Qian, Wei. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 184-199). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Thèse de doctorat--Sciences naturelles--Faculté des sciences de Paris, 1909. N°: 1346. / Notes bibliogr.

Thesis (doctoral)--Universität Bern, 1913. / Includes bibliographical references.

No description available.

Master of Science / Department of Biology / Kathrin Schrick / Steryl glucosides (SGs) are a common conjugate of sterols found in the plasma membranes of most plants and fungi, but their cellular functions remain largely unknown. Glycosylation of the C3 hydroxyl group of the sterol nucleus is catalyzed by UDP-glucose:steryl glucosyltransferase 80 (UGT80) enzymes. Two genes encoding UGT80A2 and UGT80B1 are responsible for most SG production in Arabidopsis thaliana, while UGT80C1 presents a putative third enzyme. In Arabidopsis, seed imbibition signals the epidermal seed coat cells to secrete an encapsulating mucilage that consists primarily of hydrated polysaccharides. Cellulose has been identified in the inner layer of the mucilage, providing a convenient model to study cellulose synthesis since seed mucilage is dispensable for viability and pectin and cellulose staining dyes are readily available. A reverse genetics and biochemical approach was used to characterize the role of UGT80 enzymes and their impact on cellulose synthesis in seed mucilage. ugt80B1 mucilage was found to have elongated cellulosic rays, but no defects in pectin synthesis. A double mutant of ugt80B1 and mum3-1, a mutant allele of CELLULOSE SYNTHASE 5 (CESA5), displays a novel phenotype with irregular cellulose patterning and extreme shedding of the pectinaceous layer surrounding the seed coat. The observed mucilage defects may be indicative of disrupted cellulose synthesis and a mechanistic relationship between SGs and the cellulose synthase machinery. UGT80A2 and B1 demonstrate glycosylation activity with a multitude of plant sterols. The two enzymes do display some substrate specificity, however, with UGT80A2 producing the large majority of sitosterol and stigmasterol glucoside compared to B1. UGT80C1 shows little or no sterol glucosyltransferase activity in vitro or in vivo and likely has evolved a different function from the two other genes. GFP:UGT80C1 expressed either from the constitutive 35S or from its native promoter was localized to lipid droplets and possibly chloroplasts as well, creating a new perspective on the role of the protein in plant lipid metabolism. This study extends the currently limited view of SGs as ubiquitous components of the plasma membrane to active regulators of cellulose synthesis in seeds. Evidence presented here changes the perceived role of the plant conserved protein, UGT80C1, from a putative steryl glucosyltransferase enzyme to having a function in intracellular lipid droplets.

