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Elaboration de librairies de molécules structurellement diversifiées à partir du squelette de l'acide kojique par des méthodes de synthèse micro-ondes et de flux continu / Elaboration of molecules' libraries structurelly diversified from kojic acid skeleton by microwave and continuous flow synthesis methodologiesLeleu, Ludovic 03 November 2017 (has links)
La structure de l’acide kojique et sa réactivité permet d’effectuer des transformations conduisant à la synthèse de composés polycycliques différents et structurellement très diversifiés. Ces hétérocycles sont obtenus en plusieurs étapes en s’appuyant sur des réactions typiques telles que des cycloadditions [5+2], Diels-Alder, [5+2]/[4+2] tandem, des réactions à 3 composantes ou encore l’utilisation de lithiens. L’utilisation d’appareillages spécifique tels que un réacteur micro-ondes et un synthétiseur en flux continue permettent l’obtention des molécules ciblées d’une façon plus spécifique et efficace. Les chimiothèques de molécules obtenues peuvent dès lors être testé pour une éventuelle activité biologique envers diverses structures. / The structure of kojic acid and its reactivity give the opportunity to make tranformations leading to the synthesis of different polycyclic compounds and structurally diversified. These heterocycles are obtained in several steps by typical reactions like [5+2] cycloaddtions, Diels-Alder reactions, tandem [5+2]/[4+2] cycloadditions, 3 compounds reactions or the use of lithiens. The use of specific equipments like microwave equipment and continuous-flow equipment give the opportunnity to obtain target molecules. Compounds libraries will be test for a biological activity.
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Desenvolvimento de uma fase extratora com polímeros de impressão molecular para extração em fase sólida de Venlafaxina, O-desmetilvenlafaxina e N-desmetilvenlafaxina em amostras de plasmas e análises por cromatografia líquida de ultra eficiência acoplada à espectometria de massas em tandem (UPLC-MS/MS). / Development of an extraction phase with molecularly imprinted polymers for solid phase extraction of venlafaxine, o-desmethylvenlafaxine, and n-desmethylvenlafaxine in plasma samples and analysis by Ultra Performance Liquid Chromatography-tandem mass spectrometry (UPLC-MS/MS)Miranda, Luís Felippe Cabral 18 March 2015 (has links)
A venlafaxina (VEN), em razão de sua eficácia e brandos efeitos adversos, tem sido um dos antidepressivos mais prescritos no tratamento da depressão e ansiedade. Neste trabalho, um método analítico empregando as técnicas MISPE miniaturizada e cromatografia líquida acoplada à espectrometria de massas em Tandem, foi utilizado para a determinação de VEN e seus principais metabólitos em amostras de plasma para fins de monitorização terapêutica. A fase MIP foi sintetizada via polimerização radicalar por precipitação, fazendo uso de VEN (molécula molde), ácido metacrílico (monômero funcional), etileno glicol dimetacrilato, (reagente reticulante) e 2,2 azobisisobutironitrila (iniciador radicalar) em tolueno (solvente). Para controle utilizou-se o polímero não impresso (NIP), sintetizado por procedimento análogo ao do MIP, porém sem o uso da molécula molde. A caracterização química e estrutural dos polímeros foi realizada por espectroscopia no infravermelho com transformada de fourier e microscopia eletrônica de varredura. A otimização das variáveis de MISPE miniaturizada favoreceu a detectabilidade analítica e diminuiu o efeito de memória. As extrações realizadas com MIP apresentaram taxa de recuperação de 84% para VEN e de 2-28% para os antidepressivos (clorpromazina, fluoxetina, clomipramina, imipramina e sertralina). O polímero não impresso apresentou baixa recuperação para a VEN (taxa de recuperação: 49%) e para os demais antidepressivos (taxas de recuperação menores que 40%). Estes experimentos comprovam a seletividade da fase MIP desenvolvida. O método padronizado apresentou linearidade na faixa de 3 a 700 ng mL-1 para VEN, 5 a 700 ng mL-1 para O-desmetilvenlafaxina (ODV) e de 3 a 500 ng mL-1 para N-desmetilvenlafaxina (NDV), precisão com coeficientes de variação menores que 15% e exatidão com valores de erro padrão relativo na faixa de -11,8 a 16,01 %. As concentrações correspondentes aos limites inferiores de quantificação para VEN (3 ng mL-1) e ODV ( 5 ng mL-1) foram inferiores aos intervalos terapêuticos preconizados. O método desenvolvido, quando comparado a aos métodos da literatura para determinação de VEN e metabolitos, apresentou maior seletividade, menor consumo de amostra e de solventes orgânicos e permitiu a reutilização da fase extratora. Segundo os parâmetros de validação analítica avaliados e amostras de pacientes em terapia com VEN analisadas, o método proposto é adequado para determinação de VEN, ODV e NDV em amostras de plasma para fins