Zhan, Yingqian. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 49-54). / Abstracts in English and Chinese. / Title Page --- p.i / Thesis Committee --- p.ii / Abstract (English version) --- p.iv / Abstract (Chinese version) --- p.v / Acknowledgment --- p.vi / Table of Contents --- p.vii / List of Tables --- p.ix / List of Figures --- p.x / List of Abbreviations and Symbols --- p.xiii / Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- DNA Methylation --- p.1 / Chapter 1.2 --- DNA Structure --- p.2 / Chapter 1.2.1 --- Nomenclature Scheme for DNA --- p.2 / Chapter 1.2.2 --- Conformations of DNA --- p.5 / Chapter 1.2.3 --- Base Pair Scheme --- p.5 / Chapter 1.2.4 --- Sugar Conformation --- p.7 / Chapter 1.2.5 --- Backbone Conformation --- p.7 / Chapter 1.3 --- DNA Melting --- p.7 / Chapter 1.4 --- Objective of this Work --- p.8 / Chapter 2. --- Materials and Methodology --- p.9 / Chapter 2.1 --- Sequence Design --- p.9 / Chapter 2.2 --- Sample Preparation --- p.10 / Chapter 2.3 --- Resonance Assignment --- p.10 / Chapter 2.3.1 --- Proton Resonance Assignment --- p.11 / Chapter 2.3.2 --- Phosphorous Resonance Assignment --- p.14 / Chapter 2.4 --- Determination of Sugar Conformation --- p.15 / Chapter 2.5 --- Determination of Backbone Conformation --- p.16 / Chapter 2.6 --- Thermodynamic study on DNA --- p.18 / Chapter 3. --- Effects of N1-methylated Adenine on DNA Structure and Stability --- p.21 / Chapter 3.1 --- Overview --- p.21 / Chapter 3.2 --- Resonance Assignment Results --- p.21 / Chapter 3.2.1 --- 1H Resonance Assignments --- p.21 / Chapter 3.2.2 --- 31P Resonance Assignments --- p.23 / Chapter 3.3 --- Base Pair Structures --- p.24 / Chapter 3.4 --- Sugar Conformation --- p.26 / Chapter 3.5 --- Backbone Conformation --- p.27 / Chapter 3.6 --- Thermodynamic Stability Study --- p.29 / Chapter 4. --- Effects of N1-methylated Guanine on DNA Structure and Stability --- p.31 / Chapter 4.1 --- Overview --- p.31 / Chapter 4.2 --- Resonance Assignment Results --- p.31 / Chapter 4.2.1 --- 1H Resonance Assignments --- p.31 / Chapter 4.2.2 --- 31P Resonance Assignments --- p.33 / Chapter 4.3 --- Base Pair Structures --- p.34 / Chapter 4.4 --- Sugar Conformation --- p.36 / Chapter 4.5 --- Backbone Conformation --- p.39 / Chapter 4.6 --- Thermodynamic Stability Study --- p.41 / Chapter 5. --- Conclusion and Future work --- p.43 / Appendix I H1'-H6/H8 region of NOESY spectra of TmeA at 25 °C with different mixing times --- p.44 / "Appendix II 3JH1'h2"", 3JH1'h2"", and %S of TmeA and refTA" --- p.45 / "Appendix III 3JH1' h2'，3JH1'-H2"", and %S of CmeG and refCG" --- p.46 / "Appendix IV 31P chemical shifts, 3JH3'p and %B1 of TmeA, and refTA" --- p.47 / "Appendix V 31P chemical shifts, 3JH3'P and %B1of CmeG, and refCG" --- p.48 / References --- p.49

