ESB - Dissertações de Mestrado / Master Dissertations
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Browsing ESB - Dissertações de Mestrado / Master Dissertations by Field of Science and Technology (FOS) "Engenharia e Tecnologia::Engenharia Química"
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- Cork industry waste product valorisation : bioactivity on human keratinocytesPublication . Salvador, Maria Inês Vaz; Ferreira, António César da Silva; Keating, Elisa Oliveira BragaWound healing, a complex process that involves hemostasis, inflammation, proliferation and tissue remodelling, gathers deep scientific interest, since acute and chronic wounds are common as well as pathologic under or overhealing. Cork powder is one of the major waste derived residues in the cork industry, known to possess a strong phenolic content. We herein hypothesized that the products obtained from the cork liquefaction process may have biomedical properties particularly in the wound healing process. In this context, the aim of this work was to investigate the effect of several extracts obtained by manipulation of liquefied cork upon keratinocyte viability, migration and proliferation, key cellular events in the wound healing process. To do this, liquefied (LIQ) cork was produced and it was fractionated in water soluble (WS) and organic (ORG) extracts. Additionally, a glue dispersion (GLUE) was prepared by the addiction of WS extract to distilled water, Desmodur DA-L and cork powder on a ratio of 3:20:1.2:0.2. Several dilutions of each fraction were prepared (1:1, 1:5, 1:10, 1:100 and 1:1000) and used in 24-h treatment of the human keratinocyte HaCat cell line, for viability (MTT and LDH assays), migration capacity (wound-healing assay) and proliferation (BrdU assay) assessment. Additionally, total phenolic content (FolinCiocalteu method), and antioxidant potential (FRAP and DPPH) of each fraction were assessed. The liquefaction cork process achieved a biomass conversion of 94 %. Except for the highest concentration of ORG extract, the fractions were not cytotoxic and GLUE extract increased cell survival. All the cork extracts were able to induce cell proliferation and except for the GLUE fraction, they also induced cell migration capacity. Regarding antioxidant properties, ORG fraction showed the highest reducing power and radical scavenging activity, although LIQ fraction was the richest in phenolic content. On the other hand, GLUE fraction had the lowest phenolic content and accordingly, the lowest reducing power and radical scavenging activity. In conclusion, liquefied cork and its subproducts reveal wound healing-promoting bioactivity. Overall, WS extract produced the most consistent cellular responses, being thus the most promising for a potential biomedical application in wound healing. Nevertheless, further studies are recommended, mainly in vivo tests, to validate these in vitro results.
- Development and characterization of new functional acorn beveragePublication . Costa, Maria do Rosário Leite da; Pintado, Maria Manuela EstevezAcorns are fruits from trees belonging to the genus Quercus. Portugal is one of the world's biggest producers of this fruit, however only a small part is used for animal feed, and the remaining is frequently left unharvest in the field. For many years, the acorn makes part of our diet being consumed at meals as a substitute for walnuts, peanuts or olives, incorporated into bread or cookies, or used to produce liqueurs and coffee substitutes. Acorn has high nutritional value containing components that can exert health benefits, for example the presence of non-hydrolysable starch. Given this, and knowing that today the consumer and industry seek more diverse and active functional foods, this work aimed at the development and characterization of a functional beverage made from acorn with and without incorporation of probiotic bacteria. So, acorn beverage was evaluated as matrix for incorporation of probiotic bacteria − Lactobacillus plantarum, L. acidophilus, L. paracasei and Bifidobacterium lactis B94, studying the impact of beverage supplementation with glucose, yeast extract, or both. After proving the positive effect of yeast extract on the growth of selected bacteria, the growth of each microorganism in the original beverage and beverage supplemented with yeast extract, as well as its the viability throughout 28 d of refrigerated storage was characterized. The antioxidant capacity and phenolics content as well as the protein content were evaluated in all stages. For the non-fermented acorn beverage and fermented beverage with appropriate blend of probiotics, an in vitro evaluation of the impact of beverage in a batch system with human faecal inocula of two volunteers was assessed for 48 h. In order to evaluate the potential benefits of the beverage in the balance of the intestinal microbiota and metabolites