Estudo das alterações do proteoma mitocondrial associadas à MADD

Mestrado em Bioquímica - Métodos BiomolecularesA deficiência múltipla de acil-CoA desidrogenases (MADD) é um dos défices conhecidos da β-oxidação mitocondrial, causado por mutações nos genes que codificam as proteínas ETFDH e ETF ou por alterações ainda não totalmente caracterizadas do metabolismo da riboflavina. Muito embora já se conheçam alguns estudos sobre estes défices da β-oxidação, os mecanismos e alterações subjacentes às variadas manifestações fenotípicas encontram-se ainda pouco conhecidos. De modo a contribuir para uma melhor clarificação dos mecanismos de adaptação celular à MADD, dando particular ênfase à mitocôndria, no estudo realizado no âmbito desta dissertação utilizou-se uma abordagem proteómica baseada na análise electroforética bi-dimensional seguida da identificação das proteínas por espectometria de massa, da fracção mitocondrial isolada de fibroblastos obtidos de biopsias da pele de dois doentes com MADD do tipo III, um com um défice de ETFDH (Doente 1) e o outro com um défice de ETF (Doente 2). Com esta abordagem experimental foram detectadas 248 proteínas mitocondriais, das quais 28 se encontravam diferentemente expressas no Doente 1 e 33 no Doente 2. De entre estas proteínas diferentemente expressas são de realçar a sobreexpressão de chaperones, como a Hsp90 ou a Hsp7C, e de enzimas antioxidantes, como a glutationa S-transferase e superóxido dismutase, e ainda de algumas proteínas ligadas à apoptose, como por exemplo a GAPDH. De um modo geral, este estudo proporciona uma perspectiva global das principais adaptações da mitocôndria nos casos moderados de MADD, revelando as principais vias alteradas através de variações na expressão de enzimas metabólicas, de proteínas responsáveis pela manutenção da homeostasia redox celular, pela qualidade funcional das proteínas mitocondriais e pela apoptose.The multiple acyl-CoA dehydrogenases deficiency (MADD) is one of the known deficits of the mitochondrial β-oxidation process, caused by mutations in the genes that codify for the ETFDH and ETF proteins or by some not yet totally characterized riboflavin metabolism disturbs. Although there are some studies about these deficits of the β-oxidation, the mechanisms and alterations underlying the several phenotypic manifestations are yet hardly understood. In order to contribute to a better understanding of the cellular adaptation mechanisms to MADD, giving particular emphasis to mitochondria, in this study it was used a proteomic approach based in the bi-dimensional electrophoretic analysis followed by the protein identification by mass spectometry of the mitochondrial fraction isolated from fibroblasts obtained by skin biopsy of two patients with type III MADD, one with a deficit of ETFDH (Patient 1) and the other with a deficit of ETF (Patient 2). With this experimental approach it was detected 248 mitochondrial proteins, of which 28 were differently expressed in patient 1 and 33 in Patient 2. Among these differently expressed proteins, an overexpression of chaperones, like Hsp90 or Hsp7C, antioxidants enzymes, like glutathione S-transferase and superoxide dismutase (MnSOD), and also of some proteins associated with apoptosis, like GAPDH, was observed. In general, this study provides a global perspective of the main mitochondrial adaptations in two moderate cases of MADD, revealing the main pathways involved, based on the alterations of metabolic enzymes expression and of proteins that regulate cellular homeostasis, protein quality control and apoptosis

Neuroproteomics — LC-MS Quantitative Approaches

Neuroproteomics is a scientific field that aims to study all the proteins of the central nervous system, their expression, function, and interactions. The central nervous system is intricate and heterogeneous, and the study of its proteome is consequently complex, with many biological questions still requiring deep investigation. For this, mass spectrometry approaches, most often coupled with liquid chromatography (LC-MS), have been the number one choice in proteomics, and over the years it has added many important findings to the field. At this point it is important that proteomics turns to the quantitative expression of proteins instead of only identifying which proteins are present in a given sample, much because the most important alterations may be slight alterations in the quantity of a protein in a given situation. Therefore, many LC-MS quantitative approaches have been developed relying on the labeling of the proteins or even by using label-free techniques

Disclosing proteins in the leaves of cork oak plants associated with the immune response to Phytophthora cinnamomi inoculation in the roots: a long-term proteomics approach

