Lipids are the main component of semipermeable cell membranes and linked to several important physiological processes. Shotgun lipidomics relies on the direct infusion of total lipid extracts from cells, tissues or organisms into the mass spectrometer and is a powerful tool to elucidate their molecular composition. Despite the technical advances in modern mass spectrometry the currently available software underperforms in several aspects of the lipidomics pipeline. This thesis addresses these issues by presenting a new concept for lipid identification using a customized query language for mass spectra in combination with efficient spectra alignment algorithms which are implemented in the open source kit “LipidXplorer”.

Lipids are the main component of semipermeable cell membranes and linked to several important physiological processes. Shotgun lipidomics relies on the direct infusion of total lipid extracts from cells, tissues or organisms into the mass spectrometer and is a powerful tool to elucidate their molecular composition. Despite the technical advances in modern mass spectrometry the currently available software underperforms in several aspects of the lipidomics pipeline. This thesis addresses these issues by presenting a new concept for lipid identification using a customized query language for mass spectra in combination with efficient spectra alignment algorithms which are implemented in the open source kit “LipidXplorer”.

Perfluoroalkyl substances (PFASs) are anthropogenic compounds that have been classed as persistent organic pollutants (POPs) and are found in both commercial and industrial products. PFASs have been detected in different environmental matrices and have been found to bioaccumulate in all trophic levels. The adverse effects that are associated with PFAS exposure include reduced body weight, increased liver weight, hepatocellular hypertrophy, a decrease in serum cholesterol and triglycerides. This project aims to characterize the metabolic changes in lipid metabolism in the liver after exposure to one of the well-studied PFASs, the perfluorooctane sulfonate (PFOS), during the embryo development using the domestic chicken as a model organism. The characterization of the metabolic changes was done by conducting both quantitative lipidomic analysis and semi-quantitative global profiling on extracted lipids from liver homogenates from a former related project looking at fatty acid profiles. The extracted lipids were analyzed using UHPLC/Q-TOF-MS. In the quantitative analysis, the PFOS-treated groups (0.1 ug/g and 1.0 ug/g)exhibited higher lipid concentrations when compared with the solvent control group (5% DMSO) and the untreated group leading to the conclusion that PFOS exposure disrupts the lipid metabolism. When comparing the lipid concentrations between the two PFOS-treated groups (0.1 ug/g and 1.0 ug/g), the majority of the lipids exhibited higher lipid concentrations in the 1.0 ug/g PFOS-treated groups leading to the conclusion that the effect PFOS has on the lipid metabolism is dose dependent. In the global profiling analysis, 63 lipids showed significant differences when comparing the solvent control group with samples either treated with 0.1 ug/g PFOS or 1.0 ug/g PFOS.

Submitted by Rosivalda Pereira (mrs.pereira@ufma.br) on 2017-09-29T17:19:20Z No. of bitstreams: 1 IaraNogueira.pdf: 92869 bytes, checksum: 5e68638ca4fbe0f7a83413724c91dec2 (MD5) / Made available in DSpace on 2017-09-29T17:19:20Z (GMT). No. of bitstreams: 1 IaraNogueira.pdf: 92869 bytes, checksum: 5e68638ca4fbe0f7a83413724c91dec2 (MD5) Previous issue date: 2017-05-25 / Fundação de Amparo à Pesquisa e ao Desenvolvimento Científico e Tecnológico do Maranhão / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Menopause is a physiological process that affects women during their late 40s or early 50s, but it has substantial health consequences, varying from disturbances in the lipid and glucose metabolism to changes in sleep, increasing exposure to cardiovascular disease. The aim of this study was to analyze the lipid profile and cardiovascular risk in pre and post