Cancer theranostics: multifunctional gold nanoparticles for diagnostics and therapy

Doctorate in Biology, Specialty in BiotechnologyThe use of gold nanoparticles (AuNPs) has been gaining momentum in molecular diagnostics due to their unique physico-chemical properties these systems present huge advantages, such as increased sensitivity, reduced cost and potential for single-molecule characterisation. Because of their versatility and easy of functionalisation, multifunctional AuNPs have also been proposed as optimal delivery systems for therapy (nanovectors). Being able to produce such systems would mean the dawn of a new age in theranostics (diagnostics and therapy)driven by nanotechnology vehicles. Nanotechnology can be exploit for cancer theranostics via the development of diagnostics systems such as colorimetric and imunoassays, and in therapy approaches through gene therapy, drug delivery and tumour targeting systems. The unique characteristics of nanoparticles in the nanometre range, such as high surface-tovolume ratio or shape/size-dependent optical properties, are drastically different from those of their bulk materials and hold pledge in the clinical field for disease therapeutics This PhD project intends to optimise a gold-nanoparticle based technique for the detection of oncogenes’ transcripts (c-Myc and BCR-ABL) that can be used for the evaluation of the expression profile in cancer cells, while simultaneously developing an innovative platform of multifunctional gold nanoparticles (tumour markers, cell penetrating peptides, fluorescent dyes) loaded with siRNA capable of silencing the selected proto-oncogenes, which can be used to evaluate the level of expression and determine the efficiency of silencing. This work is a part of an ongoing collaboration between Research Centre for Human Molecular Genetics, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Portugal and Biofunctional Nanoparticles and Surfaces Group, Instituto de Nanociencia de Aragón, Spain within a European project [NanoScieE+ - NANOTRUCK]. In order to achieve this goal we developed effective conjugation strategies to combine, in a highly controlled way, biomolecules to the surface of AuNPs with specific functions such as: ssDNA oligos to detect specific sequences and for mRNA quantification; Biofunctional spacers: Poly(ethylene glycol) (PEG) spacers used to increase solubility and biocompatibility and confer chemical functionality; Cell penetrating peptides: to overcome the lipophilic barrier of the cellular membranes and deliver molecules into cells using TAT peptide to achieve cytoplasm and nucleus; Quaternary ammonium: to introduce stable positively charged in gold nanoparticles surface; and RNA interference: siRNA complementary to a master regulator gene, the proto-oncogene c-Myc, that is implicated in cell growth, proliferation, loss of differentiation, and cell death. In order to establish that they are viable alternatives to the available methods, these innovative nanoparticles were extensively characterized on their chemical functionalization, ease of uptake, cellular toxicity and inflammation, and knockdown of MYC protein expression in several cancer cell lines and in in vivo models.Fundação para a Ciência e Tecnologia - (SFRH/BD/62957/2009); PTDC/BIO/66514/2006; NANOLIGHT-PTDC/QUI-QUI/112597/2009; Silencing the silencers via multifunctional gold nanoconjugates towards cancer therapy - PTDC/BBB-NAN/1812/201

