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Beta-glucans are a group of polysaccharides with intrinsic immunostimulatory properties which makes the design of new particulate vaccine adjuvants based on β-glucans very promising. The size of the particles and the antigen loading method, encapsulated into particles or adsorbed on its surface, will influence the toxicological and adjuvanticity properties of the particulate adjuvant. Herein we describe the production of glucan nanoparticles (NPs) with three different sizes, approximately 150 nm, 350 nm, and microparticles as shells (GPs) with approximately 3 μm. The association of the antigen to the particulate adjuvant is described using model protein antigens. The method can be easily adapted for real protein antigens.

Glucan (from Alcaligenes faecalis) is a polymer composed of β-1,3-linked glucose residues, and it has been addressed in different medical fields, namely in nanotechnology, as a vaccine or a drug delivery system. However, due to their small size, nanomaterials may present new risks and uncertainties. Thus, this work aims to describe the production of glucan nanoparticles (NPs) with two different sizes, and to evaluate the influence of the NPs size on immunotoxicity. Results showed that, immediately after production, glucan NPs presented average sizes of 129.7 ± 2.5 and 355.4 ± 41.0 nm. Glucan NPs of 130 nm presented greater ability to decrease human peripheral blood mononuclear cells and macrophage viability and to induce reactive oxygen species production than glucan NPs of 355 nm. Both NP sizes caused hemolysis and induced a higher metabolic activity in lymphocytes, although the concentration required to observe such effect was lower for the 130 nm glucan NPs. Regarding pro-inflammatory cytokines, only the larger glucan NPs (355 nm) were able to induce the secretion of IL-6 and TNF-α, probably due to their recognition by dectin-1. This higher immunomodulatory effect of the larger NPs was also observed in its ability to stimulate the production of nitric oxide (NO) and IL-1β. On the contrary, a small amount of Glu 130 NPs inhibited NO production. In conclusion, on the safe-by-design of glucan NPs, the size of the particles should be an important critical quality attribute to guarantee the safety and effectiveness of the nanomedicine.

Even though nanotechnology has revolutionized the biomedical research, a plethora of studies debate the nanoparticles safety. In order to contribute to these studies, we evaluated the cytotoxic and epigenetic effects of maghemite nanoparticles covered with citric acid on human submandibular gland cells. Objective: This work objective was to evaluate the cytotoxic effects and epigenetic alterations induced in human salivary gland cells after treatment with maghemite nanoparticles covered with citric acid. Methods: For that, human submandibular gland cells were cultured and treated with nanoparticles for 24 or 48 hours. To assess cytotoxicity we used lactate dehydrogenase, a general oxidative stress indicator assay and microscopy. Epigenetic status was detected by colorimetric assays and the results were confirmed by quantitative polymerase chain reaction. Results: No cytotoxic effects were detected on cells exposed to up 3.0 mgFemL-1 for 48 hours, although cytoplasmic vacuoles formation was detected by light microscopy analyses. An increased generation of reactive oxygen species in cells exposed to nanoparticles was evidenced and iron clusters accumulated in the cytoplasm of treated cells. Global DNA methylation and histones H3 and H4 acetylation were also altered in response to nanoparticles exposure, thus suggesting a reprogramming of the epigenome. Transcripts accumulation analyses showed that genes related to iron metabolism and oxidative stress were upregulated, while the gene related to epigenetic reprogramming presented reduced transcript accumulation after treatment. Conclusion: We concluded that maghemite nanoparticles covered with citric acid exposure provoked several biological responses without impairment of human submandibular gland cells viability. This is the first report on the epigenetic effects of maghemite nanoparticles on this cell lineage.

