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- Gestão inteligente e distribuída de comunidades de cidadãosPublication . SILVA, RAFAEL DUARTE PEREIRA DA; Gomes, Luís Filipe de Oliveira; Vale, Zita Maria Almeida doA utilização e integração de modelos inteligentes nos edifícios pode transformar as experiências dos utilizadores dentro do edifício, proporcionando a otimização dos espaços e formas eficientes de utilizar e interagir com os recursos do edifício. A utilização de soluções inteligentes traz alguns desafios que devem ser estudados, como a heterogeneidade entre os recursos e a necessidade de adaptar os edifícios já existentes ao conceito de edifícios inteligentes. Embora os edifícios inteligentes possam revolucionar a forma como as pessoas utilizam e interagem com os espaços, o grupo de edifícios, ao criar comunidades, traz novas oportunidades para permitir que os membros interligados atinjam objetivos comuns, modelando papéis cooperativos, colaborativos e, por vezes, competitivos. Esta nova dinâmica em que os sistemas orgânicos podem comunicar e interagir também levanta desafios quanto à modelação dos utilizadores, às suas preferências e à existência de infraestruturas comuns para permitir a implementação de modelos inteligentes ao nível da comunidade, edifício e utilizador. Esta dissertação tem como objetivo conceber, implementar, testar e validar uma infraestrutura baseada em containers, intitulada Caravels, que combina os conceitos de comunidades inteligentes e edifícios inteligentes para desenvolver uma solução sensível ao contexto que considera diferentes utilizadores e edifícios. A solução concebida emprega uma arquitetura distribuída para a gestão de comunidades inteligentes de cidadãos, onde cada membro opera como uma entidade autónoma, enquanto permanece interligado através de uma infraestrutura partilhada. A arquitetura permite serviços tanto a nível local como comunitário, sendo que um membro pode integrar serviços individuais, escolhidos especificamente para esse utilizador, ao mesmo tempo que contribui e beneficia de otimizações a nível comunitário. Central ao projeto está a modelação das preferências do utilizador em ambientes complexos, dinâmicos e multiutilizador. A dissertação explora os desafios psicológicos e cognitivos da representação de preferências, reconhecendo que os utilizadores têm dificuldades em articular ou priorizar as suas próprias preferências. Os modelos propostos podem adaptar-se ao longo do tempo, incorporando feedback e dados comportamentais para apoiar a tomada de decisões proativas e conscientes do contexto. As técnicas de inteligência artificial, incluindo a aprendizagem supervisionada, não supervisionada e por reforço, estão integradas em todo o sistema para permitir a análise preditiva, a otimização e o controlo autónomo. Para validar a arquitetura e as metodologias propostas, foram conduzidos vários estudos de caso em cenários realistas, refletindo as diferentes necessidades dos utilizadores, procura de energia e recursos distribuídos. Os resultados demonstram que o sistema pode modelar o comportamento do utilizador, apoiar a cooperação a nível comunitário e melhorar a eficiência e a inteligência geral do edifício inteligente. Os resultados desta dissertação contribuíram para seis publicações científicas, incluindo uma revista com um fator de impacto de 6,6.
- Enantioseparationof 3-chloromethcathinone by liquid chromatography at the milligram scalePublication . Langa, Ivan; Ribeiro, Cláudia; Gonçalves, Virgínia; Dias da Silva, Diana; Cravo, Sara; Tiritan, Maria; Dias da Silva, Diana CristinaThe most prominent synthetic cathinone (SCAT)is 3-chloromethcathinone (3-CMC), accounting for 34% and 63.41% of the total seized new psychoactive substances (NPS) in Europe in 2021 and 2022, respectively [1, 2].Over the latest years, since the first identification on the European drug market in September 2014 in Sweden, 3-CMC has gained significant popularity among the younger drug users [3]. Moreover, 3-CMC is chiral and its enantiomers can show different biological activity, highlighting the importance of the enantio selectivity studies in clinical, forensic and ecotoxicological context. The aim of this study was to optimize a chromatographic method for the enantiomeric separation of 3-CMC at the milligram scale for further use in in vitroand ecotoxicity assessments. The enantio separation as well as the enantiomeric purity evaluation of the 3-CMC were performed by liquid chromatography coupled to the ultraviolet-visible detector (UV/Vis), using a CHIRALPAK®AD-H 10x250mm, 5 μm, a semi-preparative column. A Dionex Ultimate 3000 automated fraction collector was used for fractions collection. Data was analyzed by Chromeleon 7.0 software. For method conditions optimization, a solution at 100 μgmL-1of 3-CMC in ethanol with diethylamine was used. The optimized method allowed the separation of the enantiomers of 3-CMCat final concentration of 3.7 mg mL-1, with an enantiomeric purity of 98 % and 95 % for the first and second eluted enantiomer, respectively. The determination of the absolute configuration of theenantiomers is ongoing by electronic circular dichroism. The isolated enantiomers will be used for the enantio selective evaluation of the 3-CMC ecotoxicity.The determination of the absolute configuration of the enantiomers will enable correlating the ecotoxicity of each enantiomer.
