Desenvolvimento de sistemas para a libertação controlada de interleucina-10

Tese de doutoramento em Engenharia Química e Biológica (área de conhecimento em Tecnologia Enzimática e Bio-separações)Therapeutic proteins are becoming available for the treatment of a wide range of diseases. A main problem limiting the efficiency of protein therapeutics is the reduced stability and short circulation half-lives after parenteral administration. Interleukin-10 (IL-10) is an anti-inflammatory cytokine, which active form is a noncovalent homodimer with two intramolecular disulphide bonds essential for its biological activity. Due to its immunoregulatory properties, IL-10 is a promising protein to be used in several clinical applications. So, it is essential to develop delivery systems that enhance the protein bioavailability and selectivity, and that enables a targeted controlled release profile. This is the main focus of the present thesis, taking IL-10 as case study. The use of Carbohydrate-Binding Modules (CBM) as a tool for protein delivery and functionalization was attempted. Proteins and peptides can be used to functionalize biomaterials used for tissue engineering or other biomedical applications, for instance to reduce inflammation (in case of IL-10) or to enhance cellular adhesion (RGD peptide). A method based on the use of a human chitin-binding module, with affinity for chitin, was tested as an alternative approach to the chemical grafting of bioactive proteins/peptides (Chapter 2) on chitin-based biomaterials. A fusion recombinant protein, containing the RGD sequence fused to a human chitin-binding module, was produced and its ability to enhance fibroblasts adhesion to reacetylated chitosan films was tested. The results show that the recombinant protein inhibits the fibroblasts adhesion and proliferation. It was also concluded that the toxic effect was mainly due to the human-chitin biding module. Several polymer protein delivery systems have been developed and described in the literature. Among them, nanometer-sized polymer hydrogels (nanogels) have attracted growing interest. By trapping proteins in a hydrated polymer-network, nanogels minimize denaturation, simultaneously allowing a slow, continuous and controlled release, ideally maintaining an effective concentration for the necessary period of time. A mutated form of murine IL-10 (rIL-10) was successfully produced and its biological activity was confirmed on endotoxin-stimulated bone marrowderived macrophages (Chapter 3). It was shown that a dextrin nanogel (previously developed) effectively incorporated, stabilized, and enabled the slow release, in vitro, of biologically active rIL-10 over time. The IL-10 delivery system based on dextrin nanogels was further analyzed (Chapter 4). Dextrin nanogels were shown to be biocompatible and, after subcutaneous injection, allowed a stable concentration of rIL-10 for at least 4 hours. Despite the low amount of rIL-10 released from the complex nanogel/rIL-10, the rIL-10 released was biologically active in vivo, in the mice. A composite hydrogel made of oxidized dextrin hydrogel with incorporated dextrin nanogels was developed (Chapter 5). The oxidized dextrin hydrogel presented acceptable mechanical properties, biocompatiblity and biodegradability. Additionally, it allowed for the controlled release of the dextrin nanogels. The dextrin nanogel permitted the incorporation of rIL-10, curcumin and β-galatosidase into the oxidized dextrin hydrogel and allowed for their release over time. In all assays, rIL-10 showed high instability and it is likely that a different release profile from either dextrin nanogel or oxidized dextrin hydrogel could be achieved using other kind of bioactive molecules. So, further studies with other bioactive and more stable proteins should be attempted in order to evaluate the potential of the dextrin nanogel and oxidized dextrin hydrogel as protein delivery systems. Even though, considering the interesting properties of dextrin, the stabilization of rIL-10 by the dextrin nanogels as well as the controlled release rate achieved with the composite hydrogel, we consider as very promising the described systems for protein delivery applications.As proteínas