Dissertations / Theses on the topic 'Lipidome profile'

La perdita di peso nei pazienti con sindrome metabolica ha effetti positivi sulle malattie cardiovascolari e sul rischio di diabete di tipo 2, ma i suoi effetti sul profilo lipidico e sul secretoma periferico sono tutt’ora poco chiari. Al fine di determinare gli effetti della perdita di peso indotta dalla dieta sui parametri metabolici sono stati analizzati il profilo lipidico e il secretoma periferico in pazienti affetti da sindrome metabolica. In questo studio sono stati arruolati 18 soggetti adulti di sesso maschile con sindrome metabolica e BMI compreso tra 25 e 35 Kg/m2, che sono stati sottoposti a dieta Mediterranea ipocalorica bilanciata per 6 mesi. Lo scopo dell'approccio dietetico era quello di indurre nei pazienti una perdita di peso di almeno il 5% del peso corporeo iniziale. Dopo la perdita di peso abbiamo osservato un miglioramento significativo del BMI, dei livelli di insulina, della glicemia a digiuno, dell’indice HOMA-I, dei livelli di trigliceridi, di LDL e HDL. L'analisi delle lipoproteine circolanti ha mostrato un cambiamento significativo nella loro composizione. In particolare, abbiamo osservato un trasferimento importante di triacilgliceroli dalle HDL alle LDL. A tale cambiamento si è associata una significativa riduzione delle citochine proinfiammatorie periferiche, come IL-6, TNF-α, IL-8 e MIP-1β. Abbiamo inoltre osservato un'interessante correlazione positiva tra i livelli di citochine e livelli periferici di CETP (cholesteryl ester transfer protein), un enzima con un ruolo chiave nel trasferimento di esteri del colesterolo tra le lipoproteine. La perdita di peso ottenuta attraverso la dieta Mediterranea ipocalorica ha determinato un miglioramento del profilo lipidico periferico, un cambiamento nella composizione delle lipoproteine e del secretoma. Questi risultati sono fondamentali per comprendere i benefici della perdita di peso e i meccanismi che possono avere un ruolo nel miglioramento del rischio cardiovascolare.
Weight loss in patients with metabolic syndrome has positive effects on cardiovascular diseases and type 2 diabetes risk, but its effects on peripheral secretome and lipidome profiles are still poorly understood. In order to determine the effects of diet-induced weight loss on metabolic parameters, lipidome and secretome profiles were evaluated. In this study, 18 adult males with metabolic syndrome and BMI between 25 and 35 Kg/m2 were enrolled, and then subjected to a balanced hypocaloric Mediterranean diet for 6 months. The aim of the dietetic approach was to induce in patients a weight loss of at least 5% of the initial body weight. After weight loss, we observed a significant improvement in BMI, insulin, fasting blood glucose, HOMA-I, triglyceridemia, LDL, and HDL levels. The analysis of circulating lipoproteins showed a significant change in their composition. In particular, a massive transfer of triacylglycerols from HDL to LDL was observed. This result was associated with a significant reduction in peripheral pro-inflammatory cytokines, such as IL-6, TNF-α, IL-8, and MIP-1β. We also observed an interesting positive correlation among cytokines levels and peripheral levels of CETP (cholesteryl ester transfer protein), an enzyme with a key role in lipid metabolism. The results achieved suggest that weight loss obtained through the hypocaloric Mediterranean diet is associated with an improvement in peripheral lipidome and secretome profiles. Furthermore, this dietetic approach stimulated changes in lipoproteins composition. These results are fundamental to understand weight loss benefits and the mechanisms that may play a role in improving cardiovascular risk.

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Prostanoids modulate the activity of human pregnant myometrium and their functional role can be appreciated through characterisation of prostanoid receptors and tissue concentration of prostanoids. We have applied a lipidomic approach to elucidate the profile of prostanoids in human non-labouring and labouring myometrium. We have identified a total of nineteen prostanoids including prostacyclin, thromboxanes, prostaglandins and dihydro-prostaglandins. Prostacyclin was the predominant prostanoid in both non-labouring and labouring myometria, with PGD2 and PGF2¿ being the second most abundant. Although the total amount of prostanoids was increased in the labouring tissue, PGE2 and 13,14-dihydro-15-keto-PGE2 were the only prostanoids to increase significantly at early and late labour (p¿0.001). Our data suggest that PGF2¿ plays an important role in parturition, whilst the increase in PGE2 could occur to facilitate cervical dilation and relaxation of the lower myometrium during labour. Although the elevation in TXA2 was less marked than expected, in terms of translation to function even a relatively small increase in the level of this potent spasmogen may have significant effects.

Mestrado em Mestre em Bioquímica, ramo da Bioquímica Clínica
Marine macroalgae, or seaweeds, have gained an increased interest in recent times for the use in various biotechnological applications, due to the added-value of their chemical constituents. Among them, glycolipids and phospholipids display several commercial applications in a wide spectrum of industries, such as food, pharmaceutical and cosmetic. In an effort to further understand the lipid composition of macroalgae, the present work reports, for the first time, the isolation and characterization of the polar lipid profile of the red macroalgae Porphyra dioica cultivated on a land-based integrated multi-trophic aquaculture (IMTA) system, using a lipidomic-based approach employing hydrophilic interaction liquid chromatography-eletrospray ionization mass spectrometry (HILIC-ESI-MS). The fatty acid profile of this species of seaweed was also determined, accounting for season variability and its life cycle. The polar lipid profile of P. dioica revealed the presence of over 69 molecular species, corresponding to glycolipids (sulfoquinovolsyldiacylglycerols, sulfoquinovosylmonoacylglycerols, digalactosyldiacylglycerols) and glycerophospholipids (lyso- and phosphatidylglycerols), lyso- and phosphatidylcholines), as well as phytyl derivatives. Some of these polar lipids contain polyunsaturated fatty acids (PUFAs), namely arachidonic acid (C20:4) and eicosapentaenoic acid (C20:5), thus revealing the ability of P. dioica to biosynthesize this long chain PUFAs. P.dioica from the winter season revealed to be richer in PUFA content, accounting for 37.0% of total fatty acid (TFA) content, as opposed to P. dioica from the summer season (25.0% of TFA content). Eicosapentaenoic acid (EPA) content was revealed to be being significantly higher in the winter season (25.2% of TFA content). The diploid sporophyte conchocelis phase of P. dioica showed to possess the highest amount of PUFAs (47.0% of TFA content), with arachidonic acid being the most abundant fatty acid (21.2% of TFA content). Several of the lipids identified have been reported to possess nutritional and health benefits, thus allowing the valorisation of P. dioica from IMTA as a source of bioactive compounds, adequate for the use in a wide range of different applications and as a functional food, rich in omega-3 fatty acids.
As macroalgas têm vindo a ganhar um interesse cada vez maior para o uso em diversas aplicações biotecnológicas, devido ao valor acrescentado dos seus diferentes constituintes. Entre estes, os glicolípidos e os fosfolípidos podem ser usados comercialmente em diferentes indústrias, tais como as indústrias alimentar, farmacêutica e cosmética. Com o objetivo de compreender melhor a composição lipídica das macroalgas, o presente trabalho relata, pela primeira vez, a caracterização do perfil de lípidos polares da macroalga vermelha Porphyra dioica, cultivada num sistema de aquacultura multi-trófica integrada (IMTA), utilizando para esse fim uma abordagem lipidómica baseada na espectrometria de massa (HILIC-ESI-MS). Foi também determinado o perfil de ácidos gordos da referida espécie de alga, tendo em consideração a variabilidade sazonal e o seu ciclo de vida. O perfil de lípidos polares da alga P. dioica revelou a presença de mais de 69 espécies moleculares diferentes, correspondendo a classes de glicolípidos (sulfoquinovosildiacilgliceróis, sulfoquinovosilmonoacilgliceróis e digalactosildiacilgliceróis), fosfolípidos (liso- e fosfatidilglicerol, liso- e fosfatidilcolinas) e derivados fitil. Alguns destes lípidos polares contêm ácidos gordos polinsaturados (PUFAs) na sua composição, nomeadamente o ácido araquidónico (C20:4) e ácido eicosapentaenóico (C20:5), revelando, assim, a capacidade da alga P. dioica em biossintetizar este tipo de ácidos gordos polinsaturados de cadeia longa. Considerando a variação sazonal do conteúdo em ácidos gordos, a P. dioica cultivada no inverno revelou ser mais rica em PUFAs, correspondendo a 37.0% do conteúdo total de ácidos gordos, contrariamente à P. dioica cultivada no verão (25.0%). O conteúdo em ácido eicosapentaenóico (EPA) é significativamente maior na estação de inverno (25.2%). O perfil em ácidos gordos também variou com o ciclo de vida P. dioica, sendo que na fase de conchocelis a quantidade de PUFA é significativamente mais elevada (47.0% de conteúdo de ácidos gordos), sendo o ácido araquidónico o ácido gordo mais abundante (21.2% de conteúdo de ácidos gordos).Várias classes de lípidos polares foram identificados como possuindo benefícios nutricionais e para a saúde, permitindo assim a valorização da alga vermelha P. dioica produzida em IMTA como uma fonte de compostos bioativos, adequados para o uso numa grande variedade de aplicações como um alimento funcional, rica em ácidos gordos polinsaturados ómega-3.