- Identification of linezolid-resistant Enterococcus spp. in calves: findings from Portuguese high-yielding dairy farmsPublication . Ribeiro, Alícia; Teixeira, Maria J.; Pinho, Luís; Campos, Carla; Novais, Carla; Peixe, Luísa; Brito, Nuno V.; Quinteira, Sandra; Miranda, Carla; Freitas, Ana R.; Campos, CarlaAntimicrobial resistance (AMR) is a global health threat requiring a One Health approach, asantibiotic-resistant bacteria canspread between animals and humans. Enterococcusspp., particularly E. faecium(Efm) and E. faecalis(Efs),are keyAMR indicators due to their role as gut commensals and their potential as reservoirs of resistance genes.They are also opportunistic pathogensthat can cause severe human infections. Cattle are a recognized reservoir of multidrug-resistant (MDR) Enterococcusspp.,yet remain among the least studied food-producing animals in this context[1,2]. To assess if contemporary faecal samples from main cattle farms in Northern Portugal carry clinically relevant antibiotic-resistant Enterococcusspp. Thirty bovine fecal swab samples were collected from 10 high-yielding farms with Holstein-Friesian dairy cattle in 2 cities during 2023[3]. Sample processing included pre-enrichment (37ºC/18h) without/with antibiotics (ampicillin 16μg/mL, vancomycin 8μg/mL or florfenicol 16μg/mL) followed by plating onto Slanetz–Bartley selective agar, without/with the same antibiotics (37°C/48h). Typical colonies weres aved for identification (MALDI-TOF MS) and antibiotic susceptibility test (disk diffusion;EUCAST/CLSI). Prevalence percentages were calculated on a per-sample basis.All samples contained Enterococcus(n=43) that were identified as Efm(n=18,60%), E. hirae(n=14, 47%), Efs(n=9, 30%), E. casseliflavus and E. durans(n=1 each, 3%); and resistant to erythromycin (44%), tetracycline (39%), chloramphenicol (20%), ampicillin, linezolid, high-level streptomycin (17%each), ciprofloxacin(13%), and high-level gentamicin (7%). MDR isolates (23%) were mostly obtained from calves rather than adults, and only found in Efs(67%) and Efm(50%) species. Linezolid-resistant isolates were only recovered from supplemented media with florfenicol while those resistant to ampicillin were better detected using culture medium with ampicillinor florfenicol. Ampicillin resistance was only detected in Efm while linezolid resistance was identified in both Efmand Efs (all calves, all MDR, 2 farms). Our study shows that dairy cattle carry MDR Enterococcus spp.,including strains resistant to critically important antibiotics in the treatment of human infections (linezolid). These findings underscore the urgent need for sustained AMR surveillance and cross-sector collaboration within a One Health framework.
- Diagnostic pitfalls in Staphylococcus spp. surveillance: lessons from a multinational university student cohortPublication . Pinheiro, Bárbara; Gomes-Sampaio, Luís M.; Pacheco, Patrícia; Campos, Carla; Braz, Maria Pranto; Coelho, Cristina; Miranda, Carla; Quinteira, Sandra; Freitas, Ana R.; Campos, CarlaStaphylococcus aureus is a leading cause of severe and hard-to-treat human infections, particularly when resistant to cefoxitin due to the presence of the mecA gene [1]. However, diagnostic challenges arise from the misidentification of S. aureus and related species when relying on classical identification methods (mannitol fermentation;coagulase production), as well as from the detection of strains carrying the mecA gene but phenotypically susceptible to cefoxitin -known as "stealth" strains [2]. Building upon a previous collection of S. aureus from healthy students’ nares [3], we aimed to expand this collection with new samples, assess the occurrence of “stealth” isolates, and further investigate cases of incongruent identification. Nasal swab samples (n=557) from 507 students (median-23-years; 9 countries) attending alarge university (Porto district) were collected between March 2022 and November 2024. They were inoculated on to mannitol-salt agar and, in parallel, enriched in brain-heart-broth with 6.5%NaCl further plated on to ChromID® MRSA-SMART. Isolates deriving from mannitol-salt (only fermenting colonies) and chromogenic (all typical colonies) agar media were stored for species identification (MALDI-TOFMS), cefoxitin-susceptibility (disk-diffusion), and mecA gene screening (PCR). Staphylococcus aureus was identified in 46% (256/557; 6 countries) of cases. Other Staphylococcus species included S. haemolyticus(n=5), S. capitis(n=3), S. warneri(n=3), S. saprophyticus(n=1), S. simulans(n=1), and S. ureilyticus(n=1). These isolates expressed variable coagulase production (7 positive, 7 negative).On another hand, a non-fermenting S. aureus was detected (cromogenic medium).Ten (1.8%) students were colonized with methicillin-resistant staphylococci species carrying mecA including S. aureus (n=6), S. haemolyticus (n=3), S.ureilyticus (n=1). The mecA gene was also detected in 4/16 (25%; 3 Portuguese, 1 Italian) S. aureus susceptible to cefoxitin, the so-called “stealth”strains. Screening is ongoing in more isolates. Our study highlights the importance of integrating both phenotypic and genotypic methods for Staphylococcus accurate identification. Furthermore, the detection of stealth strains in healthy students underscores the need for robust community-based screening, as S. aureus carriage may be underestimated. Future studies will unveil whether these strains are capable of reversion to resistência.
