Wu, Ting. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 56-70). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.Ill / ABSTRACT --- p.V / TABLE OF CONTENTS --- p.IX / LIST OF ABBREVIATIONS --- p.XII / Chapter CHAPTER 1. --- LITERATURE REVIEW --- p.1 / Chapter 1.1 --- THE DISEASE OF MALARIA --- p.1 / Chapter 1.1.1 --- Pathogenesis --- p.2 / Chapter 1.1.2 --- The treatment of malaria --- p.4 / Chapter 1.2 --- THE PLANT OF ARTEMISIA ANNUA L --- p.5 / Chapter 1.2.1. --- Horticulture --- p.5 / Chapter 1.2.2. --- Historical Importance --- p.6 / Chapter 1.3 --- ARTEMISININ --- p.7 / Chapter 1.3.1 --- The content and distribution of artemisinin --- p.7 / Chapter 1.3.2 --- The biosynthesis of artemisnin --- p.8 / Chapter 1.4 --- TRICHOMES --- p.13 / Chapter 1.4.1 --- Structure and function of trichomes --- p.13 / Chapter 1.4.2 --- Trichome investigation in A. annua --- p.14 / Chapter 1.5 --- PROTEOMICS --- p.17 / Chapter 1.5.1 --- The basic principle of proteomics --- p.17 / Chapter 1.5.2 --- Two-dimensional gel electrophoresis --- p.18 / Chapter 1.5.3 --- Mass Spectrometry --- p.19 / Chapter 1.5.4 --- Gel-free proteomics --- p.20 / Chapter 1.6 --- OBJECTIVES --- p.21 / Chapter CHAPTER 2. --- MATERIALS AND METHODS --- p.23 / Chapter 2.1 --- CHEMICALS --- p.23 / Chapter 2.2 --- PLANT MATERIALS --- p.23 / Chapter 2.3 --- ISOLATION OF GLANDULAR TRICHOMES --- p.23 / Chapter 2.4 --- PROTEIN EXTRACTION . --- p.25 / Chapter 2.5 --- Two DIMENSIONAL GEL ELECTROPHORESIS --- p.25 / Chapter 2.6 --- IMAGINE ANALYSIS --- p.26 / Chapter 2.7 --- IN GEL DIGESTION AND PROTEIN IDENTIFICAIOTN BY MASS SPECTROMETRY --- p.27 / Chapter CHAPTER 3. --- RESULTS AND DISCUSSION --- p.29 / Chapter 3.1 --- THE ISOLATION OF GLANDULAR TRICHOMES --- p.29 / Chapter 3.2 --- 2DE PATTERNS OF A. ANNUA LEAVE TRICHOMES AND LEAF TISSUE --- p.32 / Chapter 3.3 --- IDENTIFICATION OF PROTEINS IN GLANDULAR TRICHOMES --- p.34 / Chapter 3.3.1 --- Protein involved in electron transport chain --- p.47 / Chapter 3.3.2 --- Protiens invovled in metabolism --- p.48 / Chapter 3.3.2.1 --- artemisinin biosynthesis --- p.48 / Chapter 3.3.2.2 --- glycolysis --- p.49 / Chapter 3.3.2.3 --- other metabolic enzymes --- p.50 / Chapter 3.3.3 --- Proteins involved in transcription and translation --- p.51 / Chapter 3.3.4 --- Protein involved in proteolysis --- p.51 / Chapter 3.3.5 --- "Detoxificaiton, stress related protein" --- p.52 / Chapter 3.4 --- PERSPECTIVE --- p.53 / Chapter 3.5 --- CONCLUSION --- p.53 / REFERENCES --- p.56

