Pour enrichir les connaissances scientifiques sur les effets biologiques des radionucléides et risques des contaminations chroniques sur la descendance, une étude multigénérationnelle in vivo d’exposition a été réalisée à doses non toxiques d'uranium. Ce modèle, a permis de suivre les effets biologiques de l’uranium sur trois générations de rats (F0, F1 et F2) par des analyses cliniques et le suivi de marqueurs biologiques. Dans cette étude, des analyses métabolomiques, transcriptomiques et épigénomiques ont été réalisées à partir d’échantillons de sang, d’urine et de rein.Pour la première génération des rats contaminés (F0), des différences dépendant du sexe des animaux sont observables par l’analyse des niveaux d’expression géniques (ARNm et micro-ARN) dans les reins, des profils métabolomiques et biochimiques dans les reins, l’urine et le sang. Aucune modification épigénétique des profils de méthylation de l’ADN rénal n’est à noter. Pour les deux générations suivantes (F1 et F2), un effet multigénérationnel dépendant aussi du sexe des rats est observable au niveau des profils métabolomiques urinaires et rénaux ainsi qu’au niveau des profils épigénétiques de méthylation de l'ADN des reins. Une baisse de poids corporel et des reins a aussi été observée pour la troisième génération de rats chez les mâles (F2).En conclusion, les travaux de cette thèse montrent qu’une contamination chronique à faible dose d'uranium entraine des effets biologiques sur plusieurs générations de rats. Ils sont observables à différents niveaux moléculaires des systèmes de régulation cellulaires et dépendent du sexe des rats. Ces effets, étroitement liés à des systèmes biologiques intégrés, sont utiles à la compréhension des mécanismes biologiques des expositions à l'uranium et à l’évaluation des risques de nocivités à long termes. Dans le domaine de la radioprotection, ces résultats justifient la nécessité de considérer les dimorphismes sexuels des individus et les conséquences des expositions sur les générations à venir. / In order to deepen scientific knowledge regarding biological effects of radionuclides and associated risk to offspring, an in vivo multigenerational study of chronic exposure to a non-toxic dose of uranium was performed by monitoring three generation of rats (F0, F1 and F2). Clinical parameters and biological markers, including metabolomics, transcriptomics and epigenomics high throughput analysis were conducted in blood, urine and kidney samples.For the first generation of contaminated rats (F0) sex-differences to uranium effects were observed in kidney for gene expression (mRNA, miRNA) and in kidney, urine and blood for biochemical parameters and metabolomics profiles. No epigenetic modification of DNA methylation profiles was shown in kidney. For the next two generations (F1, F2), a multigenerational sex-specific effect is observed for both metabolomics and renal DNA methylation profiles of contaminated rats. Moreover, for the last generation of male rats (F2), a decrease of both total body and kidney weight was shown.In conclusion, low-dose chronic contamination of rats to uranium leads to multigenerational effects. Including sex-differences, they can be shown at different molecular levels of the cellular system. Depending of integrated system biology, data of this thesis are useful in the understanding of biological mechanisms of uranium effect and risk of delayed harmful effect. In the field of radiation protection, these results prove the requirement of considering sexual dimorphisms and consequences of such exposures to offspring.

