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V A G U S - Mobilidade e Acessibilidade para todos
Publication . Crespo, Miguel Francisco; Romãozinho, Ana Mónica Pereira Reis de Matos; Guerreiro, Alexandre Gramacho
Muitas regiões têm apostado no turismo como forma de alcançar o desenvolvimento e reconhecimento das suas culturas e tradições, história ou gastronomia, promovendo os seus pontos positivos e diminuindo os negativos, através de um pensamento económico sustentável. No entanto, por vezes a preocupação com as comunidades menos favorecidas é relegada para segundo plano, resultando assim em espaços pouco adequados para o manuseio de um equipamento de assistência técnica (como andarilhos, bengalas, entre outros) utilizados maioritariamente pela comunidade idosa. Deste modo, foram descritos e aprofundados atentamente argumentos, metodologias e opiniões, que justificam a importância do auxílio à problemática da mobilidade, da autonomia e da acessibilidade no interior de Portugal continental, com a finalidade de combater ou amenizar as patologias ligadas à perda da locomoção, da independência pessoal, da solidão e do isolamento social. À vista disso todo desenvolvimento projetual, teve como base de sustentação as metodologias do Design Thinking e do HCD (Design centrado no ser humano), introduzidas por David Kelly, com o propósito de entender a verdadeira origem da problemática e por consequência despertar a melhor solução possível para a sociedade consumidora. Assim sendo, o produto teria então como finalidade, proporcionar à persona um auxílio na atividade do dia a dia, através de um produto versátil, multifuncional, intuitivo, seguro e ajustável e que permitisse deste modo a circulação na via pública sem qualquer tipo de necessidade ou constrangimento. Estes aspetos poderão à vista disso, ser alcançados, através da preocupação com alguns elementos presentes no desenvolvimento projetual como a ergonomia, a escolha de materiais e respetivas texturas e também todos os seus componentes e combinações possíveis, com o fim de alcançar a melhor segurança e conforto possível para a persona, direcionando-se não só aos idosos mas tambem a todos os que necessitem por intermédio de uma solução de design intemporal e com linhas que se afastem do aspeto ortopédico, bastante presente em muitos equipamentos desta classe no mercado.
Evaluation of Specific Anthraquinones as New catechol-O-methyltransferase Inhibitors: Virtual Docking, Molecular Dynamics, Inhibition and Cytotoxicity In Vitro Studies
Publication . Proença, Fábio Alexandre Esteves; Passarinha, Luís António Paulino; Silvestre, Samuel Martins
Parkinson´s disease (PD) is the second most prevalent age-related neurodegenerative disorder around the world, with no cure currently in sight. This condition is caused by the gradual loss of the brain's dopaminergic neurons, in the substantia nigra pars compacta of the brain. This progressive neuronal loss leads to the typical symptoms of the disease characterized by rigidity, resting tremor, bradykinesia, and postural imbalance. Actually, the conventional therapy for this pathology consists in the administration of oral levodopa (L-DOPA), a natural dopamine precursor, and two enzymatic inhibitors: one for Catechol-O-Methyltransferase (COMT) and the other for the peripheral aromatic L-amino acid decarboxylase (AADC). In terms of central nervous system, AADC can transform the administered L-DOPA in dopamine, and both substances, at central and peripheral level, can be metabolized by COMT. In humans, the COMT enzyme is present in two isoforms, a soluble isoform (S-COMT) and the membrane-bounded isoform (MB-COMT). Despite their similarities in the primary amino acid sequences, there is a accentuated difference in the kinetic behavior of both isoforms. MB-COMT tends to have a higher affinity for the substrate (lower Km) than SCOMT. On the other hand, S-COMT has a much higher catalytic reaction capacity (Vmax) than MB-COMT. Those differences in the kinetic behavior of the isoforms determined the role of the isoenzymes, with MB-COMT being physiologically more relevant due to its role in catecholamine methylation at physiological concentrations. Typically, COMT inhibitors enhance L-DOPA and dopamine bioavailability and effectiveness. However, they are often associated with toxicity and/or limited ability to cross the blood-brain barrier (BBB). This underscored the need to discover/ develop novel molecules with greater potency, reduced toxicity, and better pharmacokinetic properties than the existing inhibitors in clinical use. This dissertation investigates the potential of anthraquinones as potential COMT inhibitors, based on their molecular skeleton similarity with tolcapone. Anthraquinones are a subclass of quinones derived from anthracene