ESS - DM - Bioquímica em Saúde
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- Relatório de estágioCord blood steam cells banking: purpose, processing and cryopreservationPublication . Pina, Joana Filipa Figueiredo; Gomes, Andreia; Oliveira, Melissa; Ferraz, RicardoStem cells are undifferentiated cells with a high proliferative capacity and the ability to differentiate into any cell type. A rich source of stem cells is the umbilical cord, which is easy, painless, safe and non-invasive for the mother and infant. After birth, instead of being discarded as biological waste, it can be used to obtain multipotent and haematopoietic stem cells. The umbilical cord stem cells are a unique collection, so they should be cryopreserved for their therapeutic potential, as they represent a valuable resource for treating more than 80 diseases. Stem cells can be stored for several years without significantly altering their properties. For that, there are two types of banks for the cryopreservation of these cells: public and private. This work consisted of enrolling in the laboratory routine of BebéVida, a Portuguese and FACTNetcord accredited bank that process, cryopreserves and stores umbilical cord blood and tissue. Therefore, it was aimed to learn and perform the related techniques to the processing and cryopreservation of umbilical cord blood, as well as processes inherent to quality assurance. Furthermore, this work also aimed to understand how ABO and Rh blood groups can influence several biological parameters, such as immune system cells, platelets, immature red blood cells and all nucleated cells, where stem cells are inserted. In conclusion, the integration into the laboratory routine was successful, with proper contact with processing techniques (AXP) and cord blood cryopreservation. Furthermore, this study also demonstrated that the AXP protocol influences the quality of the unit to be cryopreserved and that the volume collected at the time of collection should be considered. Regarding the blood group study, some blood groups showed significant differences, demonstrating that the presence and absence of A and B antigens on the cell surface may contribute to the difference between the number of immune system cells between different blood types. This highlights the potential impact of ABO blood type on several health conditions, and further research is needed to elucidate the underlying mechanisms.
- Stalling the course of Alzheimer´s disease: can cyanobacteria be a new approach toward therapy?Publication . Ramos, Maria Vitória Neto; Martins, Rosário; Reis, Mariana; Ferraz, RicardoAlzheimer's Disease (AD) is a severe, age-related neurodegenerative disease and it is the primary cause of dementia. It is marked by the progressive loss of neurons, accompanied by the presence of amyloid plaques, neurofibrillary tangles, and other pathological changes, including cholinergic deficiency. Currently, there is no cure for AD, and although treatments are available to alleviate symptoms, they do not hinder the advancement of the disease. Acetylcholinesterase inhibitors (AChEIs), such as Donepezil, Galantamine, and Rivastigmine are the main course of treatment, but new, more efficacious, and tolerable medications are needed. Blue biotechnology is leading the search for new bioactive compounds. Cyanobacteria, a heterogeneous group of photosynthetic microorganisms, are a prolific source that has shown promise in the field of AD. The Blue Biotechnology and Ecotoxicology Culture Collection (LEGE-CC) housed at CIIMAR, composed mainly of cyanobacteria strains, is a valuable resource that has been exploited for various biomedical applications. In this work, 22 cyanobacteria strains from the LEGE-CC were evaluated for their cytotoxicity in neuroblastoma cells (SH-SY5Y), endothelial cells representative of the blood-brain barrier (hCMEC/D3), and fibroblasts (3T3-L1), and for their potential to inhibit AChE. The approach included the screening of existing cyanobacterial fractions in the natural products library (LEGE-NPL), cultivating new strains to obtain biomass, preparing methanolic extracts, and HPLC fractionation. For each strain, 8 fractions (A-H) were obtained, with a total of 176 fractions tested. The cell viability results, based on the MTT assay, showed that most of the fractions are not cytotoxic at a concentration of 50 µg/mL, as only 15 showed toxicity, with inhibition of cell viability above 30%. In the in vitro AChE inhibition assay, based on Ellman's method, the fractions Spirulina sp. LEGE 11439_A and Scytonema sp. LEGE 07189_A (500 µg/mL) showed low to moderate inhibition of 29.15% and 21.73%, respectively, which indicates some potential in a preliminary screening. Further research can be undertaken to investigate additional anti-AD activities and to elucidate the chemical profiles of the fractions.