L‟acide docosahexaénoïque (DHA) s‟accumule principalement dans les membranes cérébrales humaines durant la période périnatale et les 2 premières années de la vie. Optimiser l‟incorporation des acides gras polyinsaturés à longue chaîne (AGPI-LC) n-3 dans le système nerveux central, notamment le DHA, constitue l‟un des objectifs majeurs de la nutrition néonatale et infantile. Ce projet étudie l‟effet de régimes à base de matière grasse laitière anhydre (MGLA) afin d‟améliorer la bioconversion des AGPI n-3 et l‟incorporation du DHA dans le cerveau. L‟autre objectif, au-delà des effets sur les acides gras, est d‟évaluer si l‟impact des apports destinés au jeune n‟exercent pas un effet plus large tels qu‟appréciés au niveau du lipidome du cerveau.Nous avons mis au point un modèle d‟expérimentation animale permettant d‟étudier les effets de mélanges de MGLA et d‟huiles végétales. Des mères sont nourries avec un régime déficient en acide α-linoléique (ALA) (0,4% des acides gras totaux (AGT)) pendant 6 semaines avant accouplement et pendant toute la gestation et la lactation. Après sevrage, quatre-vingts petits rats mâles et femelles ainsi carencés en AGPI n-3 sont séparés en 4 groupes et reçoivent des régimes à 10% de lipides pendant 6 semaines, soit à base d‟huile de palme mélangée avec des huiles vegétales pour un apport à 1,5%ALA (P1), ou 1,5% ALA supplementé avec 0,12%DHA et 0,4% acide arachidonique (ARA) (P2); soit à base de MGLA et d‟huiles végétales apportant 1,5%ALA (B1) ou 2,3%ALA (B2). Les acides gras cérébraux, plasmatiques et érythrocytaires ainsi que le profil lipidomique cérébral sont mesurés, et les résultats sont analysés en statistiques multivariées. Le régime B1 est supérieur aux deux régimes à base d‟huile de palme à 1,5 %ALA, pour restaurer la quantité de DHA du cerveau (augmentation de 14.38%, P < 0.05) ; le régime (B2) présente un bénéfice supplémentaire sur ce paramètre. Les concentrations cérébrales en DHA chez les rats mâles sont significativement plus faibles que chez les femelles en raison des interactions de l‟effet sexe sur l‟effet régime, mais cet effet s‟atténue avec les régimes MGLA ou l‟ajout de DHA préformés dans le régime palme (P2).Nous avons calculé un nouvel indice à l‟aide des profils en acides gras des globules rouges et du plasma pour prédire le contenu en DHA cérébral, et dont la performance est meilleure que celle des indices existants. Pour la première fois, nous avons pu mettre en évidence un effet majeur et inconnu jusqu‟alors, des régimes sur le lipidome du cerveau (analyse des espèces moléculaires de lipides), affectant près de 15% des espèces analysées. Ces changements semblent être liés, entre autres, au métabolisme du cholestérol, des acides gras et des messagers lipidiques.En conclusion, nos données sont susceptibles d„améliorer les formules infantiles. La première utilisation de l‟approche de lipidomique sans a priori que nous avons mise en oeuvre ouvre des perspectives nouvelles en nutrition infantile
The accretion of docosahexaenoic acid (DHA) in brain membranes mainly occurs around delivery and during the first two years of life. One of the main goals of neonatal nutrition is to optimize the incorporation of n-3 long chain polyinsaturate fatty acids (LC-PUFA) into the central nervous system, including DHA. Our goal was to study the impact of several kinds of diets based on dairy-fat to improve the n-3 LC-PUFA bioconversion and DHA accretion into brain. The other endpoint was to assess if beyond the brain fatty acid profiles, the dietary intakes would bring about a wider effect such as the one that can be appreciated through a lipidomic approach.We compared the nutritional effect of dairy-fat based diets to that of palm-oil based diets in the rat reproductive model. Mother rats were made deficient in α-linoleic acid (ALA) (0.4% of fatty acids (FA) for 6 weeks prior to mating and throughout gestation and lactation. After weaning, the resulting deficient 40 rat pups of either gender were split into 4 groups and received 10% fat diets made with either 1.5%ALA palm oil blend (P1), same added with 0.12% DHA and 0.4% arachidonic acid (ARA) (P2) , 1.5% ALA dairy-fat blend (B1) or 2.5% ALA dairy-fat blend (B2). The brain, red blood cell (RBC) and plasma fatty acid profiles were analyzed and treated using multivariate statistics. B1 was superior to both palm-oil based diets to improve the brain DHA contents (14.4% increase, P < 0.05). B2 brought an additional benefit for this parameter. The brain DHA contents in males were significantly lower than for the female because of a diet x gender interaction. This effect was smoothed with the dairy-fat diets or the palm-oil based diet augmented with preformed DHA.We calculated a new fatty acid index to predict the brain DHA contents, based on the fatty acid profiles measured in RBC and plasma, and which demonstrated a better performance than the existing published index. For the first time, we showed a profound and yet unsuspected effect of diets until now on the brain lipidome (lipids molecular species), affecting about 15% of the features detected. These changes were ascribed to the cholesterol and fatty acid metabolism, and to the lipid messengers, among others.In conclusion, our data appear highly relevant to improve infant formulas. The first use of the lipidomic approach in neonatal nutrition open the paths of new researches in the area of infant nutrition

Research studies indicate that up to 58% of the world adult population will be overweight or obese by 2030 [1]. Obesity is not only related to food intake, but factors such as lifestyle and genetic background contribute to its onset [3–5]. This disease [2] is commonly associated with vitamin D (Vit. D) deficiency [6,7], and genetic associations have been identified to explain this link [8]; however, differences in dietary intake, sun exposure, or Vit. D metabolism are also involved [9]. Moreover, along with Vit. D deficiency, lipids, and particularly sphingolipids (SLs) as ceramides (Cers) and sphingomyelins (SMs) have been described as involved, not only in increasing inflammation [10,11], but also in the development of cardiovascular disease and type two diabetes [12–14], two common conditions observed in obese subjects. Given the pivotal role of SLs in obesity associated co-morbidities and the association of Vit. D, dyslipidemia and obesity, our study was aimed at profiling circulating SLs in human subjects under these conditions, in order to provide hints for the identification of new biomarkers to be introduced in clinical settings. To define human SLs profile in obesity, dyslipidemia, and Vit. D associated deficiency, sera from 23 normal-weight normolipidemic (NWNL), 46 normal-weight, dyslipidemic, Vit. D deficient (NWDL) and 60 obese dyslipidemic, Vit. D deficient (ODL) Saudi Arabian subjects were analyzed with a dual approach, characterized by the use of two complementary techniques: the HPTLC-Primuline profiling and the LC-MS analysis. Furthermore, to define SLs profiles in the context of human adaptation to high altitude hypoxia, sera from 59 Vit. D deficient dyslipidemic children living at high altitude were analyzed by LC-MS. Children were grouped based on their BMI percentiles in 7 underweight (UW), 30 normal-weight (NW), 13 overweight (OW) and 9 obese (O). SLs profile analysis of NWNL and ODL Saudi Arabian subjects displayed differences in total Cer and total SM caused by dyslipidemia and vitamin D deficiency, whereas specific Cers, and SMs acyl chains characterize obese subjects, only. Gender differences were found in SLs profiles independently from dyslipidemia and Vit. D status. Obesity-associated Cers, SMs, and dihydrosphingomyelins (dhSMs) specific acyl chains were identified in the NWDL vs. ODL comparison independently from dyslipidemia and Vit D status, and are thought to be drivers of increased risk of developing obesity-associated morbidities. The analysis of SLs profiles from dyslipidemic children with Vit. D deficiency allowed to confirm the results of Saudi Arabian subjects regarding SLs association with dyslipidemia and associated Vit. D deficiency. Furthermore, SLs profile analysis led to the identification of a characteristic SLs portraits associated with BMI and related to hypoxia metabolic adaptation.

Le profilage métabolomique global de matrices biologiques dans de larges séries d'échantillon est un enjeu majeur. Dans ce contexte, notre travail vise à développer des approches LC-HRMS et outils bioinformatiques pour les analyses métabolomique et lipidomique de larges cohortes. Dans un premier temps, nous avons développé puis évalué la pertinence de 4 méthodes LC-HRMS dans l'annotation du métabolome/lipidome sérique humain. Ainsi, une base de données spectrales a été implémentée à l'aide de spectres MS, MS/MS et les temps de rétention de composés de référence afin d'assurer l'annotation de jeux de données. La combinaison de méthodes RP, HILIC et PFPP-HRMS a permis l'identification de 266 métabolites et 706 espèces lipidiques sériques répartis sur 20 et 24 classes chimiques respectivement dont 27% d'espèces isomères. Ces outils ont été appliqués, dans un second temps, à la stratification de 78 patients diabétiques. Outre le syndrome métabolique marqué (perturbation du métabolisme énergétique), nos analyses ont montré l'impact délétère de facteurs physiologiques confondants -âge et IMC-. Nous en avons évalué l'influence sur une cohorte de 227 salariés du CEA. Les empreintes lipidomiques sont robustes, néanmoins l'impact de l'IMC est marqué pour les lipides neutres. L'effet du genre démontre un catabolisme masculin important. L'effet de l'âge se manifeste par des activités enzymatiques altérées. Ces études combinent une analyse globale métabolomique et lipidomique des mêmes échantillons humains. Elles visent à construire une base de données relationnelle incluant données spectrales et biologiques servant à la caractérisation de biomarqueurs dans le cas d'études cliniques
Global metabolomic profiling of biological media in large sample sets is a major challenge. In this context, our work aims to develop LC-HRMS approaches and data mining tools for metabolomics and lipidomics analysis of large cohorts. We have first developed and evaluated the reliability of four LC-HRMS methods in the annotation of human serum metabolome and lipidome. Thus, spectral database was implemented using MS spectra, MS/MS and retention times of reference compounds to further ensure datasets annotation. The combination of RP, PFPP and HILIC-HRMS methods allowed identification of 266 metabolites and 706 lipid species in human serum over 20 to 24 chemical classes respectively including 27% of isomeric species. These analytical tools were then applied for the stratification of 78 diabetic patients. Unsurprisingly, we highlighted a metabolic syndrome (energy metabolism disruption), moreover our analyses have shown the deleterious impact of confounding physiological factors on diabetes biomarker discovery –age and BMI-. We finally evaluated their influence on a cohort of 227 CEA employees. Lipidomic fingerprints are robust, however BMI impact is marked for neutral lipids. Gender effect shows significant male catabolism and age altered enzyme activities. These studies combine an overall metabolomics and lipidomics analyses of the same human samples. They aim to build up a relational database including spectral and biological data for biomarker characterization in clinical studies

