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Metabolic Syndrome (MetS) may be defined as a clustering of risk factors for diabetes mellitus (T2DM) and cardiovascular disease (CVD) which puts individuals at increased risk of developing these conditions and consequently leads to a reduction in life expectancy and increased morbidity. Although there are a number of definitions of MetS, essentially having any three of the following five risk factors confers a diagnosis of MetS; (i) impaired fasting glucose levels, (ii) raised blood pressure, (iii) raised triglycerides, (iv) low levels of high-density lipoprotein cholesterol (HDLC), and (v) increased waist circumference. A comprehensive decision model has been developed to combine different levels of evidence in a Markov model. This model is based in the behavior of MetS and its possible progression to T2DM and CVD, in order to evaluate the potential impact of a MetS based intervention at population level. Evidence synthesis methods are going to be incorporated in the model to integrate different levels of information. Firstly, a Mixed Treatment Analysis (MTC) of Randomized Controlled Trials (RCTs), which have evaluated a number of lifestyle and pharmacological interventions in individuals with MetS was undertaken. This information also assessed the possibility of reversing a diagnosis of MetS. Secondly, a systematic review of published literature was conducted to assess the evidence related with the association between the MetS and development of T2DM and/or CVD. A Bayesian approach to the problem has also been advocated which enables flexibility to develop a Markov model of this complexity. WinBugs offers a comfortable solution for the evaluation of a Markov model, given its Gibbs sampler. Main findings of this thesis are related with large amount of uncertainty, presuming a difficulty to provide a clear decision related to the application of MetS for prevention of T2DM and CVD.

Erythropoeitic protoporphyria (EPP) is a member of the prophyria disease class and is caused by abnormal function of the enzyme ferrochelatase (Fech). In humans it has variable penetrance, but primarily leads to toxicity in the skin and liver to varying degrees. Here I have investigated the nature of EPP progression using the Fechm1PAS mouse model. This mouse contains a point mutation in the fech gene which results in reduced Fech activity to < 7% of wild type, with resultant loss of haem, anaemia and hepatic cholestasis. The phenotypic progression of Fechm1PAS/m1PAS mice was established using pathology and clinical biochemistry from 18 days gestation to 32 weeks of age. Pathological changes were found from 4 weeks with biochemical and differential gene expression (DGE) analysis showing intraheptic cholestasis from birth. Genomic data from cDNA microarrays was derived and analysed with the DGE by phenotypic anchoring. DGE was observed in all processes responsible for cell protection and epigenetic regulation. DGE analysis have led me to hypothesise that the porphyria leads to a chronic reactive oxygen species (ROS) attack, causing DNA damage, eventually leading to hepatocarinoma. This was indicated by changes in the GSH, cytochrome P450, circadian rhythm and methylation pathways. DGE in these processes included downregulation of DNA methyltransferases (Dnmt1, Dnmt6a and Dnmt6b), and upregulation of cytochrome oxidase (Cox and Por) and GSH metabolism transcription factors (Gclc and Gclm). The findings made here contribute further to the understanding of EPP progression and the relationship between phenotype and DGE in EPP.

A relationship between schizophrenia and coeliac disease (CD) has long been postulated. However, despite the substantial genetic contribution defined for both conditions, the possibility of shared genetic risk factors has not previously been addressed. In this study, key CD-associated loci were tested in a schizophrenia cohort. Where association was found, functional studies were performed; including gene expression analysis, screening for functional variants, and correlation of alleles with antibody and cytokine levels. Two principle findings were drawn from the data produced. Firstly, there was no statistically significant association between schizophrenia, and the CD-associated loci; MY09B, IL-2/21, or HLA-DQ2. Secondly, a robust association was found for 3 SNPs located in the 5' region of the gene encoding TGM2; the gluten-modifying enzyme targeted by autoantibodies in CD. Functional studies provided evidence of decreased serum IL-2, in correlation with a schizophrenia-associated SNP located in the TGM2 gene. In addition, cell culture work showed altered expression of the TGM2 gene, following treatment with 1 I-Lg/ ml of the neuroleptic drug clozapine. This study concludes that the major genetic factors underlying schizophrenia and CD are distinct, and that the gluten response observed in schizophrenia does not follow the classical CD pathway. Furthermore, this study provides evidence of a novel candidate gene for schizophrenia, TGM2, and explores how it may contribute to neurological aspects of the disease.