- Understanding the molecular bases behind congenital disorders of glycosylationPublication . Oliveira, Tiago André Cunha; Sousa, Sérgio; Ferraz, RicardoCongenital disorders of glycosylation (CDG) result from mutations affecting proteins involved in glycosylation pathways. Phosphomannomutase 2 (PMM2) deficiency (PMM2-CDG), the most common N-glycosylation disorder, induces hypoglycosylation of several proteins leading to multisystem involvement. This disease is related to a huge variety of mutante variants and a broad phenotypic spectrum, making genitype-phenotype correlations difficult. Mutations in PMM2 can affect is structure and function and may reveal a specific phenotype, One possible strategy for studying genotype-phenotype correlations would be to apply computational methods to assess the structural changes caused by amino acid substitutions. This would allow further development of personalised therapies for na untreatable disease. With the aim of developing na in silico protocol to analyse the impacto f diferente mutations on PMM2., several three-dimensional structures of mutated PMM2 were modelled and molecular Dynamics (MD) simulations were analysed to assess the impact of these variants at he energetic and molecular level on the normal activity of PMM2, including dimerization, folding, substrate-binding and structure-stability. The results predict the effect of missense mutations on the function of PMM2, in particular on the free energy of dimerization and folding of the protein.
- Avaliação da resposta imunitária a baixas doses de radiação ionizante em peixe zebraPublication . Martins, Ricardo Dinis Azevedo; Costa, Pedro; Ferraz, RicardoO sistema imune é um dos principais alvos da radiação ionizante. Os efeitos de altas doses de radiação são conhecidos e bem documentados, mas os de baixas doses (LDIR) carecem de estudos aprofundados. O peixe zebra tem sistema imune bem preservado, sendo um modelo atraente para estudar LDIR. Neste contexto, realizou-se uma revisão bibliográfica para criar um esboço inicial da interação LDIR-sistema imune. Em termos experimentais, avaliou-se os efeitos da irradiação com doses de 10 e 35 mGy em peixe zebra, estudando-se o impacto do tempo decorrido pós irradiação na expressão de proteína C-reativa (CRP) e IFN-gama, utilizando ELISA. Na análise teórica, verificou-se que LDIR em irradiações agudas induz resposta pró-inflamatória e LDIR em irradiações crónicas resposta anti-inflamatória. Após irradiação, CRP apresentou aumentos significativos no grupo de 35 mGy, do 1º ao 7º dia, enquanto 10 mGy teve aumento e decréscimo ligeiros ao 1º e 7º dia, respetivamente. Quanto a IFN-gama, verificaram-se ligeiros decréscimos no 1º dia, nas duas doses, mas uma redução significativa para 10 mGy ao 7º dia. Os resultados sugerem que irradiações com dose absorvida de 10 e 35 mGy, com baixa taxa de dose, provocam inflamação inicial, proporcional à dose, e induzem respostas anti-inflamatórias para combatê-la.
