Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-06-01T12:09:03Z No. of bitstreams: 1 ingridestevampereira.pdf: 2004254 bytes, checksum: 326e3188dc98d9fc4ddc417af89ebc1e (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-06-02T15:12:44Z (GMT) No. of bitstreams: 1 ingridestevampereira.pdf: 2004254 bytes, checksum: 326e3188dc98d9fc4ddc417af89ebc1e (MD5) / Made available in DSpace on 2017-06-02T15:12:44Z (GMT). No. of bitstreams: 1 ingridestevampereira.pdf: 2004254 bytes, checksum: 326e3188dc98d9fc4ddc417af89ebc1e (MD5) Previous issue date: 2017-02-22 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / A talidomida é uma potente droga anti-inflamatória empregada no tratamento de diversas patologias, incluindo Eritema Nodoso Leproso (ENL), câncer, doença de Crohn e outras desordens inflamatórias e vasculares. Entretanto, seus efeitos tóxicos e teratogênicos tornam sua utilização limitada e motivam pesquisas para a síntese de análogos que apresentem eficácia semelhante na imunomodulação, sem efeitos tóxicos. Diversos análogos da talidomida vêm sendo desenvolvidos no laboratório de química da UFJF. Em estudos anteriores, mostramos que a introdução de dois anidridos ftálicos na composição aumenta significativamente a atividade biológica e solubilidade em água do composto, sem aumento da toxicidade em modelos experimentais in vitro e in vivo. O presente trabalho visa a síntese de dois novos compostos análogos da talidomida, CAT-15 e CAT-16, formados por apenas um derivado anidrido ftálico aberto, mantendo um grupo amino livre, e a avaliação da sua atividade anti-inflamatória utilizando linhagem de célula HT-29 e células mononucleares de sangue periférico humano (PBMC) estimuladas com LPS. A citotoxicidade dos compostos foi avaliada pelo ensaio do MTT, com tratamento por 18 horas para células HT-29 e por 24 e 48 horas para PBMC, usando concentrações crescentes de talidomida, CAT-15, CAT-16. A dexametasona foi utilizada como controle positivo. A produção de TNF-α, CXCL-10, IL-6, IL-8 e IL-10 foi avaliada pelo método de ELISA. Os novos compostos não foram tóxicos para as células HT-29 e PBMC em nenhuma das concentrações testadas, com exceção de CAT-16 a 1600µM. Células HT-29 produziram grande quantidade de CXCL-10 em resposta ao LPS e os resultados deste trabalho mostram que a talidomida e os análogos CAT-16 e CAT-15 apresentam atividade inibitória sobre a produção desta quimiocina. O composto CAT-16 modulou a produção de CXCL-10 em concentrações menores que a talidomida em ambos os modelos de tratamento (simultâneo e prétratamento). Em contrapartida, a modulação por CAT-15 foi observada apenas no modelo de pré-tratamento. Com relação a IL-8, a talidomida e o CAT-16 inibiram a produção desta citocina por células HT-29 apenas na concentração de 100µM. Ao contrário das células HT-29, o PBMC produziu TNF-α em resposta ao LPS, tendo a talidomida e o análogo CAT-16 apresentado capacidade de inibição da produção do TNF-α em ambos os tempos de tratamento. O análogo CAT-15 não influenciou a produção de TNF-α por PBMC em nenhuma das concentrações e tempos de tratamento. Este estudo também mostra a atividade da talidomida e dos nos análogos sobre a produção de IL-6 e IL-10 por PBMC, havendo significativa inibição da produção de IL-6 por todos os compostos e tempos de tratamento e sobre IL-10 pelo composto CAT-15 após 48 horas de incubação. Nossos resultados sugerem a aplicabilidade dos novos compostos, CAT-15 e CAT-16, no controle de respostas inflamatórias uma vez que inibiram a produção de moléculas chave como TNF-α, IL6, IL-10, IL-8 e CXCL-10. Ainda, esses compostos possuem estruturas simplificadas, têm baixo custo de produção, são hidrossolúveis e não possuem centro quiral. Esses resultados podem contribuir no desenvolvimento de novas estratégias de tratamento para certas