FC - DQ | Dissertações de Mestrado e Teses de Doutoramento
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Browsing FC - DQ | Dissertações de Mestrado e Teses de Doutoramento by Sustainable Development Goals (SDG) "03:Saúde de Qualidade"
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- Backyard production systems in Portugal: exploring associated risk factors and antimicrobial resistancePublication . Baroni, Bruno Morbeck; Nunes, Alexandra Isabel Cardoso; Ferreira, Susana Margarida Paraíso; Sobral, Daniel Vieira Noro e SilvaThe One Health approach recognizes that human, animal and environmental health are interconnected and aims to create effective solutions to complex problems. Animal production is associated with the spread of zoonoses and antimicrobial resistance (AMR) throughout the food chain and the environment. However, there is limited knowledge about the impacts of backyard animals raised for self-consumption. Backyard production systems (BPS) usually have poor biosecurity implementation, with several studies identifying a diverse community of zoonotic pathogens and AMR genes in those systems. Thus, the aim of this work was to evaluate the safety and impacts of Portuguese BPS, with specific focus on zoonoses and AMR. To achieve this, the first specific objective was to apply a questionnaire to BPS owners to obtain data on husbandry and biosecurity practices implemented in BPS. The second specific objective was to evaluate, through an innovative metagenomic methodology, the presence of zoonotic pathogens and AMR genes in fecal and soil samples from BPS that raise poultry, small ruminants and pigs. In the questionnaire, a total of 283 responses were analyzed, obtaining valuable data about the characteristics of BPS in Portugal, such as that 92% of BPS owners have backyard animals for the consumption of animal products, but only 28% have training in animal production or welfare. Also, neglect of biosecurity protocols and animal health measures were reported in some BPS. For example, 43% of respondents did not use personal protection equipment when handling animals and 83% did not isolate sick animals. Some practices that could promote pathogen and AMR dissemination to the environment and other productions systems were also reported, such the use of manure as fertilizer in 95% of BPS, backyard animals being exposed to the wildlife in 39% of BPS and carcass disposal in the house waste in 6.5% of BPS. For the metagenomic analysis, fecal and soil samples were collected from 2 Portuguese BPS and submitted to DNA extraction, amplification, library preparation and sequencing. According to the bioinformatics analysis, 18 zoonotic pathogens, including Escherichia coli, Enterococcus faecium and Bacteroides fragilis, and more than 100 AMR genes were identified in all the fecal samples. This is alarming considering the presence of pathogens and AMR genes in BPS could lead to infections with limited treatment options. Although the metagenomic technology employed in this project still needs to be validated and presents some limitations, it showed potential as a powerful tool for the surveillance of zoonotic pathogens and AMR in animal production systems. In conclusion, in accordance with a One health approach, this study indicates that the risks that husbandry and consumption of backyard animals represent to the public health, animal welfare and environment cannot be overlooked. Therefore, it is critical that measures to prevent the dissemination of zoonoses and AMR, such as more rigorous biosecurity protocols, should be implemented in BPS.
- Evaluation of Specific Anthraquinones as New catechol-O-methyltransferase Inhibitors: Virtual Docking, Molecular Dynamics, Inhibition and Cytotoxicity In Vitro StudiesPublication . Proença, Fábio Alexandre Esteves; Passarinha, Luís António Paulino; Silvestre, Samuel MartinsParkinson´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 TherapeuticsPublication . Rodrigues, Madalena Filipa Geada; Passarinha, Luís António Paulino; Sousa, Ângela Maria Almeida de; Cristóvão, Ana Clara BrazGlobally, 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.