FCS - DCM | Dissertações de Mestrado e Teses de Doutoramento
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- Suspeita de Oclusão Venosa da Retina, Adaptação de Lentes de Contacto RPG em Queratocone, AmbliopiaPublication . Albino, Guilherme Soares Paulino; Monteiro, Pedro Miguel Lourenço; Nascimento, Henrique Virgílio dos SantosEste relatório, intitulado “Suspeita de Oclusão Venosa da Retina, Adaptação de Lentes de Contacto RPG em Queratocone e Ambliopia”, resume a experiência adquirida ao longo de um estágio de seis meses na clínica de optometria Optocentro, situada em Lisboa. Este estágio, iniciado em 1 de março de 2023, foi uma parte fundamental do mestrado em Optometria e Ciências da Visão da Universidade da Beira Interior. Durante este período, foram aprimoradas competências práticas que possibilitaram uma ampliação significativa do conhecimento na área da optometria, com ênfase na adaptação de lentes de contacto rígidas permeáveis a gases, na avaliação de condições oculares como a oclusão venosa da retina, e na análise de casos de ambliopia. A experiência prática incluiu a realização de exames complementares, como tomografia de coerência óptica, retinografia, topografias, retinografias, campimetrias e análise de casos de disfunções binoculares e acomodativas, permitindo uma abordagem abrangente e integrada no diagnóstico e tratamento de disfunções visuais. O relatório está organizado em três casos clínicos distintos. O primeiro aborda um paciente com suspeita de oclusão venosa da retina, uma condição que pode levar a uma diminuição significativa da acuidade visual devido à obstrução do fluxo sanguíneo nas veias retinianas. Neste caso, foram realizadas avaliações clínicas detalhadas, incluindo exames de Retinografia e OCT, para fundamentar a suspeita de patologia. O segundo caso foca na adaptação de lentes de contacto rígidas permeáveis a gases em um paciente diagnosticado com queratocone. A adaptação foi realizada com base em uma análise da topografia corneana, visando proporcionar uma correção visual eficaz e conforto ao paciente, que apresentava irregularidades na superfície corneana. O terceiro caso aborda a ambliopia numa criança de 4 anos. A paciente, sem historial de uso de óculos, foi submetida a uma avaliação optométrica completa. O tratamento prescrito incluiu a correção baseada na refração cicloplégica, complementada por oclusão terapêutica do olho dominante e, a implementação de exercícios de estimulação visual. Estas intervenções visam melhorar a acuidade visual do olho amblíope e favorecer o desenvolvimento binocular adequado.
- Exoforia Básica, Adaptação de Lentes de Contacto Multifocais, Suspeita de CatarataPublication . Damião, Daniela Sofia Moreira; Monteiro, Pedro Miguel Lourenço; Torres, Rafaela Filipa MoraisEste relatório de estágio, intitulado “Exoforia Básica, Adaptação de Lentes de Contacto Multifocais, Suspeita de Catarata” resulta do estágio curricular integrado no mestrado em Optometria e Ciências da Visão. O estágio teve a duração de 6 meses, foi realizado na Multiopticas, em Oliveira do Hospital. Durante este período, foram selecionados três casos clínicos relevantes para a execução do relatório. O primeiro caso clínico, referente ao tema visão binocular, aborda a Exoforia básica. Trata-se de uma disfunção binocular não estrábica que se manifesta com sintomas como desconforto visual, fadiga ocular e visão turva, especialmente na visão de perto. O tratamento desta condição inclui, principalmente, a correção da ametropia e terapia visual. Neste caso, foi apenas implementado o primeiro tratamento. O segundo caso clínico, Adaptação de Lentes de Contacto Multifocais, enquadra-se na temática de superfície ocular e lentes de contacto. Este tipo de lentes corrige a visão a todas as distâncias, sendo geralmente utilizado por pacientes com presbiopia, como alternativa aos óculos para visão de perto. O processo de adaptação envolve a avaliação da visão por parte do optometrista, a escolha do design da lente e a realização de testes de ajuste, com o objetivo de proporcionar uma boa acuidade visual. O terceiro caso clínico, intitulado Suspeita de Catarata, integra-se na área da saúde ocular. A catarata nuclear é a opacificação do cristalino, sendo mais comum em pessoas idosas. Esta condição provoca sintomas como visão turva, sensibilidade à luz e dificuldade de visão por exemplo, durante a condução noturna. Neste caso, o paciente, embora relativamente jovem, não apresentou sintomas. Foi encaminhado para a especialidade de Oftalmologia para acompanhamento e avaliação regular.
- Hair as a monitoring tool for psychoactive substancesPublication . Simão, Ana Aysa Rocha da; Alba, María Eugenia Gallardo; Barroso, Mário Jorge Dinis; Andraus, Maristela HaddadThe global prevalence of substance use and its severe health, social, and economic consequences remain significant public health concerns. The continuous emergence of new psychoactive substances (NPS), alongside the widespread use of traditional drugs, exacerbates the challenges faced by forensic and clinical toxicologists. These substances often evade conventional detection methods due to their rapid evolution, posing significant risks to users and complicating monitoring efforts. Understanding substance use patterns and their impacts is essential for informing prevention strategies, shaping public policies, and implementing harm reduction initiatives. Hair analysis has emerged as a powerful tool in toxicological investigations, offering unique advantages over traditional biological matrices such as blood and urine. Its extended detection window enables the assessment of chronic and historical drug use, while its resistance to adulteration ensures reliability. As a non-invasive collection method, hair analysis facilitates large-scale studies, making it particularly suitable for exploring poly-drug use and evaluating the prevalence of both classical drugs and NPS across diverse populations. Quantitative hair analysis complements self-reported data, addressing discrepancies and enhancing the accuracy of substance use research. [...]
