Development of constructs for recombinant expression of human follicle-stimulating hormone receptor in rod cells of the zebrafish

The follicle-stimulating hormone (FSH) is involved in the regulation of reproduction, by acting through a G protein-coupled receptor (GPCR) on the surface of target cells. Like most of the GPCRs, not much is known about the structure of the follicle-stimulating hormone receptor (FSHR). It is relatively difficult to purify the FSHR protein from gonadal tissues, due to its low abundance on its native cells, and therefore, to study its structure. For these reasons it is necessary to come up with a strategy that allows the production of large quantities of protein in order to use it in studies to obtain detailed structural information on full-length human FSHR. Via polymerase chain reactions, site-directed mutagenesis and other molecular biological methods, we were able to construct mutated receptors with different signal sequences and different tags, including the last fifteen amino acids of human rhodopsin which allow the receptor to be expressed on retinal rod cells of zebrafish. Mutations were designed in a manner that the receptor becomes inactive, i.e. incapable of signal transduction but still expressed at cell surface. The receptor constructs were first tested in human embryonic kidney cells (HEK 293-T) and their inserts will be used for transgenetic studies on zebrafish in the future.A hormona estimulante do folículo (FSH) está envolvida na regulação da reprodução, actuando através de um receptor acoplado a proteínas G (GPCR) localizado à superfície das células alvo. Tal como a maioria dos GPCRs, pouco se sabe acerca da estrutura do receptor da hormona estimulante do folículo (FSHR). Este receptor é relativamente difícil de purificar de tecidos gonadais, devido à pouca abundância com que se encontra nas suas células nativas, e, portanto, de estudar a sua estrutura. Por estas razões é necessário criar uma estratégia que permita a produção de grandes quantidades de proteína de modo a poder usá-la em estudos dirigidos à obtenção de informação detalhada da estrutura completa do FSHR. Através de reacções em cadeia da polimerase, mutagénese dirigida e outros métodos biomoleculares foi possível construir receptores mutantes com diferentes sequências sinal e diferentes tags, incluindo os últimos quinze aminoácidos da rodopsina humana que permitem a expressão do receptor em bastonetes da retina de peixe zebra. As mutações foram criadas de modo a que o receptor se torne inactivo, i.e. incapaz de realizar transdução de sinal mas que ainda é expresso à superfície da célula. Os receptores foram primeiro testados em células embriónicas humanas do rim (HEK 293-T) e os insertos serão usados em estudos de transgénese em peixe zebra

Avaliação do impacto das nanopartículas de prata no metabolismo celular: um estudo in vitro por metabolómica de RMN