Poly-epsilon-caprolactone (PCL) is a biodegradable polyester that has FDA and CE approval as a medical device. Nonetheless, the lack of toxicity exhibited by the polymer cannot be extrapolated to its nanomaterial conformation. Despite PCL-based NPs being widely studied in the biomedical field for their advantages as controlled drug delivery systems, little data describe PCL NPs' toxicity, particularly immunotoxicity. This work assessed different PCL-based delivery systems intended for protein delivery regarding their immunotoxicity and hemocompatibility. Two different molecular weight PCL polymers were used, as well as blends with chitosan and glucan. Results showed that the presence of NaOH during the production of PCL2 NPs and PCL2/glucan NPs induced PCL alkali hydrolysis, generating more reactive groups (carboxyl and hydroxyl) that contributed to an increased toxicity of the NPs (higher reduction in peripheral blood mononuclear cell viability and lower hemocompatibility). PCL2/glucan NPs showed an anti-inflammatory activity characterized by the inhibition of LPS stimulated nitric oxide (NO) and TNF-alpha. In conclusion, generalizations among different PCL NP delivery systems must be avoided, and immunotoxicity assessments should be performed in the early stage of product development to increase the clinical success of the nanomedicine. European Regional Development Fund (ERDF) through the COMPETE 2020-Operational Programme for Competitiveness and Internationalization Portuguese national funds via FCT-Fundacao para a Ciencia e Tecnologia [POCI-01-0145-FEDER-030331, POCI-01-0145-FEDER-032610-PTDC/MEC-DER/32610/2017, UIDB/04565/2020] info:eu-repo/semantics/publishedVersion

Adipose triglyceride lipase (ATGL) plays a key role in intracellular lipolysis, the mobilization of stored triacylglycerol. This work provides an important basis for generating reproducible and detailed data on the hydrolytic and transacylation activities of ATGL. We generated full-length and C-terminally truncated ATGL variants fused with various affinity tags and analyzed their expression in different hosts, namely E. coli, the insect cell line Sf9, and the mammalian cell line human embryonic kidney 293T. Based on this screen, we expressed a fusion protein of ATGL covering residues M1-D288 flanked with N-terminal and C-terminal purification tags. Using these fusions, we identified key steps in expression and purification protocols, including production in the E. coli strain ArcticExpress (DE3) and removal of copurified chaperones. The resulting purified ATGL variant demonstrated improved lipolytic activity compared with previously published data, and it could be stimulated by the coactivator protein comparative gene identification-58 and inhibited by the protein G0/G1 switch protein 2. Shock freezing and storage did not affect the basal activity but reduced coactivation of ATGL by comparative gene identification 58. In vitro, the truncated ATGL variant demonstrated acyl-CoA–independent transacylation activity when diacylglycerol was offered as substrate, resulting in the formation of fatty acid as well as triacylglycerol and monoacylglycerol. However, the ATGL variant showed neither hydrolytic activity nor transacylation activity upon offering of monoacylglycerol as substrate. To understand the role of ATGL in different physiological contexts, it is critical for future studies to identify all its different functions and to determine under what conditions these activities occur.

The use of particulate adjuvants offers an interesting possibility to enhance and modulate the immune responses elicited by vaccines. Aluminium salts have been extensively used as vaccine adjuvants, but they lack the capacity to induce a strong cellular and mucosal immune response. Taking this into consideration, in this study we designed a new antigen delivery system combining aluminium salts with chitosan. Chitosan-aluminium nanoparticles (CH-Al NPs) exhibited a mean diameter of 280nm and a positive surface charge. The newly developed CH-Al NPs are more stable at physiological environment than classical CH NPs, showing no cytotoxic effects and revealing potential as a delivery system for a wide range of model antigens. In vivo studies showed that mice immunized with hepatitis B surface antigen (HBsAg)-containing CH NPs display high anti-HBsAg IgG titers in the serum, as well as the highest antigen-specific IgG on vaginal washes. Furthermore, in contrast to mice receiving antigen alone, mice immunized with the particulate adjuvant were able to elicit IgG2c antibody titers and exhibited higher antigen-specific IFN-γ levels in splenocytes. In conclusion, we established that CH-Al NPs, combining two immunostimulants to enhance both humoral and cellular immune responses, are a safe and promising system for antigen delivery. Our findings point towards their potential in future vaccination approaches.