menopause, in order to better understand the possible mechanisms that increase cardiovascular risks after menopause. It was a cross-sectional analytical study, including evaluated 184 women aged to 40 to 65 years. Socio-demographic, clinical, anthropometric and laboratory data were obtained as well as comorbidities, such as diabetes, dyslipidemia, hypertension and metabolic syndrome. The stratification of cardiovascular risk in 10 years was performed using the Framingham equation and the overall risk score. A total of 40 participants blood samples of this total were randomly selected for serum lipidic analysis, using mass spectrometry. Participants mean aged of 50 (SD 6.8) years and 54.8% were defined as postmenopausal. From the Framingham score, most women were classified as low risk, with 95.1% frequency for cardiovascular disease in 10 years in premenopausal women and 74.5% in those postmenopausal, despite the high percentage of risk factors, such dyslipidemia (72.6%), metabolic syndrome (50%), insulin resistance (50.9%) and diabetes (16.7%) in postmenopausal. Stratification by the global risk score was more adequate, that is, 64.6% of premenopausal women and 29.4% of postmenopausal women were classified as low risk, whereas 22% and 62.8% were in the high risk category, respectively. In lipidic analysis, lipid species were found to have increased concentrations in postmenopausal women, with the most notable being ceramides (N.C23: 0.Cer; N.C23:0(OH).Cer and N.C24:0(OH).Cer) with Fold Change of 1.68, 1.59 and 1.58, respectively. It was also observed that 14 metabolites showed a significant difference between pre and post menopause, mainly ceramide species. Strong and positive correlations were identified between several metabolites with fasting glucose, glycated hemoglobin, total cholesterol, LDL and triglycerides. Highlight the associations between the species ceramides (N.C10:0.Cer) and lysophosphatidylethanolamine (LPE.a.C18:0) with fasting glucose (r = 0.83 and r = 0.73, p< 0.05 , respectively) and with glycated hemoglobin (r = 0.81 and r = 0.75, p <0.05, respectively). The data obtained allowed us to conclude that postmenopausal women h, ad a CVD risk that was approximately three times higher than in premenopausal women, and that the Framingham score seemed to underestimate cardiovascular risk in the climacteric, whereas the overall score stratified more adequately once which was consistent with the CVD risk factors observed in this population. However, the main findings of this study were the important lipid changes detected in postmenopausal women, especially in the class ceramides, as well as correlations with classical glycolic and lipid markers that may be useful to investigate diseases associated with this phase. / A menopausa constitui um processo fisiológico que acomete as mulheres entre 40 e 50 anos, porém traz consequências substanciais para a saúde, que variam de distúrbios no metabolismo lipídico e glicídico a alterações no sono, aumentando a exposição para as doenças cardiovasculares. Esta pesquisa teve como objetivo analisar o perfil lipidômico e o risco cardiovascular na pré e pós menopausa, para melhor entender os possíveis mecanismos que aumentam os riscos cardiovasculares após a menopausa. Foi uma pesquisa transversal analítica, que foram avaliadas 184 mulheres, com idade entre 40 e 65 anos. Dados sociodemográficos, clínicos, antropométricos e exames laboratoriais foram obtidos, bem como informações sobre comorbidades, como diabetes, dislipidemia, hipertensão e síndrome metabólica. A estratificação do risco cardiovascular em 10 anos foi realizada utilizando a equação de Framingham e o escore global de risco. Do total das participantes, foram selecionadas aleatoriamente, 40 amostras sanguíneas, para a análise lipidômica, utilizando a técnica de espectrometria de massa. As participantes tinham uma média de idade de 50 anos (DP 6,8), na qual 55% delas estavam na pós-menopausa. Pelo escore de Framingham, a maioria das mulheres foram classificadas em baixo risco, sendo que na pré menopausa a frequência foi de 95,1% e na pós menopausa de 74,5% para doença