Дисертації з теми "Food metabolome"

In recent years, omic sciences have been increasingly employed in many research fields thanks to their high-throughput capabilities and holistic approach. Among the omics sciences, metabolomics and foodomics have recently emerged for the investigation of food and nutrition and their relation to the individual health and wellness status. The analytical platforms used are ideal for non-targeted analysis, due to their capability of detecting and identifying a large set of variables (or metabolites) in complex biological samples. The most employed metabolomics techniques are mass spectrometry and nuclear magnetic resonance spectroscopy, empowered by the advent and advancement of multivariate data analysis. This thesis outlines the analytical pipeline of the foodomic approach and highlights the current challenges in the field, tracing the path of modern foodomics from the definition and description of food quality to the profiling of the human metabolome, and the investigation of the impact of food on human health, the prevention of diseases and the identification of biomarkers of health status. The impact of factors such as genetic modification or farming method was investigated in plant-based foods. And the effect of the food matrix and digestion on the stability and bioaccessibility of specific molecules was assessed. The animal metabolome was also studied, for example investigating the effect of antibiotic treatment on necrotizing enterocolitis as a model for the treatment of this condition in human newborns, too. The human metabolome (plasma, serum, urine) was then explored, firstly to develop specific algorithms for the search of dietary biomarkers in observational studies. Moreover, food intake biomarkers have been discovered in an intervention study (i.e. galactose for milk intake) and will be further investigated. Research was also carried out to investigate on specific disease-related biomarkers and to discover possible trajectories from a disease state to a healthier condition.

In the recent years, the consumer choices have been focused on health-promoting plant-based food and their preferences are oriented towards regional foodstuff from local productions. Therefore, an important factor for vegetables grown Trentino-Alto Adige (Italy) is to point out the added value of alpine farming to evaluate the nutritional values of farming products. Omics technologies (e.g. genomics, transcriptomics, proteomics and metabolomics) are aimed at investigating the assessment of different pools of molecules and how they are translated into the structure, function, and dynamics of a biological system or systems in order to provide a comprehensive characterization of a specific organism. Research use the omics techniques to exhaustively understand the functionality of food components. Several sophisticated chromatographic methods, spectroscopic techniques and chemometric tools are applied to give an insight into a comprehensive overview of the intrinsic quality, typicality and regionality of specific plant-based foods in the present PhD thesis: apples and potatoes. The quality of these foods is evaluated by quantifying the secondary metabolites to investigate their nutraceutical values. The aim of this PhD project is to use several analytical techniques (LC-MS, UV-VIS) that are capable of comprehensively characterizing the food metabolome with particular emphasis on those components with high nutritional values. The data analysis and data handling of omics data requires advanced bioinformatic, statistical, and chemometric tools. Potatoes and apples are chosen as target matrices for these studies for their relevance in the local economy and for the peculiar chemical composition of particular interest for their health-promoting proprieties. The information is acquired using several sophisticated chromatographic and spectroscopic techniques, such as ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC– MS/MS) and UV/VIS. It is integrated to chemometric approaches (principal component analysis (PCA), partial least square regression (PLS), and data fusion) to achieve a comprehensive targeted chemical characterization. The sampling procedures gathers, in the case of the potatoes study, reference cultivars that may be found in the common retailers of Trentino/Alto-Adige and different production areas, the apples of 22 cultivars were harvest from the fields of the Laimburg Research Centre (Vadena, Italy) to guaranty comparability of the obtained data. Our results may be used as solid foundation for a reliable evaluation of apples and potatoes healthy "potential" value based on cutting-edge techniques, which are capable of providing comprehensive data regarding the alpine food quality parameters with high efficiency and reliability

The present Ph.D. work shows some applications on the NMR-based metabolomic approach in food science. The investigated food matrices are largely different, from a manufactured product that undergoes only physical treatments (bottarga), to a manufactured product where biochemical transformations take place (Fiore Sardo cheese), and, finally, a raw food (Argentina sphyraena). All of these food matrices were not chosen by chance, but they represent an important piece of economy of the island of Sardinia, or might be further valorized, gaining more importance in the near future. Indeed, bottarga and Fiore Sardo are typical products exported all over the world, while Argentina sphyraena is a fish a low economic interest, finding no appreciation, at the moment, on the market. The results of this PhD study have contributed with new insights and deeper understanding of the potential perspective of the combined NMR/multivariate methods approach in food science, showing the great versatility of NMR spectroscopy and the strong synergetic relation between NMR and chemometrics. NMR revealed its extraordinary potential, when applied to natural samples and products, while chemometric analytical technique proved to be an essential tool to get information on the properties of interest (e.g., geographical origin for bottarga) based on the knowledge of other properties easily obtained (i.e. NMR spectra). The investigation performed on bottarga demonstrated that a NMR-based metabolomics technique can be a powerful tool for the detection of novel biomarkers and establishing quality control parameters for bottarga. The work presented in this study evidenced the effectiveness of metabolite fingerprinting as a tool to distinguish samples according both to the geographical origin of fish and the manufacturing process. The results relative to the Fiore Sardo showed the potential of the combination of NMR spectroscopy and chemometrics as a promising partnership for detailed cheese analysis, providing knowledge that can facilitate better monitoring of the food production chain and create new opportunities for targeted strategies for processing. Such analysis may be performed in any stage of the cheese manufacturing, allowing for thorough evaluation of every step in the process. Finally, the preliminary results relative to the metabolomic investigation of Argentina sphyraena should certainly serve as a basis for implement a research tool able to provide deeper insights on the biology of this fish species with all advantages offered by the metabolomics approach.

La globalizzazione del mercato agroalimentare ha determinato una crescente attenzione da parte dei consumatori verso i prodotti alimentari, non solo in termini di qualità e di sicurezza, ma anche di origine geografica. Infatti, il territorio d’origine ha un forte impatto sull’alimento, dovuto alle condizioni pedoclimatiche che ne determinano le caratteristiche. Poiché non esistono dei metodi analitici di routine per l’autenticazione della provenienza geografica, lo scopo del progetto di ricerca è quello di determinare l’origine geografica e l’autenticità degli alimenti mediante profiling dei composti fenolici e steroli, grazie all’applicazione di tecnologie omiche, tecniche statistiche e chemometriche. La componente fenolica e/o steroli dei campioni, viene analizzata tramite cromatografia liquida (UHPLC) accoppiata ad uno spettrometro di massa (Q-TOF-MS). I dati così ottenuti, vengono elaborati mediante statistica multivariata. L’applicazione combinata di avanzate tecnologie omiche e tecniche statistiche chemometriche ha portato come risultato l’effettiva identificazione della provenienza geografica e autenticità di numerose matrici alimentari. I dati ottenuti dimostrano che i metaboliti secondari possiedono proprietà discriminanti. L’approccio di metabolomica UHPLC/Q-TOF-MS combinato a una statistica multivariata risulta essere adeguato per identificare potenziali markers. Il lavoro attuale è focalizzato sulla ricerca di nuovi metaboliti che, insieme a fenoli e steroli possano confermare la potenza di questo approccio.
Nowadays, food traceability is a growing consumer interest worldwide. Food traceability could be considered a fundamental tool for ensuring safety and high quality of food. Food quality is based not only on the safety and integrity of food, but also on the authenticity, the genuineness of the raw material and the geographical origin. The aim of the work was to investigate the potential of untargeted metabolomics to ensure the authenticity and traceability of foods. Secondary metabolites, like polyphenols and sterols, could be conveniently used to meet this goal due to their chemical diversity and their responses to environmental stimuli. Samples were analyzed through UHPLC-ESI/QTOF-MS. The obtained data were subjected to multivariate statistical analysis. The obtained results showed that secondary metabolites can be efficiently used for authenticity and traceability purposes, with regards to cultivars and geographical origin. These information confirm the role of environmental factors in shaping the actual profile of secondary metabolites in plant foods. The markers found could be used for a target quantification method, a less expensive and less sophisticated analysis, in order to provide an efficient tool that could help to guarantee food quality on routine basis.

La globalizzazione del mercato agroalimentare ha determinato una crescente attenzione da parte dei consumatori verso i prodotti alimentari, non solo in termini di qualità e di sicurezza, ma anche di origine geografica. Infatti, il territorio d’origine ha un forte impatto sull’alimento, dovuto alle condizioni pedoclimatiche che ne determinano le caratteristiche. Poiché non esistono dei metodi analitici di routine per l’autenticazione della provenienza geografica, lo scopo del progetto di ricerca è quello di determinare l’origine geografica e l’autenticità degli alimenti mediante profiling dei composti fenolici e steroli, grazie all’applicazione di tecnologie omiche, tecniche statistiche e chemometriche. La componente fenolica e/o steroli dei campioni, viene analizzata tramite cromatografia liquida (UHPLC) accoppiata ad uno spettrometro di massa (Q-TOF-MS). I dati così ottenuti, vengono elaborati mediante statistica multivariata. L’applicazione combinata di avanzate tecnologie omiche e tecniche statistiche chemometriche ha portato come risultato l’effettiva identificazione della provenienza geografica e autenticità di numerose matrici alimentari. I dati ottenuti dimostrano che i metaboliti secondari possiedono proprietà discriminanti. L’approccio di metabolomica UHPLC/Q-TOF-MS combinato a una statistica multivariata risulta essere adeguato per identificare potenziali markers. Il lavoro attuale è focalizzato sulla ricerca di nuovi metaboliti che, insieme a fenoli e steroli possano confermare la potenza di questo approccio.
Nowadays, food traceability is a growing consumer interest worldwide. Food traceability could be considered a fundamental tool for ensuring safety and high quality of food. Food quality is based not only on the safety and integrity of food, but also on the authenticity, the genuineness of the raw material and the geographical origin. The aim of the work was to investigate the potential of untargeted metabolomics to ensure the authenticity and traceability of foods. Secondary metabolites, like polyphenols and sterols, could be conveniently used to meet this goal due to their chemical diversity and their responses to environmental stimuli. Samples were analyzed through UHPLC-ESI/QTOF-MS. The obtained data were subjected to multivariate statistical analysis. The obtained results showed that secondary metabolites can be efficiently used for authenticity and traceability purposes, with regards to cultivars and geographical origin. These information confirm the role of environmental factors in shaping the actual profile of secondary metabolites in plant foods. The markers found could be used for a target quantification method, a less expensive and less sophisticated analysis, in order to provide an efficient tool that could help to guarantee food quality on routine basis.