Classical coeliac disease (CD) is a well-defined syndrome of small bowel villous atrophy associated with abdominal pain, malabsorption, and weight loss as a result of gluten-sensitivity, reversed rapidly by gluten exclusion diet. Disease associations include dermatitis herpetiformis (DH), Addison’s disease, type 1 diabetes mellitus, autoimmune thyroid disease and a variety of neurological disorders. This thesis aims to investigate the hypothesis of the existence of a gluten sensitive neurological disease CD with coexistent neurological dysfunction is only rarely reported in a neurological setting. 23 cases were reported from the British Neurological Surveillance Unit (BNSU) over 24 months and 13 locally over 31 months. 18 sets of notes (50%) were reviewed. These patients comprise a heterogeneous group of neurological disorders including epilepsy, myelopathy, axonal neuropathy and migraine. Neurological disorders in patients with confirmed gluten sensitivity may occur simply by chance. In a cohort of 801 CD patients, 54 neurological disorders were identified in 177 patients including stroke (2.9%), migraine (2.7%), epilepsy (2.6%) and carpal tunnel syndrome (2.0%). More detailed investigation of 35 patients with DH and 53 patients with CD, confirmed a low prevalence of idiopathic neurological abnormalities (DH 11%; CD 25%). Analysis of sera from these patients did not identify the presence of anti-neuronal antibodies. A novel anti-spinal antibody was identified in over 50% of the subjects with DH but this requires further characterisation. It has been postulated that patients with idiopathic neurological disease and anti-gliadin antibody (AGA) seropositivity are gluten sensitive. However, AGA lacks disease specificity being found in 10% of healthy blood donors. Screening of 49 unselected multiple sclerosis cases found IgG AGA in 12% of patients and 13% of blood donors confirming that AGA (especially IgG isotype) can be a non-specific finding. AGA, other food antibodies and tissue transglutaminase antibody (TTG) were measured in patients with idiopathic ataxia (20), hereditary ataxia (7) and idiopathic peripheral neuropathy (32). None of the cases was positive for IgA TTG making occult CD unlikely. Cerebellar ataxia with positive AGA (so-called ‘gluten ataxia’) was rare (4 cases in 2 years from a population of 2 million). All food antibodies tested (AGA, hen’s egg albumen, and cow’s milk lactoglobulin), particularly IgG, were a common finding in both ataxia and peripheral neuropathy groups. This study found no evidence for gluten neurotoxicity. Serological tests, particularly AGA, need to be interpreted with caution. Further study is required regarding the nature of the association between neurological illness and gluten sensitivity.

Emerging evidence has shown that an abnormal postprandial accumulation of dietary tat IS atherogenic. The aim of this study is to measure triacylglycerol (TAG) kinetics in endogenous and exogenous lipoproteins in both fed and fasted states using stable isotopes.

