La multivalence est reconnue en tant qu’outil permettant d’augmenter le pouvoir d’inhibition d’un inhibiteur. La mise en évidence, en 2010, d’un effet multivalent puissant sur l’inhibition des glycosidases par un iminosucre multivalent a ouvert la voie vers la synthèse de clusters toujours plus puissants. En 2016, un composé multivalent a permis d’atteindre une inhibition 173000 fois meilleure que le composé monovalent correspondant, ce qui équivaut à une augmentation record du pouvoir d’inhibition par iminosucre de 4800. La synthèse d’une nouvelle génération d’iminosucres multivalents à ligand affin pour sa cible, pour parvenir à augmenter encore l’effet multivalent, était mon objectif de thèse. Au cours de ce travail, de nouveaux iminosucres multivalents à motif gluco- ou mannoimidazole, très affin des mannosidases, ont été synthétisés. Ces composés multivalents ont permis de travailler sur deux projets parallèles : un projet fondamental et un projet appliqué. Le premier a consisté en l’étude de l’influence de l’affinité du ligand sur l’effet multivalent par mesure du pouvoir d’inhibition sur l’enzyme commerciale répondant le mieux à l’effet multivalent, l’α-mannosidase de Jack-bean. Les résultats montrent l’existence d’un effet multivalent notable mais sa valeur n’est pas aussi importante comparée aux études précédentes. Les iminosucres multivalents pouvant également cibler les glycosidases d’intérêt thérapeutique, nous nous sommes intéressés, dans le cadre du projet appliqué, au traitement de la mucoviscidose. L’idée était de viser les mannosidases I et II du réticulum endoplasmique pour permettre de secourir la protéine CFTR déficiente impliquée dans cette maladie. Malheureusement, aucun effet correcteur ni potentiateur n’a pu être observé pour nos composés. En marge de ces axes principaux, une méthode de déshydroxylation sélective a été optimisée et développée en élargissant son champ d’application et en apportant des preuves mécanistiques. / Multivalency is known to be a tool to drive inhibitors more potent. The multivalent effect, evidenced in 2010 with multivalent iminosugar on glycosidase inhibition, paved the way to the synthesis of multimeric inhibitors even more powerful. In 2016, an iminosugar cluster was reported to be 173 000 times more potent inhibitor compared to the monovalent reference. This is equal to an inhibitory potency enhancement of 4800 per iminosugar. The goal of my PhD was the synthesis of a new generation of multivalent iminosugars very potent towards their target, to keep increasing multivalent effect. New multivalent iminosugars with glycoimidazole motifs, with high affinity towards mannosidases, were synthetised. Those multivalent compounds allowed us to work on two parallel projects: a fundamental and an applied one. The first one focused on the influence of ligand affinity on multivalent effect by measuring the inhibition potency on the most sensitive to multivalent effect commercially available enzyme: the Jack-bean α-mannosidase. Results showed that multivalent effect was maintained but at a lower level than those reported previously. Multivalent iminosugars being also able to target glycosidases of therapeutic interest, we were interested in the treatment of cystic fibrosis. Targeting endoplasmic reticulum mannosidases I and II could restore the CFTR protein whose deficiency is the root cause of the disease. Unfortunately, no corrective or potentiation effect were observed. Aside those principal lines, a selective dehydroxylation method was optimized and developed widening the scope and providing mechanistic proofs.

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第20303号 / 薬科博第72号 / 新制||薬科||8(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 川端 猛夫, 教授 高須 清誠, 教授 竹本 佳司 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DGAM

Объектами исследования являются витамины разной гидрофильности и витаминосодержащие препараты в таблетированной форме. Цель работы: исследование антиоксидантных свойств витаминных препаратов различной гидрофильности. Исследована антиоксидантная емкость модельных растворов и смесей антиоксидантов разной гидрофильности потенциометрическим методом; подобраны оптимальные условия для проведения анализа липофильного и гидрофильного соединений в совместном присутствии; исследованы фармацевтические препараты в таблетированной форме, содержащие витамины С и Е; проведено сравнение результатов с методом Фолина-Чокальтеу. Рассмотрена возможность применения методики определения АОЕ в условиях микрофлюидного устройства. / The objects of research are vitamins of different hydrophilicity and vitamin-containing preparations in tablet form. Purpose of the work: study of the antioxidant properties of vitamin preparations of various hydrophilicity. The antioxidant capacity of model solutions and mixtures of antioxidants of different hydrophilicity was investigated by potentiometric method; the optimal conditions were selected for the analysis of lipophilic and hydrophilic compounds in their joint presence; investigated pharmaceutical preparations in tablet form containing vitamins C and E; the results are compared with the Folin-Chocalteu method. The possibility of using the method for determining AOE in a microfluidic device is considered.