Les indications relatives aux origines (géographique, botanique ou animale) des aliments constituent des critères de vente persuasifs surtout lorsque la provenance est reliée étroitement à la qualité du produit. Les risques de fraudes étant non négligeables, la mise en place de méthodes d’authentification robuste est alors une nécessité. La Résonance Magnétique Nucléaire (RMN) est l’une des techniques analytiques utilisées à cette fin. Elle permet d’identifier des marqueurs d’origines au sein de la matrice en question. A cet égard, les lipides peuvent être considérés comme source quasi-universelle de biomarqueurs grâce à leur présence ubiquitaire dans les matrices alimentaires. Dans ce projet, la spectroscopie RMN a été utilisée pour la caractérisation des lipides et pour l’identification de biomarqueurs métabolomiques et isotopiques qui permettent de remonter à l’origine des produits analysés. L’œuf de poule a servi de matrice modèle pour mener nos investigations. Les analyses des triacylglycérols (TAG), des phospholipides (PL) et du cholestérol ont été réalisées après amélioration des protocoles d’extraction, d’acquisition et de traitement de leurs spectres RMN 1H et 13C. Les données métabisotopomiques (métabolomiques et isotopiques), obtenues de la déconvolution spectrale (utilisées comme variables d’entrée pour la construction de modèles), ont permis la classification des échantillons selon leur origine et selon la race et le système d’élevage des poules. Les variables métabolomiques ont aussi servi à la quantification individuelle des acides gras dans les TAG et les PL, y compris ceux présents en quantités minimes tels que les acides punicique, ruménique et d’Osbond
Origin of food products is an important criterion that affects the costumer’s choice since the food quality is determined, among other factors, by geographical and botanical or animal origins. The risk of fraud being considerable, implementation of robust authentication methods is then an urge. Nuclear Magnetic Resonance (NMR) is one of the analytical tools used for this purpose. It allows the identification of origin-related markers within the studied matrix. In this respect, lipids can be considered as a source of quasi-universal biomarkers due to their ubiquitous presence in food matrices. In this project, NMR spectroscopy was used for characterization of lipids and for identification of metabolomic and isotopic biomarkers tracing back origin of analyzed product. Hen egg served as a model matrix to conduct our investigations. Analyzes of triacylglycerols (TAG), phospholipids (PL), and cholesterol were carried out after improving extraction protocols and acquisition and processing conditions of their 1H and 13C NMR spectra. Metabisotopomic (metabolomic and isotopic) data resulting from spectral deconvolution were used as predictors in multivariate discriminant analysis allowing classification of samples according to their origin, to the hen breed, and to the farming system. Metabolomic variables were also used for the individual quantification of fatty acids within TAG and PL, even those present in minute amounts such as punicic, rumenic and Osbond acids

The effect of dietary carboxymethyl-lysine (CML) on the metabolite profile of plasma was investigated. Mice were fed one of five diets including: AIN93 diet (negative control), a 45% kcal from fat Diet Induced Obesity diet (DIO; positive control); CML0, Total Western Diet (TWD) with low CML; CML1, TWD with medium CML, and CML2, TWD with high CML. In addition, plasma glucose across the five diet groups was also quantitatively measured in this study. According to the results, 93 compounds were detected in the mouse plasma samples using Gas Chromatography-Mass Spectrometry (GC-MS). Among all 93 detected compounds, 49 of them were amino acids, fatty acids, organic acids, or other organic molecules, while 44 of them could not be identified and were considered to be “unknowns”. Four identified metabolites and 10 unknown metabolites were significantly different between the five diets. In addition, only one metabolite, lactic acid, was significantly different between the three CML diets. A principal component analysis (PCA) showed a clear separation of the CML2 diet, or the diet high in CML, from the other diets along the second principal component. The DIO and AIN93 diets were mostly separated by the third principal component. In addition, both PC1 and PC 3 affected CML0 and CML2. Overall, the metabolic profile of plasma was affected by the amount of CML in diet more than the differences between diets. However, further study is warranted to elucidate the specific mechanisms involved in the changes to the metabolome.

Thesis (MSc)--Stellenbosch University, 2002.
ENGLISH ABSTRACT: Developing countries such as South Africa are in dire need of nutritionally adequate dairy food and beverage sources that are ambient stable due to minimal access to refrigeration. One such product is Kefir, a naturally fermented milk beverage that originated in Caucasian China many centuries ago. The microorganisms responsible for fermentation of the milk are held together in a carbohydrate matrix in the form of small grains. These grains are then removed from the beverage prior to consumption, and added to fresh milk for new fermentations. This beverage holds great potential for large scale development due to the self-propagating nature of the grains, the lack of sophisticated equipment and knowledge necessary for production, and the appealing sensory characteristics of this beverage. This study was therefore performed as an initial investigation to determine the optimum fermentation conditions for large-scale grain production and optimal sensory appeal. Kefir grain production was found to be proportional to incubation temperature in the range studied (18°, 22°, 25° and 30°C), with maximum grain biomass increases of 500% for the Kefir incubated at 30°C over the 10 d trial. During fermentation of Kefir grains in milk, lactic acid and other metabolites are produced. Lactic acid results in coagulation of the milk, necessary to provide the characteristic texture and flavour of Kefir, as well as exerting a preservative effect. Lactic acid production was found to be strongly proportional to both incubation temperature and inoculum concentration. The samples containing 2% (w/v) Kefir grain inoculum concentration that were incubated at 25°C for 24 h were found to have optimum lactic acid levels for good quality Kefir (pH of 4.4 - 4.6 and TA of 1.0 - 1.15%). The other metabolites produced during Kefir fermentation are responsible for the specific flavour of Kefir, and include acetaldehyde, diacetyl, ethanol, acetone and 2-butanone. These compounds were studied using headspace gas chromatography over the fermentation period, which yielded good resolution and separation of all these compounds, however, only acetaldehyde, ethanol and acetone were found to be major metabolites in this study, These analytical results were then further compared to sensory results for key identified attributes, as obtained from a trained sensory panel, to enable recommendations for optimum fermentation conditions to be made. The studied attributes included sourness, sweetness, butteriness, creaminess, yoghurt flavour, cowiness, effervescence, yeastiness, smoothness and overall acceptability. It was apparent from this study that correlations between analytical and sensory data could be drawn, and that panellists were particularly accurate in detecting the attribute sourness resulting from the accumulated lactic acid in the Kefir. Overall acceptability also seemed to be intricately linked to the attribute creaminess, hence the regular literature references to full-cream Kefir as optimum for best sensory appeal. From this study, it was evident that Kefir with optimal sensory appeal is obtained with incubation for 18 h at moderate temperatures (22° or 25°C) and grain inoculum concentrations (0.8% w/v).
AFRIKAANSE OPSOMMING: In ontwikkelende lande soos Suid-Afrika, bestaan daar 'n groot behoefte aan voedsame suiwelprodukte wat stabiel is by kamer temperatuur aangesien 'n groot deel van die bevolking beperkte toegang tot verkoelingsfasiliteite het. Een so 'n produk is Kefir, 'n natuurlike gefermenteerde suiwelproduk wat sy oorsprong eeue gelede in China gehad het. Die mikroorganismes wat verantwoordelik is vir die fermentasie, is saamgebind in 'n koolhidraat matriks in die vorm van klein korrels. Hierdie korrels word verwyder uit die drankie voordat dit gedrink word, en word dan weer by vars melk bygevoeg vir 'n verdere fermentasie. Hierdie gefermenteerde produk het baie potensiaal vir massa-produksie, omdat die korrels natuurlik vermeerder, geen gesofistikeerde toerusting of kennis nodig is nie, en die finale produk hoogs aanvaarbare sensoriese eienskappe het. Die doel van die studie was om 'n inleidende ondersoek uit te voer om die optimum fermentasie toestande vir massakweking van korrels en die mees aanvaarbare sensoriese eienskappe te bepaal. Uit hierdie studie is gevind dat Kefirkorrel vermeerdering proporsioneel is tot die verhoging in inkubasie temperatuur in die gebied 18°, 22°, 25° en 30°C, met maksimum biomassa toenames van tot 500% vir Kefir wat vir 10 dae by 30°C geïnkubeer was. Gedurende fermentasie van Kefirkorrels in melk, word melksuur en ander metaboliete gevorm. Melksuur lei tot die verlaging van die pH van die melk, en veroorsaak stolling, wat noodsaaklik is vir die kenmerkende tekstuur en geur van Kefir, maar dien ook as 'n preserveermiddel. Daar is ook gevind dat melksuur produksie 'n direkte verband het met die inkubasie temperatuur en inokulum konsentrasie. Die monsters met Kefirkorrel inokulum konsentrasie van 2% (miv) wat vir 24 h by 25°C geïnkubeer is, het die optimale melksuur konsentrasies vir goeie kwaliteit Kefir bevat (pH van 4.4 - 4.6 en TA van 1.0 - 1.15%). Ander metaboliete wat belangrike geurkomponente van Kefir is, is asetaldehied, diasetiel, etanol, asetoon en 2-butanoon. Hierdie metaboliete is bepaal en geëvalueer met bodamp gaschromatografiese tegnieke gedurende die fermentasie, wat 'n goeie resolusie en skeiding gelewer het. In hierdie studie is slegs asetaldehied, etanol en asetoon as hoof Kefir metaboliete gevind. Die analitiese data is verder vergelyk met die sensoriese data van die hoof sensoriese komponente, soos bepaal deur 'n opgeleide sensoriese paneel, om die mees gunstigde fermentasie parameters te bepaal. Die geëvalueerde eienskappe was suurheid, soetheid, botterigheid, romerigheid, joghurt geur, koeismaak, gas inhoud, gisagtigheid, gladheid en algehele aanvaarbaarheid. Uit hierdie data is gevind dat daar wel 'n sterk korrelasie bestaan tussen die analitiese en sensoriese resultate, en dat paneellede in staat was om die suurheid, as gevolg van die gevormde melksuur, te bepaal. Algehele aanvaarbaarheid is definitief gekoppel aan romerigheid, daarom word volroommelk Kefir verkies bo die wat met afgeroomde melk berei is. Die data uit hierdie studie het ook getoon dat Kefir met optimale sensoriese eienskappe verkry is na 'n inkubasietyd van 18 h by "matige temperature" (22° of 25°C) en 'n Kefirkorrel inokulum van 0.8% (mIv).

