satisfy the needs of the minor. We concluded that this article should be subject to a review and, therefore, we proposed the terms under which this review should take place and what points can and should be reviewed.

There is an urgent need for new substances to overcome current challenges in the health sciences. Marine fungi are known producers of numerous compounds, but the manipulation of growth conditions for optimal compound production can be laborious and time-consuming. In Portugal, despite its very long coastline, there are only a few studies on marine fungi. From a collection of Portuguese marine fungi, we screened for antimicrobial, antioxidant, enzymatic, and cytotoxic activities. Mycelia aqueous extracts, obtained by high pressure-assisted extraction, and methanolic extracts of culture media showed high antioxidant, antimicrobial, and cytotoxic activities. The mycelium extracts of Cladosporium rubrum showed higher antioxidant potential compared to extracts from other fungi. Mycelia and culture media extracts of Aspergillus affinis and Penicillium lusitanum inhibited the growth of Staphylococcus aureus, Kocuria rhizophila, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa, including multiresistant strains. Penicillium lusitanum and Trichoderma aestuarinum inhibited the growth of clinical strains of Candida albicans, C. glabrata, C. parapsilosis, and C. tropicalis. All extracts from culture media were cytotoxic to Vero cells. Sea salt induced alterations in the mycelium’s chemical composition, leading to different activity profiles.

Atualmente, o cenário de competição empresarial, num mundo globalizado, trouxe a necessidade de procurar uma maior adaptação às constantes mudanças. Exemplo disso, foi a pandemia que se abateu sobre o todo o mundo nos últimos dois anos. O COVID19 confrontou o mundo com desafios sem precedentes. O modo de vida do ser humano teve de mudar drasticamente, e a sociedade em geral teve de responder com prontidão para se adaptar à situação. Apenas alguns serviços são considerados essenciais para garantir o funcionamento eficaz de infraestruturas críticas. Os elevadores e as escadas rolantes são alguns desses serviços, seja em hospitais, edifícios públicos, ou em outras instituições, o transporte vertical continua a desempenhar um papel vital na nossa vida. Neste sentido, é fundamental que os gestores disponham de ferramentas que auxiliem a tomada de decisão, e a visão global de indicadores financeiros e não financeiros do seu negócio, e que garantam o alinhamento da estratégia definida. O presente trabalho, propõe um exemplo da metodologia Balanced Scorecard, para uma empresa de Ascensores. Esta proposta irá auxiliar a alcançar os objetivos estratégicos da sucursal. Desta forma, após uma breve revisão da literatura, foram definidos os objetivos estratégicos, os indicadores, as iniciativas e as metas para as quatro perspetivas (financeira, clientes, processos internos, aprendizagem e crescimento). Foi concebido de igual forma, um mapa estratégico que considera as relações de causaefeito entre os objetivos. Por último é feita uma conclusão, consolidando toda a informação produzida ao longo do projeto.

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The effect of &gamma, aminobutyric acid (GABA) on the metabolome of two strains of Lasiodiplodia theobromae isolated from grapevine that hold a different degree of virulence to the host plant (LA-SOL3 (more virulent), LA-SV1 (less virulent)) was investigated. The culture filtrates and crude extracts from the two strains grown in the presence and absence of 10 mM of GABA were tested for phytotoxicity on tomato plant cuttings and leaves, respectively. Considering the opportunistic nature of this fungus for humans, crude extracts were also tested for cytotoxicity on mammalian cell lines. We found that culture filtrates and crude extracts have a decreased toxicity in the presence of GABA. Metabolomic analysis, conducted on both strains at both growth conditions, revealed the production of several compounds, such as indole-3-carboxylic acid (ICA, which is the main compound produced by L. theobromae), 3-indolecarboxyaldehyde, (3R,4S)-botryodiplodin, (R)-mellein. Finally, data demonstrate that GABA both induces a decrease in the amount of ICA, and a diversification of the metabolites produced by L. theobromae.

The interactome - the network of protein-protein interactions (PPIs) within a cell or organism - is technically difficult to assess. Bioinformatic tools can, not only, identify potential PPIs that can be later experimentally validated, but also be used to assign functional meaning to PPIs. Saliva's potential as a non-invasive diagnostic fluid is currently being explored by several research groups. But, in order to fully attain its potential, it is necessary to achieve the full characterization of the mechanisms that take place within this ecosystem. The onset of omics technologies, and specifically of proteomics, delivered a huge set of data that is largely underexplored. Quantitative information relative to proteins within a given context (for example a given disease) can be used by computational algorithms to generate information regarding PPIs. These PPIs can be further analyzed concerning their functional meaning and used to identify potential biomarkers, therapeutic targets, defense and pathogenicity mechanisms. We describe a computational pipeline that can be used to identify and analyze PPIs between human and microbial proteins. The pipeline was tested within the scenario of human PPIs of systemic (Zika Virus infection) and of oral conditions (Periodontal disease) and also in the context of microbial interactions (Candida-Streptococcus) and showed to successfully predict functionally relevant PPIs. The pipeline can be applied to different scientific areas, such as pharmacological research, since a functional meaningful PPI network can provide insights on potential drug targets, and even new uses for existing drugs on the market.

