A microarray-based system for the simultaneous analysis of single nucleotide polymorphisms in human genes involved in the metabolism of anti-malarial drugs

Background: In order to provide a cost-effective tool to analyse pharmacogenetic markers in malaria treatment, DNA microarray technology was compared with sequencing of polymerase chain reaction (PCR) fragments to detect single nucleotide polymorphisms (SNPs) in a larger number of samples. Methods: The microarray was developed to affordably generate SNP data of genes encoding the human cytochrome P450 enzyme family (CYP) and N-acetyltransferase-2 (NAT2) involved in antimalarial drug metabolisms and with known polymorphisms, i.e. CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP3A4, CYP3A5, and NAT2. Results: For some SNPs, i.e. CYP2A6*2, CYP2B6*5, CYP2C8*3, CYP2C9*3/*5, CYP2C19*3, CYP2D6*4 and NAT2*6/*7/*14, agreement between both techniques ranged from substantial to almost perfect (kappa index between 0.61 and 1.00), whilst for other SNPs a large variability from slight to substantial agreement (kappa index between 0.39 and 1.00) was found, e. g. CYP2D6*17 (2850C>T), CYP3A4*1B and CYP3A5*3. Conclusion: The major limit of the microarray technology for this purpose was lack of robustness and with a large number of missing data or with incorrect specificity.

PVHL is a regulator of glucose metabolism and insulin secretion in pancreatic beta cells.

Insulin secretion from pancreatic beta cells is stimulated by glucose metabolism. However, the relative importance of metabolizing glucose via mitochondrial oxidative phosphorylation versus glycolysis for insulin secretion remains unclear. von Hippel-Lindau (VHL) tumor suppressor protein, pVHL, negatively regulates hypoxia-inducible factor HIF1alpha, a transcription factor implicated in promoting a glycolytic form of metabolism. Here we report a central role for the pVHL-HIF1alpha pathway in the control of beta-cell glucose utilization, insulin secretion, and glucose homeostasis. Conditional inactivation of Vhlh in beta cells promoted a diversion of glucose away from mitochondria into lactate production, causing cells to produce high levels of glycolytically derived ATP and to secrete elevated levels of insulin at low glucose concentrations. Vhlh-deficient mice exhibited diminished glucose-stimulated changes in cytoplasmic Ca(2+) concentration, electrical activity, and insulin secretion, which culminate in impaired systemic glucose tolerance. Importantly, combined deletion of Vhlh and Hif1alpha rescued these phenotypes, implying that they are the result of HIF1alpha activation. Together, these results identify pVHL and HIF1alpha as key regulators of insulin secretion from pancreatic beta cells. They further suggest that changes in the metabolic strategy of glucose metabolism in beta cells have profound effects on whole-body glucose homeostasis.

Altered high-energy phosphate metabolism predicts contractile dysfunction and subsequent ventricular remodeling in pressure-overload hypertrophy mice.

To study the role of early energetic abnormalities in the subsequent development of heart failure, we performed serial in vivo combined magnetic resonance imaging (MRI) and (31)P magnetic resonance spectroscopy (MRS) studies in mice that underwent pressure-overload following transverse aorta constriction (TAC). After 3 wk of TAC, a significant increase in left ventricular (LV) mass (74 +/- 4 vs. 140 +/- 26 mg, control vs. TAC, respectively; P < 0.000005), size [end-diastolic volume (EDV): 48 +/- 3 vs. 61 +/- 8 microl; P < 0.005], and contractile dysfunction [ejection fraction (EF): 62 +/- 4 vs. 38 +/- 10%; P < 0.000005] was observed, as well as depressed cardiac energetics (PCr/ATP: 2.0 +/- 0.1 vs. 1.3 +/- 0.4, P < 0.0005) measured by combined MRI/MRS. After an additional 3 wk, LV mass (140 +/- 26 vs. 167 +/- 36 mg; P < 0.01) and cavity size (EDV: 61 +/- 8 vs. 76 +/- 8 microl; P < 0.001) increased further, but there was no additional decline in PCr/ATP or EF. Cardiac PCr/ATP correlated inversely with end-systolic volume and directly with EF at 6 wk but not at 3 wk, suggesting a role of sustained energetic abnormalities in evolving chamber dysfunction and remodeling. Indeed, reduced cardiac PCr/ATP observed at 3 wk strongly correlated with changes in EDV that developed over the ensuing 3 wk. These data suggest that abnormal energetics due to pressure overload predict subsequent LV remodeling and dysfunction.