- Suspeita de Membrana Epirretiniana, Adaptação de lentes de contacto RPG em Queratocone, Insuficiência de ConvergênciaPublication . Martins, Mayra da Graça Luisa; Lucas, Luís Miguel de Azevedo; Monteiro, Pedro Miguel LourençoA elaboração deste relatório afigura-se como o término do período de Estágio Curricular do 2.º Ciclo de Estudos em Optometria e Ciências da Visão, que foi desenvolvido durante 6 meses na Óptica Lucas, localizada no distrito de Castelo Branco. Com o intuito disposto acima, foram selecionados e analisados 3 casos clínicos de acordo aos critérios definidos nomeadamente, saúde ocular, superfície ocular e lentes de contacto e visão binocular, como “Suspeita de Membrana Epirretiniana, Adaptação de lentes de contacto RPG em Queratocone, Insuficiência de Convergência.” O primeiro caso é sobre uma suspeita de membrana epirretiana (ERM), que se carateriza por uma membrana proliferativa celular pré-retiniana enrugada semitranslúcida avascular, que se forma na ‘interface’ vítreo-retiniana. Neste caso, a paciente foi submetida a uma serie de exames complementares de diagnóstico e em seguida foi encaminhada para outro profissional de saúde. No segundo caso, realizou-se uma adaptação de lentes de contacto RPG em Queratocone (KC), sendo uma distrofia corneana não inflamatória, progressiva bilateral, caracterizada por adelgaçamento, e protusão da córnea, levando a astigmatismo irregular, miopia e deficiência visual. O último caso, trata-se de uma anomalia binocular não estrábica, a insuficiência de convergência (IC). O tratamento recomendado para este caso é a terapia visual, uma vez que o paciente não se mostrou motivado para a realização da mesma, essa opção foi descartada, passando assim para o segundo tratamento que é a prescrição de prisma de base in (BI).
- The role of miRNA-9 and miRNA-29 and their specific strands (-5p and -3p) in Alzheimer’s diseasePublication . Carlos, Beatriz de Almeida; Cruz, Carla Patrícia Alves Freire Madeira da; Riscado, Micaela Sofia Ribeiro; Sousa, Fani Pereira deMicroRNAs (miRNAs) are small non-coding, single-stranded RNA molecules, typically with 20-25 nucleotides in length. They play a crucial role in modulating several biological processes by regulating gene expression at the post-transcriptional level. Due to their biological role, their application is being evaluated in the context of diagnosis and treatment of neurodegenerative diseases, particularly Alzheimer's disease (AD), as biomarkers or potential therapeutic agents. AD, classified as the most common form of dementia, is an irreversible neurodegenerative disease with a high prevalence in elderly people. This disease is characterized by the accumulation of amyloid ß (Aß) peptides and hyperphosphorylated Tau protein. Several studies have demonstrated the promising effect of miRNAs for silencing the dysregulated proteins involved in the amyloid pathway, which are responsible for AD progression. With this in mind, this work aims to study the effect of miRNA-9, -29b, their specific strands (-5p and- 3p), and as well their precursor forms (pre-miRNA-9-1 and pre-miRNA-29b-1) on the proteins related with AD, such as APP, BACE1 and PS1. To proceed with this evaluation, the different forms of miRNA were encapsulated in chitosan nanoparticles and delivered in the N2a695 cell line, an AD in vitro model. After extraction of the total RNA from the cells, the mRNA levels of the target proteins were evaluated by Real-Time Polymerase Chain Reaction (qPCR). This study demonstrated a potential silencing effect by miRNA-9 on the mRNA levels of PS1 and BACE1, with pre-miRNA-9-1 via miRNA-9-1-5p being the most promising, as it induced the silencing of BACE1 mRNA in almost 50%. In conclusion, understanding how miRNAs can silence proteins involved in the amyloid pathway is crucial because these small RNAs have the potential as new therapeutic tools for treating or controlling AD progression.
- Identificação de causas genéticas do hipogonadismo hipogonadotrófico congénito utilizando a sequenciação de nova geraçãoPublication . Carriço, Josianne Nunes; Lemos, Manuel Carlos Loureiro de; Gonçalves, Catarina Inês Nunes PiresO desenvolvimento pubertário é um processo fisiológico complexo que resulta da interação entre o sistema neuroendócrino, fatores genéticos e ambientais. Inicia-se com a reativação do eixo Hipotálamo-Hipófise-Gónadas, levando à produção de gonadotrofinas, hormonas sexuais e consequente desenvolvimento dos carateres sexuais secundários. O Hipogonadismo Hipogonadotrófico Congénito (HHC) é uma doença endócrina rara que, na adolescência, se manifesta clinicamente com ausência completa ou parcial do desenvolvimento dos caracteres sexuais secundários. Na idade adulta, os sintomas incluem ausência de virilização e disfunção erétil no sexo masculino, amenorreia primária no sexo feminino, ausência de líbido e infertilidade em ambos os sexos. O HHC caracteriza-se pela ausência ou diminuição da produção, secreção ou ação da hormona libertadora de gonadotrofinas. Em 50 a 60% dos casos associa-se a anosmia/hiposmia (Síndrome de Kallmann) ou a outras anomalias congénitas. O diagnóstico do HHC baseia-se na anamnese, observação de sinais clínicos, levantamento de sintomas e exames laboratoriais e imagiológicos. Contudo, desde o início do século XXI, o conhecimento sobre as causas genéticas do HHC tem avançado na mesma medida que o desenvolvimento das tecnologias de sequenciação genética. A disponibilidade das tecnologias de sequenciação de nova geração (NGS) proporcionou grandes avanços no diagnóstico molecular destes doentes. Este estudo teve como objetivo investigar as causas genéticas do HHC em doentes portugueses, utilizando a NGS, ferramentas in silico e técnicas de biologia molecular. Foram estudados 81 doentes com HHC e 263 controlos portugueses. O ADN genómico foi extraído dos leucócitos do sangue periférico e submetido à sequenciação do exoma completo. A partir das bases de dados OMIM® e Pubmed, identificaram-se 169 genes relacionados com o fenótipo de HHC, que foram incluídos num painel virtual. As variantes encontradas nesses genes foram filtradas com base na frequência populacional e localização no genoma, sendo classificadas conforme os