Airborne environmental particles (EP) more commonly referred as particulate matter (PM) are an illustrative marker of air pollution that is associated with adverse effects on human health. Considering, PM is a complex mixture, not only in terms of its chemical composition, but also in the range of particle size, it is difficult to identify which attribute contributes more for the toxicity. Currently, there is no report about the immunotoxicological effects caused by PM with reduced content of heavy metals. This study intends to address this gap and provides a detailed characterization and immunotoxicity evaluation of PM collected in an urban area with heavy traffic congestion. Environmental particles were separated by different sizes though a sucrose gradient. This method allowed to achieve 4 sized fractions: EP f 15 % with a mean diameter of 284 nm ± 1.86 nm, EP f 25 % with a mean diameter of 461 nm ± 1.72 nm, EP f 35 % with a mean diameter of 1845 nm ± 251 nm and EP f 45 % with a mean diameter of 2204 nm ± 310 nm. Only the fractions with the smallest sizes (EP f 15 % and EP f 25 %) were subsequently studied. The chemical composition of both fractions was not substantially different, and the dominant elements were C, O, Ca and K. Only EP f 25 % showed to have a small amount of Fe. Therefore, the heavy metal elements were eliminated through centrifugation. Essentially, we found that the EP f 15 % was more cytotoxic in RAW 264.7 cells than EP f 25 %, which indicates the smaller size as the motive for the higher toxicity. In addition, both fractions of EP presented a good internalization in macrophages after 2 h exposure and induced the production of reactive oxygen species in a concentration-dependent manner. Moreover, EP f 15 % and EP f 25 % led to a strong secretion of proinflammatory cytokines (TNF-α and IL-6) in human peripheral blood mononuclear cells (hPBMCs) in the 3 concentrations tested. The inflammatory response observed was independent of the presence of heavy metals and endotoxins, since these last were suppressed by using polymyxin B sulfate. This report emphasizes the importance of an adequate physicochemical characterization and adequate controls in the experiments to achieve a right interpretation of the biological effects caused by PM.

Nanoparticles (NPs) assumed an important role in the area of drug delivery. Despite the number of studies including NPs are growing over the last years, their side effects on the immune system are often ignored or omitted. One of the most studied polymers in the nano based drug delivery system field is chitosan (Chit). In the scientific literature, although the physicochemical properties [molecular weight (MW) or deacetylation degree (DDA)] of the chitosan, endotoxin contamination and appropriate testing controls are rarely reported, they can strongly influence immunotoxicity results. The present work aimed to study the immunotoxicity of NPs produced with different DDA and MW Chit polymers and to benchmark it against the polymer itself. Chit NPs were prepared based on the ionic gelation of Chit with sodium tripolyphosphate (TPP). This method allowed the production of two different NPs: Chit 80% NPs (80% DDA) and Chit 93% NPs (93% DDA). In general, we found greater reduction in cell viability induced by Chit NPs than the respective Chit polymers when tested in vitro using human peripheral blood monocytes (PBMCs) or RAW 264.7 cell line. In addition, Chit 80% NPs were more cytotoxic for PBMCs, increased reactive oxygen species (ROS) production (above 156 μg/mL) in the RAW 264.7 cell line and interfered with the intrinsic pathway of coagulation (at 1 mg/mL) when compared to Chit 93% NPs. On the other hand, only Chit 93% NPs induced platelet aggregation (at 2 mg/mL). Although Chit NPs and Chit polymers did not stimulate the nitric oxide (NO) production in RAW 264.7 cells, they induced a decrease in lipopolysaccharide (LPS)-induced NO production at all tested concentrations. None of Chit NPs and polymers caused hemolysis, nor induced PBMCs to secrete TNF-α and IL-6 cytokines. From the obtained results we concluded that the DDA of the Chit polymer and the size of Chit NPs influence the in vitro immunotoxicity results. As the NPs are more cytotoxic than the corresponding polymers, one should be careful in the extrapolation of trends from the polymer to the NPs, and in the comparisons among delivery systems prepared with different DDA chitosans.