L’efficacité de certains agents anti-cancéreux, notamment les anthracyclines et l’oxaliplatine repose sur l'induction d’une mort cellulaire immunogène (MCI) pouvant conduire à une réponse immunitaire anti-tumorale spécifique. Les cellules succombant à ce type particulier d'apoptose vont subir certaines modifications définies par un modèle spatio-temporel précis. Celui-ci est caractérisé par la mise en place de signaux d’apparition séquentielle, dont le plus précoce est l’exposition membranaire d’une protéine du réticulum endoplasmique, la calréticuline (CRT) qui constitue un signal de danger essentiel à la phagocytose des cellules mourantes par les cellules dendritiques. Ensuite, à un stade apoptotique, la sécrétion d’adénosine triphosphate (ATP) dépendante de l'autophagie active l’inflammasome NLRP3 et induit la polarisation des cellules T CD8+ productrices d’IFN-. Enfin, au cours de la nécrose secondaire, le relargage d’un facteur pro-immunogène High-mobility group protein B1 (HMGB1) est indispensable à une présentation antigénique optimale aux cellules T CD4+ et CD8+, contribuant ainsi à l’activité tumoricide de la chimiothérapie et protégeant l’hôte d’une éventuelle rechute. De manière à identifier de nouvelles molécules capables d’induire une réponse immunitaire anti-tumorale spécifique, un criblage à haut débit de bibliothèques de composés approuvés par la FDA (Food and Drug Administration) a été réalisé grâce à l’utilisation de microscopie automatisée et de biosenseurs permettant la détection de l'exposition de la CRT, de la sécrétion d'ATP et du relargage d'HMGB1. Ce criblage multiparamétrique à haut débit a permis d’identifier les glucosides cardiotoniques (GCs), déjà bien connus pour leur activité cytotoxique préférentielle des cellules cancéreuses, comme étant des inducteurs efficaces de la MCI. Cette découverte a été validée par des méthodes alternatives in vitro, suivis d’une étude de la mécanistique d’induction de la MCI par les GCs. Les résultats ont mis en évidence une inhibition spécifique de la sous-unité α1 de la pompe Na + / K + ATPase, qui à son tour modifie l'homéostasie calcique de la cellule cible, un effet reproduit par les ionophores du Ca2 +. Nous avons ensuite montré que les CGs, en combinaison avec des chimiothérapies non immunogènes (cisplatine ou mitomycine C) pouvaient vacciner des souris syngéniques contre une ré-injection de cellules cancéreuses vivantes et que les effets antinéoplastiques de ces agents endommageants l’ADN pouvaient être potentialisés par les GCs dans les hôtes immunocompétents mais pas dans les souris immunodéficientes. Enfin, une analyse rétrospective de patients atteints de carcinomes et traités par un GC couramment utilisé en clinique dans la prise en charge de l'insuffisance cardiaque, la digoxine (n=145) a révélé une amélioration significative de la survie globale par rapport à celle de patients non traités (n=290). Les patients ont été appariés en fonction de leur âge, sexe, type de cancer et principaux paramètres pronostiques. Des analyses plus approfondies ont ensuite révélées que la digoxine n’affectait pas la survie globale des patients déjà traités par des agents chimiothérapeutiques immunogènes mais celle des patients ayant reçu des agents autres que les anthracyclines ou l’oxaliplatine. / The efficacy of some anti-cancer agents, including anthracyclines and oxaliplatin is based on their capacity to induce immunogenic cell death (ICD) in tumor cells. This peculiar type of apoptosis is defined by a sequential emission of specific immunogenic signals from the dying tumor, which in their correct spatio-temporal appearance ignite a specific immune response against therapy resistant and dormant tumor (stem) cells. Thus the early membrane exposure of the ER-resident molecular chaperone calreticulin (CRT) constitutes a critical uptake signal for the engulfment of dying tumor cells by dendritic cells (DCs). Then, at later stages, the autophagy-dependent secretion of ATP and its binding to purinergic receptors on DCs activates the NLRP3 inflammasome. Subsequent release of IL-1 by DC triggers the polarization of IFN-γ producing CD8+ T cells. Finally, during secondary necrosis, the release of the pro-immunogenic high-mobility group box 1 (HMGB1) protein and its interaction with Toll like receptor 4 (TLR4) on DCs facilitates an optimal antigen presentation to T cells. Thus ICD contributes to the tumoricidal activity of chemotherapy and protecting the host from relapse. In order to identify thus far unknown inducers of ICD, a high content screening of compound libraries approved by the Food and Drug Administration (FDA) was conducted by means of robotized automated bioimaging combined with ICD biosensors allowing for the detection of CRT relocation, ATP secretion and HMGB1 release. This multiparametric approach led to the identification of cardiac glycosides (CGs), already well known for their preferential cytotoxic activity on cancer cells, as effective inducers of ICD. The hit compounds were validated by alternative methods in vitro, followed by a mechanistic study of GC induced ICD. Results indicated an on-target inhibition of the Na+/K+ ATPase subunit 1, which in turn interfered with the Ca2+-homeostasis of the target cell, an effect that could be mimicked by Ca2+ ionophores. We then showed in different mouse models that tumor cells killed with a combination of GC and non-immunogenic chemotherapy (cisplatin or mitomycin C) have the ability to immunize syngeneic mice against rechallenge with living cells. In addition the antineoplastic effects of these DNA damaging agents in vivo were increased by GCs in immunocompetent but not in immunodeficient mice. Finally, retrospective clinical analyses revealed that the administration of the GC digoxin during chemotherapy had a significant positive impact on overall survival in cohorts of breast, colorectal, head and neck, and hepatocellular carcinoma patients, especially when they were treated with agents other than anthracyclines and oxaliplatin.