Despite the EU prohibition of anabolic substances administered to food producing animals, reports suggest that the use of growth promoting agents continues, pursued by improved animal health and subsequent financial gains. Current screening methods targeting known compounds are insufficient in discriminating endogenous levels from exogenous applications such as oestradiol. Problems also exist in the detection of corticosteroid misuse through long term, low dose dexamethasone and prednisolone administrations. Efforts have moved towards assessment of the biological response of animals to detect growth promoter exposure and methods employing proteomic'and metabolomic markers are needed. An in vivo animal study consisting of twenty-four male beef cattle randomly assigned to four groups (n=6) for experimental treatment over 40 days was conducted; a control group of untreated bovines, and three groups administered oestradiol, dexamethasone or prednisolone at levels known to reflect growth promoting practice. Plasma was collected from each animal throughout the treatment period and assessed for effect-based monitoring. An untargeted assessment of the plasma proteome utilising two-dimensional gel electrophoresis highlighted 22 proteins showing differential expression in treated cattle. Identification of protein markers via LC-MS/MS elucidated contributions to lipid and vitamin metabolism as well as the immune response. Similarly, untargeted metabolomic analysis was conducted via UHPLC-QTof-MS based on reverse phased separation of plasma under positive electrospray ionisation. Results demonstrated metabolite modifications relative to each treatment group and an OPLS-DA model was generated to predict treated from untreated cohorts based on 99 ions of interest. Further statistical analysis found 24 metabolites significantly altered within treated bovines which were putatively identified as mainly lipid components. Further verification of the biomarkers identified was conducted through targeted assessment of the relative levels in additional sample cohorts. A UHPLC-SRM-MS method was developed to detect eight plasma proteins of interest by quantification of tryptic peptides highlighting the use of vitamin-D binding protein, leucine-rich alpha-2-glycoprotein, retinol-binding protein 4 as novel markers responsive to growth promoter treatment. Additionally blood collected at the slaughterhouse was assessed for protein and metabolite perturbations to reflect authentic screening practice.

Metabolomics is defined as the systematic analysis of hundreds or thousands of small metabolites present in a living system. It has emerge as an important field of study along with other, already established ‘omics’ sciences, i.e., genomics, proteomics and transcriptomics. Metabolomics is well established in the field of medicine, drug toxicity and disease diagnosis. Among the existing analytical techniques, NMR is a fast, reproducible and non-destructive technique to construct an informative snapshot of the metabolites under certain conditions. NMR data give metabolic signature information of the samples when it is combined with data preprocessing and chemometric tools, such as multivariate statistical techniques. NMR-based metabolomics is still expanding in the field of the food chemistry. In this context, this Ph.D. thesis is focused on two major aspects, which show applications of NMR-based metabolomics in food chemistry. 1. Many nutraceutical products possess powerful antioxidant activity as demonstrated in many chemical in vitro tests and in several in vivo trials. Nevertheless, the mechanism of their activity is not completely studied in detail. Due to their poor bioavailability and fast metabolism, studies on the in vivo antioxidant effects are still needed. We performed longitudinal experiments on Sprague Dawley (SD) rats using two commonly available nutraceutical antioxidant products, namely, Curcumin (chapter 2) and Resveratrol (chapter 3). The effects of different doses of orally administered standardized antioxidant extracts in healthy rats were investigated by untargeted metabolomic analysis based on LC-MS and NMR spectrometry. The experiments were carried out over different periods of time for different antioxidants. Changes in the urinary metabolome were evaluated by monitoring the 24-hour urine composition by 1H-NMR and HPLC-MS. The two different approaches were able to detect variations in the urinary levels of antioxidant markers, leading to the observation of different metabolites thus proving the complementarity of these two analytical techniques for metabolomic purposes. Analytical tools such as MS and NMR spectroscopy in combination with chemometrics can profile the impact of time, stress, nutritional status, and environmental perturbations on hundreds of metabolites simultaneously. This results in complex, massive data sets that must be analyzed through a careful statistical protocol. Our strategy included data preprocessing, data analysis and validation of statistical models. After several data processing steps, principal component analysis (PCA) and partial least-squares (PLS) were used to identify urine biomarkers. The PLS models were validated by permutation tests and critically important variables were validated through univariate analysis. 2. The second part of this thesis (chapters 4 and 5) describe the use of NMR-based metabolomics as a fast, convenient, and effective tool for origin discrimination and biomarker discovery in food analysis. Traditionally, the determination of the floral origin of honey is made from palynological analysis. The method is based on the identification of pollen by microscopic inspection. However, melissopalynological analysis needs expertise and also it is not a very reliable technique for the discrimination of botanical origin of some types of honey. Also, honey regulation in the EU (Codex Alimentarius Commission 2001; European Commission 2002) emphasizes that the botanical and geographical origins of the product must be printed on the label in order to avoid the fraud such as adulteration with industrial sugar, selling product under false name or mixing the honey of different floral origin. Therefore, there is need to establish a method to discriminate honey from different origin. The aim of this work was to develop an NMR-based metabolomic approach that used multivariate statistical analysis to discriminate the botanical (chapter 4) and entomological (chapter 5) origin of different types of honey. Multivariate statistical analysis helped us to identify the most relevant signals to differentiate honey botanically and entomologically. The obtained data sets were useful in the search of markers responsible for the discrimination of different honey samples from different botanical species and produced by different bee species.
La metabolomica è definita come l'analisi sistematica di centinaia o migliaia di piccoli metaboliti presenti in un sistema vivente. È emerso come un importante campo di studio insieme ad altre, già affermate scienze "omiche", vale a dire, genomica, proteomica e trascrittomica. La metabolomica è ben consolidata nel campo della medicina, nello studio della tossicità di farmaci e nella diagnostica. Tra le tecniche analitiche esistenti, NMR è veloce, riproducibile e non distruttiva, utile per fornire una fotografia informativa sui metaboliti in determinate condizioni. Dati NMR forniscono informazioni metaboliche che caratterizzano i campioni quando combinati con una pre-elaborazione dei dati e con strumenti chemiometrici, come le tecniche di statistica multivariata. La metabolomica basata sull’NMR è ancora in espansione nel campo della chimica degli alimenti. In questo contesto, questa tesi di Dottorato si concentra su due aspetti principali, che mostrano applicazioni della metabolomica basata sull’NMR in chimica degli alimenti. 1. Molti prodotti nutraceutici possiedono potente attività antiossidante, come dimostrato in molti test chimici in vitro e in diverse prove in vivo. Tuttavia, il meccanismo della loro attività non è completamente studiato in dettaglio. A causa della loro scarsa biodisponibilità e metabolismo veloce, sono ancora necessari studi in vivo sugli effetti antiossidanti. Abbiamo condotto esperimenti longitudinali su ratti Sprague Dawley (SD) utilizzando due prodotti antiossidanti nutraceutici comunemente disponibili, vale a dire, curcumina (capitolo 2) e resveratrolo (capitolo 3). Gli effetti di diverse dosi di estratti antiossidanti standardizzati somministrati per via orale nei ratti sani sono stati studiati mediante analisi metabolomica non mirata (untargeted) basata su LC-MS e spettrometria NMR. Gli esperimenti sono stati eseguiti lungo diversi periodi di tempo per diversi antiossidanti. Le variazioni del metaboloma urinario sono state valutate attraverso il monitoraggio della composizione delle urine di 24 ore usando 1H-NMR e HPLC-MS. I due differenti approcci sono stati in grado di rilevare le variazioni dei livelli urinari di marcatori antiossidanti, portando all’osservazione di diversi metaboliti e dimostrando così la complementarità di queste due tecniche analitiche per scopi metabolomici. Strumenti di analisi come la spettroscopia NMR e MS in combinazione con chemiometria possono delineare l'impatto del tempo, dello stress, dello stato nutrizionale, e di perturbazioni ambientali su centinaia di metaboliti contemporaneamente. Ciò comporta complessi enormi set di dati che devono essere analizzati mediante un protocollo statistico accurato. La nostra strategia ha compreso una pre-elaborazione dei dati, l’analisi dei dati e la validazione dei modelli statistici. Dopo varie fasi di elaborazione di dati, l’analisi delle componenti principali (PCA) e l’analisi dei minimi quadrati parziali (PLS) sono state utilizzate per identificare i biomarcatori urinari. I modelli PLS sono stati convalidati dai test di permutazione e le variabili di importanza critica sono stati convalidati attraverso analisi univariata. 2. La seconda parte di questa tesi (capitoli 4 e 5) descrivono l'uso di metabolomica basata su NMR come strumento veloce, conveniente ed efficace per la discriminazione di origine e la scoperta di biomarcatori in analisi degli alimenti. Tradizionalmente, la determinazione dell'origine floreale del miele è condotta mediante analisi palinologica. Il metodo si basa sulla individuazione di polline mediante ispezione microscopica. Tuttavia, l'analisi melissopalinologica richiede perizia ed inoltre non è una tecnica molto affidabile per la discriminazione di origine botanica di alcuni titpi di miele. Inoltre, la regolamentazione del miele nell'Unione Europea (Codex Alimentarius 2001; Commissione Europea 2002) sottolinea che le origini botaniche e geografiche del prodotto devono essere stampate sull'etichetta, per evitare frodi, come l'adulterazione con zucchero industriale, vendita di prodotti sotto falso nome o aggiunte di miele di diversa origine floreale. Pertanto, vi è la necessità di stabilire un metodo per distinguere miele di diverse origini. Lo scopo di questo lavoro è stato quello di sviluppare un approccio metabolomico basato sull’NMR che ha utilizzato l'analisi statistica multivariata per discriminare l'origine botanica (capitolo 4) ed entomologica (capitolo 5) di diversi tipi di miele. statistica multivariata ci ha aiutato ad identificare i segnali più importanti per differenziare il miele sia dal punto di vista botanico che entomologico. I set di dati ottenuti sono stati utili nella ricerca di marcatori responsabili della discriminazione dei diversi campioni di miele di diverse specie botaniche e prodotti da diverse specie di api.

Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第21375号
農博第2299号
新制||農||1067(附属図書館)
学位論文||H30||N5148(農学部図書室)
京都大学大学院農学研究科食品生物科学専攻
(主査)教授 入江 一浩, 教授 橋本 渉, 准教授 後藤 剛
学位規則第4条第1項該当

La immunoteràpia nutricional es basa en fomentar la salut mitjançant el consum de compostos bioactius presents de manera natural en els aliments, com les procianidines, un tipus de flavonoid, i/o l’àcid docosahexaenoic (DHA), un àcid gras omega-3, optimitzant la funcionalitat del propi sistema immune i millorant així el seu paper com a responsable de la preservació de l’organisme enfront desestabilitzadors de la homeòstasis. La recerca que aquesta tesi abasta es centra en el paper del perfil nutricional en la regulació de la interacció immunitat-metabolisme (immunometabolisme). Amb aquesta finalitat, en la primera part del treball presentat en aquesta tesi, es va determinar si els macròfags, les cèl•lules efectores de la immunitat innata, poden percebre de manera diferencial la composició de la dieta. Hem demostrat que els compostos bioactius dels aliments modulen, a nivell molecular, l’activació dels macròfags. A més, aquest efecte immunomodulador és dependent del compost, on factors intrínsecs com les propietats químiques o nutritives són determinants per la seva bioactivitat. La segona part va tenir com a objectiu determinar el paper primordial del patró nutricional en la regulació de la interacció entre el sistema immune i el metabolisme. Utilitzant un model d’obesitat induïda per la dieta es va inferir que, mentre una dieta hipercalòrica provoca un deteriorament de l’immunometabolisme, el consum de bioactius pot enfortir-ne la relació. A la tercera part, es va analitzar el paper de les interaccions gen-dieta en l’expressió fenotípica dels trets associats amb la obesitat. Per aquest motiu, dos races de rates congènites, fenotípicament diferents, van ser sotmeses a un desequilibri immunometabòlic com a conseqüència de la ingesta d’una dieta hipercalòrica. Es va deduir que les interaccions entre els factors genètics i nutricionals són fonamentals per a la susceptibilitat d’un genotip a l’obesitat induïda per la dieta. Hem establert que el perfil nutricional és una eina poderosa per orientar la funcionalitat de l’eix immunometabòlic. A més, arribem a la conclusió que compostos bioactius presents en els aliments poden millorar-ne l’eficiència d’aquest eix, promovent així un estat saludable.
La immunoterapia nutricional se basa en fomentar la salud mediante el consumo de componentes bioactivos presentes de forma natural en los alimentos, como las procianidinas, un tipo de flavonoides, o el ácido docosahexaenoico (DHA), un ácido graso omega-3, optimizando la funcionalidad del propio sistema inmune y mejorando así su papel como responsable de la preservación del organismo frente a desestabilizadores de la homeostasis. La investigación que engloba esta tesis se centra en el papel del perfil nutricional en la regulación de la interacción inmunidad-metabolismo (immunometabolismo). Con esta finalidad, en la primera parte del trabajo presentado es esta tesis, se determinó si los macrófagos, las células efectoras de la inmunidad innata, pueden percibir de manera diferencial la composición de la dieta. Hemos demostrado que los compuestos bioactivos de los alimentos modulan, a nivel molecular, la activación de los macrófagos. Además, este efecto immunomodulador es dependiente del compuesto, donde factores intrínsecos tales como sus propiedades químicas o nutritivas son determinantes para su bioactividad. La segunda parte tuvo como objetivo determinar el papel primordial del patrón nutricional en la regulación de la interacción entre el sistema inmune y el metabolismo. Utilizando un modelo de obesidad inducida por la dieta se infirió que, mientras una dieta hipercalórica provoca un deterioro del immunometabolismo, el consumo de bioactivos pueden fortalecer su estrecha relación. En la tercera parte, se analizó el papel de las interacciones gen-dieta en la expresión fenotípica de los rasgos asociados con la obesidad. Para ello, dos razas de ratas congénitas, fenotípicamente diferentes, fueron sometidas a un desequilibrio immunometabólico como consecuencia de la ingesta de una dieta hipercalórica. Se dedujo que las interacciones entre los factores genéticos y nutricionales son fundamentales para la susceptibilidad de un genotipo a la obesidad inducida por la dieta. Hemos establecido que el perfil nutricional es una herramienta poderosa para orientar la funcionalidad del eje immunometabólico. Además, llegamos a la conclusión que compuestos bioactivos presentes en los alimentos pueden mejorar la eficiencia de este eje, promoviendo así un estado saludable.
Nutritional immunotherapy is based on promoting health through the dietary intake of natural bioactive compounds found in food, such as the procyanidins and docosahexaenoic n-3 polyunsaturated fatty acid (DHA), optimising the functionality of the host’s own immune system and improving its role in the preservation of the body against destabilisers of homeostasis. The research that this thesis encompasses is focused on the role of the nutritional profile in the regulation of immunometabolism. To accomplish this purpose, in the first part of the work presented in this thesis, we determined whether macrophages, the effector cells of innate immunity, could differentially sense dietary composition. We demonstrated that bioactive food compounds modulate, at the molecular level, the functionality of macrophages by hindering macrophage activation. Furthermore, this immunomodulatory effect is compound-dependent, relying on the intrinsic factors of each compound, such as chemical and nutritional properties, to determine its bioactivity. The second part was aimed at determining the role of the dietary pattern within the complex crosstalk of immunity and metabolism using an in vivo model of diet-induced obesity. We determined that immunometabolic regulation depends on the nutritional profile. While a diet based on foods with a high energy content can weaken immunometabolism, the presence of bioactive foods can strengthen the relationship. Within the third part, we evaluated the role of gene-diet interactions in the phenotypic expression of obesity-related complex traits. Using a diet-induced obesity model, two distinct genetic backgrounds (phenotypically different inbred rat strains) were immunometabolically challenged. Our results revealed that interactions between genetic and dietary factors are fundamental for the susceptibility of a genotype to diet-induced obesity. We established that the nutritional profile is a powerful tool to target the functionality of the immunometabolic axis. We further concluded that bioactive compounds present in food improve the efficiency of this axis, thereby promoting a healthy state.

Les Penicilliums sont des champignons filamenteux appartenant au genre Ascomycota. Ces champignons ont été utilisés par l’homme pour la production de nourriture depuis des siècles. Plus récemment, ils ont aussi été utilisés dans l’industrie biotechnologique pour la production de composés chimiques d’intérêts pharmaceutiques. Certaines espèces de Penicillium sont par ailleurs des moisissures contaminants certains aliments, d’autres sont des pathogènes de plantes, y compris de certains fruits. Leur génomique est globalement peut connue. Dans cette étude, nous avons analysé les génomes de deux espèces nouvellement séquencées, Penicillium roqueforti et Penicillium camemberti. Nous reportons ici le développement d’une nouvelle méthodologie pour l’amélioration et la validation d’assemblage de génomes en utilisant une technologie permettant l’observation de molécules d’ADN unique, le Peignage Moléculaire. En utilisant cette méthode, nous avons amélioré l’assemblage de Penicillium roqueforti. Ce manuscrit décrit aussi de multiples occurrences d’un transfert horizontal d’un ilot génomique de plus de cinq cent kilobases entre plusieurs Penicillium. Ce cas de transfert horizontal indique une fréquence d’échange latéral de matériel génétique plus forte qu’attendue. Enfin nous présentons un inventaire préliminaire du potentiel génomique pour la production de métabolites secondaires dans ces importants Penicillium alimentaires
Penicillium are filamentous fungi belonging to the Ascomycota genus. Penicillium species have been used by Man for centuries in food making processes. More recently they have also been used in the biotechnology industry for the production of compounds of pharmaceutical interest. Some Penicillium species are food spoilage agents, pathogens of plants including fruits. Aspects of their genomics are largely unknown. In this study, we analysed the genomes of two newly sequenced species, Penicillium roqueforti and Penicillium camemberti. Here we report the development of a new methodology for improving and validating genome assembly using an original single DNA molecule technology, Molecular Combing. Using this methodology we were able to produce a high quality genome assembly of Penicillium roqueforti. This work also reports the multiple and recurrent horizontal transfer of a large genomic island of over half a megabase between several Penicillium species. This horizontal transfer indicates a higher frequency of lateral genetic exchange between cheesemaking fungi than previously expected. Finally, we present an early assessment of the genomic potential for secondary metabolite production in these important food associated penicilliums

