L’obésité de l’enfant et de l’adolescent constitue un problème de santé publique important auquel sont confrontés depuis les années 1970 tous les pays industrialisés. Son origine multifactorielle, l’absence de médicaments présentant une balance bénéfice/risque favorable et l’absence de recommandations quant à la chirurgie bariatrique en font un problème thérapeutique complexe. Ceci a induit une grande variété de prises en charge thérapeutiques dont la comparaison reste difficile en absence de critère permettant de juger de leur efficacité. Ce travail avait pour but de déterminer l’efficacité d’une prise en charge multidisciplinaire dans le Centre « Les Oiseaux » pendant 8 mois, en hospitalisation complète. Une étude rétrospective incluant 1063 patients entre 1991 et 2007 a été réalisée. 746 sujets ont retourné le questionnaire 2 ans après leur sortie et 185 sujets, 5 ans après. Parmi eux plus de 75 % ont maintenu durablement au moins la moitié de leur perte de poids.Dans le but de retrouver des marqueurs précoces d’efficacité thérapeutique, nous avons aussi suivi une cohorte de 16 adolescentes et mis en place un programme de monitorage de 895 variables, données cliniques, psychologiques et biologiques ainsi que des données de pratique d’activité physique. Chaque variable a été testée pour son caractère discriminant et sa faisabilité. Une approche de métabolomique par empreintes a été mise en place sur des échantillons urinaires. Par analyse multivariée, nous avons pu montrer que le maintien 6 mois après la sortie, de la perte de poids était positivement corrélé avec la prise en charge psychologique, l’implication familiale et la capacité de conserver une activité physique suffisante, elle même sous-tendue par l’amélioration de l’estime de soi. / Obesity in childhood must be considered as a major issue in Public Health. Since the 1970s, all developed countries must have faced an increasing prevalence of this metabolic disorder. However since the epidemic of obesity has multifactorial causes, and without the availability of a positive benefit/risk balance for chemical drugs and clear-cut indications for bariatric surgery, it is easy to understand that obesity is a complex issue. This complexity induced different schemes of care the comparison of which is made difficult by the absence of clear criteria for judging therapeutic efficacy. This work was aimed to determine the efficacy of a 8-months residential treatment program performed by a multidisciplinary team in the Care Centre “Les Oiseaux”. In order to demonstrate whether the treatment was of benefit for the patients, 1063 of those treated between 1991 and 2007 have been included in a retrospective study. 746 patients returned the questionnaire after 2 years and 185 patients, 5 years later. Among these patients 75 % were actually able to control their weight since they maintained at least half their weight loss. To unveil early parameters of therapeutic efficacy, we have set up a monitoring record of more than 895 clinical, psychological, and biological parameters as well as an accurate recording of the physical activity of adolescents during their stay in the Care Centre. In addition a metabolic profiling of urine samples has been performed. Each parameter was tested for its discrepancy power as well as its practical aspects. Sixteen female adolescents have then been followed for the duration of their stay according to our monitoring program. By multivariate analysis, we deduced that the maintaining of their weight loss was positively correlated with the psychological care, the family involvement, as well as the ability to keep a sufficient physical activity, which was sustained by the recovery of their self-esteem.

