975 resultados para Biotechnology
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Dissertation to obtain master degree in Biotechnology
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Dissertation to obtain Master Degree in Biotechnology
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Hidrolases englobam um grupo de enzimas que catalisam a quebra de ligaes covalentes em reao com gua; entre elas esto as proteases, amilases, lipases, pectinases, celulases e catalases. Essas enzimas so muito importantes, com ampla utilizao na indstria em geral. O solo um ambiente muito rico e diverso em microrganismos, sendo considerado a maior fonte para obteno de substncias, enzimas e antibiticos, por exemplo. Com a metagenmica, passou a ser possvel acessar melhor esse potencial microbiano, permitindo a descoberta de novos genes e biomolculas. Neste estudo foram coletadas amostras de solos (0-10 cm de profundidade) do norte do Paran visando buscar hidrolases microbianas funcionais. Foi realizada a extrao do DNA de um Latossolo Vermelho Eutrofrrico sob quatro manejos de solo e de culturas distintos e as amostras foram submetidas ao sequenciamento utilizando a plataforma 454 (Applied Science). As sequncias de DNA foram comparadas com o banco de dados no redundante (NR) do NCBI (National Center for Biotechnology Information) e KEGG (Kyoto Encyclopedia of Genes and Genomes) para busca de similaridade com proteases, amilases, lipases, pectinases, celulases e catalases. A partir do DNA total foram realizadas reaes de PCR (Polymerase Chain Reaction) com primers degenerados direcionados para a amplificao de pectinases, celulases e lacases e os produtos de PCR foram purificados com Purelink kit (Invitrogen) e sequenciados (ABI 3500xL, Aplied Biosystems). A comparao com as sequncias do NCBI e KEGG resultou na identificao de 1.137 sequncias com grande similaridade com a enzima lacase; 16.883 sequncias para celulase; 2.001 para pectinase; 1.006 para amilase; e 3.725 para lipase. Esses resultados mostram que esses solos agrcolas representam uma fonte importante de recursos biolgicos para aplicao industrial, principalmente de enzimas celulases. At o presente momento, o sequenciamento de 26 produtos amplificados por PCR apresentou identidade para uma amostra, que foi identificada como a enzima celulase.
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Dissertation presented to obtain the Ph.D degree in Engineering and Technology Sciences-Biotechnology
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Dissertation presented to obtain the Ph.D degree in Engineering and Technology Sciences, Biotechnology.
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Cyanobacteria are photoautotrophic microorganisms with great potential for the biotechnological industry due to their low nutrient requirements, photosynthetic capacities and metabolic plasticity. In biotechnology, the energy sector is one of the main targets for their utilization, especially to produce the so called third generation biofuels, which are regarded as one of the best replacements for petroleum-based fuels. Although, several issues could be solved, others arise from the use of cyanobacteria, namely the need for high amounts of freshwater and contamination/predation by other microorganisms that affect cultivation efficiencies. The cultivation of cyanobacteria in seawater could solve this issue, since it has a very stable and rich chemical composition. Among cyanobacteria, the model microorganism Synechocystis sp. PCC 6803 is one of the most studied with its genome fully sequenced and genomic, transcriptomic and proteomic data available to better predict its phenotypic behaviors/characteristics. Despite suitable for genetic engineering and implementation as a microbial cell factory, Synechocystis growth rate is negatively affected by increasing salinity levels. Therefore, it is important to improve. To achieve this, several strategies involving the constitutive overexpression of the native genes encoding the proteins involved in the production of the compatible solute glucosylglycerol were implemented, following synthetic biology principles. A preliminary transcription analysis of selected mutants revealed that the assembled synthetic devices are functional at the transcriptional level. However, under different salinities, the mutants did not show improved robustness to salinity in terms of growth, compared with the wild-type. Nevertheless, some mutants carrying synthetic devices appear to have a better physiological response under seawaters NaCl concentration than in 0% (w/v) NaCl.