- Potential skin benefits of incorporating Prunus avium Lapins extracts into a commercially available Portuguese India Pale Ale craft beerPublication . Pereira, Maria João; Santos, Diana; Pinho, Cláudia; Oliveira, Ana Isabel; Pereira, Maria JoãoA commercially available Portuguese India Pale Ale craft beer (ALM-IPA) has shown potential skin benefits in a previous study [1]. However, the incorporation of cherry extracts into bottled beers lacks scientific evidence. To evaluate, in vitro, the benefits of incorporating aqueous (ACE) and ethanolic (ECE) cherry extracts into ALM-IPA beer, in terms of antioxidant and photoprotective activity and the viability of human keratinocytes (HaCaT cells). Experimental study, with the incorporation of ACE (infusion, 1:10) and ECE (70%) (1 mg/mL) into ALM-IPA bottles. Total phenolic content (TPC) was determined. The antioxidant potential was assessed using the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), hydrogen peroxide (H202)and iron-reducing antioxidant power (FRAP) assays. The photoprotective potential was estimated by determining the sun protection factor (SPF) and the ultraviolet absorption capacity (UV-AC). The viability of HaCaT cells was assessed using the 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Data were analysed using the one-way ANOVA test and significant differences were considered for p< 0.05. Regarding antioxidant activity, and comparing both extracts, ALM-IPA+ACE presented the lowest value of IC50for ABTS assay (86.16 ± 5.33 μg/mL) and the highest FRAP value (27.58 ± 0.42 μmol of trolox equivalents/g), which is related with the highest TPC observed (15.10 ± 0.16 mg of gallic acid/g). ALM-IPA+ECE presented the lower IC50(43.27 ± 2.14 μg/mL) compared to ALM-IPA+ACE (65.08 ± 1.69 μg/mL) for H202 assay. However, ALM-IPA beer showed higher antioxidant activity (IC50= 55.21 ± 4.68 μg/mL for ABTS; IC50= 23.54 ± 1.53 μg/mL for H202; FRAP = 53.74 ± 1.27 μmol of trolox equivalents/g). Regarding photoprotective potential, both extracts presented photoprotective potential (SPF > 6) [2]. Analyzing the viability of HaCaT cells after incubation with both extracts, ALM-IPA+ACE and ALM-IPA+ECE presented cytotoxicity, for the 24h and 48h incubation period, only for concentrations higher than 100 μg/mL (cell viability > 80%) [3]. In the 24 hincubation period, ALM-IPA cell viability was higher than ALM-IPA+ACE and ALM-IPA+ECE, and generally, ALM-IPA+ECE was superior to ALM-IPA+ACE. More studies are needed regarding the incorporation of plant extracts into commercially available beers, particularly in other stages of brewing or different styles of beer.