Zéaralénone (Zen) est un métabolite secondaire de type polykétide biosynthétisé par différentes espèces de Fusarium (Fusarium graminearum, F. culmorum, F. equiseti, et F. crookwellense). Il s'agit d'un oestrogène non-stéroïdien (ou mycoestrogène) souvent nommé phytoestrogène. La Zen est un contaminant de cultures de céréales dans le monde entier (Bennett et Klich, 2003). Elle résiste à la plupart des traitements qui ont lieu au cours de la fabrication des denrées alimentaires. En dépit de sa structure non-stéroïdienes, le zen est capable de se lier aux récepteurs des oestrogènes et provoque des altérations morphologiques et fonctionnelles des organes de reproductions. Elle interagit non seulement avec les deux types de récepteurs aux oestrogènes (Celius et al., 1999; Shier et al., 2001; Yu et al., 2004; Takemura et al., 2007), mais aussi avec les substrats dans un certain nombre d'insuffisance des enzymes hépatiques. La Zen est bien absorbée et est capable d'atteindre des cibles intracellulaires. La disparité importante des effets de la Zen entre les différentes espèces animales pourrait en partie due à la différence entre leur profil des enzymes hépatiques (Gaumy et al., 2001; Cavret et Lecoeur, 2006). Le métabolisme de la Zen est complexe, il est dominé par des réactions de conjugaison (considérées comme des voies de détoxication) et des réactions de réduction qui correspondent à l'activation biologique (Gaumy et al., 2001). Nos principaux objectifs étaient l'élucidation des effets des aliments contaminés (en particulier avec le zéaralénone) sur les enzymes de détoxication (en particulier CYP P450) et la compréhension des effets de la zéaralénone sur les différentes espèces. Dans la littérature l'effet de la Zen sur l'expression et sur l'activité des enzymes de détoxication est limité à des expériences in vitro, à notre connaissance aucune recherche in vivo n'a encore été publiée. En vue d'établir les effets in vivo de la zéaralénone sur les enzymes de détoxication nous avons effectué plusieurs expériences: tout d'abord sur le rat, qui est un modèle animal classique pour les études biologiques et sur lequel on trouve une large documentation sur les effets des xénobiotiques et en particulier sur les effets de la Zen, et ensuite sur le poulet, l'une des espèces considérée comme la plus résistante et «habituée» à la présence de la Zéaralénone. Des approches complémentaires ont été utilisées pour déterminer les effets de la zéaralénone sur les enzymes de détoxication: a) Le développement d'outils d'analyses pour des études de la pharmacocinétique des mycotoxines; b) la détermination in vivo des effets de la zéaralénone sur l'expression des enzymes de détoxication et de son devenir dans l'organisme animal c) du mécanisme moléculaire in vitro de la zéaralénone - les effets directs sur les enzymes de détoxification hépatique d) la spécificité des espèces et l'évaluation des risques humains. D'importants effets stimulants sur l'expression d'ARNm de la P-gp ont été observés in vivo chez le rat traitait avec Zéaralénone, ce qui suggère que la P-glycoprotéine pourrait être impliqué dans la voie de détoxication de la zéaralénone. L'implication de la P-glycoprotéine dans le transport de la Zen a été déterminé dans des lignées cellulaires Caco2 par Videmann et al., en 2008. La présence de zéaralénone induit des réponses métaboliques précoces et rapides, en particulier pour le CYP2C7, il pourrait jouer un rôle important dans la voie de détoxication de la Zen chez le rat. Une influence des traitements par la Zéaralénone sur l'expression des RNAm et sur l'activité des isoformes de P450, CYP2B2 et 3A a également été réalisée. CYP2C7 et CYP3A1 sont respectivement les homologues chez le rat des formes CYP2C8 et CYP3A4 humaine (Nelson, 1999), ces cytochromes sont impliqués dans la formation d'un nouveau métabolite le 8-hydroxy Zen. In vivo l'apparition de ce métabolite (le 8-hydroxy Zen) a également été démontrée. Le devenir dans le rat et le poulet de la Zéaralénone a été évalué en utilisant les nouvelles méthodes développées: HPLC-DAD et LC-MS (en utilisant comme étalon interne Zen 13C enrichi). Chez le rat, la zéaralénone est rapidement éliminée. Elle est retrouvée dans les urines, pendant les 6 premières heures après l'administration : environ 60% (47,8 ± 14,3 mg) du montant total de la zéaralénone urinaire éliminé (79,8 ± 23,9 mg) et la Zen et ses métabolites éliminés par l'urine sont d'environ 4% de la zéaralénone administrés. Dans les échantillons de muscle de poulet les niveaux d'α-Zol (13,42 mg / kg) sont plus élevés que le niveau JEFCA accepté (2 mg / kg). Ces volailles sont donc impropres à la consommation. A l'échelle mondiale l'évaluation des risques de l'exposition de l'homme à la zéaralénone, en prenant en compte les habitudes alimentaires, a été réalisée et il en résulte un important et constant risque pour la santé humaine. Aussi la nécessité d'apporter des modifications au règlement concernant la teneur maximale acceptable de la zéaralénone dans les céréales a était surligniez, et nous considérons comme acceptable la valeur 5 μg/kg.

La métabolomique permet une étude à large échelle du profil métabolique d'un individu, représentatif de son état physiologique. La comparaison de ces profils conduit à l'identification de métabolites caractéristiques d'une condition donnée. La métabolomique présente un potentiel considérable pour le diagnostic, mais également pour la compréhension des mécanismes associés aux maladies et l'identification de cibles thérapeutiques. Cependant, ces dernières applications nécessitent d'inclure ces métabolites caractéristiques dans un contexte plus large, décrivant l'ensemble des connaissances relatives au métabolisme, afin de formuler des hypothèses sur les mécanismes impliqués. Cette mise en contexte peut être réalisée à l'aide des réseaux métaboliques, qui modélisent l'ensemble des transformations biochimiques opérables par un organisme. L'une des limites de cette approche est que la métabolomique ne permet pas à ce jour de mesurer l'ensemble des métabolites, et ainsi d'offrir une vue complète du métabolome. De plus, dans le contexte plus spécifique de la santé humaine, la métabolomique est usuellement appliquée à des échantillons provenant de biofluides plutôt que des tissus, ce qui n'offre pas une observation directe des mécanismes physiologiques eux-mêmes, mais plutôt de leur résultante. Les travaux présentés dans cette thèse proposent une méthode pour pallier ces limitations, en suggérant des métabolites pertinents pouvant aider à la reconstruction de scénarios mécanistiques. Cette méthode est inspirée des systèmes de recommandations utilisés dans le cadre d'activités en ligne, notamment la suggestion d'individus d'intérêt sur les réseaux sociaux numériques. La méthode a été appliquée à la signature métabolique de patients atteints d'encéphalopathie hépatique. Elle a permis de mettre en avant des métabolites pertinents dont le lien avec la maladie est appuyé par la littérature scientifique, et a conduit à une meilleure compréhension des mécanismes sous-jacents et à la proposition de scénarios alternatifs. Elle a également orienté l'analyse approfondie des données brutes de métabolomique et enrichie par ce biais la signature de la maladie initialement obtenue. La caractérisation des modèles et des données ainsi que les développements techniques nécessaires à la création de la méthode ont également conduit à la définition d'un cadre méthodologique générique pour l'analyse topologique des réseaux métaboliques
Metabolomics allows large-scale studies of the metabolic profile of an individual, which is representative of its physiological state. Metabolic markers characterising a given condition can be obtained through the comparison of those profiles. Therefore, metabolomics reveals a great potential for the diagnosis as well as the comprehension of mechanisms behind metabolic dysregulations, and to a certain extent the identification of therapeutic targets. However, in order to raise new hypotheses, those applications need to put metabolomics results in the light of global metabolism knowledge. This contextualisation of the results can rely on metabolic networks, which gather all biochemical transformations that can be performed by an organism. The major bottleneck preventing this interpretation stems from the fact that, currently, no single metabolomic approach allows monitoring all metabolites, thus leading to a partial representation of the metabolome. Furthermore, in the context of human health related experiments, metabolomics is usually performed on bio-fluid samples. Consequently, those approaches focus on the footprints left by impacted mechanisms rather than the mechanisms themselves. This thesis proposes a new approach to overcome those limitations, through the suggestion of relevant metabolites, which could fill the gaps in a metabolomics signature. This method is inspired by recommender systems used for several on-line activities, and more specifically the recommendation of users to follow on social networks. This approach has been used for the interpretation of the metabolic signature of the hepatic encephalopathy. It allows highlighting some relevant metabolites, closely related to the disease according to the literature, and led to a better comprehension of the impaired mechanisms and as a result the proposition of new hypothetical scenario. It also improved and enriched the original signature by guiding deeper investigation of the raw data, leading to the addition of missed compounds. Models and data characterisation, alongside technical developments presented in this thesis, can also offer generic frameworks and guidelines for metabolic networks topological analysis

Por primera vez en la historia de la humanidad, una mayor proporción de la población global vive en áreas urbanas. En las próximas décadas, se espera que la mayor parte del crecimiento mundial previsto sea absorbido por las zonas urbanas costaneras del mundo en desarrollo, específicamente en las ciudades pequeñas y medianas así como en las zonas periféricas. En las últimas décadas, tanto las actividades humanas de producción y consumo de alimentos, cómo el fenómeno de la urbanización, se han identificado como actividades que contribuyen a alterar los ciclos naturales de los nutrientes del nitrógeno (N) y del fósforo (P), así como de promover el agotamiento de los recursos minerales del fósforo, generando impactos medioambientales negativos resultantes de la acumulación y pérdida de nutrientes en la tierra, el agua y en la atmosfera. Ambos macronutrientes son básicos para la seguridad alimentaria regional y global al ser insumos esenciales para la producción agrícola de los alimentos. En un mundo cada vez más urbanizado, tanto las áreas urbanas como su desarrollo se encuentan en el centro de todas las discusiones sobre sostenibilidad y desarrollo sostenible. Sin embargo, el papel que desempeñan las ciudades como motores de cambio ambiental a múltiples escalas, p.ej. como agentes en el flujo de energía y materiales, permanece poco reconocido, y por lo tanto poco estudiado, siendo aún más escasos los estudios sobre ciudades en los países en desarrollo, debido a su limitada disponibilidad, acceso y recolección de datos. La noción de metabolismo urbano (UM) proporciona un marco conceptual para el estudio del funcionamiento de las ciudades, y consecuentemente, una aproximación al análisis de la sostenibilidad de las mismas. La presente investigación, centrada en el caso de estudio de la ciudad-región de Gran Nador, en el noreste de Marruecos, tiene por objetivo el estudio del papel que desempeñan las áreas urbanas como agentes en el metabolismo de los alimentos. El estudio, por un lado, analiza la circulación de los principales flujos de nutrientes de los alimentos, el nitrógeno (N) y el fósforo (P), mediante la aplicación del método de Análisis del Flujo de Sustancias (SFA). Por otro lado, contextualiza el desarrollo sociopolítico e histórico de la región urbana de estudio. Los flujos de N y P se calcularon mediante el uso,principalmente, de datos procedentes de bases de datos estadísticas oficiales, literatura publicada e informes no publicados elaborados por las instituciones municipales y regionales de la zona. Además, se utilizaron distintas aproximaciones y “proxies”, así como la adopción de diferentes supuestos y métodos de estimación. Finalmente, se realizó un análisis de incertidumbre para mejorar la fiabilidad de los resultados. Los resultados muestran la linealidad de los flujos de N y P analizados, evidenciando así la apertura del metabolismo de los alimentos en el sistema urbano de Gran Nador. Los resultados también sugieren los fuertes indicios en relación a la influencia significativa que las historias urbanas, es decir, el contexto sociopolítico e histórico en que se desarrolla una región o área urbana, tienen sobre la estructura, el funcionamiento y los flujos metabólicos de la misma área urbana. Posteriormente, el estudio presenta distintas opciones existentes hacia una gestión más equilibrada de los nutrientes, a la vez que para reducir la pérdida y acumulación y aumentar la recirculación de los nutrientes de N y P en Gran Nador. Finalmente, el estudio introduce posibles nuevas líneas de investigación para avanzar con el presente estudio.
For the first time in history a majority of the global population live in urban areas. In the coming decades, most of the world’s population growth is expected to be absorbed by coastal urban areas -specifically in small and medium cities and urban peripheries- of the developing world. In the last decades, anthropogenic food-related activities and urbanization have been identified to contribute altering the natural nitrogen (N) and phosphorus (P) nutrient cycles, as well as exhausting P-mineral resources. These alterations have caused a myriad of pollution and environmental problems resulting from nutrient accumulation and loss to the soil, waters and air. Both macronutrients are critical for regional and global food security, by virtue of being essential inputs for agricultural food production. In an increasingly urbanized world, urban areas and their development are at the centre of all discussions on sustainability and/or sustainable development. Yet, the role played by cities as drivers of environmental change at multiple scales, e.g. as agents in the flow of energy and materials, stays poorly recognized, and thus grossly understudied, remaining even more scarce (even least available) when it comes to studies of cities in developing countries. The latter are significantly constrained by limited data availability, access, and data collection resources. The notion of Urban Metabolism (UM) provides a conceptual framework to study how a city functions, and hence, a way to address the sustainability of a city. The present research, focused in the city-region of Grand Nador in northeast Morocco as a case study, aimed to study the role played by urban areas as agents in the metabolism of food by, on the one hand, examining the circulation of the major nitrogen (N) and phosphorus (P) food-related flows by using the analytical method of Substance Flow Analysis (SFA), and on the other hand, contextualizing the politico-historical development of the urban region. N and P flows were estimated using mainly data from official statistical databases, published literature and unpublished reports authored by municipal and regional level institutions. Besides, approximations, surrogates and proxies were used, as well as the adoption of different assumptions and estimation methods. As a result of data limitations, an uncertainty analysis was performed to enhance the reliability of results. The results displayed the linearity of the N and P food-related flows studied, elucidating the openness of the food metabolism in the urban system of Grand Nador. Results also showed the strong insights of the significant influence that urban histories, namely the sociopolitical and historical context in which a region develops, have on its structure, functioning and metabolic flows. Different existing options towards more-balanced nutrient management and to reduce and enhance nutrient N and P recirculation in Grand Nador are discussed. Likewise, new lines of further research are advanced.

