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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
31

Étude métabolomique et valorisation pharmacologique et biotechnologique d'éspèces du genre Psiadia endémiques de la Réunion et de l'ile Maurice / Metabolomic study and pharmacological and biotechnological valorization of species of the Psiadia genus endemic to Reunion and Mauritius

Mahadeo, Keshika 28 February 2018 (has links)
Les travaux de thèse présentés dans ce manuscrit portent sur l’étude chimique de plantes du genre Psiadia. Trois axes de recherche ont été menés parallèlement à savoir (1) une étude chimiotaxonomique à partir de 11 espèces du genre Psiadia endémiques de La Réunion, (2) un criblage biologique réalisé sur 16 espèces du genre Psiadia dont 11 endémiques de La Réunion et 5 endémiques de Maurice et (3) une étude phytochimique ciblée sur l’espèce Psiadia arguta endémique de Maurice. Le premier axe comportant l'étude chimiotaxonomique menée par une approche métabolomique avait pour objectif d'identifier des marqueurs chimiotaxonomiques. Cette étude a été effectuée à partir des analyses CG-SM et CG-DIF des composés volatils et des analyses RMN 1H des composés non volatils de 11 espèces endémiques de La Réunion récoltées sur différents lieux géographiques et au cours des saisons estivale et hivernale. Une analyse intra-espèce a permis d'étudier la variabilité saisonnière et/ou géographique de la composition chimique de chaque espèce. Une analyse inter-espèces a conduit à deux classifications différentes des 11 espèces selon leur composition en métabolites volatils et non volatils. Le deuxième axe avait pour objectif d'identifier parmi 11 espèces du genre Psiadia endémiques de La Réunion et 5 espèces endémiques de Maurice, les espèces présentant une ou des activités biologiques prometteuses. Les cibles biologiques choisies ont été le parasite Plasmodium falciparum responsable du paludisme, la lignée cellulaire humaine cancéreuse HeLa responsable du cancer du col de l'utérus et l'enzyme HRP (Horseradish peroxydase) intervenant dans la réponse inflammatoire. À l'issue de ce criblage, 5 espèces se sont révélées prometteuses : les espèces réunionnaises P. amygdalina et P. anchusifolia et les espèces mauriciennes P. arguta et P. lithospermifolia pour l'activité antiplasmodiale, ainsi que l'espèce réunionnaise P. dentata pour les trois activités testées. Le troisième axe consacré à une étude phytochimique de P. arguta réalisée par un fractionnement bioguidé de l'extrait brut a conduit à l'isolement et à l'identification de 16 terpénoïdes : 2 triterpènes et 14 diterpènes de structure labdane dont 4 sont de structure nouvelle. Cinq diterpènes se sont révélés particulièrement actifs contre le parasite P. falciparum : l'acétate de labda-13(E)-en-8α-ol-15-yle, l'acétate de labdan-8α-ol-15-yle, le 13-épi-sclaréol, le labda-13(E)-ène-8α,15-diol et le (8R,13S)-labdane-8,15-diol. Par ailleurs, une étude métabolomique menée par RMN 1H sur des plantules de P. arguta cultivées in vitro et acclimatées a permis l'étude des facteurs influençant la production de ces composés bioactifs. / The present work describes the chemical composition of the plant genus Psiadia and focuses on three research topics: (1) a chemotaxonomic study of 11 species endemic to Reunion island, (2) a biological screening of 16 Psiadia species among which 11 are endemic to Reunion and 5 are endemic to Mauritius and (3) a phytochemical investigation of Psiadia arguta, endemic to Mauritius. The aim of the chemotaxonomic study was to identify chemical markers by a metabolomic approach using GC-MS and GC-FID for volatiles compounds and 1H NMR for non-volatiles compounds. The 11 studied species were harvested in different locations and seasons in order to analyze the seasonal or geographical variability of the chemical profile of each species. This study led to two classifications of the 11 species in terms of the composition of volatiles and non-volatiles compounds. The objective of the second research topic was to identify within 11 species endemic to Reunion island and 5 species endemic to Mauritius, the most active species for the biological activities tested. The targeted activities were antiplasmodial against Plasmodium falciparum, anticancer against the human cancer cell lines HeLa and anti-inflammatory through the inhibition of the enzyme HRP (Horseradish Peroxidase). Four species, P. amygdalina and P. anchusifolia, endemic to Reunion, and P. arguta and P. lithospermifolia, endemic to Mauritius, were particularly active against P. falciparum. Besides, P. dentata (endemic to Reunion) displayed interesting antiplasmodial, anticancer and anti-inflammatory activities. The third research topic was devoted to a phytochemical investigation of P. arguta by a bioguided fractionation and led to purification and identification of 16 terpenoids: 2 triterpenes and 4 diterpenes including 4 new compounds. The evaluation of the antiplasmodial activity of all isolated compounds allowed to highlight activities of five diterpenes: labda-13(E)-en-8α-ol-15-yle acetate, labdan-8α-ol-15-yle acetate, 13-epi-sclareol, labda-13(E)-ene-8α,15-diol and (8R,13S)-labdane-8,15-diol. Furthermore, in order to identify factors influencing the production of bioactive compounds, P. arguta has been multiplicated using in vitro culture techniques and micropropagated plants were acclimatized.