Thesis (MSc (Plant Biotechnology))--University of Stellenbosch, 2010. / Includes bibliography. / ENGLISH ABSTRACT: The study of plants on a sub-cellular level is an important, yet challenging area and its application allows for novel insight into the understanding of metabolism and its regulation. In this study I describe the development of a reverse phase liquid chromatography mass spectrometry (RPLC-MS) technique in which 29 phosphorylated and nucleotide sugars could be detected and quantified. The method was validated with the use of authentic standards and the system displayed very good linearity (Rª > 0.95), while the recovery of the standards added to the plant material before extraction was between 65 and 125%. Further, Arabidopsis thaliana wild type (Col-0) and adenylate kinase (adk1) mutant leaf discs were fed 13C labeled glucose over a period of 24 hours and harvested at defined time intervals. Non aqueous fractionation, and metabolite profiling via the above mentioned rpLC-MS method in conjunction with gas chromatography mass spectrometry (GC-MS) allowed for the detection and quantification of primary metabolites on a sub-cellular level as well as the determination of their relative isotopic label enrichments through primary carbon metabolism. Finally, a yeast complementation system was designed for the identification of tonoplast bound sucrose import proteins. The screening system identified 22 unique sequences from an Arabidopsis thaliana cDNA library. Four unknown sequences were identified, one of which displayed tonoplast membrane association upon in silico analysis. Three ATP-binding proteins were also identified as well as a sub-unit from the exocyst gene family. Further studies will include the functional characterization of the latter, as well as the development of additional cDNA libraries more suited for screening of sequences that encode sucrose importer proteins. / AFRIKAANSE OPSOMMING: Die studie van plante op a sub-sellulere vlak is ‘n belangrike maar uitdagende navorsingsarea en die toepassing daarvan dra by tot unieke insig tot ‘n beter begrip van metabolise regulasie. In die studie bespreek ek die ontwikkeling van ‘n teenoorgestelde fase vloeistof kromatografie massa spektrometrie (RPLC-MS) tegniek waarin 29 gefosforileerde en nukleotied suikers gevind en gekwantifiseer kon word. Geldigverklaring van die metode is bewerkstelling met die gebruik van oorspronklike standaarde and die systeem het baie goeie liniariteit (Rª > 0.95) getoon, terwyl die herstelbaarheid van standaarde wat bygevoeg is by die plant material voor ekstraksie tussen 65% en 125% was. Arabidopsis thaliana wilde type (Col-O) en die adenaliet kinase (adk1) mutant blaar dele is met 13C gemerkte glukose gevoed oor ‘n tydperk van 24 uur en geoes by spesifieke tydstippe. Nie-vloeibare fraksionering en metaboliet uitleg is vermag vanaf die genoemde RPLC-MS metode met behulp van gas kromotografie massa spektrometrie (GC-MS) wat die bepaling en kwantifikasie van primere metaboliete op n sub-sellulere vlak sowel as die bepaling van hul relatiewe isotropiese merker verrykers deur primere metabolisme toelaat. Verder is n gis komplementere systeem ontwerp vir die identifikasie van tonoplas gebinde sukrose invoer proteine. Die verkenningsysteem het 22 unieke volgordes opgelewer vanaf ‘n Arabidopsis thaliana kDNA biblioteek. Vier onbekende volgordes is geidentifiseer, een wat tonoplas membraan assosiasie toon met in silico analise. Drie ATP-bindings proteine is ook geidentifiseer asook ‘n sub-eenheid van die eksosyst geen familie. Verdere studies sal die funksionele karakterisering van die laaste protein insluit, asook die ontwikkeling van additionele kDNA biblioteke meer gepas vir verkenning sodiende identifiseer van volgordes wat sukrose invoer proteine vertaal.

Thesis (MSc)--Stellenbosch University, 2012 / ENGLISH ABSTRACT: The aroma and flavour of wine are important aspects that form the basis for consumers’ organoleptic experience of wine. Therefore, an understanding of the chemical composition of wine aroma is of major importance, to establish possible links between wine chemistry, sensory attributes and consumer preference for a product. For this purpose analytical chemistry and multivariate techniques are indispensable tools for the metabolic profiling of wine. Chenin blanc is one of the most important South African export white wine varieties. However, despite its importance, very limited profiling of Chenin blanc aroma compounds has been done and information is restricted to isolated and dated reports on a few chemical compounds only. Therefore, the overall aim of this study was to obtain an in-depth view of the volatile chemical profile of this cultivar. The first task was to perform targeted volatile metabolic profiling of the three dry and offdry Chenin blanc styles, fresh and fruity, rich and ripe unwooded and rich and ripe wooded. To this end, a new, simple and robust liquid-liquid extraction technique using dichloromethane was developed and validated for extraction of analytes prior to gas chromatography flame ionization