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Artemisinin compounds are the most potent anti-malarial drugs available in the market. Today, malaria treatment is largely relies on the artemisinin-based combination therapies. Artesunate (AS) is the most widely used artemisinin derivative. In this thesis, we characterized the population pharmacokinetics of AS and its active metabolite dihydroartemisinin (DHA) following oral administration of AS in different populations. In Chapter II, we developed a population pharmacokinetic model of AS and DHA in healthy subjects. These subjects received either single- or multiple-dosing of oral AS, as a monotherapy regimen or in combination with pyronaridine, with or without food. In Chapter III, we developed a population pharmacokinetic model of AS and DHA in adult and pediatric patients with uncomplicated falciparum and vivax malaria who were administered oral pyronaridine/artesunate combination once daily for 3 days. We modeled the AS and DHA data simultaneously using a parent-metabolite model that assumed complete conversion of AS to DHA. Following oral administration, AS is rapidly absorbed with maximum concentrations reached at about 0.5 hours post-dose. AS is rapidly converted to DHA. DHA then undergoes rapid metabolism, with an elimination half-life of about 0.8 hours in malarial patients. Inter-individual variability for almost all pharmacokinetic parameters and residual variability for both compounds were estimated by the models. Substantial variability was seen in the pharmacokinetic parameters between the subjects. In healthy subjects, intake of food with the dose was found to delay the absorption of AS significantly, but not the extent of absorption. Weight was also included in this model as a determinant of DHA clearance. When modeling the data from patients, we included weight as part of the model a prioria priori using an established allometric function. No other covariates examined in the analysis were statistically significant. The performance of final models was evaluated using non-parametric bootstrap technique and visual predictive check. The models were found to adequately described the data at hand, and robust with sufficient predictive power. The results can be used as the base to develop a population pharmacokinetic-pharmacodynamic model and as prior information in guiding the selection of optimal sampling schedule for future pharmacokinetic studies of AS.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2005. / Härtill 4 uppsatser.

La pompe ATPase Ca2+ du réticulum sarco-endoplasmique Plasmodium falciparum (PfATP6) est une protéine de dix transmembranes, impliqué dans la régulation de l'homéostasie du calcium dans le parasite. L'importance d'étudier cette protéine repose sur l'hypothèse d'être engagé dans le mécanisme d'action et de résistance des artémisinines. Des travaux précédents, fondé sur l'expression hétérologue dans des ovocytes de Xenopus laevis et Saccharomyces cerevisiae, ont montré des résultats opposés, générant de nombreux corollaires vérifiables. Par conséquent, des travaux supplémentaires sont nécessaires pour mieux comprendre la nature des interactions entre les artémisinines et transporteurs de type SERCA.Afin d'évaluer le caractère essentiel du gène de Plasmodium spp., une approche de génétique inverse a été utilisée. Knockout du gène, soit P. falciparum et berghei, ne pouvant pas être obtenu. La complémentation de sauvetage épisomique a été jugée impossible. Marquage à la fin 3' de PfATP6 et PbATP6 a été, également, tenté pour étudier la localisation et l'expression de la protéine chez les parasites. La manipulation des gènes à cette place n'a pas permis la survie du parasite. Nos résultats, pris ensemble, montrent que ATP6 est essentiel dans Plasmodium spp..Au cours de nos études génétiques, un phénotype stable et particulier de parasites du genre Plasmodium falciparum 3D7 a été distingué. Les étranges parasites “monstres" contiennent une vacuole digestive inhabituelle gonflées à travers toutes les étapes du développement du parasite. Caractérisation de l'insolite Plasmodium a été réalisée, montrant une sensibilité accrue à la chloroquine, mais pas à l'artémisinine ou de la méfloquine. Tenant compte de la similitude du PfATP6 avec la pompe SERCA orthologue mammifère, de nouvelles molécules, connu et synthétisé pour cibler spécifiquement la protéine chez les mammifères, ont été testés sur P. falciparum. Quatre classes différentes de composés (sHA 14-1, BHQ, chalcone et des analogues de l'ACP) a montré le blocage de la croissance in vitro du P. falciparum 3D7 et Dd2 à des concentrations inférieure au range micromolaire. En outre, une nouvelle classe de molécules (thaperoxides), conçu comme un hybride entre l'artémisinine et thapsigargine, a été testé contre le type sauvage 7G8 et la ligne muté L263E. Ce dernier porte une mutation ponctuelle unique de nucléotides dans PfATP6, déjà connu d'être impliqué dans la résistance du l'artémisinine.Compte tenu de la difficulté à manipuler les gènes du parasite, et afin de mieux caractériser PfATP6, un gène synthétique a été optimisé pour l'expression hétérologue chez S. cerevisiae. De cette façon, la complémentation d'une ligne de levure mutée (K616) sans les pompes endogènes Ca2+ de type P a été permis avec succès, montrant le sauvetage de la croissance de la levure en présence de forte concentration de calcium libre. Différents inhibiteurs de SERCA, comme la thapsigargine et l'acide cyclopiazonique, ont été testés sur la levure complémenté K616 PfATP6, afin de vérifier l'inhibition de la croissance. Tous les composés ont bloqué la croissance de levure sélectivement ciblant le PfATP6. En outre, le test a été développé comme un criblage de haute performance, afin de tester de nouvelles molécules pour leur activité. La méthode s'est révélée être un outil rapide et très fiable et reproductible pour l'identification de nouveaux composés actifs. / The Plasmodium falciparum sarco-endoplasmic reticulum ATPase Ca2+ pump (PfATP6) is a ten transmembrane protein involved in the regulation of the calcium homeostasis in the parasite. The importance of studying this protein relies on the fact that it has been hypothesized to be involved in the mechanism of action and resistance of artemisinins. Previous works, based on heterologous expression in Xenopus laevis oocytes and Saccharomyces cerevisiae, have shown contrasting results, generating many testable corollaries. Therefore, further work is needed to better understand the nature of interactions between artemisinins and SERCA-type transporters.In order to assess the essentiality of the gene in Plasmodium spp., a reverse genetics approach has been used. Knockout of the gene, in either P. falciparum and berghei, could not be achieved. Complementation for episomal rescue was found to be not possible. Tagging at the 3' end of PfATP6 and PbATP6 has been, also, attempted to study localization and expression of the protein in parasites. Manipulation of the gene at this position did not permit parasite survival. Our results, taken together, show that ATP6 is essential in Plasmodium spp..During our genetic studies, a stable and peculiar phenotype of Plasmodium falciparum 3D7 parasites has been noticed. The odd “monster” parasites contain an unusual swollen food vacuole throughout all stages of parasite development. Characterization of the unusual Plasmodium has been carried out, showing an increased sensitivity to chloroquine, but not to artemisinin or mefloquine. Taking into account the similarity of PfATP6 with the mammalian orthologue SERCA pump, new molecules, designed and synthesized to specifically target the mammalian protein, were tested on P. falciparum parasites. Four different classes of compounds (sHA 14-1, BHQ, chalcone and CPA analogues) showed to inhibit P. falciparum 3D7 and Dd2 growth in vitro at concentrations in the lower micromolar range. In addition, a novel class of molecules (thaperoxides), designed as an hybrid between artemisinin and thapsigargin, has been tested against 7G8 wild type and mutated L263E line. The latter carries a single nucleotide point mutation in PfATP6 that has been previously shown to be involved in artemisinin resistance. Considering the difficulty in manipulating the gene in the parasite and in order to better characterize PfATP6, a synthetic gene was optimized for heterologous expression in S. cerevisiae. This enabled successful complementation of a mutated yeast line (K616) lacking the endogenous P-type Ca2+ pumps, showing rescue of the yeast growth in presence of high concentration of free calcium. Different SERCA inhibitors, such as thapsigargin and cyclopiazonic acid, have been tested on K616 PfATP6 complemented yeast, in order to check for growth inhibition. All compounds showed to inhibit yeast growth selectively targeting PfATP6. In addition, the assay has been developed as a high throughput screening, in order to test new molecules for their activity. The method has proved to be a fast, highly reliable and reproducible tool for identification of new active compounds.