condições inflamatórias. / Thalidomide is a potent anti-inflammatory drug used in the treatment of various pathologies including Erythema Nodosum Leprosum (ENL), cancer, Crohn's disease and other inflammatory and vascular disorders. However, its toxic and teratogenic effects make its use limited and motivate research groups to synthesize analogues presenting similar immunomodulation efficacy, without toxic effects. Several analogs of thalidomide have been developed in the laboratory of chemistry of the UFJF. Previously, we have shown that introduction of two phthalic anhydrides into the composition significantly enhances biological activity and water solubility, without enhanced toxicity. The present work aims at the synthesis of two new analogues of thalidomide, CAT-15 and CAT-16, formed by only one open phthalic anhydride derivative, maintaining a free amino group, and the evaluation of its anti-inflammatory activity using HT- 29 and human peripheral blood mononuclear cells (PBMCs) stimulated with LPS. The cytotoxicity of the compounds was evaluated by the MTT assay, with 18 hours treatment for HT-29 cells and for 24 and 48 hours for PBMC, using increasing concentrations of thalidomide, CAT-15, CAT-16. Dexamethasone was used as a positive control. Production of TNF-α, CXCL-10, IL-6, IL-8 and IL-10 was evaluated by the ELISA method. The novel compounds were not toxic to HT-29 and PBMC cells at any of the concentrations tested, with the exception of CAT-16 at 1600μM. HT-29 cells produced large amounts of CXCL-10 in response to LPS and the results of this work show that thalidomide and the CAT-15 and CAT-16 analogs exhibit inhibitory activity on the production of this chemokine. CAT-16 compound modulated the production of CXCL-10 at lower concentrations than thalidomide in both treatment models (simultaneous and pretreatment). In contrast, CAT-15 modulation was observed only in the pre-treatment model. Regarding IL-8, thalidomide and CAT-16 inhibited their production by HT-29 cells only at the concentration of 100 μM. Unlike HT-29 cells, PBMC produced TNF-α in response to LPS, with thalidomide and CAT-16 analog being able to inhibit TNF- production at both treatment times. The CAT-15 analogue did not influence the production of TNF-α by PBMC at any of the concentrations and treatment times. This study also shows the activity of thalidomide and the analogs on IL-6 and IL-10 production by PBMC, with significant inhibition of IL-6 production by all compounds and treatment times and on IL-10 by compound CAT-15 after 48 hours of incubation. Our results suggest the applicability of the new compounds, CAT-15 and CAT-16, in the control of inflammatory responses since they inhibited the production of key molecules such as TNF-α, IL-6, IL-10, IL-8 and CXCL-10. Furthermore, these compounds have simplified structures, and low cost of production, are water soluble and have no chiral center. These results may contribute to the development of novel treatment strategies for certain inflammatory conditions.

Abstract α-Methylacyl-CoA racemase (Amacr) is an auxiliary enzyme of β-oxidation and participates in the elimination of methyl-branched fatty acids in peroxisomes and in mitochondria and in the synthesis of bile acids in peroxisomes. Amacr catalyzes in reversible manner the isomerization of fatty acyl-CoA esters with a methyl group in the R-configuration to the corresponding S-configuration, which allows them to serve as substrates for the next reaction in their metabolism. The substrates of Amacr include the acyl-CoA esters of 2R-pristanic acid, a metabolite derived from phytol, and 25R-THCA and 25R-DHCA (tri- and dihydroxycholestanoic acid), the bile acid intermediates derived from cholesterol. AMACR-deficiency in humans results in the accumulation of R-isoforms of its