Alterações no perfil lipídico de células e exossomas no enfarte agudo do miocárdio

Mestrado em Bioquímica - Bioquímica ClínicaCardiovascular Diseases are the most significant cause of death. Myocardium infarction is one of the most common of this type of diseases and it is characterized by myocardium ischemia. Ischemia occurs in consequence of simultaneous starvation and hypoxia. While ischemia represents a cellular damage, starvation is associated with a cardioprotective effect. The cell response to this injury includes either autophagy or apoptosis depending on the ability to adapt and respond to the injury and is very important for the evolution and recovery of the myocardium infarction. Autophagy is a selfdegradative process that allows cell to adapt to stress and so it is associated with cell survival. The exosomes release by cardiomyocytes is also an adaptive process which functions are related with intercellular communication. On the other hand, apoptosis is a process of programmed cell death. It is well known that lipids play an important role in cardiovascular disease although their role is not completely understood. Lipids are the major component of a cell membrane and play structural and signaling roles. Under several physiopathological conditions, the cell and exosomes lipid content can be modified. However reports on lipidome of cardiomyocytes under cardiovascular diseases are scarce. Thus, the primary aim of this work is to identify lipid profile changes in cardiomyocytes and exosomes released by them under starvation and ischemia, in order to better understand myocardial infarction and if possible to recognize new biomarkers for myocardial infarction. Cardiac cells showed that molecular species alterations in phosphatidylcholine (PC34:1 and PC36:2), phosphatidylethanolamine (PE34:1), phosphatidylserine (PS36:1), phosphatidylinositol (PI36:2, PI38:3 and PI38:5) and sphingomyelin (SM34:1) were changed in ischemia and in starvation in comparison with control group. Some differences were specific of starvation as the decrease in SM(34:1) and the increase in PS(36:1) while apoptosis, autophagy, biomarkers, cardiomyocytes, cardiovascular diseases, exosomes, ischemia, lipidomics, mass spectrometry, myocardial infarction, phospholipids, starvation others were specific of ischemia as the decrease in PC(36:2) and LPC(16:0). The molecular specie PC(34:1) showed different alterations in each condition increasing in case of ischemia and decreasing in case of starvation. For exosomes, our results showed a deviation between the lipidome of exosomes released upon ischemia and starvation for all lipid classes. Some differences matched the ones observed in cells, for example the decrease in PC(34:1) in starvation, but others were different. Since we have only performed lipidomic analysis for a smaller sample of exosomes, it requires further studies to validate the results. In conclusion, ischemia and starvation induced changes in lipid homeostasis. Our work suggests that lipids are potential tools for evaluation of cell fate, either cell death or recovery, that will be useful to improve diagnosis and prognostic of cardiovascular diseases.As doenças cardiovasculares são a principal causa de morte em todo o mundo. De entre estas, o enfarte do miocárdio é uma das doenças mais comuns, sendo caracterizado por isquemia que leva a morte de células cardíacas. A isquemia ocorre em consequência da privação simultânea de nutrientes e oxigénio. Enquanto a isquemia representa um dano celular, a privação de nutrientes está relacionada com efeitos cardioprotetores. A resposta das células a estes estímulos pode ser por indução de autofagia ou de apoptose, dependendo da sua capacidade de adaptação e resposta aos fatores indutores de isquemia. A autofagia é um processo auto-degradativo que permite à célula adaptar-se ao stresse e é, portanto, um processo associado à sobrevivência celular. A libertação de exossomas pelas células é também um mecanismo de adaptação cujas funções estão relacionadas com a comunicação intercelular. Por outro lado, a apoptose é um processo de morte celular programada. A regulação destes processos é de extrema importância para a sobrevivência e recuperação nos episódios de enfarte do miocárdio. Hoje em dia sabe-se que os lípidos têm um papel importante no desenvolvimento de doenças cardiovasculares embora o seu papel ainda não esteja completamente esclarecido. Os lípidos são os componentes maioritários da membrana celular e desempenham funções a nível estrutural e de sinalização. Quando exposto a diversas condições fisiopatológicas, o conteúdo lipídico das células e dos exossomas é modificado. No entanto, existem ainda poucas publicações sobre a avaliação do lipidoma de cardiomiócitos em patologias cardiovasculares. Assim, o objetivo principal deste trabalho é identificar alterações no perfil lipídico de cardiomiócitos e exossomas libertados por estes sob privação de nutrientes e de oxigénio, de forma a melhor compreender o enfarte do miocárdio e se possível identificar novos biomarcadores para esta patologia. apoptose, autofagia, biomarcadores, cardiomiócitos, doenças cardiovasculares, espectrometria de massa, exossomas, fosfolípidos, infarte do miocárdio, isquémia, lipidómica Nas células cardíacas verificamos que algumas espécies moleculares de fosfatidilcolina (PC34:1 e PC36:2), fosfatidiletanolamina (PE34:1), fosfatidilserina (PS36:1), fosfatidilinositol (PI36:2, PI38:3 e PI38:5) e esfingomielina (SM34:1) variam em isquemia e em privação de nutrientes em comparação com o controlo. Algumas variações foram específicas da privação de nutrientes como a diminuição de SM(34:1) e o aumento de PS(36:1) e outras foram específicas da isquemia como a diminuição de PC(36:2) e de LPC(16:0). A espécie molecular PC(34:1) foi a que se mostrou alterada de forma diferente em cada condição sendo que aumenta em caso de isquemia e diminui em caso da privação de nutrientes. No caso dos exossomas, os resultados obtidos permitiram verificar que houve um maior desvio entre o lipidoma de exossomas libertado em isquemia e privação de nutrientes em todas as classes de lípidos. Algumas alterações foram coincidentes com as observadas para as células, por exemplo a diminuição PC(34:1) em starvation mas outras foram diferentes. Uma vez que apenas se realizaram análises lipdómicas para uma dimensão reduzida de amostra de exossomas, serão necessários estudos futuros para a validação dos resultados obtidos. Em conclusão, a privação de nutrientes e a isquemia induzem alterações na homeostasia dos lípidos. Este trabalho sugere que os lípidos são potenciais ferramentas para avaliar se os cardiomiócitos estão a optar pela morte celular ou pela recuperação, que serão úteis para melhorar o diagnóstico e prognóstico de doenças cardiovasculares