Qi, Yan. / Thesis submitted in: December 2008. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 63-79). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.III / ABSTRACT --- p.IV / TABLE OF CONTENTS --- p.VII / LIST OF ABBREVIATIONS --- p.XI / Chapter CHAPTER 1. --- LITERATURE REVIEW --- p.1 / Chapter 1.1 --- the Plant of Artemisia annua L --- p.1 / Chapter 1.2 --- The disease of malaria --- p.3 / Chapter 1.2.1 --- The life cycle of Plasmodium parasites --- p.4 / Chapter 1.2.2 --- The Artemisinin-based combination therapies (ACTs) for the treatment of malaria --- p.5 / Chapter 1.3 --- Artemisinin --- p.8 / Chapter 1.3.1 --- The content and distribution of artemisinin --- p.8 / Chapter 1.3.2 --- The mechanism of artemisinin action --- p.9 / Chapter 1.3.2.1 --- The proposed non-specific mechanisms of action --- p.10 / Chapter 1.3.2.2 --- The proposed parasite-specific mechanisms of action --- p.11 / Chapter 1.3.3 --- The biosynthesis of artemisnin in vivo --- p.12 / Chapter 1.3.4 --- The biosynthesis of artemisinin in vitro --- p.16 / Chapter 1.4 --- Trichomes --- p.18 / Chapter 1.4.1 --- Non-glandular trichomes --- p.19 / Chapter 1.4.2 --- Glandular trichome --- p.20 / Chapter 1.4.3 --- Trichomes of Artemisia annua L --- p.21 / Chapter 1.5 --- DNA Sequencing Methods --- p.24 / Chapter 1.5.1 --- The basic principle of pyrosequencing --- p.25 / Chapter 1.5.2 --- 454 pyrosequencing and its application --- p.27 / Chapter CHAPTER 2. --- MATERIALS AND METHODS --- p.32 / Chapter 2.1 --- Chemicals --- p.32 / Chapter 2.2 --- Plant materials --- p.32 / Chapter 2.3 --- Preparation of the cDNA sample for 454 sequencing --- p.33 / Chapter 2.3.1 --- Scanning electron microscopy --- p.33 / Chapter 2.3.2 --- Isolation of glandular trichomes --- p.34 / Chapter 2.3.3 --- cDNA synthesis and normalization --- p.34 / Chapter 2.4 --- 454-EST SEQUENCING AND PROCESSING --- p.36 / Chapter 2.5 --- Analysis of 454 sequencing data --- p.37 / Chapter 2.6 --- Establishment of regeneration system of A. annua L --- p.37 / Chapter 2.6.1 --- Shoots induction from leaf discs --- p.37 / Chapter 2.6.2 --- The sensitivity of the explants to Kanamycin --- p.38 / Chapter 2.6.3 --- Rooting of the regenerated seedlings --- p.38 / Chapter CHAPTER 3. --- RESULTS AND DISCUSSION --- p.40 / Chapter 3.1 --- Glandular trichome isolation and cDNA preparation --- p.40 / Chapter 3.1.1 --- The distribution of glandular trichomes on A. annua --- p.40 / Chapter 3.1.2 --- The isolation of glandular trichomes --- p.42 / Chapter 3.1.3 --- The preparation of ds cDNA for 454 sequencing --- p.43 / Chapter 3.2 --- Pre-process of 454 pyrosequencing data --- p.44 / Chapter 3.3 --- Functional annotation of the 454-EST data --- p.47 / Chapter 3.4 --- Comparison of two sequencing runs --- p.49 / Chapter 3.5 --- Analysis of the 454 ESTs involved in secondary metabolisms --- p.50 / Chapter 3.6 --- Selection of the candidate genes --- p.55 / Chapter 3.7 --- Establishment of regeneration system of A. annua L --- p.57 / Chapter 3.7.1 --- Shoots induction from leaf discs --- p.57 / Chapter 3.7.2 --- Roots induction from shoots --- p.57 / Chapter 3.7.3 --- Sensitivity of A. annua to Kan --- p.59 / Chapter CHAPTER 4. --- CONCLUSION --- p.61 / REFERENCES --- p.63