substrates. Patients with adult onset AMACR-deficiency suffer from neurological disorders. The more severe infantile form of the deficiency is characterized by liver disease. Amacr-deficient mice show a bile acid pattern similar to that of human patients with accumulation of bile acid intermediates in their body. In contrast to humans, Amacr-deficient mice are clinically symptomless on a regular laboratory chow diet. Supplementation of phytol in their diet triggers the disease state with liver abnormalities. In this study it was shown that in spite of the disruption of a major metabolic pathway, Amacr-deficient mice are able to readjust their cholesterol and bile acid metabolism to a new balanced level allowing them to live a normal life span. A double knockout mouse model deficient in Amacr and MFE-1 (peroxisomal multifunctional enzyme type 1) was generated in this work. Characterization of this mouse line showed that MFE-1 can contribute to peroxisomal side-chain shortening of C27 bile acid intermediates in both Amacr-dependent and Amacr-independent pathways. In addition, this work confirmed that Amacr-deficient mice are unable to thrive when phytol is supplemented in their chow. The main cause of death was liver failure accompanied by kidney and brain abnormalities. The detoxification of phytol metabolites in liver is accompanied by activation of multiple pathways and Amacr-deficient mice are not able to respond adequately. The results of this study emphasize the indispensable role of Amacr in detoxification of α-methyl branched fatty acids. / Tiivistelmä α-Metyyliasyyli-koentsyymi-A-rasemaasi (Amacr) osallistuu metyyli-haarautuvien rasvahappojen eliminointiin peroksisomeissa ja mitokondrioissa ja sappihappojen synteesiin kolesterolista peroksisomeissa. Amacr katalysoi käänteisesti rasvahappojen asyyli-koentsyymi-A-estereiden isomerisaatio-reaktiota, jossa stereokemiallisesti R-asemassa oleva metyyliryhmä siirtyy S-asemaan. Tämä on edellytys eliminointiketjun seuraavan reaktion tapahtumiselle. Amacr-entsyymin substraatteja ovat fytolin aineenvaihdunnassa syntyvän 2R-pristaanihapon ja kolesterolista sappihapposynteesireitin välituotteina syntyvien 25R-trihydroksikolestaanihapon ja 25R-dihydroksikolestaanihapon (25R-THCA ja 25R-DHCA) asyyli-koentsyymi-A-esterit. Ihmisellä Amacr-entsyymin puutos johtaa R-muodossa olevien substraattien kertymiseen, joka aiheuttaa neurologisia oireita aikuisiässä alkavassa sairauden muodossa. Lapsuusiässä alkavassa tautimuodossa potilaille kehittyy vakava maksasairaus. Tutkimuksen tulokset osoittivat, että Amacr-poistogeenisten hiirten elinikä ei lyhene huolimatta yhden tärkeän aineenvaihduntareitin estymisestä. Tämä on hyvä esimerkki siitä, kuinka nisäkäs pystyy mukauttamaan kolesteroli- ja sappihappoaineenvaihduntaansa vastaamaan muuttunutta tilannetta aineenvaihdunnassa. Tässä työssä tuotettiin myös kaksoispoistogeeninen hiirimalli, jonka Amacr- ja peroksisomaalinen monitoiminnallinen entsyymi tyyppi 1- (MFE-1) entsyymit ovat toimimattomat. Tämä hiirimalli paljasti, että MFE-1 pystyy osallistumaan 27:ää hiiltä sisältävien sappihappovälituotteiden sivuketjun lyhentämiseen sekä Amacr entsyymin kanssa että ilman sitä. Työn tulokset myös osoittivat, että Amacr-poistogeeniset hiiret eivät ole elinkykyisiä, jos niiden ravinto sisältää fytolia. Maksan toiminnanvajaus oli näiden hiirten tärkein kuolinsyy, mutta hiirten munuaisten ja aivojen kudosrakenteissa oli myös muutoksia. Maksassa fytolin metaboliittien vaarattomaksi tekeminen aiheuttaa villityypin hiirillä useamman aineenvaihduntareitin aktivoitumisen, mutta Amacr-poistogeeniset hiiret eivät pysty reagoimaan tähän samalla tavalla. Tämä työ osoittaa, että Amacr-entsyymin elintärkeä tehtävä on osallistua ravinnon mukana elimistöön joutuvien α-metyylihaarautuvien rasvahappojen eliminaatioon.