Aplicação de SAFe® a um Projecto de Manutenção de Aviões

Maintenance, repair and overhaul (MRO) operations have a great impact on the life cycle of an aircraft (A/C). MROs organizations address various challenges on planning activities to ensure the maximum reliability of an A/C, given the amount of unscheduled maintenance. Subcontracting MRO activities by airline companies, has been continuously increasing as an alternative of performing the heavy maintenance themselves, adding a constraint on this type of industries which is to manage the customer demands. Considering the main issues, it is required to select the most suitable approach to plan and manage A/C maintenance projects. Agile Project Management (PM) could be a solution to overcome the main difficulties in this sector, managing the uncertainty throughout the project, providing customer visibility and control over the service. This work arises in a real-life case of a subcontracted MRO program in a multinational A/C manufacture enterprise, which also suffers from significant challenges of planning and managing maintenance activities throughout the project life cycle. The program has experimented agile methodologies that revealed a positive impact. In order to the whole program embrace agility and overcome the identified main problems, it was proposed the usage of an elaborate and well-defined agile framework. Scaled Agile Framework for enterprises (SAFe®) is an online knowledge base that implements diverse agile techniques to support businesses, develop and deliver solutions, achieving business agility. As SAFe® was mainly developed for software industries, due to the characteristics of the project and the type of industry where it is inserted, the application of this framework needed to be customized. Accordingly to the particularities of the project, the most suitable PM approach is a hybrid approach, where initially the scope of the project is delineated, with a contingency plan, supported by SAFe® to manage the issues that arise throughout the project. The agile methodologies allow customer centred attention, more communication channels, and by iterating over the product, planning the unscheduled work focusing on high priority tasks. Lastly, a framework in the core of the appearance of the issues was developed, to define the interconnection between the whole SAFe® concepts and to provide an extended view of how the project will progress with the new approach.As operações de manutenção, reparação e revisão produzem grande impacto no ciclo de vida de uma aeronave. As organizações que operam neste setor enfrentam vários desafios no planeamento das atividades que garantem a máxima confiabilidade de uma aeronave, dada a quantidade de manutenção não programada. A subcontratação deste tipo de atividades, por parte das companhias aéreas, tem crescido continuamente como uma alternativa à realização da própria manutenção pesada, adicionando um constrangimento para este tipo de indústrias: gerir as exigências dos clientes. Considerando os principais problemas, é necessário selecionar a abordagem mais adequada para planear e gerir projetos de manutenção de aviões. A gestão ágil de projetos poderá ser uma solução para superar as principais dificuldades deste setor, gerindo as incertezas ao longo do projeto, proporcionando visibilidade ao cliente e controlo sobre o serviço. Este trabalho surge num caso real de um programa subcontratado numa empresa multinacional de fabrico de aviões, que também sofre de desafios significativos no planeamento e gestão de atividades de manutenção ao longo do ciclo de vida do projeto. O programa experienciou metodologias ágeis que revelaram um impacto positivo. Para que todo o programa adote a agilidade e supere os principais problemas identificados, foi proposto o uso de uma elaborada e bem definida estrutura ágil. O Scaled Agile Framework para empresas é uma base de conhecimento online que implementa diversas técnicas ágeis para apoiar as empresas no desenvolvimento e entrega de soluções, alcançando a agilidade nos negócios. O SAFe® foi desenvolvido principalmente para indústrias de software, devido às características do projeto e ao tipo de indústria em que está inserido, a aplicação desta estrutura necessitou de ser personalizada. De acordo com as particularidades do projeto, a abordagem de gestão de projetos mais adequada é uma abordagem híbrida, onde inicialmente o projeto é delineado, com um plano de contingência, apoiado pelo SAFe® para gerenciar os problemas que surgem ao longo do projeto. As metodologias ágeis permitem centrar a atenção no cliente, mais canais de comunicação e, ao iterar sobre o produto, planear o trabalho não programado com foco em tarefas de alta prioridade. Por fim, foi desenvolvido um framework no cerne do surgimento dos problemas, de forma a definir as interligações entre todos os conceitos do SAFe® e fornecer uma visão ampliada de como o projeto irá progredir com a nova abordagem