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RESUMO: Os mais recentes mtodos de neuro imagem tal como a Ressonncia Magntica (RM) permitiram obter imagens detalhadas do crebro humano in vivo. Essas imagens revelam, muitas vezes, achados imprevistos face ao padro normal, com elevada propenso para os indivduos idosos e franca coexistncia com fatores de risco vascular, como caracterstica dum processo de envelhecimento normal. Embora na ltima dcada tenham surgido vrias publicaes sobre este assunto, ele continua ainda pouco explicado. Um pouco por todo o mundo tm emergido os programas de preveno da doena e promoo da sade desenvolvidos pela Sade Pblica suportadas sobretudo pelo avano das tecnologias mdicas que resultaram, entre outros impactos, num crescimento da populao idosa. Estima-se, em 2030, uma composio demogrfica com 20% de indivduos acima dos 65 anos. Neste contexto, a doena microvascular cerebral a causa mais frequente de comprometimento cognitivo vascular no idoso sendo as caractersticas senescentes na imagem por RM do tipo leses isqumicas da Substncia Branca (Leukoaraiosis) e enfartes lacunares (Status lacunar), atrofia cerebral, gliose e acumulao excessiva de ferro nos ncleos da base. Esta tese, considerando a linha de investigao de que deriva Cincias da Vida - especializao em Medicina Clinica - Biotecnologia, rene e reflete sobre trs vertentes ligadas RM e interdependentes em relao a uma problemtica comum. A primeira trata da caracterizao da Tecnologia por Ressonncia Magntica existente em Portugal, a qual inclui uma avaliao exploratria da aplicao da tcnica de Difuso Anisotrpica nos estudos cerebrais. As dimenses analticas estudadas foram a Tecnolgica, Sociodemogrfica e Econmica. Na recolha de dados recorreu-se a vrias fontes de informao e a uma metodologia exploratria faseada, validada pela triangulao dos resultados. A sua anlise obedeceu a critrios de estratificao e agrupamento segundo as mesmas dimenses analticas. Otimizao da anisotropia fronto-calosa e [RM 1,5T] no idoso normal e com risco crebro-vascular A segunda descreve o estudo antomo radiolgico que recaiu sobre parmetros de quantificao assente na temtica do crebro do idoso em cadver segundo uma metodologia experimental aplicada s mtricas da difuso por RM. Na terceira, e ltima, apresentado o estudo tcnico - radiolgico para avaliao e otimizao da imagem ponderada em difuso em estudos clnicos associados ao crebro do idoso The Usual Brain Aging ou Envelhecimento Cerebral Normal, com base metodolgica assente nos critrios e indicadores estabelecidos pelo Estudo de Imagem de Roterdo (Rotterdam Scan Study - RSS). Como principais resultados obteve-se que no existem em Portugal estruturas para avaliao dos equipamentos pesados ou Agncia de Avaliao das Tecnologias da Sade para desenvolver o importante papel da produo de estudos comparativos entre os equipamentos disponveis no mercado, a relao preo-qualidade e a sua afetao s necessidades clnico-epidemiolgicas. Constatou-se que a implementao de equipamentos de RM est fortemente assente em critrios econmicos carecendo de recomendaes e diretivas para o uso racionalizado destas tecnologias. Quanto a dados quantitativos conclumos que a maioria dos equipamentos est instalada em instituies privadas (80,2%); a intensidade de campo magntico mais frequente [1,5T] com 119 equipamentos; os equipamentos esto instalados maioritariamente nos distritos de Lisboa (55 unidades) e do Porto (39 unidades); o rcio mdio de equipamentos por habitante em Portugal de 1 para 65 195 habitantes; a amplitude de gradientes com maior expresso na amostra 30-39mT/m; a maioria dos equipamentos foi instalada no intervalo temporal [2009-2012] com 59 equipamentos; apenas 6 instituies desenvolvem investigao clinica e a maioria das bobinas para estudos de crnio so do tipo Array. O estudo de otimizao da tcnica da difuso revelou, quanto avaliao dos valoresb, que os mais baixos (b=500 s/mm2 e b=1000 s/mm2), apresentam maior IS e SNR sendo esta uma boa medida referente qualidade de imagem, no entanto, os valores-b mais elevados (b=2000 s/mm2) apresentam maior CNR e CR, face aos anteriores, o Otimizao da anisotropia fronto-calosa e [RM 1,5T] no idoso normal e com risco crebro-vascular que apesar de proporcionar inferior detalhe anatmico e consequentemente inferior qualidade de imagem, num encfalo normal, pode auxiliar na interpretao e apresentar vantagens