detection (GC-FID) analysis, to quantify 57 analytes in one rapid analytical procedure. This method was applied to profile 48 Chenin blanc wines. Very successful discrimination between the three styles, using the quantified volatile compounds, was obtained with two multivariate methods. These were partial least squares regression-discriminant analysis, (PLS-DA), as well linear-DA, using best subset selection for identifying the most important variables. According to the classification models, a higher content of maturation derived, malolactic fermentation derived and wood derived compounds were predominantly characteristic of the wooded wines. Higher content of some terpenes and ethyl esters were predominantly associated with the rich and ripe unwooded style Chenin blanc wines, while the fresh and fruity style were generally characterized by high levels of acetate esters. Secondly, untargeted analysis of 21 wines was done with gas chromatography mass spectrometry (GC-MS). Mathematical chromatography, using PARAllel FACtor analysis (PARAFAC and PARAFAC2), was applied to the GC-MS data for resolution of the complex chromatographic results by multi-way modeling, and to derive unbiased multivariate classification models of the three styles. This approach provided excellent style differentiation, without the arduous task of analysis of numerous standards and setting up of calibration curves, required by the targeted approach described above. Additionally, the data generated during this study will form part of the current South African wine aroma database, which does not contain any data regarding Chenin blanc at present. / AFRIKAANSE OPSOMMING: Die aroma en geur van wyn is belangrike aspekte aangesien dit die basis vorm van die wynverbruiker se organoleptiese ervaring van die produk. Derhalwe, is ‘n deeglike kennis van die chemiese samestelling van wynaroma baie belangrik, ten einde die korrelasies tussen wynchemie, sensoriese eienskappe en verbruikersvoorkeure te bepaal. Vir hierdie doel, is die insameling van analitiese chemiese data, tesame met multi-veranderlike tegnieke om relevante inligting uit die data te onttrek, onmisbaar vir die metaboliese profilering van wyn. Chenin blanc is huidiglik een van Suid-Afrika se belangrikste uitvoer wit wynvariëteite. Ten spyte hiervan, is daar tot hede egter baie min profilering van Chenin blanc se aromakomponente gedoen en die beskikbare inligting is beperk tot geïsoleerde en verouderde navorsingsbevindinge. In die lig van bogenoemde, is die oorkoepelende motivering vir hierdie studie dus om die vlugtige chemiese komponente se profiel in Chenin blanc wyn, in diepte te bepaal. Die eerste taak was die geteikende bepaling van die metaboliese profiel van drie droë of halfdroë Chenin blanc wynstyle, nl. vars en vrugtig, ryk en ryp ongehout, asook ryk en ryp gehout. Om dit te bereik, is ‘n nuwe eenvoudige en robuuste vloeistof-vloeistof ekstraksieprosedure met dichlorometaan ontwikkel, wat analise met gaschromatografie – vlamionisasie deteksie (GC-FID) voorafgaan, om die konsentrasies van 57 komponente in een vinnige analise te bepaal. Hierdie metode is gebruik om 48 Chenin blanc wyne te profileer. Deur gebruik te maak van multi-veranderlike data analitiese tegnieke, is die gekwantifiseerde vlugtige komponente data gebruik in diskriminant analise. Baie suksesvolle onderskeid tussen die drie style, is verkry deur gebruik te maak van twee multi-veranderlike metodes, naamlik: parsiële kleinste kwadrate regressie, diskriminant analise, asook liniêre diskriminant analise. Vir laasgenoemde analise, is die seleksie van die mees belangrike veranderlikes met beste sub-groep regressie bepaal. Volgens hierdie klassifikasie modelle is ‘n hoër inhoud van veroudering-, appelmelksuurgisting- en houtverwante aroma komponente baie kenmerkend in die houtbehandelde wyne. Hoër vlakke van sommige terpene en etiel esters was kenmerkend met betrekking tot ryk en ryp ongehoute Chenin blanc style, terwyl die vars en vrugtige style meer gekenmerk was met hoë vlakke van asetaat esters. Tweedens is ‘n seleksie van 21 wyne geanaliseer deur gebruik te maak van gaschromatografie – massa spektrometrie (GC-MS) in ‘n ongeteikende metaboliese profileringsbenadering. Wiskundige chromatografiese metodes, spesifiek, parallelle faktor analise (PARAllel FACtor analysis (PARAFAC and PARAFAC2)), was voorts gebruik om komplekse chromatogramme te prosesseer met wiskundige, multi-vlak modellering. Met hierdie nie-selektiewe benadering, is ook suksesvolle klassifikasiemodelle gegenereer vir die diskriminasie tussen die drie verskillende Chenin blanc style. Die voordeel van die ongeteikende GC-MS analise, gekoppel met die data hanterings- en prosesseringsprotokols in hierdie studie gebruik, is dat die arbeidsintensiewe taak om kalibrasiekurwes op te stel vir elke individuele komponent, soos wat vereis word in die geteikende benadering, nie nodig is nie. Die data wat ingewin is gedurende hierdie studie, sal ook bygevoeg word tot ‘n bestaande Suid-Afrikaanse aroma databasis, wat tans geen data aangaande Chenin blanc wyn bevat nie.