Relatório de estágio realizado no âmbito do mestrado em Psicologia Clínica, Universidade Lusíada de Lisboa, 2015 Exame público realizado em 16 de Novembro de 2015 Por razões éticas e deontológicas, o documento apenas se encontra disponível parcialmente. O presente relatório descreve o trabalho feito ao longo do estágio académico do Mestrado em psicologia clínica, lecionado na Faculdade de Ciências Humanas e Sociais da Universidade Lusíada de Lisboa e realizado na Fundação o Século, mais especificamente no projeto das Casas da Ponte. O estágio permitiu o primeiro contacto com a prática profissional em psicologia clínica, proporcionando assim, o aumento da autonomia do estagiário através da experiência de campo, para que este consiga crescer com a mesma e preparar-se para práticas futuras. O foco principal deste estágio foi o projeto das Casas da Ponte que consiste na utilização de apartamentos de autonomia como instrumentos promotores da autonomia de jovens provenientes de lares de acolhimento. Tendo em conta o projeto e as suas necessidades, foram definidos vários objetivos que permitissem alcançar o melhor funcionamento do mesmo, sendo que dois deles ocuparam um papel de maior destaque devido á magnitude e tempo dedicado. O primeiro consistiu na elaboração de um método que permitisse avaliar a evolução da autonomia dos jovens que pertencem e viram a pertencer a este projeto, o segundo objetivo estabelecido foi o de promover algumas competências sociais e pessoais das jovens inseridas nos apartamentos no ano 2014/2015.