This research is focused on the study of the effect of mutating proline 145 to threonine on the substrate and regiospecificity of flavonol specific 3-O-glucosyltransferase (Cp3GT). While the mutant P145T enzyme did not glucosylate anthocyanidins, it did glucosylate flavanones and flavones in addition to retaining activity with flavonols. HPLC was used for product identification and showed mutant P145T glucosylated naringenin at the 7-OH position forming naringenin-7-O-glucoside and flavonols at the 3-OH position. Homology modeling and docking was done to predict the acceptor substrate recognition pattern and models were validated by experimental results. In other related work, a thrombin cleavage site was inserted into wild type Cp3GT and recombinant P145T enzyme between the enzyme and the C-myc tags in order to be able to cleave off tags. This provides the tool needed for future efforts to crystallize these proteins for structural determination.

This study concerns the isolation, characterisation and physio-chemical properties of 8,8-methylmethine-(epi)catechin-malvidin-3-glucoside compounds found in red wines. 8,8-Methylmethine-(epi)catechin-malvidin-3-glucoside compounds were isolated via chromatographic methods developed in this study. The compounds were characterised via nuclear magnetic resonance spectrometry which, with the aid of molecular modelling, afforded their possible 3-dimensional structures. Their physio-chemical properties including ionisation and hydration constants, colour parameters and chemical stabilities were determined. The formation of 8,8-methylmethine-flavan-3-ol-malvidin-3-glucoside compounds and other pigments in wines was also studied. 8,8-Methylmethine-(epi)catechin-malvidin-3-glucoside compounds were also synthesised by condensing malvidin-3-glucoside with (epi)catechin in the presence of acetaldehyde. Diastereomers of 8,8-methylmethine-(epi)catechin-malvidin-3-glucoside pigments were isolated from the reaction using size-exclusion liquid chromatography followed by cation-exchange liquid chromatography. The structures of the four 8,8-methylmethine-catechin (and epicatechin)-malvidin-3-glucoside diastereomers were determined using mass spectrometry and one and two-dimensional nuclear magnetic resonance spectroscopy. It was found that for all four compounds, the methylmethine bridge occurs at the 8-positions of malvidin-3-glucoside and (epi)catechin and that the 3-dimensional structural differences between the diastereomers is the positioning of the (epi)catechin moiety with respect to the glucoside group. One diastereomer has the (epi)catechin on the same side, with respect to the malvidin entity whilst it is on the opposite side for the other diastereomer. The proposed structures also afforded the malvidin entity protection from nucleophilic attack via steric hindrance by the (epi)catechin moiety. 8,8-Methylmethine-(epi)catechin-malvidin-3-glucoside pigments have greater colour stability with regards to changes in pH and SO2 bleaching compared to malvidin-3-glucoside providing evidence that little or no hydration in aqueous solutions is occurring for these compounds. Further evidence for little or no hydration occurring is the presence of isosbestic points in the UV-vis spectra observed for the 8,8-methylmethine-(epi)catechin-malvidin-3-glucoside in the pH range 2 to 7. Although the 8,8-methylmethine-(epi)catechin-malvidin-3-glucoside pigments have greater colour stability to pH, SO2 and oxidation, compared to malvidin-3-glucoside, they have lower temporal stabilities and under both aerobic and anaerobic conditions they have significantly higher degradation rate constants than malvidin-3-glucoside. The ionisation constants of the 8,8-methylmethine-(epi)catechin-malvidin-3-glucoside compounds were determined using high voltage paper electrophoresis (HVPE) and UV-visible spectroscopy. The first ionisation constants (pKa1) of the 8,8-methylmethine-(epi)catechin-malvidin-3-glucoside compounds were found to be higher than that of malvidin-3-glucoside whereas the second and third ionisation constants (pKa2 and pKa3) were found to be lower. The correlation of the ionisation constants between HVPE and UV-visible spectroscopy supports the proposal that there is little or no occurrence of hydration for the 8,8-methylmethine-(epi)catechin-malvidin-3-glucoside compounds in the pH range investigated. 8,8-Methylmethine-flavan-3-ol-malvidin-3-glucoside compounds were the major pigments formed during fermentations of chemically defined grape juice media containing malvidin-3-glucoside and various flavan-3-ols. The yeast strain used for fermentation had a major influence on the levels and rates of formation of these pigments during fermentation. The yeast strain used also has an important influence on wine pigment composition, concentration and evolution during maturation thereby affecting the colour density and hue of the resultant wines. The initial formation of 8,8-methylmethine-flavan-3-ol-malvidin-3-glucoside compounds and their subsequent gradual degradation during maturation, allowed a pool of malvidin-3-glucoside to be available for the formation of other colour stable and more temporally stable pigments. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1339479 / Thesis (Ph.D.) - University of Adelaide, School of Agriculture, Food and Wine, 2008