critérios do American college of medical genetics and genomics e da Association for molecular pathology (ACMG-AMP) em cinco níveis, de benignas a patogénicas. Para avaliar o impacto de variantes com potencial para alterar o splicing, utilizou-se o método de minigene. A análise das variantes germinativas permitiu identificar a causa genética do HHC em 24 dos 81 doentes estudados (29,6%). As variantes causais estavam distribuídas por 10 dos 169 genes presentes no painel genético virtual analisado. Os genes GNRHR, FGFR1, ANOS1 e CHD7 foram os mais frequentemente afetados. Notavelmente, a ampliação do painel genético não resultou num aumentou considerável da taxa de diagnóstico. No entanto, é importante destacar que este estudo expandiu o espectro mutacional da doença, identificando várias variantes até então nunca descritas. A maioria das variantes encontradas foi classificada como de significado indeterminado (VUS), mas a taxa de VUS não diferiu significativamente entre doentes e controlos, com exceção do gene EGF, onde foram encontradas VUS exclusivamente nos doentes. Concluímos que a maioria dessas variantes raras não estão diretamente relacionadas com o fenótipo estudado, refletindo provavelmente o fundo genético da população portuguesa. Em relação à oligogenia, observou-se uma frequência de 6,2% entre os doentes (5/81), um valor estatisticamente mais elevado quando comparado com os controlos. O estudo funcional de uma variante identificada no gene CHD7 (NM_017780.4: c.4354G>T, p.Val1452Leu), resultou no aumento da taxa de diagnóstico para 30,9%, demonstrando a importância dos estudos funcionais na reclassificação de VUS. Este trabalho enfatizou a complexidade genética do HHC, reforçando a importância de uma compreensão aprofundada da doença, seja para estabelecer o diagnóstico e/ou o prognóstico, para ajudar no aconselhamento genético e tratamento, seja para o esclarecimento dos processos biológicos, que potencialmente poderão contribuir para o desenvolvimento de terapias dirigidas no futuro.
- Sweet Cherries as Health Promoters: Valuable Red Fruits with Nutritive and Functional PropertiesPublication . Gonçalves, Ana Carolina Almeida ; Silva, Luís Manuel Lopes Rodrigues da; Alves, Gilberto Lourenço; Ferreira, Amílcar Celta Falcão RamosCurrently, it is widely recognized that consuming fruits and vegetables effectively reduces the risk of morbidity and mortality caused by cardiovascular and cancerous diseases, among others, being widely recommended a daily intake of 400 g of fruits and vegetables. In fact, the potential of fruits and vegetables to treat various ailments and alleviate symptoms, such as migraines, metabolic syndrome, intestinal problems, physical pain, certain cancer types, rheumatoid arthritis, dizziness, colds, fever, psychological fatigue, and symptoms derived from rheumatoid arthritis, among others, has been known since ancient times. Nowadays, the trend is increasing, being accompanied by an emphasis on different communities. This interest is essentially since it is believed that, unlike synthetic pharmaceutics that cause undesirable side effects, natural products have few, or no side effects, and are easy to obtain and economical. Natural products have been the subject of many studies and an important topic of discussion among the medical and scientific communities. Indeed, the biological potential of a wide range of products has already been recognized, being already incorporated in many pharmaceutical drugs. These beneficial activities are directly related to their nutritional constituents, namely due to the presence of vitamins, minerals, carotenoids and fiber, as well as phenolic compounds. The combination of all these compounds is beneficial and capable of promoting the good functioning of human organisms, and hence, promoting the normalization of several parameters to basal levels and contributing to general well-being. Among the various compounds, special emphasis has been given to phenolic compounds. As far as we know, phenolic compounds derive from the secondary metabolism of plants and their main function is to protect them against abiotic (water, sunlight, and temperature) and biotic (attacks by microorganisms) factors. These are commonly divided into 2 large subclasses: (i) non-coloured phenolic compounds (e.g., hydroxybenzoic and hydroxycinnamic acids, flavan-3-ols, flavonols, among others) and (ii) coloured compounds (anthocyanins, which are largely responsible by the colors exhibited by various natural products). Recent research has demonstrated that their chemical structure gives them a remarkable ability to reduce levels of oxidative stress and interact with proinflammatory cascades, thus restoring basal levels and consequently reducing the risk of occurrence of many diseases, or alleviating your symptoms, and consequently, contributing to a better quality of life. The human body naturally possesses intracellular antioxidant enzymes (superoxide dismutase, glutathione peroxidase and catalase) that are considered essential for the survival and health of the population, once, under physiological conditions, they are capable of balancing the levels of free radicals. However, given genetic factors, unexpected decompensation, severe discomfort, and lifestyle choices, such as sedentary habits, consumption of alcohol and tobacco, and/or intake of foods very rich in fats and calories, the aforementioned enzymes become insufficient to guarantee basal levels, leading to proteins, organs and cells injury. This damage is caused by high levels of free radicals and the appearance of exacerbated inflammatory responses in the human body. Consequently, this decompensation “triggers” for the appearance and progression of many diseases whose prevalence is increasing worldwide. These data are corroborated by many studies, which describe that free radicals and pro-inflammatory species are largely related to oxidative stress, and the appearance of various types of cancer, autoimmune diseases, such as rheumatoid arthritis, and syndromes, including the metabolic one. Given the mentioned facts, it is not surprising that many studies involving cherries consumption are being carried out, since it is urgent to find accessible