An investigation into protein modification by short-chain lipid oxidation products:development of mass spectrometry-based approaches for their detection and assessment of the effect on cellular proteins structure and activity

The oxidative modification of lipids containing polyunsaturated fatty acids results in a wide diversity of reactive products, including short-chain aldehydes, which can covalently modify proteins, a process called lipoxidation, and affect their function. The detection of lipoxidation adducts is extremely challenging due to their low abundance, so there is a need for new detection methods. Using the model proteins lysozyme and human serum albumin, five reporter ions for acrolein modification, eight for 4-hydroxyhexenal and one for 2-chlorohexadecanal were found by LC-MS/MS-based label-free method. Subsequently, a targeted multiple reaction monitoring method was developed as a potential tool for the identification and characterization of these modifications of human serum albumin in biological samples. Comparison between three different chromatographic methods for the separation of intact proteins unmodified or modified by short-chain aldehydes demonstrated that anion exchange chromatography was the best method for protein isoform separation while reverse phase chromatography was the best for the separation of lipoxidized proteins. To understand the cellular effects of lipoxidation by small aldehydes, two cellular proteins were studied. The Cys328-mediated effect of short-chain aldehydes on vimentin organization was evaluated by confocal microscopy, which showed that aldehydes caused vimentin aggregation around the nucleus, and Cys328 was important in both assembly of the filament network and as a target for lipoxidation. The effect of short-chain aldehydes on the glycolytic enzyme pyruvate kinase was also studied, and this protein was found to be highly susceptible to modification; under pathophysiological concentrations acrolein, malondialdehyde and 4-hydroxyhexenal were able to inhibit its activity in a time- and dose-dependent manner. Similar inhibition was observed in breast cancer cells, altering cellular metabolism and suggesting that these aldehydes may contribute to mechanisms of tumorigenesis. Overall, this work has improved the analytical tools for detecting protein lipoxidation, as well as understanding of its functional effects

A atitude dos moradores do Bairro do Sambizanga face à reconversão urbana: relação com a vinculação ao lugar e o bem-estar subjectivo