na identificao de leses microvasculares sempre que persistirem dvidas em relao ao diagnstico diferencial de doena microvascular do tipo status lacunar ou Hiperintensidades da Substncia Branca. As alteraes deste parmetro so particularmente refletidas nas diferenas da avaliao da qualidade de imagem na regio fronto-calos Conclumos da avaliao quantitativa da concentrao mdia de ferro (26Fe) em todas as faixas etrias que os ncleos da base que apresentam maior concentrao so, por ordem decrescente: Substncia Nigra, Globus Pallidus, Putamen, Tlamo, Ncleo Rubro e Ncleo Caudado; que existe uma predominncia na concentrao de ferro (26Fe) no hemisfrio esquerdo e que os indivduos do gnero masculino apresentam mais ferro (26Fe) que os do gnero feminino nas faixas etrias [30-40[, [40-50[ e [50-60[. Como principal concluso do estudo da concentrao mdia de ferro em relao idade destacamos que a concentrao mdia de ferro (26Fe) superior nos grupos etrios superiores, logo aumenta com a idade, sobretudo na Substncia Nigra e no Ncleo Lenticular. No estudo tcnico-radiolgico encontrmos evidncias do aumento da difusibilidade da gua na substncia branca subcortical dos sujeitos idosos comparativamente aos mais jovens. Uma relao idntica foi avaliada nos tlamos. O aumento relacionado com a idade parece ser predominantemente observado em doentes com mais de 65 anos de idade o que pode refletir alteraes estruturais ligeiras associados ao envelhecimento normal. Os resultados indicam que a anlise quantitativa das imagens ponderadas em difuso fornece informaes, sobre a estrutura do crebro, as quai s no esto disponveis apenas por inspeo visual, tanto nas imagens ponderadas em difuso como em outras sequncias de aplicao clnica de rotina. Para fazer face s desvantagens dos sistemas de quantificao das HSB os quais so dispendiosos, complexos, requererem tecnologia e formao especficas, recomendamos que a aplicao automatizada GUIAL, desenvolvida ao longo do nosso trabalho de utilizao elementar e prtica para que seja introduzida nos sistemas de aquisio de imagem por RM com o fim de integrar o processamento de imagem nos indivduos portadores de fatores de risco vascular. A avaliao do ADC, nesta amostra, permitiu concluir que a variao desta varivel explicada estatisticamente pela existncia da condio clnica de status lacunar em ambos os hemisfrios ou por outras palavras, o status lacunar influencia o valor de ADC. Embora uma pequena percentagem da variao do ADC seja explicada pelo gnero, o ADC nos homens foi superior ao das mulheres o que no nos surpreende, pois so tambm os homens onde a frequncia de doena microvascular se revelou mais expressiva. Os valores do ADC, globalmente, entre os hemisfrios cerebrais no mostraram alteraes exceto na SB entre os idosos e no idosos. A SB da regio frontal mostrou valores diminudos na anisotropia e isotropia face s restantes reas anatmicas. Os estudos indicam que na idade avanada existe uma maior predisposio para suscetibilidades de estrutura com status de desconexo. A classificao das HSB foi superior em indivduos mais velhos e com status lacunar, e em menor quantidade (inferior rating de classificao) nos indivduos idosos sem status lacunar. As alteraes volumtricas foram mais frequentes no homem do que na mulher, presumivelmente devido associao com a elevada classificao de status lacunar. Um aumento do ndice de Evan correspondeu, neste estudo, ao aumento das HSB, diminuio do volume cerebral total, expanso ventrculo-sulcal frontal e ao aumento da medida do ngulo caloso. Estes resultados foram agravados pela classificao elevada de status lacunar nos indivduos que apresentaram indcios de doena de pequenos vasos, com manifesto aumento dos espaos de Virchow-Robin,enfartes lacunares ou HSB. Esses resultados foram mais expressivos no gnero masculino do que no feminino revelando uma maior vulnerabilidade sobretudo na atrofia frontal nos homens. Por sua vez as dimenses do Corpo Caloso tornaram-se reduzidas devido compresso dos ventrculos laterais e terceiro ventrculo. Estes indicadores tiveram expresso particularmente nos indivduos com mais de 65 anos. ---------------------------------------------------------------------------------------------ABSTRACT: The latest neuroimaging methods, such as Magnetic Resonance Imaging (MRI), have enabled detailed images of in vivo human brain. These images reveal often unexpected findings related to the