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- 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.
- Analysis of the Clinical Pathways of Oncological Patients: A comparison of realities for future improvement at “Unidade Local de Saude” at Guarda, PortugalPublication . Quesada, Mario Forrester; Granadeiro, Luiza Augusta Tereza Gil Breitenfeld ; Aperta, Jorge Manuel GonçalvesBackground Cancer is a leading cause of death among elderly populations, with cases continuing to rise as life expectancy increases. This growing burden has prompted a shift in oncology care from a disease-focused approach to a patient-centered model that prioritizes quality of life alongside tumor response. Ensuring safe, effective, and well-coordinated cancer treatment—along with supportive and complementary therapies—has become a key priority. Additionally, optimizing healthcare system efficiency is essential to managing costs, improving accessibility, and ensuring equitable cancer care. In this context, understanding patient demographics and the structure of oncology clinical pathways is crucial for enhancing care delivery. Methods This study examines the Oncology Clinical Pathway (OCP) at a Portuguese local health unit (Unidade Local de Saúde, ULS-Guarda) to evaluate cancer diagnosis, treatment, and management. The first phase involves a retrospective analysis of medical records from oncology patients treated between 2013 and 2017 across ULS-Guarda’s network, which includes two regional hospitals and 14 primary healthcare centers. This analysis assesses patient demographics, tumor types, and duration of care. The second phase focuses on healthcare professionals’ perspectives regarding the clinical pathway. Using a qualitative approach, structured questionnaires were distributed to key stakeholders—including physicians, nurses, hospital pharmacists, and community pharmacists—to evaluate coordination between services, management practices, process timelines, and overall perceptions of cancer care. Results and Discussion The collected data was analyzed to identify variations in experiences and perspectives among healthcare professionals. Findings highlighted the strengths and weaknesses of the oncology clinical pathway, including care transitions, coordination between different healthcare providers, and the role of available platforms in patient management. Additionally, the study explored the impact of the COVID-19 pandemic on oncology services, revealing disruptions and challenges in patient care delivery. Conclusion The study highlights significant demographic, economic, and healthcare challenges in the Beira Interior region, impacting the development of an effective Clinical Pathway. The analysis of the Oncological Clinical Pathway (OCP) revealed coordination gaps and the need for standardized protocols and multidisciplinary collaboration. The COVID-19 pandemic underscored the necessity of adaptable healthcare systems, with professionals demonstrating resilience in patient management. Disparities in access and care coordination were identified, particularly in community pharmacies. Strengthening healthcare infrastructure, improving training, and fostering collaboration among institutions are crucial to enhancing cancer care and patient outcomes in the region.
- Conhecimentos sobre Saúde Ocular: um estudo exploratórioPublication . Martins, Helena Beatriz Silva; Nunes, Amélia Maria Monteiro FernandesObjetivo: este estudo tem como principal objetivo aferir o conhecimento da população em geral relativamente a saúde ocular através de um questionário. O intuito do mesmo é entender e analisar qual a informação que cada indivíduo tem sobre a temática e no caso de a ter, identificar as principais fontes de obtenção desse conhecimento. Metodologia: foram inquiridos 190 participantes portugueses com idades compreendidas entre os 19 e os 77 anos de idade. Foram efetuadas questões genéricas sobre saúde ocular através de um questionário distribuído online e posteriormente os resultados foram agrupados por variável de estudo. Aplicou-se o teste estatístico de qui-quadrado para averiguar se o conhecimento da população difere entre grupos com características sociodemográficas semelhantes (sexo, grau de escolaridade e profissão). Resultados: verificou-se que os resultados variam em função das diferentes características sociodemográficas, identificando-se grupos com níveis de conhecimento significativamente mais baixos em relação a outros. Das cinco patologias apresentadas, a DMRI mostrou ser a que a população tem menos conhecimento, enquanto que as cataratas mostraram ser a patologia sobre a qual a população tem mais conhecimento. Apesar de se terem encontrado diferenças entre sexo e segundo o grau de escolaridade, verificámos que existem diferenças mais significativas quando a população é estratificada pela profissão. No entanto, ao analisar as respostas deste grupo, observamos que ainda uma grande parte responde “Não sei”, principalmente quando falamos de erros refrativos e cataratas, o que indica que a necessidade de aprimorar o conhecimento sobre saúde visual deve ser feito não só na população não relacionada com a área da saúde mas em também junto dos indivíduos da área da saúde. Conclusão: as características sociodemográficas da população fazem com que os conhecimentos sobre saúde ocular sejam distintos entre indivíduos. Observaram-se diferenças nos níveis de conhecimento segundo fatores como sexo, nível de escolaridade e área de profissão. Das características estudadas, o facto de trabalhar na área da saúde em geral ou não trabalhar na área da saúde mostrou ser a característica sociodemográfica mais significante, ainda que não em todas as patologias.