A influência da Dieta Mediterrânica no desenvolvimento da Aterosclerose

Introdução: A dieta é uma parte fundamental da saúde e, portanto, consegue ter uma influência diferente dos medicamentos, por ser a fonte de energia que dá origem a diversos processos metabólicos no nosso corpo. No caso da Dieta Mediterrânica, poderá ser das melhores dietas em termos cardiovasculares, visto que é bem equilibrada, tem uma grande fonte de gordura insaturada e um consumo reduzido de carnes vermelhas. Desta forma, este tipo de dieta poderá ter uma grande influência no desenvolvimento da aterosclerose. Esta tese tem como objetivo demonstrar a relação entre uma alimentação com base numa dieta mediterrânica e a evolução da placa aterosclerótica. Métodos: Após uma pesquisa na PUBMED com “mediterranean diet and atherosclerosis” como palavras-chave, os artigos encontrados foram selecionados de forma a abranger os efeitos desta dieta no desenvolvimento da aterosclerose. Uma análise extensa e sistemática destes artigos permitiu analisar os efeitos cardiovasculares de vários componentes da dieta Mediterrânica e a sua relação como um todo, não só relativamente à aterosclerose, mas também na prevenção de outras doenças cardiovasculares como, AVC, Enfarte do Miocárdio, Diabetes e Síndrome Metabólico. Resultados: Todos os artigos apontam para uma relação benéfica entre a dieta Mediterrânica e os parâmetros inflamatórios, levando a uma melhoria não só na aterosclerose, mas também de outras doenças cardiovasculares. Para além disso, também foi possível comprovar os efeitos benéficos do azeite a nível vascular, sendo esta a principal fonte de gordura na dieta. Conclusão: A dieta Mediterrânica apresenta efeitos positivos, não só a nível da prevenção de várias doenças cardiovasculares, mas também apresenta indícios de prevenção secundária nas recorrências de patologias como o AVC. Tendo em conta todos os dados obtidos em todos os artigos apresentados e tendo como base o estudo PREDIMED, foi possível concluir que a dieta mediterrânica tem benefícios superiores na saúde cardiovascular quando comparada com outras dietas recomendadas. Desta forma, esta dieta poderá ser prescrita pelos profissionais de saúde como forma de prevenção nos pacientes em risco cardiovascular.Introduction: Dietting is a fundamental part of health, and so, it can have a different approach to a person’s health than medication because it is our source of energy for the metabolism. In terms of the Mediterranean diet, it can be one of the best diets for cardiovascular health since it is very well balanced, it is a big source of unsaturated fat and it is lo won red meat. This way, this kind of diet might have a great influence on the developpment of atherosclerosis, since it is very influenced by the types of food ingested. This thesis has the objective of showing the relationship between the mediterranean diet and the evolutionof the atherosclerotic plaque. Métodos: After researching on PUBMED with the terms “mediterranean diet and atherosclerosis” as keywords, the selected articles included every mention of the relationship between the mediterranean diet and atherosclerosis. A thorough and sistematic analysis of these articles allowed to analyse the cardiovascular effects of various componentes essencial to the mediterranean diet and their relationship as a whole, not just when compared to atherosclerosis but also in the prevention of other cardiovascular diseases such as stroke, Miocardial Enfarction, Diabetes and Metabolic Syndrome. Results: Every article point to a beneficial relation between the Mediterranean diet and the inflammatory parameters, leading to na improvement not just in the case of atherosclerosis but also of other cardiovascular diseases. Besides, it was also possible to prove the beneficial effects of virgin olive oil at a vascular level, being this the main source of fat in the diet. Conclusion: The Mediterranean diet has positive effects not just on a preventive level of various cardiovascular diseases, but also in the secondary prevention of other patologies such as stroke. Having in mind all of the data collected from the studies and mostly from the PREDIMED study, it was possible to conclude that the mediterranean diet has bigger benefits to cardiovascular health than other recommended diets. This way, the diet can be perscribed by healthcare professionals as a way of prevention for the patients at cardiovascular risk

Desempenhos zootécnicos de frangos de carne alimentados com a macroalga Laminaria digitata com ou sem suplementação enzimática