Le développement de nanotransporteurs basés sur des lipides, des polymères et des nanoparticules avec des propriétés «sur mesure» pour augmenter l’efficacité de médicaments, fait l’objet de recherches intensives. Toutefois, la physico-chimie subtile des intéractions polymères-lipides and nanoparticules-lipides présente encore de larges domaines mal compris et de nombreuses questions sans réponse. Ce projet de recherche doctoral utilise des techniques de visualisation (Cryo-MET, LSCM), et de caractérisation (ITC, DSC, SAXS, SANS, QCM-D) avancées pour obtenir des informations nouvelles sur les mécanismes d’interaction entre des Cyclodextrines-α d’autre part, des polymères sensibles au pH d’autre part, et des bicouches modèle de DOPC. La forte influence de ces deux composés sur ces systèmes modèle élucide certains aspects relatifs à la toxicité vis-à-vis des membranes biologiques et suggère de nouvelles approches pour des applications pharmaceutiques. / The primary goal of nanomedicine is to improve clinical outcomes. To this end, the development of nanocarriers based on lipids, polymers and nanoparticles with tailor-made properties that enhance the in vivo potency of drugs is a subject of intense research. However, the subtle physical-chemistry of the polymer-lipid and nanoparticle-lipid interactions still present many poorly understood fields of investigation as well as unanswered questions. This doctoral research project utilizes state-of-the-art visualization (Cryo-TEM, LSCM) and characterization (ITC, DSC, SAXS, SANS, QCM-D) techniques to gain novel insights into the interaction between α-Cyclodextrins in the first hand, a pH-responsive polymer in the other hand, and model DOPC bilayers. The strong influence of both compounds on these model systems elucidate some aspects regarding biological membrane toxicity and suggests novel strategies for pharmaceutical applications.

Les ruptures imprévisibles de composants de robinetterie gaz constituent une problématique majeure de la sûreté du réseau de GrDF. Actuellement, la prévention des risques de rupture repose sur la réalisation de tests accélérés de sensibilité à la corrosion sous contrainte (CSC), préalablement à la mise en service des composants de robinetterie en laiton. Il s'agit d'éviter des accidents, au caractère potentiellement très dommageable, du fait de l'inflammabilité des gaz transportés. Or, dans certains cas, des endommagements prématurés de composants de robinetterie sont toujours constatés. Les limites du test de sensibilité actuel SROB 100 NF ont été démontrées par le CETIM ; définir un nouveau test accéléré, plus représentatif des endommagements constatés en service sur les éléments de robinetterie, et conduisant à des résultats reproductibles est donc crucial. Ce test devra être facilement adaptable sur site industriel afin de permettre aux industriels fabricants de valider la conformité des lots de pièces vis-à-vis de leur sensibilité à la CSC. Ainsi, les travaux de thèse reposent sur une démarche intégrant d'une part, la compréhension des mécanismes de CSC affectant les pièces en laiton α,β’ CuZn40Pb2 (CW617N) et d'autre part, la capacité à reproduire les endommagements observés en service, de manière accélérée. Grâce aux résultats obtenus, l'étude permet d’envisager l'industrialisation d’un nouveau test en milieu nitrate, à pH 11 sous polarisation anodique. La reproductibilité de l'endommagement obtenu et sa représentativité vis-à-vis des dommages observés en service comme le caractère discriminant du test vis-à-vis des états métallurgiques les moins résistants ont été démontrés. / The unpredictability of ruptures which occur in the gas transfer valves of the French gas network (GrDF), though sporadic, is a major security issue. Currently, the risk prevention is performed through accelerated tests (SROB100 NF) on gas transfer valves to determine their susceptibility to stress corrosion cracking (SCC). The goal behind this test protocol is to avoid serious accidents due to the flammability of the gas being transported. However, in some cases, premature ruptures of gas transfer valves are still being recorded. The limits of the current test protocol SROB 100 NF have been highlighted by the CETIM and therefore, to define a new accelerated test, more reproducible and representative of the damage observed during the service life of the pipework element is crucial. This new protocol has to be easily integrated into the current industrial set-up to allow for quick confirmation of the conformity of the pieces with regards to their susceptibility to SCC. To this end, the current study had a two-pronged approach. First, to understand the SCC mechanisms affecting brass specimen - α,β’ brass CuZn40Pb2 (CW617N); second, to reproduce the damage observed in real time, in an accelerated manner. In conclusion, the results of the study have paved the way for the industrialization of a new test in nitrate solution, at pH 11 under constant anodic polarization. This new test has proven to be reproducible and produces damage characteristics similar to those observed during service life. This study has also brought into evidence the discriminating characteristic of the test, allowing the identification of the metallurgical states of the alloy that are the least resistant to SCC.