Neofusicoccum parvum is a fungal pathogen associated with a wide range of plant hosts. Despite being widely studied, the molecular mechanism of infection of N. parvum is still far from being understood. Analysis of N. parvum genome lead to the identification of six putative genes encoding necrosis and ethylene-inducing proteins (NLPs). The sequence of NLPs genes (NprvNep 1-6) were analyzed and four of the six NLP genes were successfully cloned, expressed in E. coli and purified by affinity chromatography. Pure recombinant proteins were characterized according to their phytotoxic and cytotoxic effects to tomato leaves and to mammalian Vero cells, respectively. These assays revealed that all NprvNeps tested are cytotoxic to Vero cells and also induce cell death in tomato leaves. NprvNep2 was the most toxic to Vero cells, followed by NprvNep1 and 3. NprvNep4 induced weaker, but, nevertheless, still significant toxic effects to Vero cells. A similar trend of toxicity was observed in tomato leaves: the most toxic was NprvNep 2 and the least toxic NprvNep 4. This study describes for the first time an overview of the NLP gene family of N. parvum and provides additional insights into its pathogenicity mechanism.

In the course of investigations on the role of secondary metabolites in plant-microbe interactions, the production of secondary metabolites by Macrophomina phaseolina isolates from Eucalyptus globulus, was studied. This fungus is responsible for several plant diseases which affect crop productivity and industry. Although secondary metabolites may play a role in disease development, there are very few reports on M. phaseolina metabolomics and, as far as we know, isolates from eucalypts have not been investigated for secondary metabolites production. In the present paper, metabolites typical of fungi, from the family Botryosphaeriaceae, were identified for the first time as products of M. phaseolina. Furthermore, the isolate under examination was grown in the presence and absence of host stem tissue, and metabolite profiles were compared. Five products are reported for the first time in this species and azelaic acid was exclusively produced in the presence of eucalypt stem. Finally, phytotoxicity and cytotoxicity tests of culture filtrates and crude organic extracts were also performed. Key Contribution: Lipophilic metabolites produced by M. phaseolina might play a role in the plant-fungus interactions responsible for serious diseases of E. globulus.

The interactome – the network of protein-protein interactions (PPIs) within a cell or organism – is technically difficult to assess. Bioinformatic tools can, not only, identify potential PPIs that can be later experimentally validated, but also be used to assign functional meaning to PPIs. Saliva's potential as a non-invasive diagnostic fluid is currently being explored by several research groups. But, in order to fully attain its potential, it is necessary to achieve the full characterization of the mechanisms that take place within this ecosystem. The onset of omics technologies, and specifically of proteomics, delivered a huge set of data that is largely underexplored. Quantitative information relative to proteins within a given context (for example a given disease) can be used by computational algorithms to generate information regarding PPIs. These PPIs can be further analyzed concerning their functional meaning and used to identify potential biomarkers, therapeutic targets, defense and pathogenicity mechanisms. We describe a computational pipeline that can be used to identify and analyze PPIs between human and microbial proteins. The pipeline was tested within the scenario of human PPIs of systemic (Zika Virus infection) and of oral conditions (Periodontal disease) and also in the context of microbial interactions (Candida-Streptococcus) and showed to successfully predict functionally relevant PPIs. The pipeline can be applied to different scientific areas, such as pharmacological research, since a functional meaningful PPI network can provide insights on potential drug targets, and even new uses for existing drugs on the market.

Lasiodiplodia theobromae is a phytopathogenic fungus that causes diseases not only in a broad number of plant hosts but also occasionally in humans. The capacity of L. theobromae to produce bioactive metabolites at 25 C (environmental mean temperature) and at 37 C (body mean temperature) was investigated. Two strains, CAA019 and CBS339.90, isolated respectively from a coconut tree and a human patient were characterized. The phytotoxicity and cytotoxicity (on mammalian cells) of the secretomes of both strains of L. theobromae were investigated. Also, phytotoxicity and cytotoxicity of pure compounds were evaluated. The phytotoxicity of the secretome of strain CAA019 was higher than the phytotoxicity of the secretome of strain CBS339.90 at 25 C. However, the phytotoxicity for both strains decreased when they were grown at 37 C. Only the secretome of strain CBS339.90 grown at 37 C induced up to 90% Vero and 3T3 cell mortality. This supports the presence of different metabolites in the secretome of strains CAA019 and CBS339.90. Metabolites typical of L. theobromae were isolated and identified from organic extracts of the secretome of both strains (e.g., 3-indolecarboxylic acid, jasmonic acid, lasiodiplodin, four substituted 2-dihydrofuranones, two melleins, and cyclo-(Trp-Ala)). Also, metabolites such as scytalone, not previously reported for this species, were isolated and identified. Metabolite production is affected by strain and temperature. In fact, some metabolites are strain specific (e.g., lasiodiplodin) and some metabolites are temperature specific (e.g., jasmonic acid). Although more strains should be characterized, it may be anticipated that temperature tuning of secondary-metabolite production emerges as a putative contributing factor in the modulation of L. theobromae pathogenicity towards plants, and also towards mammalian cells.