and effective therapies for the entire population, and preferably natural based, in order to improve their quality of life. Cherry (Prunus avium Linnaeus) is a fruit highly appreciated by consumers, not only due to its organoleptic characteristics, but also due to its nutritional value and health benefits. In fact, it has been shown that this fruit is an excellent source of macro and micronutrients, and phytochemicals. In fact, this last class of compounds has been the subject of many studies, being a topic of discussion among various communities given their various positive effects on health. Among the different nutritional classes found, cherries have high contents of phenolic compounds, highlighting the presence, until now, of anthocyanins, flavan-3-ols, flavonols and hydroxycinnamic acids. Particularly, the main compounds identified are cyanidin 3-O-rutinoside and -glucoside, quercetin, rutin, kaempferol, catechin, and ρcoumaroylquinic and chlorogenic acids. Therefore, the richness of cherries in bioactive compounds has been one of the reasons for the growing interest of the scientific community in exploring the beneficial healthpromoting effects associated with their intake. Furthermore, consumers are increasingly well-informed and, once, they are looking for healthy products, such as cherries, there is verified an increase in their demand. Faced with this, the market responds by increasing their global production, especially of the cultivars that most attract consumers. To date, several scientific studies carried out on animals and humans suggest that consuming cherries reduces the risk of several inflammatory and chronic diseases, such as rheumatoid arthritis, cardiovascular diseases, diabetes and cancer. Clinical evidence has already demonstrated that consuming cherries can reduce the scale of pain caused by arthritis, gout and inflammation, possibly due to their ability to increase superoxide dismutase levels, and reduce inflammatory mediators (TNF-α, MDA and PGE-2) and serum levels of C-reactive protein levels, as well as inhibiting cyclooxygenase (COX)-2, which is one of the main proteins responsible for enhancing the pro-inflammatory response. Furthermore, cherries have also shown to be effective in reducing muscle pain, accelerating recovery and improving the performance of recreational exercisers and high-competition athletes, diminishing risk factors associated with the onset of diabetes and cardiovascular diseases, and associated with stress and anxiety, thus improving sleep and mood, memory and cognitive functions. More recently, it has been reported that the consumption of cherries can also alleviate hepatic steatosis and inhibit the activity of the α-glucosidase enzyme, thus delaying the conversion of starch and disaccharides into glucose. Furthermore, cherries also show potential to protect human erythrocytes against free radicals and to inhibit the proliferation of cancer cells. These health-promoting activities are closely related to cherry phenolic content, which is the main responsible for offering this berry, notable antioxidant and antiinflammatory capabilities. This fact is supported by several studies and correlations already performed. In recent years, the production of this fruit has increased considerably worldwide, including in Portugal. Our country produces around 20,000 tonnes per year. A large part of this production occurs in the Fundão region, and hence, it is not surprising that sweet cherries present a distinguished economic impact in this region. The availability of detailed information about their health-promoting properties could lead to an increase in consumer demand, raising their consumption and use of pharmaceutical and nutraceutical products, contributing to the valorisation of the region and the fixation of people and industrial companies. Therefore, with this doctoral project, the aim was to extensively characterize, for the first time, the quality parameters and phytochemical and mineral composition of the best-known cultivars in the region, with the intention of helping in the selection of the most promising cultivars. In total, 23 cultivars were characterized. The results obtained revealed that, among the cultivars studied, there are significant differences in the physicochemical characteristics and in the phenolic, mineral and volatile profiles, showing the verified variability between the various cherry cultivars is mainly influenced by the genotype of the cultivar. In general, Black star and Starkrimson cultivars had the highest soluble solids content, while the highest acidity value was found in Sweetheart cultivar. On the other hand, Cristalina, Kordia and Santina cultivars were those that exhibited the most intense/dark color, while the lightest ones were Sunburst and Sweetheart. Relatively to phenolic compounds, 46 phenolic compounds were identified by HPLC-DAD-ESI/MSn, including 9 hydroxycinnamic acids, 2 hydroxybenzoic acids, 13 flavonols, 5 flavan-3-ols, 2 flavanones, 1 flavanonol and 4 anthocyanins. Among the compounds, chlorogenic acids were the majority noncoloured phenolic compounds, while cyanidin 3-O-rutinoside was the most predominant coloured phenolic compound. Regarding their levels, Sunburst cultivar had the highest amounts of unstained compounds, while the Tavora, Garnet and 4-84 cultivars had the highest concentration of anthocyanins. With regard to mineral content, 27 were identified by ICP-MS and flame atomic absorption spectrometry, namely 12 essential and 15 non-essential. The element potassium (K) was the most abundant element detected in all cultivars, while Thallium (Tl) was the least abundant. On the other hand, the analysis of volatile organic compounds by SPME/GC-MS showed that cherries have a wide variety of these, having been detected a total of 66 volatiles from 8 different families, including 16 aldehydes, 23 alcohols, 6 ketones, 6 esters, 8 monoterpenes, 3 norisoprenoids, 2 hydrocarbons and 2 acids. Among the compounds, benzaldehyde, hexanal, nonanal, benzyl alcohol, (E)-2-hexen-1-ol, 1- hexanol, (Z)-2-hexen -1-ol, 2-ethyl-1-hexanol, linalool, α-terpineol and α-ionone were the main found. Based on the obtained results, from a commercial