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Caracterização proteometabolômica dos componentes da teia da aranha Nephila clavipes utilizados na estratégia de captura de presas / Proteometabolomic characterization of the spider web components Nephila clavipes used in prey capture strategy

Esteves, Franciele Grego [UNESP] 22 March 2017 (has links)
Submitted by Franciele Grego Esteves null (francielegrego@gmail.com) on 2017-04-07T15:06:37Z No. of bitstreams: 1 Dissertação de Mestrado p biblioteca.pdf: 6918114 bytes, checksum: e45b8e0514dd2fe63b041ac88e0cd8f7 (MD5) / Rejected by Luiz Galeffi (luizgaleffi@gmail.com), reason: Solicitamos que realize uma nova submissão seguindo a orientação abaixo: Incluir o número do processo de financiamento nos agradecimentos da dissertação/tese. Corrija esta informação e realize uma nova submissão com o arquivo correto. Agradecemos a compreensão. on 2017-04-17T14:12:17Z (GMT) / Submitted by Franciele Grego Esteves (francielegrego@gmail.com) on 2017-04-20T20:14:39Z No. of bitstreams: 1 Dissertação Mestrado Franciele - Autoarquivamento UNESP.pdf: 6753928 bytes, checksum: 6e5332799980f808f7228f38b18d6a7c (MD5) / Rejected by Luiz Galeffi (luizgaleffi@gmail.com), reason: Solicitamos que realize uma nova submissão seguindo a orientação abaixo: O arquivo submetido não contém o certificado de aprovação. Corrija esta informação e realize uma nova submissão com o arquivo correto. Agradecemos a compreensão. on 2017-04-25T13:55:38Z (GMT) / Submitted by Franciele Grego Esteves (francielegrego@gmail.com) on 2017-04-25T15:04:46Z No. of bitstreams: 1 Dissertação Mestrado Franciele - Autoarquivamento UNESP.pdf: 6918354 bytes, checksum: 0ea68d3a1abeca7d8514c318aef0795e (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-04-25T16:05:41Z (GMT) No. of bitstreams: 1 esteves_fg_me_rcla.pdf: 6918354 bytes, checksum: 0ea68d3a1abeca7d8514c318aef0795e (MD5) / Made available in DSpace on 2017-04-25T16:05:41Z (GMT). No. of bitstreams: 1 esteves_fg_me_rcla.pdf: 6918354 bytes, checksum: 0ea68d3a1abeca7d8514c318aef0795e (MD5) Previous issue date: 2017-03-22 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A aranha Nephila clavipes pertence ao grupo das aranhas construtoras de teias orbitais, que desenvolveram a capacidade de sintetizar fios adesivos. Esses fios adesivos são encontrados nos círculos centrais destas teias, e apresentam gotículas oleosas contendo vesículas que aprisionam em seu interior soluções de proteínas, peptídeos e muitos compostos de baixa massa molecular. Estudos da análise química dessas gotículas identificaram toxinas, ácidos graxos saturados e até alcaloides. Especula-se que quando um inseto presa é aprisionado pela teia, os ácidos graxos dessas gotículas auxiliam no processo de desestabilização da cutícula do inseto, permitindo a difusão das toxinas para o interior do corpo da presa. Também são relatados alcaloides que atuam como repelentes a predadores em algumas teias, e como inseto-toxinas em outras. A presença dessas moléculas são evidências de que a teia não é uma simples ferramenta para captura mecânica e aprisionamento de presas, mas sim uma complexa estrutura, que parece desempenhar um papel estratégico “ativo” na captura de suas presas. Considerando isso, o objetivo deste estudo foi analisar a ultraestrutura, a disposição das gotículas sobre os fios de seda, e visualizar a presença de vesículas lipídicas extraídas das gotículas da teia orbital da aranha N. clavipes através de microscopia. Além disso explorou-se a riqueza do perfil químico dos compostos de baixas massas moleculares da seda da teia através da cromatografia gasosa bidimensional abrangente acoplada a um detector de massas; e por fim investigar a riqueza de proteínas presentes na seda da teia e nas glândulas produtoras de seda, através da digestão proteolítica em solução, cromatografia líquida, e espectrometria de massas, ressaltando as possíveis toxinas envolvidas na paralisia de presas. Primeiramente, foi realizado um estudo com microscopia eletrônica de varredura das gotículas depositadas sobre os fios de seda, e com microscopia de luz das vesículas lipídicas que se encontram em suspensão, retidas dentro do conteúdo aquoso das gotículas. Posteriormente na análise da perfilagem química foram encontrados 316 compostos, dentre esses 25 foram identificados a partir de padrões químicos, sendo a maioria hidrocarbonetos saturados e alguns ácidos graxos. Este estudo também demonstrou através de bioesaios de repelência, que alguns dos ácidos graxos e hidrocarbonetos identificados nas gotículas da teia apresentaram potencial como repelentes a formigas invasoras. Enquanto que o ácido palmítico apresentou ação potencial no processo de dissolução/desintegração da camada superficial da cutícula de abelhas, para permitir a passagem das toxinas ao interior do corpo das presas da aranha N. clavipes. Na análise proteômica foram indentificados um total de 2051 proteínas na seda da teia, sendo que 163 dessas proteínas são toxinas. Também foram identificadas um total de 927, 1961, 849, e 860 proteínas nas glândulas agregada, ampulada maior, flageliforme e ampulada menor, respectivamente; sendo que desses totais, 194, 78, 32 e 30 são toxinas pertencentes a cada glândula citada acima, respectivamente. Este estudo sugere que as glândulas de seda, principalmente a glândula agregada, podem sintetizar e depositar sobre a seda da teia toxinas importantes, que são comuns a alguns venenos animais, tornando dessa forma as teias como uma estrutura ativa na captura de presas. A partir dos resultados obtidos foi possível elaborar uma hipótese que relaciona o papel das gotículas, vesículas lipídicas e dos compostos identificados como parte da estratégia química da teia na pré digestão e paralisia da presa. Portanto, este estudo forneceu uma melhor compreensão da química-ecológica da captura de presas pela teia da aranha N. clavipes, além de informações que podem possibilitar o uso desses compostos no desenvolvimento de inseticida-seletivo, ou até mesmo em possíveis aplicações farmacológicas. / Nephila clavipes belongs to the group of orb-weavings spiders that have developed the ability to synthesize adhesive threads. Such adhesive threads are found in the core circles of the orb-webs, and present oily droplets which in turn contain many vesicles in suspension, entrapping solutions of proteins, peptides and many small-molecular mass compounds. Chemical analysis studies of these droplets identified toxins, saturated fatty acids and even alkaloids. It is speculated that when an insect-prey is trapped by the web, the fatty acids of these droplets aid in the process of destabilizing the insect cuticle allowing the diffusion of the toxins into the prey body. Some studies also reported alkaloids that act as repellents to predators in some webs, and as insect-toxins in others. The presence of these molecules is evidence that the web is not a simple tool for mechanical capture and imprisonment of prey; but rather a complex structure that seems to play a strategic "active" role in capturing its prey. Considering this, the aim of this study was to analyze the ultrastructure, the disposition of the droplets on the silk fibers, and to visualize the presence of lipid vesicles extracted from the web’s droplets of N. clavipes spider, by microscopy. In addition, the richness of the chemical profile of the