In this work we developed improved bioactive glasses and glass-ceramics for biomedical applications, investigating their in vitro bioactivity, biocompatibility and antibacterial properties against E-Coli . A melt-quenched bioactive glass of the SiO 2 -CaO-P 2 O 5 -MgO system was modified with the addition of 1 and 2 mol% Ag 2 O and the 1 mol% Ag 2 O-containing glasses were then heat treated to produce glass-ceramics. Surface modifications after soaking in SBF and ionic concentration changes showed that addition of silver and crystallization did not affect bioactivity although crystalline phases promoted a decrease in the degradation rate. Biocompatibility of all Ag-containing glasses and glass-ceramics was confirmed for certain samples concentrations. The antibacterial activity of the glasses against E-Coli was generally improved with decreasing particle size or increasing Ag 2 O. The Ag-containing glass-ceramics with higher content of crystalline phase appears as a promising biocompatible biocidal material with potential applications in bone-related diseases.

Lasiodiplodia theobromae (Botryosphaeriaceae, Ascomycota) is a plant pathogen and human opportunist whose pathogenicity is modulated by temperature. The molecular effects of temperature on L. theobromae are mostly unknown, so we used a multi-omics approach to understand how temperature affects the molecular mechanisms of pathogenicity. The genome of L. theobromae LA-SOL3 was sequenced (Illumina MiSeq) and annotated. Furthermore, the transcriptome (Illumina TruSeq) and proteome (Orbitrap LC-MS/MS) of LA-SOL3 grown at 25 °C and 37 °C were analysed. Proteins related to pathogenicity (plant cell wall degradation, toxin synthesis, mitogen-activated kinases pathway and proteins involved in the velvet complex) were more abundant when the fungus grew at 25 °C. At 37 °C, proteins related to pathogenicity were less abundant than at 25 °C, while proteins related to cell wall organisation were more abundant. On the other hand, virulence factors involved in human pathogenesis, such as the SSD1 virulence protein, were expressed only at 37 °C. Taken together, our results showed that this species presents a typical phytopathogenic molecular profile that is compatible with a hemibiotrophic lifestyle. We showed that L. theobromae is equipped with the pathogenesis toolbox that enables it to infect not only plants but also animals.

Lasiodiplodia theobromae is a fungal plant pathogen that has been associated with Botryosphaeria dieback of grapevine. Despite several studies on L. theobromae, until now the production of secondary metabolites by strains isolated from grapevines has not been reported. The ability of two strains of L. theobromae isolated from grapevine to produce lipophilic metabolites was studied. Although many typical compounds of low molecular weight were identified from the crude extracts of both strains (e.g., lasiolactols, substituted 2-dihydrofuranones, melleins, jasmonic acid, 3-indolcarboxylic acid, botryodiplodins), (2R/2S,3S,4S)-3-epi-botryodiplodin was isolated for the first time as a natural compound. Furthermore, a comparative study of metabolite production was conducted at 25 and 37 C to understand temperature effects on metabolite profiles. Some metabolites were produced only by one strain (e.g., (3S,4S)-4-acetyl-3-methyl-2-dihydrofuranone produced by LA-SOL3) and others only at a specific temperature (e.g., jasmonic acid at 25 C, botryodiplodins at 37 C). Phytotoxicity and cytotoxicity of pure compounds were evaluated to clarify the influence of lipophilic metabolites on the biological activities of culture filtrates of both strains. The most toxic compound for Vero and 3T3 cells was (2R/2S,3S,4S)-3-epi-botryodiplodin.

The prevention of microbial infections associated with implantable medical devices and superficial wounds represents one of the main research strategies in the field of biomaterials. The present study reports on the development of composite membranes of Chitosan (CS)-Polyethylene glycol (PEG) matrix, incorporating particles of biphasic calcium phosphate (BCP), zinc oxide (ZnO) and copper oxide (CuO). The properties that are relevant for intended applications in tissue regeneration and antibacterial coatings of implants were assessed. It was found that the addition of 1% (w/w - relative to the mass of CS) of each metal oxide promoted satisfactory bacteriostatic activity and exhibited no cytotoxic effects towards the Vero cell line. The formation of bonds between the CS/PEG matrix and ionic species from the powders enhanced the cross-linking degree and mechanical properties of composite membranes in comparison to the non-doped membrane with the same polymer matrix (CS/PEG = 70/30%). A gradual degradation of the composite membranes over the immersion time in simulated body fluid (SBF) was accompanied by a continuous surface deposition of uniform apatite layer.