with anti-tumor, anti-inflammatory, and neuroprotective properties, among others. They are characterized by having three planar rings with two ketones groups on the second ring. The inhibition of MB-COMT by nine anthraquinone family compounds was evaluated in vitro, using an analytical method previously described by the research group. From the obtained results, four of the nine compounds in study exhibited an enzymatic inhibition capacity close to 60%, at 100 µM. After, a cytotoxicity evaluation was performed for these 4 compounds by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays using two different cell lines, normal human dermal fibroblasts (NHDF) and a rat dopaminergic neural cell line (N27). From that cytotoxic evaluation it was observed that the compounds in study displayed a cytotoxicity profile similar to the described inhibitor, tolcapone. In silico trials were performed using Autodock Vina complementing the in vitro findings by analyzing interactions and the positioning of the compounds in relation to the protein´s active center. The results were ranked based on the binding energy and key interactions with critical residues for MB-COMT´s catalytic capacity. Promising molecules were further analyzed using molecular dynamics simulations, using the commercial inhibitor tolcapone as a positive control. The principal Adsorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) properties were also predicted intending to complement the pharmacokinetic analysis and toxicity of the molecules. The in vitro and in silico obtained results suggest that purpurin, alizarin, 3-nitroalizarin and lucidin are promising candidates as COMT inhibitors, however, half maximal inhibitory concentration (IC50) assays for the enzymatic inhibition and for cytotoxicity, need to be performed to properly evaluate the potential of this molecules.
New delivery systems based on gellan gum nanoparticles for Parkinson Diseases Therapeutics
Publication . Rodrigues, Madalena Filipa Geada; Passarinha, Luís António Paulino; Sousa, Ângela Maria Almeida de; Cristóvão, Ana Clara Braz
Globally, there are up to 10 million people diagnosed with Parkinson's Disease, a chronic neurodegenerative disorder without a cure. Physiologically, it is characterized by the progressive loss of dopaminergic neurons in the substantia nigra. Clinically, patients with this disease reveal several motor symptoms, such as tremors, tension, and postural instability, and non-motors, such as depression and anxiety. To date, the most effective drug combination used in the treatment of Parkinson's disease is the administration of levodopa (L-Dopa) combined with catechol-O-methyltransferase (COMT) inhibitors and monoamine oxidase inhibitors to restore dopaminergic brain levels. However, the commercially available inhibitors have low capabilities to cross the blood-brain barrier and, thus, low bioavailability in the brain. Also, the prolonged use of these drugs is associated with high hepatotoxicity, which currently limits their use. Therefore, the discovery of molecules with the potential to inhibit the COMT and the development of new delivery systems for these drugs are crucial elements to improve the effectiveness of existing therapies. Using polymeric nanoparticles as antiparkinsonian drug carriers presents numerous advantages considering the current therapies. These nanosystems can cross biological barriers due to their chemical properties and small size. Furthermore, they can achieve the therapeutic target more efficiently, increasing the bioavailability of the drug in restricted environments, such as the brain. Also, the application of polysaccharides in developing this type of delivery system presents advantages such as lower economic costs and better biocompatibility and biodegradability compared to oral and intravenous therapies. The discovery of the molecules 3,4-dihydroxy-5-nitrobenzonitrile (ZINC035) and 2- bromo-3,4-dihydroxy-S-nitrobenzaldehyde (ZINC496) as new COMT inhibitors, combined with the potential of nanoparticles based on natural polymers, such as gellan and chitosan, as drug delivery systems, have proven to be the impulse for this work. In the first part of this work, an analytical method using High-Performance Liquid Chromatography (HPLC) was developed to detect and quantify these new inhibitors in brain tissue samples from adult Wistar rats. In this study, the biological samples were spiked with each inhibitor and, subsequently, analyzed using two different types of detection, in which electrochemical detection (HPLC-ECD) was more effective for biological matrices than diode-array detection (HPLC-DAD). Additionally, a study was realized using different mobile phases, varying from 9 to 18% (v/v) of the organic compound (acetonitrile) to optimize the experimental