Os estudos de metabolômica ganham importância a cada dia, por ajudarem a explicar processos biológicos em situações normais (fisiológicas) ou patológicas. Oferecem uma nova visão sobre o impacto funcional da expressão gênica, complementando os estudos de sequenciamento gênico, que negligenciam o impacto da exposição ao meio ambiente na etiologia de doenças. A metabolômica tem sido aplicada na toxicologia, mostrando que o perfil metabólico comparativo de duas situações, controle e teste, pode desempenhar um papel importante na descoberta e validação de biomarcadores, além de contribuir para o entendimento e consequente interpretação dos mecanismos de ação tóxica de xenobióticos. Este projeto de pesquisa tem por objetivo utilizar uma abordagem metabolômica para avaliar o impacto de alterações metabólicas no cérebro da prole de camundongos, ocorridas após exposição (via inalação) à fumaça decorrente da queima de maconha (Cannabis sativa). Fêmeas gestantes foram expostas a doses diárias de Cannabis sativa ou ar filtrado durante todo o período gestacional. Após o nascimento, os filhotes machos ao atingirem a idade para o desmame, considerado como fase adolescente, foram separados de suas respectivas mães e expostos à Cannabis sativa ou ar filtrado por mais 60 dias. As amostras de cérebro da prole foram submetidas a análises por cromatografia em fase gasosa acoplada a espectrometria de massas (CG-MS) numa abordagem metabolômica global (untargeted metabolomics). Os perfis metabólicos dos cérebros dos animais expostos (grupo teste) foram comparados com os obtidos na análise do grupo controle (não expostos), sendo identificados os metabólitos discriminadores candidatos. Estavam alterados os metabólitos: isoleucina, uréia, leucina, GABA, ácido succínico, ácido fumárico, serina, treonina, creatinina, ácido glutâmico, ácido acetilaspártico, glicerol -1 -fosfato, ácido ascórbico, tirosina, ácido cítrico, adenina, hipoxantina, inosina e uracila. A partir dos resultados apresentados, pode-se observar, que tanto a exposição gestacional à Cannabis sativa, quanto a exposição da prole na fase adolescente, provocam alterações metabólicas importantes. Os metabólitos significativamente alterados estão envolvidos no ciclo do ácido tricarboxílico, responsável pela respiração mitocondrial, na produção de energia, atuam na biossíntese de aminoácidos, glicólise, estresse oxidativo, podendo alterar o desenvolvimento e maturação do cérebro. Os resultados são preliminares, mas contribuem para o melhor entendimento dos mecanismos toxicológicos envolvidos na exposição crônica causada por essa droga, contribuindo para a prevenção e diagnóstico de danos no desenvolvimento fetal e adolescente. / Metabolomics studies gain importance each day because they help explain biological processes in normal (physiological) or pathological situations. They provide a new insight into the functional impact of environmental gene expression, complementing gene sequencing studies that neglect the impact of environmental exposure on the etiology of diseases. Metabolomics has been applied in toxicology, showing that the comparative metabolic profile of two situations, control and test, can play an important role in the discovery and validation of biomarkers, in addition to contributing to the understanding and interpretation of the toxic action mechanisms of xenobiotics. This research project aims to use a metabolomic approach to evaluate the impact of metabolic changes in the brain of the offspring of mice, which occurred after exposure (via inhalation) to the smoke from the burning Cannabis sativa. Pregnant females were exposed to daily doses of Cannabis sativa or filtered air throughout the gestational period. After birth, male offspring were reached adolescents weaning age were separated from their respective mothers and exposed to Cannabis sativa or to the filtrate for another 60 days. Offspring brain samples were analyzed by gas chromatography coupled to mass spectrometry (GC-MS) in a global metabolomic approach (non-target metabolomics). Metabolic profiles of the exposed animals brains (test group) were compared with those obtained in the control group (non-exposed), and the candidate discriminant metabolites were identified. The metabolites were altered: isoleucine, urea, leucine, GABA, succinic acid, fumaric acid, serine, threonine, creatine, glutamic acid, acetyl aspartic acid, glycerol-1-phosphate, ascorbic acid, tyrosine, citric acid, adenine, hypoxanthine, inosine and uracil were altered. From the results presented, it can be observed that both the gestational exposure in Cannabis sativa and the exposure of the adolescent phase exposure, induced important metabolic alterations. Significantly altered metabolites are involved in the tricarboxylic acid, which are responsible for mitochondrial respiration, in energy production, act at the amino acid biosynthesis, glycolysis, oxidative stress, which may alter the development and maturation of the brain. The results are preliminary but contribute to a better understanding of the mechanisms of toxicity and of fetal and adolescent infection.