point of view, Cristalina, Saco, Tavora, 4-84, Bigalise, Celeste and Satin cultivars might be considered some of the most interesting cultivars, since they offer a better flavor and a higher percentage of edible fruit, and consequently, major intake of phytochemicals, mainly due to their size, weight, and phenolic, mineral and volatile contents. The microbial ecology of the Saco cultivar was also explored for the first time. In total, 22 different bacteria and 33 fungi were isolated. The genera of Pseudomonas spp. (27.273%) and Ralstonia spp. (18.182%) were the most dominant bacteria, followed by Bacillus spp., Staphylococcus spp., Erwinia spp., Tatumella spp. and Dermacoccus spp. (each with 9.091%). Regarding fungi, Metschnikowia spp. (39.394%) was the most abundant genus, followed by Aureobasidium spp. (27.273%) and Hanseniaspora spp. (18.182%). In the initial stages of fruit development, Erwinia Tasmaniensis, Peudomonas viridiflava and Pseudomonas syringae bacteria are the first to emerge, while Ralstonia pickettii, Bacillus altitudinis, Enterococcus Rotai, Tatumellla terrea, Pseudomonas qingdaonensis, Pseudomonas gramininis, Dermacoccus nishinomiyaensis and Buttiauxella ferragutiae appear in the final stages of fruit ripening. Regarding fungi, Metschnikowia spp. (39.39%) was the most abundant genus, followed by Aureobasidium spp. (27.27%) and Hanseniaspora spp. (18.18%). The majority of fungi were detected in the final stages of fruit ripening, particularly the fungi Hanseniaspora uvarum, Metschnikowia pulcherrima, Hanseniaspora pseudoguilliermondii, Penicillium crustosum, Hanseniaspora meyeri, Aureobasidium proteae and Aureobasidium pullulans. The study of the microbial ecology of fruits and vegetables is vital because they can be a potential vector of foodborne pathogenic diseases and/or an important reservoir of microorganisms capable of improving the quality, characteristics and nutritional value of foods, and exert positive effects on human health. The isolation of certain microorganisms can also be an added value at an industrial level. Additionally, the in vitro biological properties of Saco cultivar were also evaluated, namely its potential to reduce free radicals and pro-inflammatory levels, as well as its potential to protect human blood samples against hemolysis and hemoglobin oxidation, interfere with the growth of cancer cells and with the activity of P-glycoprotein (P-gp), one of the main proteins related to resistance seen against several drugs, as well of αglucosidase enzyme. It was also observed that both fractions and the total extract can also inhibit the activity of α-glucosidase enzyme. Saco cultivar was chosen to carry out these assays, since it already showed to possess considerable biological properties and also for being one of the most produced cultivars in Portugal, possessing inclusive, protected geographical indication. Therefore, with the intention of increasing knowledge of the biological potential of the various phenolic compounds, and for the first time, 2 fractions rich in phenolic compounds were extracted using a solid phase C18 column, a fraction I, rich in noncoloured phenolic compounds, and fraction II, rich in anthocyanins. For comparison purposes, a total extract (III) rich in both phenolic subclasses was also tested and the results were further compared between them and with positive controls. In general, the three extracts showed a remarkable ability to capture free radicals and ferric species, as well as to interfere with the activity of proteins related to inflammation (iNOS and COX-2), and with the transmembrane transport protein, P-gp, as well as with the activity of α-glucosidase enzyme, in dose-dependent manner. It was also possible to verify that the three extracts demonstrated effectiveness in inhibiting the proliferation of cancer cells, namely colon, stomach and liver cancer cells, causing necrosis at the highest concentration (800 µg/mL for colon and stomach cells) and 100 µg/mL for liver cells). Both fractions and the total extract also showed the ability to reduce induced-oxidative stress in cancer cells, as well as in neuroblastoma model cells. From the obtained data, it is important to highlight the biological potential of the fraction rich in anthocyanins, which is in accordance with the literature. In fact, anthocyanins have been a target of many studies, due to their chemical structure. Indeed, anthocyanins are composed of several hydroxyl groups, which gives them a remarkable biological potential, namely, to reduce levels of free radicals and inflammation. Furthermore, it was also verified that the interaction between different phenolic compounds, which was observed in the total extract, was an added value in the majority of the assays done. In order to deepen the results obtained, the antioxidant activity of the main individual phenolic compounds present in cherries was evaluated against DPPH, nitric oxide and superoxide radicals. The obtained values for the DPPH radical revealed that anthocyanins, (-)-epicatechin and kaempferol 3-O-rutinoside were the most active phenolic compounds against this radical, while isorhamnetin 3-O-glucoside was the least. On the other hand, anthocyanins, (-)-epicatechin, quercetin 3-O-glucoside and caffeic acid proved to be the most effective in scavenging nitric oxide radicals, while ρhydroxybenzoic acid was the least efficient. In relation to the superoxide radical, quercetin and its derivatives showed the highest capacity, while cyanidin aglycone did not show the potential to intercept this radical at the concentrations tested. Additionally, molecular docking and absorption, distribution, metabolism, and excretion (ADME) studies were carried out, and it was observed that compounds with lower molecular masses, such as kaempferol, can easily interact with proteins related to oxidative stress, interfering in their activity, and thus, contributing to lower the concentration of free radicals to basal levels. The results obtained are highly promising and encourage translation into clinical trials, as well as the incorporation of cherries and/or their extracts into new medicines, cosmetic products, food supplements and nutraceuticals.