Lack of knowledge regarding, and the stigma associated with, mental disorders have been identified as major obstacles for the promotion of mental health and early intervention. The present study aimed to evaluate the effectiveness of a school-based intervention program focused on the promotion of mental health literacy (MHL) in young people (&ldquo, Finding Space for Mental Health&rdquo, ). A sample of 543 students (22 classes), aged between 12 and 14 years old, participated in the study. Each class of students was randomly assigned to the control group (CG, n = 284, 11 classes) or the experimental group (EG, n = 259, 11 classes). MHL was assessed using the Mental Health Literacy questionnaire (MHLq), which is comprised of three dimensions&mdash, Knowledge/Stereotypes, First Aid Skills and Help Seeking, and Self-Help Strategies. The scores on these dimensions can also be combined to give an overall or total score. Participants from the EG attended the MHL promotion program (two sessions, 90 min each) delivered at one-week intervals. Sessions followed an interactive methodology, using group dynamics, music, and videos adapted to the target group. All participants responded to the MHLq at three points in time: pre-intervention assessment (one week prior to the intervention), post-intervention assessment (one week after the intervention) and follow-up assessment (six months after the intervention). The intervention effectiveness and the differential impact of sociodemographic variables on the effectiveness of the program were studied using a Generalized Estimation Equation (GEE). Results revealed that participants from the EG demonstrated, on average, significantly higher improvement in MHL from pre-intervention to follow-up when compared to participants from the CG. Different sociodemographic variables affected the effectiveness of the program on distinct dimensions of the MHLq. Overall, &ldquo, showed efficacy as a short-term promotion program for improving MHL in schools.

Phytopathogenic fungi are known to produce several types of enzymes usually involved in plant cell wall degradation and pathogenesis. The increasing of global temperature may induce fungi, such as Lasiodiplodia theobromae (L. theobromae), to alter its behavior. Nonetheless, there is only limited information regarding the effect of temperature on L. theobromae production of enzymes. The need for new, thermostable enzymes, that are biotechnologically relevant, led us to investigate the effect of temperature on the production of several extracellular enzymatic activities by different L. theobromae strains. Fungi were grown at 25 °C, 30 °C and 37 °C and the enzymatic activities were detected by plate assays, quantified by spectrophotometric methods and characterized by zymography. The thermostability (25–80 °C) of the enzymes produced was also tested. Strains CAA019, CBS339.90, LA-SOL3, LA-SV1 and LA-MA-1 produced amylases, gelatinases, caseinases, cellulases, lipases, laccases, xylanases, pectinases and pectin liases. Temperature modulated the expression of the enzymes, and this effect was more visible when fungi were grown at 37 °C than at lower temperatures. Contrary to proteolytic and endoglucanolytic activities, whose highest activities were detected when fungi were grown at 30 °C, lipolytic activity was not detected at this growth temperature. Profiles of proteases and endoglucanases of fungi grown at different temperatures were characterized by zymography. Enzymes were shown to be more thermostable when fungi were grown at 30 °C. Proteases were active up to 50 °C and endoglucanases up to 70 °C. Lipases were the least stable, with activities detected up to 45 °C. The enzymatic profiles detected for L. theobromae strains tested showed to be temperature and strain-dependent, making this species a good target for biotechnological applications.

Bacterial phenotypes, such as biofilm formation, antibiotic resistance and virulence expression, are associated with quorum sensing. Quorum sensing is a density-dependent regulatory system of gene expression controlled by specific signal molecules, such as N-acyl homoserine lactones (AHLs), produced and released by bacteria. This study reports the development of linear polymers capable to attenuate quorum sensing by adsorption of AHLs. Linear polymers were synthesized using MMA as backbone monomer and methacrylic acid and itaconic acid as functional monomers. Two different quorum sensing-controlled phenotypes, Vibrio fischeri bioluminescence and Aeromonas hydrophila biofilm formation, were evaluated to test the polymers' efficiency. Results showed that both phenotypes were significantly affected by the polymers, with the itaconic acid-containing material being more effective than the methacrylic acid one. The polymer inhibitory effects were reverted by the addition of lactones, confirming attenuation of quorum sensing through sequestration of signal molecules. The polymers also showed no cytotoxicity when tested using a mammalian cell line.

This study builds on successful episodes of improvisation in the prehospital emergency context to investigate how protocols can be articulated with unexpected events. An inductive approach shows that it is crucial for the flexible treatment of protocols to confront the disparity among mental models and the reality presented. Prehospital care practitioners accomplish this through benefit and harm assessment, risk assessment and resources attributes processes. Different types of unexpected events reflect in different improvisational processes: re-evaluation of cognitive resources, unrelated knowledge sources and re-interpretation of resources. Experience, resources, contextual factors and other operators are important factors in the decision process.