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2009. / Härtill 6 uppsatser.

Thesis (M.S.P.H.)--University of South Florida, 2009. / Title from PDF of title page. Document formatted into pages; contains 57 pages. Includes bibliographical references.

Ambient mass spectrometry (MS) is a new and growing sub-field in MS which has opened new research avenues, particularly for applications relating to the analysis of solid samples. Results on the implementation and application of ambient MS techniques including: desorption electrospray ionization (DESI) and direct analysis in real time (DART) indicated that these techniques could serve as complementary tools for the rapid qualitative screening of pharmaceuticals, allowing up to two orders of magnitude improvement in throughput compared to traditional methods such as liquid chromatography MS. The selectivity of DESI could be enhanced by performing the experiment in the reactive mode. In this mode, complexation reactions between reagents added to the spray solvent and analytes on the sample surface resulted in analyte stabilization, inhibiting fragmentation. They also resulted in a concomitant enhancement in the analyte surface activity, facilitating their evaporation from secondary droplets culminating in an improvement in sensitivity. Also for drug tablets analysis, the analyte signal dependency on DESI geometrical set-up variables could be mitigated following the careful and controlled addition of an isotopically labeled internal standard (IS) to the sample or by spraying samples with a pair of reagents with different affinities for the analyte. Either of these approaches resulted in an analyte-to-IS signal ratio (in the former) or an analyte complex ratio (in the later), which was largely independent of DESI experimental variables allowing quantitative analysis using this technique. DESI MS was also observed to be a very powerful tool for determining the 2-D distribution of various pharmaceutically important compounds on tablet and tissue surfaces. The ability to map the distribution of molecules of interest by DESI MS has very great implications in drug tablet quality control and in determining the role of chemical signals presented on tissue surfaces. DESI was observed to be limited to ionizing molecules of medium to high polarities without much limitation in terms of mass range, whereas DART was better suited for the analysis of molecules within a broader range of polarities, but within a more limited mass range (up to 800 Da approximately). These limitations were circumvented by implementing a novel multimode ambient ion source, desorption electrospray/metastable-induced ionization (DEMI), which combines various aspects of DESI and DART. Initial experiments with the DEMI ion source demonstrated its ability to enable the simultaneous analysis of molecules within a broader range of polarities and masses than DESI and DART alone.

Thesis (M.S.P.H.)--University of South Florida, 2009. / Title from PDF of title page. Document formatted into pages; contains 114 pages. Includes bibliographical references.