An exceptionally important stress response of Pseudomonas putida strains to toxic chemicals is the induction of efflux pumps that extrude solvents, as well as other toxicants, into the surrounding medium. However, the bacterial tolerance mechanisms are still not fully understood, thus in this thesis metabolomic approaches were used to detect and identify metabolites involved in P. putida DOT-T1E tolerance to abiotic stresses, in particular focussing on the role of efflux pumps. To elucidate any metabolome alterations several strains of P. putida, including the wild type DOT-T1E, and the efflux pump knockouts DOT-T1E-PS28 and DOT-T1E-18, were challenged with different levels of propranolol. Fourier-transform infrared (FT-IR) spectroscopy, which provided a rapid, high-throughput metabolic fingerprint of P. putida strains, was used to investigate any phenotypic changes resulting from exposure to propranolol. FT-IR data illustrated phenotypic changes associated with the presence of propranolol within the cell that could be assigned to the bacterial protein components. To complement this phenotypic fingerprinting approach metabolic profiling on the same samples was performed using gas chromatography mass spectrometry (GC-MS) to identify metabolites of interest during growth of bacteria following this toxic perturbation with propranolol. GC-MS revealed significant changes in ornithine levels which can be directly linked to bacterial tolerance mechanisms, and alterations in the levels of several other metabolites which were also modified in response to propranolol exposure. Moreover, the effect of the organic solvent toluene was also investigated using the same approach. Examination of FT-IR data indicated that protein and fatty acids were the most affected components of P. putida strains due to the presence of toluene within the cell. Moreover, application of GC-MS allowed for the identification and quantification of several metabolites which were differentially produced or consumed in the presence of toluene. To investigate the role of efflux pumps in P. putida DOT-T1E, several analytical techniques were employed including Raman spectroscopy, gas and liquid chromatography to identify and quantify the level of propranolol or toluene in P. putida cells. These analyses showed that propranolol and toluene accumulated in the mutant P. putida DOT-T1E-18 (lacking the TtgABC pump) at higher levels in comparison with the levels found in the wild-type DOT-T1E and the mutant DOT-T1E-PS28 (lacking the TtgGHI pump), indicating the key role of efflux pumps in solvent tolerance. Furthermore, the effect of Mg2+ and Ca2+ on the stabilisation of the toluene tolerance of P. putida DOT-T1E strains was examined in order to elucidate whether divalent cations interact with efflux pumps or other resistant mechanisms to improve solvent tolerance. FT-IR analysis suggested that the influence of divalent cations on the stabilisation of the toluene tolerance could be due to the contribution of metal ions towards other tolerance mechanisms such as lipopolysaccharide (LPS) instead of enhancing the activity of efflux pumps. In conclusion, this thesis presents evidence that phenotypic fingerprinting and metabolic profiling approaches in combination with chemometric methods can generate valuable information on phenotypic responses occurring within microbial cultures subjected to abiotic stress.