- Caraterização de Intoxicações da Urgência da ULS da Cova da BeiraPublication . Sena, Naylia Bento de; Rosado, Tiago Alexandre Pires; Alba, Maria Eugénia Gallardo; Saraiva, Rosa Maria PereiraO presente relatório de estágio, elaborado para a obtenção do grau de mestre, encontrase dividido em três capítulos. O primeiro capítulo aborda a vertente de investigação, o segundo refere-se à experiência profissional em Farmácia Comunitária e o terceiro trata da experiência de estágio em Farmácia Hospitalar. O capítulo I é referente ao estudo retrospetivo e descritivo dos doentes que recorreram as urgências da Unidade Local de Saúde (ULS) da Cova da Beira por possíveis intoxicações no ano de 2022, ano de pandemia. Através deste estudo é possível expandir o conhecimento sobre as intoxicações que ocorrem na área de intervenção da ULS Cova da Beira, sendo o seu principal objetivo a melhoraria do atendimento aos utentes que recorrem a esta unidade. Durante o ano de 2022, o Serviço de Urgência da ULS da Cova da Beira registou 52 casos de intoxicação em adultos e 11 em menores. Em ambos os grupos, a maioria das intoxicações ocorreu em indivíduos do sexo feminino, nas faixas etárias de 50 a 59 anos e 16 a 17 anos, respetivamente. É importante destacar que o agente tóxico mais frequente foi o medicamento. Tanto adultos quanto crianças foram levados às urgências devido à manifestação de sintomas, sendo oral a via de intoxicação predominante, com as intoxicações a ocorrerem de forma involuntária. No caso das crianças, 36,4% foram internadas, enquanto que entre os adultos, 21,2% foram encaminhados para o centro de saúde. No capítulo II são descritas as principais atividades realizadas e as competências adquiridas ao longo do estágio na Farmácia São Cosme, que ocorreu de 5 de fevereiro a 26 de abril de 2024, sob a orientação do Dr. Carlos Tavares. Este estágio em farmácia comunitária destacou a importância fundamental da profissão farmacêutica na sociedade. O capítulo III relata a minha experiência e conhecimento adquiridos ao longo do estágio nos Serviços Farmacêuticos da Unidade Local de Saúde de Castelo Branco (ULSCB), nas instalações do Hospital Amato Lusitano, sito em Castelo Branco, sob a orientação da Dr.ª Sandra Queimado. O estágio decorreu no período de 29 de abril a 19 de junho de 2024.
- Conjugados Anticorpo-Fármaco na Terapêutica FarmacológicaPublication . Silva, Sónia Catarina Ribeiro da; Morgado, Manuel Augusto Nunes Vicente PassosNo âmbito da Unidade Curricular “Estágio” para obtenção do grau de Mestre em Ciências Farmacêuticas, surge esta presente dissertação, que se completa por três distintos capítulos: componente de Investigação Científica (Capítulo I), componente da experiência profissionalizante em Farmácia Hospitalar (Capítulo II) e, por fim, a componente da experiência profissionalizante em Farmácia Comunitária (Capítulo III). O Capítulo I intitulado por “Conjugados Anticorpo-Fármaco na Terapêutica Farmacológica” diz respeito à revisão narrativa realizada. Esta teve como principal objetivo revisar os vários antibody-drug conjugates (ADCs) aprovados e em uso a nível nacional e internacional, assim como descrever as suas indicações terapêuticas. Para isso, foram analisados vários artigos científicos disponíveis na base de dados PubMed entre 2020 e 2023 e ainda intercetada com informação disponível nas bases de dados do INFARMED, European Medicines Agency (EMA) e U.S. Food and Drug Administration (FDA), pois foi segundo as aprovações destas entidades que a investigação se baseou. Ao momento do início desta investigação, encontram-se, no total, aprovados 12 ADCs, que dão, na sua maioria, resposta a patologias oncológicas (quer tumores sólidos quer hematológicos), através do seu mecanismo de ação diferenciador, e que promove um direcionamento mais preciso ao alvo terapêutico. Ainda num futuro próximo pretende-se alargar essas indicações terapêuticas face ao uso de ADCs, existindo já alguns em fase de desenvolvimento nesse sentido. O Capítulo II desta dissertação trata-se de um relatório onde é descrita a minha experiência profissionalizante em Farmácia Hospitalar que decorreu no Centro Hospitalar Universitário Cova da Beira, durante o período de 8 semanas. Nele é abordado todo