Los principales objetivos de esta Tesis fueron investigar la base genética de la composición y deposito de la grasa en cerdos, e identificar los factores genéticos involucrados en el establecimiento de los patrones de pigmentación rubia vs roja en cerdos Mangalitza, mediante el uso de herramientas genómicas y transcriptómicas. En el primer estudio comparamos los patrones de expresión del músculo esquelético en dos grupos de cerdos Duroc, con diferentes perfiles de crecimiento y engrasamiento (HIGH: elevado espesor del tocino dorsal, grasa intramuscular, contenido de ácidos grasos saturados e insaturados y lípidos séricos vs LOW: fenotipos opuestos). Mediante el uso de la técnica RNA-seq, hemos encontrado que 96 genes se expresan diferencialmente en el músculo gluteus medius de cerdos HIGH y LOW. Varios de estos genes están relacionados con el metabolismo lipídico (p.ej, SLC27A4, SFRP5, y CES1) y el factor de transcripción PPARG parece ser un regulador clave del engrasamiento en porcino. También hemos observado que muy pocos RNAs no codificantes se expresan diferencialmente en estos dos grupos de cerdos, lo que sugiere que el transcriptoma no codificante tiene un efecto limitado sobre el establecimiento de los fenotipos HIGH y LOW. En el segundo estudio, analizamos la expresión de isoformas de mRNA en cerdos HIGH y LOW y demostramos la expresión diferencial de isoformas específicas de cuatro genes muy relacionados con la obesidad (ITGA5, LITAF, TIMP1 y ANXA2). La expresión diferencial de estas isoformas podría tener efectos sobre la estructura del transcrito, así como sobre la secuencia de la proteína. En el tercer estudio, hemos analizado la expresión diferencial de genes que codifican mRNA en respuesta a la ingestión de alimentos. Este objetivo se ha logrado al comparar los patrones de expresión muscular de cerdas Duroc antes de comer (T0), 5 h. (T1) y 7 h. (T2) después de comer. Además de los genes con un papel bien conocido en la homeostasis energética (p.ej, PFKFB3 y G0S2), hemos identificado varios genes con un rol plausible pero mal caracterizado en el metabolismo (p.ej, MIGA2, SDC4 y CSRNP1). También hemos observado un enriquecimiento de un conjunto de genes expresados ​​diferencialmente antes y después de comer que engloba diversos factores de transcripción así como genes implicados en el estrés oxidativo, la angiogénesis y los ritmos circadianos. Teniendo en cuenta estos resultados, en el cuarto estudio hemos desarrollado un experimento basado en RT-qPCR para descubrir cómo la expresión de 8 genes circadianos (ARNTL, BHLHE40, CRY2, NPAS2, NR1D1, PER1, PER2 y SIK1) se modifica en respuesta a la ingestión de alimentos en cinco tejidos porcinos (músculo esquelético, hipotálamo, hígado, intestino y grasa dorsal). Nuestros resultados indican que la expresión de los genes circadianos no cambia en el hipotálamo, el tejido que contiene el reloj central influenciado por la luz. Por el contrario, dicha expresión sí que presenta fuertes variaciones en los otros cuatro tejidos. Este hallazgo demuestra que la nutrición cambia la expresión de los genes circadianos integrados en los relojes periféricos. Finalmente, en el quinto estudio, hemos analizado, en colaboración con investigadores del Research Institute for Animal Breeding and Nutrition (Hungría) y la Universidad de Cluj-Napoca (Rumanía), la base genética del color de la capa (rojo vs rubio) en cerdos Mangalitza. Combinando un barrido de selección y un estudio de asociación del genoma completo, hemos encontrado que el gen SLC45A2 probablemente esté involucrado en la determinación genética de la pigmentación roja y rubia de los cerdos Mangalitza, un resultado que concuerda bien con estudios previos que demuestran la implicación de este locus en los patrones de color de múltiples especies de mamíferos, incluyendo la especie humana. Más específicamente, dos SNP con efecto no-sinónimo, c.806G>A (p.Gly269Glu) y c.956G>A (p.Arg319His), situados en el gen SLC45A2, están fuertemente asociados con los colores rojo y rubio, no obstante dicha asociación no es completa por lo que cabe deducir la existencia de factores genéticos adicionales en la pigmentación de los cerdos Mangalitza.
The main objectives of this Thesis were to investigate the genetic basis of fatness in pigs and to identify the genetic factors involved in the establishment of blond vs red pigmentation patterns in Mangalitza pigs by using genomic and transcriptomic tools. In the first study of the Thesis (Chapter 3), we compare the skeletal muscle expression patterns of two groups of Duroc pigs with different growth and fatness profiles (HIGH: high backfat thickness, intramuscular fat, saturated and unsaturated fatty acid content and serum lipids vs LOW: opposite phenotypes). By using a RNA-Seq technology, we identified 96 genes differentially expressed. Several of these genes are related to lipid metabolism (e.g. SLC27A4, SFRP5 and CES1) and the transcription factor PPARG appears to be a key regulator of porcine fatness. We have also observed that very few non-coding RNAs are differentially expressed in these two groups of pigs, suggesting that the non-coding transcriptome has a limited effect on the establishment of the HIGH and LOW phenotypes. In the second study of the Thesis, we demonstrate the differential expression of specific mRNA isoforms of four genes with a known role in obesity (ITGA5, LITAF, TIMP1 and ANXA2) in HIGH vs LOW pigs. The differential expression of these isoforms may have effects on transcript structure as well as on the protein sequence. In the third study, we aimed to investigate the differential expression of mRNA encoding genes in response to food ingestion. This goal has been achieved by comparing the muscle mRNA expression patterns of Duroc sows before feeding (T0) and 5 h. (T1) and 7 h. (T2) after feeding. Besides genes with a well-known role in energy homeostasis (e.g. PFKFB3 and G0S2), we have identified several genes with a plausible but poorly characterized role in metabolism (e.g. MIGA2, SDC4, and CSRNP1). We have also observed that the set of genes differentially expressed before and after feeding is enriched in transcription factors and pathways related to oxidative stress, angiogenesis, and circadian rhythms. Considering these results, in the fourth study we use quantitative RT-qPCR technique to find out how the expression of 8 circadian genes (ARNTL, BHLHE40, CRY2, NPAS2, NR1D1, PER1, PER2 and SIK1) changes in response to food ingestion in five porcine tissues i.e. skeletal muscle, hypothalamus, liver, intestine and dorsal fat. Our results indicate that the expression of the Clock genes does not change in the hypothalamus, the tissue containing the central clock entrained by light, but in contrast, it is strongly modified in the other four tissues. This finding demonstrates that nutrition changes the expression of circadian genes integrated in peripheral clocks. Finally, in the fifth study, we have analysed, in collaboration with researchers of the Research Institute for Animal Breeding and Nutrition (Hungary) and the University of Cluj-Napoca (Romania), the genetic basis of coat color (red vs blond) of Mangalitza pigs. By combining a selection scan and a genome-wide association study, we have found that the SLC45A2 gene is probably involved in the genetic determination of pigmentation in Mangalitza pigs, a result that agrees well with previous studies demonstrating the implication of this locus on the color patterns of multiple mammalian species including humans. More specifically, two missense SNPs c.806G>A (p.Gly269Glu) and c.956G>A (p.Arg319His) in the SLC45A2 locus appear to be strongly but not fully associated with the red and blond coat colors of Mangalitza pigs. This finding suggests the existence of addiitonal genetic factors regulating the pigmentation of Mangalitza pigs.

L'analyse de la performance de haut niveau en ski de fond nécessite l'utilisation de méthodes non invasives telle que l'analyse du geste en 3D sans utilisation de marqueurs passifs ou actifs sur le skieur (approche écologique de l'analyse). Les objectifs de ce travail sont les suivants : - mise en évidence de comportements typiques propres à chacune des trois disciplines nordiques utilisant le pas de patineur : le ski de fond, le biathlon et le combiné nordique. Cette analyse, effectuée à partir de données enregistrées lors de compétitions de coupe du monde, permet de comparer les disciplines entres elles (analyse interdisciplinaire) et les skieurs au sein d'une même discipline (analyse intradisciplinaire). - la comparaison de deux systèmes d'analyse 3D du mouvement : cliqbmp et animan3d qui mettent en oeuvre respectivement une modélisation filaire (représentation de l'individu en fil de fer) et volumique (représentation du volume corporel de l'individu). - L'étude de cas à propos de l'évaluation de l'ordre de grandeur du coût d'énergie mécanique engendré par des fautes techniques volontaires ou involontaires lors de cycle de pas en pas alternatif et en pas de patineur 2 temps combiné. La bonne technique dans les disciplines nordiques passe, selon nous, par une adaptation optimale de la gestuelle aux caractéristiques de l'individu (physiologiques, biomécaniques etc) mais aussi aux contraintes de son environnement (logique interne de la discipline, état du terrain d'évolution, etc. ). Un traitement didactique des résultats de ce travail permet de faire des propositions de contenus d'entraînement pour les entraîneurs en charge d'athlètes de haut niveau en ski nordique.