La sporulation est un phénomène présent chez de nombreux microorganismes, généralement impliqué dans les mécanismes de dispersion et/ou résistance en conditions environnementales défavorables. La sporulation observée chez certaines souches de Frankia (genre actinobactérien fixateur d'azote) lors de leur interaction symbiotique avec les plantes actinorhiziennes est donc paradoxale dans un contexte où la bactérie bénéficie d'une niche écologique favorable à son développement. Ces souches particulières de Frankia, dites Sp+, représentent un modèle unique de symbiote capable de sporulation au sein même des cellules de son hôte. Le rôle écologique et le sens évolutif de cette sporulation in-planta reste à ce jour peu élucidé. Les deux principaux objectifs de ce travail de thèse visent donc à (i) comprendre l'influence de la sporulation in-planta sur les capacités symbiotiques des souches Sp+, en termes d'infectivité et de compétitivité et (ii) appréhender l'impact de cette sporulation sur le fonctionnement du complexe symbiotique par une méthode de profilage métabolique. Ces travaux ont permis de confirmer les particularités symbiotiques des souches Sp+ (infectivité et compétitivité accrues) et de montrer des différences significatives dans le métabolisme primaire et secondaire du complexe symbiotique associées à la présence de spores de Frankia / Sporulation is a phenomenon present in many microorganisms, usually involved in the mechanisms of dispersion and/or resistance to unfavorable environmental conditions. Sporulation occurs in some Frankia strains (a diazotrophic actinobacteria) during their symbiotic interaction with actinorhizal plants, which is paradoxical in a context where the bacterium has a favorable ecological niche for its development. These particular Frankia strains, called Sp+, represent a unique model of symbiont capable of sporulation within the host cells. The ecological role and the evolutionary meanings of this in-planta sporulation still remain understood. The two main objectives of this thesis aimed to (i) understand the influence of in-planta sporulation on the symbiotic capacity of Sp+ strains in terms of infectivity and competitiveness and (ii) understand the impact of this sporulation on the functioning of the symbiotic complex by a metabolic profiling approach. These studies have confirmed the symbiotic characteristics of Sp+ strains (greater infectivity and competitiveness) and have shown significant differences in the primary and secondary metabolism of the symbiotic complex associated with the presence of Frankia spores

Background: Tumor cells are highly dependent on glucose even in the presence of oxygen. This concept called the Warburg effect is a hallmark of cancer and strategies are considered to therapeutically exploit the phenomenon such as ketogenic diets. The success of such strategies is dependent on a profound understanding of tumor cell metabolism. With new techniques it is now possible to thoroughly analyze the metabolic responses to the withdrawal of substrates and their substitution by others. In the present study we used gas chromatography coupled to mass spectrometry (GC-MS) to analyze how glioblastoma brain tumor cells respond metabolically when glucose is withdrawn and substituted by pyruvate. Methods: Glioblastoma brain tumor cells were cultivated in medium with high (25 mM), medium (11 mM) or low (5.5 mM) glucose concentration or with pyruvate (5 mM). After 24 h GC-MS metabolite profiling was performed. Results: The abundances of most metabolites were dependent on the supply of glucose in tendency but not in a linear manner indicating saturation at high glucose. Noteworthy, a high level of sorbitol production and release was observed at high concentrations of glucose and high release of alanine, aspartate and citrate were observed when glucose was substituted by pyruvate. Intermediates of the TCA cycle were present under all nutritional conditions and evidence was found that cells may perform gluconeogenesis from pyruvate. Conclusions: Our experiments reveal a high plasticity of glioblastoma cells to changes in nutritional supply which has to be taken into account in clinical trials in which specific diets are considered for therapy.

Diabetes mellitus currently affects over 422 million people globally and over 80% of patients with diabetes will develop diabetic retinopathy. Patients with diabetic retinopathy initially develop background retinopathy, which does not cause significant deterioration of visual function; however, background retinopathy may progress and lead to proliferative diabetic retinopathy and diabetic macular oedema, both of which cause severe visual dysfunction if left untreated. Current therapies for diabetic retinopathy include invasive intravitreal injections and laser photocoagulation; however these treatments only attenuate the progression of proliferative diabetic retinopathy and diabetic macular oedema. Aside from prevention by maintaining good blood glucose and blood pressure control, there are currently no treatments to prevent progression to late-stage diabetic retinopathy and new innovations in the field have not significantly progressed. For this reason, we have used untargeted –omics approaches to identify previously unknown pathological pathways in diabetes. In this thesis, I have analysed a range of trace metals in donor retinas and found that total copper was increased in diabetic retinas compared with non-diabetic. This result was replicated in streptozotocin-induced diabetic rat retinas and further evidenced by upregulation of metallothioneins and caeruloplasmin in diabetic rat retinas compared with non-diabetic. Treatment with the copper chelator triethylenetetramine modulated these changes, the downstream effects of which require further study. This is the first description, to our knowledge, of dysregulated copper homeostasis in the diabetic retina. I have also mapped metabolic changes in streptozotocin-induced diabetic rat retinas and found previously undescribed metabolite changes such as diabetes-induced downregulation of scyllo inositol. This coincided with substantial changes to retinal lipids during diabetes and changes to individual lipids were consistent within their respective class. I have also found a pattern whereby regardless of the extent of change to a lipid class in diabetes, lipids containing docosahexaenoic acid (22:6 carbon chain) were consistently downregulated. This is thought to be the first study to describe this range of metabolite changes in the diabetic retina but also the first study to describe this range of metabolite analysis concomitantly within the same tissue sample. The data from this study provides new insights into metallomic and metabolic dysfunction in diabetic retinopathy and shown that these data are reproducible. We suggest that there is plenty of scope for further research to investigate mechanisms behind copper dysregulation, how this affects pathogenesis of diabetic retinopathy along with new insights into dysregulated metabolic pathways.