Relatório da Prática de Ensino Supervisionada, Mestrado em Ensino de História no 3º Ciclo do Ensino Básico e no Ensino Secundário, Universidade de Lisboa, Instituto de Educação, 2017 Submitted by Biblioteca FPIE-ULisboa (bibliorul@fpie.ulisboa.pt) on 2017-11-29T15:39:27Z No. of bitstreams: 2 ulfpie052007_tm_tese.pdf: 4186423 bytes, checksum: 4d6c0b7281cd97969437acaa83cf55e5 (MD5) ulfpie052007_tm_anexos.pdf: 28303842 bytes, checksum: 2f7480a85b120a9ebe0e94ba7ae76431 (MD5) Made available in DSpace on 2017-11-29T15:40:24Z (GMT). No. of bitstreams: 2 ulfpie052007_tm_tese.pdf: 4186423 bytes, checksum: 4d6c0b7281cd97969437acaa83cf55e5 (MD5) ulfpie052007_tm_anexos.pdf: 28303842 bytes, checksum: 2f7480a85b120a9ebe0e94ba7ae76431 (MD5) Previous issue date: 2017

Bacterial collagenases are metalloproteinases involved in the degradation of the extracellular matrices of animal cells, due to their ability to digest native collagen. These enzymes are important virulence factors in a variety of pathogenic bacteria. Nonetheless, there is a lack of scientific consensus for a proper and well-defined classification of these enzymes and a vast controversy regarding the correct identification of collagenases. Clostridial collagenases were the first ones to be identified and characterized and are the reference enzymes for comparison of newly discovered collagenolytic enzymes. In this review we present the most recent data regarding bacterial collagenases and overview the functional and structural diversity of bacterial collagenases. An overall picture of the molecular diversity and distribution of these proteins in nature will also be given. Particular aspects of the different proteolytic activities will be contextualized within relevant areas of application, mainly biotechnological processes and therapeutic uses. At last, we will present a new classification guide for bacterial collagenases that will allow the correct and straightforward classification of these enzymes.

Environmental alterations modulate host-microorganism interactions. Little is known about how climate changes can trigger pathogenic features on symbiont or mutualistic microorganisms. Current climate models predict increased environmental temperatures.The exposing of phytopathogens to these changing conditions can have particularly relevant consequences for economically important species and for humans. The impact on pathogen/host interaction and the shift on their biogeographical range can induce different levels of virulence in new hosts, allowing massive losses in agricultural and health fields. Lasiodiplodia theobromae is a phytopathogenic fungus responsible for a number of diseases in various plants. It has also been described as an opportunist pathogen in humans, causing infections with different levels of severity. Lasiodiplodia theobromae has a high capacity of adaptation to different environments, such as woody plants, moist argillaceous soils or even humans, being able to grow and infect hosts in a wide range of temperatures (9oC-39oC). Nonetheless, the effect of an increase of temperature, as predicted in climate change models, on L. theobromae is unknown. Here we explore the effect of temperature on two strains of L. theobromae – an environmental strain CAA019, and a clinical strain, CBS339.90.We show that both strains are cytotoxic to mammalian cells but while the environmental strain is cytotoxic mainly at 25oC, the clinical strain is cytotoxic mainly at 30oC and 37oC. Extracellular gelatinolytic, xylanolytic, amylolytic and cellulolytic activities at 25oC and 37oC were characterized by zymography and the secretome of both strains grown at 25oC, 30oC and 37oC were characterized by electrophoresis and by Orbitrap LC-MS/MS. More than 75% of the proteins were identified, mostly enzymes (glycosyl hydrolases and proteases). The strains showed different protein profiles, which were affected by growth temperature. Also, strain specific proteins were identified, such as a putative f5/8 type c domain protein, known for being involved in pathogenesis, by strain CAA019 and a putative tripeptidyl-peptidase 1 protein, by strain CBS339.90. We showed that temperature modulates the secretome of L. theobromae. This modulation may be associated with host-specificity requirements. We show that the study of abiotic factors, such as temperature, is crucial to understand host/pathogen interactions and its impacts on disease.

Dissertation presented as the partial requirement for obtaining a Master's degree in Statistics and Information Management, specialization in Marketing Research and CRM Vários estudos têm reconhecido o impacto dos activos intangíveis, como o valor da marca, a satisfação e lealdade do cliente, etc., na performance financeira das empresas, sobretudo em contextos sectoriais muito competitivos. É por isso natural que o mundo corporativo dedique cada vez mais importância à satisfação dos clientes e invista avultadas quantias nos factores considerados determinantes no aumento da satisfação. No entanto, numa altura em que o sector empresarial é confrontado com a necessidade de reduzir os custos, a eficácia destes investimentos tem vindo a ser questionada, tornando cada vez mais pertinente quantificar de que forma os investimentos realizados na melhoria da satisfação se traduzem, de facto, em receitas para a empresa. Circunscrevemos a análise a apenas um sector de actividade – sector bancário nacional, e correlacionámos os índices de satisfação de 4 instituições bancárias com a variabilidade do preço das suas acções em bolsa, para a década 2004-2014. Aplicaram-se modelos de regressão linear simples e múltipla para perceber se: a) É possível estabelecer uma relação de causa-efeito entre as variações dos bancos nos índices de satisfação e a oscilação do preço das suas acções no PSI20; b) Existem diferenças na relação de causalidade para cada banco individualmente; c) É possível identificar o efeito moderador de outra(s) variáveis na relação entre satisfação e cotação das acções; Foi testada outra variável para representar o cenário macroeconómico dos bancos: a média global do índice PSI20. Isoladamente, a satisfação dos clientes não demonstra qualquer efeito na variação do preço das acções, no entanto quando associada ao PSI20 mostrou-se estatisticamente significativa em 2 bancos.

