C-glycoside synthesis towards innovation in antimicrobials

Tese de mestrado, Química (Química, Saúde e Nutrição) Universidade de Lisboa, Faculdade de Ciências, 2020Bacillus anthracis, a spore forming Gram (+) bacterium, is considered one of the most lethal biological weapons due to its highly pathogenic nature, easy production, preservation, and to the possibility of being aerosolized and sprayed. The fact that antibiotics are ineffective against the spores and the released toxins, makes it a serious threat 1,2.In addition, development of antimicrobial resistance has become a worldwide problem, as the impact of antibiotics is diminishing due to progressive rise of resistance to the available antimicrobial drugs 3. It was previously described the importance of the bacteria membrane’s integrity in its life - the cell envelope cannot change without significant consequences to the bacteria life. Also, the development of resistance becomes extremely difficult 4. For this reason, the essentiality of cell membrane makes it a promising target for new antibiotics. In order to target B. anthracis membrane, our research group has investigated the use of carbohydrate-based surfactants as potential antibiotics since these amphiphilic compounds are known to be able to interact with the cell membrane lipid bilayer 5. Hence, a library of alkyl glycosides was synthesized varying deoxygenation pattern, alkyl chain, sugar stereochemistry, anomeric configuration and atom linking the alkyl chain to the saccharide moiety 6–10. The compound that showed the best results was an O-glycoside, the dodecyl 4,6-dideoxy-α-D-xylo-hexopyranoside, which was active against B. cereus, E. faecalis and B. anthracis (strains: pathogenic, sterne and ovine) with MIC = 12.6 μM. Comparing to chloramphenicol, the control used in the biological assays, the MIC value revealed to be half of the MIC showed by the control (MIC = 25 μM). Also C-glycosides were found less toxic compounds on Caco-2 cells than the corresponding O- and S-glycosides 9. The aim of the presenting dissertation was to explore the first synthetic route towards dodecyl 4,6-dideoxy-α-D-xylo-hexopyranoside, the C-glycoside analogue to the O-glycoside previously described. Three synthetic pathways were investigated, using either the naturally occurring D-glucose or its methyl glucoside as starting materials. When starting from D-glucose, it was regioselectively protected with a 4,6-O-benzylidene group while benzyl groups were protecting positions 1, 2 and 3. Since the reaction outcome of the acetal protecting group with N-iodosuccinimide was not successful possibly due to the reactivity of the benzyl protecting groups, other attempts were tried and the desired compound – the C-glycoside with the 4,6-dideoxy pattern (19) was obtained in 13% overall yield. Cytotoxicity tests are to be performed and biological assays are on-going in order to determine this C-glycoside efficacy towards B. anthracis.Bacillus anthracis, uma bactéria Gram (+) existente na forma de esporos, é considerada uma das armas biológicas de maior carácter letal devido à sua natureza altamente patogénica, à facilidade da sua produção e preservação bem como a possibilidade de os esporos serem pulverizados. O facto de os antibióticos não serem efetivos quando usados contra os esporos ou contra as toxinas libertadas pela bactéria, torna-a um problema sério 1,2. Para além disso, o desenvolvimento de resistência a antibacterianos tornou-se um problema grave à escala mundial, com o impacto da evolução dos antibióticos a diminuir face ao aumento progressivo da resistência dos patógenos às terapêuticas disponíveis 3. A importância da integridade da membrana bacteriana foi descrita anteriormente, onde se afirmou que o envelope celular não muda sem gerar consequências significativas na vida da bactéria. Ainda, o desenvolvimento de mecanismos de resistência torna-se extremamente difícil. Por esta razão, a essencialidade da membrana celular torna-a um alvo promissor para novos antibióticos 4. Tendo a membrana da bactéria B. anthracis como alvo a atingir, o nosso grupo de investigação está focado no desenvolvimento de carboidratos surfactantes como potenciais antibióticos, uma vez que se sabe que este tipo de moléculas anfifílicas interage com a membrana lipídica das células 5. Deste modo, foi sintetizada uma pequena biblioteca de C-glicósidos de alquilo com padrão de desoxigenação variável, diferentes cadeias alquílicas, estereoquímica do monossacárido, configuração anomérica e átomo que liga a cadeia alquílica ao glicósido 6–10. O composto que demonstrou melhores resultados foi um O-glicósido, 4,6-didesoxi-α-D-xylo-hexopiranósido de dodecilo, que revelou ser ativo contra as bactérias B. cereus, E. faecalis e B. anthracis (estirpes: patogénica, sterne e ovino) com MIC = 12.6 μM. Comparando com os resultados demonstrados aquando do uso de cloranfenicol, controlo usado nos testes biológicos, o valor de MIC do O-glicósido revelou ser cerca de metade do valor de MIC do controlo (MIC controlo = 25 μM). Ainda, os C-glicósidos sintetizados demonstraram ser menos tóxicos para a linha celular Caco-2 do que os correspondentes O- e S-glicósidos 9. O objetivo da dissertação apresentada foi o da exploração da primeira via sintética do composto 4,6-didesoxi-α-D-xylo-hexopiranósido de dodecilo, o C-glicósido análogo ao O-glicósido previamente descrito. Foram investigados três esquemas sintéticos, usando a D-glucose ou o seu análogo metilado como produtos de partida. O esquema sintético A partiu da D-glucose, cuja proteção dos grupos -OH livres foi conseguida com a introdução regiosseletiva do grupo 4,6-O-benzilideno e de grupos benzilo nas posições 1, 2 e 3. Uma vez que a redução do grupo benzilideno com N-iodosuccinimida não foi conseguida com sucesso, possivelmente devido à reatividade dos grupos benzilo, o esquema sintético B foi adotado. Após várias reações de proteção/desproteção dos hidroxilos da glícona, C-glicosilação da posição anomérica e elongação da cadeia alquílica, o derivado de glicósido desejado – o C-glicósido com o padrão de desoxigenação 4,6-didesoxi (19) foi obtido com um rendimento total de 13%. Os resultados dos testes de citotoxicidade são esperados de modo a confirmar a hipótese, inicialmente proposta, de que o C-glicósido é menos tóxico que o seu análogo O-glicósido. Além disso, os testes biológicos estão em curso para que seja determinada a eficácia deste C-glicósido contra a bactéria B. anthracis