terapêuticas representam uma nova classe de fármacos para o tratamento de diversas doenças. Um dos principais problemas é a estabilidade reduzida e consequentemente o curto tempo de semi-vida das mesmas, após administração parenteral. A interleucina 10 (IL-10) é uma citocina anti-inflamatória, cuja forma activa é composta por um homodímero com duas ligações intramoleculares dissulfureto, que são essenciais para a sua actividade biológica. Devido às suas propriedades imunoreguladoras, a IL-10 apresenta grandes potencialidades em diversas aplicações clínicas. Portanto, é essencial o desenvolvimento de sistemas que permitam não só a libertação controlada da proteína mas também aumentem a biodisponibilidade da mesma. Este é o objectivo principal desta tese. Um domínio de ligação a carbohidratos (CBM) foi usado como ferramenta para veicular proteínas e funcionalizar biomateriais. As proteínas e péptidos podem ser usados para funcionalizar biomateriais, por exemplo exercendo actividade antiinflamatória (IL-10) ou aumentando a adesão celular (péptido RGD). Um método baseado no uso de domínio de ligação à quitina foi utilizado neste trabalho como alternativa ao enxerto químico de proteínas ou péptidos (Chaper 2) em biomateriais de quitina. Foi produzida e testada a capacidade de uma proteína recombinante de fusão, contendo uma sequência RGD fundida com um domínio humano de ligação a quitina, no aumento da adesão de fibroblastos a filmes de quitosano reacetilado. Os resultados mostram que as proteínas recombinantes afectam negativamente os fibroblastos, inibindo a sua adesão e proliferação. Concluiu-se também que este efeito negativo se deve, essencialmente, ao domínio humano de ligação à quitina. Diversos sistemas de libertação baseados em polímeros têm sido desenvolvidos e descritos na literatura. Entre eles, os hidrogéis de tamanho nanométrico (nanogéis) têm atraído muito interesse. Incorporando as proteínas numa rede hidratada, os nanogéis minimizam a desnaturação, e simultaneamente permitem uma libertação lenta, contínua e controlada, mantendo uma concentração efectiva da proteína pelo tempo necessário. Uma forma mutada de IL-10 (rIL-10) de ratinho foi produzida com sucesso e a sua actividade biológica confirmada em macrófagos derivados de medula óssea activados (Chapter 3). Demonstrou-se que um nanogel de dextrino (desenvolvido anteriormente) foi capaz de efectivamente incorporar e estabilizar proteína, possibilitando a libertação controlada, in vitro, de rIL-10 biologicamente activa. Os nanogéis como sistema controlado de libertação de IL-10 foram caracterizados detalhadamente (Chapter 4). Os nanogéis são biocompatíveis e, após injecção subcutânea, permitiram que fosse mantida uma concentração estável de IL-10 pelo menos por 4 horas. Apesar das baixas quantidades de rIL-10 libertada do nanogel, a rIL-10 livre é biologicamente activa in vivo, em ratinhos. Foi também desenvolvido um biomaterial composto por um hidrogel de dextrino oxidado com incorporação de nanogéis de dextrino (Chapter 5). O hidrogel de dextrino oxidado apresenta boas propriedades mecânicas, é biocompatível e biodegradável. Adicionalmente, foi verificada a libertação controlada dos nanogéis de dextrino incorporados. O nanogel permitiu a incorporação no hidrogel de dextrino oxidado de rIL-10, curcumina e β-galatosidase e verificou-se a sua libertação ao longo do tempo. Em todos os ensaios foi verificada uma elevada instabilidade da rIL-10 e é provável que usando outro tipo de moléculas bioactivas seja possível obter um perfil de libertação diferente, tanto dos nanogéis de dextrino como dos hidrogéis de dextrino oxidado. Portanto, de forma a avaliar as potencialidades dos sistemas descritos para a libertação de proteínas, mais estudos deverão ser feitos com proteínas bioactivas mais estáveis. Considerando as propriedades do dextrino, interessantes do ponto de vista do desenvolvimento de aplicações biomédicas, a estabilização da rIL-10 pelos nanogéis assim como a libertação controlada observada nos hidrogéis compostos, podem classificar-se de muito promissores os sistemas descritos para a libertação de proteínas.Fundação para a Ciência e Tecnologia (FCT) - SFRH/BD/27359/2006; POCTI/BIO/45356/2002; PTDC/BIO/67160/200