cardiovascular em 10 anos, apesar do percentual elevado dos fatores de risco, como dislipidemia (72,6%), síndrome metabólica (50%), resistência insulínica (50,9%) e diabetes (16,7%), na pós menopausa. Já a estratificação pelo escore global de risco mostrou que 64,6% das mulheres na pré-menopausa e 29,4% na pós-menopausa foram classificadas como baixo risco, enquanto que 22% e 62,8% estavam na categoria de alto risco, respectivamente. Na análise lipidômica, verificou-se que espécies lipídicas apresentavam concentrações aumentadas na pós menopausa, destacando-se as ceramidas (N.C23:0.Cer; N.C23:0(OH).Cer e N.C24:0(OH).Cer) com Fold Change de 1,68, 1,59 e 1,58, respectivamente. Observou-se também que 14 metabólitos apresentaram diferença significativa entre pré e pós menopausa, principalmente espécies de ceramidas. Foram identificadas correlações fortes e positivas entre vários metabólitos com glicemia em jejum, hemoglobina glicada, colesterol total, LDL e triglicerídeos. Destacamse as associações entre as espécies de ceramidas (N.C10:0.Cer) e lisofosfatidiletanolamina (LPE.a.C18:0) com a glicemia em jejum (r=0,83 e r=0,73; p< 0,05, respectivamente) e com a hemoglobina glicada (r=0,81 e r=0,75; p< 0,05, respectivamente). Os dados obtidos nos permitiram concluir que as mulheres na pós menopausa apresentavam um risco para DCV aproximadamente três vezes maior que na pré menopausa e que o escore de Framingham parece subestimar o risco cardiovascular no climatério, enquanto que o escore global estratifica mais adequadamente, uma vez que foi condizente com os fatores de risco para DCV observados nesta população. Mas, o principal achado deste estudo foram as importantes alterações nos lipídeos detectadas na pós menopausa, especialmente na classe das ceramidas, além das correlações com marcadores glicídicos e lipídicos clássicos que poderão ser úteis para investigar doenças associadas a esta fase.

Cell and organelle membranes consist of a complex mixture of phospholipids that determine their size, shape, and function. Among the distinct types of phospholipids found in membranes of living organisms, phosphatidylcholine (PC) is the most abundant. The rate-limiting step of the predominant pathway for PC synthesis in eukaryotic cells is catalysed by the enzyme, CTP: phosphocholine cytidylyltransferase α (CCTα or PCYT1A). CCTα has a critical role in lipid metabolism and also has direct clinical relevance as mutations in CCTα result in an interesting spectrum of human diseases, such as lipodystrophy with fatty liver, growth plate dysplasia and cone-rod related dystrophy. Numerous biochemical and structural studies on purified CCTα have revealed its membrane-bound activation and suggested that it acts as a lipid compositional sensor, yet the in vivo mechanism of how CCTα senses and regulates PC levels in membranes remains unclear. Here I show that in budding yeast Saccharomyces cerevisiae, Pct1, the yeast homolog of CCTα, is intranuclear and translocates to the nuclear membrane in response to changes in membrane properties and the need for membrane PC synthesis. By aligning imaging with lipidomic analysis and data-driven modelling, Pct1 membrane association is demonstrated to correlate with membrane stored curvature elastic stress estimates. Furthermore, this process occurs inside the nucleus, although nuclear localization signal mutants can compensate for the loss of endogenous Pct1. These data suggest an ancient mechanism by which CCTα senses lipid packing defects and regulates phospholipid homeostasis from the nucleus. Additionally, I identified the importance of mammalian CCTα in early adipogenesis and investigated the enzymatic function of PCYT1A mutants in fibroblasts from lipodystrophic patients. The allele Val142Met is evaluated to be the main cause of loss-of-function in the compound heterozygous mutations by using yeast survival assay. These results collectively provide preliminary evidence for the pathogenicity of PCYT1A mutations in adipose tissue. From yeast to humans, this study uncovers the critical role of Pct1/CCTα in maintaining the internal membrane environment.