Doutoramento em Nanociências e NanotecnologiaFace ao uso disseminado e enorme potencial terapêutico das nanopartículas de prata (AgNPs), o estudo dos seus efeitos biológicos é um assunto relevante e atual. O trabalho apresentado nesta tese teve como objetivo aprofundar o conhecimento existente sobre o impacto das AgNPs no metabolismo celular, usando a metabolómica por espectroscopia de ressonância magnética nuclear (RMN). Os tipos celulares escolhidos para este estudo foram queratinócitos da epiderme humana, células de hepatoma humano e macrófagos sanguíneos, por serem relevantes, respetivamente, ao nível da entrada, acumulação e captação de nanopartículas no organismo. O Capítulo 1 introduz as principais propriedades das AgNPs, a sua atividade biológica e potencial toxicidade, e descreve a abordagem metabolómica, incluindo uma breve revisão bibliográfica das suas aplicações em nanotoxicologia. O âmbito e os objetivos desta tese são, também, apresentados. O Capítulo 2 descreve os métodos experimentais utilizados ao longo deste trabalho, incluindo a caracterização das AgNPs, os procedimentos usados na cultura celular e nos ensaios biológicos, a colheita e preparação de amostras, a análise por RMN e o tratamento estatístico dos dados. No Capítulo 3, a atividade metabólica e a composição dos três tipos de células usados neste trabalho (queratinócitos HaCaT, células de hepatoma HepG2 e macrófagos RAW 264.7) são descritas com base na análise por RMN dos sobrenadantes dos meios de cultura (exometaboloma) e dos extratos celulares polares e lipofílicos (endometaboloma). O Capítulo 4 apresenta a análise metabolómica das células HaCaT expostas a AgNPs de diferentes tamanhos (10, 30 ou 60 nm de diâmetro) e revestimentos (citrato, polietilenoglicol ou albumina de soro bovino). Verificou-se que o metaboloma celular foi afetado mesmo a concentrações subtóxicas de AgNPs, sugerindo: aumento da glicólise e glutaminólise, alteração na atividade do ciclo dos ácidos tricarboxílicos (TCA) e nos processos de produção e transferência de energia, degradação de proteínas, síntese de glutationa (GSH), modificações a nível das membranas e do equilíbrio osmótico. Apesar de muitas variações serem comuns a todas as nanopartículas testadas, as AgNPs de 10 nm causaram os efeitos mais distintos, nomeadamente no que diz respeito à glicólise e à síntese/utilização de GSH. Além disso, a exposição celular a prata iónica (Ag+) confirmou o importante papel dos iões prata no mecanismo de ação das AgNPs, enquanto a comparação com o peróxido de hidrogénio (H2O2) permitiu destacar os efeitos relacionados com o stress oxidativo. No Capítulo 5 são apresentadas as respostas metabólicas das células de fígado HepG2 a dois tipos de AgNPs, umas obtidas por redução química e estabilizadas em citrato e as outras obtidas por síntese verde na presença de um extrato vegetal (Cit30 e GS30, respetivamente), ambas com centros metálicos de 30 nm. Os resultados sugeriram adaptações metabólicas em processos de produção de energia (metabolismo da glucose e sistema da fosfocreatina), autofagia e metabolismo lipídico, refletindo possivelmente a ativação de mecanismos de proteção. Ainda que os dois tipos de AgNPs tenham induzido muitos efeitos semelhantes, as Cit30 pareceram causar um maior impacto no ciclo TCA e na degradação de proteínas, enquanto as GS30 aparentaram induzir uma diminuição mais forte na síntese de fosfolípidos. A assinatura metabólica da prata iónica foi bastante semelhante à das AgNPs, sugerindo, no entanto, uma menor capacidade das células expostas a Ag+ extracelular para lidar com o stress oxidativo. O Capítulo 6 descreve a avaliação do impacto das AgNPs Cit30 no metaboloma de macrófagos de murganho RAW 264.7, a concentrações subtóxicas (decréscimos de ~5 e 20% na viabilidade celular). As alterações encontradas apontaram para: estimulação da glicólise (a baixa concentração de exposição), reprogramação do ciclo TCA (resultando numa intensa produção de itaconato e succinato e numa marcada depleção de ATP, consistentes com uma resposta pro-inflamatória), ativação da gluconeogénese, promoção da síntese de GSH e acumulação de creatina/fosfocreatina. Foram, ainda, observadas variações possivelmente relacionadas com a osmorregulação e a modificação membranar. De notar que os macrófagos expostos a Ag+ mostraram características semelhantes aos expostos a AgNPs (por ex., aumento da glucose intracelular – gluconeogénese), mas também revelaram efeitos distintos, nomeadamente em metabolitos envolvidos no ciclo TCA, em processos de transferência de energia e no metabolismo lipídico. Adicionalmente, viu-se que o metaboloma dos macrófagos respondeu de maneira diferente à exposição a H2O2 (por ex., tendência para diminuição da glicólise e sem efeitos observados na ativação da gluconeogénese ou da síntese de GSH), indicando que muitos dos efeitos