o processo relacionado com o circuito do medicamento dentro de uma instituição hospitalar, com principal enfoque na dinamização dos vários setores existentes: logística hospitalar, farmacotecnia, distribuição em ambulatório e distribuição individual em dose unitária. Ao longo de todo o relatório é evidenciado o papel crucial do farmacêutico e as suas responsabilidades, assim como a importância de uma multidisciplinariedade, que nos últimos anos tem vindo a ganhar, cada vez mais, espaço dentro da dinâmica diária do hospital, tendo em vista o ganho em saúde dos doentes. O Capítulo III é referente à minha experiência profissionalizante em Farmácia Comunitária, na Vila de Lousada. Neste capítulo encontra-se, uma vez mais, descrito, o circuito do medicamento e produtos de saúde, assim como o papel ativo do farmacêutico e as suas responsabilidades, mas num contexto diferente do mencionado no capítulo anterior. Durante este período de 12 semanas em que decorreu este estágio pude acompanhar transversalmente todas as áreas da farmácia comunitária e destacar a importância do farmacêutico junto da comunidade, pois é o profissional de saúde próximo do utente e na qual este confia, devendo por isso ser criado uma relação de respeito, mas também de compromisso para quem o procura, pois mostrou-me a necessidade de um acompanhamento atento para que haja uma maior adesão à terapêutica e maiores ganhos em saúde. Destaco assim, a importância que a Farmácia Fonseca teve neste percurso, uma vez que me proporcionou um contacto próximo e ativo junto da comunidade abrangente deste estabelecimento de saúde, podendo terminar este período com um leque diversificado de atividades desenvolvidas.
- Desenvolvimento de uma emulsão cosmética O/A com Ulva lactuca para a Dermatite AtópicaPublication . Louro, Sofia Alexandra Pinto Gamboa; Oliveira, Rita Manuela Palmeira de; Gama, Ana Rita; Gomes, Carolina ProençaO presente Relatório aqui apresentado divide-se em três partes: uma correspondente à componente de investigação e duas relativas às componentes de estágios em farmácia hospitalar e em farmácia comunitária. O primeiro capítulo aborda a componente de investigação, realizada no Centro de Investigação em Ciências da Saúde da Universidade da Beira Interior. O estudo teve como objetivo o desenvolvimento de uma emulsão cosmética O/A com Ulva lactuca para a Dermatite Atópica. A Dermatite Atópica (DA) é uma doença inflamatória crónica da pele cujo controlo torna necessário recorrer à utilização de produtos cosméticos. Com vista a incorporar extratos de algas como ingredientes cosméticos, o trabalho desta dissertação avaliou as propriedades de quatro extratos da alga Ulva lactuca, relativamente à sua biocompatibilidade in vitro com células da pele através de um ensaio MTT, poder antioxidante através de um ensaio de DPPH e poder reparador da pele com recurso a um ensaio de migração. Os resultados demonstraram que o extrato etanólico a 40% foi o mais biocompatível, o metanólico o que demonstrou maior poder cicatrizante e que todos demonstraram fraca atividade antioxidante. Paralelamente, desenvolveuse uma emulsão com ingredientes com ações benéficas na DA, com vista à incorporação de um dos extratos. O protótipo escolhido foi formulado a quente. O produto final foi sujeito a testes de estabilidade acelerada encontrando-se a 400C durante 3 meses, quando apresentou alteração da cor e manutenção das restantes características organoléticas. Concluiu-se que estes extratos de algas podem apresentar potencial aplicação cosmética graças à sua baixa toxicidade celular e ao seu poder cicatrizante. O segundo capítulo refere-se à experiência profissionalizante em farmácia hospitalar, cujo estágio decorreu na Unidade Local de Saúde Cova da Beira, durante o período de tempo compreendido entre 5 de fevereiro e 29 de março de 2024. O terceiro capítulo refere-se se à experiência profissionalizante em farmácia comunitária, cujo estágio decorreu na farmácia São Cosme, durante o período de tempo compreendido entre 1 de abril e 21 de junho de 2024. Nos capítulos 2 e 3 serão apontadas as atividades realizadas, assim como o conhecimento adquirido ao longo dos períodos correspondentes aos estágios curriculares mencionados anteriormente.