Mass spectrometry has emerged as having a vital role in various applications to biochemical fields. In this thesis, we have utilized a variety of mass spectrometry techniques for both bacteriophage proteomics and colostrum and milk lipidomics studies. Our first study was the proteome characterization of Great Salt Lake bacteriophage NS01 with SDS-PAGE GEL to separate the viral proteins and high performance liquid chromatography (HPLC) coupled with an LTQ Orbitrap to identify the proteins after in-gel digestion. In this project, we have successfully identified 11 proteins with high confidence, p-values < 0.01, including coat protein gp88 with a coverage of 91% and tail protein gp86 with a coverage of 40.96%, which facilitated the classification of NS01 as a T7-like phage. Our second study was the discovery of colostrum and milk biomarkers that can be used to predict the likelihood of development of production-related metabolic diseases (PRMDs) in dairy cows through a lipidomics approach. In this study, an electrospray ionization, time-of-flight mass spectrometer was applied to lipid profiling, quantification and significant biomolecule selection. A Q-Star quadrupole, orthogonal time-of-flight mass spectrometer and an Agilent 6530 accurate-mass quadrupole/time-of flight mass spectrometer were both used for lipid biomarker fragmentation and identification. According to linear discriminative statistical modeling, three panels of biomarkers were defined. A combination of 2 milk lipid predictors, including DG18:0/18:0 and TG 18:0/18:0/18:1, provided PRMD predictions with 75.0% sensitivity at 90.0% specificity. A combination of 3 colostrum lipid predictors, including TG16:0/18:1/18:3, DG16:0/16:0 and C40H60NO, provided PRMD prediction with 90.0% sensitivity at 86.4% specificity. Furthermore, a combination of 7 colostrum and milk biomarkers, including calculated differences between 'shared' markers found to be significantly different in both colostrum and milk, provided a predictive sensitivity of 87.5% at a specificity of 100%. Thus, three panels of lipid biomarkers have been discovered in 1-4 day postparturient dairy cow colostrum and milk that can be used to predict resistance or susceptibility prior to onset of clinically apparent PRMDs. These novel lipids could be used as important diagnostic predictors in the future. Therefore, mass spectrometry based proteomics and lipidomics approaches have been efficient tools in the biochemical research described in this thesis.

In biological systems, small changes can have significant impacts. It is, therefore, very important to be able to identify these changes in order to understand what is occurring in the organism. In many cases, this is not an easy task. Mass spectrometry has proven to be a very useful tool in elucidating biological changes even at a very small scale. Several different mass spectrometry based techniques have been developed to discover and investigate complex biological changes. Some of these techniques, such as proteomics, have been through years of development and have advanced to the point that anyone can complete complex analyses of global protein identification and measurement with relative ease. Other techniques are still developing and still have some ground to cover in terms of experimental outcome and ease of execution. Herein we show improvements we have made in high-throughput high-resolution mass spectrometry based techniques to identify and quantify small molecules that are involved in significant biological changes. To begin, we show that our improved high-resolution mass spectrometry based lipidomics techniques are capable of identifying small changes in diseased states that are associated with inflammation, mitochondrial shape and function, and cancer. With our techniques we have been able to extract, identify, and quantify several thousand unique lipid species from complex samples with confidence. Our initial studies looked at global lipidome profiles of differing tissue types from human and mouse biopsies. This was then adapted to compare the global lipidomes of diseased states against healthy states in asthmatic lung tissue, cigarette smoke treated cells, high fat high sugar (HFHS) stressed animals (with and without additional treatment), and in signaling lipids associated with cell death resistance and growth signaling in pancreatic cancer. As a result of our success with lipidomic method improvement we then adapted our techniques and knowledge for use in elucidating small molecule signaling peptides and oxidation changes in proteins. We were able to show that our improved liquid chromatography mass spectrometry based small molecule assays are capable of identifying and quantifying small peptides and protein modifications that would otherwise be undetectable using traditional techniques. This work resulted in the development of a scalable method to detect and quantify the small iron-regulatory hormone known as hepcidin from a variety of samples such as blood, urine, and cell-culture media. We were also instrumental in evaluating and revising a new ultra-high pressure liquid chromatography (UHPLC) system that allows for better separation of analytes from complex mixtures for identification and quantification. Through these advances we hope to aid researchers and clinicians to enable them to use mass spectrometry to further our knowledge about the small but significant changes that regulate complex biological systems.