The establishment of primary cell cultures is invaluable for studying cell and molecular biological questions. Although primary cell cultures more closely resemble and function like in the native environment, during the culture establishment the cells undergo several changes including the damage sustained during their removal from original tissue. The resultant cells have to rebalance the expression of their processing molecules to ascertain matrix signalling that ensure cell adaptation and consequent proliferation. Hence, we used cardosin, a novel plant enzyme for tissue disaggregation, for isolating and culturing neuronal cells from embryonic rats. The present investigation reports the molecular events, mainly related with matrix metalloproteinases (MMPs)/tissue inhibitor of metalloproteinase (TIMPs) expression, which could substantiate the superior neurite outgrowth and dendritic extension previously described. It was observed that 24 h after primary culture establishment, MMP-2 and MMP-9 messenger RNA (mRNA) are significantly upregulated, while the expression of TIMP-1 and TIMP-2 is unaltered. Regarding the role of laminin in neuronal pathfinding, it was found that the use of anti-laminin antibody and arginine–glycine–aspartate (RGD) peptide exerted inhibitory effects on neurite outgrowth after mechanical lesion where the expression of MMP-9 and TIMP-1 is upregulated under non-permissive conditions in response to mechanical injury.

UNLABELLED The aim of this study was to investigate the presence and the phenotypic expression of a gene coding for a putative collagenase. This gene (AHA_0517) was identified in Aeromonas hydrophila ATCC 7966 genome and named colAh. We constructed and characterized an Aeromonas piscicola AH-3::colAh knockout mutant. Collagenolytic activity of the wild-type and mutant strains was determined, demonstrating that colAh encodes for a collagenase. ColAh-collagen interaction was assayed by Far-Western blot, and cytopathic effects were investigated in Vero cells. We demonstrated that ColAh is a gluzincin metallopeptidase (approx. 100 kDa), able to cleave and physically interact with collagen, that contributes for Aeromonas collagenolytic activity and cytotoxicity. ColAh possess the consensus HEXXH sequence and a glutamic acid as the third zinc binding positioned downstream the HEXXH motif, but has low sequence similarity and distinct domain architecture to the well-known clostridial collagenases. In addition, these results highlight the importance of exploring new microbial collagenases that may have significant relevance for the health and biotechnological industries. SIGNIFICANCE AND IMPACT OF THE STUDY Collagenases play a central role in processes where collagen digestion is needed, for example host invasion by pathogenic micro-organisms. We identified a new collagenase from Aeromonas using an integrated in silico/in vitro strategy. This enzyme is able to bind and cleave collagen, contributes for AH-3 cytotoxicity and shares low similarity with known bacterial collagenases. This is the first report of an enzyme belonging to the gluzincin subfamily of the M9 family of peptidases in Aeromonas. This study increases the current knowledge on collagenolytic enzymes bringing new perspectives for biotechnology/medical purposes.

Cardosin A is an aspartic proteinase, like pepsin, purified from the cardoon, Cynara cardunculus L. and has been traditionally used in Portugal in the manufacture of traditional cheeses, due to its milk clotting activity. This enzyme is a fine example of a small number of plant aspartic proteinases that have been isolated, purified and characterised both in aqueous and in non-aqueous media. The present work deals with the effect of organic solvents on cardosin A specificity towards complex substrates such as κ-, β- and α-caseins and human type I collagen. At the same time fluorescence studies were carried out to correlate reaction rates with possible structural alterations induced by the organic solvents on the substrates. Results show a strong dependence of the velocity of hydrolysis on the medium composition and additionally, it is shown that none of the organic solvents used in this work, induced alterations of cardosin A specificity. Furthermore, it is shown that cardosin A in the presence of organic solvents can be used as a catalyst in order to obtain reaction intermediary products that are, otherwise, rapidly consumed in aqueous buffer.