The pathological interaction between oak trees and Phytophthora cinnamomi has implications in the cork oak decline observed over the last decades in the Iberian Peninsula. During host colonization, the phytopathogen secretes effector molecules like elicitins to increase disease effectiveness. The objective of this study was to unravel the proteome changes associated with the cork oak immune response triggered by P. cinnamomi inoculation in a long-term assay, through SWATH-MS quantitative proteomics performed in the oak leaves. Using the Arabidopis proteome database as a reference, 424 proteins were confidently quantified in cork oak leaves, of which 80 proteins showed a p-value below 0.05 or a fold-change greater than 2 or less than 0.5 in their levels between inoculated and control samples being considered as altered. The inoculation of cork oak roots with P. cinnamomi increased the levels of proteins associated with protein-DNA complex assembly, lipid oxidation, response to endoplasmic reticulum stress, and pyridine-containing compound metabolic process in the leaves. In opposition, several proteins associated with cellular metabolic compound salvage and monosaccharide catabolic process had significantly decreased abundances. The most significant abundance variations were observed for the Ribulose 1,5-Bisphosphate Carboxylase small subunit (RBCS1A), Heat Shock protein 90-1 (Hsp90-1), Lipoxygenase 2 (LOX2) and Histone superfamily protein H3.3 (A8MRLO/At4G40030) revealing a pertinent role for these proteins in the host-pathogen interaction mechanism. This work represents the first SWATH-MS analysis performed in cork oak plants inoculated with P. cinnamomi and highlights host proteins that have a relevant action in the homeostatic states that emerge from the interaction between the oomycete and the host in the long term and in a distal organ.FCT:UID/Multi/00631/2019 and UIDB/00631/2020 CEOT BASE to CEOT and ACC, GS and RP; UIDB/ 04326/2020 to CCMAR; Norma Transitoria- DL57/2016/CP1361/CT0015 to PP; contract NIBAP (ALG-01-0247-FEDER-037303) to RP; projects POCI-01-0145-FEDER-007440 (Ref. UIDB/04539/ 2020), POCI-01-0145-FEDER-016428 (Ref. SAICTPAC/0010/2015), POCI-01-0145- FEDER-029311 (Ref. PTDC/BTM-TEC/29311/ 2017), POCI-01-0145-FEDER-30943 (Ref. PTDC/ MECPSQ/30943/2017) and PTDC/MED-NEU/ 27946/2017 to CNC, BM and CS. The work at CNC was also funded by the National Mass Spectrometry Network (RNEM) under contract POCI-01-0145-FEDER-402-022125info:eu-repo/semantics/publishedVersio

Role of mitochondrial antioxidant defense systems in fatty acid β-oxidation defects

Mitochondrial fatty acid oxidation (FAO) plays a pivotal role in energy homeostasis, namely during periods of fasting or metabolic stress. FAO defects are a group of inherited metabolic disorders that encompass at least twelve distinct enzyme or transporter deficiencies, and can present with a wide range of clinical symptoms with various degrees of severity. Besides recent advances, many doubts still remain on the degree and characteristics of mitochondrial dysfunction on FAOD and its contribution to the clinical phenotype

A Pathogen and a Non-pathogen Spotted Fever Group Rickettsia Trigger Differential Proteome Signatures in Macrophages

We have previously reported that Rickettsia conorii and Rickettsia montanensis have distinct intracellular fates within THP-1 macrophages, suggesting that the ability to proliferate within macrophages may be a distinguishable factor between pathogenic and non-pathogenic Spotted fever group (SFG) members. To start unraveling the molecular mechanisms underlying the capacity (or not) of SFG Rickettsia to establish their replicative niche in macrophages, we have herein used quantitative proteomics by SWATH-MS to profile the alterations resulted by the challenge of THP-1 macrophages with R. conorii and R. montanensis. We show that the pathogenic, R. conorii, and the non-pathogenic, R. montanensis, member of SFG Rickettsia trigger differential proteomic signatures in macrophage-like cells upon infection. R. conorii specifically induced the accumulation of several enzymes of the tricarboxylic acid cycle, oxidative phosphorylation, fatty acid β-oxidation, and glutaminolysis, as well as of several inner and outer membrane mitochondrial transporters. These results suggest a profound metabolic rewriting of macrophages by R. conorii toward a metabolic signature of an M2-like, anti-inflammatory activation program. Moreover, several subunits forming the proteasome and immunoproteasome are found in lower abundance upon infection with both rickettsial species, which may help bacteria to escape immune surveillance. R. conorii-infection specifically induced the accumulation of several host proteins implicated in protein processing and quality control in ER, suggesting that this pathogenic Rickettsia may be able to increase the ER protein folding capacity. This work reveals novel aspects of macrophage-Rickettsia interactions, expanding our knowledge of how pathogenic rickettsiae explore host cells to their advantage