S-adenosyl-L-homocysteine (AdoHcy) hydrolase effects hydrolytic cleavage of AdoHcy to produce both adenosine and L-homocysteine and is a feedback inhibitor of S-adenosyl-L-methionine (SAM). Nucleoside analogues bearing an alkenyl or fluoroalkenyl chain between sulfur and C5′ utilizing Negishi coupling reactions were synthesized. Palladium-catalyzed cross-coupling between the 5′-deoxy-5′-(iodomethylene) nucleosides and alkylzinc bromides gives analogues with the alkenyl unit. Palladium-catalyzed selective monoalkylation of 5′-(bromofluoromethylene)-5′-deoxy-adenosine with alkylzinc bromide afford adenosylhomocysteine analogues with a 6′-(fluoro)vinyl motif. The vinylic adenine nucleosides produced time-dependent inactivation of the S-adenosyl-L-homocysteine hydrolases. Stannydesulfonylation reaction is a critical step in the synthesis of E-fluorovinyl cytidine (Tezacitabine) a ribonucleoside reductase inhibitor with a potent anticancer activity. The synthesis involves the removal of the sulfonyl group by a radical-mediated stannyldesulfonylation reaction using tributyltin hydride. In order to eliminate the toxicity of tin, I developed a radical-mediated germyldesulonylation utilizing less toxic germane hydrides. Treatment of the protected (E)-5'-deoxy-5'-[(p-toluenesulfonyl)-methylene]uridine and adenosine derivatives with tributyl- or triphenylgermane hydride effected radical-mediated germyldesulfonylations to give 5'-(tributyl- or triphenylgermyl)methylene-5'-deoxynucleoside derivatives as single (E)-isomers. Analogous treatment of 2'-deoxy-2'-[(phenylsulfonyl)methylene]uridine with Ph3GeH afforded the corresponding vinyl triphenylgermane product. Stereoselective halodegermylation of the (E)-5'-(tributylgermyl)-methylene-5'-deoxy nucleosides with NIS or NBS provided the Wittig-type (E)-5'-deoxy-5'-(halomethylene) nucleosides quantitatively. Radical-mediated thiodesulfonylation of the readily available vinyl and (α-fluoro) vinyl sulfones with aryl thiols in organic or aqueous medium to provide a bench and environmentally friendly protocol to access (α-fluoro)vinyl sulfides were developed. Methylation of the vinyl or (α-fluoro)vinyl phenyl sulfide gave access to the corresponding vinyl or (α-fluoro)vinyl sulfonium salts. These sulfonium ions were tested as possible methyl group donors during reactions with thiols, phenols or amino groups which are commonly present in natural amino acids.

Liver mitochondria play a crucial role in intermediary metabolism and main metabolic pathways. We evaluated the pharmacological effect on liver mitochondria in vitro using two novel anticancer drugs: 3-bromopyruvate and α-tocopheryl succinate. Metabolic influence on liver mitochondria was performed in vivo by high fat and high cholesterol diet. Toxicity of both drugs was evaluated in cell cultures of hepatocytes isolated from rat and mouse liver. The effect of anticancer drugs on liver mitochondrial functions in vitro was studied on suspensions of isolated liver mitochondria, tissue homogenate and permeabilized hepatocytes. Mitochondrial respiration was measured using high-resolution respirometry. 3-bromopyruvate caused morphological and functional damage of primary rat and mouse hepatocytes in cell cultures; this toxic effect was accompanied by an increase of reactive oxygen species production and mitochondrial dysfunction. 3-bromopyruvate decreased the oxygen consumption of mitochondria energized by substrates for complex I and complex II. α-Tocopheryl succinate caused a decrease of succinate-dependent respiration in all experimental models both in coupled and in uncoupled states. The most pronounced effect of α-tocopheryl succinate was apparent in isolated mitochondria and the least pronounced...

The phenotype-linked fertility hypothesis predicts that both, male carotenoid-based sexual ornamentation and their spermatozoa are phenotypically plastic and may be co-affected by the environment. One of the factors affecting their phenotype may be oxidative stress and the ability of organism to eliminate its effect. Oxidative stress may reduce sperm quality because sperm lack the ability to repair DNA, but it can also affect spermatogenesis itself. However, some substances may function as antioxidants, and thus eliminate effect of reactive oxygen species (oxidative stress) in the body. In this study, adult zebra finch males (Taeniopygia guttata) originating from the domesticated and recently wild-derived populations were exposed to the diquat (D), which enhances the oxidative stress, and carotenoid lutein (L), which could have an antioxidant function. Experimental design had factorial character 2x2 with a control (group L, D, LD, control). Neither oxidative stress, carotenoids, nor their interactions affected sperm morphology or velocity and it also did not increase abnormal sperm proportion in the ejaculate. However, the differences were observed at the molecular level, where by inducing the oxidative stress, the sperm had reduced signal intensity of acetylated α-tubulin in the sperm tails....