Le diabète de type 2 (DT2) est associé à plusieurs altérations du métabolisme lipidique ainsi qu’à l’incidence de complications cardiovasculaires. L’hypertriglycéridémie (HTG), caractérisée par une augmentation de la concentration des lipoprotéines riches en triglycérides (LRT), à jeun et en postprandial, ainsi que l’hyperactivité plaquettaire sont des facteurs de risque d’athérothrombose chez les patients DT2. La composition lipidomique des lipoprotéines peut également être modifiée dans un contexte de DT2, ce qui peut, par conséquent, affecter leur fonctionnalité. Notre objectif était de déterminer l’effet des LRT de patientes DT2 à jeun et suite à un repas contenant une matière grasse de source différente (végétale vs laitière) sur l’agrégation de plaquettes saines, ainsi que de déterminer la composition lipidomique, plus particulièrement en sphingolipides (SL), des LRT. D’abord, nous avons montré que les LRT de patients DT2 à jeun activent la voie de signalisation de l’acide arachidonique dans les plaquettes et augmentent leur concentration de thromboxane B2 (TxB2) et leur agrégation. Nous avons ensuite mis en place un protocole clinique en parallèle randomisé visant à étudier les effets des LRT de 30 patientes DT2 à jeun et 4h après l’ingestion d’un petit déjeuner contenant 20g de lipides de source laitière (beurre) ou végétale (pâte à tartiner cacao-noisette) sur les plaquettes et la composition en SL de ces lipoprotéines. Nous avons démontré que l’ingestion de 20g de lipides cause une augmentation des triglycérides et de l’ApoB-48 plasmatiques à 4h et modifie la composition en acides gras (AG) des LRT. De plus, les LRT isolés à jeun et en postprandial augmentent de manière similaire l’agrégation et la concentration de TxB2 plaquettaire. Concernant la composition en SL, la consommation d’une matière grasse riche en AG saturés a augmenté les concentrations en céramides (Cer) totales des LRT, plus précisément au niveau des espèces Cer 16:0, 22:0, 24:1 et 24:0, comparativement aux concentrations à jeun. La composition en SL des LRT de DT2 à jeun est également différente de celle de volontaires saines (augmentation des sphingomyélines, gangliosides GM3, de la sphingosine-1-phosphate et de la lyso sphingomyéline). Ces travaux mettent en évidence l’implication possible des LRT post-prandiales dans le développement de l’athérothrombose chez les patients DT2, ainsi que l’intérêt de l’étude du lipidome des lipoprotéines dans une optique de prévention des complications cardiovasculaires associées au DT2. Les mécanismes sous-tendant les effets de la composition en SL des LRT sur leur interaction avec les plaquettes restent à élucider
Type 2 diabetes (T2D) is associated with impaired lipid metabolism as well as higher incidence of cardiovascular complications. Hypertriglyceridemia (HTG), defined by an increase of triglyceride-rich lipoproteins (TGRL) in the fasting and postprandial states, and platelet hyperactivity are risk factors for atherothrombosis development in T2D individuals. Lipidomic composition of lipoproteins can also be modified in the context of T2D, and therefor affect their functionality. Our objective was to determine the effect of TGRL from T2D patients in the fasting state and after a breakfast containing a fat rich in saturated fatty acids (SFA) from a different source (dairy vs vegetal oil) on aggregation of healthy platelets, as well as assessing the lipidomic composition, especially of sphingolipids (SL), of TGRL. We first showed that TGRL from fasting T2D patients activate the arachidonic acid signaling pathway in platelets and increase their thromboxane B2 (TxB2) concentration and aggregation. We then designed a randomized parallel clinical trial that aimed to study the effect of TGRL from thirty T2D patients in fasting state and 4h after the ingestion of a breakfast containing 20g of lipids from dairy or vegetal oil on platelets, as well as the SL composition of the particles. We showed that the ingestion of 20g of saturated lipids induce an increase in plasma TG and ApoB-48 at 4h and modify the fatty acid (FA) composition of TGRL. Moreover, TGRL isolated in the fasting and postprandial state increase platelet aggregation and TxB2 concentration similarly. Regarding TGRL SL composition, consumption of a fat rich in SFA led to an increase in total ceramides (Cer) content in TGRL, more precisely of Cer16:0, 22:0, 24:1 and 24:0 molecular species. There are also differences in the SL composition of TGRL from fasting T2D patients compared to particles from controls (increase in sphingomyelins, GM3 gangliosides, sphingosine-1-phosphate and lyso sphingomyelin contents).This work highlights the probable implication of TGRL in atherothrombosis development in T2D patients, as well as the relevance of studying lipoproteins lipidome to identify new strategies for the prevention of cardiovascular complications associated with T2D. The mechanisms underlying the effects of TGRL SL composition and their interaction with platelets remain to be elucidated

CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior
FundaÃÃo de Amparo a Pesquisa do Estado do PiauÃ
Objetivo: Investigar o potencial biotecnolÃgico do bagaÃo de uva, variedade Benitaka, cultivada no municÃpio de SÃo JoÃo do PiauÃ, PiauÃ, por meio de metodologias analÃticas cientificamente reconhecidas, buscando reconhecer os principais grupos de metabÃlitos responsÃveis por atividade antioxidante. Metodologia: As uvas (Vitis vinifera L.), variedade Benitaka, foram resultantes da safra 2011/2012 e coletadas no Polo de Viticultura do Assentamento Marrecas, no MunicÃpio de SÃo JoÃo do PiauÃ/PI e posteriormente higienizadas, prensadas em despolpadeira para separaÃÃo do bagaÃo, o qual foi submetido à desidrataÃÃo, trituraÃÃo, peneiramento e formaÃÃo do bagaÃo de uva em pÃ. A partir do bagaÃo de uva em pà foram realizadas anÃlises da composiÃÃo centesimal, conteÃdo de minerais, fibra dietÃtica total, solÃvel e insolÃvel, determinaÃÃo de Vitamina C, conteÃdo de antocianinas, perfil de Ãcidos graxos e investigaÃÃo de sua qualidade microbiolÃgica. Foram tambÃm elaborados extratos a partir de diferentes solventes, os quais foram analisados quanto à toxicidade frente à Artemia salina sp., conteÃdo de fenÃlicos totais, flavonÃides totais e taninos totais; atividade antioxidante in vitro pelos mÃtodos de DPPHâ e autooxidaÃÃo do sistema β-caroteno/Ãcido linoleico; estabilidade oxidativa em Ãleo de soja e identificaÃÃo e quantificaÃÃo de polifenÃis por HPLC-UV. Os resultados foram expressos como mÃdia e desvio padrÃo (n=3) e utilizou-se o programa estatÃstico SAS para AnÃlise de VariÃncia e Teste de Tukey. Adotou-se o nÃvel de significÃncia de 5% (p<0,05). Resultados: Os resultados mostraram que no bagaÃo de uva em pà a quantidade de fibra dietÃtica total (46,17g/100g) se destacou quantitativamente em relaÃÃo aos carboidratos (29,2g/100g), proteÃnas (8,49g/100g) e lipÃdeos (8,16g/100g). O valor energÃtico total encontrado foi de 224Kcal/100g. A fraÃÃo fibra insolÃvel (79%) foi superior à fraÃÃo solÃvel (21%). O conteÃdo de Vitamina C foi de 26,25mg de Ãcido ascÃrbico/100g e de antocianinas, 131mg/100g. Os minerais ferro (18,08mg/100g), potÃssio (1,40mg/100g), zinco (0,98mg/100g), manganÃs (0,82mg/100g) e cÃlcio (0,44mg/100g) estavam presentes em maiores concentraÃÃes. A fraÃÃo lipÃdica foi composta principalmente por Ãcido linoleico (89,61%) e o teor de PUFA (89,61%) >MUFA (21,37%) >SFA (18,46%). O rendimento dos extratos variou de 6,85% a 45,5%, dependendo do solvente de extraÃÃo, sendo que o menor rendimento foi observado no extrato acetÃnico e o maior no extrato metanÃlico. O conteÃdo de fenÃlicos totais, flavonoides totais e taninos totais variaram em funÃÃo do solvente de extraÃÃo. Os extratos etanÃlico e acetÃnico conseguiram estabilizar o radical DPPHâ de forma eficiente, com valores de EC50 de 0,31 Âg/mL e 0,39 Âg/mL, os quais nÃo diferiram estatisticamente dos padrÃes quercetina (0,22 Âg/mL) e BHT (0,11 Âg/mL). Em relaÃÃo à avaliaÃÃo da atividade antioxidante pelo mÃtodo de autooxidaÃÃo do sistema β-caroteno/Ãcido linolÃico, os extratos agiram de forma similar ao antioxidante sintÃtico BHT, com mÃdias de EC50 de 0.34 Âg/mL a 0.36 Âg/mL. O extrato etanÃlico aumentou a vida de prateleira do Ãleo de soja de forma similar ao BHT. O composto fenÃlico presente em maior concentraÃÃo foi isoquercitrina (12,94 mg/100g), seguido de rutina (7,54 mg/100g), quercetina (5,4 mg/100g) e resveratrol (2,5 mg/100g). ConclusÃo: Os resultados mostraram que o bagaÃo de uva representa uma fonte potencialmente importante de nutrientes e compostos fenÃlicos; alÃm de elevado potencial antioxidante, o que contribui para o seu elevado valor como um subproduto de frutos, com possibilidades de comercializaÃÃo como antioxidante natural.