O presente trabalho pretende analisar a atitude dos moradores do bairro do Sambizanga face à reconversão urbana, à sua relação com a vinculação ao lugar; e o respectivo bem-estar subjectivo das populações deste bairro. Os estudos sobre vinculação ao lugar permitem uma associação positiva de afectos entre indivíduos e os seus ambientes residenciais (Shumaker& Taylor, 1983), ou seja, uma associação que cria sentimentos de conforto e segurança (Rivlin, 1982). Este estudo contou com a participação de 68 moradores residentes no bairro do Sambizanga, especificamente, os que foram abrangidos na primeira fase de reconversão. Deste modo, foi utilizada uma metodologia quantitativa que permitiu analisar as escalas de vinculação ao lugar de Hernández, et al. (2007), os indicadores do bem-estar subjectivo de Diener (2000), e de satisfação residencial de Amérigo (1995). Os resultados obtidos indicaram uma relação de significância negativa entre a atitude face à reconversão urbana e a vinculação ao lugar. A casa e o bairro foram objectos de menor apego por parte dos indivíduos; estes apresentaram-se pouco satisfeitos com as suas residências. Os indivíduos que se percepcionaram mais saudáveis são os que estão mais satisfeitos com a residência e mais vinculados com a casa e não com o bairro. E os com menor percepção de saúde são os que apresentam pior atitude face à reconversão urbana e ao futuro do bairro. A presente pesquisa contribuiu para alargar a investigação inexistente sobre os conceitos relacionados no estudo, demonstrando a importância dos mesmos na vida das populações em situações de mudança residencial.The present study aims to examine the attitude of the residents of the neighborhood of Sambizanga regarding the urban redevelopment, their relationship with the attachment to the place, and their subjective well-being. Studies about attachment to a place, allow a positive association between individuals and their residential environments (Shumaker & Taylor, 1983), that is, an association that creates feelings of comfort and safety (Rivlin, 1982). This study involved the participation of 68 residents living in the neighborhood of Sambizanga, specifically, those that were covered in the first phase of redevelopment. Thus, we used a quantitative methodology that allowed us to analyze the scales of place attachment (Hernandez, et al, 2007), indicators of subjective well-being of Diener (2000), and residential satisfaction by Amérigo (1995). The results indicated significant relationship between negative attitude towards urban regeneration and attachment to place. The house and neighborhood were objects of lower attachment by the individuals; they present themselves unsatisfied with their homes. Those individuals who perceived healthier are the most satisfied with their home and more connected with the house and not with the neighborhood. On the other hand, those with a lower perception of health are those who present a worse attitude towards urban redevelopment and the future of the neighborhood. This research helped to extend the inexistent research on the concepts related in this study, demonstrating their importance in the lives of people in situations of residential change

Novos complexos micelares de metais de transição para aplicação na terapia dirigida contra o cancro

O cancro é a segunda causa de mortalidade a nível mundial. Um dos problemas nos tratamentos atuais de quimioterapia relaciona-se com os efeitos secundários causados pela falta de seletividade dos fármacos utilizados. Assim, um dos desafios atuais é o desenvolvimento de sistemas terapêuticos que permitam potenciar o efeito dos fármacos e diminuir os seus efeitos nocivos. Neste contexto, neste trabalho, foram sintetizados complexos de Fe, Co e Zn com fórmula geral [M(bipy-R)3]x+, a partir dos quais foi possível obter sistemas micelares formados in situ pela adição de um copolímero de PLA-b-PEG ou por introdução de macroligandos poliméricos anfifílicos. Os resultados mostram que as nanopartículas formadas apresentam tamanho e características adequadas para aplicações na terapia dirigida contra o cancro. Os resultados preliminares de citotoxicidade na linha tumoral humana da mama MDAMB231 indicam que todos os compostos estudados apresentam atividade citotóxica relevante, sendo os compostos micelares os mais promissores; Abstract: New micellar transition metals complexes for targeted therapy of cancer Cancer is the second leading cause of mortality worldwide. One problem in the current chemotherapy treatments is related to the severe side effects caused by the lack of selectivity of the drugs in clinical use. Thus, one of the current challenges is the development of new therapeutic systems that allow maximizing the effect of the drugs and reducing their harmful effects. In this context, several Fe, Co and Zn compounds of the general formula [M (bipy-R)3]x+ were synthesized in order to obtain micellar systems, either by in situ addition of a PLA-b-PEG copolymer or by introducing amphiphilic macroligands. The results show that the formed nanoparticles have size and characteristics suitable for applications in targeted therapy against cancer. Preliminary results of cytotoxicity in human breast cancer line MDAMB231 indicate that all the studied compounds show significant cytotoxic activity, being the micellar compounds the most promising agents