induzidos pelas AgNPs não foram necessariamente mediados por stress oxidativo. Finalmente, com base na integração dos resultados apresentados ao longo dos capítulos anteriores, as principais conclusões deste trabalho são apresentadas e discutidas no Capítulo 7.The wide dissemination and promising therapeutic potential of silver nanoparticles (AgNPs) make the study of their biological effects a relevant upto- date subject. The work presented in this thesis aimed at deepening current understanding of the impact of AgNPs on cell metabolism, using NMR metabolomics of cultured mammalian cells. Epidermis keratinocytes, hepatoma cells and blood macrophages, relevant, respectively, to nanoparticle entry, accumulation and uptake, have been selected for the study. Chapter 1 introduces the main properties of AgNPs, including their biological activity and toxicological potential, and describes the metabolomics approach, briefly reviewing its applications in the field of nanotoxicology. Also, the scope and aims of this thesis are presented. Chapter 2 covers the experimental methods adopted during the course of this work, including the sources and characterisation of AgNPs, the procedures used in cell culture and biological assays, sample collection and preparation methods, NMR analyses and statistical data treatment. In Chapter 3, the metabolic activity and composition of the three cell types used in this work (HaCaT keratinocytes, HepG2 hepatoma cells and RAW 264.7 macrophages) are described based on the NMR analysis of culture medium supernatants (exometabolome) and polar/lipophilic cell extracts (endometabolome). Chapter 4 presents the metabolomic analysis of HaCaT skin cells exposed to AgNPs of different sizes (10, 30 or 60 nm in diameter) and coatings (citrate, polyethylene glycol or bovine serum albumin). The cellular metabolome was found to be affected even at sub-toxic concentrations of AgNPs, suggesting: upregulation of glycolysis and glutaminolysis, altered tricarboxylic acid (TCA) cycle activity, protein degradation, disruption of energy-producing pathways, glutathione (GSH) synthesis, membrane modification and changes in osmotic balance. Although several metabolic variations were common to all tested nanoparticles, the 10 nm AgNPs showed the most distinct effects, namely in regard to glycolysis and GSH synthesis/utilisation. Furthermore, cell exposure to ionic silver (Ag+) confirmed the major role of silver ions in AgNPs mode of action, while comparison with hydrogen peroxide (H2O2) allowed the effects related to oxidative stress to be highlighted. In Chapter 5, the metabolic responses of liver HepG2 cells to two types of 30 nm AgNPs, obtained by chemical reduction and stabilised with citrate or produced by green synthesis in the presence of a plant extract (Cit30 and GS30, respectively) are presented. Sub-toxic concentrations of AgNPs were proposed to induce metabolic adaptations in energy production processes (glucose metabolism and the phosphocreatine system), autophagy and lipid metabolism, possibly reflecting the activation of metabolism-mediated protective mechanisms. Although the two types of AgNPs induced many common effects, Cit30 appeared to have a greater impact on the TCA cycle and protein degradation, whereas GS30 seemed to induce stronger downregulation of phospholipid synthesis. The metabolic signature of Ag+ was largely similar to that of AgNPs, although suggesting a lower ability of cells exposed to extracellular Ag+ to cope with oxidative stress. Chapter 6 addresses the impact of Cit30 AgNPs on the metabolome of murine RAW 264.7 macrophages, at concentrations causing minimal (~5 and 20%) decreases in cell viability. Exposed cells were suggested to upregulate glycolysis (at the low exposure concentration), to reprogram the TCA cycle (resulting in marked production of itaconate and succinate and in ATP depletion, consistent with a pro-inflammatory response), to activate gluconeogenesis, to promote GSH synthesis, and to increase the creatine/phosphocreatine pool. Changes putatively related to osmoregulation and membrane modification were also observed. Notably, macrophages exposed to Ag+ showed common features to AgNPs-exposed cells (e.g. increased intracellular glucose suggesting gluconeogenesis), but also several distinct effects, for instance in metabolites involved in the TCA cycle, energy transfer processes or lipid metabolism. Furthermore, the cellular metabolome responded differently to H2O2 exposure (e.g. trend for downregulated glycolysis, no evidence of gluconeogenesis activation or of GSH upregulation), indicating that many of the AgNPs-induced effects were not necessarily mediated by oxidative stress. Finally, based on the integration of the results presented along the previous chapters, the main conclusions of this work are presented and discussed in Chapter 7

