Thesis (Ph.D.)--Virginia Commonwealth University, 2008. / Prepared for: Dept. of Medicinal Chemistry. Title from thesis description page.

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submitted by Chair Siu Fan. / Thesis (M.Sc.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 75-83). / Chapter 1 --- INTRODUCTION --- p.1 / Chapter 2 --- LITERATIRE REVIEW --- p.3 / Chapter 2.1 --- Causes and clinical syndromes in MPS --- p.3 / Chapter 2.1.1 --- MPS --- p.4 / Chapter 2.1.2 --- Classification of MPS --- p.4 / Chapter 2.1.2.1 --- MPS I (Hurler's syndrome) --- p.5 / Chapter 2.1.2.2 --- MPS IS (Scheie syndrome) --- p.5 / Chapter 2 .1.2.3 --- MPS II (Hunter's disease) --- p.6 / Chapter 2.1.2.4 --- MPS Type III (The Sanfilippo diseases) --- p.6 / Chapter 2.1.2.5 --- MPS Type IV (Morquio's disease) --- p.6 / Chapter 2.1.2.6 --- MPS Type VI (Maroteaux 一 Lamy syndrome) --- p.7 / Chapter 2.1.2.7 --- MPS Type VII (Sly syndrome) --- p.8 / Chapter 2.1.3 --- Treatment and prospects for MPS --- p.8 / Chapter 2.1.3.1 --- To manage the handicaps and disabilities --- p.8 / Chapter 2.1.3.2 --- Enzyme replacement --- p.9 / Chapter 2.1.3.3 --- Bone marrow transplantation --- p.10 / Chapter 2.2 --- Basic aspects of GAG --- p.10 / Chapter 2.2.1 --- Distributions of GAG --- p.12 / Chapter 2.2.2 --- Functions and Roles of GAG --- p.12 / Chapter 2.2.3 --- Stepwise degradation of GAGs --- p.12 / Chapter 2.2.4 --- Source of urinary GAG --- p.13 / Chapter 2.2.5 --- Common features of GAGS --- p.14 / Chapter 2.2.6 --- Factors affecting the excretion pattern ot GAG --- p.16 / Chapter 2.3 --- Methods for MPS Diagnosis --- p.16 / Chapter 2.3.1 --- Qualitative urine methods for screening and typing --- p.16 / Chapter 2.3.1.1 --- Spot tests --- p.16 / Chapter 2.3.1.2 --- Precipitation methods --- p.16 / Chapter 2.3.1.3 --- One-dimensional electrophoresis --- p.17 / Chapter 2.3.1.4 --- Two-dimensional electrophoresis --- p.17 / Chapter 2.3.1.5 --- Thin layer chromatography --- p.17 / Chapter 2.3.2 --- Quantitative methods for urinary GAG --- p.17 / Chapter 2.3.2.1 --- Measurement of hexuronic acid --- p.18 / Chapter 2.3.2.2 --- HPLC or Column chromatography --- p.18 / Chapter 2.3.2.3 --- Dye-binding methods --- p.19 / Chapter 2.3.3 --- Cytological studies --- p.19 / Chapter 2.3.4 --- Tissue culture --- p.20 / Chapter 2.3.5 --- Tissue biopsy --- p.20 / Chapter 2.3.6 --- Prenatal diagnosis of the MPS --- p.21 / Chapter 2.4 --- Bacterial GAG hydrolytic enzymes --- p.23 / Chapter 2.5 --- Summary of Literature Review --- p.25 / Chapter 3. --- AIMS OF STUDY --- p.26 / Chapter 4. --- MATERIALS AND METHODS --- p.26 / Chapter 4.1 --- Sample collection --- p.26 / Chapter 4.2 --- Materials &-Equipment --- p.26 / Chapter 4.3 --- Preparation of Reagents and Standards --- p.27 / Chapter 4.3.1 --- Stock DMB reagent solutions --- p.27 / Chapter 4.3.2 --- Working DMB solution --- p.27 / Chapter 4.3.3 --- GAG standards --- p.27 / Chapter 4.3.4 --- Reagents for electrophoresis --- p.27 / Chapter 4.3.4.1 --- 0.1M barium acetate