INTRODUCCIÓN: La obesidad en uno de los mayores factores de riesgo para desordenes metabólicos y cardiovasculares. El exceso de peso ganado durante la infancia aumenta el riesgo de enfermedad en el adulto, sin embargo revirtiendo esta condición tempranamente se reduce el riesgo futuro mejorando la calidad de vida de los individuos. OBJETIVOS: Una intervención en el estilo de vida de individuos prepuberales con obesidad podría provocar cambios en signaturas metabólicas en paralelo con mejoras del IMC. Nuestro objetivo principal fue determinar los efectos de una intervención en el estilo de vida en el perfil metabolómico tanto del plasma como de la orina en individuos prepuberales con obesidad. MÉTODOS: Estudio longitudinal prospectivo en 40 individuos prepuberales con obesidad (IMC > 2 D.E.) entre las edades de 7 a 10 años. La intervención fue principalmente educacional, focalizada a los individuos y sus familias durante 6 meses. Se analizaron parámetros nutricionales, antropométricos y bioquímicos antes y después de la intervención. También se realizó metabolómica no dirigida para muestras de plasma mediante LC-MS y dirigida para muestras de orina mediante RMN, obteniendo así un perfil metabólico completo antes y después de la intervención. RESULTADOS: La intervención disminuyó el IMC de 3,55 D.E. (3,30 - 3,79) a 3,09 D.E. (2,86 - 3,32), la circunferencia de cintura de 83 cm (79,6 - 85,8) a 81 cm (77,8- 84,5) y los niveles de HbA1c de 5,4% (5,3 - 5,4) a 5,2% (5,2 - 5,3), las variables se analizaron mediante Test t-Student pareado. Se disminuyó la ingesta de calorías, carbohidratos, azúcares libres y grasas. No se logró modificar las horas de actividad física. En el perfil metabólico del plasma se identificaron 2581 picos y se aplicó el Análisis de Componentes Principales para consolidarlos en 8 componentes (PC). El PC1 fue el único factor que tuvo diferencia después de la intervención (p= 0,008) incluso ajustando por comparaciones múltiples. El PC1 está compuesto por metabolitos relacionados con el metabolismo de esfingolípidos observando una disminución de los niveles circulantes de las especies identificadas. En relación al perfil metabólico de las muestras de orina, se identificaron 32 metabolitos. Los niveles de TMAO fueron significativamente menores después de la intervención (FDR q < 0,05). El cambio en los niveles de TMAO se relacionaron inversamente con el cambio en la ingesta de fibra. CONCLUSIONES: Una intervención en el estilo de vida reduce el IMC D.E. y modifica el metaboloma del plasma y de la orina. En particular, la intervención disminuyó los niveles de ceramidas, asociadas estrechamente con enfermedades metabólicas. Además, se redujeron los niveles de TMAO, biomarcador de riesgo cardiovascular. Por lo tanto podemos sugerir que una intervención en el estilo de vida de individuos prepuberales con obesidad puede ser un importante mecanismo para reducir el riesgo de enfermedades metabólicas y cardiovasculares.
BACKGROUND: Obesity is one of the major risk factor for metabolic disorders, and its global prevalence has increased exponentially in the last decades. Excessive weight gained during early childhood increases long-term risk; however, reversing this condition during early-life reduces risk, improving children’s quality of life. OBJECTIVE: We hypothesized that a lifestyle intervention in obese prepubertal children would result in differential metabolic signatures, in parallel to improvements in BMI. Our aim was to determine the changes in the plasma and urine metabolomics profiles induced by the intervention. METHOD: Longitudinal prospective study of 40 obese (BMI >2 SDS) prepubertal children ages 7 to 10. The lifestyle intervention was primarily educational, focused on children and family for 6 months. We analyzed nutritional, anthropometrics and biochemical parameters before and after intervention program. Untargeted metabolomics was applied to analyze plasma samples by LC-MS and targeted metabolomics to urine samples by nuclear magnetic resonance and to obtain a comprehensive metabolic profile at baseline and after intervention. RESULTS: The intervention decreased BMI 3.55 SDS (3.30-3.79) vs 3.09 SDS (2.86-3.32), waist- circumference 83 cm (79.6-85.8) vs 81 cm (77.8-84.5), and HbA1c levels 5.4% (5.3-5.4) vs 5.2% (5.2-5.3) using 2-tails paired student t-test. After 6 month decreased calories, carbohydrates, sugars and fat intakes. Was not achieved modify the physical activity hours. In relation with plasma metabolic profile, identified 2581 features and principal component analysis was applied to consolidate them into 8 principal factors (PC). PC1 differed between pre- and post- intervention (p=0.008), and significance was maintained after adjusting for multiple comparisons; PC1 was characterized by metabolites related with sphingolipid metabolism decreasing its levels after intervention. Urine metabolomics identified 32 metabolites. Trimethylamineoxide (TMAO) levels were significantly lower after intervention (FDR q< 0.05). The change in TMAO was inversely relationated with changes in fiber intake CONCLUSION: A 6-month lifestyle intervention able to reduce BMI-SDS and change the plasma and urine metabolome. In particular, the intervention reduced ceramides levels related to metabolic diseases and TMAO levels, a major cardiovascular risk factor. Therefore, these data suggest that the lifestyle intervention improves the metabolic and cardiovascular risk profiles in prepubertal obese children.

Global obesity is a pandemic status, estimated to affect over 2 billion people, and has resulted in an enormous strain on healthcare systems worldwide. The situation is compounded by the fact that apart from the direct costs associated with overweight pathology, obesity presents itself with a number of comorbidities, including an increased risk for the development of neurodegenerative disorders. Alzheimer disease (AD), the main cause of senile dementia, is no exception. Spectacular failure of the pharmaceutical industry to come up with effective AD treatment strategies is forcing scientific community to rethink the underlying molecular mechanisms leading to cognitive decline. Research described in this doctoral thesis is focused on the effects of a high-fat diet (HFD) versus control chow in C57/Bl6 Wild-type (WT) and APPswe/PS1dE9 (APP/PS1) mice, a well-established mouse model of familial AD. WT and APP/PS1 mice were fed control chow from the time of weaning (21 days) until the age of 3 months. In addition, a separate group of animals were fed either a control or a HFD from the time of weaning until 6 months of age. We have detected tau hyperphosphorylation, amyloidogenesis and impaired mitochondrial signaling in the hippocampi of 3-month-old APP/PS1 mice. By the time the mice reached 6 months of age this phenotype was exacerbated. In addition, 6-month-old APP/PS1 mice present with cognitive deficits, hippocampal insulin signaling abnormalities and impairments in peripheral glucose and insulin tolerance. On a HFD, diet-induced obesity and metabolic syndrome were present both in the WT and APP/PS1 groups. Both groups demonstrate memory loss and impaired peripheral glucose metabolism as well as abnormal hippocampal insulin signaling and mitochondrial metabolism, with APP/PS1 mice exhibiting a nearly diabetic phenotype. Significantly higher levels of insoluble Aβ (1-42) were detected in the cortical extracts of HFD-fed APP/PS1 mice versus APP/PS1 animals on a control chow. In transgenic animals predisposed to AD development, the introduction of hypercaloric diet significantly worsened existing phenotype both at the periphery and in the hippocampal networks. Overall, our results suggest that metabolic syndrome, diabetes and related comorbidities clearly do have a potential to significantly worsen the symptoms of AD disease.
La obesidad es una pandemia mundial, la cual según estudios recientes afecta a más de 2.000 millones de personas en todo el mundo y conlleva una fuerte presión de los sistemas sanitarios globales. El problema se ve aumentado no sólo por los costes directos asociados al sobrepeso, sino también por las comorbilidades relacionadas, entre ellas el incremento del riesgo de desarrollo de enfermedades neurodegenerativas, como por ejemplo la enfermedad de Alzheimer (EA), principal causa de demencia senil. Hasta el momento, la industria farmacéutica no ha sido capaz de desarrollar estrategias eficaces para el tratamiento de la EA. Por ello, ha surgido la necesidad de plantearse nuevos mecanismos moleculares que puedieran explicar el declive cognitivo de esta enfermedad. La presente tesis doctoral trata sobre los efectos provocados por la ingesta de una dieta rica en grasa (high-fat diet, HFD) en ratones salvajes C57/Bl6 (wild-type, WT) y en ratones APPswe/PS1dE9 (APP/PS1), como modelo experimental de EA familiar. Dichos ratones fueron alimentados con dieta estándar a partir del destete (21 días) hasta los 3 meses. Por otro lado se estableció un nueva línea de investigación en la cual los animales fueran alimentados con dieta estándar o HFD hasta los 6 meses de edad. Se detectó hiperfosforilación de la proteína tau, amiloidogénesis y deterioro de la señalización mitocondrial en el hipocampo de los ratones APP/PS1 a 3 meses de edad. Por otro lado, los ratones presentaban un fenotipo exacerbado a 6 meses. Además, presentaban defectos cognitivos, anomalías en la vía de señalización de la insulina en el hipocampo y alteraciones en el metabolismo periférico de la glucosa y de la insulina. La ingesta de la HFD indujo obesidad y desarrollo de síndrome metabólico, tanto en el grupo de ratones WT como en APP/PS1. Ambos grupos presentaron pérdida de memoria, alteración del metabolismo periférico de la glucosa, así como anomalías en la vía de señalización de la insulina y en el metabolismo mitocondrial en hipocampo, desarrollando un fenotipo casi diabético en el caso de los ratones APP/PS1. En los extractos corticales de estos animales alimentados con HFD se detectaron niveles de Aβ insoluble (1-42) significativamente más elevados en comparación con los alimentados con dieta estándar. En los animales transgénicos con predisposición al desarrollo de EA, la introducción de una dieta hipercalórica provocó un empeoramiento significativo del fenotipo, tanto en la periferia como en el hipocampo. En general, nuestros resultados parecen indicar que el síndrome metabólico, la diabetes y las comorbilidades asociadas podrían causar un empeoramiento significativo de los síntomas de EA.