Mestrado em Engenharia Zootécnica - Produção Animal. Universidade de Lisboa, Instituto Superior de Agronomia, Faculdade de Medicina VeterináriaO milho e o bagaço de soja são as matérias-primas principais na alimentação de frangos. A procura de diferentes matérias-primas é importante para diminuir a dependência destes. Recentemente, a utilização de algas na alimentação de frangos de carne tem sido um alvo de estudo, sendo a macroalga Laminaria digitata uma potencial matéria-prima a ser estudada. Foram selecionados 120 frangos da estirpe Ross 308 e alimentados com quatro diferentes tratamentos, formulados para frangos de crescimento, entre os 21-35 dias de idade. Estes diferentes tratamentos consistiram em: 1) CON, sem incorporação de Laminaria digitata nem suplementação enzimática; 2) LAM, com incorporação de Laminaria digitata (15%) sem suplementação enzimática; 3) LAMROV, com incorporação de Laminaria digitata (15%) e suplementação com a mistura enzimática Rovabio®; 4) LAMREC, com incorporação de Laminaria digitata (15%) e suplementação com a mistura enzimática recombinante. Foi analisado o peso vivo (PV), ganho médio diário (GMD), ingestão de alimento, índice de conversão (IC), dimensão de órgãos do sistema digestivo e viscosidade dos conteúdos intestinais. Os tratamentos LAM e LAMROV levaram a menor GMD e maior IC. A dimensão dos órgãos digestivos foi influenciada pela incorporação de Laminaria digitata, levou a moelas menos pesadas e a órgãos dos segmentos posteriores do intestino delgado e do intestino grosso de maiores dimensões, em peso e em comprimento. A viscosidade dos conteúdos intestinais do íleo em LAM e LAMROV foi significativamente maior do que em CON e LAMREC. Este estudo mostrou que a mistura enzimática recombinante tem potencial na suplementação de alimento para frangos de carne com incorporação de Laminaria digitata. A mistura enzimática Rovabio® não mostrou eficácia na digestão dos hidratos de carbono da Laminaria digitata, especialmente os polissacarídeos não amiláceos (PNAs). A Laminaria digitata mostra algum potencial como matéria-prima alternativa na alimentação de frangos de carne quando suplementada com a mistura enzimática recombinante de liase de algina.Corn and soybean meal are the main ingredients of brolier feeds. Finding different ingredients is important to decrease the dependence on corn and soybean meal. The use of algae in broiler feeds has been a target of research. The macroalgae Laminaria digitata is a potential ingredient to study. In the present study, 120 Ross 308 broilers were selected and fed four different diets, formulated for growing broilers, and given between days 21-35. These different diets consisted of: 1) CON, without incorporation of Laminaria digitata and without enzyme suplementation; 2) LAM, with incorporation of Laminaria digitata (15%) but without enzyme suplementation; 3) LAMROV, with incorporation of Laminaria digitata (15%) and suplementation with Rovabio® enzyme mixture; 4) LAMREC, with incorporation of Laminaria digitata (15%) and suplementation with recombinant enzyme mixture. Body weight (BW), average daily gain (ADG), feed intake, feed conversion rate (FCR), dimension of organs of the digestive tract and viscosity of the duodenum+jejun and ileum were analysed. The diets LAM and LAMROV led to lower ADG and higher FCR. The organ size was influenced by the addition of Laminaria digitata, it led to a lighter gizzard and bigger posterior small intestine segment and large intestine organs, both in weight and in length. Intestine content viscosity was significantly higher in broilers fed with LAM and LAMROV than in broilers under CON and LAMREC diets. Collectively, the present study showed that the recombinant enzyme mixture has potential as a suplement of broiler feed with incorporation of Laminaria digitata. The enzyme mixture Rovabio® doesn’t show effectiveness in the digestion of Laminaria digitata carbohydrates, especially non-starch polysaccharides (NSPs). The macroalgae Laminaria digitata shows some potential as an alternative ingredient for broiler feed when suplemented with the alginate lyase recombinant enzyme mixture.N/

Integrated Omics analysis of pig muscle metabolism under the effects of dietary Chlorella vulgaris and exogenous enzymes

Monogastric feeding is dependent on costly conventional feedstuffs. Microalgae such as Chlorella vulgaris are a sustainable alternative; however, its recalcitrant cell wall hinders monogastric digestion. Carbohydrate Active Enzyme (CAZyme) supplementation is a possible solution. The objective of this work was to evaluate the effect of 5% dietary C. vulgaris (CV) and enzymatic supplementation (CV + R— Rovabio® Excel AP; CV + M—four CAZyme mix) on muscle transcriptome and proteome of finishing pigs, in an integrated approach. Control pigs increased the abundance of contractile apparatus (MYH1, MYH2, MYH4) and energy metabolism (CKMT1, NDUFS3) proteins, demonstrating increased nutrient availability. They had increased expression of SCD, characteristic of increased glucose availability, via the activation of SREBP-1c and ChREBP. CV and CV + R pigs upregulated proteolytic and apoptotic genes (BAX, DDA1), whilst increasing the abundance of glucose (UQCRFS1) and fatty acid catabolism (ACADS) proteins. CV + R pigs upregulated ACOT8 and SIRT3 genes as a response to reduced nutrient availability, maintaining energy homeostasis. The cell wall specific CAZyme mix, CV + M, was able to comparatively reduce Omics alterations in the muscle, thereby reducing endogenous nutrient catabolism compared to the CV + R and CVinfo:eu-repo/semantics/publishedVersio

