Estudos de metabolismo in vitro de extractos aquosos de São Roberto, Geranium robertianum: aplicações terapêuticas na doença de Alzheimer

Tese de mestrado, Bioquímica, Universidade de Lisboa, Faculdade de Ciências, 2009O objectivo deste trabalho foi o estudo do metabolismo in vitro de extractos aquosos de Geranium robertianum e actividades biológicas com aplicações terapêuticas na Doença de Alzheimer. O aumento das doenças neurodegenerativas na população leva à necessidade de encontrar novas terapêuticas. Uma área em expansão é o estudo de plantas medicinais, como o G. robertianum, utilizado na medicina tradicional portuguesa e cuja caracterização exaustiva permitirá a compreensão das suas potencialidades como agente terapêutico. Determinou-se a concentração de G. robertianum necessária para inibir 50% da actividade do enzima acetilcolinesterase, obtendo-se 765,88±15,39 μg/mL. Simultaneamente, calculou-se a concentração de extracto aquoso responsável por 50% de extinção do radical DPPH, 6,53±0,58 μg/mL. A análise destas actividades biológicas para cada fracção maioritária mostrou que o seu efeito sinergístico promove as actividades globais apresentadas. Seguidamente, caracterizou-se a planta relativamente ao tipo e estrutura dos principais compostos. Quantificaram-se os fenóis totais, obtendo-se um valor de 106,96±2,37 μg de equivalentes de pirogalol / mg de extracto. A análise por espectrometria de massa permitiu concluir que as principais moléculas presentes em G. robertianum são o ácido sinápico glucuronado, quercitrina, ácido gálhico, ácido elágico e cianidina. O estudo do metabolismo in vitro de extractos aquosos de G. robertianum mostrou que não há metabolização pelos sucos gástrico e pancreático, bem como por células Caco-2. A digestão por β-glucuronidase de E. coli permitiu comprovar a existência de ácido sinápico glucuronado. Adicionalmente verificou-se que durante todo o metabolismo o extracto aquoso mantém as suas actividades biológicas de relevância. O estudo da reacção de glucuronidação, típica de metabolismo hepático, permitiu concluir que os polifenóis existentes no extracto não sofrem glucuronidação e, mais uma vez, mostrou a presença do composto glucuronado no extracto. Finalmente estudou-se a actividade antimicrobiana de G. robertianum, concluindo-se que 12,99 mg/mL e 19,70 mg/mL de extracto inibem 50% do crescimento de S. mutans e S. sobrinus, respectivamente. Simultaneamente estudou-se a inibição do enzima glucosiltransferase isolado das estirpes bacterianas, verificando-se que 8,27 mg/mL e mais de 10 mg/mL inibem 50% da actividade do enzima extraído de S. mutans e S. sobrinus, respectivamente. Assim, concluiu-se que o extracto aquoso de G. robertianum possui actividades biológicas passíveis de aplicação na Doença de Alzheimer. Estudos adicionais poderão indicar como garantir a chegada do extracto ou seus componentes aos tecidos-alvo e elucidar acerca de mecanismos celulares e moleculares através dos quais este exerce os seus efeitos biológicos.This work focused on the study of the in vitro metabolism of aqueous extracts of Geranium robertianum and its biological activities, relevant to the Alzheimer’s disease therapeutics. The increase of neurodegenerative diseases within the world population urges the research of new therapeutic strategies. A field of research is that of medicinal plants, such as G. robertianum, widely used in Portuguese folk medicine. However, an extensive characterization of this plant is required, in order to understand its potentialities as a therapeutic agent. G. robertianum was screened for its acetylcholinesterase inhibitory and antioxidant activities. At a concentration of 765,88±15,39 μg/mL the enzyme activity was inhibited in 50% and at a value of 6,53±0,58 μg/mL 50% of the free radical DPPH was scavenged. The analysis of these biological activities, for each major component of the aqueous extract, showed that a synergetic effect between them accounts for the global activities determined. Afterwards, the plant was characterized according to the type and chemical structure of its main components. It was determined that there were 106,96±2,37 μg pyrogallol equivalents of phenolic compounds in 1 mg of aqueous extract. Mass spectrometry analysis showed that G. robertianum contained glucuronized sinapic acid, quercitrin, gallic acid, ellagic acid and cyanidin. The study of the in vitro metabolism of aqueous extracts of G. robertianum showed that there is no metabolization by the gastric and pancreatic juices, as well as by Caco-2 cells. Additionally, the digestion by an E. coli β-glucuronidase allowed the confirmation of the glucuronized sinapic acid presence. Furthermore, the biological activities of interest maintain their values during the entire metabolic pathway. The study of the hepatic metabolism showed that the polyphenols present do not under go glucuronidation and, once again, confirmed the presence of a glucuronide in the aqueous extract. Finally the antimicrobial activity of G. robertianum was studied. At a concentration of 12,99 mg/mL and 19,70 mg/mL of extract 50% of the growth of S. mutans and S. sobrinus was inhibited, respectively. Simultaneously the inhibition of the glucosyltransferase enzyme, isolated from the bacterial strains, was determined. At a concentration of 8,27 mg/mL (S. mutans) and over 10 mg/mL (S. sobrinus) 50% of enzyme activity was inhibited. We have concluded that the aqueous extract of G. robertianum has biological activities with interest for Alzheimer’s disease. Additional studies will be necessary to understand how the aqueous extract can reach the tissues and determine through which cellular and molecular mechanisms the aqueous extract can exert its biological effects