small-molecular mass compounds in the web-silk was explored through comprehensive two-dimensional gas chromatography coupled to a mass detector; and finally to investigate the richness of proteins present in web-silk and silk-producing glands through in solution proteolytic digestion, liquid chromatography, and mass spectrometry, highlighting the possible toxins involved in the prey paralysis. First, was performed scanning electron microscopy study of the droplets deposited on the web-silk and light microscopy of the suspended lipid vesicles retained within the aqueous contents of the droplets. Posteriorly in the profiling chemical analysis were identified 316 compounds, among these 25 were identified from chemical standards, most of them are hydrocarbons saturated and some fatty acids. This study also demonstrated through repellence bioassays, that some of the fatty acids and hydrocarbons identified in the web's droplets presented potential as repellents to invasive ants. While the palmitic acid presented potential action in the process of dissolution / disintegration superficial layer of cuticle bees, to allow the difusion of toxins into the prey body. In the proteomic analysis a total of 2051 proteins were identified in the web-silk, of which 163 of these proteins are toxins. A total of 927, 1961, 849, and 860 proteins were also identified in the aggregate, major ampullate, flagelliform and minor ampullate glands, respectively; and of these totals, 194, 78, 32 and 30 are toxins belonging to each gland mentioned above, respectively. This study suggests that silkproducing glands, especially the aggregate gland, can synthesize and deposit on the silk-web important toxins, which are common to some animal venoms, thereby making the webs an active structure in the prey capture. From the obtained results it was possible to elaborate a hypothesis that relates the role of the droplets, lipid vesicles and the compounds identified as part of the chemical strategy of the web in the pre-digestion and prey paralysis. Thus, this study provided a better understanding of the chemical-ecological prey capture by N. clavipes spider web, in addition to information that may enable the use of these compounds in the development of insecticide-selective or even possible pharmacological applications. / FAPESP: 2011/51684-1 / FAPESP: 2015/14220-8
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Métodos rápidos para análise de ácidos graxos em alimentos e seleção de biomarcadores para aterosclerose

Porto, Brenda Lee Simas 16 July 2015 (has links)
Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-05-09T13:54:13Z No. of bitstreams: 1 brendaleesimasporto.pdf: 2937738 bytes, checksum: 5bab0afa2d60b1556e96b14ca4858ece (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-05-17T14:14:41Z (GMT) No. of bitstreams: 1 brendaleesimasporto.pdf: 2937738 bytes, checksum: 5bab0afa2d60b1556e96b14ca4858ece (MD5) / Made available in DSpace on 2017-05-17T14:14:41Z (GMT). No. of bitstreams: 1 brendaleesimasporto.pdf: 2937738 bytes, checksum: 5bab0afa2d60b1556e96b14ca4858ece (MD5) Previous issue date: 2015-07-16 / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / Foi proposto um método alternativo para a determinação de ácidos graxos trans expressos em ácido elaídico por CZE com detecção direta no UV. A solução de eletrólito consistiu de 12,0 mmol L-1 de tetraborato de sódio, 12,0 mmol L-1 de brij 35 e MeOH/ACN na proporção de 33:17 v:v. Os ácidos graxos trans foram quantificados com sucesso em amostras de bala de caramelo, mistura para bolo, waffer recheado, chocolate e mistura para pão de queijo em 13 minutos de análise com a estatística baseada no cálculo de uma fator de resposta usando o ácido cis-nonadecaenóico como padrão interno. A comparação estatística entre CZE-UV e o método clássico de análise por GC-FID não apresentou diferenças significativas no intervalo de 95% de confiança. Também foi proposto um método alternativo para análise target de CLA por fast GC usando a coluna SLB-IL111 com 15 m de comprimento e 0,10 mm de diâmetro interno. A separação ocorreu em isoterma a 168 º C e os isômeros de CLA cis-9, trans-11 e trans-10, cis-12 eluiram em 4,6 e 4,7 minutos, respectivamente. Duas amostras de leite com diferentes teores de CLA em sua composição foram analisadas e quantificadas por adição de padrão. A comparação estatística entre o método proposto com a coluna SLB-IL11 e o método clássico de análise por GC-FID com a coluna CPSil-88 não apresentou diferenças significativas no intervalo de 95% de confiança. O terceiro foco deste trabalho envolve a busca de biomarcadores para aterosclerose instável, utilizando a abordagem de fingerprint em metabolomica. Foi selecionado um grupo de 38 pacientes, entre homens e mulheres, metade diagnosticada com aterosclerose instável e metade com aterosclerose estável. A partir da análise do plasma deses pacientes, por LC-MS e GC-MS, foi possível selecionar 25 metabólitos como candidatos a biomarcadores para a enfermidade. Destes, apenas 18 puderam ser identificados e classificados como ácidos graxos, aminoácidos e carnitinas. Sua variação entre as amostras testadas sugere que a alimentação humana pode estar relacionada com as diferenças no metabolismo de pacientes diagnosticados com aterosclerose estável e instável. / An alternative method for determination of trans fatty acids expressed as elaidic acid in CZE-UV with direct detection was proposed. The background electrolyte used consisted of 12,0 mmol L-1 of sodium tetraborate buffer, 12,0 mmol L-1 of brij 35 and MeOH/ACN in ratio 33:17 v:v. Trans fatty acids in different samples such as butter toffee, cake mix, stuffed wafers, chocolate and a mix for brazilian cheese bread were successfully quantified within an analysis time of 13 min, considering the statistical approach based on response factor calculation using cis-nonadecaenoic acid as internal standard. The statistical comparison between CZE-UV and the classical GC method for the analyzed samples did not present significant differences within the 95% confidence interval. Also was proposed an alternative method for the target analysis of CLA by fast GC using the SLB-IL111 column with 15 m length and 0.10 mm in internal diameter. The separation occurred with isotherm at 168 ° C and the CLA isomers cis-9, trans-11 and trans10, cis-12 eluting at 4.6 and 4.7 minutes, respectively. Two samples of milk with different CLA content in its composition was analyzed and quantified by standard addiction. The statistical comparison between the proposed method with the column SLB-IL111 and the classical GC method with the column CPSil-88 did not present significant differences within the 95% confidence interval. The third focus of this work involves the search of biomarkers for unstable atherosclerosis, using fingerprint approach in metabolomics. It selected a group of 38 patients, men and women, half diagnosed with unstable atherosclerosis and half with stable atherosclerosis. From the plasma analysis of these patients, by LC-MS and GC-MS, 25 metabolites as biomarkers candidate for this disease were selected. In which, only 18 could be identified and classified as fatty acids, amino acids, and carnitines. Its variation between samples suggests that human food may be related to the differences in the metabolism of patients diagnosed with stable and unstable atherosclerosis.