Hepatitis C virus (HCV) is a leading cause of chronic liver disease. In the developed world, injection drug use (IDU) through sharing of infected needles and other paraphernalia remains the principal risk factor for HCV transmission. Effective but expensive treatment is now possible but there remains a pressing need for a vaccine. A proportion of people who inject drugs (PWIDs) remain uninfected despite HCV exposure from a long history of sharing needles and other paraphernalia. These cases are termed exposed but uninfected (EU) and test negative for both HCV antibodies and RNA and exhibit a phenotype of resistance to HCV infection. Improved understanding of the mechanisms that confer resistance in the EUs has the potential to aid development of an effective vaccine and novel therapeutic strategies. This thesis reports on the findings from 3 different strategies to identify characteristics of HCV resistance. I used urinary metabolomics, serum lipidomics and the study of adaptive and innate immune responses. Each of these methods has demonstrated clear differences between EU cases and healthy controls and/or spontaneous resolvers of HCV infection. Urinary metabolomics suggest a potential role of the gut microbiome, the serum lipidomics showed marked differences in lipid profiles in EU cases pointing towards a perturbed lipid/virus interaction, and the immune studies confirmed previous work identifying low level T cell responses in many EU cases but has also identified a marked upregulation of interferon alpha production to low dose viral RNA in EU cases utilising ELISA assay. In conclusion, this thesis reports data that identifies a number of new findings that provide insight into mechanisms of resistance to HCV infection. My findings suggest that the complex interplay between the virus and lipids together with an upregulated innate immune response may together help determine the outcome following HCV exposure. In summary, studies performed in this thesis have demonstrated that there are different pathways that define the EU phenotype. Despite being a heterogenous subgroup of PWIDs, the EUs are clearly distinct from a healthy control population.

Les plaquettes sont des cellules sanguines anucléées impliquées dans les phénomènes d'hémostase et de thrombose. La majorité des fonctions plaquettaires dépend de leur membrane cellulaire, qui contient de nombreux microdomaines lipidiques ordonnés appelés radeaux lipidiques. Ces microdomaines jouent un rôle central dans toutes les phases de l'hémostase médiées par les plaquettes. Les radeaux lipidiques sont essentiels pour le fonctionnement des récepteurs responsables de l'activation des plaquettes et de la transduction du signal. Le rôle des plaquettes dans la thrombose artérielle est crucial et explique l'intérêt continu de la recherche dans la thérapie antiplaquettaire. Dans ce contexte, nous avons cherché à étudier les radeaux lipidiques comme lieu d'assemblage principal des récepteurs membranaires. Nous avons également cherché à identifier des protéines impliquées dans la fonction des plaquettes, en vue de proposer de nouvelles cibles thérapeutiques. Nous avons utilisé des analyses lipidomiques et protéomiques ainsi que des analyses d'immunoblotting pour identifier les radeaux lipidiques de la membrane des plaquettes et étudier leur organisation dans les plaquettes non stimulées, stimulées et traitées par des antiagrégants plaquettaires. Des détergents, l'ultracentrifugation et les gradients de sucrase ont été utilisés principalement pour le fractionnement de la membrane et l'isolement des radeaux lipidiques. Les principaux résultats de notre travail sont: 1) Élaboration d'une méthodologie pour l'étude des radeaux lipidiques des plaquettes ; 2) Présentation d'un profil global de la composition lipidique et protéique des radeaux lipidiques ; 3) Démonstration de l'impact de l'activation plaquettaire et des antiagrégants plaquettaires sur la réorganisation des radeaux lipidiques ; Et 4) Proposition de nouvelles cibles thérapeutiques potentielles par protéomique et identification de réseau interactif de protéines autour notamment du facteur XIII (FXIII) et de la phosphoprotéine stimulée par vasodilatateur (V ASP). Nos résultats montrent que les radeaux lipidiques peuvent potentiellement être considérés comme nouvelles cibles thérapeutiques pour la découverte de nouveaux antiagrégants plaquettaires. Les études "Omics" sont importantes pour élargir nos connaissances dans ce domaine / Latelets are blood ce lis at the crossroads of both haemostasis and thrombosis. The majority of platelet functions depend on their membrane, which contains numerous, ordered