- Fatores de risco genético para adenomas hipofisários: Uma análise nacional, multicêntrica, genética e clínicaPublication . Gaspar, Leonor Isabel Mesquita ; Lemos, Manuel Carlos Loureiro de; Gonçalves, Catarina Inês Nunes PiresOs adenomas hipofisários representam, aproximadamente, 10-15% do total dos tumores intracranianos. A prevalência destes tumores foi estimada em 1:1000 na população geral, sendo mais frequentemente diagnosticados entre os 40-60 anos de idade. Estes tumores são monoclonais, tipicamente benignos e de crescimento lento, no entanto podem estar associados a um aumento da morbilidade e mortalidade através da sobreprodução hormonal e dos efeitos de massa resultantes da compressão das estruturas adjacentes ao tumor. Os tumores hipofisários mais frequentes são os prolactinomas, seguido pelos adenomas hipofisários não funcionantes. Os mecanismos subjacentes à tumorigénese hipofisária não são ainda totalmente conhecidos, pelo que uma melhor compreensão desta questão ajudará a gerir a doença. O aumento do risco associado a mutações em genes como o AIP, MEN1, CDKN1B e PRKAR1A, fornece evidências de uma predisposição genética para adenomas hipofisários familiares. A grande maioria dos adenomas hipofisários (cerca de 95%) ocorre num contexto esporádico e na ausência de predisposição genética conhecida. No entanto, três polimorfismos (rs2359536, rs10763170 e rs17083838) foram significativamente associados a adenomas hipofisários esporádicos na população Chinesa Han. O objetivo geral desta tese foi realizar um estudo de âmbito multicêntrico nacional, acerca dos fatores de risco genético para o desenvolvimento de adenomas hipofisários familiares e esporádicos, de forma a ampliar o conhecimento sobre a tumorigénese hipofisária. Numa primeira fase desta tese, foi construída uma base de dados com todas as variantes germinativas identificadas no gene AIP publicadas em casos esporádicos e familiares de adenomas hipofisários, até à data, a nível mundial. Nesta revisão, foram identificadas e avaliadas, ao nível da sua patogenicidade, um total de 158 mutações germinativas entre 562 doentes com adenomas hipofisários esporádicos ou familiares. Estas variantes estavam localizadas em toda a região codificadora e nas regiões de splicing do gene AIP. A patogenicidade de todas as variantes germinativas publicadas foi categorizada de acordo com os critérios da American College of Medical Genetic and Genomics (ACMG), utilizando todos os dados disponíveis. Do número total de doentes, 35,4% apresentavam variantes patogénicas e 24,0% apresentavam variantes provavelmente patogénicas. Na segunda fase desta tese foi determinada a frequência de mutações germinativas do gene AIP em doentes portugueses com macroadenomas hipofisários esporádicos de início precoce. Para isso, foi sequenciado o gene AIP em 218 doentes com macroadenomas hipofisários esporádicos diagnosticados antes dos 40 anos. Foram identificadas variantes raras em heterozigotia neste gene em 18 (8,3%) doentes. No entanto, apenas quatro (1,8%) doentes apresentavam variantes patogénicas. Estas variantes compreendiam duas mutações já conhecidas (p.Arg81* e p.Leu115Trpfs*41) e duas mutações novas (p.Ser53Thrfs*36, e p.Glu246*). Estes quatro doentes tinham sido diagnosticados com somatotrofinoma em idades compreendidas entre os 14 e os 25 anos. A frequência de variantes patogénicas no gene AIP em doentes com idade inferior a 30 anos foi de 3,4% e com idade inferior a 18 anos foi de 5%, respetivamente. A frequência de mutações no gene AIP nesta coorte de doentes portugueses foi inferior à de outros estudos. A identificação de novas variantes no gene AIP expande o espetro das causas genéticas dos adenomas hipofisários e pode ajudar a compreender o papel das mutações neste gene nos mecanismos moleculares subjacentes à tumorigénese hipofisária. A terceira fase desta tese consistiu em identificar mutações germinativas num conjunto específico de 29 genes, descritos na literatura como tendo mutações germinativas em doentes com adenomas hipofisários, numa coorte de doentes portugueses diagnosticados com adenomas hipofisários esporádicos de início precoce. Para isso, foi feita a sequenciação completa do exoma em 225 doentes com macroadenomas hipofisários esporádicos diagnosticados até aos 40 anos de idade. Foram identificadas 154 variantes raras em 25 dos 29 genes. Destas foram identificadas três variantes patogénicas e 13 variantes provavelmente patogénicas, nos genes AIP, CDH23, MEN1, MSH2, PMS2, SDHB, TP53 e VHL, em 7,1% dos doentes. Nos doentes diagnosticados com idades inferiores a 30 e 18 anos, a frequência de mutações foi de 9,0% e 12%, respectivamente. Esta é, até à data, a maior análise multigénica de doentes com macroadenomas hipofisários esporádicos de início jovem. Confirmámos que o AIP é o gene mais frequentemente envolvido, mas também descobrimos causas genéticas mais raras de adenomas hipofisários, incluindo a primeira confirmação independente de um papel do gene CDH23. Na última fase desta tese foi avaliada a associação de três polimorfismos comuns próximos dos genes NEBL (rs2359536), PCDH15 (rs10763170) e CDK8 (rs17083838) à suscetibilidade a adenomas hipofisários esporádicos na população portuguesa. Foram determinadas as frequências genotípicas e alélicas de 570 casos e 546 controlos. O alelo minor CDK8 rs17083838 (alelo A) foi significativamente associado a adenomas hipofisários esporádicos. As variantes NEBL rs2359536 e PCDH15 rs10763170 não foram associadas a risco geral para a doença, embora tenha sido observada uma associação significativa entre o alelo minor PCDH15 rs10763170 (alelo T) e somatotrofinomas. Estes resultados sugerem que a variante CDK8 rs17083838, e possivelmente a variante PCDH15 rs10763170, podem aumentar a suscetibilidade a adenomas hipofisários esporádicos na população portuguesa. Concluindo, diferentes estratégias foram desenvolvidas e implementadas, ao longo desta tese, de forma a determinar quais os fatores de risco genético mais associados ao desenvolvimento de adenomas hipofisários esporádicos e familiares. Estes resultados são importantes sob o ponto de vista científico não só para uma melhor compreensão do panorama genético dos adenomas hipofisários, como também abrem portas para novas estratégias de rastreio genético direcionadas, oferecendo conhecimentos fundamentais para a gestão personalizada dos macroadenomas hipofisários de início precoce.