The coastal zone management plan of Santa Maria as a chance for fossiliferous outcrops management

The Azores Environment and Sea Agency through its Regional Department of Land Management and Water Resources, together with the Agency of Land Management of the Autonomous Government of The Canary Islands and the Regional Cabinet of Transport and Social Equipment of the Autonomous Government of Madeira carry on the project “Sustainable Management of the Social, Economic and Ecological Development of the coastal areas of Macaronesia within the community initiative INTERREG III B 2000-2006,Açores-Madeira-Canary Islands, which has been designated as LITOSOST. The LITOSOST project aims at achieving land management of a coastal area which focuses on reducing the urban and infra-structural pressure and regenerating, recovering and converting it for public fruition (UNESCO, 1997; Mota et al.,2004; DROTRH/SRAM, 2006; MAOTDR, 2006). The general goal of the project consists on stimulating practices to the sustainable management of the coastal areas of the Azores, Madeira and Canary Islands. As specific objectives, the following stand out: to strengthen the cooperation between the Macaronesia authorities; to identify the problems and the common potential of the coastal areas of the islands; to train human resources: Management and Preservation of Resources; to contribute for the improvement of the state of the coastal ecosystems of the EU islands

An ancestral HIV-2/simian immunodeficiency virus peptide with potent HIV-1 and HIV-2 fusion inhibitor activity