- Uso de probióticos na proteção contra o cancro colorrectalPublication . Santos, Rejane Viana dos; Pereira, Ana Cláudia; Fernandes, Rúben; Baylina, PilarA obesidade é uma doença metabólica crónica definida pelo excesso de gordura e hipertrofia do tecido adiposo e está associada a elevados riscos de saúde. Na atualidade, a obesidade é considerada um dos mais prevalentes problemas de saúde pública e com taxas de mortalidade a disparar. Autores sugerem que a obesidade é uma doença “adquirida” com fatores etiológicos complexos. Estudos recentes demonstraram que mais de 40% dos pacientes com cancro gastrointestinal estão acima do peso e que a obesidade é o principal fator de risco extrínseco para cancro colorrectal. Pois, a metainflamação sustentada pelo tecido adiposo fornece lípidos circulantes, espécies reativas de oxigénio e metabolitos pró-inflamatórios cruciais no desenvolvimento e agressividade tumoral. De igual modo, existe forte associação entre doenças metabólicas e a microbiota intestinal, podendo esta funcionar como fator de predileção ou proteção para o CCR. Alterações positivas na microbiota intestinal com uso de probióticos pode produzir resposta protetora contra o CCR, assim, o objetivo deste trabalho é investigar o efeito de um probiótico de uso terapêutico e a sua atividade antitumoral em células de tumor intestinal expostas a condições de adiposidade. Inicialmente foram produzidos dois tipos de extratos: 1) conteúdo proveniente de um cocktail de bactérias probióticas, e 2) secretoma da diferenciação da linha celular 3T3-L1, pré-adipócitos de ratinhos. Para otimizar a diferenciação foram utilizados 10 μM de Pioglitazona num dos ramos da diferenciação dos pré-adipócitos. Seguidamente, foram realizados ensaios in vitro de viabilidade celular, migração e testes antioxidantes em linha celular Caco2, adenocarcinoma de cólon, utilizando os extratos referidos. Os ensaios foram realizados em triplicado, com 95% de IC. Os resultados apontaram que os dois extratos aumentaram a viabilidade no linha tumoral Caco2, sem identificação de efeito benéfico na proteção anti-tumoral neste modelo de doença. Contudo, o probiótico antepadamente à suplementação do secretoma previniu um potencial efeito agressor de migração celular, principalmente em contextos que melhor mimetizam obesidade (****p<0,0001). Além disso, observou-se efeito de proteção antioxidade em tumor de cólon, especialmente em duas condições a) que mimetizam tecido adiposo “normal” (** p<0,05) e b) adiposidade com inflamação (*p<0,05). Discussão: A utilização de probióticos promove alterações físico-químicas na microbiota intestinal, seja pelo aumento na produção de ácidos gordos de cadeia curta (AGCC) ou aumento de componentes antitumorais e antimutagénicos, que influenciam positivamente na modulação imunológica. A suplementação probiótica ativa as defesas antioxidades, e é um dos fatores associados ao tumor de cólon, pois microbiota disbiótica promove estimulação de inflamação crónica pela produção acentuada de ROS que induzem danos ao DNA, ativação de oncogenes e inativação de genes supressores tumorais. Por fim, a Pioglitazona, apesar de ser um fármaco anti-diabético que reduz a resistência insulínica, está associado com a redução modesta de 9% no risco de desenvolvimento de CCR em pacientes com DMT2 com este tratamento. Este trabalho-piloto traz à luz alguns achados que poderão responder o porquê da modulação da microbiota intestinal com uso de probióticos ser uma estratégia alternativa para levar a melhorias da condição e estadio do tumor de cólon. Estudos adicionais são necessários para esclarecer o papel da Pioglitazona neste tipo de tumor e os metabolitos da obesidade que são atenuados pelo uso do probiótico.