Characterization of widespread proteome aggregation through aging in mammals

Proteome and proteostasis network disruptions accompany proteostasis alterations, leading to accumulation of protein aggregates, characteristic of several age-related diseases. Previous work in Caenorhabditis elegans and zebrafish unveiled asymptomatic protein aggregation (ARPA), characterized by generalized increased accumulation of insoluble proteins through aging. However, the consequences of protein homeostasis imbalances in the context of healthy aging in mammals remains mostly unexplored. To elucidate if accumulation of insoluble proteins also occurs through aging in mammals, C57BL/6 mice with different ages corresponding to young (6 months old), adult (13 months old), and old stages (18 and 24 months old) were used. Detergent-insoluble fractions were isolated from total protein extracts of tissues, followed by characterization of both total and detergent-insoluble protein profiles. Our results show tissue-specific proteome alterations through aging. For example, the insoluble fractions increase through aging in brain-derived tissues, but muscular tissues do not display significant alterations until 18 months old. We are now performing SWATH-MS analysis to identify differential proteome signatures of aging and which proteins are more prone to aggregate to further elucidate the functions and biological processes affected by ARPA.publishe

Proteome dataset of sea bass (Dicentrarchus labrax) skin-scales exposed to fluoxetine and estradiol

Contamination of aquatic ecosystems with anthropogenic pollutants, including pharmaceutical drugs, is a major concern worldwide. Aquatic organisms such as fish are particularly at risk of exposure to pollutants. The surface of fish is the first point of contact with pollutants, but few studies have considered the impact of pollutants on the skin-scale barrier. The present proteome data are the basis of the findings discussed in the associated research article "Proteomics of sea bass skin-scales exposed to the emerging pollutant fluoxetine compared to estradiol" [1]. Juvenile sea bass were exposed by intraperitoneal injections to: a) the antidepressant fluoxetine (FLX), a widely prescribed psychotropic drug and an emerging pollutant; b) the natural estrogen 17 beta-estradiol (E2) and c) the vehicle, coconut oil (control). The scale proteome of fish exposed to these compounds for 5 days was analysed using quantitative label-free proteomics technology SWATH-MS (sequential windowed data-independent a cquisition of the total high-resolution-mass spectra). The proteome data generated was submitted to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD020983. LC-MS data from pooled protein extracts from the scales of all experimental groups was acquired using information-dependent acquisition (IDA) and 1,254 proteins were identified by searching against the sea bass genome database. 715 proteins were quantified by SWATH acquisition, and 213 proteins had modified levels (p < 0.05) between the E2- or FLX-exposed fish compared to the control. The main biological processes and KEGG pathways affected by E2 or FLX treatments were identified using Cytoscape/ClueGO enrichment analyses. (c) 2022 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)info:eu-repo/semantics/publishedVersio

Low-Voltage High-Speed Ring Oscillator with a-InGaZnO TFTs

ECR/2017/000931 POCI-01-0145-FEDER-007688 PTDC/NAN-MAT/30812/2017This paper presents a high-speed ring oscillator (RO) with amorphous Indium-Gallium-Zinc-Oxide Thin-film transistors (a-IGZO TFTs). The proposed RO reduces the delay of a single stage inverter using intermediate signals generated within the RO, hence, improving the speed. To validate the proposed idea, two conventional ROs (with diode-load load inverter and bootstrapped pseudo-CMOS inverter) and the proposed RO were fabricated at a temperature ≤ 180°C. Measured results of the proposed RO have shown a frequency and power-delay-product (PDP) of 173.2 kHz and 0.7 nJ at a supply voltage of 6V. Further, it shows approximately 155% (44%) increase in frequency and 14% (24.5%) decrease in PDP compared to diode-load inverter (bootstrapped pseudo-CMOS inverter) based ROs. Therefore, the proposed RO finds applications in low-voltage and high speed designs for timing signal generation.publishersversionpublishe