de monitorização terapêutica. / Venlafaxine elicits a small number of adverse effects, so it is one of the most frequently prescribed drugs to treat major depression, generalized anxiety, and social anxiety disorders in adults. In this study, venlafaxine (VEN), O-desmethylvenlafaxine (ODV), and N-desmethylvenlafaxine (NDV) were pre-concentrated with the aid of miniaturized SPE based on MIPs as extraction phase. MIPs are synthetic polymers with cavities specifically designed to hold a target molecule or structurally similar compounds. The molecularly imprinted polymers were prepared by addition of VEN, metacrylic acid (MAA, monomer), ethylene glycol dimethacrylate (EGDMA, cross-linker), and 2,2-azobisisobutyronitrile (AIBN, initiator) to toluene (solvent). The non-imprinted polymer (NIP), used for comparison, was also synthesized by following exactly the same procedure, but excluding the template VEN from the formulation. The polymer was characterized by Fourier transform infrared spectroscopy and scanning electron microscopy (SEM). Optimization of the MIP phase extraction variables favored miniaturized analytical detectability and reduced the memory effect. The extractions performed with the synthesized MIP showed recovery rate of 84% for VEN and 2-28% for other antidepressants (chlorpromazine, fluoxetine, clomipramine, imipramine, and sertraline). The non-imprinted polymer provided low recovery of VEN (recovery rate: 49%) and other antidepressants (recovery rates lower than 40%). These experiments demonstrated the selectivity of the developed MIP phase. The standardized method was linear in the range of 300 - 700 ng mL-1 for VEN, 5-700 ng mL-1 for ODV, and 3 to 500 ng mL-1 for NDV. Precision had coefficients of variation smaller than 15%; the accuracy standard error values ranged from -11.8 to 16.01%. Compared with literature methods, the developed method was more selective for determination of VEN and metabolites, required lower consumption of sample and organic solvents, and enabled reuse of the extraction phase. According to the assessed analytical validation parameters and to the analysis of samples obtained from patients undergoing therapy with VEN, the proposed method is suitable to determine VEN, NDV, and ODV in plasma samples for therapeutic drug monitoring.
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Activation de liaisons C-H au moyen d’un système catalytique bio-inspiré pour la synthèse d’hétérocycles d’intérêt pharmacologique / Activation of C-H bonds through a bioinspired catalytic system for the synthesis of pharmacologically relevant heterocyclesNguyen, Khac Minh Huy 10 February 2016 (has links)
Les métalloenzymes d’origine naturelle constituent une riche source d’inspiration pour la conception de catalyseurs synthétiques en raison de leur capacité à réaliser des réactions d’oxydation sélective dans des conditions douces. Parmi ces métalloenzymes, les amine-oxydases à cuivre (CuAOs) permettent l’oxydation sélective des amines primaires grâce à la coopération d’un catalyseur organique quinonique (topaquinone) et d’un ion cuivrique. Récemment, un regain d’intérêt s’est manifesté pour le développement de catalyseurs biomimétiques permettant l’oxydation des amines en imines à l’air ambiant, en raison de l’importance des imines comme intermédiaires de synthèse en chimie fine et en pharmacologie. Au laboratoire, un système co-catalytique mimant l’activité des CuAOs a été décrit pour l’oxydation, à l’air ambiant, des amines primaires en imines, permettant une forte économie d’atomes. Le procédé catalytique comprend deux couples redox comparables à ceux des CuAOs : le catalyseur organique o-iminoquinonique 1ox, généré in situ à partir de l’o-aminophénol correspondant 1red, est le véritable catalyseur de l’oxydation de l’amine substrat, tandis que le sel de cuivre (II) sert de médiateur redox. Il est intéressant de noter que de faibles quantités de sel de cuivre (II) biocompatible et de catalyseur organique 1ox suffisent à activer la liaison C-H située en α de la fonction NH2 des amines primaires aliphatiques, qui sont converties, à l’air ambiant, en imines issues du couplage hétérolytique, à l’issue d’un processus de transamination qui conduit à l’imine résultant du couplage homolytique, suivi d’une réaction de transimination. Les conditions douces utilisées sont particulièrement intéressantes d’un point de vue synthétique, notamment pour engager les alkylimines instables in situ dans des réactions subséquentes. Aussi, avons-nous envisagé d’utiliser ce système co-catalytique bioinspiré dans la synthèse one-pot d’hétérocycles d’intérêt pharmacologique. Dans la première partie de la thèse, nous avions l’intention d’utiliser le système co-catalytique Cu(II)/1ox dans la synthèse de nouveaux dérivés de la 1,4-benzoxazine. Dans le cas particulier des amines primaires de type R1R2CHCH2NH2, le processus catalytique se