Brown macroalgae elicited great interest due to their nutritional properties and biochemical composition rich in compounds with bioactive properties that can be used in pharmaceutical, nutritional and cosmetical applications. Among the lipids in algae, polar lipids such phospholipids and glycolipids have been associated to anti-inflammatory and antioxidant properties that have triggered the interest of the scientific community. However, the polar lipid composition of brown macrolagae is poorly known. Therefore, this work intended to identify the polar lipid composition of brown macroalgae Sargassum muticum and Bifurcaria bifurcata from the Portuguese coast, and to evaluate their antioxidant properties. The characterization of the polar lipid profile was performed using mass spectrometry coupled with liquid chromatography, HILICESI-MS and MS/MS. The fatty acid profile of these polar lipids was determined by CG-MS. The polar lipid profile of Sargassum muticum and Bifurcaria bifurcata revealed the presence of 176 and 141different molecular lipid species respectively, distributed between glycolipids, phospholipids and betaine lipids. Some of the identified lipid species were previously associated with bioactive properties. Fatty acid profile present in these lipid classes includes polyunsaturated fatty acids (PUFA), namely 18:3 n-3, 18:4 n-3, 20:4 n-6 and 20:5 n-3 that have nutritional value. Both lipidic extracts revealed antioxidant activity, but with better results in the case of S. muticum highlighting best antioxidant results. In a general way, the results obtained showed that both lipid extracts can be promising for pharmaceutical, biomedical and nutritional applications, contributing to the valorisation of these two macroalgae as a source of bioactive and added-value compounds.
As macroalgas castanhas têm vindo a despertar um grande interesse pelas suas propriedades nutricionais e pela sua composição bioquímica nomeadamente pela presença de compostos com propriedades bioativas e por isso, com potencial aplicação nas indústrias farmacêutica nutracêutica e cosmética. Entre estes, os lípidos polares tais como os fosfolípidos e glicolípidos têm sido associados a propriedades antiinflamatórias, antioxidantes, entre outras. No entanto a composição lipídica das algas castanhas é ainda pouco conhecida. Assim, este trabalho teve como objetivo identificar a composição em lípidos polares das macroalgas castanhas Sargassum muticum e Bifurcaria bifurcata presentes na costa Portuguesa e avaliar as suas propriedades antioxidantes. A identificação do perfil lipídico destas algas foi realizada usando uma abordagem lipidómica baseada em espectrometria de massa e acoplada a cromatografia líquida HILIC-ESI-MS e MS/MS. O perfil de ácidos gordos esterificados foi analisado por GC-MS. O perfil de lípidos polares do Sargassum muticum e da Bifurcaria bifurcata revelou a presença de 176 e 141espécies moleculares lipídicas diferentes, respetivamente, distribuídas entre as classes dos glicolípidos, fosfolípidos e betaínas. Entre as espécies lipídicas identificadas, algumas foram já associadas a propriedades bioativas. O perfil em ácidos gordos presente nestas classes lipídicas inclui ácidos gordos polinsaturados (PUFA), nomeadamente o 18:3 n-3, 18:4 n-3, 20:4 n-6 e o 20:5 n-3 com grande valor nutricional. Ambos os extratos lipídicos evidenciaram atividade antioxidante tendo o S. muticum evidenciado valores mais promissores. De uma forma geral, os extratos lipídicos destas duas macroalgas revelaram ser muito promissores para aplicações farmacêuticas, biomédicas e nutricionais, permitindo assim a valorização destas duas macroalgas como fonte de compostos bioativos.
Mestrado em Bioquímica

A Dermatite de Contacto Alérgica, uma das doenças ocupacionais mais comuns, é a patologia resultante de uma resposta imune alérgica após a exposição cutânea a um grande subconjunto de químicos. Actualmente, o potencial sensibilizador cutâneo de químicos é apenas avaliado através de testes in vivo que utilizam animais. Contudo, face à legislação europeia em vigor, que impõe a adopção de novas abordagens, é imperativo desenvolver testes in vitro alternativos e eficazes para a avaliação da sensibilização cutânea. O objectivo deste estudo foi identificar o perfil lipídico de células dendríticas (DCs) e queratinócitos (KCs) e avaliar as alterações que ocorrem no seu perfil após a exposição ao potente estímulo pró-inflamatório, o lipopolissacarídeo (LPS), e ao forte sensibilizador cutâneo (DNFB), a fim de demonstrar o papel dos lípidos e as alterações lipídicas que ocorrem durante a maturação das DCs e identificar bons biomarcadores para posteriormente usar numa abordagem in vitro alternativa. O perfil lipídico foi avaliado por espectrometria de massa, usando uma abordagem lipidómica, que combina TLC e posterior análise por ESI-MS e ESI-MS/MS. Os resultados obtidos neste estudo permitem observar que durante a maturação DC e após exposição ao alergénio ocorrem alterações no conteúdo total de ceramidas e no seu perfil, ocorrendo um aumento das ceramidas C16, C24:1 e C24:0. A identificação e caracterização das alterações lipídicas desencadeadas pelo LPS e sensibilizador cutâneo nos KCs revelam modificações importantes no conteúdo total de fosfatidilserinas e alterações no seu perfil, promovendo uma drástica redução numa das mais abundantes fosfatidilserinas presentes nos queratinócitos, PS(16:0/18:1 ou 16:1/18:0). O padrão de alteração destes lípidos fornece uma fonte extremamente rica de informação para avaliar a modulação de espécies específicas de lípidos induzida pela exposição das DCs e KCs a um sensibilizador cutâneo e ao LPS, que pode ser integrado numa estratégia com vista à redução do uso de animais para avaliar o potencial de sensibilização cutânea.
Allergic contact dermatitis (ACD), one of the commonest occupational diseases, is the clinical condition that can result from an allergic immune response following skin exposure to a large subset of chemicals. There are currently no validated non-animal approaches for the prediction of skin sensitization potential of contact allergens. However, existing and forthcoming European legislation imposes the adoption of new alternative approaches; thus, it is imperative to develop an effective alternative in vitro approach for skin sensitization evaluation. The aim of this study was to identify the lipidomic profile of dendritic cells (DCs) and keratinocytes (KCs), and evaluate the changes that occur in their profile after exposure to the potent pro-inflammatory stimulus, lipopolysaccharide (LPS) and to the strong skin sensitizer (DNFB), in order to demonstrate the lipid role and even lipid changes that occur during DC maturation and disclose good biomarkers for further use in an alternative in vitro approach. The lipid profile was evaluated by mass spectrometry, using a lipidomic approach, that combine TLC and further analysis by ESI-MS and ESI-MS/MS. The results obtained in this study allow observing that during DC maturation and after allergen exposure occur significant changes in the cellular levels of both signalling and structural lipids, mainly in total content of ceramides and in their profile, increasing the C16, C24:1 and C24:0 ceramides. The identification and characterization of lipidic changes triggered by LPS and skin sensitizer in KCs reveal major changes in total levels of phosphatidylserine and their profile, promoting a drastic reduction in one of the most abundant PS presents in KCs, PS(16:0/18:1 or 16:1/18:0). The pattern of change of these lipids give an extremely rich source of data for evaluating modulation of specific lipid species triggered during DCs and KCs exposure to a skin sensitizer and LPS, which could be used in an integrative test strategy towards the reduction of animals use for skin sensitization prediction.