"Objectives: To produce new fusion inhibitor peptides for HIV-1 and HIV-2 based on ancestral envelope sequences. Methods: HIV-2/simian immunodeficiency virus (SIV) ancestral transmembrane protein sequences were reconstructed and ancestral peptides were derived from the helical region 2 (HR2). The activity of one ancestral peptide (named P3) was examined against a panel of HIV-1 and HIV-2 primary isolates in TZM-bl cells and peripheral blood mononuclear cells and compared to T-20. Peptide secondary structure was analyzed by circular dichroism. Resistant viruses were selected and resistance mutations were identified by sequencing the env gene. Results: P3 has 34 residues and overlaps the N-terminal pocket-binding region and heptad repeat core of HR2. In contrast to T-20, P3 forms a typical a-helical structure in solution, binds strongly to the transmembrane protein, and potently inhibits both HIV-2 (mean IC50, 63.8 nmol/l) and HIV-1 (11 nmol/l) infection, including T-20-resistant isolates. The N43K mutation in the HR1 region of HIV-1 leads to 120-fold resistance to P3 indicating that the HR1 region in transmembrane glycoprotein is the target of P3. No HIV-2-resistant mutations could be selected by P3 suggesting that the genetic barrier to resistance is higher in HIV-2 than in HIV-1. HIV-1-infected patients presented significantly lower P3-specific antibody reactivity compared to T-20. Conclusion: P3 is an HIV-2/SIV ancestral peptide with low antigenicity, high stability, and potent activity against both HIV-1, including variants resistant to T-20, and HIV-2. Similar evolutionary biology strategies should be explored to enhance the production of antiviral peptide drugs, microbicides, and vaccines.

Marine endophytic fungi associated with Halopteris scoparia (Linnaeus) Sauvageau as producers of bioactive secondary metabolites with potential dermocosmetic application

Marine fungi and, particularly, endophytic species have been recognised as one of the most prolific sources of structurally new and diverse bioactive secondary metabolites with multiple biotechnological applications. Despite the increasing number of bioprospecting studies, very few have already evaluated the cosmeceutical potential of marine fungal compounds. Thus, this study focused on a frequent seaweed in the Portuguese coast, Halopteris scoparia, to identify the endophytic marine fungi associated with this host, and assess their ability to biosynthesise secondary metabolites with antioxidative, enzymatic inhibitory (hyaluronidase, collagenase, elastase and tyrosinase), anti-inflammatory, photoprotective, and antimicrobial (Cutibacterium acnes, Staphylococcus epidermidis and Malassezia furfur) activities. The results revealed eight fungal taxa included in the Ascomycota, and in the most representative taxonomic classes in marine ecosystems (Eurotiomycetes, Sordariomycetes and Dothideomycetes). These fungi were reported for the first time in Portugal and in association with H. scoparia, as far as it is known. The screening analyses showed that most of these endophytic fungi were producers of compounds with relevant biological activities, though those biosynthesised by Penicillium sect. Exilicaulis and Aspergillus chevalieri proved to be the most promising ones for being further exploited by dermocosmetic industry. The chemical analysis of the crude extract from an isolate of A. chevalieri revealed the presence of two bioactive compounds, echinulin and neoechinulin A, which might explain the high antioxidant and UV photoprotective capacities exhibited by the extract. These noteworthy results emphasised the importance of screening the secondary metabolites produced by these marine endophytic fungal strains for other potential bioactivities, and the relevance of investing more efforts in understanding the ecology of halo/osmotolerant fungi.info:eu-repo/semantics/publishedVersio

Relação entre contagem de leucócitos, adiposidade e aptidão cardiorrespiratória em adolescentes púberes