trouvait bloqué après un certain nombre de cycles catalytiques en raison de l’engagement du catalyseur 1ox dans une réaction de Diels-Alder à demande électronique inverse avec la forme énamine tautomère de l’imine éliminée au cours du processus catalytique, conduisant ainsi aux dérivés de la 1,4-benzoxazine. Toutefois, l’utilisation de ce procédé s’avéra un échec, les énamines générées étant trop instables à l’air ambiant pour permettre l’isolement des dérivés de la 1,4-benzoxazine avec des rendements acceptables. Nous avons ainsi été amenés à développer une réaction alternative en tandem : les dérivés o-aminophénols sont oxydés dans le méthanol sous atmosphère d’azote, à l’aide d’une quantité stoechiométrique de dioxyde de manganèse activé, en o-iminoquinones. Ces hétérodiènes sont ensuite piégés in situ par différentes énamines diénophiles pour conduire aux dérivés de la 1,4-benzoxazine attendus, dans des conditions douces. La possibilité d’introduire des éléments de diversité dans chacun des partenaires de la cycloaddition permet de préparer des dérivés de la 1,4-benzoxazine hautement substitués. Parmi ces composés, un dérivé présentant deux groupements phényle en position 3 s’est avéré présenter une activité neuroprotectrice notable chez la souris nouveau-né, faisant de lui un candidat potentiel pour le traitement et la prévention de la paralysie cérébrale du nouveau-né. Dans la seconde partie de la thèse, le système co-catalytique Cu(II)/1ox est utilisé dans une réaction de couplage oxydatif d’amines primaires, activées ou non, avec des o-aminoanilines conduisant ainsi à des dérivés du benzimidazole d’intérêt biologique au travers d’un procédé multi-étapes. (...) / Naturally occurring metalloenzymes constitute a rich source of inspiration for the design of synthetic catalysts because of their ability to perform controlled aerobic oxidations under very mild conditions. Among metalloenzymes, copper amine oxidases (CuAOs) promote selective aerobic oxidation of primary amines through the cooperation of a quinone-based cofactor (topaquinone) and a copper ion. Recently, there has been a boost in the development of biomimetic catalysts for the aerobic oxidation of amines to imines owing to the importance of imines as pivotal intermediates in the synthesis of fine chemicals and pharmaceuticals. In our laboratory, a CuAOs-like homogeneous co-catalytic system has been described for the atom-economical oxidation of primary amines to imines, under ambient air. The catalytic process combines two redox couples in a way reminiscent of CuAOs: the o-iminoquinone organocatalyst 1ox, generated in situ from the corresponding o-aminophenol 1red, is the substrate-selective catalyst, whereas the copper (II) salt serves as an electron transfer mediator. Interestingly, low loadings of biocompatible CuII and organocatalyst 1ox are sufficient to activate the α-C-H bond of primary aliphatic amines, which are converted, under ambient air, into cross-coupled imines through a transamination process that leads to the homocoupled imine intermediate, followed by dynamic transimination. The mild reaction conditions are highly favorable from a synthetic viewpoint, in particular for trapping the unstable alkylimines in situ for further reactions. So, we have envisioned the use of this bioinspired co-catalytic system in the one-pot synthesis of heterocycles of pharmacological interest. In the first part of the thesis, we envisioned that the Cu(II)/1ox cooperative system might be utilized to synthesize novel 1,4-benzoxazine derivatives. In the specific case of R1R2CHCH2NH2 amines, the catalytic process should stop after a few turnovers, because the catalyst 1ox should be trapped through inverse-electron-demand Diels-Alder (IEDDA) reaction with the simultaneously in situ generated tautomeric enamine form of the alkylimine extruded during the catalytic process, leading to 1,4-benzoxazine derivatives. Unfortunately, this protocol failed to produce the expected cycloadducts in acceptable yields as enamines rapidly decomposed under ambient air. For this reason, we have developed a tandem oxidation-inverse electron demand Diels-Alder reaction as an alternative: a stoichiometric amount of activated MnO2, in deaerated methanol, was then sufficient to convert various o-aminophenol derivatives into o-iminoquinone heterodienes which were trapped in situ by different enamine dienophiles leading to the expected 1,4-benzoxazine derivatives under mild conditions. The possibility of introducing variations in both cycloaddition partners led to highly substituted 1,4-benzoxazine cycloadducts with up to five elements of diversity. Among these compounds, a 3,3-diphenyl-substituted-1,4-benzoxazine derivative was identified as an effective neuroprotective agent in newborn mice, suggesting that it could be a potential candidate for the treatment and prevention of cerebral palsy. In the second part of the thesis, the Cu(II)/1ox cooperative system has been successfully used for the catalytic oxidative coupling of a diverse range of activated and non-activated primary amines with o-amino-anilines under ambient air leading to benzimidazoles of biological interest through multistep oxidation and nucleophilic addition reactions. Through the variation of both solvent and coupling partners, MeOH proved to be the best solvent for this transformation because it provided the ideal balance of 1ox solvation and reaction rate, except when reactive N-alkyl o-aminoanilines were used as in situ imine traps, due to the concomitant formation of a benzimidazole byproduct originated from MeOH itself. (...)