In order to improve the production of acarbose, the fermentation medium of acarbose-producing strain Actinoplanes sp. KCTC 9161 – L14 mutant was optimized in this internship. Fractional factorial design was employ to investigate the influences of glucose, maltose and corn power on acarbose production (by a-glucosidase inhibitory ability). Two significant factors: glucose and maltose have significant and positive effects on acarbose amount. In addition, a model was obtained from the regression results of fractional factorial experiment. Other success, we demonstrated that chromatography by active charcoal column can used to purify acarbose from fermentation broth. Acarbose amount in purification solution was 191.5 g/L and an acarbose - purification process was inducted. / Nhằm mục đích nâng cao khả năng sinh tổng hợp hoạt chất acarbose từ chủng đột biến Actinoplanes sp. KCTC 9161-L14, môi trường lên men của chủng dùng để sản xuất acarbose đã được tối ưu hóa. Một phần mềm thiết kế đã được thiết lập để khảo sát ảnh hưởng của glucose, maltose và bột ngô đến khả năng sản xuất acarbose (thông qua hoạt tính ức chế a-glucosidase). Kết quả đã cho thấy, hai yếu tố glucose và maltose có ý nghĩa quan trọng và ảnh hưởng trực tiếp đến khả năng sinh tổng hợp acarbose. Một phương trình đã được hình thành từ kết quả tối ưu. Bên cạnh đó, chúng tôi đã chứng minh được cột sắc ký sử dụng than hoạt tính có thể tinh sạch acarbose từ dịch lên men. Hàm lượng acarbose trong dung dịch tinh sạch đạt 191,5 g/l và một quy trình tinh sạch acarbose được đề xuất.

Les maladies neurodégénératives d’Alzheimer et de Parkinson sont caractérisées par des agrégats des peptides des amyloïdes-beta (Aβ) et l’alpha synucléine (αSyn), respectivement. L’agrégation de ces peptides solubles donne les matériels insolubles (les amyloïdes) sous forme de fibrilles et de plaques. L’agrégation de ces amyloïdes insolubles cause de la toxicité cellulaire dans le cerveau provoquant les effets neurodégénératifs. Les D, L-α-cyclopeptides synthétiques (e. g., CP-2, c-[leu-Nle-trp-His-ser-Lys]) furent synthétisés précédemment par le groupe de Rahimipour. Ils sont capables de former des nanotubes par pont hydrogène capables d’inhiber l’activité néfaste des amyloïdes. Les azapeptides utilisent de semicarbazide comme un substituant d’amide aminé. Insérés dans le D, L-α-cyclopeptide CP-2, les aza-résidus permettent d’améliorer la basicité de Lewis et l’acidité de Bronsted du peptide pour obtenir des ponts hydrogènes plus forts. Les azapeptides [azaNle3]-CP-2, [azaHse6]-CP-2 et [azaGly6]-CP-2 offrent d’excellents résultats au niveau des tests biologiques pour inhiber l’activité des amyloïdes ainsi que pour réduire la mortalité cellulaire en présence d’amyloïdes. À partir de ces résultats prometteurs, l’insertion de deux azapeptides dans un D, L-α-cyclopeptide a été effectué. En remplaçant l’histidine par l’alanine pour éviter des problèmes d’épimérisation, quatre nouveaux diazacyclopeptides ont été synthétisés en insérant les aza-résidus : l’aza-norleucine (azaNle), l’aza-homoserine (azaHse) et l’azaglycine (azaGly). Leur synthèse ainsi que leur caractérisation biophysique préliminaire seront présentés. / Alzheimer's and Parkinson's diseases are neurodegenerative diseases characterized by the aggregation of the peptides amyloid-beta (Aβ) and alpha-synuclein (Synα), respectively. The aggregation of these soluble peptides gives insoluble peptides (amyloids) in the forms of fibrils and plaques. Aggregation of insoluble amyloids can cause cellular toxicity in the brain resulting in neurodegenerative effects. Certain synthetic cyclic D,L-alpha-peptides (e.g., CP-2, c-[leu-Nle-trp-His-ser-Lys]) were previously synthesized by the Rahimipour laboratory and shown to form intermolecular hydrogen bonds and self-assemble into nanotubes capable of inhibiting the aggregation and harmful effects of amyloids. Azapeptides employ semicarbazides as amino amide surrogates. Insertion of aza-residues into the D,L-α-cyclopeptide CP-2 was explored to alter the Lewis basicity and Bronsted acidity of the peptide and improve hydrogen bonding potential. The azapeptides [azaNle3]-CP-2, [azaHse6]-CP-2, and [azaGly6]-CP-2 inhibited effectively amyloid aggregation and reduced amyloid-induced cell death. Based on the promising results of single aza-residue substitutions, the insertion of two aza-residues into D,L-α- cyclopeptide CP-2 analogs was investigated. Histidine was replaced with alanine to minimize epimerization. Four new diazacyclopeptides were synthesized by inserting aza-norleucine (azaNle), aza-homoserine (azaHse) and aza-glycine (azaGly). The syntheses and preliminary biophysical characterizations of the diazacyclopeptides will be presented.