A importância da comunicação museológica : o caso do Museu da Guarda Nacional Republicana

Este Relatório de Estágio reflecte o trabalho de 6 meses de estágio no Museu da GNR, mais precisamente na área da Inventariação. Esta foi uma oportunidade valiosa de entrar no mundo dos museus e aprender na prática como é feita a gestão entre necessidades, recursos e objectivos. O Museu da GNR sendo recente, mas com muita história e património a conhecer, assumiu desde o início uma atitude atenda e cuidadosa face ao seu espólio e à forma de fazer chegar a sua mensagem ao visitante. Apostou numa museografia original e bastante informativa não esquecendo as traduções para pelo menos uma língua estrangeira. No âmbito da Inventariação procurou registar detalhadamente as peças de maior realce e, aceitando a minha presença, alastrou a sua base de dados. No decorrer do estágio pude integrar no programa Matriz cerca de 47 fichas de inventário, a maioria respeitante à primeira parte da exposição permanente. Foi neste trabalho de inventário que obtive as bases precisas para conseguir criar o produto essencial deste relatório – o Roteiro de exposição. Isto porque este estágio centra-se no sucesso que as fichas de inventário e uma linguagem acessível têm na comunicação do museu com o seu público. Será sublinhado várias vezes que depois de se inventariar as peças expostas surgem informações de excelente pertinência e interesse que o público gostará de saber. Se se recorrer a níveis de linguagem ligeira para passar o testemunho intelectual, o museu verá o seu público cativado e estimulado a querer saber mais.This Internship Report reflects the work of 6 months of internship in the GNR Museum, more precisely in the area of Inventory. This was a valuable opportunity to enter the world of museums and learn in practice how management is done between needs, resources and objectives. The GNR Museum, being recent, but with a lot of history and heritage to discover, has assumed since the beginning an attitude of attention and care towards its collection and the way to get its message to the visitor. It has invested in an original and very informative museology, not forgetting the translations into at least one foreign language. Within the scope of the Inventory, she sought to record in detail the most prominent pieces and, accepting my presence, spread the database. During the internship, I was able to add into the Matrix programme around 47 inventory sheets, most of which related to the first part of the permanent exhibition. It was in this inventory work that I obtained the precise basis to be able to create the essential product of this report - the Exhibition Roadmap. This is because this internship focuses on the success of the inventory sheets and accessible language in communicating with the museum's audience. It will be stressed several times that after inventorying the exhibits, there is information of excellent relevance and interest that the public will like to know. If a lighter language is used to convey intellectual testimony, the museum will see its audience captivated and encouraged to want to know more

Rho-kinase as a therapeutic target for nonalcoholic fatty liver diseases

Nonalcoholic fatty liver disease (NAFLD) is a major public health problem and the most common form of chronic liver disease, affecting 25% of the global population. Although NAFLD is closely linked with obesity, insulin resistance, and type 2 diabetes mellitus, knowledge on its pathogenesis remains incomplete. Emerging data have underscored the importance of Rho-kinase (Rho-associated coiled-coil-containing kinase [ROCK]) action in the maintenance of normal hepatic lipid homeostasis. In particular, pharmacological blockade of ROCK in hepatocytes or hepatic stellate cells prevents the progression of liver diseases such as NAFLD and fibrosis. Moreover, mice lacking hepatic ROCK1 are protected against obesity-induced fatty liver diseases by suppressing hepatic de novo lipogenesis. Here we review the roles of ROCK as an indispensable regulator of obesity-induced fatty liver disease and highlight the key cellular pathway governing hepatic lipid accumulation, with focus on de novo lipogenesis and its impact on therapeutic potential. Consequently, a comprehensive understanding of the metabolic milieu linking to liver dysfunction triggered by ROCK activation may help identify new targets for treating fatty liver diseases such as NAFLD.publishersversionpublishe