Professional awareness among English language teacher educators: individual and collaborative reflective practice

Studies involving reflective practice (RP) in Education generally focus on student or novice teachers. The research reported on here involved higher level language teacher educators, reflecting as individuals and as a community. The researcher investigates the role and limitations of cooperation in the refinement of professional awareness. The participants engaged in a non-longitudinal process of self-evaluation, supported by three data-led guidance activities, as external frames of reference, namely, self and peer observation, post observation peer-feedback and group socialization. Blocks of discourse, studied as sub-corpora were analyzed as independent units and also in reference to other existent corpora. The analysis combined quantitative and qualitative research methods, taking a predominantly qualitative approach involving descriptive, exploratory and explanatory analysis. The analytical methods combine conversation and discourse analysis supported by corpus linguistics. The results showed that the impact of collaborative modes of work on individual professional self-awareness depend heavily on the nature of the interactions, individual levels of experience, previous RP engagement and resistance to change. Moreover, the results highlight the need for RP research to be more frequently extended to all levels of teaching experience, as a way to maintain continual development at any stage of an individual’s professional career.  Estudos envolvendo prática reflexiva em educação geralmente focam em professores-alunos ou noviços. A pesquisa aqui reportada envolveu educadores de professores em nível superior, refletindo como indivíduos e como uma comunidade, enquanto a pesquisadora investiga o papel e as limitações da cooperação para o refino da auto-percepção profissional. Os participantes engajaram-se em um processo não-longitudinal de auto-avaliação, apoiados por três atividades-guias: auto observação e observação em pares, sessão de feedback e encontro de grupo. Assim, blocos de discurso, estudados como sub-corpora, foram analisados em si e em refererência a outros corpora existentes. A análise combinou métodos quantitativos e qualitativos, com abordagem predominantemente qualitativa, envolvendo análise descritiva, exploratória e explanatória. Os métodos analíticos combinaram análise do discurso e da conversação com suporte da linguística de corpus. Os resultados mostraram que o impacto de modos colaborativos de prática reflexiva sobre a auto-percepção individual depende pesadamente da natureza das interações, níveis de experiência, engajamento prévio em prática reflexiva e resistência a mudanças. Além disso, os resultados enfatizam a necessidade de as pesquisas envolverem mais frequentemente todos os níveis de experiência, como forma de manter o desenvolvimento contínuo do profissional em quaisquer estágios de sua carreira

Study of the potential toxicity of adrenaline to neurons, using the SH-SY5Y human cellular model

Prolonged overexposure to catecholamines causes toxicity, usually credited to continuous adrenoceptor stimulation, autoxidation, and the formation of reactive pro-oxidant species. Non-differentiated SH-SY5Y cells were used to study the possible contribution of oxidative stress in adrenaline (ADR)-induced neurotoxicity, as a model to predict the toxicity of this catecholamine to peripheral nerves. Cells were exposed to several concentrations of ADR (0.1, 0.25, 0.5 and 1mM) and two cytotoxicity assays [lactate dehydrogenase (LDH) release and 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction] were performed at several time-points (24, 48, and 96h). The cytotoxicity of ADR was concentration- and time-dependent in both assays, since the lowest concentration tested (0.1mM) also caused significant cytotoxicity at 96h. N-acetyl-cysteine (1mM), a precursor of glutathione synthesis, prevented ADR-induced toxicity elicited by 0.5mM and 0.25mM ADR following a 96-h exposure, while the antioxidant Tiron (100µM) was non-protective. In conclusion, ADR led to mitochondrial distress and ultimately cell death in non-differentiated SH-SY5Y cells, possibly because of ADR oxidation products. The involvement of such processes in the catecholamine-induced peripheral neuropathy requires further analysis