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, Programa de Pós-graduação em Biologia Molecular, 2015. Introdução: Diversos estudos discutem a segurança do uso de nanopartículas já que estas são capazes de promover alterações na expressão gênica, padrão epigenético, homeostase do ferro e estresse oxidativo em ensaios in vitro. Uma gama de estudos aponta diferentes comportamentos celulares em resposta à exposição a nanopartículas, mas ainda não existe um consenso sobre os reais benefícios e riscos que estes nanomateriais podem apresentar em um sistema biológico complexo. Portanto, torna-se necessário estudar estes riscos a fim de contribuir com o desenvolvimento de nanopartículas mais adequadas para aplicações biológicas. Objetivo: Avaliar e caracterizar os possíveis efeitos citotóxicos e epigenéticos em células de carcinoma de glândula submandibular humana (HSG) submetidas a exposição de nanopartículas de maghemita funcionalizadas com cobertura de citrato (NPs). Analisar possíveis alterações na expressão gênica mediante a quantificação do número de transcritos para determinados genes alvo comprometidos com a regulação epigenética e estresse oxidativo. Materiais e métodos: As NPM-citrato foram sintetizadas pelo método de coprecipitação de Fe (II) e Fe (III) e adição direta de ácido cítrico. Os ensaios de citotoxicidade foram realizados por detecção da lactato desidrogenase (LDH) e ensaio de MTT. A morfologia celular foi analisada por coloração panótica InstaProv em microscópio de luz. A detecção de ferro intracelular foi realizada pelo ensaio do Azul da Prússia. As quantificações de metilação global de DNA e de acetilação das histonas H3 e H4 foram realizados por ensaio colorimétrico. A expressão dos genes FERRH, FERRL, DNMT1, DNMT3a, HDAC1, HDAC2 e COX-2 foi realizada por qRT-PCR. Para análise da produção de espécies reativas de oxigênio foi utilizado protocolo de H2DCFA. Resultados: As NPM-citrato nas concentrações testadas de 0,5 mgFe/mL e 3,0 mgFe/mL não apresentaram efeito citotóxico pela metodologia empregadas. Contudo, foram observadas alterações na morfologia celular, como vacuolização citoplasmática nas células expostas a nanopartículas por 24h e 48h, presença de ferro intracelular mesmo após a retirada do tratamento, intensa produção de espécies reativas de oxigênio dose e tempo dependentes, diminuição no perfil global de metilação de DNA e acetilação das histonas H3 e H4: a acetilação da H3 diminui para 38% e 62% após os tratamentos com 0,5 mgFe/mL e 3,0 mgFe/mL, respectivamente. Para a H4 a hipoacetilação foi mais acentuada, diminuindo de 82% para 4% e 10% após os tratamentos com 0,5 mgFe/mL e 3,0 mgFe/mL, respectivamente. Diferenças entre o acúmulo de transcritos dos genes estudados também foram observadas: transcritos referentes aos genes de COX-2, FERRL, e FERRH encontraram-se aumentados, enquanto que DNMT3a sofreu uma diminuição no número de transcritos. Conclusão: Concluímos que as nanopartículas de maghemita recobertas com citrato são capazes de induzir vacuolização citoplasmática, acúmulo de ROS, diminuição do padrão de metilação global e de acetilação de H3 e H4, alteração do acúmulo dos transcritos COX-2, FERRH, FERRL, DNMT3a e HDAC1 sem comprometer a viabilidade das células HSG. Esse é o primeiro relato sobre os efeitos genéticos e epigenéticos das NPM-citrato sobre células HSG. Introduction: A plethora of studies debate about the safety of the use of nanoparticles due to their ability to promote alterations in gene expression, epigenetic patterns, iron homeostasis, and oxidative stress in vitro. Several papers report these different cell behaviours to different nanoparticles, but there is still no consensus on the real benefits and risks associated to nanoparticle exposure on a complex biological system. Hence, it is necessary to elucidate these risks in order to contribute with adequate therapy development and biological applications. Objective: To evaluate and distinguish possible cytotoxic and epigenetic effects on human submandibular gland (HSG) cancer cells exposed to maghemite nanoparticles functionalised with citric acid. Methodology: The NPM-citrate were synthesized by coprecipitation of Fe (II) and Fe (III) method and direct addition of citric acid. LDH and MTT were performed in order to evaluate cytotoxicity. Cell morphology was analysed by panoptic staining. Intracellular iron detection was assayed by the Prussian Blue. Methylation of DNA and Histone acetylation quantifications were performed by colorimetric assays. Gene expression of FERRH, FERRL, DNMT1, DNMT3a, HDAC1, HDAC2 e COX-2 was performed by qRT-PCR. For ROS production assay protocol for H2DCFA was performed. Results: No cytotoxic effects were observed on cells exposed to the tested concentrations of 0.5 mgFe/mL and 3.0 mgFe/mL. Although, cell morphology was found altered by both concentrations after 24h and 48h of exposure. Intracellular iron presence was also observed even after cease of exposure as well as intense generation of ROS. Furthermore, global DNA methylation and acetylation of histones H3 and H4 were also found to be altered. Histone 3 acetylation decreased to 38% and 62% after treatments with 0.5 mgFe/mL and 3.0 mgFe/mL, respectively. Hipoacetylation for H4 was even more accentuated and decreased from 82% to 4% and 10% after treatments of 0.5 mgFe/mL e 3.0 mgFe/mL, respectively. Differences between the gene transcripts accumulation were also reported: COX-2, FERRL and FERRH were found to be overexpresses, while DNMT3a decreased its transcript accumulation. Conclusion: We conclude that maghemite nanoparticles functionalized with citric acid were able to promote cytoplasmatic vacuolization, ROS generation, decreasing of global methylation pattern and acetylation of H3 and H4 and alteration on transcript accumulation of COX-2, FERRH, FERRL, DNMT3a and HDAC1 without impairment of HSG cells viability. This is the first report on the genetic and epigenetic effects of maghemite nanoparticles functionalized with citric acid on HSG cells.