Dans la rhizosphère du riz (Oryza sativa), certaines bactéries qualifiées de PGPR (Plant Growth Promoting Rhizobacteria), dont Azospirillum, sont capables de stimuler la croissance de cette céréale. Les effets bénéfiques de l’interaction PGPR-plante semblent conditionnés par la capacité de PGPR à interagir de façon précoce avec son hôte et d’induire chez ce dernier une réponse physiologique spécifique. Pour autant, peu d’informations sont disponibles quant à la nature et le degré de spécificité de la réponse physiologique de la plante à son interaction avec une PGPR. L’objectif de cette étude a donc été d’analyser, par profilage métabolique, la réponse physiologique du riz à son interaction avec Azospirillum et d’évaluer le degré de spécificité de cette réponse en fonction des partenaires de l’interaction. Dans ce but, nous avons comparé les profils chromatographiques des métabolites secondaires de deux cultivars de riz, Cigalon et Nipponbare, après inoculation ou non par les PGPR Azospirillum lipoferum 4B et Azospirillum sp. B510. Les résultats ont été comparés à ceux obtenus en présence d’une bactérie pathogène Burkholderia glumae AU6208 et d’une bactérie commensale Escherichia coli B6. En parallèle, la croissance végétale et l’architecture racinaire des cultivars ont été comparées, et la colonisation des systèmes racinaires par les PGPR a été suivie. Nous avons montré que chaque souche d’Azospirillum est capable de coloniser les deux cultivars, et qu’elle augmente préférentiellement la croissance du cultivar dont elle a été isolée. Le métabolisme secondaire du riz est profondément modifié en réponse à chacune des interactions biotiques testées. Dans ce cas des interactions riz-PGPR, la réponse est souche-cultivar dépendante. L’ensemble des résultats suggèrent l’existence d’une spécificité d’interaction entre la souche d’Azospirillum et son cultivar d’origine / In rice (Oryza sativa) rhizosphere, some bacteria designed as PGPR (Plant Growth Promoting Rhizobacteria) like Azospirillum, are able to enhance the growth of this crop. The beneficial effects of PGPR-plant interactions seem to be influenced by the ability of PGPR to interact at an early stage with its host and induce a specific physiological response. However, little information is available about the nature and degree of specificity of the physiological response of the plant to its interaction with PGPR. Thus, the aim of this study was to analyse, by using a metabolic profiling approach, the physiological response of rice to its interaction with Azospirillum and to assess the degree of specificity of this response according to interaction partners. To this purpose, chromatographic profiles of secondary metabolites of two rice cultivars, Nipponbare and Cigalon, were compared in non-inoculated plants and in plants inoculated by the PGPR Azospirillum lipoferum 4B and Azospirillum sp. B510. The results were compared with those obtained in the presence of pathogenic bacteria Burkholderia glumae AU6208 and a commensal bacteria Escherichia coli B6. In parallel, plant growth and root architecture of rice cultivars were compared, and root system colonization by PGPR was followed. We showed that each strain of Azospirillum is able to colonize both cultivars, and to preferentially increase the growth of the cultivar from which it was isolated. In addition, rice secondary metabolism is profoundly altered in response to each tested biotic interaction. In the case of rice-PGPR interactions, the response is strain-cultivar dependent. The overall results of this work suggest the existence of interaction specificity between Azospirillum strain and its cultivar of origin

No description available.

No description available.