A group of Actinobacteria, termed the mycolata within the order Corynebacteriales, are of industrial interest due to their potential to catabolise diverse organic and inorganic compounds (including toxins), tolerate high substrate concentrations in production-scale fermentation, and utilise waste materials to produce value-added products. The defining feature of this group is possession of a thick layer of long-chained lipids, the mycolic acids (MA), in their cell wall. MA are formed uniquely in the Corynebacteriales by the head-tohead condensation of fatty acids (FA), which occur in the cell wall as free or covalently bound to arabinogalactan. Together with other surface lipids, MA form a barrier which protects cells from noxious chemicals and enzymatic assault. Some members of this group are known to accumulate large amounts of triacylglycerols (TAG) as a storage compound, which makes them attractive targets for the manufacture of biodiesel. To find potential TAG producers which may have attractive biotechnological traits, six sub-Antarctic bacterial isolates from Macquarie Island were selected for further biochemical and genetic characterisation. These strains were previously screened for lysozyme resistance and were presumptive Actinobacteria. 16S rRNA gene sequencing determined that the strains belonged to two genera, Williamsia and Rhodococcus. Bacterial growth rates were determined at culture temperatures of 0 to 45 ˚C and growth temperature ranges were predicted using a nonlinear model. All six isolates were psychrotolerant, with predicted Tmin and Tmax ranges of -12 to 37.5 ˚C. Carbohydrate and nitrogen utilisation testing indicated that all strains exhibited a preference for D-fructose and better growth was observed with ammonium sulphate as N source, whilst four strains could utilise glycerol, a common industrial waste product and therefore an abundant carbon source suitable for industrial-scale fermentation. Carbon chain lengths were between 40-60 for MA and 30-60 for TAG analysed using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), and 12-23 for FA analysed using gas chromatography-mass spectrometry (GC-MS). The genomes of all six bacterial strains were sequenced using MiSeq Illumina technology after optimising culture conditions which facilitated the extraction of high molecular weight DNA. Whole genome sequences of all six strains were assembled using ABySS software and annotated using RAST and the NCBI Prokaryotic Genome Annotation Pipeline. Based on whole genome cluster analysis (pfam function), average nucleotide identity (ANI) and biochemical characteristics, the two Williamsia strains, 1135 and 1138, and two Rhodococcus strains, 1163 and 1168, were identified as novel species; another two Rhodococcus strains, 1139 and 1159, were identified as R. qingshengii and R. erythropolis, respectively. Genome sequences of R. qingshengii 1139 and Williamsia sp. 1138 were analysed manually to identify the genes involved in carbohydrate, nitrogen and MA transportation system. It was hypothesized that lack of glucose-specific phosphotransferase systems (PTS) in the genome of Williamsia sp. 1138 could be the reason for lower growth rate and biomass yield of the bacterial strain on D-glucose-supplemented medium. As some Rhodococcus species accumulate very high amounts of TAG, and they also have MA in the waxy cell envelop as well as membrane phospholipids (PL), it is important to understand how carbon is distributed across different lipid classes if attempting to optimise TAG biosynthesis under controlled fermentation conditions. Most analytical methods for identifying and quantifying different lipid classes involve different extraction, derivatisation, separation and detection techniques for each lipid class, so quantification of all rhodococcal lipids during TAG optimisation is normally not undertaken. Prior to lipid analysis on cells grown under different fermentation conditions, a single-step solvent system was developed using thin layer chromatography-flame ionisation detection (TLC-FID) to separate the major lipid classes in all six bacterial strains. Each lipid class was then quantified by calibrating with commercial standards, using surrogates for compounds if commercial standards were not available. A broad non-polar peak was observed as the major lipid class for all bacterial strains and the controls, Mycobacterium phlei and Corynebacterium glutamicum, which did not elute with the same retention time as the commercial free MA standard prepared from M. tuberculosis. The compounds in this peak were later identified using UPLC-MS/MS and nuclear magnetic resonance (NMR) analyses as MA bound to sugar fragments (likely arabinogalactan). The amount of carbohydrate in the non-polar lipid peak was extremely low (from NMR signal responses) so quantification of MA from peak area detected by TLC-FID was possible using a hydrocarbon surrogate as the standard. R. qingshengii 1139 was selected for proteomic and lipidomic analyses based on its rapid growth rate and higher biomass yields in shake-flask cultures, ability to utilise a broad range of carbon sources and synthesis of relatively high total lipids compared to other strains studied (total lipid of 29.8% of cell dry weight produced by strain 1139 compared to 22.6- 31.4% for the other strains, all of which grew more slowly with lower final biomass yields). Optimisation of carbon source concentration was undertaken in shake-flask cultures prior to culturing in controlled bioreactors using 4% D-fructose as carbon source and 2 g/L (NH\(_4\))2SO\(_4\) as N source. The experimental strategy was to determine the changes in the proteomes across the growth cycle as a preliminary study for future TAG optimisation. Results from TLC-FID analysis demonstrated that bound MA were the dominant lipid over the growth cycle with increasing concentrations per cell dry weight from mid-log to stationary phase but decreasing at late stationary phase. The concentration of polar lipids increased up to late log phase, then dropped at stationary and late stationary phases. In contrast, TAG concentration increased from mid-log to late stationary phase. Major changes were observed for the minor components of odd-chain-length FA analysed using GC-MS, however, the composition of MA and TAG analysed using UPLC-MS/MS did not change significantly over the growth cycle. A total of 1651 protein were detected from the tryptic digest of a cleared lysate of cells disrupted by bead beating (cell free extract) using nanoLC-MS/MS. A total 1297 out of 1651 proteins were obtained after filtering using Perseus software; two-sided t-test was performed for the proteins identified from late log, stationary and late stationary phase samples against mid-log phase samples. The proteins were grouped into 33 classes according to their function (based on BLAST/NCBI/RAST annotation) and a global heat map was constructed using the t-test values of proteins which demonstrated that lipid metabolism, amino acid degradation, nucleic acid degradation and detoxification systems were highly upregulated over the growth cycle. Detailed analysis of the highly upregulated protein groups indicated changes in relative abundance of proteins involved in the underlying pathways, which allowed predictions to be made about a suite of metabolic changes in cells as they moved from growth into stationary phase. These predictions suggest that amino acid and nucleic acid degradation supports bacterial survival at stationary phase, given that no residual D-fructose was detected by stationary phase, and also provides carbon flow into glycolysis to provide energy for further other metabolic pathways, including synthesis of TAG. It was also apparent that lipid degradation was occurring, which may support the hypothesis that cells recycled structural lipids for TAG production at the later stage of growth cycle under the assigned growth conditions. Furthermore, a nitrogen starvation condition was predicted in growth which induced the ureide pathway, which provides the NH3 through uric acid degradation also producesglyoxylate, as the majority of the enzymes of this pathway were detected only at stationary and late stationary phases. Proteins associated with the glyoxylate shunt were either detected or upregulated, suggesting that glyoxylate may be further metabolised through the glyoxylate shunt and other pathways to provide energy to cells. In addition to urea hydrolysing enzymes, a urea ABC transporter system was found highly upregulated with cell age indicating management of excess urea via cellular export. Analysis of proteins involved in defence systems revealed that this bacterial strain uses general stress proteins and glutathione-dependent glyoxalase systems to protect cells from oxidative stress, although chaperone proteins (DnaK and GroES/L) systems were not obviously upregulated at later stages of growth. This study helped us to understand how this oleaginous strain responds under nutritional stress conditions at the later stages of the growth cycle and how a shift of lipid classes between structural and storage lipids may be occurring in response to nutritional availability. The current study is not exhaustive, and additional information on the impacts of stationary phase transition can be gained from the current proteomic data sets; this is a matter for future further analysis, particularly relating to regulators detected in the data. Most of the previous studies demonstrated that TAG is the major lipid produced by Rhodococcus and related species under stressed conditions as quantified by fatty acid methyl esters analysis using GC-MS. However, our study demonstrated that bound MA were more abundant than other lipid classes under the culture conditions studied; this is the first study where all major lipid classes were quantified over the growth cycle. However, we could not separate trehalose dimycolates (TDM) from PL using TLC-FID system developed here, so further studies are required to improve the solvent system used for their separation. MA are known to be the major component of the mycobacterial lipid from many reports in the literature, compromising around 40% of cellular dry weight. In addition to TAG, MA could be potential feedstock for chemicals and fuels which are currently based on petrochemical processing, given that MA structures resemble aliphatic hydrocarbons. Further study is required to improve the solvent system to separate the bound and unbound MA which could also be helpful for better understanding of MA metabolism, the drug target for pathogenic mycobacterium and related species. This is a subject of active interest in medical microbiology. Overall, lipid profiling with proteomic analyses provided a better understanding of the physiological changes which occur over the growth cycle of oleaginous bacterial strain R. qingshengii 1139 and the genome analysis helped us to annotate and predict the function of some unannotated proteins. This study could also be useful for providing insight into how Rhodococcus and related bacterial species survive long periods in the environment under nutritionally-limited condition, as occurs on Macquarie Island from whence the strains were isolated. Furthermore, the conditions for TAG production were not optimised in the current study for the sub-Antarctic strains selected. Further study is required to optimise the culture conditions of Williamsia species for TAG production which has not yet been reported. The Williamsia species are of interest as they grow well on glycerol.

Insects are the most diverse animal group, but there are several aspects of their ecology and physiology that remain unknown or undervalued. The use of insects in animal feed is one such aspect. Over the past five years, this sector has undergone major developments in the western world, with insects currently being used as a source of protein and lipids in aquaculture. The production of marine organisms, in particular, is highly dependent on the supply of omega-3, which comprise a group of fatty acids crucial for these animals and for good human health. While traditionally used insect species are poor in these compounds, aquatic and coastal insects are often rich in polyunsaturated fatty acids, including omega-3. Thus, the objective of this work was to evaluate the lipid profile of two insect species collected at different sites in the Ria de Aveiro lagoon (Portugal), to study their potential in a context of sustainable exploitation of marine resources. This study was performed with a lipidomic approach, utilizing chromatographic techniques coupled to mass spectrometry (LC-MS and GC-MS). Two insects were investigated, namely the bush-cricket Conocephalus fuscus and the fly Machaerium maritimae, the latter in greater detail. The results revealed distinct profiles, but both interesting for different reasons. M. maritimae is particularly rich in omega-3 fatty acids and its lipidome has an exceptionally high molecular diversity. The profile of C. fuscus is less complex but still rich in polyunsaturated fatty acids. Intra-specific variations in the fatty acid profile are partially explained by factors such as geographic distribution and gender. The proximal composition of these insects is also presented. Finally, these results are discussed in the context of the species ecology and its biotechnological application as feed ingredients for aquaculture animals, with a focus on fishes.
Os insetos são o grupo animal mais diverso, mas existem vários aspetos da sua ecologia e fisiologia que permanecem desconhecidos ou desvalorizados. A utilização de insetos como ingredientes na alimentação animal é um desses aspetos. Nos últimos anos, este sector sofreu importantes desenvolvimentos no mundo ocidental, sendo os insetos atualmente utilizados como fonte de proteína e lípidos em aquacultura. A produção de organismos marinhos, em particular, está muito dependente de fontes de ómega-3, os quais constituem um grupo de ácidos gordos fundamentais para estes animais e para a dieta humana. No entanto, as espécies de insetos tradicionalmente utilizadas são pobres nestes compostos, embora existam evidências de que os insetos aquáticos e costeiros são ricos em ácidos gordos polinsaturados, incluindo ómega-3. Assim, o objetivo deste trabalho foi avaliar o perfil lipídico de duas espécies de insetos, recolhidos em diferentes locais na Ria de Aveiro, com o objetivo de explorar as suas potencialidades num contexto de exploração sustentável dos recursos marinhos. Este estudo foi realizado através de uma abordagem lipidómica, recorrendo ao uso de técnicas cromatográficas acopladas a espectrometria de massa (LC-MS e GC-MS). As espécies investigadas foram o grilo Conocephalus fuscus e a mosca Machaerium maritimae, esta última com maior detalhe. Os resultados revelaram perfis distintos. A M. maritimae é particularmente rica em ácidos gordos ómega-3, e apresenta uma diversidade molecular excecionalmente elevada no seu lipidoma. O perfil do C. fuscus, embora sendo mais simples, é também rico em ácidos gordos polinsaturados. As variações intraespecíficas no perfil de ácidos gordos são parcialmente explicadas por fatores como a distribuição geográfica e o sexo dos indivíduos. A composição proximal destes insetos é igualmente apresentada. Por fim, os resultados são discutidos no contexto da ecologia das espécies e sua potencial aplicação biotecnológica como ingredientes na alimentação de organismos marinhos de aquacultura, com foco em peixes.
Mestrado em Ecologia Aplicada

Liczba stron - 199, Liczba tabel - 18, Liczba rycin - 6, Liczba wykresów - 22, Suplement - 23, Bibliografia – 232 pozycji
Tempo metabolizmu podstawowego (BMR) jest cechą ilościową, tj. warunkowaną przez wiele genów oraz zmienną zarówno na poziomie między-, jak i wewnątrzgatunkowym. W 1999 roku Hulbert i Else zaproponowali swoją teorię metronomu błonowego, zgodnie z którą BMR zależy od składu lipidowego błon komórkowych. Mimo iż teoria ta potwierdza się na poziomie międzygatunkowym to w obrębie gatunków zwierząt stałocieplnych jej przewidywania mogą być niespełnione ze względu na mniejszy zakres obserwowanej zmienności, jak i negatywne skutki zwiększonej peroksydacji błon biologicznych. Głównym celem niniejszych badań było ustalenie, czy obserwowane na poziomie wewnątrzgatunkowym różnice w profilu lipidowym błon komórkowych i BMR są spowodowane wpływem polimorfizmu w genach, kodujących enzymy szlaku przemian kwasów tłuszczowych (Fads1, Fads2, Scd1, Elovl1-3,5,6 i Srebf1) oraz sprawdzenie, czy różnice te zmniejszają podatność membran na peroksydację, przy jednoczesnym zachowaniu dużej płynności błon komórkowych, niezbędnej do utrzymania wysokiego BMR. Zsekwencjonowałam wszystkie 9 genów wśród 120 myszy selekcjonowanych na niskie i wysokie BMR. W genach Fads2 oraz Scd1 wykryłam polimorfizmy, które miały wpływ na BMR oraz aktywność kodowanych enzymów, ale nie zmieniały indeksów nienasycenia, oraz peroksydacji kwasów tłuszczowych. Przeprowadzony w niniejszych badaniach test teorii metronomu błonowego nie zdołał jej potwierdzić na poziomie wewnątrzgatunkowym, ale podkreśla rolę desaturacji w szlaku metabolicznych przemian kwasów tłuszczowych i jej wpływ na BMR.
Basal metabolic rate (BMR) is a quantitative trait, i.e. affected by many genes and variable at the inter-, as well as at the intraspecific level. In 1999 Hulbert and Else proposed their “membrane pacemaker theory of metabolism”, according to which BMR is dependent on the lipids content in biological bilayers. Although the theory has been confirmed at the interspecific level, their predictions within a single endothermic species could not be fulfil because of the much narrower range of the observed variation of BMR, as well as the negative results of the increasing of the biological membranes peroxidation. The main goal of present study was to establish whether differences observed at the intraspecific level in the lipid profile of cell membranes and BMR are caused by polymorphism(s) in genes encoding enzymes of the fatty acids metabolic pathway (Fads1, Fads2, Scd1, Elovl1-3,5,6 and Srebf1) and confirmation if these differences decrease the biological bilayers susceptibility to peroxidation with keeping the great cell membranes fluidity, which is necessary in maintaining high level of BMR. I sequenced all 9 genes among 120 mice selected to high and low BMR. I identified polymorphisms in the Scd1 and Fads2 genes witch affected BMR and activity of encoding enzymes, but they did not change the unsaturation and peroxidation indexes. The data presented here did not confirm the membrane pacemaker theory of metabolism, but it supported the role of desaturation in fatty acids metabolic pathway and their impact on BMR.
Wydział Biologiczno-Chemiczny, Instytut Biologii