Objective To compare the total and differential leukocyte count in obese and normal-weight adolescents, and to verify their possible relations with cardiorespiratory fitness and adiposity indicators. Methods A cross-sectional study conducted with 139 adolescents (107 obese and 32 normal weight) aged between 13 and 18 years. Cardiorespiratory fitness was determined by direct gas analysis during an incremental treadmill test. Total leukocytes and subsets were estimated by flow cytometry. Body composition was assessed by dual-energy X-ray absorptiometry. The t-test for independent samples was used for comparison between groups. The relation between leukocytes, cardiorespiratory fitness and adiposity indicators was verified by Pearson’s correlation and multiple linear regression (adjusted for age and body mass index) tests. Results Obese adolescents had higher leukocyte (8.12±2.36u/L x 103; p=0.001), neutrophil (4.33±1.86u/L x 103; p=0.002), and monocyte (0.70±0.22u/L x 103; p=0.002) counts compared to the levels of normal weight subjects. After the necessary adjustments, cardiorespiratory fitness had a negative association with leukocytes, neutrophils, and monocytes in boys. Conclusion Obese adolescents had higher total and differential leucocyte count when compared to normal weight individuals. We also observed a weak positive association between adiposity and total leukocyte, monocyte, and neutrophil counts, and in boys, a negative association between cardiorespiratory fitness and total count of leukocytes, monocytes, and neutrophils.Objetivo Comparar a contagem total e diferencial de leucócitos de adolescentes obesos e eutróficos, e verificar suas possíveis relações com a aptidão cardiorrespiratória e indicadores de adiposidade. Métodos Estudo transversal realizado com 139 adolescentes (107 obesos e 32 eutróficos), com idades entre 13 e 18 anos. A aptidão cardiorrespiratória foi determinada por análise direta de gases durante um teste incremental em esteira rolante. As contagens de leucócitos totais e subconjuntos foram estimadas por citometria de fluxo. A composição corporal foi avaliada pelo método de dupla energia de raios X. O teste t para amostras independentes foi utilizado para comparação entre os grupos. A relação entre leucócitos, aptidão cardiorrespiratória e indicadores de adiposidade foi verificada por meio dos testes de correlação de Pearson e regressão linear múltipla (ajustado para idade e índice de massa corporal). Resultados Os adolescentes obesos apresentaram leucócitos (8,12±2,36u/L x 103; p=0,001), neutrófilos (4,33±1,86u/L x 103; p=0,002) e monócitos (0,70±0,22u/L x 103; p=0,002) mais elevados em comparação com os níveis dos eutróficos. Após os ajustes necessários, a aptidão cardiorrespiratória foi negativamente associada com leucócitos, neutrófilos e monócitos em meninos. Conclusão Adolescentes obesos apresentaram maior contagem de leucócitos totais e subpopulações, quando comparados aos eutróficos. Observou-se também uma fraca relação positiva entre adiposidade e leucócitos totais, monócitos e neutrófilos, e em meninos, uma relação negativa entre aptidão cardiorrespiratória e leucócitos totais, monócitos e neutrófilos.Universidade de PernambucoUniversidade Federal de São Paulo (UNIFESP)UNIFESPSciEL

Effect of empagliflozin beyond glycemic control: cardiovascular benefit in patients with type 2 diabetes and established cardiovascular disease