A pesquisa bioquímica no campo da Metabolômica/Metabonômica tem se intensificado consideravelmente nos últimos anos, por sua capacidade de adquirir uma grande quantidade de informação a respeito do comportamento de um organismo através de seu metabolismo. Para isso, frequentemente faz uso da aplicação das mais diversas técnicas analíticas, como a Ressonância Magnética Nuclear. A Ivermectina é um fármaco de amplo uso no Brasil, dada a sua eficiência no controle de verminoses e pragas em gado (e humanos) e está aqui inserida no contexto metabolômico/metabonômico dadas as inúmeras violações ocorridas na carne brasileira exportada. A não observância dos períodos adequados de carência para abate dos animais tratados pode refletir seriamente na qualidade destes produtos. Assim, utilizou-se a Ivermectina como forma de provocar mudanças no metabolismo de bovinos e camundongos, procurando-se correlacionar as variações encontradas à dose aplicada. Através de ferramentas auxiliares, como RMN-2D e ferramentas quimiométricas exploratórias, fez-se a avaliação de amostras de plasma sanguíneo e urina bovinos, e plasma sanguíneo de camundongos Balb-C, após administração de Ivermectina. Os resultados obtidos mostram que a Ivermectina tem influência no balanço energético do organismo, interferindo nos níveis de lactato e β-hidróxibutirato, podendo estar ligada ao aparecimento de uma condição metabólica crítica em mamíferos, relacionada à alta concentração de corpos cetônicos na corrente sanguínea dos mesmos. / The biochemical research in the field of Metabolomics/ Metabonomics has grown considerably in recent years because its capability of acquiring a large amount of information about the behavior of an organism through its metabolism. For this, it often applies several analytical techniques such as Nuclear Magnetic Resonance. Ivermectin is a drug widely used in Brazil, for its effectiveness in controlling verminosis and pests in livestock (and humans) and is here inserted in the metabolomic/metabonomic context because of the numerous breaches occurred in brazilian beef exports. Failures to comply with the appropriate withdrawal periods for slaughtering treated animals may reflect seriously on the quality of these products. Thus, we used Ivermectin as a metabolism change inducer in cattle and mice, trying to correlate these variations to the applied dose. Through auxiliary tools such as 2D-NMR and chemometric exploratory tools, we evaluated samples of bovine blood plasma and urine, and blood plasma of Balb-C mice, after Ivermectin administration. The results show that Ivermectin has influence on the organism\'s energy balance, interfering with lactate and β-hydroxybutyrate which can be connected to the onset of a critical metabolic condition in mammals, related to the high concentration of ketone bodies in their blood stream.

A análise da composição química das uvas é de grande importância para avaliar a data da colheita e a produção de vinhos de qualidade. O estudo do metabolismo das uvas é essencial para definirem-se em quais etapas os metabólitos são produzidos, assim como as proteínas e genes que regulam esses processos. Açúcares, polifenóis e ácidos orgânicos são importantes classes de metabólitos relacionados com o desenvolvimento de uvas. Os açúcares são os compostos que primeiramente indicam a data de colheita, sendo substâncias-chave em diversos processos biológicos. Os polifenóis tem se destacado por sua atividade antioxidante, além de participarem dos mecanismos de defesa da planta. Os ácidos orgânicos são responsáveis pela qualidade organoléptica e estão envolvidos em vários processos metabólicos. As proteínas não contribuem de forma significativa para o valor nutricional, mas são importantes marcadores de variedade para verificar adulterações de vinhos. O Rio Grande do Sul é responsável por grande parte das uvas produzidas no país, e recentemente o Vale do São Francisco tem se destacado na produção destas frutas. Em regiões do Sudeste um manejo de podas diferenciado vem sendo feito para a obtenção de uvas com bons índices de maturação e resistência a doenças fúngicas. Uvas produzidas em Água Vermelha e Louveira, interior de São Paulo, foram estudadas durante a maturação com relação a análises físico-químicas, perfil proteômico, por eletroforese bidimensional e espectrometria de massas, e perfil metabolômico de antocianinas, polifenóis não-antociânicos, ácidos orgânicos e açúcares por técnicas cromatográficas e eletroforéticas acopladas a detectores por arranjo de diodos e/ou espectrometria de massas. Os resultados