- Beer enriched with “Lapins” cherry extracts: antioxidant activity and liver toxicityPublication . Santos, Diana; Pereira, Maria João Sequeira; Oliveira, Ana Isabel; Pinho, Cláudia; Pinho, CláudiaBeer can be considered a functional beverage and integrate innovative ingredients, namely sweet cherries, with different properties, such as antioxidant activity[1,2]. To evaluate the antioxidant activity, in vitro, and liver toxicity, in human hepatocarcinoma cells (HepG2), in beer after incorporation of aqueous (CAE) and ethanolic (CEE) extracts of cherry variety "Lapins". CAEand CEE(1mg/mL) were incorporated into commercial bottles of Imperial Stout beer (IS-N). The total phenolic content(TPC), expressed in mg of gallicacid equivalents (GAE)/g, was determined. The antioxidant capacity was evaluated by the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) neutralization and hydrogen peroxide (H₂O₂) assays, both expressed in the concentration required to inhibit the activity by 50% (IC50). Also, the ferricreducing antioxidant power (FRAP) assay was performed and expressed in μmol of trolox equivalent (TE)/mg. Cell toxicity was studied in HepG2 cells, with assessment of metabolic activity by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Data were analysed using GraphPad Prism software, and significant differences were considered for p< 0.05.The incorporation of CEEto IS-N beer significantly decreased TPC (4.2±0.1mg GAE/g for IS-N+CEE; 8.3 ± 0.2mg GAE/g for IS-N;p<0.05).There was an increase in the antioxidant capacity by the ABTS assay(IC50= 80.1±1.1μg/mLfor IS-N; IC50= 60.5 ±1.5 μg/mLfor IS-N + CAE; IC50= 48.0 ±0.6 μg/mLfor IS-N + CEE); however, the incorporation of cherry extracts was not promising in the H2O2(27.0±1.5μg/mLfor IS-N; IC50= 58.7 ±0.4 μg/mLfor IS-N + CEE; IC50= 78.8 ±1.6 μg/mLfor IS-N + CAE;) and FRAP (44.4±0.0μmol/gfor IS-N; 42.7 ±0.0μmol/gfor IS-N + CAE; and 39.7 ±0.0μmol/gfor IS-N + CEE) assays. Further more, IS-N+CEE showed greater antioxidant capacity than IS-N+CAE. After the incorporation of cherry extracts, cytotoxicity was observed in concentrations higher than 10 mg/mL (for IS-N + CAE, 24h incubation) and at the concentration of 500mg/mL (for IS-N + CAE and IS-N + CEE, 48h incubation). IS-N + CEE showed the greatest increase in cell viability. The addition of cherry extracts to beer increased the antioxidant capacity by the ABTS assay, while TPC was reduced with the addition of CEE. The incorporation of both extracts showed promising potential, with low cytotoxicity in HepG2.
- Ivermectin decreases the expression of ALDH1 in ovarian cancer cell lines in combination with chemotherapyPublication . Santos, Cláudia; Oliveira, Érica; Ally, Sofia; Nunes, Mariana; Resende, Albina; Ricardo, SaraCell Microarray (CMA) technology has been established as an essential tool for advancing cancer studies, allowing for the simultaneous protein analysis of multiple samples [1]. This method facilitates the exploration of various biomarkers associated with diagnosis, prognosis, and therapeutic response, making evaluation processes more efficient and comprehensive [1]. Carboplatin and paclitaxel are commonly used chemotherapy drugs for ovarian cancer treatment. Numerous studies have demonstrated that patients often develop resistance, underscoring the need to discover more effective therapeutic options to improve patient outcomes [2]. In oncology, drug repurposing has shown promising results in achieving this goal. Ivermectin, an antiparasitic drug, has been shown to enhance the efficacy of carboplatin and create a synergistic effect when combined with paclitaxel [3]. Aldehyde Dehydrogenase 1 (ALDH1) is an aldehyde catalyzer that plays a crucial role in drug metabolism. The study of this biomarker could be vital to the understanding of treatment resistance found in advanced ovarian cancer. Using the CMA approach, the aim is to explore the ALDH1 expression pattern in two ovarian cancer cell lines, after in vitro treatment with carboplatin, paclitaxel and ivermectin. Two ovarian cancer cell lines were used: OVCAR8, characterized by resistance to Carboplatin, and OVCAR8 PTX R C, which exhibits resistance to both Carboplatin and Paclitaxel [2]. The cells were exposed to chemotherapeutic agents for 48 hours, administered either alone or in combination with Ivermectin. After the incubation period, the cells were collected, formalin-fixed and embedded in Histogel®. Then the samples were paraffin-embedded and cut into glass slides to perform ALDH1 protein detection using immunocytochemistry. In general, ALDH1 expression was found in the cytoplasm of the cells with a dot pattern. Our results show that in both OVCAR8 and OVCAR8 PTX R C cell lines, the treatment with the combinations of carboplatin/paclitaxel plus Ivermectin, whether in 2D or 3D environments, leads to a notable decrease in ALDH1 expression compared to treatments in monotherapy. High levels of ALDH1 are associated with chemoresistance. The low levels of ALDH1 found in ovarian cancer cells treated with Ivermectin plus carboplatin or paclitaxel reveal a more sensitive profile, which could be a promising alternative for ovarian cancer treatment.