Dissertação apresentada na Escola Superior de Educação de Lisboa, para obtenção do grau de Mestre em Ciências da Educação - Especialidade Intervenção Precoce Na esteira dos estudos sobre a relação creche-família e sobre o modelo Touchpoints, surgiu o interesse de elaborar uma investigação-ação, no qual se pretende implementar práticas reflexivas Touchpoints e avaliar o seu impacto na satisfação dos pais, da educadora, no desenvolvimento infantil, na qualidade do contexto educativo, nas rotinas da família e no desenvolvimento das crianças. Para o efeito e assumindo um duplo papel de educadora e investigadora, foram convidados a participar neste estudo 11 pais de 12 crianças com idades compreendidas entre os 24- 36, que frequentam a sala de creche onde exerço a minha atividade profissional. Esta investigação assentou em quatro fases distintas de trabalho, de forma a cumprir os objetivos propostos: 1ª) a recolha de dados, antes da formação em Touchpoints, recorrendo aos seguintes instrumentos e observações: Escala de Desenvolvimento Infantil Growing Skills II, observação da interação pais-criança com a escala Care-Index, avaliação da satisfação dos pais e do educador com a Parent Caregiver Relationship Scale (PCRS), avaliação do contexto educativo com a ITERSR, e os Mapas de Rotinas Semanais da família; 2ª) formação Touchpoints com uma equipa interdisciplinar; 3ª) Práticas reflexivas Touchpoints – o educador agente de mudança que reflete sobre as suas práticas com guiões prévios e coaching (supervisão com formadores que visitaram a creche, a equipa educativa e a sala da educadora); 4ª) repetição da recolha de dados no final do projeto. O estudo indicou que o desenvolvimento infantil, as relações pais-filhos, as rotinas familiares, a satisfação pais-educadora e educadora-pais assim como a própria prática da educadora, mudaram após o treino com as práticas reflexivas em Touchpoints. Embora estes resultados não possam ser generalizados e o controlo das variáveis seja afetada por uma metodologia de investigação-ação (portanto, não possamos inferir que todos estes ganhos se devem às praticas reflexivas Touchpoints), os principais resultados obtidos neste estudo permitem-nos repensar formas de relacionamento entre a creche e a família e sobre o impacto que a prática reflexiva do educador pode ter na qualidade destas relações. Julgamos que a presente investigação suscita questões sobre a relação educadores-pais e sobre o contributo das práticas reflexivas em Touchpoints nas práticas de creche. Abstract This study is based on an intervention research project with focus on the relationship between center-based child care and family through the Touchpoints model. The main aim of this study is to implement the Touchpoints reflexive practices and assess their impact on: parents’ levels of satisfaction, early childhood teachers practice, child care quality, family routines and children’s development. Eleven parents of children aged between 24 and 36 months, which currently attend center-based child care, participated in this study and me as an early childhood teacher and investigator of the present study. This research followed four stages: 1) data collection, before training Touchpoints, using the following instruments and observations: Scale of Infant Development Growing Skills II, observing parent-child interaction with the Care-Index scale, accessing parents and teachers satisfaction with Parent Caregiver Relationship scale (PCRS), accessing child care quality with the ITERS -R, and using weekly maps of family routines; 2) training in touchpoints reflexive practices with an interdisciplinary team; 3) Touchpoints reflexive practices according to guidelines and coaching; 4) repeat the initial data collection at the end of the project. This study suggests that the children development; the parents-child relationships; the family routines; the parents-early childhood teacher and early childhood teacher-parents levels of satisfaction; and the early childhood teacher educational practice have significantly changed after the Touchpoints Reflexive Practices training programme. However, several factors can contribute for these results and our findings only report our experience. Nevertheless, the study raises important questions concerning school-family relationship and the impact of reflexive practice in this relationship.

Bacterial phenotypes, such as biofilm formation, antibiotic resistance and virulence expression, are associated with quorum sensing. Quorum sensing is a density-dependent regulatory system of gene expression controlled by specific signal molecules, such as N-acyl homoserine lactones (AHLs), produced and released by bacteria. This study reports the development of linear polymers capable to attenuate quorum sensing by adsorption of AHLs. Linear polymers were synthesized using MMA as backbone monomer and methacrylic acid and itaconic acid as functional monomers. Two different quorum sensing-controlled phenotypes, Vibrio fischeri bioluminescence and Aeromonas hydrophila biofilm formation, were evaluated to test the polymers' efficiency. Results showed that both phenotypes were significantly affected by the polymers, with the itaconic acid-containing material being more effective than the methacrylic acid one. The polymer inhibitory effects were reverted by the addition of lactones, confirming attenuation of quorum sensing through sequestration of signal molecules. The polymers also showed no cytotoxicity when tested using a mammalian cell line.

Mestrado, Enfermagem de Saúde Infantil e Pediatria, 2014, Escola Superior de Enfermagem de Lisboa Made available in DSpace on 2016-11-25T09:44:25Z (GMT). No. of bitstreams: 0 Previous issue date: 2014

Mestrado, Enfermagem de Saúde Infantil e Pediatria, 2014, Escola Superior de Enfermagem de Lisboa O Enfermeiro Especialista em Enfermagem de Saúde da Criança e do Jovem apresenta-se como um profissional que se encontra presente em vários contextos da prestação de cuidados de saúde, deparando-se frequentemente com o cliente pediátrico (criança/jovem e cuidadores familiares) em qualquer fase do seu processo de saúde-doença. Este relatório retrata todo o percurso formativo que permitiu a aquisição de competências na área de especialização referida, que abrangeu diversos contextos da prática em enfermagem pediátrica. O processo de aquisição de competências teve por base o desenvolvimento de um projeto de estágio, cuja problemática da doença crónica em idade pediátrica foi objeto de estudo. A metodologia de trabalho utilizada foi a descritiva, analítica e reflexiva, sustentada na evidência científica de enfermagem e de outras disciplinas, mobilizada ao longo da experiência formativa. Ao considerar que os cuidados de enfermagem têm em conta a totalidade e a individualidade da pessoa, qualquer que seja o cliente alvo, foi mobilizado o Modelo de Sistemas da Teórica de Enfermagem de Betty Neuman como conceção teórica dominante. Este referencial defende que os clientes são vistos como um todo cujas partes estão em interação dinâmica, considerando que, simultaneamente, existem cinco variáveis que podem ser afetadas no sistema-cliente por uma diversidade de fatores de stress associados aos processos de saúde-doença. As atividades realizadas em cada um dos contextos de estágio estão espelhadas neste relatório, e permitiram a identificação e apropriação das intervenções de enfermagem direcionadas a cada uma das variáveis do sistema-cliente pediátrico e numa lógica de cuidado humano e holístico. O contacto com crianças e jovens com doença crónica e seus cuidadores familiares possibilitou o desenvolvimento de competências no âmbito da problemática em estudo.