procedure time. The results obtained using an HPLC-ECD analysis system interestingly showed that the molecules under study have interaction capabilities with brain tissues, and due to their similarity with commercial inhibitors, their potential use in Parkinson's therapies has been proven. In the progress of this work, a new polymeric delivery system has been developed to improve metabolism and absorption, increasing their chemical stability, decreasing their susceptibility to enzymatic degradation, and improving bioavailability of the commercial drugs. This delivery system results from the complexation of two natural polymers with opposite charges, the gellan gum (GG) and the chitosan (CH). These polymeric complexes were formulated, testing different conditions, varying the molecular weight of chitosan (5 kDa and low molecular weight) and the ratio and concentration of the polymers (0.05-1 mg/mL). After optimization, the GG/CH systems were prepared with the inclusion of the selected COMT inhibitor (ZINC035) or L-Dopa. In order to obtain the encapsulation efficiency of the nanoparticles, a purification system was developed, testing three different methodologies (centrifugation, filtration and molecular exclusion chromatography) and varying some conditions, such as centrifugation speeds (8000- 12500 rpm), the size of filter pore (0.22 and 0.45 µm) and the elution buffers (sodium acetate, sodium chloride and phosphate-buffered saline). The best GG/CH delivery system had a size of 238.52 nm; a polydispersity index (PDI) value of 0.449 and a zeta potential (ZP) of +30.2 mV. Also, GG/CH nanoparticles loaded with ZINC035 showed a mean size of 163.5 nm; a PDI of 0.355; a zeta potential of +20.6 mV and an encapsulation efficiency of 67.04%, which are stable up to 48h after formulation. Finally, the L-Dopa-loaded delivery systems achieved a size of 177.01 nm, a PDI of 0.392, and an encapsulation efficiency of approximately 26%. Overall, the HPLC system with electrochemical detection developed in this work is an effective and innovative methodology for studying COMT inhibitors in biological tissues. Furthermore, nanoparticulate polymeric systems have unique properties that can be used according to the desired intranasal delivery application and will certainly present advantages over conventional therapies.
Development of DNA nanovaccines based on functionalized RALA and Chitosan nanoparticles bearing HPV-16 oncogenes
Publication . Giusti, Andressa Moreira; Sousa, Ângela Maria Almeida de; Eusébio, Dalinda Isabel da Silva; Cui, Zhengrong
Cervical cancer (CC), a leading cause of cancer mortality among women, is mainly caused by persistent high-risk human papillomavirus infections, particularly HPV-16 and -18. These viruses possess the oncoproteins E6 and E7, which interfere with p53 and retinoblastoma protein (pRB), respectively, contributing to tumorigenesis. Current vaccines prevent infection but have no therapeutic effect. Moreover, the aggressiveness and lack of specificity of current treatments require innovative targeted therapeutics. DNA vaccines targeting the E6 and E7 oncogenes can be a safer and more promising option for CC eradication, providing preventive and therapeutic effects. The optimal DNA vaccine scenario includes the use of minicircle DNA (mcDNA), a safer and efficient vector than the conventional plasmid DNA (pDNA). So, in this study, we explored the complexation of mcDNA encoding one or both mutated HPV-16 oncogenes (E7mut or E6mut) with biocompatible materials, such as cellpenetrating peptides (CPPs) like RALA, and chitosan (CS). These delivery systems were functionalized with ligands of R8-mannose (R8M), which can enhance DNA delivery and targeting to antigen-presenting cells (APCs). Additionally, we investigated the powder conversion of the DNA/CS-based vaccine using thin-film freeze-drying (TFFD) to enhance vaccine stability. Pure mcDNA (2 µg) was used to optimize the amine-to-phosphate (N/P) ratios of RALA, with and without R8M, and nanoparticles (NPs) were characterized for size, polydispersity index (PDI), zeta potential, complexation efficiency (CE), stability, morphology, and Fourier transform infrared spectroscopy (FTIR). In vitro studies assessed biocompatibility and gene expression in JAWSII dendritic cells after 24 h transfection. For CS-based systems, NPs were prepared with CS, sodium tripolyphosphate (TPP), and 2 µg of parental plasmid (PP)/pDNA or mcDNA encoding both genes, and R8M was also included. The same characterization and biocompatibility assays were performed. Powder conversion of CS NPs was done using TFFD, optimizing conditions for suitable and stable powders. For RALA-based NPs, a N/P ratio of 1.25 was optimal, resulting in homogeneous NPs under 150 nm, negative surface charge, and high CE (>97%). Their morphology was spherical/oval, and incorporation of components was confirmed by FTIR. The NPs were stable under cell culture conditions and biocompatible with JAWSII cells. Expression of the E6 gene was significantly higher in RALA-mannosylated systems, with no differences between the E6mut and multigenic vectors’ gene expression. For CS-based NPs, promising characteristics were obtained, with sizes under 120 nm, homogeneity, positive charges (>20 mV), and CE >98%. Incorporation of PP or mcDNA and R8M did not affect NP properties, indicating feasibility for formulation. The NPs exhibited spherical/oval morphology, and FTIR confirmed the presence of all components. The systems were stable under cell culture conditions and biocompatible with JAWSII cells. Powder conversion of CS-based NPs was optimized with sucrose (1% solid content) as a lyoprotectant, yielding the best results with 0.5 mL per vial. For scale-up with higher NP batches, a solid content of 5.05% with sucrose and leucine was optimal, where R8M incorporation and mcDNA usage showed consistent results, with no changes in NP properties after TFFD. The powders had porous, brittle matrices typical of TFFD. Stability tests over 14 days showed the best result of the powder vaccine at 4°C, supporting improved vaccine storage and distribution in low-resource settings. In conclusion, R8M functionalization enhanced cellular transfection and gene expression in RALA-based systems. Both peptide- and polymer-based delivery systems, with and without R8M, exhibited suitable physicochemical characteristics and biocompatibility. TFFD successfully converted liquid vaccines into stable powders while preserving NPs properties and producing highly porous powders with the potential for good aerosol properties. These findings support further studies exploring intranasal administration for cervical cancer immunization.
O plexo coroide como porta de entrada para metástases cerebrais
Publication . Capoti, Érica de Sousa; Santos, Cecília Reis Alves dos; Duarte, Ana Catarina Abreu
O plexo coroide (PC) é uma estrutura vascularizada localizada nos ventrículos cerebrais que desempenha um papel crucial na produção e regulação do líquido cefalorraquidiano (LCR). Essa função é essencial para a proteção do sistema nervoso central (SNC), regulação da homeostase cerebral e transmissão de sinais neuronais. O PC é composto por células epiteliais que formam a BCSFB, caracterizada por junções apertadas que controlam seletivamente o fluxo de moléculas e células entre o sangue e o LCR. Anatomicamente, o PC apresenta configurações distintas nos diferentes ventrículos cerebrais, estende-se ao longo do assoalho ventricular nos ventrículos laterais e forma aglomerados no teto do terceiro e quarto ventrículos. A estrutura básica do PC inclui vasos sanguíneos envolvidos por tecido estromal e uma camada epitelial rica em junções de oclusão, constituídas por ocludinas e claudinas, que desempenham um papel crucial na manutenção da integridade da BCSFB. Além das funções na produção de LCR e na BCSFB, o PC atua na proteção antioxidante do SNC contra o estresse oxidativo, utilizando enzimas como superóxido dismutase e glutationa peroxidase. Esses sistemas enzimáticos são fundamentais para neutralizar espécies reativas de oxigênio que podem ser prejudiciais ao cérebro. Em contextos patológicos, como o cancro cerebral, o PC pode desempenhar um papel importante na propagação de metástases cerebrais, embora esse processo ainda não esteja completamente compreendido. Há indícios de que células tumorais, como as de cancro de pulmão, mama e melanoma, possam utilizar a via do PC para entrar no SNC, atravessando a BCSFB. No entanto, a capacidade de invasão por meio desta via e as interações moleculares envolvidas, como aquelas mediadas por E-caderina e ICAM2 nas células endoteliais do PC, ainda carecem de estudos mais aprofundados. Por isso, investigar esse mecanismo é crucial para esclarecer o papel do PC na invasão de células tumorais no cérebro. Para a realização deste trabalho foram utilizadas linhas celulares HIBCPP (que mimetizam a BCSFB) e A549 de cancro do pulmão para modelar a BCSFB in vitro e estudar a transmigração celular. As células foram cultivadas em meio DMEM/F12 suplementado com soro fetal bovino e antibióticos, mantidas em condições de 37°C e 5% de CO2. Os inserts foram revestidos com colagénio para melhorar a adesão das células HIBCPP, cuja integridade da barreira foi avaliada pela medição da resistência elétrica transepitelial (TEER). Ensaios de transmigração com células A549 marcadas com CellTracker Green CMFDA foram feitos com o intuito de investigar os possíveis efeitos na função da BCSFB e poder contribuir para a compreensão das interações celulares em condições fisiológicas e patológicas do SNC.