Conceptualisation et emploi du passé composé et de l'imparfait en français langue étrangère (L3) au Sri Lanka : l'impact du singhalais langue maternelle (L1) et de l'anglais langue seconde (L2)

Résumé: Notre étude chevauche deux domaines de recherche quasi indissociables : ceux de la linguistique et de la didactique des langues. Comme l'indique le sujet, elle examine la conceptualisation et l'emploi de deux notions aspecto-temporelles du français (le passé composé et l'imparfait), sous l'impact des connaissances grammaticales déjà acquises sur deux autres langues : le singhalais et l'anglais. Notre recherche relève des domaines de la psycholinguistique, de la linguistique acquisitionnelle et de la linguistique comparative. Toutefois, dans le cadre de cette étude, nous examinons ces notions grammaticales françaises et leurs équivalents présumés dans les deux autres langues comme étant des concepts relevant des langues à statuts sociaux spécifiques [à savoir, langue maternelle (L1), langue seconde (L2) et langue étrangère (L3)], dans un contexte particulier d'enseignement/apprentissage et d'acquisition de langue [à savoir, le contexte d'enseignement/apprentissage et d'acquisition du français langue étrangère (FLE) au Sri Lanka]. En ce sens, notre étude est également liée aux domaines de la sociolinguistique et de la didactique des langues, notamment, étrangères. Ce qui pourrait probablement distinguer cette recherche des autres, c'est qu'elle aborde certaines questions linguistiques et didactiques peu étudiées jusqu'ici. Entre autres, l'influence de deux langues sur l'enseignement/apprentissage d'une L3, l'enseignement/apprentissage des langues dans des contextes exolingues et le rôle des transferts dans la conceptualisation des notions grammaticales. Pourtant, lorsque nous avons choisi le contexte d'apprentissage du FLE au Sri Lanka comme terrain de recherche, nous avons également visé d'autres objectifs : examiner les systèmes verbaux de trois langues dont l'imbrication n'a pas encore été objet d'étude ; examiner le système verbal aspecto-temporel peu explicité du singhalais à la lumière des descriptions linguistiques occidentales ; vérifier certains préjugés concernant les liens de proximité et de distance entre les trois langues choisies et étudier les causes de ces préjugés. Notre corpus provient de plusieurs classes de FLE au Sri Lanka. Le public observé était constitué d'adolescents ou d'adultes bilingues ayant le singhalais en L1 et l'anglais en L2. Les cours choisis se distinguaient les uns des autres par plusieurs critères, mais travaillaient tous sur les notions du passé composé et de l'imparfait. A la conclusion de notre étude, nous avons constaté qu'un nombre important de nos hypothèses initiales se sont avérées véridiques. A titre d'exemples, les transferts entre les langues premières et la langue cible sont récurrents et non négligeables chez l'écrasante majorité des apprenants exolingues observés, et parfois, même chez leurs enseignants; si ces apprenants recourent à ces langues pour étayer leur apprentissage, ni leurs enseignants ni leurs manuels provenant de l'étranger ne les guident dans ce travail; les transferts ayant l'anglais pour origine l'emportent considérablement sur ceux provenant du singhalais. De même, suite à l'analyse contrastive des trois systèmes verbaux aspecto-temporels et à l'analyse du corpus, nous avons également eu un résultat imprévu : contrairement à une représentation répandue chez les apprenants singhalais, il existe des points convergents entre leur L1 et le français ; du moins, au niveau de l'emploi de certains temps du passé. Un fait dont on était jusqu'ici ignorants mais dont on peut sûrement profiter dans les cours de FLE au Sri Lanka. Suite à ces observations et à la fin de notre thèse, nous avons fait quelques recommandations didactiques afin d'améliorer les conditions d'enseignement/apprentissage des langues étrangères, au Sri Lanka et ailleurs. Abstract: Our research is related to the fields of both linguistics and didactics, two research areas which are almost inseparable. As the title