solution --- p.27 / Chapter 4.3.4.2 --- 15% ethanolic barium acetate --- p.28 / Chapter 4.3.4.3 --- 50% ethanolic barium acetate --- p.28 / Chapter 4.3.4.4 --- Alcian blue working solution --- p.28 / Chapter 4.3.4.5 --- 0.1M Tris Buffer --- p.28 / Chapter 4.3.4.6 --- CTB Tris solution --- p.28 / Chapter 4.3.4.7 --- 2.0M lithium chloride --- p.28 / Chapter 4.3.5 --- Reagents for enzymatic degradation --- p.28 / Chapter 4.3.5.1 --- Reconstitution of CSE enzyme --- p.28 / Chapter 4.3.5.2 --- Reconstitution of DSE enzyme --- p.29 / Chapter 4.3.5.3 --- Reconstitution ofHSE I enzyme --- p.29 / Chapter 4.4 --- Methods --- p.31 / Chapter 4.4.1 --- Cobas Bio DMB method --- p.31 / Chapter 4.4.2 --- Cobas Fara DMB method --- p.31 / Chapter 4.4.3 --- Evaluation of methods --- p.31 / Chapter 4.4.3.1 --- To study the matrix effect --- p.31 / Chapter 4.4.3.2 --- Calibration --- p.31 / Chapter 4.4.3.3 --- Precision performance --- p.34 / Chapter 4.4.3.4 --- Linearity check --- p.34 / Chapter 4.4.3.5 --- Detection Limit --- p.34 / Chapter 4.4.3.6 --- Recovery study --- p.35 / Chapter 4.4.3.7 --- Correlation with Cobas Bio to develop the reference range --- p.35 / Chapter 4.4.4 --- Electrophoresis method --- p.36 / Chapter 4.4.4.1 --- Sample preparation --- p.36 / Chapter 4.4.4.2 --- Electrophoresis procedure --- p.36 / Chapter 4.4.5 --- Enzymatic degradation method --- p.37 / Chapter 4.4.5.1 --- Digestion of GAG in aqueous and urine matrix --- p.37 / Chapter 4.4.5.2 --- To optimize the amount of enzyme used to degrade GAG --- p.38 / Chapter 4.4.5.3 --- To study the specificity of GAG degrading enzyme --- p.39 / Chapter 4.4.5.4 --- To study the interaction of GAG --- p.40 / Chapter 4.4.5.5 --- To study the stability of enzyme CSE and DSE --- p.40 / Chapter 4.4.5.6 --- Study MPS patient sample --- p.41 / Chapter 5 --- Results --- p.42 / Chapter 5.1 --- Performance characteristics of the DMB method --- p.42 / Chapter 5.1.1 --- Matrix effect --- p.42 / Chapter 5.1.2 --- Calibration --- p.42 / Chapter 5.1.3 --- Precision performance --- p.42 / Chapter 5.1.4 --- Linearity Range --- p.42 / Chapter 5.1.5 --- Detection limit --- p.42 / Chapter 5.1.6 --- Recovery --- p.47 / Chapter 5.1.7 --- Correlation of Cobas Fara with Cobas Bio --- p.47 / Chapter 5.2 --- Results of GAG enzymatic degradation --- p.50 / Chapter 5.2.2 --- To optimise the amount of enzyme for GAG degradation --- p.57 / Chapter 5.2.3 --- The specificity of GAG degrading enzymes --- p.57 / Chapter 5.2.4 --- The interaction of GAG --- p.57 / Chapter 5.2.5 --- The stability of enzymes --- p.57 / Chapter 5.2.6 --- MPS patient study --- p.57 / Chapter 5.2.6.1 --- Type I/II/VI/VII --- p.57 / Chapter 5.2.6.2 --- MPS Type III patient 1 --- p.64 / Chapter 5.2.6.3 --- MPS Type IIIC patient 2 --- p.64 / Chapter 6. --- DISCUSSION --- p.67 / Chapter 6.1 --- Automated DMB method on Cobas Fara --- p.67 / Chapter 6 2 --- GAG specific degradation enzymes --- p.70 / Chapter 7. --- CONCLUSION & SUGGESTION FOR FUTURE STUDIES --- p.73 / Chapter 8. --- REFERENCES --- p.75