- Riscos Associados ao Uso dos Inibidores da 5a- Redutase: Análise de Reações Adversas Notificadas à EudraVigilancePublication . Alves, Ricardo Paulo Moreira; Monteiro, Cristina Sofia de Jesus; Silvestre, Samuel MartinsEste relatório encontra-se estruturado em dois capítulos. O primeiro capítulo aborda a vertente de investigação, na qual foi conduzido um estudo sobre as reações adversas associadas aos inibidores da 5a-redutase (5ARI) reportadas à EudraVigilance, durante o período compreendido entre 1 de janeiro de 2005 e 27 de março de 2023. O segundo capítulo é dedicado ao relatório do estágio curricular realizado em Farmácia Comunitária. O primeiro capítulo, intitulado "Riscos Associados à Utilização dos Inibidores da 5aRedutase: Análise de Reações Adversas Notificadas à EudraVigilance", aborda a segurança dos inibidores da 5a-redutase no contexto da farmacovigilância. Desde os tempos mais remotos, o combate às doenças tem sido uma prioridade para o ser humano, sendo os medicamentos instrumentos essenciais nesse processo. Contudo, nenhum medicamento está isento de riscos. Neste âmbito, a farmacovigilância assume um papel fundamental na monitorização e controlo das reações adversas a medicamentos (RAM), contribuindo para a prevenção e minimização dos riscos inerentes ao seu uso, com o consequente impacto positivo na saúde pública. Entre os diversos fármacos desenvolvidos, os 5ARI destacam-se pelo seu papel crucial na prevenção, controlo e tratamento de patologias como a hiperplasia benigna da próstata (HBP) e a alopecia androgénica (AGA). Estes fármacos têm demonstrado eficácia no tratamento destas patologias, contudo, a sua utilização está associada a reações adversas que necessitam de uma contínua avaliação para garantir um equilíbrio entre os benefícios e os riscos do tratamento. Os 5ARI analisados neste estudo foram a finasterida e a dutasterida, tanto de forma isolada como em combinação. Assim, foram avaliados os dados relativos às RAM associadas aos 5ARI notificadas à EudraVigilance, no período compreendido entre 1 de janeiro de 2005 e 27 de março de 2023. Para este estudo, foram selecionadas 7777 notificações, nas quais se analisaram várias variáveis, incluindo o número de notificações registadas ao longo dos anos, com discriminação entre os diferentes 5ARI, o tipo de notificador e a faixa etária da população afetada pelas RAM. As RAM foram caracterizadas de acordo com a sua gravidade e respetivos critérios de avaliação, a sua evolução ao longo do tempo, e a relação entre a gravidade, o tipo de 5ARI utilizado e a faixa etária dos indivíduos afetados. Adicionalmente, as RAM mais prevalentes foram classificadas em termos de presença ou ausência no Resumo das Características do Medicamento (RCM). As RAM mais frequentes também foram categorizadas com base no tipo de 5ARI suspeito, utilizando termos da DME list (Do Inglês, Designated Medical Event) e avaliadas quanto à sua inclusão no RCM. A faixa etária da população mais afetada, excluindo a categoria "Não Especificado", foi a de "18-64 anos". De modo geral, o 5ARI mais frequentemente notificado foi a finasterida. As RAM reportadas foram, na sua maioria, classificadas como “Grave”, com uma evolução predominante para persistência sem recuperação, e, em grande parte, não estavam descritas no RCM do respetivo 5ARI. Relativamente às RAM consideradas “Grave”, o critério de gravidade mais reportado foi "Clinicamente importante". Com base nestes resultados, conclui-se que a monitorização contínua destes medicamentos é essencial para prevenir e mitigar as RAM, de modo a proteger a população e a promover a saúde pública. Além disso, futuros estudos são necessários para verificar se as RAM não descritas no RCM poderão constituir novos sinais de segurança. O segundo capítulo descreve a experiência profissionalizante vivida durante o estágio curricular em Farmácia Comunitária, realizado na Farmácia Sant’Ana, localizada na Covilhã, sob a orientação e supervisão da Dra. Paula Bártolo, no período compreendido entre 6 de fevereiro e 30 de junho de 2023. Este período revelou-se fundamental para consolidar o conhecimento teórico adquirido ao longo da formação académica, desenvolver novas competências profissionais e compreender de forma aprofundada o papel essencial que o farmacêutico comunitário desempenha na sociedade, tanto no aconselhamento dos utentes como na promoção da saúde pública.