34

Analyse intégrée de la réponse de la vigne à l'infection par Plasmopara viticola : par l'étude d'un cas de contournement de résistance / Integrated analysis of the grapevine response to Plasmopara viticola infection : through study of breakdown resistance

Negrel, Lise 27 May 2016 (has links)
Un déploiement optimal de variétés résistantes nécessite une excellente connaissance des relations entre la vigne et P. viticola. Ces connaissances fondamentales pourront ensuite alimenter les stratégies pour le développement de variétés durablement résistantes au vignoble. Bianca est une variété de vigne résistante au mildiou qui possède le gène résistance Rpv3. Cette variété est résistante à la plupart des souches de P. viticola. Cependant, une souche virulente capable de l’infecter a été isolée. Dans ce projet, un pathosystème original, fondé sur la variété Bianca confrontée à une souche avirulente et à une souche virulente de P. viticola, a été utilisé pour obtenir une image complète de l'impact sur la vigne de l'infection par P. viticola en situation compatible et incompatible, en combinant des études de physiopathologie avec des analyses métabolomiques par spectrométrie de masse à haute résolution et par résonance magnétique nucléaire. Parallèlement, l'identification de métabolites et de séquences géniques spécifiques de P. viticola a permis le développement de méthodes de suivi dynamique de l'infection, grâce à la PCR quantitative et à la quantification de lipides caractéristiques de l'agent pathogène. / Optimal deployment of resistant varieties requires an excellent knowledge of the relationship between grapevine and P. viticola. This fundamental knowledge can then feed the strategies for the development of grapevine varieties with sustainable resistance. Bianca is a downy mildew-resistant grapevine variety, due its Rpv3 resistance gene. This variety is resistant to most strains of P. viticola. However, a virulent strain capable of infecting Bianca has recently been isolated. In this project, we use this original pathosystem to obtain a complete picture of the impact P. viticola infection on grapevine, by combining physiopathological studies with metabolomic analyses. In addition, the identification of specific metabolites and gene sequences from P. viticola has allowed the development of original methods for dynamic monitoring of the infection process, through quantitative PCR and quantification of specific lipids.
35

MASS SPECTROMETRY TO CHARACTERIZE SIGNIFICANT PROCESSES: FROM CHIRAL ENRICHMENT TO DISEASE METABOLISM

Rong Chen (9702269) 12 October 2022 (has links)
<p>Mass spectrometry (MS) can provide rapid, sensitive, and specific analysis, making it a valuable tool to characterize biomolecules, especially their dynamic changes when involved in significant processes.  Compared to other analytical techniques, which mostly focus on solution-phase or solid-phase characterization, MS enjoys a more general and efficient detection of gas-phase analytes since it ultimately measures abundances of bare ions in vacuum. This unique detection capability of MS has been demonstrated, in this dissertation, by characterizing the neutral serine octamer, a gas-phase amino acid cluster that has been detected by MS only so far. Besides its existence, the progress of chiral enrichment has also been monitored and quantified by MS during octamer formation. The acquired MS data is crucial to interpreting the mechanism of chiral enrichment achieved by serine octamer and might suggest its involvement in the prebiotic world to eventually achieve biohomochirality. The work also showcases the capability of detecting neutral compounds by MS, which breaks the stereotype that MS is exclusively an ion-based technique. </p> <p><br></p> <p>Besides process monitoring in the open air, MS also monitors the highly complicated metabolism processes inside biosamples, primarily benefiting from its excellent sensitivity, specificity, and throughput of ion detection. Since altered cellular metabolism is being recognized as a hallmark of cancer, MS is suitable for cancer diagnostics, whose performance of diagnosing glioma, a common brain cancer, has been tested.  Desorption electrospray ionization(DESI) has been used as it avoids sample preparation and allows direct characterization of raw tissue, therefore well suited for on-site analysis such as in the operating room. In short, we have applied intraoperative DESI-MS analysis on raw brain biopsies to provide glioma diagnostics within 5 min. Specifically, the molecular features revealed by MS are translated into pathological information of analyzed tissue, like genetic mutations and tumor concentrations, which is highly desired during surgeries to guide tumor resection and improve patient management. </p> <p><br></p> <p>Knowledge of diagnostic biomarkers is essential to the translation from MS data to pathology, which can be obtained by metabolic profiling using MS. Despite the tradeoff between comprehensive characterization and analysis time, we have extensively explored endogenous metabolites by using tandem MS and expedited analysis by avoiding the use of chromatography. After fast profiling, statistical analysis of all MS features has been applied to discover diagnostic markers to distinguish healthy brain tissue from cancerous tissue. DESI-MS methods have been developed to facilitate a simple and rapid characterization of these biomarkers in tissue for a smooth clinical transition. </p> <p><br></p> <p>However, the complete characterization of endogenous metabolites in a complicated biomixture, like tissue, is challenging, especially without the orthogonal separation provided by chromatography. This unmet demand calls for the development of novel MS scans to improve the metabolite coverage. For lipidomics by direct infusion MS, the MS scans used for lipid profiling have not been greatly expanded since its introduction. These conventionalMS scans only target one structural moiety of lipids and leave the rest unresolved, which limits the structure elucidation and biological interpretation of diagnostic lipids. We have introduced additional lipid scans that target both the lipid headgroup and one fatty acyl chain, leaving the other fatty acyl chain flexible. These scans with higher specificity can further alleviate the matrix effect by uncovering fewer ions in each scan and provide more structural information to support lipid identification. As a proof-of-concept, we have used them to profile both common phospholipids and the rarer ether lipids that display significant variations between healthy mice tissue and those with metabolic syndrome. The additional structural information provided by these scans ensures a clear message expressed by the disease metabolism and potentially indicates invention points and therapeutic candidates.</p>
