Relatório de Estágio Profissional

Este relatório de estágio foi realizado com a intenção de organizar toda a informação relativa ao estágio profissional que decorreu no Mestrado em Educação Pré-Escolar. Pretende relatar, interpretar, refletir e fundamentar sobre as experiências vividas e observadas durante o estágio, e também afirmar a aquisição e partilha de conhecimentos entre colegas, educadoras e supervisoras da Escola Superior de Educação João de Deus. O presente relatório está dividido em 4 capítulos, sendo o 1.º denominado como Relatos de Estágio pois engloba 10 relatos de estágio, onde se encontram atividades e estratégias desenvolvidas por mim e pelas educadoras dos grupos onde estagiei. Destes 10 relatos, 7 são de experiências observadas nas salas de atividades de grupos nas faixas etárias entre 3 e 5 anos e 3 de atividades realizadas por mim e avaliadas pelas educadoras cooperantes e orientadoras da prática pedagógica. O 2.º capítulo é referente a planificações de atividades que elaborei para crianças entre os 3 e os 5 anos. É constituído então por 6 planificações referentes a atividades integradas nas diferentes Áreas e Domínios das Orientações Curriculares: Área de Formação Pessoal e Social, Área de Expressão e Comunicação e Área do Conhecimento do Mundo utilizando diferentes materiais e estratégias, fundamentado com autores a importância das mesmas para as crianças. No 3.º capítulo são apresentados 3 dispositivos de avaliação baseados em 3 atividades colocadas em prática nos três grupos etários, a primeira foi aplicada no grupo de 4 anos, a segunda na faixa etária dos 5 anos e a terceira e última atividade no grupo dos 3 anos. Foi realizada a avaliação das mesmas e analisados os resultados. O 4.º e último capítulo apresenta uma proposta de projeto que elaborei com o objetivo de sensibilizar as crianças de que as árvores são nossas amigas e devemos cuidar delas e preservá-las, realçando a mensagem de que devemos preservar a natureza e evitar os incêndios. Revelou-se deveras importante a realização deste relatório para o meu crescimento pessoal e profissional, pelo quanto me obrigou a repensar atividades, estratégias e procedimentos sempre numa perspetiva de melhorar o meu desempenho

Development of Cyanine 813@Imidazole-Based Doped Supported Devices for Divalent Metal Ions Detection

PM003/2016 IF/00007/2015 CEECIND/00648/2017A NIR cyanine@imidazole derivative Cy1 was synthesized and evaluated as a metal ion sensor in solution. Cy1 was shown to be very sensitive to all metal ions tested, presenting a blue shift in the absorption from 668 nm to 633 nm, followed by a change in colour from pale green to blue with Zn2+, Cd2+, Co2+, Ni2+ and Hg2+ ions. Despite the blue shift in the absorption, a decrease at 633 nm (with a colour change from pale green to colourless), as well as a quenching in the emission intensity at 785 nm were observed for Cu2+ ions. The results show the formation of sandwich complexes of two ligands per metal ion with the highest association constant observed for Cu2+ (Log Kass.abs = 14.76 ± 0.09; Log Kass.emis. = 14.79 ± 0.06). The minimal detectable amounts were found to be 31 nM and 37 nM, with a naked eye detection of 2.9 ppm and 2.1 ppm for Hg2+ and Cu2+ ions, respectively. These results prompted us to explore the applicability of Cy1 by its combination with nanomaterials. Thus, Cy1@ doped MNs and Cy1@ doped PMMA nanoparticles were synthesized. Both nanosystems were shown to be very sensitive to Cu2+ ions in water, allowing a naked-eye detection of at least 1 ppm for Cy1@ doped MNs and 7 ppm for Cy1@ doped PMMA. This colourimetric response is an easy and inexpensive way to assess the presence of metals in aqueous media with no need for further instrumentation.publishersversionpublishe