Extra-curricular activities and the prevention of drug abuse: a polemic question

Data obtained from 16,117 high school students in fifteen Brazilian cities, relating to participation in a number of extracurricular activities and consumption of drugs and alcohol, are described. In the great majority of cases, no association was found between participation in artistic, community or sports-related activities and the use of these substances. On the other hand, a weak but constant negative correlation was found between alcohol/drug consumption and involvement in religious activities. The authors discuss these findings in the light of some of the current preconceptions prevailing in Brazilian society: namely, that such activities constitute effective strategies for drug use prevention; and that idle young persons tend to be potential drug users. They also discuss the implications of the fact among students involved in religious activities drug and alcohol consumption has been shown to be slightly lower.O presente trabalho descreve dados colhidos entre 16.117 estudantes de primeiro e segundo graus, de quinze cidades brasileiras, sobre a prática de algumas atividades não curriculares e o consumo de álcool e drogas. Não foi encontrada, na ampla maioria dos casos, nenhuma associação entre praticar esportes, artes e atividades comunitárias e o consumo dessas substâncias. Mas foi encontrada correlação negativa fraca, mas constante, entre consumo de álcool e drogas e freqüência a atividades religiosas. Os achados são discutidos à luz de alguns preconceitos correntes na sociedade brasileira, que rotula o jovem sem ocupação definida como drogado em potencial. Discutem também as implicações do fato de entre os jovens praticantes de atividades religiosas haver uma discreta diminuição do uso de álcool e drogas.Escola Paulista de Medicina Departamento de Psicobiologia Centro Brasileiro de Informações sobre Drogas PsicotrópicasUNIFESP, EPM, Depto. de Psicobiologia Centro Brasileiro de Informações sobre Drogas PsicotrópicasSciEL

Mature women : life histories and depressive complaints

O trabalho é parte da dissertação de mestrado da primeira autora, realizada sob orientação da segunda.O presente trabalho buscou investigar a queixa depressiva em mulheres na maturidade, a partir de eventos significativos ao longo de sua história de vida. Foram realizadas entrevistas clínicas com nove mulheres com diagnóstico de depressão. A análise das entrevistas indicou que as participantes enfrentaram situações de perda ou abandono dos pais, humilhações e maus-tratos, bem como privações econômicas em sua infância, que tiveram impacto sobre sua saúde física e mental. Já na vida adulta, algumas perderam filhos prematuramente. Há casos de violência conjugal. A queixa depressiva parece estar também relacionada a imagens autodepreciativas sobre o exercício dos papéis de mãe e esposa, com sentimentos de culpa e fracasso. O estudo aponta a necessidade de atenção clínica à história de mulheres que apresentam sintomas depressivos na maturidade. Indica-se ainda a discussão da categoria gênero na área da Saúde Mental, para a revisão de concepções históricas e culturais sobre o ciclo de vida feminino. ________________________________________________________________________________________ ABSTRACTThe present study investigated depressive symptoms in mature women, based on significant events in their life history. Clinical interviews were carried out with nine women diagnosed with depression. The data analysis showed that participants had experienced parents’ loss or abandonment, humiliations, ill-treatment, and were financially deprived during their childhood. Such events had a profound impact on their physical and mental health. As adults, some women have lost their children prematurely. There are also cases of marital violence. Depressive complaints seem to be related to low self-esteem images concerning their role as mothers and wives, with feelings of guilt and failure. The study, points out the need of clinical care related to the history of mature women facing depressive symptoms. It is also suggested a discussion of gender category in the field of Mental Health, in order to review historical and cultural concepts of women’s life cycle

Fatores associados ao aumento da espessura do septo atrial em lactentes com Síndrome da Hipoplasia do Coração Esquerdo (SHCE): implicações para septostomia percutânea