lipid microdomains named lipid rafts. These microdomains play a pivotai role in all phases of platelet­mediated haemostasis. Lipid rafts are a prerequisite for the functioning of receptors in charge of platelet activation and signal transduction. The role of platelets in thrombotic diseases is crucial, and underpins the continue research interest in antiplatelet therapy. ln this context, we aimed to study the lipid rafts of platelet membranes as the principal assembly place of known receptors, and likely also other, unknown elements that participate in the thrombotic function of platelets, with a view to proposing new therapeutic targets. We used lipidomics and proteomics as well as immunoblot analysis to identify lipid rafts and investigate the organization of lipid rafts in resting, stimulated and antiplatelet-treated platelets. Detergents, ultracentrifugation and sucrose gradients were used mainly for membrane fractionatio and isolation of lipid rafts. The main findings of our work are: 1) Development of a framework or guidelines for platelet lipid raft investigation; 2) Presentation of a global profile of the lipid and protein composition of plate let lipid rafts; 3) Demonstration of the impact of activators and inhibitors on the reorganization of platelet lipid rafts; and 4) Suggestion for potential new therapeutic targets by proteomics analysis through interactive network analyzing of coagulation factor XIII (FXIII) and Vasodilator-Stimulated Phosphoprotein (VASP). Our results show that lipid rafts have potential as new therapeutic targets in pharmacological research in antiplatelets. "Omics" studies are important to expand our knowledge in this field

The 21st Century has seen a rise in incidence of complex diseases such as cancer and in the quest to develop essential new therapeutic options, the study of drug-cell interactions can yield powerful information. Acute myeloid leukaemia (AML) is an aggressive cancer that causes life-threatening deficits of functional blood cells in humans for which current treatment options are highly toxic and often poorly tolerated. A combination of two existing drugs, bezafibrate and medroxyprogesterone acetate in a drug redeployment situation has shown promise in vitro and in vivo and further investigations are crucial to elucidate the mode of action of this treatment. This project investigated the mechanistic action of BaP at a cellular level. Orthogonal spectroscopic and mass spectrometric platforms were employed to probe the biochemical composition of two AML cell lines, HL60 and K562 in the presence and absence of this combined drug treatment. Analysis was performed on single living cells, dehydrated cells, fixed cells and cell extracts to give a large and detailed data set. A consideration of the main spectral differences obtained by Synchrotron-FTIR and ATR-FTIR in conjunction with multivariate statistical analysis revealed a significant change to the cellular lipid composition with drug treatment; furthermore, this response was not caused by cell apoptosis. In particular, the ratio of CH2:CH3 was observed to increase with BaP treatment and this was determined to be a significant change in both cell lines (p &lt;0.05). An overall increase in lipid unsaturation suggests that BaP targets cellular lipid biosynthesis. Raman microspectroscopy added a further dimension to the spectroscopic study by providing spatial information of lipid distribution which suggested that BaP-induced saturation change is uniform across a single cell. UHPLC-MS was employed for global metabolomics analysis of AML cell extracts and revealed a number of biochemical pathways that were indicated as targets of BaP therapy in both cell lines. Univariate and multivariate analysis determined statistically significant metabolites for which putative identifications were made. Pyrimidine metabolism was the most significant pathway identified for changes consistent in both HL60 and K562 cell lines. The complementarity of ToF-SIMS and UHPLC-MS provided large coverage of the lipidome of AML cells through untargeted and targeted approaches. For data derived by both techniques, a general increase in polyunsaturated species for BaP treated cell extracts was observed which correlated well with findings from spectroscopic investigations. Adopting a multi-disciplinary approach to cell analysis can afford a powerful insight into understanding drug mode of action at a cellular level and novel information regarding BaP mechanistic action in AML cell lines was revealed. This analytical approach could be extended to the future study of drug-cell interactions for other oncological systems.