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Forrageamento de Pachycondyla striata Smith, 1858 (Hymenoptera: Formicidae: Ponerinae) em ambiente urbano / Foraging of Pachycondyla striata Smith, 1858 (Hymenoptera: Formicidae: Ponerinae) in environmentSilva, Janiele Pereira da 30 November 2017 (has links)
As formigas se adaptam as diferentes situações que encontram no seu ambiente em parte por apresentarem flexibilidade comportamental. Um exemplo é o uso de mais de uma estratégia durante a exploração de um recurso alimentar. No caso da Ponerinae Pachycondyla striata, as formigas podem forragear solitariamente ou fazer recrutamento por tandem running. Apesar desta espécie estar presente em diversas áreas verdes em ambiente urbano, pouco se sabe sobre o seu comportamento durante o forrageamento nessas áreas. Por isso, o objetivo deste trabalho foi analisar as estratégias de forrageamento e os comportamentos de P. striata em ambiente urbano. O estudo foi realizado em um jardim da Cidade Universitária (USP, campus Butantã). No local foram observadas 96 formigas de 12 colônias. Como iscas alimentares foram usadas proteína (atum) e carboidrato (maçã com mel) em duas quantidades (3g e 7g) e em duas distâncias do ninho (0,5 m e 4,0 m). Durante 90 minutos foram registrados: as estratégias de forrageamento; os comportamentos das forrageadoras; as interações com espécies competidoras; o tempo de trajeto entre o ninho e a isca. Verificou-se que o forrageamento solitário foi a principal estratégia, sendo utilizada por todas as forrageadoras e que a atividade solitária aumentava quando o alimento próximo ao ninho era proteína. O recrutamento foi realizado por 81% das forrageadoras, mas as formigas perderam o contato em 27% dos recrutamentos. As chances de uma forrageadora recrutar eram maiores em três situações: quando o alimento era proteína; estava perto do ninho; e a umidade do ar era alta (70% UR). Cerca de 72% das forrageadoras tiveram competição nas iscas, sendo a competição interespecífica mais frequente que a intraespecífica. Durante as interações com as competidoras, as forrageadoras apresentaram, principalmente, comportamento agressivo. Quanto ao tempo de trajeto, o forrageamento solitário era percorrido em menos tempo que o recrutamento, independente da distância. Por fim, verificou-se uma correlação negativa entre a ordem das viagens e o tempo do trajeto em ambas as distâncias e estratégias de forrageamento. Conclui-se que os dados coletados neste trabalho reforçam a prevalência do forrageamento solitário como principal estratégia da espécie e também trazem novas informações, como a tomada de decisão baseada no tipo do alimento, a variação na atividade de forrageamento devido a fatores abióticos, as interações competitivas no ambiente urbano e o aprendizado individual e social entre as forrageadoras / The ants adapt to different environmental contexts exhibiting behavioral flexibility. An example of behavioral flexibility is the use of more than one foraging strategy. In the case of Ponerinae Pachycondyla striata, the ants can forage solitarily or recruit and guide nestmates to a food sorce by tandem running. This species is found in various green areas in urban environment, but little is known about its foraging behaviour in this area. The aim of this research was to analyze the foraging strategies and the behaviors of P. striata in urban environment. The study was performed in a garden of the University City (USP campus Butantã). At this site we observed 96 ants of 12 colonies. As feeding baits were used protein (tuna) and carbohydrate (apple with honey) in two quantities (3g and 7g) and at two distances from the nest (0,5m and 4,0m). During 90 minutes we registered: the foraging strategies; the behaviors of the foragers; the interactions with competing species; and the travel time from the nest to the bait. It was found that the solitary foraging was the main strategy used by all the foragers. The solitary foraging activity was especially frequent when protein was close to the nest. The recruitment was performed by 81% of the foragers, but the ants lost contact in 27% of the recruitments. The frequency of recruitment increased in