foram analisados por ferramentas de análise multivariada e comparados com uvas maduras das regiões Sul e Nordeste. Foram observadas tendências de agrupamento das uvas verdes devido à maior acidez e das uvas maduras devido à maior concentração de antocianinas e açúcares, sendo que o perfil de antocianinas pode ser utilizado como marcador de origem varietal. Em termos do perfil proteômico foi possível estabelecer diferenças entre variedades de uvas, além de uma tendência com relação à origem geográfica. Foram identificadas 74 proteínas relacionadas principalmente, às funções de defesa e resposta a stress, metabolismo de carboidratos e metabolismo energético. / Analysis of the chemical composition of grapes is very important to evaluate harvest time and production of high quality wines. The study of grape metabolism is essential to define in which stages metabolites are produced, as well as proteins and genes that regulate these processes. Sugars, polyphenols and organic acids are important classes of metabolites related with grape development. Sugars are compounds that primarily indicate harvest time, and they are key substances in various biological processes. Polyphenols have been noted for its antioxidant activity, also for taking part in mechanisms of plant defense. Organic acids are responsible for organoleptic quality, and they are evolved in diverse metabolic processes. Proteins do not contribute significantly to the nutritional value, but they are important variety markers to verify adulterations of wines. Rio Grande do Sul is responsible for most of the grapes produced in Brazil, and recently Vale do São Francisco has received attention because of the production of these fruits. In some regions of Southeast a different pruning handle has started to obtain grapes with good levels of ripeness and resistance in developing fungal diseases. Grapes cultured in Água Vermelha and Louveira, São Paulo State, were studied during ripening in relation to physical-chemical analysis, proteomic profile, by two-dimensional electrophoresis and mass spectrometry; the metabolomic profile of anthocyanins, non-anthocyanin polyphenols, organic acids and sugars by chromatographic and electrophoretic techniques coupled to diode array and/or mass spectrometry detectors. The results were analyzed by multivariate analysis and compared with those of mature grapes from South and Northeast regions. The data show clustering of green grapes due to higher acidity and clusters of mature grapes due to higher anthocyanin and sugars concentrations, and the profile of anthocyanins can be used as a varietal marker. Considering the proteomic profile, it was possible to group different varieties of grapes with a trend in relation to geographical origin being also observed. It was identified 74 proteins related, mainly, to defense and stress response, carbohydrate metabolism and energetic metabolism.

O câncer de bexiga é uma neoplasia urogenital que acomete homens e mulheres, sendo que somente no Brasil 8.600 novos casos ao ano são diagnosticados. Cistoscopia transuretral é a conduta padrão no diagnóstico e acompanhamento do câncer de bexiga. Entretanto, tal procedimento é extremamente invasivo e doloroso além de ter elevado custo e não garantir todos os resultados. Assim, busca-se por marcadores moleculares que possam auxiliar no diagnóstico e progressão do câncer de bexiga, bem como diminuir a necessidade de exames invasivos no acompanhamento de pacientes tratados. Nesse sentido, a urina tem papel de destaque como fonte de biomarcadores devido principalmente ao seu caráter não invasivo. <br /> Nesse trabalho foram utilizadas duas abordagens \'ômicas\': proteômica e metabolômica, para a busca de biomarcadores em urina para o diagnóstico e prognóstico do câncer de bexiga, respectivamente. Com a abordagem proteômica buscou-se apenas por biomarcadores para o diagnóstico da doença e, utilizando as técnicas de eletroforese 2-DE, OFFGEL e MS, juntamente com análise estatística multivariada, foi possível identificar 32 proteínas que apresentam-se como potenciais marcadores para o câncer de bexiga. A abordagem metabolômica foi empregada para a busca de biomarcadores para reincidência e progressão da doença. As técnicas analíticas utilizadas nessa abordagem, LC-MS e CE-MS, mostraram-se complementares e, dos resultados obtidos com ambas e avaliados com análise estatística multivariada foi possível indicar 19 metabólitos para reincidência e 23 metabólitos para progressão do câncer de bexiga. <br /> Assim, neste trabalho explorou-se como as ciências \'ômicas\', a qual abrange técnicas analíticas de high-throughput, estatística multivariada e ferramentas de bioinformática auxiliando na descoberta de potenciais biomarcadores não invasivos para o diagnóstico e prognóstico do câncer de bexiga. Portanto, o presente estudo foi de grande importância e relevância à medida que ilustrou como tais técnicas podem potencialmente auxiliar no diagnóstico e prognóstico de doenças e contribuir para tratamentos personalizados no futuro, indicando a potencialidade de estudos dessa natureza. / Bladder cancer is an urogenital cancer affecting men and women, and just in Brazil 8,600 new cases are diagnosed annually. Transurethral cystoscopy is a standard conduct in the diagnosis and monitoring of bladder cancer. However, this procedure is extremely invasive, painful, expensive and does not guarantee the best results. Thus, the searching for molecular markers may assist in the diagnosis and monitoring of bladder cancer, as well as decreasing the need for invasive tests in the monitoring of patients treatment. In this way, urine shows an important role as a source of biomarkers, mainly due to its non-invasive nature. <br /> In this work we used two \'omics\' approaches: proteomics and metabolomics, to search for biomarkers in urine for the diagnosis and progression of bladder cancer, respectively. The proteomics approach was explored for biomarkers for diagnosing disease. Using 2-DE, OFFGEL electrophoresis, and MS techniques, as well multivariate statistical analysis, they were identified 32 proteins that may be pointed as potential markers for bladder cancer. The metabolomics approach was used to search for biomarkers for progression and recurrence of the disease. The analytical techniques used for this approach, LC-MS and CE-MS, were complementary to each other and the results evaluated with multivariate statistical analysis indicated that 19 metabolites for recurrence and 23 metabolites for progression of bladder cancer could possibly be used for validation studies. <br /> Thus, we demonstrated how the \'omics\' sciences, which include high- throughput analytical techniques, multivariate statistical analysis, and bioinformatics tools, aid in the discovery of potential biomarkers for noninvasive diagnosis, evaluate recurrence and monitor progression of bladder cancer. Therefore, this study was of high relevance to demonstrate the potential of such techniques to contribute to studies of personalized medicine.

Changes to the metabolism of the cardiomyocyte are driven by complex signaling pathways in order to adjust to stress. For instance, HIF-1α is classically known to upregulate glycolytic metabolism to compensate for oxygen deficiency. Other important effects upon glucose metabolism, which we investigate here more extensively, were also observed. Hearts derived from mice with the cardiac-restricted expression of a stabilized form of HIF-1α are remarkably ischemia stress-tolerant. Here, stable isotope-resolved metabolomic analyses were utilized to investigate glucose cardiometabolism remodeling by HIF-1αduring ischemia. We found that 13C-lactate accumulation was significantly elevated in HIF-1α expressing hearts while paradoxically glycogen was maintained to a remarkable extent during an ischemic time course. These findings suggested an unexpected source of glucose in HIF-1α hearts during global ischemia. Accordingly, the presence of gluconeogenesis in hearts was evaluated. Indeed, gluconeogenic intermediates (i.e. m+3) including glucose-6-phosphate [m+3], fructose-6-phosphate [m+3], and fructose 1,6-bisphosphate [m+3] were observed at significantly elevated levels in the ischemic HIF-1α heart. Collectively, these data establish the surprising finding that HIF-1α supports active gluconeogenesis in the heart during ischemia. As less is known regarding the effects of CTRP3 we first tested whether CTRP3 overexpression would protect the ischemic heart. Our data indicate that CTRP3 failed to confer ischemic tolerance in heart ex vivo. However,we were able to show that CTRP3 protected the liver from lipid-induced stress and prevented hepatic lipid accumulation. To further investigate the mechanisms of hepatic protective effect mediated by CTRP3, we identified the receptor and established that CTRP3 increases oxygen consumption in response to lipid overloaded. Lysosomal-associated membrane protein 1 (LAMP-1), In summary, these data indicate that targeted metabolic rearrangements within cardiomyocyte/hepatocyte holds promise for the alleviation of common pathological conditions.