Effect of Dietary Laminaria digitata with Carbohydrases on Broiler Production Performance and Meat Quality, Lipid Profile, and Mineral Composition

This article belongs to the Section Animal Nutrition.Simple Summary: Seaweeds represent promising alternatives to unsustainable conventional feed sources, such as cereals, incorporated in poultry diets. Brown macroalgae (e.g., Laminaria digitata) correspond to the largest cultured algal biomass worldwide and are rich in bioactive polysaccharides, minerals, and antioxidant pigments. However, their utilization as feed ingredients is limited due to the presence of an intricate gel-forming cell wall composed of indigestible carbohydrates, mainly alginate and fucose-containing sulfated polysaccharides. Therefore, supplementation with carbohydrate-active enzymes is required to disrupt the cell wall and allow seaweed nutrients to be digested and absorbed in poultry gut. The present study aimed to evaluate if the dietary inclusion of 15% L. digitata, supplemented or not with carbohydrases, could improve the nutritional value of poultry meat without impairing growth performance of broiler chickens. The results show that L. digitata increases antioxidant pigments and n-3 long-chain polyunsaturated fatty acids in meat, thus improving meat nutritional and health values. On the other hand, feeding algae at a high incorporation level impaired growth performance. Feed enzymatic supplementation had only residual effects, although alginate lyase decreased intestinal viscosity caused by dietary L. digitata with potential benefits for broiler digestibility.Abstract: We hypothesized that dietary inclusion of 15% Laminaria digitata, supplemented or not with carbohydrases, could improve the nutritional value of poultry meat without impairing animal growth performance. A total of 120 22-day old broilers were fed the following dietary treatments (n = 10) for 14 days: cereal-based diet (control); control diet with 15% L. digitata (LA); LA diet with 0.005% Rovabio® Excel AP (LAR); LA diet with 0.01% alginate lyase (LAE). Final body weight was lower and feed conversion ratio higher with LA diet than with the control. The ileal viscosity increased with LA and LAR diets relative to control but without differences between LAE and control. The pH of thigh meat was higher, and the redness value of breast was lower with LA diet than with control. Meat overall acceptability was positively scored for all treatments. The γ-tocopherol decreased, whereas total chlorophylls and carotenoids increased in meat with alga diets relative to control. The percentage of n-3 polyunsaturated fatty acids (PUFA) and accumulation of bromine and iodine in meat increased with alga diets compared with control. Feeding 15% of L. digitata to broilers impaired growth performance but enhanced meat quality by increasing antioxidant pigments, with beneficial effects on n-3 PUFA and iodine.The study was funded by the Fundação para a Ciência e a Tecnologia, Portugal, through the PTDC/CAL-ZOO/30238/2017 grant, associated with a postdoctoral contract to M.M.C. and a PhD fellowship to D.C. (SFRH/BD/126198/2016), as well as CIISA (UIDB/00276/2020), AL4AnimalS (LA/P/0059/2020), and LEAF (UIDB/04129/2020) grants.info:eu-repo/semantics/publishedVersio

Effect of Dietary Laminaria digitata with Carbohydrases on Broiler Production Performance and Meat Quality, Lipid Profile, and Mineral Composition

Research Areas: Agriculture; Veterinary Sciences; ZoologyABSTRACT - We hypothesized that dietary inclusion of 15% Laminaria digitata, supplemented or not with carbohydrases, could improve the nutritional value of poultry meat without impairing animal growth performance. A total of 120 22-day old broilers were fed the following dietary treatments (n = 10) for 14 days: cereal-based diet (control); control diet with 15% L. digitata (LA); LA diet with 0.005% Rovabio® Excel AP (LAR); LA diet with 0.01% alginate lyase (LAE). Final body weight was lower and feed conversion ratio higher with LA diet than with the control. The ileal viscosity increased with LA and LAR diets relative to control but without differences between LAE and control. The pH of thigh meat was higher, and the redness value of breast was lower with LA diet than with control. Meat overall acceptability was positively scored for all treatments. The γ-tocopherol decreased, whereas total chlorophylls and carotenoids increased in meat with alga diets relative to control. The percentage of n-3 polyunsaturated fatty acids (PUFA) and accumulation of bromine and iodine in meat increased with alga diets compared with control. Feeding 15% of L. digitata to broilers impaired growth performance but enhanced meat quality by increasing antioxidant pigments, with beneficial effects on n-3 PUFA and iodineinfo:eu-repo/semantics/publishedVersio