Un certain nombre de maladies neurodégénératives, comme la maladie de Creutzfeld-Jacob chez l'homme ou la maladie dite de "la vache folle" chez les bovins, sont dues au mauvais repliement d'une protéine cellulaire dite prion. Plusieurs protéines prions ont été identifiées chez des microorganismes comme Ure2p chez Saccharomyces cerevisiae ou HET-s chez Podospora anserina. Ces protéines sont non pathogènes pour l'homme et se comportent comme le prion de mammifères c'est à dire en s'assemblant également en fibres amyloïdes. Chez S. cerevisiae, la protéine Ure2p est associée au phénotype prion [URE3] et est agrégée dans ce type de cellule. Dans un premier temps, nous avons montré que les agrégats [URE3] obtenu in vivo étaient d'une nature différente des fibres amyloïdes produites in vitro. Dans un second temps, la technicité acquise au cours de l'étude biochimique d'Ure2p m'a permis d'aborder celle d'un orthologue, Ure2p de Saccharomyces paradoxus (Ure2p-Sp), qui n'est pas un prion "in vivo". Après avoir montré que les deux protéines sous forme soluble présentent les mêmes caractéristiques biochimiques, je montre qu'elles possèdent la même propension à former des fibres amyloïdes. Dans le cadre de cette étude nous montrons également que ces fibres sont infectieuses laissant penser qu'in vivo cette incapacité de la protéine Ure2p-Sp à basculer sous forme prion est plutôt due à une défaillance du mécanisme de propagation des prions plutôt qu'à une différence de la structure amyloïde proprement dite. Enfin, nous avons pu montrer qu'il était possible de transformer une protéine amyloïde non toxique en une protéine toxique en ne changeant que quelques acides aminés. En poursuivant cette étude par une étude in vitro, j'ai également pu observer que la protéine toxique s'organisait in vitro en de courtes fibres amyloïdes qui pouvaient mimer les intermédiaires responsables de la toxicité au cours des maladies neurodégénératives. Si dans cette première partie l'apport de la biochimie dans l'étude ciblée de différentes protéines amyloïdes de champignons ascomycètes est évident, l'étude des protéines peut également se faire par une approche biochimique globale sans a priori comme dans l'étude de la réponse protéique d'une plante à un stress environnemental. Grâce au séquençage des génomes, la biochimie post-génomique connait un essor exceptionnel et permet de mettre en évidence de nouvelles protéines dans le cadre d'études fonctionnelles. Dans ce but, j'ai mis en oeuvre une technique de protéomique différentielle : l'électrophorèse bidimensionnelle de type DiGE (Differential in-Gel Electrophoresis) dans le cadre de l'étude du protéome d'une plante invasive, le solidage Solidago canadensis, soumise à un stress d'origine anthropique. Cette étude protéomique vient en complément d'une étude phytosociologique des plantes capables de coloniser un sol très dégradé mais aussi de données de biodiversité, croissance et de mesures de la réponse anti-oxydante de ces végétaux. Cette analyse a révélé que sur des sols pollués, le solidage parvient non seulement à produire l'énergie nécessaire à sa croissance mais il parvient également à synthétiser les intermédiaires de biosynthèse du glutathion et des phytochélatines. Ainsi, le solidage est capable de s'adapter à ce milieu ce qui lui permet d'être tolérant à la pollution. Mes premiers résultats de biochimie post-génomique offrent de nouvelles bases pour la compréhension des mécanismes de tolérance au milieu de certaines plantes permettant le développement de technologies innovantes de phytorémédiation afin de restaurer les sols pollués / A number of neurodegenerative illnesses such as Creutzfeld-Jacob disease in humans or the disease known as mad cow disease in bovine, are due to misfolding of a cellular protein called prion. These proteins are not pathogenic for human and can form amyloid fibers like mammalian prion. They are therefore a useful tool to study molecular events responsible for the emergence and propagation of prions. In S. cerevisiae, the Ure2p protein is associated with [URE3] prion phenotype and is aggregated in this cell. Initially, I developed the conditions to characterize Ure2p amyloid fibers by proteolysis and we showed that [URE3] aggregates obtained in vivo were different than amyloid fibers produced in vitro. In a second phase, the technicity acquired during the biochemical study of Ure2p allowed me to approach the study of an ortholog, Saccharomyces paradoxus Ure2p (Ure2p-Sp), which is not an "in vivo" prion. After showing that soluble forms of the two proteins have the same biochemical characteristics, I show that they have the same propensity to form amyloid fibers. In this study we show that these fibers are infectious in vivo, suggesting that the incapacity of Ure2p-Sp protein to switch into a prion form is rather due to a failure in prion propagation mechanism rather than differences in amyloid structure itself.Then using all technologies that I introduced in the laboratory, I studied the relationship between amyloid structure and toxicity in S. cerevisiae. We showed that it was possible to transform a non-toxic amyloid protein in a toxic form by changing only a few numbers of amino acids. An in vitro study has also shown that the toxic protein was organized into short amyloid fibers that could mimic the intermediates responsible for toxicity in neurodegenerative diseases.The last chapter of this manuscript focuses on research that I developed in the Laboratory of Ecotoxicology Interactions,Biodiversity, Ecosystems, Metz. Within this laboratory, we aim at identifying effects of disturbances and physico-chemical analysis of the mechanisms involved at different scales of observation, from organisms to ecosystems.Progress in genomes sequencing allows an exceptional development in post-genomic biochemistry and can highlight new proteins through functional studies. In order to better understand molecular events responsible for these responses, I introduce a differential technique of proteomics: a DiGE two-dimensional electrophoresis (Differential In-Gel Electrophoresis). I developed this technics in a proteome study of an invasive plant, the goldenrod Solidago canadensis, under metal stress. This proteomic study supplements a phyto-sociological study of plants competent in colonizing a very poor soil but also of biodiversity data, growth and measures of the antioxidant response of these plants. This analysis revealed that in polluted soils, the goldenrod achieves not only to produce energy for its growth but it also achieves to biosynthesize intermediates of glutathion and phytochelatins. Thus, the goldenrod is able to adapt to its environment, which allows it to be tolerant to pollution. My first results of post-genomic biochemistry provide new bases for understanding the molecular mechanisms behind environmental tolerances. These studies may help us to identify the most adapted plants to these environments and why they are. They may help us to understand the effects of these pollutants and allow the development of innovative technologies for phytoremediation to restore polluted soils