- Mitochondrial Gene Therapy: Development of a mitochondrial targeted peptide/plasmid DNA vectorPublication . Faria, Rúben Miguel Ribeiro ; Costa, Diana Rita Barata; Sousa, Ângela Maria Almeida de; Boisguérin, PriscaMitochondria are cellular organelles measuring approximately 1 micron that can be found in large numbers in eukaryotic cells. These small organelles play a crucial role in cellular activity, being essential in intracellular signaling processes, apoptosis mechanisms, and energy production, among others. Mitochondria generate 90% of all energy consumed in cells, through the oxidative phosphorylation system that produces energy in the form of adenosine triphosphate (ATP) molecules. Mitochondria, similar to the nucleus, have their own genome, called mitochondrial DNA (mtDNA). Human mtDNA is composed of double-stranded circular DNA molecules, with each strand having its own composition and encoding different ribonucleic acids (RNA). The guanine-rich strand encodes 14 tRNAs, 2 rRNAs, and 12 polypeptides, while the lighter strand has information to transcribe only 8 tRNAs and one polypeptide. In total, mtDNA consists of just 37 genes that encode 13 mRNA, giving rise to 13 proteins that are part of the electron transport system and the ATPase complex. The oxidative phosphorylation system is composed of 5 complexes (NADH-ubiquinone reductase complex (complex I); succinate dehydrogenase complex (complex II); ubiquinol-cytochrome c oxidoreductase (complex III); cytochrome-C oxidase complex (complex IV) and ATP synthase (complex V)), which form the respiratory chain. mtDNA is much more susceptible to mutations when compared to the nuclear genome. Changes in mtDNA compromise the normal functioning of cells, mainly affecting neuronal and muscle tissues. The higher frequency of mutations in mtDNA can be explained by the fact that it does not have telomeres or introns in its constitution. Mitochondrial dysfunctions lead to the emergence of multisystem diseases, which can affect the normal functioning of the immune response, motor and brain function, and metabolic regulation and lead to aging. The vast majority of pathologies originating from mitochondria are inherited from maternal mtDNA. However, environmental factors such as stress and the consequent presence of reactive oxygen species, also contribute to the emergence of mutations in mtDNA. The most common mitochondrial diseases are Leber's hereditary optic neuropathy (LHON), mitochondrial encephalomyopathy, Pearson's syndrome, Parkinson's, Huntington's disease, Alzheimer's, and some types of cancer (breast, kidney, and colorectal). Complex I of the mitochondrial respiratory chain is the main entry point for electrons into the electron transport chain. Due to this fact, this complex is very important in the normal functioning of mitochondria. It is in complex I that the transfer of electrons from Nicotinamide Adenine Dinucleotide + Hydrogen (NADH) to ubiquinone occurs, the transport of protons across the inner mitochondrial membrane and is the main source of reactive oxygen species (ROS). Mutations in mitochondrial genes responsible for structural and assembly proteins of this complex lead to increased ROS production and loss of functions. One of these genes is the mitochondrial gene ND1 (NADH dehydrogenase 1). The mt-ND1 protein plays a crucial role in the structure of complex I. Mutations in mt-ND1 are associated with the emergence of LHON; Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS); progressive cardiomyopathy, and some types of cancer. Data from 2020 revealed that 1 in every 250 people have mutations in mtDNA and that 1 in every 5,000 have serious pathologies associated with mitochondrial dysfunction. However, currently, the medications available on the market only serve to mitigate the symptoms. No drug approved by the FDA or in development has been able to cure or slow the progression of mitochondrial diseases. Although there are approaches such as the use of antioxidant agents and other drugs to alleviate symptoms, the ineffectiveness of current medications highlights the urgent need for more effective treatments. Mitochondrial gene therapy is a promising approach that can focus its action directly on the cause of mitochondrial diseases and develop therapies tailored to the type of mutation. Gene therapy consists of the application of recombinant DNA techniques in which functional genes are used to replace defective genes and restore their normal functioning. As most mitochondrial diseases originate from mutations in mtDNA, mitochondrial gene therapy appears as a very promising strategy for treating this type of disease. Mitochondrial gene therapy makes it possible to attack the problem at its source and restore normal function to the affected mitochondrial gene. However, this type of therapy needs delivery systems that are effective in protecting and delivering genetic material to target cells/organelles. The greatest difficulty in applying gene therapy has been the development of nanocarriers that can effectively deliver genetic material. For mitochondrial gene therapy, the difficulty has been even greater, as the systems need to cross more barriers and be able to deliver only to that organelle. Thus, the main objective of this thesis is to develop delivery systems that have an affinity for mitochondria and can effectively deliver mitochondrial genes for the treatment of mitochondria-associated pathologies. The work carried out consisted of the development of delivery systems based on peptides (cell-penetrating peptides (CPP)) and polymers (polyethylenimine (PEI)), to deliver the mitochondrially encoded NADH dehydrogenase 1 protein (ND1) gene. To achieve this, these delivery systems were functionalized with ligands that allow specific targeting of mitochondria. The ligands used were triphenylphosphonium (TPP) and dequalinium chloride (DQA) to functionalize PEI and for CPP a mitochondrial targeting sequence (MTS) was used. The first step for the PEI-TPP/pND1 polymeric systems was to evaluate, through an experimental design, the optimal conditions for the formulation of nanoparticles. These systems were then characterized in terms of size, surface charge, and morphology. These delivery systems demonstrated the ability to internalize into cells and, through confocal microscopy, their preferential accumulation in mitochondria was demonstrated. Furthermore, these systems have demonstrated the ability to deliver the ND1 gene to mitochondria and lead to its