- Umbilical cord mesenchymal stem cells effect on melanoma progressionPublication . Fernandes, Pablo Rende; Coelho, Pedro; Gomes, Andreia; Ferraz, RicardoMelanoma, a skin cancer originating from malignant transformation of melanocytes, is charcaterized by its agressive nature and high tendency to metastasize, rendering melanoma management challenging. The few existing treatments for metastatic melanoma have limited effectiveness, underlying a demand for novel therapeutic strategies. In preclinical cancer models, huma umbilical cord mesenchymal stem cells (huCMSCs) have show promising anti-cancer effects, hampering both the proliferation and metastasis of malignant cells. In this work, we investigated the impacto f conditioned media (CM) obtained from huCMSCs on viability, proliferation, cell cycle, migration, and ahesion of melanoma cells using in vitro cell culture models. huCMSCs, isolated from the umbilical cordo f seven healthy neonates by enzymatic digestion followed by direct plastic adherance method, were cultivated and their CM stored for subsequente assays. Then, malignant melanoma B16F10 cells were treated with final concentration of 100% of CM or standard sérum-free media (control)for 24 hours. Cell viability was assessed using MTT assay and 7-aminoactinomycin D (7-ADD) staining assay. When treated wich huCMSCs secretome, B16F10 cells decreased their viability by 32% and 11% as shown by the MTT assay and 7-AAD exclusion stain, respectively (p˂0.05, n=7). Next proliferation and cell cycle progression were evaluated by flow cytometry analysis with Ki-67 and propidium iodide (PI) stains, respectively. The expression of Ki-67 proliferation marker was reduced by 14% (p˂0.05, n=7) with a concomitante increase in the number of cells in G0/G1 phase arrest (6%). huCMSCs-CM reuced cell migration, assessed by scratch assay, by 24% (p˂0.05 vs control, n=7) while enhancing cell adhesion capacity, evaluated bgy the crystal violet assay, by 16% (p˂0.05 vs control, n=7). Our results showed that huCMSCs secretome reduced the viability and proliferation capacity of malignant melanoma cells with a concomitante arresto f cells in G0/G1 cell cycle stage. This study suggests huCMSCs paracrine signaling induces melanoma cells into a quiescente growth inhibition state with a concomitante decrease in overall cell viability and survial. Further research is required to characterize the molecular pathways of these promising effects of huCMSCs secretome in inhibiting melanoma cells porliferation and establish their safety and efficacy. Altogether, our findings support huCMSCs paracrine componente therapies for melanoma.
- Ultrasound-assisted extration of goji berries: bioactive composition and pro-healthy propertiesPublication . Teixeira, Filipa Marisa Ribeiro; Rodrigues, Francisca; Silva, Ana Margarida; Ferraz, RicardoLycium barbarum L., also known as goji or wolfberries, have a remarkable chemical composition and extraordinary biological activities, allowing it to be classified as a "superfruit". The main objective was to optimize the Ultrasound-Assisted Extraction (UAE) of goji berries using the Response Surface Methodology, and to characterize the optimal extract. The optimal UAE conditions established were a solid:liquid ratio of 8.75%, for 56.21 min and an intensity of 59.05 W/m2. The optimal extract demonstrated a high total phenolic content (20.91 mg GAE/g dw) as well as exceptional antioxidant/antiradical properties (ABTS = 14.95 mg AAE/g dw; DPPH = 10.29 mg TE/g dw; FRAP = 106.79 µmol FSE/g dw). LC/DAD-ESI-MS analysis revealed a diverse phytochemical profile that included compounds such as feruloylquinic acid, 3,5-dicaffeoylquinic acid, rutin, and numerous derivatives. Furthermore, the optimal extract demonstrated strong scavenging activities, particularly against HOCl (IC50 = 12.99 μg/mL). Cell viability studies on intestinal cell lines (Caco-2 and HT29-MTX) revealed that doses up to 500 µg/mL were safe, however 1000 µg/mL significantly reducing Caco-2 (60.00%) and HT29-MTX (17.02%) viability. Thus, a concentration of 500 g/mL was used in the intestinal co-culture experiment, which showed that most compounds from the optimal extract crossed the intestinal cell monolayer and exerted their pro-healthy properties.