Si vol consolidar-se com a explotació econòmicament i ecològicament sostenible, la piscicultura ha de deslligar-se de la pesca mitjançant la reducció del contingut de farina de peix als pinsos. Aquesta fita pot ser assolida ajustant la proporció de proteïna de la dieta als requeriments nutricionals dels peixos, o bé emprant fonts de proteïna vegetal. Per a optimitzar l'ús de la proteïna en dietes per a orada (Sparus aurata) es varen usar tres suplements alimentaris: el glutamat, la glutamina i la taurina. En orades alimentades a base de farina de peix, el glutamat i la glutamina són més eficaços que el midó millorant l'ús de la proteïna ingerida per a creixement. La inclusió d'un 4% de glutamat en dieta a base de farina de peix o proteïna vegetal millora la retenció proteica en promoure l'activitat glucolítica al fetge de les orades. L'augment de la retenció proteica, conjuntament amb l'estimulació de la ingesta de pinso per part del glutamat, resulta en un major creixement en orades alimentades amb proteïna majoritàriament d'origen vegetal (el 90% de la proteïna de la dieta). Els nostres resultats de creixement i eficiència alimentaria assenyalen a la taurina com a nutrient essencial en dietes amb una elevada o total substitució de farina de peix per proteïna d'origen vegetal. En base al creixement, orades alimentades amb proteïna majoritàriament vegetal requereixen una inclusió de taurina en dieta d'entre el 0.52 i el 0.91%. En base a l'eficiència alimentària, aquest requeriment seria del 0.93%. La suplementació d'un 1% de taurina millora el creixement d'orades alimentades amb pinso a base de proteïna vegetal. Aquest increment del creixement deriva de l'estimulació de la ingesta de pinso i la millora de l'eficiència alimentària. La taurina també millora la digestibilitat del fòsfor de les dietes vegetals i, per tant, la ingesta de fòsfor digerible per part de les orades. A nivell metabòlic, la taurina incrementa l'activitat glucoquinasa al fetge de les orades alimentades amb dietes vegetals, alhora que disminueix la glucèmia. La taurina promou la lipòlisi en orada atès que en redueix la proporció de greix corporal. La presència en la dieta d'un 10% de proteïna procedent de farina contraresta l'acció lipolítica de la taurina, alhora que accentua els beneficis de la taurina sobre el creixement i l'eficiència alimentària. La inclusió d'un 6.5-6.6% d'hidrolitzat de proteïna de soja a la dieta afavoreix una major absorció de nutrients i millora el creixement d'orades alimentades amb proteïna exclusivament vegetal. En conclusió, segons els nostres resultats, el glutamat i la taurina representen dos additiu alimentaris de gran valor per a la substitució de farina de peix per proteïnes d'origen vegetal i carbohidrats en dietes per a orada.
To be economic and ecologically viable, fish farming must be freed from it its dependence on fish meal. This goal can be achieved either by adjusting dietary protein content to fish nutritional requirements or by replacing fish meal by plant proteins. In order to optimize protein utilization in gilthead sea bream (Sparus aurata) we tested three different feed additives: glutamate, glutamine and taurine. In gilthead sea bream fed on fish meal-based diets, glutamate and glutamine are more effective than starch improving dietary protein utilization for growth. The supplementation of 4% glutamate in diet based on fish meal or plant protein improves protein retention through the promotion of glycolysis in the liver of gilthead sea bream. Protein retention improvement, together with the stimulation of feed intake by glutamate supplementation, results in higher growth rates in fish fed on plant protein sources. Our results point to taurine as an essential nutrient to be included in diets with high or total fish meal replacement. On a growth basis, the optimal dietary taurine supplementation of gilthead sea bream is 0.52-0.91%. On a feed efficiency basis, gilthead sea bream have a dietary taurine requirement of 0.93%. The supplementation of 1% taurine improved the growth of sea bream fed on plant-based diets. This growth promotion is due to feed intake stimulation and the improvement of feed efficiency. Taurine also increases phosphorous digestibility in plant-based diets. At a metabolic level, taurine up-regulates glucokinase activity and decreases glycemia. Taurine also promotes lipolysis since it reduces the body fat content of gilthead sea bream. The presence of 10% dietary protein coming from fish meal counteracts taurine lipolytic action, but accentuates taurine benefits on fish growth and feed efficiency. The inclusion of 6.5-6.6% soy protein hydrolysate in plant-based diet leads to a higher nutrient absorption and increases fish growth. In sum, according to our results, glutamate and taurine are two useful feed additives for fish meal replacement with plant protein sources and carbohydrates in diets for gilthead sea bream.

The aim of this PhD work was to apply the NMR based metabolomic approach to the study of complex matrices such as several food plants (pepper, celery, tomatoes, hemp, baobab, teas, blueberries and olive oils). A comprehensive description of the chemical composition in term of primary and secondary metabolites obtained by means of 1D and 2D experiments was reported and information regarding specific aspects (variety, type of production etc) were obtained. The study of stool samples of patients with liver cirrhosis was also carried out confirming the important contribution of the NMR approach in the disease investigation.

In the last years, the research in food science and nutrition has grown parallel to the consumers’ concern about the quality and safety of the food they eat. As a result, ensuring food safety, food quality and investigating on nutrition has never been more necessary than today. More powerful analytical procedures are now required to detect and identify undesired and toxic compounds. Moreover, consumers demand is now moving toward the valorization of food with potential health benefits. The identification of bioactive compounds is crucial to provide customers a healthy diet. Finally, one of the main challenges is to improve our limited understanding of the roles of nutritional compounds at molecular level. Food scientists have to face a large number of challenges to adequately answer the new emerging questions of food science. In this context, mass spectrometry showed to be a powerful tool for the investigation on food quality, safety and nutrition. The technical developments of chromatography coupled to mass spectrometry allowed several progresses for the detection of both endogenous compounds and contaminants in food. However, the main progress was due to high resolution mass spectrometry, a promising technique that has opened new horizons in screening and identification of a wide range of unknowns compounds. Mass spectrometry-based metabolomics is a key tool that has been involved, nowadays, in the study of the food and nutrition domains. As per definition, metabolomics includes the exhaustive study of the whole small metabolite composition of a particular system, the food metabolome is not an exception, being rich of endogenous and exogenous compounds with different properties and abundances. Nowadays, three main approaches for the screening of substances can be pointed out: targeted screening (analysis of few known compounds), suspected screening (analysis of a class of expected compounds) and untargeted screening (analysis of a wide range of unknown and unexpected compounds). In this thesis, the three approaches have been studied in details to understand their difficulties, advantages and disadvantages for the analysis of food metabolome. Several steps have been proved to be necessary for the development of a reliable analysis such as sample preparation, chromatographic and mass spectrometric method, data analysis and identification. The thesis is then divided in three main sections: targeted approach for food safety, suspected approach for food quality, untargeted approach for nutrition. In each section, one of the three main approaches used in food metabolomics has been studied for its application on one of the three different food analysis fields. Moreover, each topic has been treated focusing the attention on a specific step of the method development. The topic of metabolomics for food safety has been studied in a targeted approach by developing a method for the analysis of secondary metabolites of fungi, namely mycotoxins, in food (Paper I and II). A particular material for the clean-up of mycotoxins for their quantification in milk and cereals was presented. The topic of metabolomics for food quality has been treated in a suspected approach by developing a method for the analysis of several classes of secondary metabolites of plants such as phenolic acids, flavonoids, and glucosinolates in food (Paper III, IV and V). The development of a chromatographic and mass spectrometric method for the metabolic profiling of strawberry and cauliflower was presented. The topic of metabolomics for nutrition has been treated in an untargeted approach by developing a method for the analysis of the human urinary metabolome after the consumption of meat and dairy products. Several data analysis approaches have been shown for the investigation of the whole urinary metabolome. Ultimately, it has been shown how several aspects should be taken into account when analyzing complex matrices like food through different approaches. Sample preparation, chromatographic and mass spectrometric methods and data analysis have to be treated in different ways based on the used approach. In each case, the advantages of each technique can be exploited based on the purpose of the study. However, aside from the challenges that have to be faced, mass spectrometry-based metabolomics can definitely represent a powerful tool for food safety, quality and nutrition.

Studies described in this thesis were performed to investigate associations among season, diet, pituitary pars intermedia dysfunction (PPID) and blood concentrations of adrenocorticotropic hormone (ACTH), insulin, glucose, and leptin in horses. In the first study, higher ACTH concentrations were detected in horses affected with PPID. A seasonal increase in plasma ACTH concentration was detected in the late summer and early fall, but PPID did not affect the timing or duration of this increase. Pasture grazing raised glucose and insulin concentrations with a peak in September, at the same time that horses had higher ACTH concentrations, and this convergence of risk factors may raise the risk of laminitis. All of the horses included in this study were from the same farm. The second study was performed to determine whether horses from different locations within the same region exhibited the same seasonal increase in ACTH concentrations. Results of this study indicate that the seasonal increase in plasma ACTH concentrations occurs in horses from different farms with varying management practices. The third study investigated the effects of season on plasma leptin concentrations in the horses from the first study. We hypothesized that higher leptin concentrations would be detected in advance of the seasonal increase in plasma ACTH concentrations. Results did not support our hypothesis because leptin concentrations increased after ACTH concentrations peaked in September. Our findings suggest that the seasonal increase in ACTH concentrations induced leptin resistance, which might facilitate weight gain in the autumn. Alternatively, leptin concentrations increased as a result of weight gain or change in body fat composition. In summary, season appears to signal upregulation of the hypothalamic-pituitary-adrenal axis in horses, in an effort to prepare for winter. This upregulation is retained in horses with PPID, a disorder associated with loss of dopaminergic inhibition to the pars intermedia of the pituitary. The seasonal rise in plasma ACTH concentrations is followed by an increase in leptin concentrations, which suggests the development of leptin resistance or an increase in adiposity.