Validation of a Standard Luminescence Method for the Fast Determination of the Antimicrobial Activity of Nanoparticles in Escherichia coli

Publisher Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland.The use of nanoparticles in multiple industries has raised concerned voices about the assessment of their toxicity/antimicrobial activity and the development of standardized handling protocols. Issues emerge during the antimicrobial assaying of multiple cargo, colorimetric, colloidal nanoformulations, as standard protocols often rely on visual evaluations, or optical density (OD) measurements, leading to high variance inhibitory concentrations (MIC). Thus, a fast, luminescence-based assay for the effective assessment of the antimicrobial activity of nanoparticles is herein reported, using the bioluminescence of an in-house E. coli ATCC® 8739™ construct with the pMV306G13 + Lux plasmid (E. coli Lux). The new strain’s sensitivity to ofloxacin as a standard antibiotic was confirmed, and the methodology robustness verified against multiple nanoparticles and colorimetric drugs. The reduction of incubation from 24 to only 8 h, and the sole use of luminescence (LUX490) to accurately determine and distinguish MIC50 and MIC90, are two main advantages of the method. By discarding OD measurements, one can avoid turbidity and color interferences when calculating bacterial growth. This approach is an important tool that contributes to the standardization of methods, reducing samples’ background interference and focusing on luminescence as a direct probe for bacterial metabolic activity, growth and, most importantly, the correct assessment of nanomaterials’ antimicrobial activity.publishersversionpublishe

Magneto-Fluorescent Mesoporous Nanocarriers for the Dual-Delivery of Ofloxacin and Doxorubicin to Tackle Opportunistic Bacterial Infections in Colorectal Cancer

Funding Information: This work was funded by the Associate Laboratory for Green Chemistry—LAQV which is financed by national funds from FCT/MCTES (UIDB/50006/2020 and UIDP/50006/2020) as well as the Scientific Society PROTEOMASS (Portugal) for funding support (General Funding Grant 2021). G.M thanks to FCT/MEC (Portugal) for his doctoral grant PD/BD/142865/2018. E.O thanks FCT/MEC (Portugal) for the individual contract, CEECIND/00648/2017. This work was also funded by FCT—Foundation for Science and Technology, I.P., through projects UIDB/04077/2020, UIDP/04077/2020, OrMagNa—PTDC/NAN-MAT/28785/2017, BioISI—UID/Multi/04046/2019, UIDB/04046/2020 and UIDP/04046/2020 Centre grants (BioISI) and NECL infrastructure. Publisher Copyright: © 2022 by the authors.Cancer-related opportunistic bacterial infections are one major barrier for successful clinical therapies, often correlated to the production of genotoxic factors and higher cancer incidence. Although dual anticancer and antimicrobial therapies are a growing therapeutic fashion, they still fall short when it comes to specific delivery and local action in in vivo systems. Nanoparticles are seen as potential therapeutic vectors, be it by means of their intrinsic antibacterial properties and effective delivery capacity, or by means of their repeatedly reported modulation and maneuverability. Herein we report on the production of a biocompatible, antimicrobial magneto-fluorescent nanosystem (NANO3) for the delivery of a dual doxorubicin–ofloxacin formulation against cancer-related bacterial infections. The drug delivery capacity, rendered by its mesoporous silica matrix, is confirmed by the high loading capacity and stimuli-driven release of both drugs, with preference for tumor-like acidic media. The pH-dependent emission of its surface fluorescent SiQDs, provides an insight into NANO3 surface behavior and pore availability, with the SiQDs working as pore gates. Hyperthermia induces heat generation to febrile temperatures, doubling drug release. NANO3-loaded systems demonstrate significant antimicrobial activity, specifically after the application of hyperthermia conditions. NANO3 structure and antimicrobial properties confirm their potential use in a future dual anticancer and antimicrobial therapeutical vector, due to their drug loading capacity and their surface availability for further modification with bioactive, targeting species.publishersversionpublishe

Can Biofluids Metabolic Profiling Help to Improve Healthcare during Pregnancy?