“Vencer a asma 2017” campaign: A community-based screening

A asma é uma das doenças crónicas não transmissíveis mais frequentes afetando cerca de 700 000 portugueses atualmente. Os rastreios constituem uma oportunidade de prestar esclarecimentos sobre a doença à população, alertar a população para a importância do controlo da asma e reconhecimento dos seus sintomas. Objetivos, material e métodos: Caracterização sociodemográfica e descritiva dos resultados da campanha “Vencer a Asma 2017”. Estudo transversal em 8 cidades de Portugal continental numa iniciativa pública, gratuita e de inclusão voluntária da população a propósito do Dia Mundial da Asma. Aplicação de questões adaptadas do questionário GA2LEN e realização de espirometria simples. Resultados: 1086 participantes (idade 52,5±19,7 anos; 62,4 % do género feminino; 20,5 % fumadores e 62,6 % observados na região Norte). Dos participantes, 299 (28,0 %; IC95 % 25,3-30,7) declararam ter asma atual ou no passado (autorreportada) e, entre estes, 80 (26,8 %; IC95 % 21,7-31,8) reportaram pelo menos uma hospitalização por asma, 181 (60,5 %; IC95 % 55,0-66,1) afirmaram estar medicados para a asma; 498 (46,6 %; IC95 % 43,6-49,6) mencionaram ter tido um episódio de sibilância nos últimos 12 meses. Durante o rastreio, 549 participantes (50,6%; IC95 % 47,6-53,5) realizaram espirometria simples e, destes, 186 (34,1 %; IC95 % 30,1-38,1) apresentaram alte-rações dos parâmetros espirométricos avaliados. A análise de subgrupos revelou uma prevalência de “asma atual” de 19,9 % (IC95 %; 17,5-22,2). Conclusões: O rastreio permitiu, apesar das suas limitações, identificar um grande número de indivíduos na comunidade num curto espaço de tempo. No futuro, será útil que estas iniciativas incluam questionários de resultados reportados pelo doente, testes cutâneos por picada, prova de broncodilatação ou fração exalada de óxido nítrico, que permitam uma melhor caracterização da população estudada bem como a perceção de necessidade de avaliação clínica posterior nos casos em que não haja um adequado acompanhamento médico.GlaxoSmithKline™; LPM Consultadoria, MEDIDA, Lda,info:eu-repo/semantics/publishedVersio

Influence of EPICardial adipose tissue in HEART diseases (EPICHEART) study: Protocol for a translational study in coronary atherosclerosis

Accumulation of epicardial adipose tissue (EAT) is associated with coronary artery disease (CAD) and increased risk of coronary events in asymptomatic subjects and low-risk patients, suggesting that EAT promotes atherosclerosis in its early stage. Recent studies have shown that the presence of CAD affects the properties of adjacent EAT, leading to dynamic changes in the molecular players involved in the interplay between EAT and the coronary arteries over the history of the disease. The role of EAT in late-stage CAD has not been investigated.coronárioResumoIntroduc¸ão:Acumulac¸ão de tecido adiposo epicárdico (TAE) tem sido associado a doenc¸acoronária aterosclerótica (DC) e aumento do risco de eventos coronários em indivíduos ass-intomáticos e doentes de baixo risco, sugerindo que o TAE pode promover fases precoces daDC. Estudo recentes mostraram que a presenc¸a de DC afeta as características do TAE adja-cente levando a modificac¸ões dinâmicas nos mediadores envolvidos na comunicac¸ão entre oTAE e as artérias coronárias ao longo da história da DC. O papel doTAE nas fases avanc¸adas daaterosclerose coronária não foi investigado.Objetivos: Através de análise comparativa com o tecido adiposo mediastínico e subcutâneo,pretendemos investigar se o volume do TAE, avaliado por tomografia computadorizada (TC), eo seu proteoma, avaliado por espectrometria de massa técnica de SWATH, estão associados aestadios avanc¸ados da DC numa coorte de estenose aórtica grave.Métodos: O estudo EPICHEART (NCT03280433) é um estudo prospetivo que inclui doentescom estenose aórtica grave referenciados para substituic¸ão eletiva da válvula aórtica, cujoprotocolo envolve avaliac¸ão pré-operatória clínica, nutricional, ecocardiográfica, por TC eangiografia coronária invasiva. Durante a cirurgia cardíaca, colhemos amostras de tecido adi-poso epicárdico, mediastínico e subcutâneo para análise do seu proteoma por espectrometriade massa técnica de SWATH. Adicionalmente, colhemos líquido pericárdico, sangue venoso per-iférico e do seio coronário para investigar mediadores de DC derivados do TAE na circulac¸ãosistémica e local.Conclusão: Desenhámos um estudo de translac¸ão para explorar a associac¸ão da quantidade equalidade do TAE com a DC tardia. Esperamos identificar mediadores da comunicac¸ão recíprocaentre o TAE e as artérias coronárias que estão envolvidos na patogénese das fases avanc¸adas daDC, especialmente, calcificac¸ão coronária, os quais podem servir como novos alvos terapêuticose soluc¸ões de engenharia biomédica para visualizac¸ão da DC