Die vorliegende Untersuchung hatte zur Zielsetzung, bei Kühen die Unterschiede zwischen hypokalzämischen und normokalzämischen Festliegern zu analysieren. Es sollte geklärt werden, welche klinischen und labordiagnostischen Veränderungen, außer der Kalzium- (Ca) Konzentration, dem normokalzämische Festliegen zugrunde liegen. Des Weiteren wurden die TNF-α-, Haptoglobin- (Hp-) und TEAC-Konzentrationen in Beziehung zum Festliegen, den Mineralstoffkonzentrationen sowie hinsichtlich diagnostischer Information geprüft. Außerdem wurden die Festlieger mit Nachgeburtsverhaltung (Ret. sec.) und die Kühe mit Exitus letalis labordiagnostisch genauer analysiert.

Brown rice is a food ingredient which has high nutritious values. During germination, some nutritious and functional components are increased such as lysine, vitamin E, B1, B6, magnesium, calcium, iron… and especially γ – amino butyric acid. Enzyme activity will also change during the germination of the grains. The α – amylase activity of ungerminated grains is very low, only 34.91 UI/g. This is because the enzyme is hibernating and not activated. During germination, enzyme activity will increase. Submerge the Anhdao brown glutinous rice for 6 hours at 30oC in solutions with different pH values (2.0, 3.0, 4.0, 5.0, 6.0). The results show that at pH 3.0 the activity of α – amylase enzyme reaches the highest value of 82.93 UI/g. After the submersion, incubate the germinated brown rice in unentirely anaerobic condition at different temperature of 25, 30, 35, 37oC. The result showed that at 35oC after 24 hours of incubation, the α – amylase activity reaches the highest value of 89.82 UI/g. Examine the dynamic of changes of α – amylase activity against time at 35oC, we can see that in the first 28 hours the α – amylase activity increased significantly. Highest α – amylase activity reaches 97.10 UI/g after 28 hours of incubation. In reality, people usually use enzyme from germinated grains for many food manufacturing industries. α – amylase activity increases during incubation, which can bring promising prospects for processing sugar syrup and prebiotics food from germinated rice. / Gạo lứt là một nguyên liệu thực phẩm có giá trị dinh dưỡng cao. Trong quá trình nảy mầm các thành phần dinh dưỡng và chức năng của hạt gạo lứt được tăng lên ví dụ như lysine, các vitamin E, B1, B6, magie, canxi, sắt… và đặc biệt là γ – amino butyric acid. Hoạt tính của hệ enzyme sẽ thay đổi trong suốt quá trình nảy mầm của hạt gạo. Hoạt tính enzyme α – amylase của hạt gạo chưa nảy mầm là rất thấp, chỉ đạt 34,91UI/g, do enzyme của hạt đang ở trạng thái ngủ chưa được kích hoạt. Trong quá trình nảy mầm thì hoạt tính của α – amylase tăng lên. Tiến hành ngâm gạo lứt giống nếp Anh Đào trong 6 tiếng, ở 30oC trong nước ngâm có pH khác nhau (pH 2.0, 3.0, 4.0, 5.0, 6.0). Kết quả cho thấy, ở pH 3.0 hoạt độ enzyme α – amylase cao nhất đạt 82,93 UI/g. Sau quá trình ngâm, tiến hành ủ nẩy mầm gạo lứt yếm khí không hoàn toàn ở những nhiệt độ khác nhau 25, 30, 35, 37oC, kết quả ở 35oC sau 24 giờ ủ hoạt độ enzyme α – amylase đạt cao nhất là 89,82 UI/g. Khảo sát động học sự thay đổi của hoạt độ enzyme α – amylase theo thời gian ở nhiệt độ ủ 35oC, kết quả cho thấy,trong 28 giờ đầu hoạt độ của α – amylase tăng mạnh. Hoạt độ α – amylase cao nhất đạt 97,10 UI/g sau 28 giờ ủ. Trong thực tế người ta đã sử dụng enzyme từ hạt nảy mầm cho rất nhiều ngành sản xuất thực phẩm. Hoạt độ của α-amylase sau quá trình ủ mầm tăng lên, có thể đem lại triển vọng sử dụng để chế biến dịch đường và các sản phẩm có tính prebiotic từ gạo lứt nảy mầm.