This paper describes a metabonomics study of 2nd trimester biofluids (amniotic fluid, maternal urine, and blood plasma), in an attempt to correlate biofluid metabolic changes with suspected/diagnosed fetal malformations (FM) and chromosomal disorders as well as with later occurring gestational diabetes mellitus (GDM), preterm delivery (PTD), and premature rupture of membranes (PROM). The global biochemical picture given by the threesome of biofluids should enable the definition of potential disease signatures and unveil potential metabolite markers for clinical use in predictive prenatal diagnostics. Results show that relatively strong metabolic disturbances accompany FM, reflected in all three biofluids and thus suggesting the involvement of both fetal and maternal metabolisms. Regarding GDM, amniotic fluid and maternal urine seem potential good media to detect early metabolic changes, and PTD subjects show small metabolite changes in the same biofluids, undergoing work being focused on plasma composition. Chromosomal disorders show an interestingly marked effect on maternal urine, whereas no statistically relevant early changes have been observed for PROM subjects. Interestingly, in the case of FM and chromosomal disorders, maternal biofluids show some sensitivity to disorder type, for example, for central nervous system malformations and trisomy 21, respectively. These results show the usefulness of biofluid metabonomics to probe overall metabolic disturbances in relation to prenatal disorders

Development of Cyanine 813@Imidazole-Based Doped Supported Devices for Divalent Metal Ions Detection

A NIR cyanine@imidazole derivative Cy1 was synthesized and evaluated as a metal ion sensor in solution. Cy1 was shown to be very sensitive to all metal ions tested, presenting a blue shift in the absorption from 668 nm to 633 nm, followed by a change in colour from pale green to blue with Zn2+, Cd2+, Co2+, Ni2+ and Hg2+ ions. Despite the blue shift in the absorption, a decrease at 633 nm (with a colour change from pale green to colourless), as well as a quenching in the emission intensity at 785 nm were observed for Cu2+ ions. The results show the formation of sandwich complexes of two ligands per metal ion with the highest association constant observed for Cu2+ (Log Kass.abs = 14.76 ± 0.09; Log Kass.emis. = 14.79 ± 0.06). The minimal detectable amounts were found to be 31 nM and 37 nM, with a naked eye detection of 2.9 ppm and 2.1 ppm for Hg2+ and Cu2+ ions, respectively. These results prompted us to explore the applicability of Cy1 by its combination with nanomaterials. Thus, Cy1@ doped MNs and Cy1@ doped PMMA nanoparticles were synthesized. Both nanosystems were shown to be very sensitive to Cu2+ ions in water, allowing a naked-eye detection of at least 1 ppm for Cy1@ doped MNs and 7 ppm for Cy1@ doped PMMA. This colourimetric response is an easy and inexpensive way to assess the presence of metals in aqueous media with no need for further instrumentation