Στην εργασία διερευνήθηκε πώς η έλλειψη του υποδοχέα της χαμηλής πυκνότητας λιποπρωτεϊνών (Ldlr-/-) τροποποιεί τις επιδράσεις της τεστοστερόνης στην παχυσαρκία και στις συναφείς μεταβολικές δυσλειτουργίες. ‘Εμμεση θερμιδομετρική ανάλυση έδειξε πως ο υπογοναδισμός σε μυς με έλλειψη του Ldlr συσχετίζεται με μείωση του βάρους του σώματος, και παράλληλα, αυξημένο μεταβολικό ρυθμό. Η έκφραση του κυτοχρώματος C και της UCP1 των μιτοχονδρίων ήταν αυξημένη στο λευκό λιπώδη ιστό, υποδεικνύοντας ότι η αυξημένη μεταβολική δραστηριότητα αντανακλά σε αυξημένο αριθμό μιτοχονδρίων με επίσης αυξημένη ικανότητα θερμογένεσης. Η αποκατάσταση της τεστοστερόνης σε ορχεκτομηθέντες μυς Ldlr-/- για διάστημα οχτώ εβδομάδων οδήγησε σε παχυσαρκία επαγόμενη από δίαιτα, υποδεικνύοντας την άμεση σχέση της τεστοστερόνης με τον παρατηρούμενο φαινότυπο. Η θεραπεία ψευδοχειρουργημένων μυών Ldlr-/- με εξεμεστάνη, έναν αναστολέα της αρωματάσης, για οχτώ εβδομάδες, έδειξε πως η αντίσταση στην παχυσαρκία των ορχεκτομηθέντων Ldlr-/- μυών είναι ανεξάρτητη από την δράση των οιστρογόνων. Συμπερασματικά, η εργασία δείχνει πως ο LDLr συσχετίζεται με μεταβολικές αλλαγές σε υπογοναδικούς μυς, οι οποίες δεν οφείλονται σε οιστρογονικές δράσεις, αλλά.στην ανεπάρκεια τεστοστερόνης. / In this work we investigated how low-density lipoprotein receptor deficiency (Ldlr-/-) modulates the effects of testosterone on obesity and related metabolic disorders. Indirect calorimetric analysis indicated that hypogonadism in Ldlr deficient mice correlates with a decrease in body weight and an increased metabolic rate. The expression of cytochrome C and UCP1 in mitochondria was increased in white adipose tissue, indicating that the increased metabolic activity reflects an increased number of mitochondria with an increased ability for thermogenesis. Testosterone replacement in orchectomized Ldlr-/- mice for a period of eight weeks led to diet induced obesity, suggesting a direct relationship between testosterone and the observed phenotype. Treatment of sham-operated Ldlr-/- mice with exemestane, an aromatase inhibitor, for eight weeks, showed that the obesity of orchectomized Ldlr-/- mice is independent of estrogen effects. In conclusion, this work demonstrates how the LDLr is associated with metabolic changes in hypogonadal mice which are not related to estrogenic effects but to testosterone deficiency.

Ο λιπώδης ιστός δεν θεωρείται πλέον αποκλειστικά παθητικός αποταμιευτικός ιστός, αλλά εκκρίνει ποικίλα βιοδραστικά πεπτίδια, γνωστά ως λιποκίνες. Η εμπλοκή των τελευταίων στην παθογένεια του μεταβολικού συνδρόμου και των επιπλοκών του τις καθιστά μόρια-στόχους που δύνανται να συμβάλουν στη θεραπευτική προσέγγιση του μεταβολικού συνδρόμου. / Adipose tissue is no more considered a passive tissue with storage function, but it has proved to be a source of a variety of bioactive peptides, described as adipokines. The implication of adipokines in the pathogenesis of metabolic syndrome and its consequences renders many of them putative target molecules in a new therapeutic approach of this syndrome.