three foraging contexts: when the food was protein; was close to the nest; and with high air humidity (70% UR). About 72% of the foragers found competitors at the baits, and interspecific competition was more frequent than intraspecific competition. The foragers presented during the interactions with the competitors, mainly, aggressive behavior. Traveling time during solitary foraging foraging went through in less time that recruitment, regardless the distance. Lastly, it was verified a negative correlation between the traveling order and the traveling time at both distances and foraging strategies. Our data lead us to conclude that the solitary foraging is the main strategy of P. striata, adding new information as the decision making based on food characteristics, the variation in foraging activity due to abiotic factors and the competing interactions in urban environment and individual and social learning between ants
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Aplicação da cromatografia a gás associada à espectrometria de massas em tandem no diagnóstico da deficiência de 3β-hidroxidesidrogenase / Application of gas chromatography coupled to tandem mass spectrometry in the diagnosis of 3β-hidroxidesidrogenase deficiencyPresutti, Thais Rodrigues 10 April 2017 (has links)
Pregnenolona (PREG) e 17-alfa-hidroxipregnenolona (17OHPREG) são dois esteroides produzidos pela glândula adrenal e precursores de vários hormônios esteroidais. A dosagem desses compostos tem aplicações clínicas, como o diagnóstico de doenças relacionadas aos corticoesteroides e mineralocorticóides e especialmente na avaliação da atividade da enzima 3-β-hidroxidesidrogenase que é decisiva no diagnóstico de um dos tipos de hiperplasia da glândula adrenal que causa defeitos severos na síntese de esteroides. Métodos cromatográficos associados à espectrometria de massas superaram a especificidade reduzida dos imunoensaios e tem sido crescentemente utilizados na quantificação de esteroides. Os últimos anos tem sido marcados pela hegemonia da cromatografia líquida acoplada à espectrometria de massas em tandem (LC-MS/MS) em grande parte devido à velocidade e possibilidade da análise direta de vários analitos. Porém, no caso específico dos esteroides de tipo 3-hidroxi-5-eno, que apresentam baixa afinidade protônica e, portanto, baixa eficiência de ionização, são necessárias muitas etapas para a conversão em derivados mais detectáveis. Embora desfavorecida em relação ao LC-MS/MS nos últimos anos, a cromatografia gasosa acoplada à espectrometria de massas (CG-MS) apresenta várias características favoráveis para a análise de esteroides como a eficiência cromatográfica ainda insuperável. Adicionalmente, a incorporação da espectrometria de massas em tandem ao CG (CG-MS/MS) torna a técnica tão seletiva quanto LC-MS/MS. No presente trabalho, foi desenvolvido um novo método que permite a extração e derivatização simultâneas da PREG e 17OHPREG de amostras de soro tornando o método de preparo da amostra tão simples quanto os descritos para LC-MS/MS. O método de detecção desenvolvido baseado em ionização química no modo negativo obteve a sensibilidade necessária para o diagnóstico da deficiência da enzima 3-beta-hidroxidesidrogenase utilizando apenas 250 µ:L de amostra. / Pregnenolone (PREG) and 17α-hydroxypregnenolone (17OHPREG) are two steroid precursors produced by the adrenal gland. The quantification of these compounds is essential for the evaluation of 3-β-hidroxidesidrogenase enzyme activity, which promotes the conversion of PREG in 17OHPREG. The 3-β:-hidroxidesidrogenase deficiency is a rare but severe type of adrenal hyperplasia that causes serious defects in steroid synthesis. Chromatographic methods coupled to mass spectrometry overcame immunoassays limitations such as reduced specificity, and have been widely used for steroids quantification. Recent years have been marked by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) hegemony due to the speed and possibility to analyze directly several analytes. However, in the case of type 3-hydroxy-5-ene steroids, which have low affinity for protons and, therefore, low ionization efficiency, many steps are required for conversion to detectable products. Notwithstanding, gas chromatography coupled to mass spectrometry (GC-MS) has some favorable features for steroid analysis such as unbeatable chromatographic efficiency. In addition, the incorporation of tandem mass spectrometry (GC-MS/MS) makes it as selective as LC-MS/MS. In this study, a new method for simultaneous extraction and derivatization of PREG and 17OHPREG from serum was developed. This procedure makes sample preparation for GC-MS/MS as simples as those described for LC-MS/MS. The detection method based on negative mode chemical ionization achieved the sensitivity required for the diagnosis of 3-β-hidroxidesidrogenase defficiency using only 250 µL of sample.