Mesoporous silica based devices as new delivery tools in bacteria

In this present work, mesoporous silica nanoparticles were evaluated as possible drug-delivery platforms. Three different types of mesoporous silica nanoparticles, MNs, MNs@EPI and SPION@2D-MNs, were synthesized and characterized. These nanomaterials were then subjected to loading assays of epirubicin, doxorubicin and ofloxacin, both in a single and dual-loading perspective. Both epirubicin and doxorubicin presented the highest loading percentages and encapsulation efficacies, when in comparison with ofloxacin. These different loading percentages are tightly connected with electrostatic interactions between the drugs and the matrix. These loaded nanomaterials were then subjected to in vitro release assays at both pH 7.4 and pH 4.0, which revealed increased release percentages at more acidic pH values, indicating the system presents pH sensitivity for drug release. Antibacterial assays with both Gram-negative and Gram-positive bacteria were also performed for all previously obtained nanomaterials. The most effective formulation was ofloxacin loaded MNs@EPI (MNs@EPI-OFLO) due to the presence of synergetic effects between the drug and the matrix. Synergy was also detected for cocktails containing epirubicin and ofloxacin in formulations with MNs and SPION@2D-MNs as matrixes. A new protocol for assessment of bacterial viability was also developed. Herein Escherichia coli (ATCC®8739TM) was transformed with a plasmid that confers constitutive bioluminescence to bacteria. Phenotypic characteristics confirmation, antibiotic susceptibility and temporal growth assays were performed to ensure the integrity of the strain and optimize the conditions in which the assay was conducted. Subsequently, the luminescent E.coli was employed for the assessment of the antibacterial activity of previously assayed nanomaterials. Results obtained with luminescent bacteria were similar to the ones obtained with non-luminescent E.coli, verifying the reliability of the assay. Luminescence measurements allow for an easy and quick detection of metabolic response in bacteria, enabling a thorough detection of antibacterial activity of a wide variety of samples.Neste trabalho, nanopartículas mesoporosas de sílica foram avaliadas como possíveis plataformas para drug-delivery. Três diferentes tipos de nanopartículas mesoporosas de sílica, MNs, MNs@EPI e SPION@2D-MNs foram sintetizadas e caracterizadas. Estes nanomateriais foram sujeitos a ensaios de carregamento de epirubicina, doxorubicina e ofloxacina, tanto numa perspetiva singular como dupla. Tanto a epirubicina como a doxorubicina apresentaram percentagens de carregamento e eficácia de encapsulamento mais elevadas, em comparação com a ofloxacina. Estas percentagens de carregamento diferentes estão estreitamente relacionadas com interações eletrostáticas entre os fármacos e a matriz. Estes nanomateriais carregados foram então sujeitos a ensaios de libertação in vitro a pH 7.4 e pH 4.0, o que revelou um incremento nas percentagens de libertação a valores de pH mais acídicos, indicando que o sistema exibe sensitividade ao pH quanto à libertação de fármacos. A atividade antibacteriana de todos os nanomateriais anteriormente obtidos foi testada em bactérias, tanto Gram-positivas como Gram-negativas. A formulação mais eficaz foi MNs@EPI carregada com ofloxacina (MNs@EPI-OFLO), devido à presença de efeitos sinérgicos entre os fármacos e a matriz. Foi igualmente verificado um efeito de sinergia em cocktails contendo epirubicina e ofloxacina em formulações apresentando MNs e SPION@2D-MNs como matrizes. Um novo protocolo para a determinação de viabilidade bacteriana foi também desenvolvido. Para esse fim, procedeu-se à transformação de Escherichia coli (ATCC® 8739TM), bactéria utilizada nos ensaios de atividade antimicrobiana, com um plasmídeo que confere bioluminescência constitutiva. Após a transformação foram efetuados ensaios de confirmação das características fenotípicas e de suscetibilidade a antibióticos, bem como curvas de crescimento temporal para assegurar a integridade da estirpe e otimizar as condições nas quais o ensaio foi realizado. Subsequentemente, as bactérias E. coli luminescentes foram utilizadas para a determinação do potencial antimicrobiano dos nanomateriais anteriormente testados. Os resultados obtidos nestes ensaios foram semelhantes aos obtidos com as bactérias não-luminescentes, verificando a fiabilidade do mesmo. As medições de luminescência permitem uma fácil e rápida deteção de respostas metabólicas em bactérias, possibilitando uma deteção da atividade antibacteriana de uma grande variedade de amostras

Development of New Targeted Nanotherapy Combined with Magneto-Fluorescent Nanoparticles against Colorectal Cancer