produced by it, several groups of bacteria were quantified in faecal samples by implementing the qPCR technique, as well as the measurement of the content of short-chain fatty acids (SCFA) generated during 48 h. It was found that the plain beverage maintain the growth of each microorganism tested, reaching 107 CFU/mL after 24 h, which was maintained throughout the 28 days of refrigerated storage. The antioxidant capacity of this beverage was 0.501 ± 0.00 g of AAE/L, the phenolic content 0.312 ± 0.01 g of GAE/L and protein content 0.893 ± 0.00 g/L. Along the 28 days of refrigerated storage there was the reduction of antioxidant activity to 0.287 ± 0.06 g of AAE/L. The positive impact of fermented beverage and non-fermented beverage on the intestinal microbiota was confirmed mainly by the selective increase of SCFA, since the implemented qPCR technique did not allow reliable results in the selective quantification of representing groups from microbiota. Thus, the beneficial effect of non-fermented and fermented beverage was demonstrated by the significant increase of SCFA (propionic acid and acetic acid) and especially lactic acid, with increments even superior to those observed for the inulin, used as a positive control. Thus, it was possible to develop an acorn beverage with prebiotic properties and a symbiotic acorn beverage by incorporating probiotics, demonstrating both the potential ability to modulate the intestinal microbiota, in addition to its antioxidant potential.
- Development of a reade-in routine to aid quantitative analysis of cardiac 1H-MRS DataPublication . Santos, José Tiago Costa; Schneider, JurgenA Espectroscopia por Ressonância Magnética (ERM) é a única técnica não-invasiva e não radioactiva que permite investigar o metabolismo dos tecidos vivos. A ERM do protão 1H, que proporciona a maior sensibilidade de todos os núcleos visíveis por RM, é um método capaz de detectar e quantificar biomoléculas cardíacas específicas. No entanto, os metabolitos estudados com ERM estão presentes em concentrações que são várias ordens de grandeza inferiores às dos protões da água, o que faz com que a 1H-ERM não seja ainda utilizada na prática clínica devido a desafios metodológicos. Muitos estudos têm vindo a ser realizados a fim de melhorar os meios de quantificação e o BMRU tem estado na vanguarda do desenvolvimento de uma plataforma de análise de dados de ERM. Portanto, este projecto tem como objectivo desenvolver uma rotina capaz de ler dados de ERM da Siemens no formato específico TWIX e realizar uma análise piloto sobre dados 1H-ERM provenientes de coração humano. O trabalho realizado comtemplou diversas etapas. Inicialmente foi feito um estudo detalhado em relação aos ficheiros TWIX da Siemens, recorrendo a um algoritmo escrito em Matlab que tem o propósito de ler este tipo de ficheiro. De seguida, foi desenvolvida em IDL (Interactive Data Language) a rotina para leitura de ficheiros TWIX da Siemens que devolve os dados de ERM não processados no domínio do tempo. Foram analisados em jMRUI 14 conjuntos de dados de Espectroscopia por Resonância Magnética 1H relativos a coração humano, no domínio do tempo, usando Lorentzian line shape, e comparou-se os resultados para o sinal de água com os obtidos anteriormente por Rial et al. (2011), para os mesmos dados. Os mesmos espectros foram re-analisados utilizando o mesmo modelo mas no domínio da frequência, utilizando o software bmru_mrsw, onde foi incorporada a rotina desenvolvida. Finalmente foram analisados os mesmos conjuntos de dados usando Voigt line shape, no domínio da frequência, novamente com o software bmru_mrsw. Houve conformidade entre os resultados obtidos na análise dos dados de ERM com o sofware jMRUI e os publicados por Rial et al. (2011), com um coeficiente de correlação de 0,997. A diferença média entre as duas medições do sinal de água foi de 0,06, segundo o gráfico Bland-Altman. A análise dos dados, no domínio da frequência, com o software bmru_mrsw demonstrou concordância com os resultados obtidos pelo jMRUI, tendo-se obtido um coeficiente de correlação de 0,716 entre os resultados. A quantificação do sinal de água utilizando Voigt line shape, no software bmru_mrsw, demonstrou diminuição dos resíduos gerados. Deste modo, foi possível com este projecto a criação de uma rotina capaz de ler data de Espectroscopia por Ressonância Magnética da Siemens no formato específico TWIX. Esta rotina conferiu ao software bmru_mrsw compatibilidade com este formato de ficheiros, tendo deste modo contribuído para o aperfeiçoamento das técnicas de quantificação de sinais de espectroscopia. A potencialidade da abordagem de análise no domínio do tempo e frequência foi também demonstrada. Os objectivos propostos neste projecto foram alcançados com sucesso, podendo este ter contribuído para o papel da Espectroscopia por Ressonância Magnética no campo da cardiologia clínica.