Avaliação sensorial de bacalhau com natas

A Análise Sensorial é hoje considerada com um elo de ligação entre a pesquisa e o desenvolvimento, pois a ela estão associadas um conjunto de técnicas capazes de obter resultados precisos tendo em conta o objetivo em causa. Assim, foram empregues no presente trabalho, algumas técnicas sensoriais descritivas capazes de determinar o perfil sensorial de uma refeição pré-preparada, nomeadamente Bacalhau com Natas. O presente trabalho foi desenvolvido ao longo de seis meses na empresa Sense Test – Sociedade de Estudos de Análise Sensorial a Produtos Alimentares, Lda. Desta forma, e seguindo a metodologia inerente à análise sensorial, o trabalho encontra-se estruturado em duas partes distintas. Numa primeira fase, é feita uma abordagem e descrição do local de estágio, sendo também realizada uma descrição superficial de todas as etapas necessárias para a avaliação sensorial, desde a estruturação de uma sala de provas até à seleção e treino de provadores. E finalmente, numa segunda fase é realizado um trabalho de investigação, evocando-se de forma aprofundada o uso de algumas técnicas descritivas convencionais e rápidas. São, assim, apresentados resultados específicos relativamente à Aceitação Global, ao QDA (Quantitative Descriptive Analysis), ao Flash Profile e por fim ao Preference Mapping. Deste trabalho conclui-se que os consumidores preferem amostras caracterizadas pelo desfiado e quantidade de bacalhau (visual), pela intensidade de odor e sabor global e pela presença de molho (visual). As técnicas abordadas revelaram ser ferramentas adequadas à descrição do produto em estudo, demonstrando resultados capazes de assegurar o seu valor. Palavras-chave: Análise Sensorial, técnicas descritivas convencionais e rápidas, Aceitação, QDA, Flash Profile, Preference MappingSensory analysis is, nowadays, considered with a connecting link between research and development, because, to it, are associated a set of techniques capable of obtaining accurate results, taking into account the problem in question. Therefore, in this study, some descriptive sensory techniques were employed to determinate the sensory profile of a pre-prepared meal, in this case, cod with cream. This work was developed during six months in a company called 'Sense Test - Sociedade de Estudos de Análise Sensorial a Produtos Alimentares, Lda. Thereby, following the procedures inherent to sensory analysis, the work is divided in two distinct parts. On one hand, in the initial phase, a superficial description of all required steps for the sensory evaluation is made, from structuring a tasting room to selecting and training the assessors. On the other hand, in the final phase, there's the research work, encompassing the use of some conventional and rapid descriptive techniques in detail. In this way, specific results are shown relatively to the global acceptance to the QDA (Quantitative Descriptive Analysis), to the Flash profile, and finally to the Preference mapping. With this investigation, was conclude that consumers prefer samples that are characterized by shredded and quantity of cod (visual),odor ant overall flavor intensity, and by the presence of sauce (visual). The techniques used have proved to be appropriate tools to the description of the product in question, showing results that will ensure its value. Key-words: Sensory Analysis, conventional and fast descriptive techniques, Acceptation, QDA, Flash Profile, Preference Mappin

Digital mental health interventions for children and youth

The period from childhood to adolescence is critical for mental health promotion, as it is estimated that, worldwide, approximately 10% to 20% of individuals in this age group have mental health problems that may lead to mental disorders that may persist throughout adulthood. Furthermore, recent studies show that mental health problems during childhood and adolescence contribute to a decrease in academic performance and an increase of risk-taking behaviors, self-injury, and suicide, with consequences into adulthood. Thus, preventing mental health problems in children and adolescents is essential to promote positive lifelong outcomes for young people. Schools are a privileged context for creating favorable environments for the implementation of mental health promotion programs, effectively and with long-term benefits. This context allows for an early intervention during the phase of development of socioemotional skills, thus enhancing the results of the programs themselves, contributing to the healthy development of children and youth and to a higher academic achievement of students.info:eu-repo/semantics/publishedVersio

Pain intensity and mental health quality of life in veterans with mental illnesses: the intermediary role of physical health and the ability to participate in activities

© 2020, Springer Nature Switzerland AG. Purpose: The purpose of this study was to examine the intermediary role of physical health quality of life and ability to participate social roles and activities in the relationship between pain intensity and mental health quality of life in veterans with mental illnesses. Methods: This is a cross-sectional correlational design study. Our participants are 156 veterans with self-reported mental illness (Mage = 37.85; SDage = 10.74). Descriptive, correlation, and mediation analyses were conducted for the current study. Results: Pain intensity was negatively correlated with physical health QOL, ability to participate in social roles and activities, and mental health QOL. Physical health QOL and ability to participate in social roles and activities were positively associated with mental health QOL, respectively. Physical health QOL was positively correlated with a ability to participate in social roles and activities. Study results indicate that the effect of pain intensity on mental health QOL can be explained by physical health QOL and ability to participate. Conclusions: Specific recommendations for practitioners include implementing treatment goals that simultaneously focus on physical health and ability to participate in social roles and activities for clients who present with both physical pain and low mental health QOL