Estrutura fatorial, fidedignidade e parâmetros de itens da versão em português brasileiro do questionário GAD-7

This study examined the psychometric properties of the Brazilian-Portuguese version of the Generalized Anxiety Disorder GAD-7 questionnaire in a community sample (n = 206) of Brazilian adults. The sample was 41% female, with a mean age of 21.10 (SD = 4.49), 75.6% from colleges/universities. Results of a confi rmatory factor analysis provided support to the original unidimensional model of the GAD-7 in the Brazilian context. Analyses of Variance (ANOVA) showed that the GAD-7 scores were signifi cantly different between males and females, with females scoring higher than males. The scale demonstrated good reliability evidence; both Cronbach’s alpha coeffi cient (α = .916) and rho composite reliability coeffi cient (ρ = .909) were adequate. Item parameter analysis showed items 5 and 7 presented the highest severity thresholds for the generalized anxiety latent trait, whereas item 1 presented the lowest ones. Our fi ndings suggest that the Brazilian-Portuguese version of the GAD-7 is suitable for assessing Generalized Anxiety Disorder symptoms in Brazilian adults in community settings.Este estudo analisou as propriedades psicométricas da versão em Português Brasileiro do questionário de “Transtorno de Ansiedade Generalizada - GAD-7 em uma amostra de comunidade (n = 206) da população adulta brasileira. A amostra foi composta por de 41% de participantes do sexo feminino, com idade média de 21,10 (DP = 4,49), e 75,6% em instituições de ensino superior. Os resultados da análise fatorial confi rmatória forneceram apoio para o modelo unidimensional original do GAD-7 no contexto brasileiro. As análises de variância (ANOVA) mostraram que os escores do GAD-7 foram signifi cativamente diferentes entre homens e mulheres, com as mulheres apresentando maiores escores que os homens. A escala demonstrou uma boa fi dedignidade; sendo que tanto o coefi ciente de Cronbach alfa (α = 0,916) e rho coefi ciente de confi abilidade composta (ρ = 0,909) foram adequados. A análise dos parâmetros dos itens mostrou itens 5 e 7 apresentaram os limiares mais elevados de severidade para o traço-latente de ansiedade generalizada, enquanto que o item 1 apresentou as menores. Nossos resultados sugerem que a versão Português Brasileiro do GAD-7 é adequada para avaliar os sintomas do transtorno de ansiedade generalizada em adultos brasileiros em ambientes comunitários.Este estudio examinó las propiedades psicométricas de la versión de portugués brasileño del cuestionario de Desorden de Ansiedad Generalizada GAD-7, en una muestra comunitaria (n = 206) de adultos brasileños. La muestra se compone de 41% de mujeres, con una edad media de 21,10 (SD = 4,49), y 75,6% estudiantes universitarios. El análisis factorial confi rmatorio proporcionó evidencias que soportan el modelo unidimensional original del GAD-7 en el contexto brasileño. El análisis de variancia (ANOVA) mostró que las puntuaciones fueron signifi cativamente diferentes entre hombres y mujeres, siendo que las mujeres obtuvieron puntuaciones más altas que los hombres. La escala demostró tener una buena confi abilidad; el coefi ciente de alpha de Cronbach (α = .916) y el coefi ciente rho de fi abilidad (ρ = .909) fueron adecuados. El análisis de datos mostró los ítems 5 y 7 tuvieron los umbrales de gravedad más altos en el trazo de ansiedad generalizada, mientras el ítem 1 tuvo el más bajo. Nuestros hallazgos sugieren que la versión en portugués brasileño de GAD-7 es adecuado para la evaluación de síntomas de trastorno de ansiedad generalizada en adultos brasileños en contextos comunitarios