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Développement d’une approche analytique pour la caractérisation du sélénoprotéome in vivo / Development of analytical methodology for selenoproteomicsBianga, Juliusz 21 February 2013 (has links)
Le sélénium est un micronutriment essentiel pour des nombreux organismes vivants, y compris l’homme. Son rôle est lié à sa présence dans des sélénoprotéines sous forme d’un acide aminé, génétiquement encodé – la sélénocystéine. Il y a 25 sélénoprotéines encodées dans le génome humain. Leurs fonctions, la cinétique et la hiérarchie d'expression se trouvent au cœur des problématiques de recherche concernant le sélénium et la santé humaine. Il existe également un autre type de protéines où le sélénium est inséré par un remplacement partiel du soufre dans la méthionine mais aussi, potentiellement, dans la cystéine. Ces protéines suscitent l’intérêt dans les sciences de nutrition comme source de sélénium biodisponible dans l’alimentation naturelle et supplémentée. L'objectif de cette thèse a été la mise au point de méthodologies analytiques visant la spéciation du sélénium incorporé dans les protéines à l’échelle du protéome entier. Une procédure inédite a été développée pour la détection globale de protéines séléniées dans des gels d’électrophorèse bidimensionnelle par l’imagerie d’ablation laser ICP MS (spectrométrie de masse plasma à couplage inductif) permettant de s’affranchir de l’utilisation de l’isotope radioactif 75Se. Les autres avancées comprennent la mise en place d’un couplage robuste de HPLC capillaire avec l’ICP MS pour la détection des sélénopeptides dans des microvolumes de digestats trypsiques des protéines extraites du gel ainsi que la mise en place des protocoles d’identification des protéines séléniées par la spectrométrie de masse électrospray en tandem utilisant la trappe orbitale (Orbitrap). Les méthodes développées ont permis (i) la caractérisation de la part du protéome sélénié contenant la sélénocystéine chez la levure séléniée, (ii) l’identification des protéines majeures qui accumulent le sélénium dans le blé, et (iii) le dosage semi quantitatif et la caractérisation globale des sélénoprotéomes (GPx1, GPx4, TRxR1, TRxR2, Sel15kDa) dans les lignées cellulaires. / Selenium is an essential micronutrient for many living organisms including man. Its role is related to selenoproteins which contain genetically encoded selenocysteine. There are 25 selenoproteins encoded in the human genome. Their function, expression kinetics and hierarchy have been a topic of intense research in life sciences. There is another type of proteins which contain selenium inserted non-specifically by partly replacing sulphur in methionine and, potentially, cysteine. They are of interest in nutrition science as source of bio-available selenium in natural and supplemented foods. The goal of this Ph.D. was the development of methodologies for the analysis of selenium-containing proteins on the entire proteome scale. A novel procedure was developed for their global detection in 2D electrophoretic gels par laser ablation inductively coupled plasma mass spectrometry (ICP MS) imaging permitting to avoid the use of the radioactive 75Se. The other developments included (i) a robust capillary HPLC – ICP MS coupling allowing the detection of Se-containing peptides in microliter volumes of the digests of proteins extracted from the gel and (ii) protocols allowing the targeted identification of the Se-containing proteins by a parallel capillary HPLC - electrospray Orbitrap MS/MS. The methods developed allowed (i) the characterisation of the selenocystein-containing part of the selenoproteome of Se-enriched yeast, (ii) identification of the major Se-accumulating proteins in wheat, and (iii) semiquatitive analysis and global identification of the selenoproteomes (GPx1, GPx4, TRxR1, TRxR2, Sel15kDa) expressed in different human cell lines.