L'hypercholestérolémie et la forme pathologique qui en résulte, l'athérosclérose, sont fréquemment impliquées dans les accidents cardiovasculaires, actuellement première cause de mortalité dans les pays développés. L'hypercholestérolémie se caractérise par un taux anormalement élevé de cholestérol dans le sang. Le taux de cholestérol sanguin dépend principalement de l'équilibre entre les lipoprotéines de faible densité, les LDL et les lipoprotéines de hautes densités, les HDL. Les apolipoprotéines A-I, B et E (apoA-I, apoB et apoE respectivement) sont les éléments essentiels de la structure des lipoprotéines. Des altérations du métabolisme des apolipoprotéines sont associées à l'hypercholestérolémie et au développement de l'athérosclérose chez l'adulte. Cependant, plusieurs études ont montré que l'athérosclérose pouvait se développer à un âge plus précoce, pendant la période de vie foetale. Il est alors légitime de penser que le métabolisme des apolipoprotéines pendant la grossesse pourrait être impliqué dans le développement de l'athérosclérose au niveau foetal. Les macromolécules comme les lipoprotéines ne peuvent traverser le placenta librement. La synthèse d'apolipoprotéines par le placenta est donc indispensable pour fournir le cholestérol au foetus. Comme la grossesse est caractérisée par une élévation du cholestérol maternel, l'étude de l'expression des apolipoprotéines placentaires suivant le taux de cholestérol maternel est indispensable pour déterminer leur implication dans l'athérosclérose au niveau foetal. Pour réaliser ce projet, à partir de placentas humains prélevés à l'accouchement:, l'expression génétique de l'apoA-I, l'apoB et de l'apoE a été évaluée dans le tissu par la technique de polymérisation en chaîne en temps réel. Puis, la concentration tissulaire en protéine a été déterminée par dosage immuno-enzymatique. Nos résultats ont montré que les mères ayant un taux de cholestérol élevé avaient également une augmentation de cholestérol associé aux LDL et d'apoB dans leur circulation. Dans le placenta, l'hypercholestérolémie maternelle n'affecte ni la transcription des gènes ni les concentrations tissulaires de l'apoA-I et de l'apoE. L'absence de régulation de l'apoA-I et de l'apoE placentaires par le cholestérol maternel est surprenante, cependant, l'exacerbation du système endocrinien pendant la grossesse, notamment des hormones stéroïdiennes, pourrait avoir une influence sur l'expression des apolipoprotéines dans le placenta. Par contre, l'apoB est régulée à la hausse dans le placenta des mères hypercholestérolémiques. Un des mécanismes probable de cette régulation pourrait être l'augmentation, par le cholestérol, de l'activité de la protéine microsomale de transfert des triglycérides. Cette protéine est un élément essentiel dans la régulation de l'expression de l'apoB. De plus, l'augmentation de l'apoB est corrélée au cholestérol total maternel et à la concentration d'apoB dans la circulation maternelle et foetale. Ces résultats suggèrent que l'excès de cholestérol maternel est incorporé majoritairement dans les LDL sans affecter le métabolisme des HDL. Cette étude suggère un rôle potentiel de l'apoB placentaire dans le développement de l'athérosclérose au niveau foetal. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Placenta, Grossesse, Apolipoprotéines, Lipides, Profil lipidique.

Bacillus licheniformis I89 is a Gram-positive endospore-forming bacterium that has the ability to produce various compounds with biotechnological application including proteases, amylases, surfactants and antibacterial. There is no information on the lipid composition of this strain. Some published studies on B. licheniformis species report the composition in fatty acids. However, some branched fatty acids, already described in Gram positive bacteria, have also been detected and are predominant constituents of B. licheniformis I89. These branched fatty acids have been described as beneficial to human health and useful in disease prevention, and thus have potential biotechnological applications. In the present work we studied the lipidome of B. licheniformis I89. The main objectives of the study were: a) to identify the fatty acid (FA) profile of B. licheniformis strain I89 and to evaluate the adaptation of the fatty acid profile in response to the growth temperature (37 and 50ºC) and in the different growth phases (lag, exponential and stationary), b) characterize the lipidome of B. licheniformis I89 in the different phases of growth (lag, exponential and stationary) at 37 ºC and c) evaluate the effect of the antibiotic vancomycin on the alteration of the lipid composition profile of B. licheniformis I89 in the lag and exponential growth phases at 37 ° C. In order to reach the proposed objectives, gas chromatography coupled to mass spectrometry (GC-MS) was used to identify and quantify the FAs and normal phase liquid chromatography using a hydrophilic interaction column coupled to mass spectrometry (HILIC-ESI-MS) and MS/MS was used to identify the polar lipid profile. In addition, GC-MS and HILIC-ESI-MS were also used to analyze the effect of the antibiotic vancomycin on the lipid profile change. The FAs profile of B. licheniformis I89 obtained by GC-MS revealed the predominance of branched FAs of the iso and anteiso series (i-15:0, ai-15:0, i-16:0, i-17:0 e ai-17:0) and a low abundance of saturated FAs (14:0, 16:0 and 18:0) in all the growth conditions.The FA profile showed variation with temperature and also with the growth phases. From lag phase to stationary phase at 50 ° C, there was a decrease of the FAs ai-17: 0 and i-16:0, while the FA i-15: 0 increased, whereas at 37 ° C, there was an increase of FA i-15:0 and i-16: 0 and a decrease of the FA ai-15:0 and ai-17:0. On the other hand, the lipidome of B. licheniformis I89 was identified for the first time by HILIC-ESI-MS and MS/MS. In the lipidome of B. licheniformis I89, four classes of phospholipids were identified: phosphatidylethanolamine, phosphatidylglycerol, lysylphosphatidylglycerol and cardiolipin; two classes of glycolipids: monoglycosyldiglycerol and diglycosyldiglycerol; and two classes of phosphoglyceroglycolipids: mono-alanylated lipoteichoic acid primer and lipoteichoic acid primer. All lipid classes were identified in the three growth phases analyzed, with a significant increase in the lipid species with 30:0 and a significant decrease in the lipid species with 32:0, between the exponential and stationary phases, when compared to the lag phase. In addition, a change in the composition of the lipid profile of B. licheniformis I89 was observed in the presence of vancomycin in the two growth phases (lag and exponential) at 37 ° C with the reduction of the levels of some PG molecular species. Vancomycin acts at the level of inhibition of the synthesis of the cell wall. B. licheniformis I89 is sensitive to this antibiotic and therefore, it seems to affect the membrane lipids. In this particular context the lipidomic approach employed is a very promising tool to study bacterial lipid composition. Since this allows to accurately analyze changes in lipid profile in response to different growth conditions, namely, those observed in the presence of antibiotics. Branched fatty acids have been described as having antitumor activity. Considering that B. licheniformis I89 is rich in branched fatty acids, this bacterium may be used as the source of this type of FA. However, it is still necessary to investigate a possible biotechnological application of these compounds as therapeutic agents.
Bacillus licheniformis I89 é uma bactéria de Gram positivo formadora de endósporos, que possui a capacidade de produzir vários compostos de interesse biotecnológico incluindo proteases, amilases, surfactantes e antibacterianos. Não existe informação sobre a composição lipídica desta estirpe. Alguns estudos publicados sobre outras espécies B. licheniformis reportam apenas a composição em ácidos gordos. Entre os ácidos gordos identificados, estão descritos ácidos gordos ramificados, também encontrados em outras bactérias de Gram positivo. Estes ácidos gordos ramificados estão descritos como sendo benéficos para a saúde humana e úteis na prevenção de doenças, para além das potenciais aplicações biotecnológicas. Assim, o presente trabalho teve como objetivo alargar o conhecimento sobre o lipidoma de B. licheniformis I89, nomeadamente a) identificar o perfil de ácidos gordos de estirpe B. licheniformis I89 e analisar a adaptação do perfil de ácidos gordos em função da temperatura de crescimento (37 e 50 ºC) e nas diferentes fases do crescimento (lag, exponencial e estacionária), b) caracterizar o lipidoma de B. licheniformis I89 nas diferentes fases de crescimento (lag, exponencial e estacionária) a 37 ºC, c) avaliar o efeito do antibiótico vancomicina na alteração do perfil de lípidos de B. licheniformis I89, nas fases lag e exponencial de crescimento a 37 ºC. Para alcançar os objetivos propostos foram utilizadas as metodologias de cromatografia gasosa acoplada à espectrometria de massa (GC-MS) para identificação e quantificação de ácidos gordos e a cromatografia líquida de fase normal, usando uma coluna de interação hidrofílica, acoplada a espectrometria de massa (HILIC-ESI-MS) e MS/MS, para a identificação do perfil de lípidos polares. As mesmas metodologias foram também aplicadas para analisar o efeito de antibiótico vancomicina na alteração de perfil de lípidos da estirpe. O perfil de ácidos gordos (FAs) de B. licheniformis I89 obtido por GC-MS revelou a predominância de FAs ramificados das séries iso e anteiso (i-15:0, ai-15:0, i- 16:0, i-17:0 e ai-17:0) e menor quantidade de ácidos gordos saturados (14:0, 16:0 e 18:0) em todas as condições de crescimento. O perfil de FAs variou com a temperatura e também com as fases de crescimento. Da fase lag para a fase estacionária, a 50 ºC, houve uma diminuição dos ácidos gordos ai-17: 0 e i-16: 0, enquanto que o ácido gordo i-15: 0 aumentou. Para a temperatura de 37 ºC, observou-se um aumento dos ácidos gordos i-15: 0 e i-16: 0 e uma diminuição dos ácidos gordos ai-15: 0 e ai-17: 0. A análise do extrato lipídico por HILIC-ESIMS e MS / MS permitiu identificar o lipidoma de B. licheniformis I89, o qual ainda não tinha sido descrito até a data.No lipidoma de B. licheniformis I89 foram identificadas quatro classes de fosfolípidos: fosfatidiletanolamina, fosfatidilglicerol, lisil-fosfatidilglicerol e cardiolipina; duas classes de glicolípidos: monoglicosildiacilglicerol e diglicosildiacilglicerol; e duas classes de fosfogliceroglicolípidos: primer de ácido lipoteicóico monoalanilado e primer de ácido lipoteicóico. Todas as classes de lípidos foram identificadas nas três fases de crescimento analisadas, tendo-se observado variações na abundância em algumas espécies moleculares. Entre as fases exponencial e estacionária observou-se um aumento significativo nas espécies lipídicas 30:0 e uma diminuição significativa nas espécies lipídicas 32: 0, quando comparadas com as da fase lag. Para além disso, estudou-se ainda a alteração na composição do perfil lipídico de B. licheniformis I89 na presença de vancomicina nas duas fases de crescimento (lag e exponencial) a 37 ºC. Os resultados obtidos permitiram observar uma redução de algumas espécies moleculares de fosfatidilglicerol (PG), em resposta à presença da vancomicina. Uma vez que B. licheniformis I89 é sensível a este antibiótico, esta alteração pode ser justificada pelo facto da vancomicina atuar ao nível da inibição da síntese da parede celular podendo, por isso, afetar os lípidos que a constituem. Neste contexto, a abordagem lipidómica revelou-se uma ferramenta muito promissora no estudo da composição lipídica bacteriana, uma vez que esta permite analisar com precisão a alterações do perfil lipídico em resposta a diferentes condições de crescimento, nomeadamente, as que se observam na presença de antibióticos. Por outro lado, e dado a sua composição em ácidos gordos iso e anteiso, esta espécie de Bacillus pode vir a ser usada como fonte de ácidos gordos ramificados, uma vez que estes têm sido descritos como podendo ter atividade anti-tumoral. Muito embora essa atividade ainda tenha que ser investigada, os resultados obtidos fazem prever uma possível aplicação biotecnológica destes compostos com atividades terapêuticas.
Programa Doutoral em Bioquímica