- Estudo da eficácia antimicrobiana de revestimentos de dióxido de titânio (TiO2) com propriedades fotocatalíticasPublication . Quintas, Vitória Raquel Ferreira; Pintado, Maria Manuela Estevez; Teixeira, PaulaEm diferentes áreas, há necessidade de garantir ambientes higienicamente seguros que permitam a proteção contínua através da redução de contaminantes microbianos e agentes patogénicos. Novas soluções têm sido desenvolvidas nos últimos anos para resolver este problema, entre as quais se incluem os sistemas de fotocatálise. O sistema Pure health consiste em revestimentos com dióxido de titânio (TiO2). A atividade antibacteriana de TiO2 fotocatalítico baseia-se na reação de oxidação produzida pela sua irradiação, utilizando lâmpadas fluorescentes de espetro solar, a interação entre a radiação e o revestimento origina radicais hidroxilo e iões superóxido que são eficazes contra bactérias. Estes materiais podem ser aplicados em ambientes ao ar livre, em espaços interiores e áreas públicas (praças, hospitais, escolas, aeroportos). Neste estudo, a eficiência (redução microbiana) de materiais como PVC e cerâmica revestidos com TiO2 foi testada, contra vários agentes patogénicos relevantes, frequentemente encontrados em ambientes clínicos e alimentares: Escherichia coli, Staphylococcus aureus, Acinetobacter baumannii, Listeria monocytogenes, Pseudomonas aeruginosa e Salmonella spp.. As amostras de PVC com e sem revestimento foram analisadas por microscopia eletrónica de varrimento. As imagens obtidas evidenciam uma “ película” no material com revestimento, a qual não é observada no material controlo. O material em estudo foi implementado numa cantina, sendo efetuadas amostragens, ao longo de duas semanas, para avaliar o impacto do revestimento em ambiente real. Tendo como principal objetivo determinar a eficácia antimicrobiana dos revestimentos de diferentes matrizes (PVC e cerâmica), foram realizados ensaios experimentais utilizando como metodologia a ISO 22196:2007. Em geral, de 0,4 ml de um inoculo padrão (105 UFC / cm2) de cada microrganismo foi aplicado sobre uma área fixa do material e submetido à luz, durante 24 horas. A redução da carga microbiana durante este período foi monitorada e a eficiência antibacteriana determinada. Ao mesmo tempo foram testados: um controlo não exposto à luz com revestimento ativo e dois controlos, com o mesmo material sem revestimento ativo (com e sem luz). Para todos os microrganismos investigados, foram observadas reduções significativas, entre 1 e 2 ciclos logarítmicos dependendo do material e bactéria em estudo.
