ESS - DM - Bioquímica em Saúde
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Browsing ESS - DM - Bioquímica em Saúde by Field of Science and Technology (FOS) "Ciências da Saúde"
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- Development of a method based on HPLC-DAD for the detection and quantification of glutamate and gamma-aminobutyric acidPublication . Machado, Patrícia Alexandra Peixoto; Vieira, Mónica; Prudêncio, CristinaIntroduction: With the increasing incidence of neurodegenerative diseases, the need arose to study several molecules that may be useful to the study of new therapies and to treat the symptoms that come with these diseases, as is the case of depression. GABA and glutamate have a very important role in the homeostasis of the organism and molecules of interest and potential from the point of view of diagnosis are revealed. HPLC is a method that allows the detection and quantification of various analytes in different biological matrices, allowing rapid results with high precision, sensitivity and specificity. Objectives: Perform a meta-analysis to understand the role of depression in neurodegenerative diseases and develop a simple, fast, non-derivatizing method based on HPLC-DAD for the detection and quantification of GABA and glutamate. To test its applicability in several biological matrices, so that it can be used as an additional tool for the study of neurodegenerative pathologies. Materials and methods: The validation of the developed method was performed according to ICH guidelines, which was tested in standard solutions of GABA and glutamate and in samples of serum, urine and yeast extract. All assays were performed using a Hitachi LaChrom Elite HPLC system, with separation on a Lichrospher LiChroCART 250-4 100 (5 m) RP-18 column and with DAD detection. Results: At the end of several assays, the chosen method has as mobile phase H2O and acetonitrile, eluted in gradient, at a flow rate of 1 mL/min for 10 minutes. The column temperature was 25 and the detection was performed at 210 nm. This method not only allows a rapid analysis but also a quantification in the order of g/mL. Although the quantification was possible in the standard solutions, the same did not occur in the biological matrices tested. Conclusion: Although it still needs to be optimized for biological matrices, the method developed allows an easy, fast and economically sustainable analysis of GABA and glutamate. It is, to date, the only method with DAD detection that allows the simultaneous detection of GABA and glutamate without recourse to derivatization of the sample.
- Estudo da atividade biológica de sais orgânicos baseados no ácido betulínico e antimaláricos e sais orgânicos de beta-lactâmicosPublication . Cerqueira, Maria João Amorim; Costa-Rodrigues, João; Ferraz, Ricardo; Prudêncio, CristinaO interesse acerca dos líquidos iónicos (LIs) tem crescido muito nos últimos anos e as suas áreas de aplicação têm vindo a ser alargadas, nomeadamente ao nível das ciências biológicas. A introdução de LIs nas áreas próximas da saúde ainda é recente e por isso há poucos estudos sobre a toxicidade e segurança dos LIs, o que causa alguma dificuldade da aplicação dos LIs nestas mesmas áreas. Os LIs são formados por um catião orgânico e um anião orgânico ou inorgânico e são estáveis acima do seu ponto de fusão. As suas estruturas e interações conferem-lhes propriedades únicas, potencialmente úteis em áreas como a indústria química, biotecnológica e farmacêutica. Com este trabalho pretendeu-se estudar o efeito de alguns LIs baseados em fármacos (LIs acoplados a Ingredientes Farmacêuticos Ativos) em diversos tipos de células, com complexidade crescente, nomeadamente, bactérias Gram-Positivas (Staphylococcus aureus) e Gram-Negativas (Escherichia coli) - células procariontes, leveduras Saccharomyces cerevisiae (células eucariontes) e linhas tumorais humanas (HepG2 – cancro do fígado, MG63 – osteossarcoma, T47D – cancro da mama, A459 – cancro do pulmão e RKO – cancro do cólon). Os compostos testados foram o ampicilinato de cetilpiridinio, o betulinato da primaquina, o betulinato da quinacrina, o betulinado do catião tri-hexiltetradecilfosfónio e o betulinato da cloroquina. Usaram-se sempre os fármacos para perceber em que medida os novos compostos melhoravam os existentes. Os compostos já existentes utilizados foram a ampicilina de sódio, o fosfato da primaquina, o fosfato de cloroquina, o cloreto de quinacrina e o cloreto de tri-hexil tetradecil fosfónio. Para tal, foram utilizados métodos de caracterização da viabilidade celular adequados a cada modelo celular. Os resultados deste estudo mostraram que dentro desta gama de líquidos iónicos se poderão encontrar compostos com potencial interesse no combate a infeções bacterianas e no combate ao cancro.
- The effect of adipocyte secretome in bacterial growth within na hyperglicemic environmentPublication . Martins, António Miguel Bronze Monteiro; Fernandes, RúbenThis project aims to check whether there is an influence on the bacterial growth due to the presence of the adipocyte’s secretome by creating an in vitro model modulating obesity. Due to the rise of obesity all over the world, to discover a link between it and bacterial infections is crucial to better understand exactly what mechanisms hide behind it. Several different bacterial strains will be used to test the model. The strains to be used in this project were selected taking in consideration the most common infections, ranging from enterobacteria, gram positive and gram negative (Esherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 10145, Kebsiela pneumoniae ATCC BAA-1705, Salmonella enterica ATCC 13076, Proteus mirabilis ATCC 25933, Sthapylococcus aureus ATCC 25923, Sthapylococcus epidermis ATCC 14990 and Mycobacterium smegmatis ATCC 19420). In order to mimic the adipocyte’s secretome 3T3-L1 pre-adipocytes were selected to be grown and differentiated, which will later be incubated in serum-free DMEM, by harvesting the medium after a period of 24 hours a medium composed by DMEM and the 3T3-L1 adipocyte’s is obtained. The bacterial strains will be grown in liquid M9 minimal medium and then placed in 96 wells microplates. Each strain will be placed within 3 different mediums, M9, to ensure the bacteria is alive, used as a control, DMEM and CM. With these last two mediums we will be able to verify the difference in growth caused by the adipocyte’s secretome. The growth will be monitored through optical density, at a wavelength of 620nm during an expected period of 2 weeks, which may be adjusted impending on the results obtained.