Aeromonas molluscorum Av27 is an estuarine bacterium highly resistant to tributyltin (TBT). Also, the strain is able to degrade TBT into the less toxic compounds dibutyltin and monobutyltin. Therefore, this bacterium has potential to be employed in bioremediation processes. In this context, defining its biological safety is crucial. With that purpose a number of intrinsic characteristics, usually present/associated with virulent strains, were investigated. Few virulence factors were detected in strain Av27. For instance, a DNase gene is present, but it is not apparently expressed in vitro. Motility, adherence factor and phospholipase activity were also detected. Additionally, cytotoxicity to Vero cells was negative. Resistance to penicillin (10 μg ml(-1)), amoxicillin/clavulanic acid (30 μg ml(-1)) and cephalothin (30 μg ml(-1)) and also to the vibriostatic agent O/129 was observed. Five plasmids (4, 7, 10, 100 kb and one greater than 100 kb) were identified. No Class I and II integrons were detected. Study of the optimal growth conditions showed that Av27 easily adapts to different environmental conditions. Overall, the results suggest that A. molluscorum Av27 can be considered safe to use to bioremediate TBT in contaminated environments.

Several instruments assess constructs related to mental health literacy and to the stigma associated with mental health problems. However, most of them have conceptualisations that may limit the assessment of the mental health literacy concept in a more up-to-date and comprehensive perspective. Furthermore, some of the instruments’ structure may limit their use in large scale samples, in a short period of time and with repeated measures. This article presents the development of the Mental Health Literacy questionnaire (MHLq) and the studies to assess its psychometric properties. After item-pool generation, content validity was assessed by experts and a think aloud procedure. The MHLq was tested in two samples of students (study 1 n=239; aged between 12 and 15 years old; study 2 n=737; aged between 11 and 17 years old) to determine its psychometric properties. Factor analysis procedures favoured a three-factor solution (with 33 items) of the MHLq. The questionnaire showed good internal consistency (total score α=0.84; Factor 1 first aid skills and help seeking - α=0.79; Factor 2 knowledge/stereotypes - α=0.78; Factor 3 self-help strategies - α=0.72); and excellent test-retest reliability, the ICC for the total score of MHLq was 0.88 and for the three dimensions of MHLq was 0.80 (Factor 1), 0.90 (Factor 2) and 0.86 (Factor 3). The MHLq is a practical, valid and reliable tool for identifying gaps in knowledge, beliefs and behavioural intentions, in large samples, allowing the development and evaluation of interventions aimed at promoting mental health in young people.

Biofilm production and expression of virulence factors are correlated with Quorum Sensing (QS), a density-dependent regulation of gene expression controlled by specific signal molecules produced by bacteria. Acyl-homoserine lactones (AHLs) are the most common signal molecules produced by Gram-Negative bacteria for QS. We have developed synthetic polymers that inhibit QS by sequestering the bacterial signal molecules. These synthetic polymers are methacrylate based and their composition was optimized by computer modeling. After synthesis, the binding capacity to AHLs in water was tested by equilibrium experiments and HPCL-MS. The polymers were able to absorb 0.1–0.3 mg (per gram of polymer) of 3-oxo-C6-AHL. Vibrio fischeri, that is bioluminescent through QS, was used to assess the performance of the polymers in a “real” environment. In the presence of polymers, the bioluminescence of V. fischeri was drastically reduced, without any noticeable effect on growth. In conclusion, these new polymers can sequester a signal molecule of V. fischeri and prevent QS-controlled phenotypes, thus representing a new solution for disease outbreaks in systems such as aquacultures. published

Tese de mestrado, Ciências da Educação (Formação de Professores), Universidade de Lisboa, Instituto de Educação, 2011 Submitted by Jorge Revez (jrevez@fpie.ul.pt) on 2012-05-09T17:29:10Z No. of bitstreams: 1 ulfpie040047_tm.pdf: 2295848 bytes, checksum: f368f6147ca8cb129f35d141d1d0f99c (MD5) Made available in DSpace on 2012-05-09T17:29:17Z (GMT). No. of bitstreams: 1 ulfpie040047_tm.pdf: 2295848 bytes, checksum: f368f6147ca8cb129f35d141d1d0f99c (MD5) Previous issue date: 2011