RECRUTAMENTO E SELECÇÃO DE DOCENTES NO ENSINO SUPERIOR: ESTRATÉGIAS ADOTADAS PELAS INSTITUIÇÕES PRIVADAS DO ENSINO SUPERIOR EM LUANDA (ANGOLA)

This study aims to understand how the process of recruitment and selection of teachers in the Universities of Luanda is carried out. The research uses the quantitative method and reflects a descriptive study that uses as a data collection instrument, the questionnaire survey. The sample consists of professors from six different universities: Catholic University; Gregório Semedo University; Independent University of Angola; Methodist University; Piaget University; Utanga University. It is concluded that each university has its own process of recruitment and selection of teachers, however, there are some similarities. Keywords: Human Resource Management; Higher education institutions; Recruitment and Selection of Teachers.Este estudo procura perceber como é realizado o processo de recrutamento e de seleção de docentes nas Universidades de Luanda.A investigação recorre ao método quantitativo e reflete um estudo descritivo que utiliza como instrumento de recolha de dados, o inquérito por questionário. A amostra é composta por seis universidades diferentes: Universidade Católica; Universidade Gregório Semedo; Universidade Independente de Angola; Universidade Metodista; Universidade Piaget; Universidade de Utanga. Conclui-se que cada universidade tem o seu próprio processo de recrutamento e de seleção de docentes, no entanto, existem algumas similaridades. Palavras-chave: Gestão de Recursos Humanos; Instituições de Ensino Superior; Recrutamento e Seleção de Docentes

Chemistry and analysis of HNE and other prominent carbonyl-containing lipid oxidation compounds

The process of lipid oxidation generates a diverse array of small aldehydes and carbonyl-containing compounds, which may occur in free form or esterified within phospholipids and cholesterol esters. These aldehydes mostly result from fragmentation of fatty acyl chains following radical oxidation, and the products can be subdivided into alkanals, alkenals (usually α,β-unsaturated), γ-substituted alkenals and bis-aldehydes. Isolevuglandins are non-fragmented di-carbonyl compounds derived from H2-isoprostanes, and oxidation of the ω−3-fatty acid docosahexenoic acid yield analogous 22 carbon neuroketals. Non-radical oxidation by hypochlorous acid can generate α-chlorofatty aldehydes from plasmenyl phospholipids. Most of these compounds are reactive and have generally been considered as toxic products of a deleterious process. The reactivity is especially high for the α,β-unsaturated alkenals, such as acrolein and crotonaldehyde, and for γ-substituted alkenals, of which 4-hydroxy-2-nonenal and 4-oxo-2-nonenal are best known. Nevertheless, in recent years several previously neglected aldehydes have been investigated and also found to have significant reactivity and biological effects; notable examples are 4-hydroxy-2-hexenal and 4-hydroxy-dodecadienal. This has led to substantial interest in the biological effects of all of these lipid oxidation products and their roles in disease, including proposals that HNE is a second messenger or signalling molecule. However, it is becoming clear that many of the effects elicited by these compounds relate to their propensity for forming adducts with nucleophilic groups on proteins, DNA and specific phospholipids. This emphasizes the need for good analytical methods, not just for free lipid oxidation products but also for the resulting adducts with biomolecules. The most informative methods are those utilizing HPLC separations and mass spectrometry, although analysis of the wide variety of possible adducts is very challenging. Nevertheless, evidence for the occurrence of lipid-derived aldehyde adducts in biological and clinical samples is building, and offers an exciting area of future research

Ventilação Inteligente Modos e suportes inteligentes em Ventilação Mecânica Invasiva