Nicotinamide adenine dinucleotide (NAD+) is a cofactor in hydride transfer reactions and consumed substrate of several classes of glycohydrolyitc enzymes, including sirtuins. NAD+, its biosynthetic intermediates, breakdown products, and related nucleotides (the NAD metabolome) is altered in many metabolic disorders, such as aging and obesity. Supplementation with the novel NAD+ precursor, nicotinamide riboside (NR), ameliorates these alterations and opposes systemic metabolic dysfunctions in rodent models. Based on the hypothesis that perturbations of the NAD metabolome are both a symptom and cause of metabolic disease, accurate assessment of the abundance of these metabolites is expected to provide insight into the biology of diseases and the mechanism of action of NR in promoting metabolic health. Current quantitative methods, such as HPLC, lack specificity and sensitivity to detect distinct alterations to the NAD metabolome. In this thesis, I developed novel sensitive, accurate, robust liquid chromatography mass spectrometry methodologies to quantify the NAD metabolome and applied these methods to determine the effects of disease states and NR supplementation on NAD+ metabolism. My investigations indicate that NR robustly increases the NAD metabolome, especially NAD+ in a manner kinetically different than any other NAD+ precursor. I provide the first evidence of effective NAD+ supplementation from NR in a healthy, 52 year old human male, suggesting the metabolic promoting qualities of NR uncovered in rodent studies are translatable to humans. During my investigation of NR supplementation, my work establishes an unexpected robust, dramatic increase in deamino–NAD+, NAAD, directly from NR, which I argue could serve as an accessible biomarker for efficacious NAD+ supplementation and the effect of disease upon the NAD metabolome. Lastly, I further establish NR as a general therapeutic against metabolic disorder by detailing its ability to oppose aspects of chronic alcoholism and diabetes mellitus.

A variety of unicellular green algae have the ability to photo-produce molecular hydrogen (H2). Using sunlight to power the production of H2 from water is attractive due to the abundant supply of both resources and the potential for the technology to address global warming and energy supply concerns. Increasing levels of H2 production from those currently achievable with algal systems is a necessity for the technology to become economically feasible. Green unicellular algae are rare amongst organisms in that some have an ability to switch to an H2-producing metabolism when environmental conditions become anaerobic. The process of H2 production is greatly accentuated in the light due to the role of the photosynthetic apparatus directing electron flow to hydrogenase enzymes located in the chloroplast. Difficulties in maintaining continuous systems of H2 production largely result from the O2 sensitivity of hydrogenase enzymes. As O2 is generally produced through photosynthesis, the process of H2 production has always been short-lived. Recently, a process of inducing H2 production for several days was accomplished by depriving the growth medium of sulphur (Melis et al., 2000). Lacking sulphur, photosystem II activity diminishes to a point where any O2 evolved is consumed by respiration; this leads to the culture becoming anaerobic and to the onset of H2 production. The method of sulphur depletion has proven to be very useful for studies of H2 production due to enhanced rates over longer time periods being possible. This work was performed to search for new H2-producing Australian algal species and to shed light upon the molecular and biochemical interactions occurring when algal species move from aerobic photosynthetic growth to an anaerobic H2-producing status. An assay to test new species for an H2-producing ability was developed and implemented; leading to the isolation of new H2-producing species from Australian waters. The assay involved purging algal cultures in the dark with N2, sealing them in bioreactors and then exposing them to light. Metabolic profiling performed during this assay revealed cells to rapidly enter a fermentative metabolism upon the onset of anoxia. Acetate, formate and ethanol were key metabolites produced alongside H2 during this period. Metabolomics was used as a tool to understand the biochemical interactions occurring during 120 h of sulphur depleted H2 production. Extraction protocols were developed that allowed the detection and identification of over 100 metabolites using gas chromatography coupled to mass spectrometry, nuclear magnetic resonance spectroscopy and thin layer chromatography. Shifts in primary energy metabolism when cells switch from O2 production to H2 production were revealed. Indications are that both starch and triacylglyceride accumulate during the first 24 h of sulphur depletion prior to anoxia. Following the onset of anoxia, fermentative metabolism begins, H2 is produced and amino acids generally increase. A build-up of toxic fermentative end products and a lack of sulphur are believed to cause the termination of H2 production, rather than a lack of energy reserves. Key achievements of this work have been: • The establishment of an assay that can be used for future bio-prospecting work aimed at finding H2-producing algal species. • The isolation of new H2-producing green algal species from Australian waters. • The establishment of protocols for the extraction of metabolites from small volumes (1 ml) of Chlamydomonas reinhardtii cultures for analysis on a variety of analytical platforms. • The mapping of changes in metabolism of C. reinhardtii during the switch from an aerobic environment to an anaerobic H2-producing environment. • A range of recommendations for future research that may lead to higher H2 production.