Circulating lipids play an important role in human physiology and pathophysiology. Lipids, as the major components in various cellular membranes, are involved in homeostatic regulation, particularly in relation to immune function and inflammatory mechanisms. With the growing global prevalence of lifestyle-related diseases, including obesity, diabetes, cardiovascular disorders and cancers, dietary lipids have received a great attention. Long-chain omega-3 polyunsaturated fatty acids (LC n-3 PUFA) have been associated with a broad range of health benefits. The three main LC n-3 PUFA are eicosapentaenoic acid (EPA, 20:5n-3), docosahexaenoic acid (DHA, 22:6n-3) and docosapentaenoic acids (DPA, 22:5n-3). Fish oil and krill oil are currently the most available sources of EPA and DHA as over-the-counter supplements, although other marine sources such as algae oil are also rich in EPA and DHA. Krill oil, derived from Antarctic krill (Euphausia Superba), is rich in EPA and DHA found in phospholipids (predominantly phosphatidylcholine) rather than triacylglycerol (TAG), in which EPA and DHA in fish oil are found. Krill oil also contains astaxanthin, a carotenoid contributing to its red colour which may also have beneficial health effects (Barros et al. 2014a, Pashkow et al. 2008). Despite a number of studies examining the effects of krill oil compared with fish oil on the incorporation of LC n-3 PUFA into different tissues, the outcomes have been conflicting, which might be associated with the different study designs using different chemical forms of fish oil and/or different doses of LC n-3 PUFA, and focusing at different target tissues. The research presented in this thesis consists of nine chapters covering a literature review (Chapter two) and two intervention studies in humans (Chapters four, five, six and seven) which have examined the effect of krill oil compared with fish oil on the incorporation of LC n-3 PUFA into plasma lipid fractions. There were a postprandial and a longer-term (30 days) intervention studies, and both clinical studies were randomised crossover designs involved healthy women (n = 10 and n = 11, respectively). All participants were instructed to maintain the habitual dietary intake and habitual physical activity throughout the interventions. The aim of the postprandial study was to compare the incorporation of LC n-3 PUFA into the plasma and circulating lipids in plasma and chylomicron fractions from five capsules (1 g each) of krill oil compared with five capsules (1g each) of fish oil and 5 g of the olive oil (control) over a 5-hour postprandial period. The second study aimed to investigate the longer-term effect of krill oil supplementation (containing 1,269 mg/d of LC n-3 PUFA including EPA, DHA and DPA) on the plasma LC n-3 PUFA, plasma circulating TAG and inflammatory biomarkers compared with fish oil supplementation (containing the closest possible match to these fatty acids from the capsules, 1,441 mg/d) over a 30-day intervention period. In both studies, lipidomics, was applied to identify the differences in plasma lipid molecular responses between krill oil and fish oil supplementation. Using this technique, a number of plasma lipid classes, and lipid molecular species containing EPA and DHA were identified and quantified. In the 5-hour postprandial study (Chapters four and five), there were no significant differences in the levels of TAG or cholesterol in plasma or chylomicron between the three study oil interventions, although the expected increases in chylomicron TAG were observed in all groups. In comparison to the olive oil, both krill oil (containing 907 mg of LC n-3 PUFA) and fish oil (containing 1,441 mg of LC n-3 PUFA) supplementation significantly increased the level of plasma EPA, which plateaued after three hours; there were no significant differences in the plasma EPA levels between krill oil and fish oil supplementation groups. There were no significant changes in either DHA or DPA between the three groups. Krill oil, with a lower dose of EPA in this study, showed a similar incorporation outcome of EPA into plasma lipids as fish oil. Given that there were 31% less EPA from krill oil, these results indicate a differential extent of incorporation of EPA between krill oil and fish oil, suggesting that EPA from krill oil may be more efficiently incorporated into the plasma than fish oil. The advanced technique for lipidomics was performed by high-performance liquid chromatography-mass spectrometer analysis (HPLC MS/MS), which was able to identify and quantify changes in various lipid molecular species containing LC n-3 PUFA in both the postprandial and the longer-term studies. Therefore, the HPLC MS/MS facilitated a comparison between differences in the individual lipid molecular species between krill oil and fish oil supplementation. A more sensitive setting of HPLC MS/MS was applied to the postprandial data than the longer-term data, based on the settings applied by the research laboratory at Baker Heart and Diabetes Institute where these analyses were conducted. In Chapter five, the postprandial plasma lipidomic changes are reported at hours zero (baseline), 3 and 5. A total of 29 lipid classes (≥ 500 pmol/mL) (for example: TAG, diacylglycerol (DAG), phosphatidylcholine (PC), cholesterol esther (CE)) were identified; six of these including O-linked phosphatidylethanolamine classes had significantly greater the incremental area under the curve from baseline (net iAUC 0-5 h) after krill oil supplementation compared with fish oil supplementation. Over the postprandial period, 56 EPA-containing and 76 DHA-containing molecular species (for example 16:0-20:5-PC, 16:0-18:1-20:5-TAG, 16:0-22:6-PC, 16:0-18:1-22:6-TAG) were significantly increased after both krill oil and fish oil supplementation. There were 33 phospholipid molecular species containing EPA, and 16 of these molecular species, including six ether-phospholipid molecular species had significantly greater increased net iAUC 0-5 h after krill oil than fish oil supplementation. In contrast, for TAG and DAG molecular species containing EPA, seven out of a total of 21 showed significantly increased net iAUC 0-5 h for fish oil compared with krill oil. Put simply, the EPA from krill oil was associated with increases in phospholipid EPA-molecular species, while the EPA from fish oil was associated with increased TAG and DAG EPA-molecular species. There were 49 phospholipid molecular species containing DHA, and 11 of these including six ether-phospholipid molecular species, had significantly greater increased net iAUC 0-5 h after krill oil supplementatin than fish oil supplementation. In a total of 61 AA-containing molecular species (for example 16:0-20:4-PC, 16:1-20:4-DAG) identified, there were 51 phospholipid molecular species containing AA, and seven of these including six ether-phospholipid molecular species, had significantly greater increased net iAUC 0-5 h after krill oil supplementation than fish oil. A novel finding from this postprandial study was that there was a consistent trend that ether-phospholipid classes (O-linked (containing an alkyl bond) or P-linked (containing an alkenyl bond) phosphatidylcholine and phosphatidylethanolamine) were significantly increased after krill oil supplementation, but decreased after fish oil supplementation. Consistently, it was found that EPA- and DHA-containing ether-phosphatidylethanolamines were significantly increased after the krill oil supplementation, but decreased after the fish oil supplementation. While the significance of this finding is not clear, it is worth noting that plasma levels of O- and P-linked phosphatidylethanolamine have been reported to be decreased in a number of disease states including Alzheimer’s disease. Little is known about the origin of these ether-phospholipids in plasma, but the fact that krill oil increased their post-prandial levels and fish oil decreased them is a clear differentiation between these two omega-3 oils. In the longer-term study (Chapters six and seven), EPA, DHA and DPA were significantly increased after both krill oil and fish oil supplementation over the 30-day period (p < 0.001). The main response to the 30-day krill oil supplementation was that the increase of plasma EPA level was significantly greater in the net iAUC 0-30 d than that of fish oil supplementation (p < 0.05). Both krill oil and fish oil significantly reduced plasma TAG over the intervention period (p < 0.05 and p < 0.01, respectively), but no significant differences were observed between the two groups. Over the 30-day intervention period, some plasma pro-inflammatory cytokines including IL-1β, IL-10, IL-4 and IL-5 (p ≤ 0.05) were significantly reduced after krill oil supplementation, while no such changes were found after fish oil supplementation. In Chapter seven, the long-term lipidomic changes (≥ 500 pmol/mL), at days zero (baseline), 15 and 30, are reported. Twenty three EPA-containing and 46 DHA-containing molecular species were significantly increased after both krill oil and fish oil supplementation over the 30-day supplementation period. Among EPA-, DHA-, and DPA-containing molecular species, there were 20 cases of net iAUC 0-30 d significant differences between the two supplementation. Fourteen of these molecular species in phospholipid species, including 12 ether-phospholipid species, had significantly greater increased net iAUC 0-30 d after krill oil than fish oil (p ≤ 0.05) supplementation. Consistently, it was found that EPA- and DHA-containing ether-phospholipid species, including six ether-phosphatidylethanolamines, were significantly increased after the krill oil supplementation, and decreased after the fish oil supplementation. The changes in the ether-phospholipids in the long-term trial were consistent with the changes described in the postprandial trial (Chapter five). These results support strongly the differentiation between krill oil and fish oil although there are still many unanswered questions flowing from this novel finding. What is known about plasmalogens is that they play a role in anti-inflammatory response, which might be linked to the significant decrease in pro-inflammatory cytokines observed in the present study. Overall, both postprandial and longer-term studies demonstrated that EPA from krill oil is efficiently incorporated into plasma, has a similar effect on the plasma TAG-lowering and a greater efficacy on the plasma inflammatory biomarkers when compared with fish oil. No previous studies have investigated plasma lipidomic responses to krill oil and fish oil supplementation in humans. There were significant increases in molecular species containing EPA and DHA following supplementation with krill oil and fish oil over both the postprandial and the longer-term periods. The plasma lipidomic changes of net iAUC over both intervention periods were significantly different between krill oil and fish oil supplementation, particularly for phospholipids (krill oil resulted in a greater increase than fish oil) and TAG (fish oil resulted in a greater increase than krill oil, as described in Chapter five). A novel aspect identified in this study was that krill oil increased ether-phospholipids, particularly ether-linked phosphatidylethanolamine, whereas fish oil decreased ether-phospholipids. The biological relevance of this novel lipidomic finding has yet to be fully explored.