- Electrochemical genosensor for the detection of Dynophisis spp. dinoflagellatesPublication . Pereira, Eduarda Almeida Rufo; Santos, Marlene; Barroso, Fátima; Silva, Nádia; Vieira, MónicaHarmful algal blooms (HABs) pose a significant threat to the environment and public health. To minimize their impact, it is necessary to develop technological devices towards their fast and precise detection. Therefore, the aim of this work was to develop an analytical approach based on an electrochemical genosensor device in order to create a low-cost platform to monitor the presence of dinoflagellates from the genus Dinophysis. The design of this deoxyribonucleic acid (DNA)-based sensor consists in three steps: (i) Sensing phase, where a mixed self-assembled monolayer composed by a linear DNA-capture probe and mercaptohexanol onto the electrode’s surface is generated; (ii) Hybridization of complementary DNA sequence through a “sandwich” hybridization assay; and (iii) Electrochemical detection by chronoamperometry, taking advantage of an enzymatic scheme to amplify the signal. The best analytical conditions were used to study the relationship between the electrochemical signal and the DNA-target concentration, to produce the best electrochemical genosensor device. Polymerase chain reaction (PCR) was used for validation of the electrochemical genosensor. These advancements contribute to the ongoing efforts in environment and public health’s protection by providing effective tools for mitigating the risks of HABs. Further research and implementation of these methods hold great potential in ensuring the safety and sustainability of aquatic ecosystems.
- From the gut to the brain: is microbiome a new paradigm for Parkinson´s disease treatment?Publication . Vilela, Cristiana Andreia Sousa; Teixeira, Fábio; Vieira, MónicaParkinson's disease (PD) is recognized as the second most prevalent primary chronic neurodegenerative disorder of the Central Nervous System (CNS). Clinically, PD is characterized as a movement disorder, exhibiting an incidence and mortality rate that is increasing faster than any other neurological condition. In recent years, there has been a significant interest in investigating the role of the gut microbiome in the etiology and pathophysiology of PD. The establishment of a brain-gut microbiome axis is now real, with evidence denoting a bidirectional communication between the brain and the gut microbiota, which operates through various signaling pathways and mechanisms encompassing metabolic, immune, neuronal, and endocrine pathways. Among these, the vagus nerve represents the most direct form of communication. Given the intricate interactions between bacteria and drugs, it has been observed that treatments for PD can have an impact on the composition of the microbiome. Current treatments just provide effective management of motor symptoms and do not modify the course of the disease. For that, new routes have been considered, for instance, N-acetyl cysteine (NAC) is a naturally occurring molecule derived from the natural amino acid L-cysteine, associated with both antioxidant, mucolytic, and anti-inflammatory properties. It has demonstrated beneficial effects in modulating the symptomatology of PD, given that this medication is capable of restoring one of the major antioxidants of neurons, glutathione (GSH). Bearing this in mind, the main objective of the present project will be to understand if a real connection between the gut microbiome and PD exists and if the application of NAC has any influence on PD gut microbiota, thereby leading to cellular, molecular, and functional improvements. Such approach could open new therapeutical opportunities, leading to development of microbiome-based disease-modifying strategies for PD.
- Avaliação de um ingrediente cosmético na proteção química da pele contra poluiçãoPublication . Kringel, Cândida Cristine; Monteiro, Marta; Vieira, Mónica AlmeidaPara garantir a segurança do consumidor, está em vigor na Europa o Regulamento que estabelece as especificações concretas que os produtos cosméticos devem cumprir (Regulamento (CE) nº 1223/2009) e as suas alegações devem ser objetivas e claras (Regulamento (EU) nº 655/2013). Os produtos cosméticos estão cada vez mais presentes no nosso dia a dia e a indústria cosmética vem crescendo cada vez mais, seja em quantidade de produtos disponíveis no mercado, seja em inovação no seu desenvolvimento. Este relatório descreve, o estágio realizado na Inovapotek Pharmaceutical Research & Development para a conclusão do meu mestrado na Escola Superior de Saúde (ESS), onde detalho o desenvolvimento de um estudo clínico que tem como objetivo avaliar a eficácia de um ingrediente cosmético na proteção química da pele contra a poluição no Departamento de Ensaios de Eficácia e Segurança e também sugestões de melhoria ao equipamento utilizado para o estudo.