- Novel hyaluronan-based hydrogels obtained by supramolecular crosslinking with peptidesPublication . Faria, Ana Luísa de Oliveira Leite; Azevedo, Helena; Radvar, ElhamIn recent years, self-assembled biomaterials using peptides as building blocks have been developed as a solution for several medical challenges. Peptides offer a great diversity of biochemical properties and, through the combination of different amino acids, it is possible to produce a variety of peptide-based structures with specific proprieties with interest for regenerative medicine and tissue engineering. In the present project, novel hyaluronan base hydrogels obtained by supramolecular crosslinking with peptides were developed for biomedical application. Thus, different designs of peptides, containing a positively charged residue at one end and an aromatic residue at other end, were synthesized to form supramolecular interactions with hyaluronic acid (HA). The mass of the synthesised peptides was confirmed by Electrospray Ionization Mass Spectrometry (ESI-MS), their purity by High Performance Liquid Chromatography (HPLC) and their secondary structure by Circular Dichroism (CD) spectroscopy. In order to investigate gel formation, the peptides, in powder form and in solution of different concentrations at neutral and acidic pH, were combined with 1% and 2% w/v HA solutions of different molecular weights, following different strategies. Observation of gel formation was expected through electrostatic interactions between the positively charge residue of the peptide with HA (negatively charged) and π-π stacking between aromatic residues of the peptides. The resulted hydrogels were characterized by Scanning Electron Microscopy (SEM), revealing a network of nasnofibrous structures. The concentration of the HA solutions, different molecular weights HA and the peptide design showed to have an effect on the density, organization and packing of the fibers.
- Production and molecular characterization of enzymes related with the decolourisation ability of synthetic reactive dyes by yeast strainsPublication . Canaza Jorges, Alejandro; Pintado, Maria Manuela Estevez; Moreira, Patricia; Castro, PaulaThe present research work focused on the decolourisation of reactive dyes mediated by yeast. It comprises the decolourisation of the dyes Remazol Black B-A, Remazol Yellow RR, Levafix Blue CA, Levafix Red CA, Everzol Yellow, Sumifix Yellow, Sumifix Red, Everzol Red, Samofix Black, Navy Everzol ED, Sumifix Blue ESC, Sumifix Blue and Everzol Blue LED, mediated individually by yeast strains LIIIST7 (Candida tropicalis), LIVST11 (Saccharomycete sp), HOMOGS20 (Candida parapsilosis), HOMOGST27 (Yarrowia lipolytica), HOMOGST27A (Yarrowia lipolytica), LIIIS36 (Candida pseudoglaebosa) isolated from wastewater treatment plant and strain HOMOGST31 (Kluyveromyces marxianus) isolated from cheese. The strains were morphologically characterized in PDA and species identification was performed by molecular biology methods using both ITS (ITS 4 – ITS 5) and 26S rDNA (NL 1 – NL 4) set of primers. Colour reduction was assessed in Normal Solid Decolourisation Media NSDM and Normal Decolourisation Media NDM supplied with individual dyes and also with a mix of most of the dyes. The strains exhibited different processes of decolourisation, which occurred through mechanisms of biosorption (colourful yeast cells pellet) or through true decolourisation mechanisms. The presence of colourless yeast cell pellet associated with supernatant reduction of colour indicates an underlying biodegradation mechanism. LIIIS36 strain exhibited high tolerance and decolourisation capacity in solid media and the highest efficiency in decolourisation for all the dyes tested in a period of time ranging between 12 – 24 h. Enzymatic activities of manganese peroxidase (MnP), azoreductase, oxidoreductase and laccase were detected for LIIIS36, with the reductase activity being the most relevant. The intracellular extract was used to perform protein separation trough FPLC, which allowed the purification of protein fractions that afterwards showed oxidoreductase activity. Phytotoxicity response for the decolourisation products from LIIIS36 cultures was performed using a Trifolium pratense assay resulting in the effective concentration (EC) similar both for the decolourisation supernatant and the supernatant of yeast growth without dyes. Candida pseudoglaebosa LIIIS36 has revealed important traits which make it a promising strain for a yeast-based biological decolourisation process for reactive dyes.