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Quantification des contraintes métaboliques et physiologiques liées à la surproduction de protéines recombinantes par Escherichia coli : amélioration des performances et de la robustesse du système d'expression et du procédé de production / Quantification of metabolics and physiologics contraints related to overexpression of recombinants proteins in Escherichia coli : Optimisation of performances and robustness of expression system and production process

Patacq, Clement 23 October 2018 (has links)
La production de protéines hétérologues permet de développer une nouvelle génération de vaccins. La bactérie Escherichia coli est l’un des organismes hôtes les plus utilisés pour la production de protéines hétérologues, appelées également protéines recombinantes. Le déclenchement de la production de protéine altère la croissance bactérienne en réponse à la réallocation des ressources métaboliques vers la synthèse de la protéine ; ce qui peut conduire à l’arrêt complet de la croissance. Le maintien de la croissance bactérienne durant la production de la protéine recombinante est pourtant essentiel pour améliorer significativement la quantité et la fonctionnalité des protéines produites. Dans une démarche rationnelle visant à développer un système biologique robuste et performant pour la production d’une grande diversité de protéines recombinantes chez E. coli, les contraintes métaboliques liées à leur production ont été quantifiées. A partir de ces résultats, le système d’expression T7 a été intégré à la régulation métabolique et traductionnelle de la bactérie E. coli BL21 (DE3) afin d’adapter la vitesse de production avec les capacités métaboliques de la souche. Ce nouveau système biologique de production a ainsi permis d’augmenter considérablement les quantités de protéines produites et offre la possibilité de développer de nouveaux procédés performants de production semi-continus et continus en milieu chimiquement défini. / The production of heterologous proteins offers the ability to develop a new generation of vaccines. The most used organism for the production of heterologous proteins, also called recombinant proteins, is the bacterium Escherichia coli. However, the induction of the production often alleviates the bacterial growth by the new allocation of metabolic resources toward the production of the recombinant protein. Even, this may also lead to growth arrest. The production of high quantities of functional recombinant proteins requires a good balance between of bacterial growth and production of the recombinant protein.In order to rationally develop a robust and an efficient biological system for the production of a large variety of recombinant proteins in E. coli, the metabolic constraints associated to their production have been quantified. From this observation, the T7 expression system has been integrated into the metabolic and translational regulation of the E. coli BL21 (DE3) in order to adjust as perfect as possible the protein production rate to the metabolic capacities of the strain. This new biological production system has made it possible to significantly increase the quantities of proteins produced and opens up the possibility of developing performant semi-continuous and continuous production processes in a chemically defined medium.
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Réponse de la forêt à des scénarios de sécheresse appliqués à moyen et long terme en milieu naturel : étude des COVB du chêne pubescent, principal émetteur d’isoprène en région méditerranéenne / Response of mediterranean forest to applied drought scenarios in natural area : study of BVOC emitted by Quercus Pubescens, main emitter of isoprene in mediterranean region

Saunier, Amélie 16 May 2017 (has links)
Les Composés Organiques Volatils d’origine Biogénique (COVB) émis par la végétation représentent 1PgC.an-1 à l’échelle globale. Ces COVB, une fois émis dans l’atmosphère, peuvent participer à la formation d’ozone troposphérique ainsi qu’à la formation d’aérosols organiques secondaires et donc à la pollution atmosphérique. C’est pourquoi, il est important de quantifier le plus précisément possible les taux d’émissions de COVB et de mieux comprendre quels sont les facteurs environnementaux qui contrôlent ces émissions. Il est bien connu que les émissions de COVB sont contrôlées par la lumière et/ou la température mais elles peuvent également être influencées par d’autres facteurs comme le stress hydrique, bien que son impact soit encore mal compris. En effet, il a été montré que le déficit hydrique pouvait augmenter ou diminuer les émissions de COVB selon son intensité, sa durée et l’espèce étudiée. Dans le cadre du changement climatique, une intensification de la sécheresse est attendue en région Méditerranéenne avec une augmentation de la température, une diminution des pluies ainsi qu’une prolongation de la période de sécheresse. Ce changement climatique pourrait donc modifier les émissions de COVB. De plus, les effets d’une sécheresse appliquée sur plusieurs années sont encore mal connus. Dans cette étude, nous nous sommes intéressés à la réponse des émissions de COVB du chêne pubescent (Quercus pubcescens Willd.)face au stress hydrique attendu en région méditerrannéenne avec le changement climatique. / Biogenic Volatile Organic Compounds (BVOC) emitted by vegetation represent 1PgC.yr-1 at the global scale. These BVOC, once emitted into the atmosphere, can participate in the troposheric ozone formation as well as secondary oragnic aerosols and, consequently, on the atmospheric pollution. That’s why, it is very important to quantify, as accurately as possible, the BVOC emissions and to improbe the knowledge about the environmental factors which drive these emissions. It is well known that BVOC emissions are controlled by the light and the temperature but they can be impacted by other factors such as water stress. Nevertheless, these mechanisms are not well understood yet, since it has been shown that water stress can increase or decrease BVOC emissions according to the intensity and the duration of stress. In a context of climate change, we can expected an intensification of summer drought in Mediteranean area with an incerase of temperature, a decrease of rainfall as well as an elongation of stress period. This climate change could modify BVOC emissions. Moreover, the effects of a water stress applied during several years are not known. In this study, we wanted to evaluate the impact of water stress, expected with climate change, on BVOC emitted by Downy oak (Quercus pubescens Willd.), main isoprene emitter of Mediterranean region.