Introdução: Nos procedimentos usualmente realizados em recém-nascidos acometidos pela Síndrome da Hipoplasia do Coração Esquerdo (SHCE), variações fenotípicas influenciam o sucesso do tratamento, principalmente as características relacionadas ao septo atrial e ao forame oval (FO) no caso de septostomia por cateter. Objetivos: Analisar características macroscópicas do septo atrial de corações com SHCE, a fim de definir e orientar procedimentos terapêuticos nessa estrutura. Métodos: Foram avaliados 18 corações de pacientes falecidos e diagnosticados com SHCE quanto à perviedade e tamanho do FO, abaulamento da lâmina, atresia ou perviedade da válvula mitral e calibre da aorta ascendente e tronco pulmonar. Cortes histológicos do septo atrial foram feitos para medidas da espessura máxima e mínima da lâmina. Resultados: A idade média dos pacientes foi de 34,5 dias (57% do sexo masculino), com espessura do septo atrial médio de 1,90 mm (0,63 - 4,09 mm). O diâmetro médio do tronco e da aorta pulmonar foi de 1,16 cm e 0,22 cm, respectivamente. O FO era patente em 39% dos casos. A valva mitral era atrésica em 21% das amostras. Houve diferença significativa na espessura do septo atrial nos casos com FO patente ou fechada, sendo maior nos casos em que a FO era fechada (p = 0,047). A relação FO / idade apresentou correlação negativa estatisticamente significante com a espessura do septo atrial (r = -0,76 ep <0,05). Conclusões: Os resultados indicam que a perviedade e o tamanho do forame oval têm repercussões na espessura do septo atrial, sugerindo que esse fator pode limitar o sucesso de intervenções terapêuticas, principalmente na septostomia por cateter.Introduction: In the usual procedures performed on newborns affect by Hypoplastic Left Heart Syndrome (HLHS), phenotypic variations influence the success of the treatment, especially the characteristics related to the atrial septum and foramen ovale (FO) in case of catheter septostomy. Objectives: To analyze macroscopics features of the atrial septum of hearts with HLHS in order to define and guide therapeutic procedures on this structure. Methods: 18 hearts of deceased patients diagnosed with HLHS were evaluated for FO patency and size, bulging of its blade, atresia or patency of the mitral valve and caliber of the ascending aorta and pulmonary trunk. Histological sections of the atrial septum were made for measurements of maximum and minimum thickness of the lamina. Results: The mean age of the patients was 34.5 days (57% male), with a mean atrial septal thickness of 1.90 mm (0.63 - 4.09 mm). The mean diameter of the pulmonary trunk and aorta were 1.16 cm and 0.22 cm, respectively. FO was patent in 39% of cases. The mitral valve was atresic in 21% of the specimens. There was a significant difference in the thickness of the atrial septum in cases with patent or closed FO, being greater in cases where the FO was closed (p = 0.047). The ratio FO/age presented a statistically significant negative correlation with atrial septum thickness (r = -0.76 and p <0.05). Conclusions: The results indicate that the patency and size of the oval foramen have repercussions on the thickness of the atrial septum, suggesting that this factor may limit the success of therapeutic interventions, especially catheter septostomy

Factors associated with increased atrial septum thickness in infants with left heart hypoplasia syndrome: implications for percutaneous septostomy

Introduction: Hypoplastic Left Heart Syndrome (HLHS) encompasses a spectrum of cardiac malformations characterized by underdevelopment of the left heart structures and aorta that, despite the low incidence among cases of congenital heart disease have high mortality rates. Phenotypic variations of the anomaly may influence the success of the interventional and surgical procedures, especially the characteristics related to the atrial septum and foramen ovale (FO), in cases where balloon catheter septostomy is necessary to maintain the interatrial shunt.Objectives: To analyze the gross features of the atrial septum of hearts with HLHS in order to define and guide therapeutic procedures on this structure, correlating them with other morphological alterations of the syndrome, such as the anatomy of the heart valves. Methods: We studied 18 hearts of patients diagnosed and deceased with HLHS, all belonging to the anatomic collection of the Laboratory of Pathology of the Heart Institute (InCor) HCFMUSP. The atrial septum was evaluated for FO patency and size, as well as for the presence of bulging of its lamina to one or the other atrial cavity. Other morphological features of the anatomical specimen such as atresia or patency of the mitral valve and caliber of the ascending aorta and pulmonary trunk were annotated. After resection of the atrial septum, histological sections were made for microscopic measurements of the maximum and minimum thickness of the lamina (atrial septum).Results: The mean age of the patients was 34.5 days (57% male), with a mean atrial septal thickness of 1.90 mm (0.63-4.09 mm). The mean diameter of the pulmonary trunk and aorta were 1.16 cm and 0.22 cm, respectively. FO was patent in 39% of cases (mean diameter 4,4 mm, range 4 to 8 mm), with bulging of the lamina to one of the atrial cavities in half of the cases. The mitral valve was atretic in 21% of the specimens. There was a significant difference in the mean thickness of the atrial septum in cases with patent versus closed FO, being thicker in cases where the FO was closed (p = 0.047). The ratio FO/age presented a statistically significant negative correlation with the atrial septum thickness (r = -0.76 and p <0.05).Discussion and Conclusions: The results indicate that the patency and size of the oval foramen may influence the thickness of the atrial septum, suggesting that this factor may limit the success of therapeutic interventions, especially balloon catheter septostomy