- Regulation of the pore-forming translocon of the type III secretion system by the EspC Serine Protease in Enteopathogenic Escherichia ColiPublication . Gomes, Leticia Tavares; Nhieu, Guy Tran VanAs bactérias do grupo Escherichia coli enteropatogénica (EPEC) são uma das maiores causas de diarreia fatal em crianças com menos de cinco ano nos países em desenvolvimento. A virulência de EPEC depende da presença do sistema de secreção do tipo III (SST3). O SST3 é constituído por um corpo basal situado entre a membrana externa e interna da bactéria, prolongado por uma agulha que é projetada a partir da superfície da bactéria. No caso de EPEC, a agulha do SST3 é prolongada por um filamento formado pela proteína hidrofílica, EspA. Depois de contactar com a célula hospedeira, o sistema ativa a secreção de duas proteínas hidrofóbicas, EspD e EspB. Estas proteínas inserem-se na membrana plasmática da célula hospedeira para formar o transclocão. O translocão é uma estrutura chave para formar o poro na membrana da célula hospedeira pelo qual os efetores são inseridos na célula. O laboratório onde se realizou este trabalho estuda o papel da proteína EspC, que pertence à família SPATE (serine protease autotransporter of Enterobacteriaceae), na regulação e funcionamento do translocão. EspC é secretada pelo sistema de secreção do tipo V, sendo independente do SST3. Foi demonstrado que EspC tem um papel importante no controlo da formação do poro membranar e da virulência mediada pelo SST3. EspC tem preferencialmente como alvo os complexos de EspA-EspD, envolvidos na formação do poro membranar, presumivelmente após o destacamento do translocão do filamento da agulha. Este trabalho de estágio utilizou procedimentos de purificação para isolar o complexo EspA-EspD sensível à proteólise de EspC com o objetivo de realizar a caracterização estrutural deste complexo por Microscopia Eletrónica. Implementamos várias fases de purificação distintas para aumentar a pureza das amostras contendo os complexos desejados. Conseguimos isolar complexos homogéneos contendo EspA que mostram um estrutura em forma de anel com um diâmetro externo de 10 nm e um interno de 4 nm.
- Synthesis and characterization of new oligosaccharides with prebiotic activityPublication . Montenegro, Maria Inês Pereira; Pintado, Maria Manuela Estevez; Cardelle-Cobas, Alejandra; Gullón, BeatrizThe importance of human intestinal microbiota in maintaining host health is well-known and in the past few decades, the consumer’s awareness for healthier foods has increased. There are several strategies to stimulate the proliferation of beneficial intestinal bacteria, including the consumption of prebiotics. Currently, there is a range of prebiotic carbohydrates on the market, most of them isolated from plant polysaccharides such as inulin and fructooligosaccharides (FOS) but there is an increasing interest in the development of new prebiotics, with added functionality. In this sense, chitosan, which is a polysaccharide composed of glucosamine (GlcN) and N-acetyl glucosamine (GlcNAc) units, linked by β (1→4) bonds, presents a structure very similar to prebiotic glucooligosaccharides. The main difference is the presence of amino groups, which are the cause of antimicrobial effect of chitosan. Chemical modification of chitosan by substitution of its amino groups could eliminate the antimicrobial effect and convert chitosan in a new interesting prebiotic ingredient. Thus, the objective of the present work is to chemically modify chitosan to be used in the food industry as prebiotic ingredient. In order to achieve this objective, the optimization of the synthesis of chitosan derivatives with glucose by the Maillard reaction and enzymatic hydrolysis, was conducted and the obtained purified derivatives were assayed for molar mass distribution and structural characterization by Size Exclusion Chromatography (SEC), Fourier Transform Infrared Spectroscopy (FT-IR), Proton Nuclear Magnetic Resonance (1H-NMR) and colloid titration. The purified product was assayed for its prebiotic potential by means of in vitro fermentability assays performed with individual microbial strains and human faecal inocula. The experimental data showed that the refined chitooligosaccharide (COS) derivatives obtained in this work had potential prebiotic effects, inducing changes in both the pattern of generated metabolic products and the count of Bifidobacterium, which might contribute to a healthy intestinal environment. Finally, the in vitro cytotoxicity of the COS derivatives synthesized was performed by flow cytometry. The results obtained demonstrated that the COS derivatives are biocompatible molecules. Also, the assay showed that the substitution of the amino groups decreased the cytotoxicity of the COS derivatives when compared to unmodified COS. Nevertheless, further studies are recommended, mainly in vivo tests, to eventually confirm these in vitro results.