The need for non-invasive therapies capable of conserving drug efficiency and stability while having specific targetability against colorectal cancer (CRC), has made nanoparticles preferable vehicles and principal building blocks for the development of complex and multi-action anti-tumoral approaches. For that purpose, we herein report the production of a combinatory anti-tumoral nanotherapy using the production of a new targeting towards CRC lines. To do so, Magneto-fluorescent NANO3 nanoparticles were used as nanocarriers for a combination of the drugs doxorubicin (DOX) and ofloxacin (OFLO). NANO3 nanoparticles’ surface was modified with two different targeting agents, a newly synthesized (anti-CA IX acetazolamide derivative (AZM-SH)) and a commercially available (anti-epidermal growth factor receptor (EGFR), Cetuximab). The cytotoxicity revealed that only DOX-containing nanosystems showed significant and even competitive cytotoxicity when compared to that of free DOX. Interestingly, surface modification with AZM-SH promoted an increased cellular uptake in the HCT116 cell line, surpassing even those functionalized with Cetuximab. The results show that the new target has high potential to be used as a nanotherapy agent for CRC cells, surpassing commercial targets. As a proof-of-concept, an oral administration form of NANO3 systems was successfully combined with Eudragit® enteric coating and studied under extreme conditions

Tailoring Mesoporous Silica-Coated Silver and Gold Nanoparticles and Polyurethane-Doped Films for Enhanced Antimicrobial Applications.

The global increase in multidrug-resistant bacteria poses a challenge to public health and requires the development of new antibacterial materials. In this study, we examined the bactericidal properties of mesoporous silica-coated silver nanoparticles, varying the core sizes (28 nm and 51 nm). We also investigated gold nanoparticles (26 nm) coated with mesoporous silica coating as possible inert metal cores. To investigate the modification of antimicrobial activity after the surface charge change, we used silver nanoparticles with a 28 nm core coated with a mesoporous shell (16 nm) and functionalized with a terminal amine group. Furthermore, we developed a facile method to create Ag@mSiO2 doped films using polyurethane (IROGRAN) as a polymer matrix. The antibacterial effects of silver nanoparticles with different core sizes were analyzed against Gram-negative and Gram-positive bacteria relevant to the healthcare and food industry. The results demonstrated that gold nanoparticles were inert, while silver nanoparticles exhibited antibacterial effects against Gram-negative (Escherichia coli and Salmonella enterica subsp. enterica serovar Choleraesuis) and Gram-positive (Bacillus cereus) strains. In particular, the larger Ag@mSiO2 nanoparticles showed a minimum inhibitory concentration (MIC) and a minimum bactericidal concentration (MBC) of 18 µg/mL in the Salmonella strain. Furthermore, upon terminal amine functionalization, reversing the surface charge to positive values, there was a significant increase in the antibacterial activity of the NPs compared to their negative counterparts. Finally, the antimicrobial properties of the nanoparticle-doped polyurethane films revealed a substantial improvement in antibacterial efficacy. This study provides valuable information on the potential of mesoporous silica-coated silver nanoparticles and their applications in fighting multidrug-resistant bacteria, especially in the healthcare and food industries

Human plasma stability during handling and storage: impact on NMR metabolomics

This work contributes to fill in some existing gaps in the knowledge of human plasma degradability during handling and storage, a paramount issue in Nuclear Magnetic Resonance (NMR) metabolomics. Regarding the comparison between heparin and EDTA anti-coagulant collection tubes, the former showed no interference of the polysaccharide, while conserving full spectral information. In relation to time/temperature conditions, room temperature was seen to have a large impact on lipoproteins and choline compounds from 2.5 hours. In addition, short-term storage at -20 degrees C was found suitable up to 7 days but, for periods up to 1 month, -80 degrees C was recommended. Furthermore, in the case of reusing plasma samples, no more than 3 consecutive freeze-thaw cycles were found advisable. Finally, the impact of long-term -80 degrees C storage (up to 2.5 years) was found almost negligible, as evaluated on a partially matched non-fasting cohort (n = 49), after having investigated the possible confounding nature of the particular non-fasting conditions employed