normal pattern, with high predisposition for the elderly people with forthright coexistence with vascular risk factors such as characteristics of a normal aging process. Although it has been, in the last decade, several publications on this subject, it is still little explained. All over the world have emerged disease prevention programs and health promotion developed by the Public Health sector, supported mainly by the advancement of medical technologies that have resulted, among other impacts, in a growing of the elderly population. It is estimated, in 2030, a demographic composition with 20% o people over 65 years. In this context, microvascular disease is the most common cause of cognitive vascular impairment in the elderly and senescent characteristics in the MRI trough ischemic lesions of the white matter (Leukoaraiosis) and lacunar infarcts (lacunar status), cerebral atrophy, gliosis and iron accumulation in the basal ganglia in excess.This thesis, considering the research line that stems - Life Sciences - specialization in Clinical Medicine, Biotechnology, gathers and reflects on three aspects linked to MR, interdependent and related to a common problem. The first deals with the Magnetic Resonance Technology characterization in Portugal, which includes an exploratory evaluation of the implementation of Anisotropic Diffusion technique in brain studies. The analytical dimensions studied were the Technologic, Socio-demographic and Economics. Collecting data was supported by different sources of information and was applied an exploratory methodology whose results were validated by triangulation. The research method was grouped and stratified criteria under the same analytical dimensions. The second describes the anatomical study - which was focused on radiological measurement parameters, based on the brains specimen under an experimental methodology applied to MRI diffusion metrics. Radiological evaluation and optimization of the weighted image diffusion in clinical studies were associated with the brain of the elderly "The Usual Brain Aging" with methodological basis based on established criteria and indicators by Rotterdam Scan Study (RSS). The main results obtained reveal the inexistence frameworks in Portugal for evaluation of equipments or Agency of Health Technology to produce studies comparing the equipment available on the market, the value for money and its allocation to clinical and epidemiological needs. It was found that the implementation of MRI equipment is strongly based on economic criteria lacking recommendations and guidelines for the rationalized use of these technologies. As the quantitative data we conclude that most of the scanners are located in private clinical institutions (80,2%); the most frequent magnetic field intensity is [1.5T] with 119 scanners; the scanners are mainly installed in Lisbon (55 units) and Porto (39 units) districts; the average ratio of equipment per capita in Portugal is 1 to 65 195 people; the gradient power with higher expression in the sample is 30-39mT / m;most of the scanners were installed in the years range [2009-2012 years] with 59 equipment; only 6 clinical placements develop clinical research and the most coils for brain studies are of Array type. The optimization study of diffusion technique revealed, as the assessment of the bvalues, the lower (b = 500 s / mm2 and b = 1000 s / mm 2), promotes an increase in the SI and SNR being this measure related to a higher image quality, however the highest b values (b = 2000 s / mm 2) have a higher CNR (Contrast to Noise-Ratio) and CR (Contrast Ratio), compared to the previous ones. This may provide less anatomical details and, thus, ower image quality, of a normal brain, however can help the interpretation and have advantages in identifying microvascular injuries when doubts persist regarding the differential diagnosis of microvascular disease of lacunar or WMH (White Matter Hyperintensities) status type. Changes on this parameter are Otimizao da anisotropia fronto-calosa e [RM 1,5T] no idoso normal e com risco crebro-vascular particularly reflected in the differences of image quality evaluation in the frontocallosum anatomical area. We conclude from the quantitative assessment of the average concentration of iron (26Fe), in all age groups to the basal ganglia, that the higher concentrations are, in descending order: Nigral Substantia, Globus pallidus, Putamen, Thalamus, Rubio nucleus and Caudate nucleus; that there is a predominance in the concentration of iron (26Fe) in the left hemisphere and that male gender show higher iron (26Fe) level tha females, in the age groups [30-40 [[40-50 [and [50- 60 [. Regarding a main conclusion of the mean concentration study of iron, in terms of age we point out that the average concentration of iron (26Fe) is higher among older groups and increases with age, especially in Nigral Substantia and Lenticular Nucleus. On the technical and radiological study we found evidence of an increased in water /diffusivity in the ubcortical white matter of the elderly compared with younger subjects. A similar relationship was assessed in the Thalamus. The increase agerelated seems to be predominantly observed in patients over 65 years which may reflect minor structural changes associated with normal aging. The results indicate that quantitative analysis of diffusion weighted imaging can provide information about the structure of the brain which is not reached only by visual inspection or standard sequences applied in clinical routine. To address the disadvantages the systems of quantification of WMH which the authors state that are costly, complex, require specific technology and training, we recommend that the automated application GUIAL, developed over our work is basic and practical to use and to be introduced in MR image systems acquisition in order to integrate image processing in patients with vascular risk factors. The evaluation of the ADC showed that its variation is statistically explained by the existence of the medical condition of lacunar status, in both hemispheres, or in other words, the lacunar status influences the ADC value. Although a small percentage of the ADC variation is explained by gender, the ADC in men was higher than women which Otimizao da anisotropia fronto-calosa e [RM 1,5T] no idoso normal e com risco crebro-vascular do not surprise us, since they are also men where the frequency of microvasculardisease has proved more significant. The values of ADC, overall, between the cerebral hemispheres showed no changes but were different in WM among the elderly and non-elderly subjects.The WM's forehead showed decreased values in anisotropy and isotropy face the other anatomical areas. The studies indicate that in old age there is a greater tendency to higher susceptibility to disconnection- status framework. The classification of WMH was higher in elderly people and lacunar status, and fewer (lower classification rating) in the elderly without lacunar status. volumetric changes were more frequent in men than in women, most probably because of its association with high lacunar status rating. An increase of Evan index corresponded, in this study, to an increase in WMH, to a decreased of total brain volume, to a ventricle sulcal frontal and callous angle expansion. These results were wound up by high ranking of lacunar status in subjects who had small vessel disease, clear increase in spaces of Virchow-Robin, lacunar infarctions or WMH. These results were more significant in males than in females revealing vulnerability particularly in the frontal atrophy in men. In turn the size of Corpus Callosum because reduced due to the compression of the lateral and third ventricles. These indicators had expression particularly in individuals over 65 years.
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Dissertao de mestrado em Estudos de Gesto
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Triple negative breast cancer (TNBC) is a particular immunopathological subtype of breast cancer that lacks expression of estrogen and progesterone receptors (ER/PR) and amplification of the human epidermal growth factor receptor 2 (HER2) gene. Characterized by aggressive and metastatic phenotypes and high rates of relapse, TNBC is the only breast cancer subgroup still lacking effective therapeutic options, thus presenting the worst prognosis. The development of targeted therapies, as well as early diagnosis methods, is vital to ensure an adequate and timely therapeutic intervention in patients with TNBC. This review intends to discuss potentially emerging approaches for the diagnosis and treatment of TNBC patients, with a special focus on nano-based solutions that actively target these particular tumors.