<p>Metabolites, small endogenous molecules existing in every living cell, tissue or organism, play a vital role for maintaining life. The collective group of all metabolites, the metabolome, is a consequence of the biochemistry and biochemical pathways that a cell or tissue uses to promote survival. Analysis of the metabolome can be done to reveal changes of specific metabolites which can be a manifestation, a reason or a consequence of for example a disease. The physical chemical diversity amongst these components is tremendous and it poses a large analytical challenge to measure and quantify all of them. Targeting sub groups of the meta­bolome such as specific functional classes has shown potential for increasing metabolite coverage. Group selective labeling with biotin-tags followed by high affinity avidin capture is a well established purification strategy for protein purification.</p><p>The purpose with this project is to explore if it is possible to transfer the avidin biotin approach to metabolomics and use this method for small mole­cules purification. Specifically, this investigation aims to see if it is achievable to make a bio­tinylation of specific functional groups, to increase the sensitivity through reduction of sample complexity in liquid chromatography mass spectrometry metabolomics analyses after high affinity avidin capture. By purifying the analyte of interest and thereby reducing the sample complexity there will be a reduction in ion suppression. The aim is to increase the analytical sensitivity through a reduction in ion suppression during liquid chromatography mass spectrometry analysis.</p><p>Delimitations have been done to only investigate the possibility to obtain a biotinylation of primary amines and amides. As model compounds phenylalanine, spermi­dine, histamine and nicotinamide have been selected.</p><p>The result from this study indicates that it is possible to increase metabolite coverage through biotin labeling followed by high affinity avidin capture. It is a gain in analytical sensitivity of selected model compounds when comparing biotinylation strategy with a control non­biotinylation approach in a complex sample. A broader study of additional model compounds and a method development of this strategy are necessary to optimize a potential future method.</p>

<p>Lung cancer is the cancer form that has the highest mortality worldwide and inaddition the survival of lung cancer is very low. Only 15% of the patients are alivefive years from set diagnosis. More research is needed to understand the biologyof lung cancer and thus make it possible to discover the disease at an early stage.Early diagnosis leads to an increased chance of survival. In this thesis 179 lungcancer- and 116 control samples of blood serum were analyzed for identificationof metabolomic biomarkers. The control samples were derived from patients withbenign lung diseases.Data was gained from GC/TOF-MS analysis and analyzed with the help ofthe multivariate analysis methods PCA and OPLS/OPLS-DA. In this thesis it isinvestigated how to pre-treat and analyze the data in the best way in order todiscover biomarkers. One part of the aim was to give directions for how to selectsamples from a biobank for further biological validation of suspected biomarkers.Models for different stages of lung cancer versus control samples were computedand validated. The most influencing metabolites in the models were selected andconfoundings with other clinical characteristics like gender and hemoglobin levelswere studied. 13 lung cancer biomakers were identified and validated by raw dataand new OPLS models based solely upon the biomarkers.In summary the identified biomarkers are able to separate fairly good betweencontrol samples and late lung cancer, but are poor for separation of early lungcancer from control samples. The recommendation is to select controls and latelung cancer samples from the biobank for further confirmation of the biomarkers.NyckelordLung cancer is the cancer form that has the highest mortality worldwide and inaddition the survival of lung cancer is very low. Only 15% of the patients are alivefive years from set diagnosis. More research is needed to understand the biologyof lung cancer and thus make it possible to discover the disease at an early stage.Early diagnosis leads to an increased chance of survival. In this thesis 179 lungcancer- and 116 control samples of blood serum were analyzed for identificationof metabolomic biomarkers. The control samples were derived from patients withbenign lung diseases.Data was gained from GC/TOF-MS analysis and analyzed with the help ofthe multivariate analysis methods PCA and OPLS/OPLS-DA. In this thesis it isinvestigated how to pre-treat and analyze the data in the best way in order todiscover biomarkers. One part of the aim was to give directions for how to selectsamples from a biobank for further biological validation of suspected biomarkers.Models for different stages of lung cancer versus control samples were computedand validated. The most influencing metabolites in the models were selected andconfoundings with other clinical characteristics like gender and hemoglobin levelswere studied. 13 lung cancer biomakers were identified and validated by raw dataand new OPLS models based solely upon the biomarkers.In summary the identified biomarkers are able to separate fairly good betweencontrol samples and late lung cancer, but are poor for separation of early lungcancer from control samples. The recommendation is to select controls and latelung cancer samples from the biobank for further confirmation of the biomarkers.Nyckelord</p>