The effect of an RGD-Human Chitin-Binding Domain fusion protein on the adhesion of fibroblasts to reacetylated chitosan films

Biomaterials used for tissue engineering applications must provide a structural support for the tissue development and also actively interact with cells, promoting adhesion, proliferation and differentiation. To achieve this goal, adhesion molecules may be used, such as the tripeptide Arg-Gly-Asp (RGD). RGD was found to be the major functional amino acid sequence responsible for cellular adhesion. This sequence can be used to elicit specific cellular responses and it has been extensively demonstrated that RGD sequence improves cell adhesion, spreading and proliferation in different materials. Chitosan and chitin represent a family of biopolymers, made up of b(14)-linked N-acetyl-Dglucosamine and D-glucosamine subunits. Due to their biodegradability and biocompatibility, chitin and chitosan, are widely studied for biomedical applications. A method based on the use of a human Carbohydrate-Binding Module, with affinity for chitin, was tested as an alternative approach to the chemical grafting of bioactive peptides. This approach would simultaneously allow the production of recombinant peptides (alternatively to peptide synthesis) and provide a simple way for the specific and strong adsorption of the peptides to the biomaterial. A fusion recombinant protein, containing the RGD sequence fused to a human chitin-binding module (ChBM), was expressed in E. coli. The adhesion of fibroblasts to reacetylated chitosan (RC) films was the model system selected to analyse the properties of the obtained proteins. Thus, the evaluation of cell attachment and proliferation on polystyrene surfaces and reacetylated chitosan films, coated with the recombinant proteins, was performed using mouse embryo fibroblasts 3T3. The results show that the recombinant proteins affect negatively fibroblasts anchorage to the materials surface, inhibiting its adhesion and proliferation. We also conclude that this negative effect is fundamentally due to the human chitinbinding domain

h3 gourmet: not so fast food

The h3 case study focuses on the development and offer of a new chain of hamburger restaurants that began in Portugal and is spreading to other countries like Spain, Brazil and Poland. The general idea of the business venture is to grow quickly and occupy an unconventional place in the fast food burger business: a food chain with a gourmet touch, taking a high profile approach with natural ingredients, customization and quality service, where hamburgers are served on heated plates with cutlery and may be accompanied by a glass of wine, homemade lemonade or iced tea. The client can finish the meal with ice cream or chocolate coulant desserts with quality ingredients. H3 now has more than 40 restaurants; it is preparing to conquer the world and challenge the big players both in malls and street restaurants. Competitive advantages and critical success factors will play a vital role in this case

Impact of grade retention and school engagement on student intentions to enrol in higher education in Portugal

Grade retention and inequalities that derive from grade retention can influence student school trajectories and careers. Grade retention can discourage students from education, and increase school failure and dropout. This study explored the relationship between grade retention and student intentions to enrol in higher education. We also studied the role of school identification and behavioural engagement in this relationship. The analysis is based on a sample of 1,089 students (grades 6 to 10) from Portugal, one of the European countries with the highest rates of grade retention. We employed multilevel probit regression modelling with random intercept and fixed slopes to explore both the individual and school level effects of grade retention. The analyses showed that retained students had a lower probability of intent to enrol in higher education and that there was a contextual effect of the number of retained students in the school, on students' probability of intent to enrol in higher education. This association was partially explained at the individual level by students' school identification. Retained students presented lower levels of school identification, which in turn results in lower probabilities of enrolling in higher education. Student behavioural engagement was not associated with grade retention nor student intentions. Our findings suggest the need for interventions that foster students' school identification to overcome the adverse effects of grade retention.info:eu-repo/semantics/publishedVersio