Les expériences suivantes testent l’hypothèse que la sérine/thréonine kinase p38α/β MAPK inhibe la rentrée dans le cycle cellulaire des cardiomyocytes ventriculaires de rats nouveau-nés (CVRNs), et induit l’expression d’un panel de cytokines/chimiokines inflammatoires. Le traitement des CVRNs par le phorbol 12,13-butyrate (PDBu), activateur de la protéine kinase C (PKC), aboutit au recrutement de l’isoforme conventionnelle (PKC-α) et des isoformes nouvelles (PKC-δ et PKC-ε) de PKC en l’absence de la rentrée dans le cycle cellulaire. Cette absence d’entrée dans le cycle cellulaire à la suite du traitement par PDBu est associée à une augmentation d’expression des ARNm des gènes qui bloquent la rentrée dans le cycle cellulaire. Les gènes comprennent Runx1(Runt-related transcription factor 1) et CDKN2a (cyclin-dependent kinase inhibitor 2A) également connu sous le nom de p16, inhibiteur du cycle cellulaire. En présence de l’inhibiteur de p38α/β MAPK, SB203580, le traitement PDBu induit une entrée dans le cycle cellulaire de deux populations distinctes de cardiomyocytes caractérisées par l’absence ou l’expression de novo de la protéine filamenteuse Nestine. En parallèle, le co-traitement PDBu/SB203580 atténue l’augmentation du niveau d’expression de l’ARNm de Runx1 et CDKN2a. L’inhibition pharmacologique du recrutement de PKC-α par GF109203X, inhibe sélectivement la rentrée dans le cycle cellulaire des CVRNs qui présentent une expression de novo de Nestine. En parallèle, le traitement par PDBu augmente le niveau d’ARNm d’un panel de cytokines inflammatoires et la co-administration de SB203580 inhibe cette réponse. Ces données révèlent que le cœur des rats nouveau-nés contient deux sous-populations distinctes de cardiomyocytes ventriculaires qui rentrent dans le cycle cellulaire à la suite d’un co-traitement PDBu / SB203580, et que la réponse proliférative est associée à une diminution des cytokines inflammatoires. Collectivement, ces résultats mettent en relief une nouvelle prémisse selon laquelle le recrutement de p38α/β MAPK médié par PKC-α joue un rôle central dans l’inhibition de l’entrée dans le cycle cellulaire et induit une réponse inflammatoire robuste par les CRVNs. / The following experiments test the hypothesis that the serine/threonine kinase p38α/β MAPK inhibits the cell cycle re-entry of neonatal rat ventricular cardiomyocytes (NNVMs) and induces the expression of a panel of inflammatory cytokines/chemokines. Treatment of NNVMs with phorbol 12,13-butyrate (PDBu), an activator of protein kinase C (PKC), results in the recruitment of the conventional isoform (PKC-α) and novel isoforms (PKC-δ and PKC-ε) of PKC in the absence of cell cycle re-entry. This lack of cell cycle re-entry following PDBu treatment is associated with an increase in the expression of mRNA of genes that inhibit cell cycle re-entry. These genes include Runx1 (Runt-related transcription factor 1) and CDKN2a (cyclin-dependent kinase inhibitor 2A), also known as p16, a cell cycle inhibitor. In the presence of the p38α/β MAPK inhibitor, SB203580, PDBu treatment induces cell cycle re-entry in two distinct populations of cardiomyocytes characterized by the absence or de novo expression of the filamentous protein Nestin. In parallel, co-treatment with PDBu/SB203580 attenuates the increase in Runx1 and CDKN2a mRNA levels. Pharmacological inhibition of PKC-α recruitment by GF109203X selectively inhibits cell cycle re-entry of NNVMs exhibiting de novo Nestin expression. Additionally, PDBu treatment increases the mRNA levels of a panel of inflammatory cytokines, and co-administration of SB203580 inhibits this response. These data reveal that the heart of neonatal rats contain two distinct subpopulations of ventricular cardiomyocytes that re-enter the cell cycle following PDBu/SB203580 co-treatment, and that the proliferative response is associated with a decrease in inflammatory cytokines. Collectively, these results highlight a novel premise whereby p38α/β MAPK recruitment mediated by PKC-α plays a central role in inhibiting cell cycle re-entry and induces a robust inflammatory response by NNVMs.

Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: The study of α clustering is a well-established topic of research in nuclear physics. Recent experimental evidence has revealed the first 2+ excitation of the Hoyle state in 12C, which is known to have a strong α cluster structure. The idea of multi-particle α cluster structures in light nuclear matter has received much attention from theoretical investigations of late. This research has profound implications in the fields of both nuclear structure and nuclear astrophysics. The 20Ne nucleus is a good example for α clustering, since many of its states are known to have α clustering structures. Few low spin states are known at high excitation energies of this nucleus. It is predicted that this nucleus contains a 0+ 5-α cluster state, a so-called ‘Hoyle analogue state’, above its 5-α break-up threshold at Ex = 19.17 MeV. This thesis presents a study of the 20Ne nucleus with the 22Ne(p,t)20Ne reaction at laboratory angles θlab =(0◦, 7◦, 16◦, 27◦). The iThemba LABS K600 magnetic spectrometer was employed with a beam of energy Elab = 60 MeV, incident upon a 22Ne gas target held intact by Aramid foils. The aim was to search for low spin states in 20Ne at excitation energies above Ex = 15 MeV, and, possibly, to find an indication of the 5-α cluster state. Three narrow states were discovered at energies of Ex = 20.59 MeV, Ex = 21.16 MeV and Ex = 21.80 MeV. Calculations performed with the isobaric multiplet mass equation indicate that these states may be T = 2 isobaric analogue states of three known states in 20O. However, shell-model calculations indicate that these states may also have T = 0 or T = 1 isospin values. There is also evidence of a new state at Ex = 17.67 MeV and, possibly, of a collection of new states which could not be resolved at Ex = 18.84 MeV. A tentative candidate for the desired 5-α cluster state was observed, but this will require another measurement with cleaner background to be confirmed. / AFRIKAANSE OPSOMMING: Alfa bondelvorming is ’n gevestigde navorsingsonderwerp in kernfisika. Daar is onlangse bewyse vir die ontdekking van die 2+ opwekking van die Hoyle toestand in 12C, wat ’n erkende alfa bondelstruktuur het. Die idee van multi-alfa bondelstrukture in ligte kerne het onlangs baie aandag geniet in teoretiese ondersoeke. Hierdie navorsing het besondere implikasies vir kernstruktuur, sowel as vir kernastrofisika. Die 20Ne kern bied ‘n ideale voorbeeld vir alfa bondelvorming aangesien dit bekend is dat baie van die kern se toestande alfa bondelstrukture het. Min lae-spin toestande is by ho¨e opwekenergie¨e bekend in hierdie kern. Daar word voorspel dat ’n 0+ 5-alfa bondeltoestand, ’n sogenaamde ‘Hoyle analoogtoestand’, bo die drumpel vir 5-alfa verval by Ex = 19.17 MeV bestaan. Hierdie tesis beskryf ‘n studie van die 20Ne kern met die 22Ne(p,t)20Ne reaksie by laboratorium hoeke van θlab =(0◦, 7◦, 16◦, 27◦). Die K600 magnetiese spektrometer van iThemba LABS is gebruik met ‘n proton bundel, by ‘n energie van Elab = 60 MeV, wat op ‘n 22Ne gas teiken omhul met aramid foelies gerig is. Die doel was om lae-spin toestande in 20Ne by opwekenergie¨e bo Ex = 15 MeV op te spoor, en om moontlik ook ‘n aanduiding van die 5-alfa bondeltoestand te vind. Drie smal toestande is by energie¨e van Ex = 20.59 MeV, Ex = 21.16 MeV en Ex = 21.80 MeV opgespoor. Berekeninge wat met die isobariese multiplet massa vergelyking uitgevoer is, dui daarop dat hierdie toestande T = 2 isobariese analoogtoestande van drie bekende toestande in 20O kan wees, hoewel skilmodel berekeninge ook T = 0 en T = 1 kandidate aandui. Daar is ook bewyse van ’n nuwe toestand by Ex = 17.67 MeV, en moontlik van ’n versameling nuwe toestande rondom Ex = 18.84 MeV wat nie uitmekaar geken kon word nie. ‘n Tentatiewe kandidaat vir die gesogte 5-alfa bondel toestand is waargeneem, maar nog ‘n meting met ’n skoner agtergrond word vir bevestiging benodig.