Novel and safe single-dose treatment of cutaneous leishmaniasis with implantable amphotericin B-Loaded microparticles

International audienceThe development of an effective amphotericin B (AmB) topical formulation to replace the systemically toxic injections currently used in cutaneous leishmaniasis (CL) treatment is challenging due to poor absorption through the skin. Aiming at an effective local chemotherapy, we designed PLGA (poly lactide-co-glycolide acid) microspheres loaded with deoxycholate amphotericin B (d-AmB) for both macrophage intracellular targeting and sustained extracellular release. For that, d-AmB/PLGA microspheres with sizes ranging from 0.5 μm to 20 μm were synthesized and tested both in vitro and in vivo. In vitro, d-AmB/PLGA was more selectively active against intracellular amastigotes of Leishmania amazonensis than free d-AmB (selectivity index = 50 and 25, respectively). In vivo, the efficacy of a single intralesional (i.l) injection with d-AmB/PLGA was determined in early and established BALB/c mouse ear lesions. In early lesions, a single injection given on day 10 of infection was more effective in controlling parasite growth than eight i.l. injections with free d-AmB, as measured on day 120. Such d-AmB/PLGA injection was also effective in established lesions (day 30), leading to 97% parasite burden reduction, as compared with d-AmB or liposomal AmB (Ambisome®) i.l. injection containing the same AmB dose. Pharmacokinetic studies showed that following d-AmB/PLGA injection, AmB leaked slower from non-infected than infected ears, yet remaining in the ear tissue for as long as 30 days. Of interest, AmB was not detectable in the circulating plasma for at least two weeks of d-AmB/PLGA injection, contrasting with the rapid and durable (2 days) detection after free d-AmB injection. Despite the transient ear swelling and local cell infiltration, no alterations in AST, ALT and creatinine serum levels was induced by d-AmB/PLGA. For its approved components, local efficacy, and single-dose applicability, this novel and safe AmB microsphere depot formulation has strong potential as a new therapy for human CL

Vivir con una ileostomía: un estudio de caso sobre el proceso de transición

Background: An ileostomy affects the person’s identity, leading to a process of transition. The person’s response to transition, as the subject of nursing study, should be explored, as well as the context in which the transition is experienced. Objective: To describe and understand the transition phenomenon experienced by a person with an ileostomy and diagnosed with familial adenomatous polyposis, based on Afaf Meleis’ Transition Model. Methodology: An instrumental case study was conducted using the qualitative research paradigm. The case was selected through a convenience sampling strategy and data were collected through observation, analysis of the electronic nursing records, and semi-structured interviews. Results: The person and the context involved in the transition process are two non-recurring realities that influence its course. Conclusion: Afaf Meleis’ Transition Model allows understanding the transition process experienced by the ileostomy patient in terms of nature, conditions, and response patterns of transitions.Enquadramento: A confeção de uma ileostomia constitui um evento capaz de afetar a identidade da pessoa, levando- -a a experimentar um processo de transição. As respostas humanas envolvidas nas transições, como objeto de estudo da enfermagem, necessitam de ser exploradas, na pessoa e no contexto de quem as vive. Objetivos: Descrever e compreender o fenómeno de transição vivido por uma pessoa com ileostomia com polipose adenomatosa familiar, a partir do modelo de transição de Afaf Meleis. Metodologia: Estudo de caso único, do tipo instrumental, conduzido sob o paradigma de investigação qualitativo. O caso foi selecionado por conveniência e a informação recolhida através da observação, da análise aos registos eletrónicos de enfermagem e de uma entrevista semiestruturada. Resultados: A pessoa e o contexto envolvidos numa transição são duas realidades que não se repetem e que influenciam o curso do processo de transição. Conclusão: O modelo de transição de Afaf Meleis permite compreender o processo de transição vivido pela pessoa com ileostomia quanto à natureza, condições e padrões de resposta envolvidos.Marco contextual: Una ileostomía puede afectar a la identidad de la persona y llevarla a experimentar un proceso de transición. Es necesario explorar las respuestas humanas involucradas en las transiciones, como objeto de estudio de la enfermería, en la persona y en el contexto de quienes las viven. Objetivos: Describir y comprender el fenómeno de transición vivido por una persona con ileostomía con poliposis adenomatosa familiar, a partir del modelo de transición de Afaf Meleis. Metodología: Estudio de caso único, de tipo instrumental, conducido de acuerdo con el paradigma de investigación cualitativo. El caso se seleccionó por conveniencia y la información se recogió a través de la observación, el análisis a los registros electrónicos de enfermería y una entrevista semiestructurada. Resultados: La persona y el contexto involucrados en una transición son dos realidades que no se repiten y que influyen en el transcurso del proceso de transición. Conclusión: El modelo de transición de Afaf Meleis permite comprender el proceso de transición vivido por la persona con ileostomía en cuanto a la naturaleza, las condiciones y los patrones de respuesta involucrados.info:eu-repo/semantics/publishedVersio