A prevalência da diabetes mellitus tipo 2 (DMT2) continua a aumentar e a sua associação com a doença cardiovascular (CV) tem levado à incorporação e valorização de endpoints CV nos ensaios clínicos sobre o tratamento da DMT2. Este artigo faz uma revisão dos vários ensaios já realizados e em desenvolvimento, neste âmbito, com especial enfoque no estudo EMPA‐REG OUTCOME. Neste estudo, a empagliflozina, um inibidor do cotransportador tipo 2 de sódio/glicose (iSGLT2), demonstrou efeitos benéficos na redução do risco CV em doentes com DMT2 e doença CV estabelecida, para além de segurança CV e diminuição dos valores de HbA1c, representando uma mudança de paradigma com impacto ao nível das recomendações internacionais para o tratamento da DMT2. Estes resultados mantiveram‐se em análises de subgrupos posteriores, nomeadamente na insuficiência cardíaca, doença renal crónica e doença arterial periférica, ainda que muitas questões se coloquem sobre os mecanismos envolvidos nestes efeitos – se efeitos hemodinâmicos, efeitos metabólicos ou se a diminuição das concentrações de sódio citoplasmático no miocárdio. Com a diminuição do risco de eventos CV major em doentes com DMT2, os resultados do estudo EMPA‐REG OUTCOME demonstraram, pela primeira vez, proteção CV associada ao efeito de um fármaco anti‐hiperglicémico e iniciaram uma nova era no tratamento e gestão da DMT2. Este estudo levou ao desenvolvimento de outros ensaios, ainda a decorrer, que permitirão estabelecer quais os doentes que mais beneficiarão desta terapêutica, nomeadamente na relação à existência de comorbilidades.ABSTRACT - The prevalence of type 2 diabetes (T2D) continues to increase, and its association with cardiovascular (CV) disease has led to the inclusion of CV endpoints in clinical trials on the treatment of T2D. This article explores the various trials already performed and underdevelopment in this field, with particular focus on the EMPA‐REG OUTCOME trial. In this trial, empagliflozin, a sodium‐glucose co‐transporter 2 inhibitor, demonstrated a reduction in CV risk in patients with T2D and established CV disease, in addition to CV safety and a decrease in glycated hemoglobin. This represents a paradigm shift that has led to changes in the international guidelines for the treatment of T2D. These results were maintained in subsequent subgroup analysis for heart failure, chronic kidney disease, and peripheral arterial disease, although there are many questions concerning the mechanisms involved in these effects, including whether they are hemodynamic, metabolic or due to decreased myocardial cytoplasmic sodium concentrations. With this reduction in risk for major CV events in patients with T2D, the EMPA‐REG OUTCOME trial demonstrated CV protection from a hypoglycemic drug for the first time and opened a new era in the treatment and management of T2D. This study has led to the development of ongoing trials that will establish which patients will benefit most from this therapy, particularly with regard to comorbidities.info:eu-repo/semantics/publishedVersio

Cysteine as a Multifaceted Player in Kidney, the Cysteine-Related Thiolome and Its Implications for Precision Medicine

Funding Information: This research was supported by Fundação para a Ciência e Tecnologia (PTDC/MED-TOX/30418/2017) and iNOVA4Health (UID/Multi/04462/2013). M.J.C., D.G.F.F. and J.M. were supported by FCT (PhD grant SFRH/BD/131331/2017, PhD grant PD/BD/135484/2018 and postdoctoral contract PTDC/MED-TOX/30418/2017, respectively).In this review encouraged by original data, we first provided in vivo evidence that the kidney, comparative to the liver or brain, is an organ particularly rich in cysteine. In the kidney, the total availability of cysteine was higher in cortex tissue than in the medulla and distributed in free reduced, free oxidized and protein-bound fractions (in descending order). Next, we provided a comprehensive integrated review on the evidence that supports the reliance on cysteine of the kidney beyond cysteine antioxidant properties, highlighting the relevance of cysteine and its renal metabolism in the control of cysteine excess in the body as a pivotal source of metabolites to kidney biomass and bioenergetics and a promoter of adaptive responses to stressors. This view might translate into novel perspectives on the mechanisms of kidney function and blood pressure regulation and on clinical implications of the cysteine-related thiolome as a tool in precision medicine.publishersversionpublishe

Construindo projectos: vivências no Jardim-de-infância

A presente comunicação tem por objectivo apresentar a vivência por projectos realizada durante um ano lectivo em contexto de Jardim-de-infância. O trabalho foi desenvolvido pelos educadores de infância de cinco salas dum Jardim-de-infância da rede pública, em colaboração com os educadores-estagiários e a professora de prática pedagógica que os acompanhava. Apesar de toda a equipa estar consciente do trabalho que envolvia a metodologia de projecto observam-se algumas necessidades, na estruturação da acção educativa decorrentes da emergência dos projectos. Assim, um dos principais desafios encontrado foi o de encetar diálogo com algumas gramáticas pedagógicas sustentadoras de modos de acção centrados na identificação de problemas, onde a criança fosse um elemento activo, no levantamento das questões de pesquisa, planificação da acção a desenvolver e na avaliação dos percursos efectuado