shows, the thesis examines the issue of conceptualizing and using of two grammatical (aspectual and temporal) concepts of the French language (le passé composé and l'imparfait), under the influence of previously acquired grammatical knowledge of two other languages: Sinhalese and English. Thus, our research is linked to the domains of psycholinguistics, acquisitional linguistics and comparative linguistics. However, within the framework of this study, we will consider the above-mentioned two French grammatical concepts and their presumed equivalents in the other two languages as concepts belonging to three languages with specific social status [i.e. first language (L1), second language (L2) and foreign language (L3)], taught/learnt/acquired in a particular language teaching/learning context [the context of teaching/learning of French as a foreign language (FFL) in Sri Lanka]. In that sense, our study is also associated with the fields of sociolinguistics and language teaching, especially foreign language teaching. What could probably make this study outstanding is that it studies certain linguistic and didactic issues which have not yet been studied. For example, it examines, among other issues, the following: the influence of two languages (i.e. mother tongue -L1 & second language -L2) on the teaching/learning process of a third language (i.e. foreign language- L3); foreign language teaching and learning in an exolingual context (where the target language is not spoken outside the classroom); the role of language transfers in the process of grammatical notion conceptualization. However, in selecting the FFL teaching/learning context in Sri Lanka as our field of research, we had further objectives in mind : i.e. 1) studying the verb systems of three languages whose combination has never been studied before ; 2) studying the aspectual-temporal formation of the Sinhalese verb system (which is hardly taught explicitly) in the light of the linguistic descriptions of dominant European languages; 3) verifying certain preconceived ideas regarding the proximity and the distance between the three chosen languages, and 4) studying the causes for these preconceptions. Our corpus is obtained from a number of FFL classes in Sri Lanka. The observed student groups consisted of bilingual adolescents and adults whose first language (L1) was Sinhalese and the second language (L2) was English. The observed classes differed in many ways but in each of those classes, a common factor was that the students had been learning some aspect of the two grammatical concepts, le passé composé and l'imparfait. Having completed our study, we now see that a considerable number of our initial hypotheses are proven correct. For example, in the exolingual French language teaching/learning context in Sri Lanka where we carried out our research, language transfers between the first and target languages were recurrent and numerous in the work of the greater majority of the observed language learners, and even their teachers; these transfers were so frequent that they could hardly be ignored during the teaching/learning process ; although learners turned to their first languages to facilitate the learning process of a new language, neither their teachers, nor their text books helped them in this task; the transfers originating from English were far too numerous than those originating from Sinhalese; however, contrary to the popular belief among many Sinhalese learners of French, the contrastive analysis of the three aspectual-temporal verb systems and the study of our corpus helped us in proving that there are common linguistic features between the Sinhalese and the French languages ; at least, when it comes to using some of their past tenses. This is a fact which had been ignored up to now but which could probably be used to improve French teaching/learning in Sri Lanka. Taking all observations into account, we made some pedagogical recommendations in the concluding part of our thesis with the view of improving foreign language teaching/learning in Sri Lanka, and elsewhere.