published_or_final_version / Physiology / Master / Master of Philosophy

Les glycosaminoglycanes (GAGs), des polysaccharides bio-actifs exprimés à la surface des cellules et dans la matrice extracellulaire, sont à l’origine d’un grand nombre de processus physiologiques et pathologiques tels le développement embryonnaire, la croissance cellulaire, l’homéostasie, etc. Parmi les biopolymères, ils offrent le plus grand potentiel d’information grâce à la variété de combinaisons et de modifications régio-sélectives des monosaccharides les constituant. Leur analyse structurale représente ainsi l’un des défis des glycosciences les plus difficiles à relever. De nouvelles approches basées sur la détection à l’échelle de la molécule unique permettent l’observation directe et la nano-manipulation de biomolécules. Principalement utilisées pour les acides nucléiques ou les protéines, ces approches ont rarement été appliquées à l’étude des polysaccharides. Nous présentons ici la détection à l'échelle de la molécule unique d’oligo-glycosaminoglycanes individuels confinés dans un nanopore protéique d’aérolysine et d’α-hémolysine. Nos résultats montrent la capacité de cette technique à discriminer les oligosaccharides d’acide hyaluronique selon leur degré de polymérisation, d’après la durée et la fréquence des blocages de courants. La preuve de la translocation a été montrée par spectrométrie de masse. Cette approche nous a permis de suivre la dépolymérisation enzymatique de l’acide hyaluronique et de déterminer ses paramètres cinétiques. D’autres oligosaccharides (l'héparine, le dermatane sulfate et le dextrane sulfate) ont été étudiés, présentant des signatures caractéristiques différentes, mettant en évidence des différences de structures et/ou conformations. / Glycosaminoglycans (GAGs) are bio-active polysaccharide expressed at the cell surface and in the extra-cellular matrix, which mediate cell-cell and cell-matrix interactions at the origin of a variety of physiological and pathological activities such as in embryonic development, cell growth, homeostasis, etc. Among all biopolymers, they offer the largest potential of information owing the incomparable variety of combinations and region-selective modifications of their building monosaccharides. The structural analysis of such complex carbohydrates is recognized as one of the most challenging task of glycosciences. New approaches based on single-molecule detection are currently arousing great interest in biology as it allows the direct observation and nano-manipulation of bio-molecules. Mainly applied to nucleic acids and proteins, these approaches have been not often used for the study of carbohydrates. We report here the detection of individual glycosaminoglycan oligosaccharides confined in aerolysin and α-hemolysin proteic nanopores. Our results show the capability of this new approach to discriminate hyaluronic acid (HA) oligosaccharides according to their polymerization degree based on the analysis of duration and frequency of the current blockades. This feature prompted us to apply this approach to the enzyme monitoring of the hyaluronidase-catalyzed depolymerization of HA and the determination of its kinetic parameters. Translocation has also been proved by mass spectrometry. Other oligosaccharides like heparin, dermatan sulfate and dextran sulfate, have also been studied, showing different characteristic “fingerprints”, due to structure and/or conformation differences.

Cancer est avere etre une dissemination metastatique et touts ses etapes sont obligatoirement commis par des interactions specifiques entre lescellules tumorales et leur microenvironement. ECM est specifiquement structuree et hautement responsable de ces interactions et de la signalisation de celui-ci. En raison des interactions accordes fines, il peut en resulter dans l' expression du gene altere, et contribuer dans le remodelage de l' ECM dans les conditions pathologiques. Proteoglycanes constituent une categorie cledes proteines glycosylees specials, qui outre le role de structure, sont egalement impliques dans la croissance et la metastase du cancer. SLRPs constituent des molecules biologiquement actifs et sont generalemnt characterises comme les voies de signalisation multifonctions. Lumicane, molecule cle de cette these, appartient aus SLRPs et son effet anticacereux seront etudiees efficaciment. L'objectif de cette these, est de decouvrir la voie de signalisation, dans lequel lumicane actes, est encore reste inconue. / Cancer is proved to be a metastatic dissemination and all its stages are obligatorily perpetrated through specific interactions between tumor cells and their microenvironment. Specifically structured ECM is highly responsible for these interactions and the signalling thereof. Because of the fine tuned interactions,it can result in altered gene expression and contribute to remodeling of ECM under pathological conditions. Proteoglycans constitute a key category of special glycosylated proteins, which apart from the structural role, are also involved in all steps of cancer growth and metastasis. SLRPs constitute biologically active molecules of ECM and ususally are characterised as multifunctional signalling molecules. Lumican, key molecule of the thesis, belongs to the SLRPs and its anticancer effect will extensively be studied. The goal of the thesis is to discover the signalling pathway, in which lumican acts,and still remains unknown.

No description available.

Thesis (M. Phil.)--University of Hong Kong, 2008. / Includes bibliographical references (leaf 108-131) Also available in print.

Thesis (M. Phil.)--University of Hong Kong, 2008. / Includes bibliographical references (leaf 75-83) Also available in print.