Metabolism of N-ethylhexedrone and buphedrone: An in vivo study in mice using HPLC-MS/MS

N-ethylhexedrone (NEH) and buphedrone (BUPH) are synthetic drugs structurally related to natural cathinone. These synthetic cathinones (SC) are members of the heterogenous family of new psychoactive substances (NPS), which have caused major concern in scientific and forensic communities over the past years, due to their widespread consume. Thus, there is a constant need for monitoring the use of these new substances and gather knowledge on their metabolism and excretion profiles, in order to try to identify markers of NPS consumption. This study aimed at the identification and quantification of NEH, BUPH and selected phase I metabolites using HPLC-MS/MS. NEH, BUPH and some related metabolites were synthesized in-house and quantified in 24 h mice urine, following single dose administration of each drug (64 mg kg−1, i.p.). NEH and BUPH were quantified in mice urine at 58.3 ± 14.4 and 146.2 ± 14.9 µg mL−1, respectively. Similar metabolic pathways were observed for both drugs. Among the metabolites studied, the most excreted ones derived from N-dealkylation of either NEH or BUPH (at around 80 µg mL−1 of urine). Other metabolites resulting from ketone reduction and ketone reduction combined with N-dealkylation or 4-aryl hydroxylation (detected for the first time in non-ring substituted SC) were also identified and quantified. Urine samples were screened using liquid chromatography-high resolution mass spectrometry and various phase II metabolites, including N-acetylated, glucuronides and dicarboxylic acid conjugates were tentatively identified, some of them for the first time. This work is a contribution to the identification of metabolites from SC that can become potential markers to estimate drug consumption

Evaluation of a quality improvement intervention to reduce anastomotic leak following right colectomy (EAGLE): pragmatic, batched stepped-wedge, cluster-randomized trial in 64 countries

Background Anastomotic leak affects 8 per cent of patients after right colectomy with a 10-fold increased risk of postoperative death. The EAGLE study aimed to develop and test whether an international, standardized quality improvement intervention could reduce anastomotic leaks. Methods The internationally intended protocol, iteratively co-developed by a multistage Delphi process, comprised an online educational module introducing risk stratification, an intraoperative checklist, and harmonized surgical techniques. Clusters (hospital teams) were randomized to one of three arms with varied sequences of intervention/data collection by a derived stepped-wedge batch design (at least 18 hospital teams per batch). Patients were blinded to the study allocation. Low- and middle-income country enrolment was encouraged. The primary outcome (assessed by intention to treat) was anastomotic leak rate, and subgroup analyses by module completion (at least 80 per cent of surgeons, high engagement; less than 50 per cent, low engagement) were preplanned. Results A total 355 hospital teams registered, with 332 from 64 countries (39.2 per cent low and middle income) included in the final analysis. The online modules were completed by half of the surgeons (2143 of 4411). The primary analysis included 3039 of the 3268 patients recruited (206 patients had no anastomosis and 23 were lost to follow-up), with anastomotic leaks arising before and after the intervention in 10.1 and 9.6 per cent respectively (adjusted OR 0.87, 95 per cent c.i. 0.59 to 1.30; P = 0.498). The proportion of surgeons completing the educational modules was an influence: the leak rate decreased from 12.2 per cent (61 of 500) before intervention to 5.1 per cent (24 of 473) after intervention in high-engagement centres (adjusted OR 0.36, 0.20 to 0.64; P < 0.001), but this was not observed in low-engagement hospitals (8.3 per cent (59 of 714) and 13.8 per cent (61 of 443) respectively; adjusted OR 2.09, 1.31 to 3.31). Conclusion Completion of globally available digital training by engaged teams can alter anastomotic leak rates. Registration number: NCT04270721 (http://www.clinicaltrials.gov)