The use of enzymes as catalysts for chemical transformations has emerged as a “greener” alternative to traditional organic synthesis. An issue to solve though, is that enzymes are designed by nature to catalyze reactions in a living cell and therefore, in many cases, do not meet the requirements of a suitable biocatalyst. By mimicking Darwinian evolution these problems can be addressed in vitro by different types of directed evolution strategies. α-Hydroxy aldehydes and α-hydroxy ketones are important building blocks in the synthesis of natural products, fine chemicals and pharmaceuticals. In this thesis, two alcohol dehydrogenases, FucO and ADH-A, have been studied. Their potentials to serve as useful biocatalysts for the production of these classes of molecules have been investigated, and shown to be good. FucO for its strict regiospecificity towards primary alcohols and that it strongly prefers the S-enantiomer of diol substrates. ADH-A for its regiospecificity towards secondary alcohols, its enantioselectivity and that is has the ability to use a wide variety of bulky substrates. The kinetic mechanisms of these enzymes were investigated using pre-steady state kinetics, product inhibition, kinetic isotope effects and solvent viscosity effects, and in both cases, the rate limiting steps were pin-pointed to conformational changes occurring at the enzyme-nucleotide complex state. These characterizations provide an important foundation for further studies on these two enzymes.   FucO is specialized for activity with small aliphatic substrates but is virtually inactive with aryl-substituted compounds. By the use of iterative saturation mutagenesis, FucO was re-engineered and several enzyme variants active with S-3-phenylpropane-1,2-diol and phenylacetaldehyde were obtained. It was shown that these variants capability to act on larger substrates are mainly due to an enlargement of the active site cavity. Furthermore, several amino acids which are important for catalysis and specificity were identified. Phe254 interacts with aryl-substituted substrates through π-π stacking and may be essential for activity with these larger substrates. One mutation caused a loss in the interactions made between the enzyme and the nucleotide and thereby enhanced the turnover number for the preferred substrate

No description available.

Edible potatoes originated in the Andes and were brought to Europe in the 16th century. Their introduction spurred both the European population growth and economic development. Being the world's fourth-largest food crop, potatoes continue to shape the global economy and world history. Glycoalkaloids are natural insect deterrents generated by potatoes, and are known for their toxic effects as well as potential medicinal utilities. Demissidine, the aglycone of the primary glycoalkaloids, represents one major Solanum alkaloid. Its unique indolizidine framework presents a challenging synthetic target in organic chemistry. Our synthesis of demissidine starts from readily available epiandrosterone and takes advantage of a Lewis acid-mediated fragmentation of a γ-silyloxy-β-hydroxy-α-diazoester; the D-ring of a diazo ester derivative of epiandrosterone was efficiently ruptured to provide an aldehyde tethered ynoate product. In combination with a subsequent azomethine ylide 1,3-dipolar cycloaddition and a transition metal catalyzed oxidation/reduction, the core indolizidine framework of demissidine was successfully prepared in a stereoselective manner. In addition, the syntheses of two amino acids, 5-methylenepipecolic acid and (5S)-5-methylpipecolic acid were explored; they are used for the installation of the α-oriented C25 methyl group on demissidine. The successful preparation of demissidine was supported by NMR analysis of the synthetic compound in comparison with a natural sample. As an efficient and stereoselective synthesis, our efforts toward demissidine illuminate a strategy to indolizidine frameworks that could be applied in the preparation of other polycyclic amine natural products.