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Catalyse tandem pour la protection régiosélective de saccharides : vers l’élaboration de sulfoglycolipides mycobactériens / Regioselective protection of saccharides by tandem catalysis : toward the synthesis of mycobacterial sulfoglycolipidsLemétais, Aurélie 25 November 2011 (has links)
L’accès par voie chimique à des oligosaccharides nécessite souvent le recours à de nombreuses étapes de protection-déprotection. Au cours de ce projet de thèse, une méthodologie pour la protection régiosélective et orthogonale des fonctions alcool de disaccharides dérivant de la biomasse a tout d’abord été développée. Les glycopyranosides protégés ont été préparés par catalyse tandem au FeCl3∙6H2O en réalisant dans le même pot des réactions d’acétalation, d’éthérification réductrice, d’acétylation et/ou d’ouverture réductrice régiosélective d’acétals. Dans un second temps, une stratégie de synthèse flexible, rapide et performante pour accéder à des sulfoglycolipides diacylés et tétraacylés comportant un cœur tréhalose a été mise au point. Ces molécules sont produites par Mycobacterium tuberculosis, l’agent pathogène responsable de la tuberculose, et les sulfoglycolipides diacylés pourraient permettre l’élaboration d’un nouveau vaccin contre cette maladie. Des sulfoglycolipides diacylés et tétraacylés comportant des chaînes monométhylées chirales ont été obtenus. Les précurseurs des acides gras chiraux utilisés au cours de la synthèse ont été analysés par spectroscopie RMN du deutérium en abondance naturelle dans des cristaux liquides chiraux. / The synthesis of oligosaccharides often requires long sequences of protection-deprotection steps. For a rapid access to suitably protected glycopyranosides, we have developed a one-pot regioselective protection strategy based on FeCl3∙6H2O-tandem catalyzed reactions (acetalation, acetylation, reductive etherification, regioselective ring opening of acetal). This procedure was applied to persilylated disaccharides derived from biomass. This methodology allowed the development of a fast, efficient and flexible access to diacylated and tetraacylated sulfoglycolipids based on a trehalose core. These molecules are found in the cell wall of Mycobacterium tuberculosis and the diacylated sulfoglycolipids appear to be promising candidates for the development of a new tuberculosis vaccine. Synthetics diacylated and tetraacylated sulfoglycolipids bearing chiral monomethylated fatty chains were produced. The chiral fatty-acid precursors, used in the procedure, were synthetized and analyzed by NMR spectroscopy of natural abundance deuterium in chiral liquid crystals.
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Développement de méthodes de séparation des chitooligosaccharides obtenus par déacétylation enzymatiqueTang, Marie-Christine January 2008 (has links)
Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.
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De l’usage du polymorphisme de répétitions en tandem pour l’étude des populations bactériennes : mise au point et validation d’un système de génotypage automatisé utilisant la technique de MLVA / The use of tandem repeats polymorphism for bacterial populations study : conception and validation of a MLVA-based automated genotyping systemSobral, Daniel 02 May 2012 (has links)
Les espèces bactériennes exhibent plusieurs états de structure de populations pouvant varier de clonale à panmictique selon l'importance des transferts horizontaux et la nature de leur écosystème. Dans mon travail de thèse, je me suis intéressé à trois espèces bactériennes, Staphylococcus aureus, Legionella pneumophila et Pseudomonas aeruginosa qui reflètent trois situations différentes. Afin de pouvoir décrire de façon rapide de grandes collections de souches, j'ai utilisé comme marqueurs de diversité le polymorphisme de séquences répétées en tandem appelées VNTRs, pour Variable Number Tandem Repeat. La méthode MLVA, ou Multiple Loci VNTR Analysis, est une méthode de typage moléculaire qui s’appuie sur l’étude concomitante du polymorphisme de plusieurs loci VNTRs. Dans un premier temps, j'ai conçu des protocoles de typage automatisés pour les trois espèces considérées, puis j'ai appliqué ces outils pour traiter de questions d'épidémiologie. S. aureus, espèce à structure clonale, est un pathogène majeur responsable notamment de toxi-infections alimentaires collectives (TIAC). Les travaux réalisés ont permis de démontrer la spécificité d’hôte de certains complexes clonaux et l’origine humaine des cas de TIAC. L. pneumophila est un pathogène de l’environnement dont la structure de population est atypique : présumée panmictique dans la nature, la bactérie semble connaitre une évolution clonale lorsque son écosystème est restreint, dans un milieu anthropique par exemple. L’étude épidémiologique menée sur la population de L. pneumophila dans la ville de Rennes a mis en