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Étude de la synthèse des furocoumarines chez le panais par des approches d'ingénierie métabolique et de multi-omique / Study of furocoumarin synthesis in parsnip using metabolic engineering and multi-omic approaches

Galati, Gianni 17 July 2019 (has links)
Les plantes sont soumises durant leur vie à de nombreux stress environnementaux. Face à ces contraintes, les végétaux ont développé au cours de l'évolution différentes stratégies. La plus emblématique est la mise en place du métabolisme spécialisé, représenté par une grande diversité chimique et fonctionnelle. Bien que ce métabolisme soit de plus en plus étudié ces dernières années, de nombreuses lacunes persistes à son propos, liées notamment (i) à la complexité des modifications métabolomiques engendrées par la perception de stress, (ii) aux coûts et avantages que ces métabolites imputent à la plante les accumulant, et (iii) aux voies métaboliques menant à cette diversité de composés. Pour appréhender ces différentes problématiques, nous avons adopté une stratégie combinant des approches de phytochimie, de biologie moléculaire et de génétique. Dans un premier temps, nous avons étudié les changements métaboliques globaux engendrés par l’application de deux stress environnementaux, l’ozone et la blessure mécanique, sur une plante modèle au laboratoire, le panais, en fonction du temps. Les résultats de ces travaux nous ont permis d’identifier 40 métabolites différentiellement accumulés dans ces conditions, dont certaines furocoumarines. Par la suite, nous avons focalisé notre étude sur ces molécules en évaluant leurs profils d’accumulation, en condition de stress par blessures mécaniques, par la biais d’analyses différentielles. A partir de ces données, nous avons initié la recherche et l'identification de gènes candidats potentiellement impliqués dans cette voie à partir de plusieurs banques transcriptomiques et génomiques de panais. La fonction des gènes sélectionnés a été évalué par des approches d'expression hétérologue dans la levure. En parallèle de ces travaux, nous avons développé une stratégie destinée à mieux comprendre le coût métabolique de la synthèse de métabolites spécialisés. Pour ce faire, nous avons adapté aux furocoumarines une technique de clonage multigénique permettant de transférer dans une plante, et en une seule opération, plusieurs gènes impliqués dans la même voie de biosynthèse. Cette méthode nous a permis d'initier la génération de lignées stables ayant intégré les deux premiers gènes de la voie. Ces plantes seront comparées à des plantes sauvages et permettront ainsi d’étudier les coûts métaboliques et physiologiques de l’introduction de cette nouvelle voie de biosynthèse ainsi que ses bénéfices en termes de défense de la plante. / Plants are subjected to many environmental stresses during their life. Faced with these constraints, plants have developed different strategies during their evolution. The most emblematic is the establishment of a specialized metabolism, represented by a great chemical and functional diversity. Although this metabolism has been studied more and more in recent years, many gaps remain, related in particular (i) to the complexity of the metabolomic changes generated by the perception of stress, (ii) to the costs and benefits that these metabolites impute to the producing plant, and (iii) to the metabolic pathways leading to the diversity of compounds. To cope with these different issues, we adopted a strategy combining approaches of phytochemistry, molecular biology and genetics. First, we studied global metabolic changes caused by the application of two environmental stresses, ozone and mechanical wounding, on parsnip. The obtained results allowed us to identify 40 metabolites differentially accumulated under these conditions, including some furocoumarins. Subsequently, we focused our study on these molecules by evaluating their accumulation profiles under mechanical wounding stress condition, using differential analyzes. From this data, we initiated the search and identification of candidate genes potentially involved in this pathway based on transcriptomic and genomic parsnip libraries analyses. The function of the selected genes was evaluated by heterologous expression approach in yeast. In parallel to this work, we have developed a strategy to better understand the metabolic cost of specialized metabolites synthesis. To do this, we have adapted a multigene cloning method to furocoumarines, allowing to transfer several genes involved in the same pathway in a plant, in a single operation. This method allowed us to initiate the generation of stable lines having integrated the first two genes of the pathway. These plants will be compared to wild plants and will thus allow to study the metabolic and physiological costs of the introduction of this new biosynthetic pathway and its benefits in terms of plant defense.