Improving the performance of a SME in the cutlery sector using lean thinking and digital transformation

The main purpose of this paper is to show that if three specific contextual factors are present in a company, it is possible to achieve great performance improvements with a lean and industry 4.0 implementation. In terms of research methods, a case study was carried out of a project to implement digitalization and Lean practices in a cutlery company, which in fact encompassed a project of master’s degree in engineering and industrial management. Thus, the research question is: “It is possible to achieve major improvements in a lean and industry 4.0 implementation if three specific contextual factors are present in the company, namely (i) commitment of top management, (ii) knowledge on digitalization and lean, and (iii) very low Value-Added Ratio?”. Regarding the company project, action-research was adopted, and the project team began by mapping and diagnosing the production processes of the two product families (knives and spoons/forks). High levels of work in process, long throughput times, poor flow planning and control, and high stocks of finished products, quickly stood out in both families. Improvement proposals were developed and implemented, namely: (i) creation of a production scheduling and control system, (ii) improvement of the warehouse stock management system, and (iii) adoption of new routines, management tools, visual management, and kaizen meetings. The results achieved were excellent (e.g., throughput time reduced by 27.6% and productivity increased by 36.5%) and aligned with Sustainable Development Goals SDG 9 and 12. The findings of this study corroborate that exceptional results in the company performance can be achieved through a lean and industry 4.0 intervention, if the three referred contextual factors occur.This work has been supported by FCT—Fundação para a Ciência e Tecnologia within the R&D Units Project Scope: UIDB/00319/2020

S-nitrosoglutathione reverts dietary sucrose-induced insulin resistance

The liver is a fundamental organ to ensure whole-body homeostasis, allowing for a proper increase in insulin sensitivity from the fast to the postprandial status. Hepatic regulation of glucose metabolism is crucial and has been shown to be modulated by glutathione (GSH) and nitric oxide (NO). However, knowledge of the metabolic action of GSH and NO in glucose homeostasis remains incomplete. The current study was designed to test the hypothesis that treatment with S-nitrosoglutathione is sufficient to revert insulin resistance induced by a high-sucrose diet. Male Wistar rats were divided in a control or high-sucrose group. Insulin sensitivity was determined: (i) in the fast state; (ii) after a standardized test meal; (iii) after GSH + NO; and after (iv) S-nitrosoglutathione (GSNO) administration. The fasting glucose level was not different between the control and high-sucrose group. In the liver, the high-sucrose model shows increased NO and unchanged GSH levels. In control animals, insulin sensitivity increased after a meal or administration of GSH+NO/GSNO, but this was abrogated by sucrose feeding. GSNO was able to revert insulin resistance induced by sucrose feeding, in a dose-dependent manner, suggesting that they have an insulin-sensitizing effect in vivo. These effects are associated with an increased insulin receptor and Akt phosphorylation in muscle cells. Our findings demonstrate that GSNO promotes insulin sensitivity in a sucrose-induced insulin-resistant animal model and further implicates that this antioxidant molecule may act as a potential pharmacological tool for the treatment of insulin resistance in obesity and type 2 diabetes.publishersversionpublishe

A review of capture-recapture methods and its possibilities in ophthalmology and vision sciences

Epidemiological information is expected to be used to develop key aspects of eye care such as to control and minimise the impact of diseases, to allocate resources, to monitor public health actions, to determine the best treatment options and to forecast the consequence of diseases in populations. Epidemiological studies are expected to provide information about the prevalence and/or incidence of eye diseases or conditions. To determine prevalence is necessary to perform a cross-sectional screening of the population at risk to ascertain the number of cases.The aim of this review is to describe and evaluate capture-recapture methods (or models) to ascertaining the number of individuals with a disease (e.g. diabetic retinopathy) or condition (e.g. vision impairment) in the population.The review covers the fundamental aspects of capture-recapture methods that would enable non-experts in epidemiology to use it in ophthalmic studies. The review provides information about theoretical aspects of the method with examples of studies in ophthalmology in which it has been used. We also provide a problem/solution approach for limitations arising from the lists obtained from registers or other reliable sources.We concluded that capture-recapture models can be considered reliable to estimate the total number of cases with eye conditions using incomplete information from registers. Accordingly, the method may be used to maintain updated epidemiological information about eye conditions helping to tackle the lack of surveillance information in many regions of the globe.- This study was supported by FCT (COMPETE/QREN) grant reference [PTDC/DPT-EPI/0412/2012] in the context of the Prevalence and Costs of Visual Impairment in Portugal: a hospital-based study (PCVIP-study) and FCT Strategic Funding UID/FIS/04650/2013. PLR is funded by FCT (COMPETE/QREN) grant reference [SFRH/BD/119420/2016]