Nitroxidized lipids are recognized as important bioactive molecules in physiological and pathophysiological conditions. They are generated in vivo by the reaction of lipids with reactive nitrogen species (RNS) under nitroxidative stress conditions. Nitrated fatty acids (NO2FAs) are the most studied nitroxidized lipids. NO2FAs were well-characterized in vitro and detected in vivo, in tissues and biofluids. They are associated with relevant biological roles, such as mediating anti-inflammatory, antioxidant, vasculoprotective, anti-hyperglycemic, antitumoral and cytoprotective activities. One of the main mechanisms for these activities is the covalent interaction of NO2FAs with proteins. Nitroxidized phospholipids were recently identified in vivo, namely in phosphatidylcholines and phosphatidylethanolamines. However, this subject remains largely unaddressed, although the first studies indicate that nitroxidized phospholipids also own significant biological activity. Moreover, the ability of nitroxidized phospholipids to form covalent adducts with peptides or proteins is not yet reported. Thus, the main goal of the work developed in this PhD thesis has been to contribute for the advancement of mass spectrometry (MS)-based analysis of nitroxidized phospholipids and their covalent adducts with proteins, by using in vitro models for the generation of new species. Also, the impact of nitroxidative stress in vivo lipidome has was studied by using liquid chromatography (LC)-MS and tandem mass spectrometry (MS/MS) analysis. The data obtained using these approaches were then correlated with the studied pathological conditions, i.e., acute myocardial infarction and cancer-induced cachexia. In this work, state-of-the-art LC-MS and MS/MS platforms were used analysing nitroxidized phospholipids. In vitro assays were used to characterize for the first time nitroxidized species of cardiolipin, and adducts between nitro-phospholipids and peptides representing potential targets in proteins. Up to ten different nitroxidative modifications were identified for cardiolipin by LC-MS and MS/MS, by using their chromatographic behaviour, exact mass measurements and MS/MS fragmentation pathways. Adduction between nitro-phospholipids and glutathione was demonstrated, as confirmed by MS/MS analysis of the adducts using different MS instruments. Analysis of biological samples where nitroxidative stress was expected to occur, namely myocardial infarction and cachexia, was performed by HILIC-LC-MS and MS/MS lipidomics. These studies revealed a strong impact in the lipidic phenotype that allowed to classify between health and disease status. It also allowed to point out molecular species responsive to onset and progress of the disease, as PUFA-containing phospholipids, plasmalogens, and cardiolipins in myocardial infarction, or cardiolipins and phosphatidylserines in cachexia.In conclusion, these results contribute to the study of the effect of nitroxidative stress in the lipidome in different ways. They provide a database and protocols for implementation of in vivo detection of new nitroxidized phospholipids and their adducts with proteins. The profiling of the lipidome in biological samples under nitroxidative stress constitutes a reference to undertaking the challenge of detecting nitroxidized phospholipid in vivo and provided a perspective of the changes occurring in lipidome in clinical situations related to nitroxidative stress. These results are also useful to direct future investigation and contribute for the discussion on the clinical relevance of the modulation of the lipidome in health and disease. Overall, this work provides a step forward in the MS-based analysis and understanding of the effect of nitroxidative stress in lipids and the lipidome, and its potential biological significance, diagnostic value and therapeutic utility.
Os lípidos nitroxidados são moléculas bioativas que desempenham funções importantes em condições fisiológicas e fisiopatológicas. Estes lípidos nitroxidados são formados in vivo pela reação de lípidos com espécies reativas de nitrogénio (RNS), em condições de stress nitroxidativo. Os ácidos gordos nitrados (NO2FAs) são os lipídos nitroxidados mais estudados. Os NO2FAs já foram caracterizados in vitro, e têm sido detetados in vivo, em tecidos e biofluidos. Estas moléculas têm sido associadas a funções biológicas relevantes, mediando atividades anti-inflamatórias, antioxidantes, vasculoprotetoras, anti-hiperglicémicas, antitumorais e citoprotetoras. Um dos principais mecanismos relacionados com estas atividades é a interação covalente dos NO2FAs com proteínas. Os fosfolípidos nitroxidados foram recentemente identificados in vivo, nomeadamente as fosfatidilcolinas e as fosfatidiletanolaminas. No entanto, este campo de investigação permanece pouco explorado, apesar de os primeiros estudos indicarem que os fosfolipídos nitroxidados também possuem atividade biológica significativa. Além disso, a capacidade dos fosfolipídos nitroxidados formarem aductos covalentes com peptídeos ou proteínas ainda não foi reportada. Assim, o principal objetivo do trabalho desenvolvido nesta tese de doutoramento foi o desenvolvimento da análise de fosfolipídos nitroxidados e seus aductos covalentes com proteínas utilizando a espectrometria de massa (MS) e modelos in vitro para a geração de novas espécies. Além disso, foi estudado o impacto do stress nitroxidativo in vivo no lipidoma por análise cromatografia liquída associada à MS (LC-MS) e e espectrometria de massa tandem (MS/MS). Os resultados foram correlacionados com condições patológicas em estdo,i.e., o enfarte agudo do miocárdio e a caquexia induzida por cancro. Assim, neste trabalho, foram utilizadas plataformas de LC-MS e MS/MS de última geração para a análise de fosfolipídos nitroxidados. Foram desenvolvidos ensaios in vitro para caracterizar, pela primeira vez, espécies de cardiolipina nitroxidada bem como aductos entre nitro-fosfolipídos e péptidos representando potenciais alvos em proteínas. Foram identificadas dez modificações nitroxidativas diferentes para a cardiolipina, utilizando a LC-MS e MS/MS utilizando diversos critérios tais como o seu comportamento cromatográfico, medições exatas de massa e as vias de fragmentação em MS/MS. A adição entre nitro-fosfolipídios e a glutationa foi demonstrada, como confirmado pela análise MS/MS dos aductos usando diferentes espectrómetros de massa. O estudo de amostras biológicas ondeé esperada a presença de stress nitroxidativo, nomeadamente, enfarte do miocárdio e caquexia, foi realizado através da análise lipidómica com LC-MS e MS/MS. Esses estudos revelaram um forte impacto do stress nitroxidativo no fenótipo lipídico, que permitiu a diferenciação entre o estado saudável e patológico. Também foi possívelidentificar espécies moleculares que respondem ao início e progressão da doença, como os fosfolipídos contendo ácidos gordos poliinsaturados, plasmalogénios e cardiolipinas em caso de enfarte do miocárdio, ou as cardiolipinas e fosfatidilserinas na caquexia. Em conclusão, estes resultados contribuem para o desenvolvimento da estudo do efeito do stress nitroxidativo no lipidoma em diversas condiçoes fisiológicas e patológicas e constituem um banco de dados e protocolos para a implementação da deteção in vivo de novos fosfolipídios nitroxidados e seus adutos com proteínas. Adicionalmente, a caracterização do lipidoma em amostras biológicas sob stress nitroxidativo supõe uma referência para detetar fosfolípidos nitroxidizados in vivo e fornece uma perspetiva das mudanças que ocorrem no lipidoma, em situações clínicas relacionadas com o stress nitroxidativo. Estes resultados são também úteis para investigações futuras e uma contribuição importante para a discussão da relevância clínica de alteração do lipidoma e sua modelação em situações de stress nitroxidativo. No geral, este trabalho constitui avanço na análise e compreensão do efeito do stress nitroxidativo nos lípidos e no lipidoma, e devido ao seu potencial significado biológico, do seu possível valor em diagnóstico e em terapêutica.
Programa Doutoral em Bioquímica