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Μεταβολομική ανάλυση κυττάρων HeLa μετά από υπερέκφραση της πρωτεΐνης DGCR14, ενός παράγοντα που σχετίζεται με το σωματίδιο συναρμογής (spliceosome)

Καυκιά, Ελένη 02 March 2015 (has links)
Στην μετα-γονιδιωματική εποχή, την εποχή της συστημικής βιολογίας, η κατανόηση της πολυπλοκότητας της κυτταρικής φυσιολογίας απαιτεί την ανάλυση της δυναμικής των δικτύων βιομοριακών αλληλεπιδράσεων σε όλα τα μοριακά επίπεδα κυτταρικής λειτουργίας. Με τη σειρά της, η λειτουργική γονιδιωματική, ένας θεμελιώδης λίθος της συστημικής βιολογίας, στοχεύει στον πολυδιάστατο χαρακτηρισμό ενός γονιδίου, συνδυάζοντας δεδομένα από τις τεχνολογίες υψηλής απόδοσης. Είναι αυτή ακριβώς η ενοποίηση όλων των μοριακών προτύπων για ένα διαταραγμένο βιολογικό σύστημα που μπορεί να δώσει πληροφορίες αναφορικά με την λειτουργία ενός αγνώστου γονιδίου. Στο πλαίσιο αυτό, η παρούσα Διπλωματική Εργασία αποτελεί μέρος της ολιστικής λειτουργικής ανάλυσης δύο αλληλεπιδρώντων, αγνώστου βιολογικού ρόλου, πρωτεϊνών, της DGCR14 και της FRA10AC1, οι οποίες έχουν απομονωθεί ως συστατικά του σωματιδίου συναρμογής και έχουν συσχετιστεί με νευρολογικές ασθένειες. Η παρούσα εργασία επικεντρώνεται στην μεταβολομική μελέτη των μοριακών επιπτώσεων της υπερέκφρασης της DGCR14 σε ένα ανθρώπινο κυτταρικό μοντέλο, τα κύτταρα HeLa, με την χρήση της αέριας χρωματογραφίας - φασματομετρία μάζας. Ωστόσο, για να επιτευχθεί αυτό, θέματα σχετικά με τις δυνατότητες ποσοτικοποίησης των πολυβηματικών ομικών αναλύσεων έπρεπε να επιλυθούν. Μια σημαντική παράμετρος αφορά στην γρήγορη αδρανοποίηση των ενζυματικών διεργασιών έτσι ώστε οι αποκτηθέντες μετρήσεις να αντικατοπτρίζουν την πραγματική κυτταρική φυσιολογία. Για τον σκοπό αυτό, ο πειραματικός σχεδιασμός πρέπει να τροποποιείται κατάλληλα έτσι ώστε οποιεσδήποτε απαιτούμενες προ-αναλυτικές διαδικασίες χειρισμού των κυττάρων να έχουν ελάχιστη επίδραση στην φυσιολογία τους. Μελετήσαμε συνεπώς την επίδραση τεσσάρων πρωτοκόλλων συλλογής προσκολλημένων κυττάρων και δύο διαφορετικών διαλυμάτων έκπλυσης στο μεταβολικό πρότυπο κυττάρων HeLa. Τα μεταβολομικά δεδομένα αξιολογήθηκαν στο πλαίσιο της καρκινικής μεταβολικής φυσιολογίας και το πρωτόκολλο με την ελάχιστη δυνατή επίδραση στην κυτταρική φυσιολογία καθορίστηκε. Μεταξύ των αποτελεσμάτων αυτής της μελέτης, πολύτιμες πληροφορίες σχετικά με την μεταβολική φυσιολογία των αθανατοποιημένων κυτταρικών σειρών προέκυψαν, οι οποίες ενίσχυσαν σημαντικά την υπάρχουσα γνώση γύρω από τον καρκινικό μεταβολισμό, σε σταθερές ή μεταβαλλόμενες περιβαλλοντικές συνθήκες. Επακόλουθα, η βελτιστοποίηση της διαδικασίας συλλογής είχε ως αποτέλεσμα την δημιουργία ενός αντιπροσωπευτικού μεταβολικού προτύπου κυττάρων HeLa πάνω στο οποίο πραγματοποιήθηκε η αξιολόγηση της υπερέκφρασης της πρωτεΐνης DGCR14 χωρίς να επισκιάζεται από πειραματικές αποκλίσεις εισαγόμενες από την διαδικασία χειρισμού των κυττάρων. Αναφορικά με τα κύτταρα που υπερεκφράζουν την DGCR14, η μεταβολομική ανάλυση εντόπισε μια αλλαγή φυσιολογίας συνδεόμενη με συγκεκριμένα μεταβολικά μονοπάτια τα οποία υποδηλώνουν έντονο μεταβολικό στρες. Για να διερευνήσουμε την συσχέτιση της υπερέκφρασης της DGCR14 με τον παραπάνω μεταβολικό φαινότυπο, χρησιμοποιήσαμε το ανακατασκευασμένο δίκτυο πρωτεϊνικών αλληλεπιδράσεων του σωματιδίου συναρμογής στον άνθρωπο και το δίκτυο πρωτεϊνικών αλληλεπιδράσεων στον άνθρωπο από την μετα-βάση δεδομένων PICKLE, προκειμένου να αντλήσουμε επιπλέον πληροφορίες για τον ρόλο της DGCR14 βάσει της θέσης της σε σχέση με άλλους κόμβους και υπερ-κόμβους. Μια πιθανή λειτουργική συσχέτιση της DGCR14 με αυτοφαγικούς και λυσοσωμικούς μηχανισμούς βρέθηκε, η οποία θα αξιολογηθεί και μελλοντικά μέσω της ανάλυσης, ξεχωριστά και συνδυαστικά, των μοριακών συνεπειών της υπερ- και υπο-έκφρασης σε όλα τα μοριακά επίπεδα κυτταρικής λειτουργίας. / In the post-genomic, systems biology era, developing a systems level understanding of a physiological process requires the analysis of biomolecular network dynamics at all molecular levels of cellular function. Likewise, functional genomics, an essential foundation of systems biology research, aims to define and analyze gene function at a global level by integrating data obtained from multiple high-throughput technologies. It is the integration of all the molecular profiles for a systematically perturbed system that can provide insight about the function of unknown genes. Along these lines, the present study is part of the systematic functional analysis of two interacting, but yet of unknown biological role, spliceosomal proteins, DGCR14 and FRA10AC1, that have both been implicated in neurological diseases. The present work focuses on studying the molecular consequences of DGCR14 overexpression in a human cell model, HeLa cells, at the metabolic level using Gas Chromatography-(ion trap) Mass Spectrometry. However, to succeed in this, issues regarding the quantification capabilities of the multistep omic analysis procedures needed to be resolved. A major concern refers to the fast quenching of any enzymatic processes, so that the acquired measurements indeed reflect the cellular physiology in vivo. To this end, the experimental design should be appropriately adjusted so that any required sample handling actions before quenching have a minimal effect on cellular physiology. Thus, we investigated the effect of four cell collection protocols and two different washing solutions on the intracellular metabolic profile measurements of a HeLa cell culture. The measurements were interpreted in the context of the known cancer cell metabolic physiology and the protocol with the minimum possible effect on cellular physiology was specified. Among the results of this study, valuable information about the metabolic physiology of the immortal cell line arise, which improved our knowledge about cancer metabolism under steady or varying environmental conditions. Subsequently, the optimization of the collection procedure enabled us to establish a representative metabolic profile of HeLa cells against which the overexpression of DGCR14 was evaluated without being obscured by the effect of the sample handling. Regarding the overexpressing cells, the metabolomic analysis detected a trend of physiological change connected to specific metabolic pathways indicating strong metabolic stress. To understand how DGCR14 overexpression generates this particular metabolic phenotype, we used the human spliceosomal complex protein-protein interaction (PPI) network and the integrated human PPI meta-database PICKLE to extract additional information about DGCR14 role based on its location with respect to other nodes and hubs. A possible functional correlation of DGCR14 to autophagic and lysosomal mechanisms was established, that will be further evaluated in the future through the analysis, separately and in combination, of the consequences of DGCR14 over- and under-expression at all molecular levels of cellular function.