L'efficacité de certains agents anti-cancéreux, notamment les anthracyclines et l'oxaliplatine repose sur l'induction d'une mort cellulaire immunogène (MCI) pouvant conduire à une réponse immunitaire anti-tumorale spécifique. Les cellules succombant à ce type particulier d'apoptose vont subir certaines modifications définies par un modèle spatio-temporel précis. Celui-ci est caractérisé par la mise en place de signaux d'apparition séquentielle, dont le plus précoce est l'exposition membranaire d'une protéine du réticulum endoplasmique, la calréticuline (CRT) qui constitue un signal de danger essentiel à la phagocytose des cellules mourantes par les cellules dendritiques. Ensuite, à un stade apoptotique, la sécrétion d'adénosine triphosphate (ATP) dépendante de l'autophagie active l'inflammasome NLRP3 et induit la polarisation des cellules T CD8+ productrices d'IFN-. Enfin, au cours de la nécrose secondaire, le relargage d'un facteur pro-immunogène High-mobility group protein B1 (HMGB1) est indispensable à une présentation antigénique optimale aux cellules T CD4+ et CD8+, contribuant ainsi à l'activité tumoricide de la chimiothérapie et protégeant l'hôte d'une éventuelle rechute. De manière à identifier de nouvelles molécules capables d'induire une réponse immunitaire anti-tumorale spécifique, un criblage à haut débit de bibliothèques de composés approuvés par la FDA (Food and Drug Administration) a été réalisé grâce à l'utilisation de microscopie automatisée et de biosenseurs permettant la détection de l'exposition de la CRT, de la sécrétion d'ATP et du relargage d'HMGB1. Ce criblage multiparamétrique à haut débit a permis d'identifier les glucosides cardiotoniques (GCs), déjà bien connus pour leur activité cytotoxique préférentielle des cellules cancéreuses, comme étant des inducteurs efficaces de la MCI. Cette découverte a été validée par des méthodes alternatives in vitro, suivis d'une étude de la mécanistique d'induction de la MCI par les GCs. Les résultats ont mis en évidence une inhibition spécifique de la sous-unité α1 de la pompe Na + / K + ATPase, qui à son tour modifie l'homéostasie calcique de la cellule cible, un effet reproduit par les ionophores du Ca2 +. Nous avons ensuite montré que les CGs, en combinaison avec des chimiothérapies non immunogènes (cisplatine ou mitomycine C) pouvaient vacciner des souris syngéniques contre une ré-injection de cellules cancéreuses vivantes et que les effets antinéoplastiques de ces agents endommageants l'ADN pouvaient être potentialisés par les GCs dans les hôtes immunocompétents mais pas dans les souris immunodéficientes. Enfin, une analyse rétrospective de patients atteints de carcinomes et traités par un GC couramment utilisé en clinique dans la prise en charge de l'insuffisance cardiaque, la digoxine (n=145) a révélé une amélioration significative de la survie globale par rapport à celle de patients non traités (n=290). Les patients ont été appariés en fonction de leur âge, sexe, type de cancer et principaux paramètres pronostiques. Des analyses plus approfondies ont ensuite révélées que la digoxine n'affectait pas la survie globale des patients déjà traités par des agents chimiothérapeutiques immunogènes mais celle des patients ayant reçu des agents autres que les anthracyclines ou l'oxaliplatine.

No description available.