évidence la présence d’un écotype, non impliqué dans les cas cliniques épidémiques, particulièrement adapté aux réseaux d’eau. P. aeruginosa, modèle de bactérie panmictique, colonise les bronches de patients atteints de mucoviscidose. Le suivi longitudinal de patients indique que les souches installées sont persistantes et quasi-exclusive de la niche qu’elles occupent. L’exploration de cette diversité du monde bactérien est un préalable à l’investigation épidémiologique des maladies infectieuses. Avec un même outil moléculaire de première intention, cette thèse retrace l’épidémiologie et la structure de trois espèces bactériennes très différentes. L’adaptation à un nouvel environnement (hôte animal, niche écologique, organe) est l'occasion d'expansions clonales. / Bacterial species exhibit diversity in their population structure varying from clonal to panmictic according to the abundance of horizontal transfer and the nature of their ecosystem. During my PhD, I focused on three bacterial species, Staphylococcus aureus, Pseudomonas aeruginosa and Legionella pneumophila, which reflect three different situations. To perform the characterisation of large strain collections, I studied the polymorphism of molecular markers called VNTRs for Variable Number Tandem Repeat. MLVA (Multiple Loci VNTR Analysis) is a PCR based typing method that relies on the concomitant analysis of several VNTRs loci. Initially, I designed automated typing protocols for the three species, then I applied these tools to address issues of epidemiology. S. aureus, a clonal species, is a major cause of food poisoning. The present work confirmed the existence of host-specific clonal complexes and demonstrated the predominantly human origin of foodborne disease cases. L. pneumophila is an environmental pathogen whose population structure is atypical: it is presumed panmictic in the environment but the bacterium expands clonally when the ecosystem is restricted, in an anthropogenic habitat for instance. A long-term epidemiological monitoring of L. pneumophila populations in the city of Rennes highlighted the presence of an ecotype, not involved in epidemic cases, particularly adapted to hot water supply systems. P. aeruginosa, a well-described panmictic bacterium, colonizes CF patients’ airways. The longitudinal monitoring of patients provided evidence that the settled strains were persistent and exhibited strong exclusivity for the occupied niche. Exploring the bacterial world diversity is a prerequisite for epidemiological investigation of infectious diseases. Using a first-line molecular tool, these works trace the epidemiology and the population structure of three bacterial species. The adaptation to a new environment (animal host, ecological niche, organ) generally results in clonal expansions.
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Diversitat genòmica a les poblacions del Nord d'ÀfricaBosch Fusté, Elena 18 February 2000 (has links)
S'ha estudiat la variabilitat genètica de les poblacions del nord d'Àfrica a partir de l'anàlisi de diverses regions genòmiques per tal d'entendre les poblacions analitzades d'una banda, i comprendre la dinàmica del genoma per l'altra. Els resultats obtinguts ens han permès verificar diferents hipòtesis sobre la història de les poblacions d'aquesta regió com són l'efecte paral·lel i independent de l'onada de difusió del neolític des de l'Orient Mitjà al llarg d'ambdues ribes de la Mediterrànea; i l'efecte de l'arabització. S'ha pogut estimar també la contribució genètica masculina nord africana a la península ibèrica i detectat certa contribució genètica del pobles sub-saharians a les poblacions nordafricanes. Per altra banda, el tipatge de marcadors genètics que evolucionen a velocitats diferents al cromosoma Y ha permès mostrar que el background genètic predomina sobre el background poblacional en l'estructura de la variació genètica dels microsatèl·lits en la regió no recombinant del cromosoma Y humà. / The genetic variability of the North African populations has been studied through the analysis of different genomic regions in order to understand both the analysed populations and the dynamics of the genome. The obtained results allow us to verify different hypotheses about the population history of this region including the parallel and independent effect of the Neolithic wave of advance from the Middle East and along both Mediterranean coasts; and the effect of Arabization phenomena. We also tried to estimate the North African male genetic contribution to the Iberian peninsula and detected Sub-Saharian genetic influences to the North African peoples. Moreover, the typing of genetic markers with different evolutionary rates on the Y chromosome allowed us to demonstrate that variation in microsatellites is deeply structured by genetic background on the non-recombining region of the human Y chromosome.
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