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Candida bracarensis is an uncommon Candida species found during an epidemiological study of candidiasis performed in Braga, Portugal. Initially, it was identified as C. glabrata, but recently detailed analyses pointed out their differences. So, little information is still available about C. bracarensis virulence factors and antifungal susceptibilities. Therefore, the main goal of this work is to evaluate the ability of C. bracarensis to form biofilms, to produce hydrolytic enzymes (proteases, phospholipases and hemolysins), as well as its susceptibility to amphotericin B and fluconazole. It was shown, for the first time, that all C. bracarensis strains were able to form biofilms and display proteinase and hemolytic activities. Moreover, although planktonic cells presented antifungal susceptibility, amphotericin B and fluconazole were unable to inhibit biofilm formation and eradicate pre-formed biofilms. Due to the propensity of C. bracarensis to display antifungal resistance and virulence attributes, the control of these emerging pathogens is recommended.
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Major advances in the development and use of antimicrobial textiles to control bacterial proliferation on wound beds continue. However, wound dressings are, in general, not included in standardized regimens for measuring and monitoring their antimicrobial effectiveness. This work adapts these methods to assess the antibacterial activity of textiles designed for wound healing purposes. Environmental conditions representative of those present at the wound site (i.e., moisture levels, infection, and available nutrients) were evaluated. This work shows that moisture levels were the environmental factor that had the greatest influence on the antimicrobial agent activities tested. These results suggest that it is possible to use the more representative environmental conditions present on the wound bed for in vitro screening of textile antimicrobial activity.
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Yarrowia lipolytica, a yeast strain with a huge biotechnological potential, capable to produce metabolites such as -decalactone, citric acid, intracellular lipids and enzymes, possesses the ability to change its morphology in response to environmental conditions. In the present study, a quantitative image analysis (QIA) procedure was developed for the identification and quantification of Y. lipolytica W29 and MTLY40-2P strains dimorphic growth, cultivated in batch cultures on hydrophilic (glucose and N-acetylglucosamine (GlcNAc) and hydrophobic (olive oil and castor oil) media. The morphological characterization of yeast cells by QIA techniques revealed that hydrophobic carbon sources, namely castor oil, should be preferred for both strains growth in the yeast single cell morphotype. On the other hand, hydrophilic sugars, namely glucose and GlcNAc caused a dimorphic transition growth towards the hyphae morphotype. Experiments for -decalactone production with MTLY40-2P strain in two distinct morphotypes (yeast single cells and hyphae cells) were also performed. The obtained results showed the adequacy of the proposed morphology monitoring tool in relation to each morphotype on the aroma production ability. The present work allowed establishing that QIA techniques can be a valuable tool for the identification of the best culture conditions for industrial processes implementation.
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Context: Caffeic acid is described as antibacterial, but this bioactive molecule has some issues regarding solubility and stability to environmental stress. Thus, encapsulation devices are required. Objective: The aim of this work was to study the effect of the caffeic acid encapsulation by cyclodextrins on its antibacterial activity. Materials and methods: The interactions between the caffeic acid and three cyclodextrins (-cyclodextrin (CD), 2-hydroxypropyl--cyclodextrin (HPCD) and methyl--cyclodextrin were study. Results and discussion: The formation of an aqueous soluble inclusion complex was confirmed for CD and HPCD with a 1:1 stoichiometry. The CD/caffeic acid complex showed higher stability than HPCD/caffeic acid. Caffeic acid antibacterial activity was similar at pH 3 and pH 5 against the three bacteria (K. pneumoniae, S. epidermidis and S. aureus). Conclusions: The antibacterial activity of the inclusion complexes was described here for the first time and it was shown that the caffeic acid activity was remarkably enhanced by the cyclodextrins encapsulation.
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PhD thesis in Bioengineering
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Tese de Doutoramento em Cincias - Especialidade em Biologia