Circadian clock orchestration of signaling pathways influences mouse metabolism

Circadian clocks, present in organisms leaving in a rhythmic environment, constitute the mechanisms allowing anticipation and adaptation of behavior and physiology in response to these environmental variations. As a consequence, most aspects of metabolism and behavior are under the control of this circadian clock. At a molecular level, in all the studied species, the rhythmic expression of the genes involved are generated by interconnected transcriptional and translational feedback loops. In mammals, the heterodimer composed of BMAL1 and its partners CLOCK or NPAS2 constitutes a transcriptional activator regulating transcription of Per and Cry genes. These genes encode for repressors of the activity of BMAL1:CLOCK or BMAL1: NPAS2 heterodimers, thus closing a negative feedback loop that generates rhythms of approximately 24 hours. The aim of my doctoral work consisted in the investigation of the role of circadian clock in the regulation of different aspects of mouse metabolism through the rhythmic activation of signaling pathways. First, we showed that one way how the circadian clock exerts its function as an oscillator is through the regulation of mRNA translation. Indeed, we present evidence showing that circadian clock influences the temporal translation of a subset of mRNAs involved in ribosome biogenesis by controlling the transcription of translation initiation factors as well as the clock-dependent rhythmic activation of signaling pathways involved in their regulation. Moreover, the circadian oscillator regulates the transcription of ribosomal protein mRNAs and ribosomal RNAs. Thus the circadian clock exerts a major role in coordinating transcription and translation steps underlying ribosome biogenesis. In the second part, we showed the involvement of the circadian clock in lipid metabolism. Indeed, the three PAR bZip transcription factors DBP, TEF and HLF, are regulated by the molecular clock and play key roles in the control of lipid metabolism. Here we present evidence concerning the circadian expression and activity of PPARα via the circadian transcription of genes involved in the release of fatty acids, natural ligands of PPARα. It leads to the rhythmic activation of PPARα itself which could then play its role in the transcription of genes encoding proteins involved in lipid, cholesterol and glucose metabolism. In addition, we considered the possible role of lipid transporters, here SCP2, in the modulation of circadian activation of signaling pathways such as TORC1, PPARα and SREBP, linked to metabolism, and its feedback on the circadian clock. In the last part of this work, we studied the effects of these circadian clock-orchestrated pathways in physiology, as clock disruptions have been shown to be linked to metabolic disorders. We performed in vivo experiments on genetically and high-fat induced obese mice devoid of functional circadian clock. The results obtained showed that clock disruption leads to impaired triglycerides and glucose homeostasis in addition to insulin secretion and sensitivity. -- Les rythmes circadiens, présents chez tout organisme vivant dans un environnement rythmique, constituent l'ensemble de mécanismes permettant des réponses comportementales et physiologiques anticipées et adaptées aux variations environnementales. De ce fait, la plupart des aspects liés au métabolisme et au comportement de ces organismes apparaissent être sous le contrôle de l'horloge circadienne contrôlant ces rythmes. Au niveau moléculaire, dans toutes les espèces étudiées, l'expression rythmique de gènes impliqués sont générés par l'interconnexion de boucles de contrôle transcriptionnelles et traductionnelles. Chez les mammifères, l'hétérodimère composé de BMAL1 et de ses partenaires CLOCK ou NPAS2 constitue un activateur transcriptionnel régulant la transcription des gènes Per et Cry. Ces gènes codent pour des répresseurs de l'activité des hétérodimères BMAL1:CLOCK ou BMAL1:NPAS2. Cela a pour effet de fermer la boucle négative, générant ainsi des rythmes d'environ 24 heures. Le but de mon travail de thèse a consisté en l'investigation du rôle de l'horloge circadienne dans la régulation de certains aspects du métabolisme chez la souris via la régulation de l'activation rythmique des voies de signalisation. Nous avons tout d'abord montré que l'horloge circadienne exerce sa fonction d'oscillateur notamment au niveau de la régulation de la traduction des ARNm. En effet, nous présentons des preuves montrant que l'horloge circadienne influence la traduction temporelle d'un groupe d'ARNm impliqués dans la biogénèse des ribosomes en contrôlant la transcription de facteurs d'initiation de la traduction ainsi que l'activation rythmique des voies de signalisation qui sont impliquées dans leur régulation. De plus, l'oscillateur circadien régule la transcription d'ARNm codant pour les protéines ribosomales et d'ARN ribosomaux. De cette façon, l'horloge circadienne exerce un rôle majeur dans la coordination des étapes de transcription et traduction permettant la biogénèse des ribosomes. Dans la deuxième partie, nous montrons les implications de l'horloge circadienne dans le métabolisme des lipides. En effet, DBP, TEF et HLF, trois facteurs de transcription de la famille des PAR bZip qui sont régulés par l'horloge circadienne, jouent un rôle clé dans le contrôle du métabolisme des lipides par l'horloge circadienne. Nous apportons ici des preuves concernant l'expression et l'activité rythmiques de PPARα via la transcription circadienne de gènes impliqués dans le relargage d'acides gras, ligands naturels de PPARα, conduisant à l'activation circadienne de PPARα lui-même, pouvant ainsi jouer son rôle de facteur de transcription de gènes codant pour des protéines impliquées dans le métabolisme des lipides, du cholestérol et du glucose. De plus, nous nous sommes penchés sur le rôle possible de transporteurs de lipides, ici SCP2, dans la modulation de l'activation circadienne de voies de signalisation, telles que TORC1, PPARα et SREBP, qui sont liées au métabolisme, ainsi que son impact sur l'horloge elle-même. Dans la dernière partie de ce travail, nous avons étudié les effets de l'activation de ces voies de signalisation régulées par l'horloge circadienne dans le contexte physiologique puisqu'il a été montré que la perturbation de l'horloge pouvait être associée à des désordres métaboliques. Pour ce faire, nous avons fait des expériences in vivo sur des souris déficientes pour l'horloge moléculaire pour lesquelles l'obésité est induite génétiquement ou induite par la nourriture riche en lipides. Les résultats que nous obtenons montrent des dérèglements au niveau de l'homéostasie des triglycérides et du glucose ainsi que sur l'expression et la réponse à l'insuline.