transcription. The PEI-DQA/pND1 polymeric systems developed also demonstrated excellent physicochemical properties, showing the ability to transfect and internalize into cells. These nanocarriers delivered the ND1 gene directly into the mitochondria, leading to transcription of the gene of interest and production of the ND1 protein. However, the peptide-based systems (MTS-CPP) exhibited superior performance in terms of cellular internalization and targeting to mitochondria. Its greater ability to complex pND1 led to the formulation of nanoparticles with smaller sizes and consequently greater delivery of the gene of interest and protein expression. Showing better in vitro results, the MTS-CPP systems were tested in in vivo models (zebrafish embryos (ZF)). The peptide systems demonstrate the ability to internalize and distribute throughout the ZF organism, without causing any toxicity in these in vivo models. In short, the work carried out during this doctoral thesis sought to find solutions to the lack of effective delivery systems in mitochondrial gene therapy, to make this therapy viable for the treatment of mitochondrial diseases. The results obtained during the thesis demonstrate that the delivery systems developed are very promising for the development of mitochondrial gene therapy protocols. This work contributed to progress and innovation in an area of research that is still little explored, such as mitochondrial gene therapy. In the case of peptide-based systems, these systems have the potential to be considered in future investigations, to evaluate their translation to the clinic. The nanocarriers developed during this thesis were optimized for the delivery of the mitochondrial ND1 gene, however, these systems can be easily adapted for the delivery of any mitochondrial genes that are involved in pathologies associated with mtDNA mutations.
- Chronotherapy of Brain Diseases: Assessment of the Circadian Rhythms of Efflux Transporters at the Blood-cerebrospinal Fluid BarrierPublication . Furtado, André Filipe Lino ; Paixão, Telma Alexandra Quintela; Santos, Cecília Reis Alves; Gallardo Alba, Maria EugéniaThe choroid plexus (CP) is an integral part of the blood cerebrospinal-fluid barrier (BCSFB). The CP is formed by a monolayer of cuboidal epithelial cells united by tight junctions. On the apical side, these cells present microvilli and are in contact with the cerebrospinal fluid (CSF). On the basal membrane, these cells are surrounded by a vast network of capillary blood vessels. The CP is responsible for several functions that are vital to the homeostasis of the central nervous system (CNS) where we include the production of the CSF, synthesis of several proteins, CNS protection against foreign elements, CSF detoxification from noxious compounds that result from normal cell metabolism and the transport of multiple molecules across the BCSFB. The CP has an essential role on the transport across the BCSFB of therapeutic molecules targeting the CNS. For that, it expresses multiple membrane transporters that have been described in the literature as essential for the transport of therapeutic compounds across CNS biological barriers. Recently, a functional molecular clock was described in the CP. This means that the biological functions of this structure might have a circadian rhythmicity associated. There's the possibility that this circadian clock influences membrane transporters' expression and activity at the CP which would result in circadian changes of the bioavailability of therapeutic compounds in the CNS depending on the time of administration. As such, the main goal of this doctoral thesis was to analyse the influence of circadian rhythms on the expression of multiple membrane transporters on the CP. Additionally, we used therapeutic compounds, namely methotrexate (MTX) and donepezil (DNPZ) to assess the relation between the CP's membrane transporters circadian expression and their drug transport function across the BCSFB. One of the objectives of this project, as mentioned earlier, was to assess the circadian expression of multiple CP’s membrane transporters. For that, CP primary cell cultures of neonate rats were used. We concluded that rSlc9a1 and rSlc1a5 expression was rhythmic during a 24-hour period while rSlc47a1 did not reveal a circadian pattern. This work also aimed at disclosing the influence of sex on the daily expression oscillations of several ABC and SLC membrane transporters expressed by the CP. For this we used CPs from male, female, ovariectomized and sham-operated female rats. The results showed that the membrane transporter rAbcc1 is expressed in a circadian manner in the CP of male rats, while rAbcg2 presented circadian rhythmic expression in the CP of female rats. Both rAbcc4 and rOat3 were rhythmically expressed in the CP of male and female rats. Next, we used an in vitro model of the CP in order to evaluate the relevance of Abcc4’s circadian expression in the transport of MTX across the BCSFB. We demonstrated that MTX transport across the BCSFB was rhythmic. Besides, we also concluded that Abcc4 circadian expression might influence the MTX circadian transport across the BCSFB. Finally, this project also aimed to describe the impact of circadian rhythms on CP Abcg2 expression and also on the circadian transport profile of DNPZ across the BCSFB. Using CP primary cell cultures of neonate rats, we demonstrated the presence of rAbcg2 circadian expression. Next, using primary cell cultures, an in vitro model of the BCSFB was established and we discovered that DNPZ transport across the BCSFB presents circadian rhythmicity. Furthermore, it was also proposed that besides rABCG2, SLC22A4 could also be involved in the DNPZ circadian transport across the BCSFB. The results obtained in this project demonstrate that membrane transporters present circadian expression in the BCSFB. Moreover, the transport of therapeutic compounds, such as MTX and DNPZ, across the BCSFB is also influenced by the circadian rhythm of CP membrane transporters. In the future, it is essential to further exploit the role of circadian rhythms on the expression of membrane transporters at the CP and its influence on the transport of therapeutic compounds across the BCSFB. This information might prove vital in the treatment of CNS diseases. By timing drug administration with the period when they are more prone to reach the target tissue at the CNS, we are ensuring their maximum target tissue concentration, and a reduction in side effects.