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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.
- Effects of Clobetasol on Human Skin Lipids: An Ex Vivo Study - Insights into barrier disruption and regenerative potentialPublication . Ramos, Ana Carolina da Silva; Oliveira, Rita Manuela Palmeira de; Paraskevopoulou, AnnaThe skin is the largest organ of the body, playing a vital role in the protection of the human organism. The stratum corneum (SC) is the outermost layer, composed of corneocytes embedded in a lipid matrix. Lipid ratio disruptions lead to an increase in water loss and a weaker skin barrier, causing different skin diseases. One of them, atopic dermatitis (AD), is a chronic inflammatory skin condition, impacting 15% to 20% of children and 1% to 3% of adults worldwide. As a common treatment for AD, glucocorticosteroids are used, but their transient efficacy and non-specific action often requires additional treatments, increasing the risks for adverse effects. In this project, the effect of glucocorticosteroids on the lipids of SC was investigated. To achieve this, donated human skin from plastic surgery (abdomen) was cultivated for 10 days, while applying topically clobetasol 0.05% in a suitable vehicle (propylene glycol:ethanol (7:3)) for 7 consecutive days. The viability of skin explants was tracked by the TTC assay. To gain a better understanding of the skin barrier and lipid alterations, the skin and the SC lipids were analysed using various methods, including transepidermal water loss measurement, infrared spectroscopy, alongside Liquid Chromatography with Mass Spectrometry. The results showed an increase in transepidermal water loss after treatment with clobetasol. In terms of skin viability, lipid composition, and lipid arrangement, the skin remained mostly stable. This demonstrates that short-term clobetasol treatment does not markedly alter the lipid composition of the SC. Overall, these findings help us understand how corticosteroids alter the skin barrier and SC lipids, and may inform future studies focused on creating safer, more targeted approaches to treating AD.
- 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.
- Injectable and implantable hydrogels for localized delivery of drugs and nanomaterials for cancer chemotherapy: A reviewPublication . Pouso, Manuel António do Rosário ; Melo, Bruna Daniela Lopes ; Gonçalves, Joaquim; Louro, Ricardo; Mendonça, António; Correia, Ilídio Joaquim Sobreira ; de Melo-Diogo, DuarteMultiple chemotherapeutic strategies have been developed to tackle the complexity of cancer. Still, the outcome of chemotherapeutic regimens remains impaired by the drugs’ weak solubility, unspecific biodistribution and poor tumor accumulation after systemic administration. Such constraints triggered the development of nanomaterials to encapsulate and deliver anticancer drugs. In fact, the loading of drugs into nanoparticles can overcome most of the solubility concerns. However, the ability of systemically administered drug-loaded nanomaterials to reach the tumor site has been vastly overestimated, limiting their clinical translation. The drugs’ and drug-loaded nanomaterials’ systemic administration issues have propelled the development of hydrogels capable of performing their direct/local delivery into the tumor site. The use of these macroscale systems to mediate a tumor-confined delivery of the drugs/drugs-loaded nanomaterials grants an improved therapeutic efficacy and, simultaneously, a reduction of the side effects. The manufacture of these hydrogels requires the careful selection and tailoring of specific polymers/materials as well as the choice of appropriate physical and/or chemical crosslinking interactions. Depending on their administration route and assembling process, these matrices can be classified as injectable in situ forming hydrogels, injectable shear-thinning/selfhealing hydrogels, and implantable hydrogels, each type bringing a plethora of advantages for the intended biomedical application. This review provides the reader with an insight into the application of injectable and implantable hydrogels for performing the tumor-confined delivery of drugs and drug-loaded nanomaterials.
- 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.