Brain energy metabolism measured by (13) C magnetic resonance spectroscopy in vivo upon infusion of [3-(13) C]lactate.

The brain uses lactate produced by glycolysis as an energy source. How lactate originated from the blood stream is used to fuel brain metabolism is not clear. The current study measures brain metabolic fluxes and estimates the amount of pyruvate that becomes labeled in glial and neuronal compartments upon infusion of [3-(13) C]lactate. For that, labeling incorporation into carbons of glutamate and glutamine was measured by (13) C magnetic resonance spectroscopy at 14.1 T and analyzed with a two-compartment model of brain metabolism to estimate rates of mitochondrial oxidation, glial pyruvate carboxylation, and the glutamate-glutamine cycle as well as pyruvate fractional enrichments. Extracerebral lactate at supraphysiological levels contributes at least two-fold more to replenish the neuronal than the glial pyruvate pools. The rates of mitochondrial oxidation in neurons and glia, pyruvate carboxylase, and glutamate-glutamine cycles were similar to those estimated by administration of (13) C-enriched glucose, the main fuel of brain energy metabolism. These results are in agreement with primary utilization of exogenous lactate in neurons rather than astrocytes. © 2014 Wiley Periodicals, Inc.

A peroxisomal glutathione transferase of Saccharomyces cerevisiae is functionally related to sulfur amino acid metabolism

Saccharomyces cerevisiae cells contain three omega-class glutathione transferases with glutaredoxin activity (Gto1, Gto2, and Gto3), in addition to two glutathione transferases (Gtt1 and Gtt2) not classifiable into standard classes. Gto1 is located at the peroxisomes, where it is targeted through a PTS1-type sequence, whereas Gto2 and Gto3 are in the cytosol. Among the GTO genes, GTO2 shows the strongest induction of expression by agents such as diamide, 1-chloro-2,4-dinitrobenzene, tert-butyl hydroperoxide or cadmium, in a manner that is dependent on transcriptional factors Yap1 and/or Msn2/4. Diamide and 1-chloro-2,4-dinitrobenzene (causing depletion of reduced glutathione) also induce expression of GTO1 over basal levels. Phenotypic analyses with single and multiple mutants in the S. cerevisiae glutathione transferase genes show that, in the absence of Gto1 and the two Gtt proteins, cells display increased sensitivity to cadmium. A gto1-null mutant also shows growth defects on oleic acid-based medium, which is indicative of abnormal peroxisomal functions, and altered expression of genes related to sulfur amino acid metabolism. As a consequence, growth of the gto1 mutant is delayed in growth medium without lysine, serine, or threonine, and the mutant cells have low levels of reduced glutathione. The role of Gto1 at the S. cerevisiae peroxisomes could be related to the redox regulation of the Str3 cystathionine -lyase protein. This protein is also located at the peroxisomes in S. cerevisiae, where it is involved in transulfuration of cysteine into homocysteine, and requires a conserved cysteine residue for its biological activity.

A Cellular Perspective on Brain Energy Metabolism and Functional Imaging.

The energy demands of the brain are high: they account for at least 20% of the body's energy consumption. Evolutionary studies indicate that the emergence of higher cognitive functions in humans is associated with an increased glucose utilization and expression of energy metabolism genes. Functional brain imaging techniques such as fMRI and PET, which are widely used in human neuroscience studies, detect signals that monitor energy delivery and use in register with neuronal activity. Recent technological advances in metabolic studies with cellular resolution have afforded decisive insights into the understanding of the cellular and molecular bases of the coupling between neuronal activity and energy metabolism and point at a key role of neuron-astrocyte metabolic interactions. This article reviews some of the most salient features emerging from recent studies and aims at providing an integration of brain energy metabolism across resolution scales.