INTRODUÇÃO: A ventilação mecânica invasiva (VMI) tem um papel central em UCI no suporte orgânico. A sua implementação e parametrização seguras, adequada às patologias em curso representam um desafio diário ao corpo clínico que trabalha em medicina intensiva. OBJETIVO: Esta monografia tem como objetivo compilar os modos não convencionais que existem atualmente e como funcionam, bem como os seus benefícios. METODOLOGIA: Pesquisa pelos sites das empresas atualmente no mercado e posterior aprofundamento, sem esquema de metodologia definido, sobre cada um dos modos encontrados. RESULTADOS: Das 10 empresas principais no mercado, só 4 empresas têm modos inteligentes de circuitos fechados que atuam por feedback de parâmetros dos pacientes e interagem com eles de diferentes formas, sincronizando de forma eficaz. CONCLUSÕES: Os benefícios dos modos não convencionais para os pacientes são inegáveis. Podemos referir a diminuição da morbilidade, diminuição do tempo de entubação, prevenção de lesões e também benefícios a nível de trabalho profissional.BACKGROUND: Invasive mechanical ventilation (IMV) plays a central role in ICU in organ support. Its safe implementation and parameterization, suitable for ongoing pathologies, represent a daily challenge for the clinical staff working in intensive care medicine. OBJECTIVE: This monograph aims to compile the nonconventional modes that currently exist and how they work, as well as their benefits. METHOD: Search through the websites of the companies currently n the market and further deepen, withouta defined methodology scheme, on each of the modes found. RESULTS: Out of the top 10 companies in the market, only 4 companies have intelligence closed-loop modes that act by feedback of patient parameters and interact with them in different ways, effectively synchronizing. CONCLUSIONS: The benefits of non-conventional modes for patients are undeniable. We can reder to the reduction of morbidity, reduction of intubation time, prevention pf injuries and also benefits in terms of professional work

A mass spectrometry approach for the identification and localization of small aldehyde modifications of proteins

Lipids containing polyunsaturated fatty acids are primary targets of oxidation, which produces reactive short-chain aldehydes that can covalently modify proteins, a process called lipoxidation. Improved mass spectrometry (MS) methods for the analysis of these adducts in complex biological systems are needed. Lysozyme and human serum albumin (HSA) were used as model proteins to investigate lipoxidation products formed by two short-chain aldehydes, acrolein and pentanal, which are unsaturated and saturated aldehydes respectively. The adducts formed were stabilized by NaBH4 or NaBH3CN reduction and analysed by MS. Analysis of intact modified lysozyme showed a pentanal modification resulting from Schiff's base formation (+70 Da), and up to 8 acrolein adducts, all resulting from Michael addition (+58 Da). Analysis of tryptic digests identified specific histidine, cysteine and lysine residues modified in both lysozyme and HSA, and determined characteristic amino acid-specific fragmentations. Eight different internal fragment ions were found that could be used as general diagnostic ions for pentanal- and acrolein-modified amino acids. The combined use of intact protein analysis and LC-MS/MS methods provided a powerful tool for the identification and localization of aldehyde-protein adduct, and the diagnostic ions will facilitate the development of targeted MS methods for analysis of adducts in more complex samples

Short-chain lipid peroxidation products form covalent adducts with pyruvate kinase and inhibit its activity in vitro and in breast cancer cells

Pyruvate kinase catalyses the last step in glycolysis and has been suggested to contribute to the regulation of aerobic glycolysis in cancer cells. It can be inhibited by oxidation of cysteine residues in vitro and in vivo, which is relevant to the more pro-oxidant state in cancer and proliferating tissues. These conditions also favour lipid peroxidation and the formation of electrophilic fragmentation products, including short-chain aldehydes that can covalently modify proteins. However, as yet few studies have investigated their interactions with pyruvate kinase, so we investigated the effects of three different aldehydes, acrolein, malondialdehyde and 4-hydroxy-2(E)-hexenal (HHE), on the structure and activity of the enzyme. Analysis by LC-MS/MS showed unique modification profiles for each aldehyde, but Cys152, Cys423 and Cys474 were the residues most susceptible to electrophilic modification. Analysis of enzymatic activity under these conditions showed that acrolein was the strongest inhibitor, and at incubation times longer than 2 h, pathophysiological concentrations induced significant effects. Treatment of MCF-7 cells with the aldehydes caused similar losses of pyruvate kinase activity to those observed in vitro, and at lower concentrations than those required to cause cell death, with time and dose-dependent effects; acrolein adducts on Cys152 and Cys358 were detected. Cys358 and Cys474 are located at or near the allosteric or active sites, and formation of adducts on these residues probably contributes to loss of activity at low treatment concentrations. This study provides the first detailed analysis of the structure-activity relationship of C3 and C6 aldehydes with pyruvate kinase, and suggests that reactive short-chain aldehydes generated in diseases with an oxidative aetiology or from environmental exposure such as smoking could be involved in the metabolic alterations observed in cancer cells, through alteration of pyruvate kinase activity