Experimental characterization of a microfluidic device based on passive crossflow filters for blood fractionation

The separation of red blood cells (RBCs) from blood plasma and the analysis of individual RBCs are of great importance, as they provide valuable information regarding the health of their donor. Recent developments in microfluidics and microfabrication have contributed to the fabrication of microsystems with complex features to promote the separation and analysis of RBCs. In this work, the separation capacity of a multi-step crossflow microfluidic device was evaluated by using a blood analogue fluid made by Brij L4 micelles and human RBCs separated from whole blood, suspended in a solution with hematocrits (Ht) of 0.5 and 1%. All the samples collected at the outlets of the device were experimentally analyzed and compared. The absorbance spectrum was also measured for the prepared blood samples. The results indicate that the tested blood analogue fluid has exhibited a flow behavior similar to that of blood. In addition, the optical absorbance spectrophotometry revealed that it was possible to evaluate the separation efficiency of the microfluidic device, concluding that the concentration of cells was lower at the most lateral outside outlets of the microchannel due to the cumulative effect of the multiple cross-flow filters

An integrated view

Funding Information: This work was supported by “Fundação para a Ciência e a Tecnologia”—FCT MJM (PD/BD/114256/2016), MPM (PTDC/BIM-MET/4265/2014 and PTDC/MEC-MET/29314/2017), MGA (PTDC/BIM-MET/4712/2014), iNOVA4Health (UIDB/Multi/04462/2020), by the European Commission Marie Skłodowska-Curie Action H2020 (mtFOIE GRAS, grant agreement n. 734719), by the Sociedade Portuguesa de Diabetologia, and by the research infrastructure CONGENTO, project LISBOA-01-0145-FEDER-022170, co-financed by Lisboa Regional Operational Programme (Lisboa2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund and by Fundação para a Ciência e Tecnologia (Portugal). NMR data were collected at the UC-NMR facility which is supported in part by FEDER – European Regional Development Fund through the COMPETE Programme (Operational Programme for Competitiveness) and by National Funds through FCT – Fundação para a Ciência e a Tecnologia (Portuguese Foundation for Science and Technology) through grants REEQ/481/QUI/2006, RECI/QEQ-QFI/0168/2012, CENTRO-07-CT62-FEDER-002012, and Rede Nacional de Ressonância Magnética Nuclear (RNRMN). Publisher Copyright: Copyright © 2022 Meneses, Sousa-Lima, Jarak, Raposo, Alves and Macedo.Objective: In the last years, changes in dietary habits have contributed to the increasing prevalence of metabolic disorders, such as non-alcoholic fatty liver disease (NAFLD) and type 2 diabetes mellitus (T2DM). The differential burden of lipids and fructose on distinct organs needs to be unveiled. Herein, we hypothesized that high-fat and high-fructose diets differentially affect the metabolome of insulin-sensitive organs such as the liver, muscle, and different adipose tissue depots. Methods: We have studied the impact of 12 weeks of a control (11.50% calories from fat, 26.93% from protein, and 61.57% from carbohydrates), high-fat/sucrose (HFat), or high-fructose (HFruct) feeding on C57Bl/6J male mice. Besides glucose homeostasis, we analyzed the hepatic levels of glucose and lipid-metabolism-related genes and the metabolome of the liver, the muscle, and white (WAT) and brown adipose tissue (BAT) depots. Results: HFat diet led to a more profound impact on hepatic glucose and lipid metabolism than HFruct, with mice presenting glucose intolerance, increased saturated fatty acids, and no glycogen pool, yet both HFat and HFruct presented hepatic insulin resistance. HFat diet promoted a decrease in glucose and lactate pools in the muscle and an increase in glutamate levels. While HFat had alterations in BAT metabolites that indicate increased thermogenesis, HFruct led to an increase in betaine, a protective metabolite against fructose-induced inflammation. Conclusions: Our data illustrate that HFat and HFruct have a negative but distinct impact on the metabolome of the liver, muscle, WAT, and BAT.publishersversionpublishe