Lors de ces travaux de thèse, nous nous sommes intéressés à de nouveaux développements méthodologiques en cycloaddition 1,3-dipolaire de nitrones suivant deux axes principaux :<br />• l'étude de la réactivité des N-alcényloxazolidin-2-ones en tant que nouveaux dipolarophiles aza-substitués en cycloaddition 1,3-dipolaire à demande inverse vis-à-vis de nitrones.<br />• la mise en jeu de nitrones aspartiques originales en cycloaddition vis-à-vis de différents alcènes pour fournir des précurseurs d'aspartates α-substitués par une chaîne fonctionnelle.<br />Dans un premier temps, nous avons mis au point deux méthodes inspirées de celles de la littérature en partant d'une oxazolidin-2-ones pour synthétiser les N-alcényloxazolidin-2-ones: (i) vinylation cupro-catalysée utilisant un bromure d'alcényle, (ii) condensation avec un aldéhyde. Ces méthodes simples, générales, directes et à hauts rendements nous ont permis d'accéder aux N-alcényloxazolidin-2-ones de structure diverse.<br />Ces N-alcényloxazolidin-2-ones ont montré une grande réactivité en tant que dipolarophile vis-à-vis de diverses nitrones dans différentes conditions : thermiques (avec/sans solvant) et promues par TMSOTf. Les 5-aza-isoxazolidines diversement substituées originales ont été obtenues avec des rendements élevés mais de faibles stéréosélectivités. Cette limitation − due à l'instabilité configurationnelle des nitrones activées et à la flexibilité conformationnelle des N-alcényloxazolidin-2-ones − a été résolue par utilisation de la nitrone chirale à géométrie fixe de Tamura : les adduits trans-β sont obtenus avec d'excellents sélectivités<br />La tranformation des adduits issus de la N-benzyl-α-carbonyloxyéthylnitrone en dérivés aspartates carboxy-différenciés a été ensuite étudiée par une séquence en deux étapes (i) ouverture du cycle isoxazolidinique en aspartimide via N-quaternarisation par benzylation (ii) attaque chimiosélective d'un hétéronucléophile sur la fonction amide. Les aspartates carboxy-différenciés ont été obtenus avec de bons rendements, des excès énantiomériques élevés en version non-racémique, et un grand degré de diversité fontionnelle en ω (ester, amide, acide).<br />L'étude a été étendu à la cycloaddition 1,3-dipolaire des N-vinyloxazolidin-2-ones β,β-difluorées originales avec la N-benzyl-α-carbonyloxyéthylnitrone : les 4,4-difluoro-5-aza-isoxazolidines sont obtenues avec de bons rendements malgré une faible stéréosélectivité probablement due à un mécanisme non-concerté.<br />Notre étude a été complétée par l'accès à des dérivés d'α-aminoacides α,α-disubstitués via cycloaddition 1,3-dipolaire mettant en jeu de nouvelles nitrones aspartiques d'une stablilité configurationnelle inédite. Ce type de nitrones − préparé facilement par addition d'une N-benzylhydroxylamine sur un acétylènedicarboxylate – a montré une grande réactivité vis-à-vis d'une large gamme d'alcènes de différents natures électroniques. Les adduits issus des éthers vinyliques ont été obtenus avec de hauts sélectivités trans. L'extension asymétrique utilisant soit un éther vinylique chiral , soit une nitrone chirale a été effectuée et a permis l'obtention facile des adduits diastéréomériquement enrichis. A partie de ces adduits, un premier accès réussi aux dérivés aminoacides α,α-disubstitués a été réalisé en trois étapes. La diversification de cette méthodologie a été effectuée en utilisant une nitrone aspartique carboxy-différenciée.