- Non-canonical DNA secondary structures as a therapeutic strategy for lung cancerPublication . Miranda, André Filipe Rodrigues ; Cruz, Carla Patrícia Alves Freire Madeira; Mergny, Jean-Louis; Oliveira, Paula Alexandra Martins deNucleic acids store and control genetic information and, beyond the canonical B-form DNA double helix, can adopt a variety of non-canonical architectures, including G-quadruplex (G4s), i-motifs (iMs), triplexes, R-loops, and Z-DNA/Z-RNA. These structures are widespread across genomes and transcriptomes and play important biological roles (transcription, replication, translation, and genome stability). They are recognized as regulatory hotspots enriched at promoters, enhancers, telomeres, untranslated regions, and viral genomes and their involvement in cancer, viral infection, neurodegeneration, and inflammatory disorders. Their in vivo functions have also opened therapeutic avenues both as drug targets and therapeutic tools. As drug targets, these non-canonical structures can be modulated to alter cellular behavior, while as therapeutic agents, nucleic acids can be engineered to adopt non-canonical folds that bind key cellular partners and exert biological effects. This dual role is unusual for other approaches and positions non-canonical structures at the forefront of modern drug discovery. These structures are emerging as important tools in oncological research and therapeutic development. Proto-oncogenes encode proteins essential to normal physiology; however, when mutated or amplified become oncogenes, they drive unchecked growth and survival. The transcription factors (TFs) are a particularly important class of proto-oncogenes that bind to DNA and recruit co-regulators, orchestrating broad gene-expression programs. Their sweeping control of transcription makes them powerful cancer drivers but also challenging drug targets. Unlike enzymes with well-defined catalytic pockets, TFs often rely on flexible, intrinsically disordered regions for interactions, contributing to their reputation as “undruggable.” Numerous TFs have been reported as dysregulated in cancer and are frequently correlated with poor prognosis and resistance to chemotherapy. Other TF are gaining relevance, such as B-MYB (MYBL2), a transcription factor from the MYB family required for normal cell-cycle progression, which acts as an oncogene when overexpressed or deregulated. Functionally, the B-MYB protein serves as a master cell-cycle regulator by integrating into multiprotein assemblies, most notably the DREAM and MMB complexes, that coordinate cell-cycle gene expression. Beyond these physiological functions, B-MYB is upregulated in multiple cancers, such as breast and lung, and its overexpression is correlated with aggressive disease, treatment resistance, and unfavorable prognosis. The molecular mechanisms linking B-MYB to tumorigenesis include gene amplification, cell cycle deregulation, genomic instability, apoptosis suppression, post-transcriptional and post-translational modifications, or can contribute to epithelial-to-mesenchymal transition. Thus, B-MYB TF can be seen as a relevant clinical biomarker and one of the main orchestrators of carcinogenesis; however, until now, no pharmacological therapy against B-MYB has been developed, also related to its “undruggable” profile. Also, it’s known, by bioinformatic analysis of the human genome, that oncogene promoter regions are G/C rich around transcription start sites (TSS), which allows the formation of structures called G4 or iM. The G4s are formed by the self-association of four guanine bases in a quasi-planar arrangement via Hoogsteen bonds and are described as having a gene regulatory function, namely at the transcriptional level, while the iM is formed by Hoogsteen bonds between protonated and deprotonated cytosines. Motivated by these observations, the central objective of this thesis was to explore alternative routes to target the B-MYB oncogene using non-canonical nucleic-acid structures: first as drug targets and later as therapeutics. The thesis started by the identification of G-rich sequences at the B-MYB promoter capable of forming G4 structures. The identification of G-rich sequences was performed using the G4Hunter algorithm, and their conservation across mammalian species was also verified. The experimental validation of their formation was performed by combining 10 biophysical and biochemical methods. Later, the in-cell relevance of G4 structures was evaluated employing the G4access method, which reveals that from the predicted sequences, only the most stable one (B-MYB 43R) was shown to be significantly formed in cells, evidencing a potential impact on the transcription of this gene to its location closest to the TSS. Next, the ability of C-rich sequences to form iM structure was evaluated. Again, the iM-forming sequences were predicted using the G4Hunter algorithm and the experimental validation started by circular dichroism (CD) and nuclear magnetic resonance (NMR) to determine if the sequences fold into an iM structure. Then, stability parameters such as pHT (pH transitional midpoint) were determined by acquiring spectra at different pH values (between pH=5 and pH=8; 0.25-unit increments). The thermal stability and thermodynamic parameters were also calculated using the denaturation and renaturation melting curves. Then, the in-cell formation was assessed using iM-CUT&Tag experiments in HEK293T, which revealed the formation of the iB-MYB 43 that was characterized to have the highest Tm and the highest pHT among the studied sequences. After this characterization, the interaction of small molecules with iB-MYB structures was assessed. As a general tendency, the ligands did not affect the CD spectral shape; however, some of them evidenced changes in secondary structure or thermal destabilization. After evaluating the capacity to form G4 or iM, we continued to validate the G4 formation within a cellular environment using a G4-triggered fluorogenic hybridization probe. This strategy circumvents the limitations of the antibodies and small-molecule probes, which show a lack of structural selectivity labeling diverse DNA and RNA G4s indiscriminately, and cannot specifically target the desired G4. The molecular probe was composed of G4-recognizing light-up ligand (acridine derivative) with an antisense oligonucleotide that hybridizes adjacent region of B-MYB G4. Before probe synthesis using a click chemistry approach, the ligands were photophysically characterized, biophysically evaluated against G4, and further validated spectroscopically and in cells. Cellular studies confirmed the co-localization between the molecular probe and B-MYB G4 in-cell, offering promise for future applications in cancer research in terms of targeted therapies and monitoring of G4. Although G4s are highly dynamic and topologically diverse, promoter G4s are commonly parallel, whereas antiparallel forms are underreported and less characterized. In earlier work, we identified a B-MYB promoter sequence (B-MYB 26RA), that forms an antiparallel G4. Thus, a biophysical pipeline, starting in solution NMR spectroscopy alongside in-cell NMR studies, was made to predict a three-dimensional model (antiparallel quadruplex-duplex junction) and to assess the formation in a live complex environment of B-MYB 26RA. Interestingly, B-MYB 26RA evidenced an ionic sensitivity to K+ and Na+, increasing their topological dynamics, and demonstrated that, when associated with PhenDC3 ligand, a conformational shift from hybrid to antiparallel topology happens. Then, we moved to B-MYB 43R G4 to discuss the therapeutic relevance and the capacity to be targeted by small molecules. Thus, using spectroscopic methods, the G4 formation and its interaction with a panel of G4-stabilizing ligands were confirmed. From the tested ligands, PhenDC3 and TMPyP4 demonstrated the highest binding affinity and stabilization and revealed that both ligands inhibited proliferation and migration in two lung cancer cell lines (A549 and H1299), with PhenDC3 showing potent cytotoxic and cytostatic effects. Furthermore, PhenDC3 upregulated B-MYB expression despite its strong phenotypic effects, highlighting the complexity of G4-targeting mechanisms and supporting the growing evidence that ligands do not always downregulate their target genes. Finally, we explored the use of Polypurine Reverse-Hoogsteen (PPRH) hairpins as an alternative therapeutic approach to target the B-MYB promoter. PPRH are DNA oligonucleotides that fold into intramolecular hairpins and bind complementary polypyrimidine, forming stable DNA triplexes, impeding transcription, and displacing the G-rich strand, inducing G4 formation, enabling dual-level regulation of oncogene expression. Thus, using a bioinformatic tool (TFO Searching Tool), a PPRH was designed against the G4-forming region at the B-MYB promoter (B-MYB 43R), and then experimentally assessed the triplex formation using biophysical methods. The biological effects of designed PPRH, evaluated in lung cellular models (A549, H1299 and MRC-5), demonstrated a low cell viability and clonogenic capacity accompanied by mRNA expression reduction and proteomic profile alterations. Thus, the combination of triplex-based approaches with G4 biology could be a future venue for therapy. Overall, this thesis establishes non-canonical nucleic-acid structures as both actionable targets and therapeutic agents. As a target, the formation of G4 and iM structures at the promoter region of the B-MYB oncogene; was provided new insights about structure and topological dynamics according to the surrounding environment; was validated their formation in-cell, as well as explored the interaction with small molecules. On the other side, the therapeutic potential was explored using PPRH, which revealed a selective and powerful tool to target B-MYB. Together, these findings provide fundamental insights and open new avenues for translational research on the B-MYB oncogene.