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Caractérisation métabolomique des tissus épilectogènes par spectroscopie RMN à haute résolution à l'angle magique (RMN HRMAS) : applications à l'épilepsie temporale humaine et animale / Metabolomic profile of cerebral biopsies in temporal lobe epilepsy (TLE) using High Resolution Nuclear Magnetic Resonance Spectroscopy at Magic Angle Spinning (HRMAS NMR) : applications to human and animal model of TLE.

Detour, Julien 02 October 2013 (has links)
La métabolomique a pour objet l’identification et la quantification de métabolites dans un échantillon biologique. Cette discipline s’inscrit dans une approche du vivant connue sous le terme de « biologie des systèmes ». La spectroscopie par résonance magnétique nucléaire haute résolution à l’angle magique (RMN HRMAS) est une méthode de choix pour l’obtention de ce type de profilage métabolique. L’épilepsie du lobe temporal (ELT) est une épilepsie focale fréquente associée le plus souvent à des pertes neuronales sélectives, une gliose réactionnelle et une plasticité cellulaire spécifique. Bien que restant débattue, une origine neurométabolique reste un axe de recherche majeur. A ce jour une caractérisation métabolomique des tissus épileptogènes par RMN HRMAS reste à effectuer. Notre travail a consisté dans un premier temps à caractériser, chez le rat, les effets des méthodes de prélèvement et de fixation sur le métabolome cérébral dans le cadre des acquisitions RMN HRMAS. Dans un second temps, nous avons travaillé sur le modèle animal lithium-pilocarpine d’ELT. Nous avons pu décrire le métabolome issu des données RMN 1H HRMAS de différentes structures cérébrales impliquéesdans l’épileptogénèse. Des analyses multivariées de type PLS-DA ont pu mettre en évidence des profils métaboliques pathologiques au sein du cortex entorhinal et de l’hippocampe. A l’aide de substrats marqués au carbone 13 ([1-13C]glucose et de [1,2-13C]acétate) nous avons étudié les voies métaboliques neuronales et gliales. Nos résultats suggèrent l’absence d’anomalies métaboliques au sein des astrocytes. Enfin dans un dernier temps, nous avons effectué des analyses RMN 1H HRMAS sur près de 200 échantillons cérébraux de patients atteints d’ELT. Une analyse multivariée a permis de distinguer les profils métaboliques des hippocampes sclérosés et non sclérosés. En revanche la construction de modèles sur la base d’hypothèses clinico métaboliques (durée de la maladie, fréquence de crises, antécédents de convulsions fébriles) n’a pas permis d’identifier de profils métaboliques spécifiques. L’ensemble de ces données suggère l’existence de profils métabolomiques distincts en fonction des caractéristiques neuropathologiques des patients atteints d’ELT. Notre travail confirme la nécessité d’une approche intégrée de type « biologie des systèmes » pour l’étude de l’ELT aussi bien chez l’homme que dans des modèles animaux. / Metabolomics relates to the identification and quantification of metabolites in biological samples. This discipline is part of an approach known under the term of "systems biology". High Resolution Nuclear Magnetic Resonance Spectroscopy at Magic Angle Spinning (HRMAS NMR) is a method for obtaining metabolic profiling in such sample. Temporal Lobe Epilepsy (TLE ) is a common focal epilepsy often associated with selective neuronal loss, reactive gliosis and specific cellular plasticity. A neurometabolic origin of this epilepsy is a major area of research. To date no characterization of human cerebral biopsy from TLE patients has been conducted using HRMAS NMR. In the present work we aimed first at characterizing, in rats, the effects of sampling methods and fixation on brain metabolome under HRMAS NMR acquisitions. In a second step, we studied the lithium-pilocarpine model of TLE. In this model, we could describe the metabolome from HRMAS 1H NMR data of different brain structures involved in epileptogenesis. Multivariate analysis could highlight pathological metabolic profiles in the entorhinal cortex and hippocampus. Using substrates labeled with carbon 13 ( [1 -13C ]-glucose and [1,2-13C ]-acetate) we studied neuronal and glial metabolic pathways. Our results suggest the absence of metabolic abnormalities in astrocytes metabolism as previously reported. Finally, we conducted HRMAS 1H NMR analysis in nearly 200 brain samples from TLEpatients. Multivariate analysis was able to distinguish metabolic profiles between sclerotic and non sclerotic hippocampi. However mutlivariate models based on clinico- metabolic assumptions (disease duration, frequency of seizures, history of febrile seizures ) did not identify specific metabolic profile. All these data suggest the existence of distinct metabolomic profile based on neuropathological features of patients with TLE. Our work confirm the need of an integrated approach such as " systems biology" for the study of TLE in humans as long as in animal models.

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