Purines as a class of compounds have been implicated in many biological systems, including as adenosine receptor antagonists. A method of synthesising 2,6,9-trisubstituted purines would be useful to produce small libraries of compounds for probing adenosine receptor selectivity. A library of trisubstituted purines has been achieved using a solid-phase methodology. The electronic properties of the substrate were found to result in difficulties with the loading of substrate onto the resin. Theoretical calculations provided the basis for mono-substitution in order to activate the substrate. This modified substrate has loaded onto the resin in reproducible and high yields. Amine and thiol, on-resin, C-2 substitution was shown to proceed at room temperature. This represents significantly milder conditions than are generally seen in the literature. This is due to the activating effect of the carbamate linker chosen on the pyrimidine ring. This also results in a faster reaction rate than is seen in the corresponding solution-phase reaction. This study showed that the electronic profile of the loaded substrate was responsible for the low alkylation on the carbamate nitrogen of loaded dichloro- or C-6 substituted chloropyrimidines. This reaction was modified by activating the pyrimidine ring via C-2 substitution and has been shown to go to completion with three different alkyl groups to give a clean product direct from resin cleavage. On-resin nitro reduction had been planned. The resin bound product would then be carried on to the next step of resin cleavage and cyclisation of the imidazole ring to give the final purine products. On resin reduction could not be achieved, however, cleavage of the compound from the resin and reduction in solution was found to be efficient as the cyclisation reagents could be included in this step without interfering with yield or purity of products and so this represents a clear improvement upon the planned synthesis. Efforts to fully characterise the library brought up issues of purine NMR. Extremely broad signals were observed in the proton spectra of many of the compounds making assignments difficult. Broad 13C NMR signals have also been observed. Restricted rotation about the substituent N-C bond is responsible for these problems. Crystal structure data has confirmed the double bond character of this bond with one of the substituted pyrimidines. High temperature NMR experiments have demonstrated how this can be overcome and the fine structure of the spectra observed. HMBC and COSY correlations have been used alongside the 1H and 13C spectra to allow full characterisation of the compounds wherever possible. Receptor homology models were created and updated for all four adenosine receptor subtypes. Known adenosine agonists and antagonists were created and minimised for use in docking experiments. Receptor docking experimental data is reported. Binding assays are being carried out by a third party and will be submitted for publication at a later date. A small library of 2,6,9-trisubstituted purines has been synthesised, exemplifying an efficient and robust method to achieve pure compounds for biological evaluation. A good level of diversity has been achieved at each combinatorial position (two substitutions and an N-alkylation). Final compounds have been isolated in good yields with a high level of purity.

No description available.

La voie de biosynthèse des purines est une voie métabolique conservée et essentielle. Chez l’Homme, des mutations dans plusieurs gènes impliqués dans cette voie provoquent de sévères maladies neuro-musculaires à composante développementale. Cependant, le lien entre génotypes et phénotypes n’est pas connu. Afin de mieux comprendre le rôle des gènes de la voie des purines au cours du développement, nous avons utilisé Xenopus laevis comme modèle vertébré. Les principaux gènes de la voie des purines du xénope n’étaient pas connus, ils ont donc tout d’abord été identifiés in sillico, puis les fonctions enzymatiques pour lesquels ils codent ont été validées in vivo en système hétérologue chez S. cerevisiae. Des analyses d’expression spatiotemporelle chez l’embryon de xénope ont montré que ces gènes sont exprimés tout au long du développement et en particulier dans les tissus neuro-musculaires, suggérant un rôle dans le développement de ces tissus. Le knock-down des gènes, ppat, hprt ou adsl, trois gènes clés de la voie des purines, conduit dans chaque cas à de sévères altérations des muscles squelettiques et en particulier des somites et des muscles hypaxiaux des embryons. Ces phénotypes musculaires sont la conséquence d’une altération précoce de l’expression des gènes MRF (Myogenic Regulatory Factors) myoD et myf5. Un défaut de migration des myoblastes précurseurs des muscles hypaxiaux a également été mis en évidence. Pour conclure, X. laevis est un modèle pertinent qui apporte de nouvelles connaissances permettant de mieux comprendre la cause des altérations musculaires développementales associées aux déficiences en purines. / The purine biosynthesis pathway is a conserved metabolic pathway essential for many cell functions. In Human, several mutations in genes involved in this pathway lead to severe neuromuscular diseases, which are at least in part caused by unknown developmental impairments. We established a Xenopus laevis model to decipher the role of the purine biosynthesis genes during vertebrate development. As no data was available regarding this pathway, the main Xenopus purine genes were first identified in silico and functionally validated in vivo using the yeast Saccharomyces cerevisiae as a heterologous system. Spatio-temporal analyses revealed that these genes are expressed all along the development, especially in neuromuscular tissues, suggesting an important role during their formation. The knock-down of ppat, adsl or hprt, three key purine genes, leads in each case to severe defects in skeletal muscles embryonic defects, in particular in somites and hypaxial muscles. These muscular phenotypes are the consequence of an early alteration in expression of some crucial Myogenic Regulatory Factors (MRF), such as myoD and myf5. Moreover, an alteration of the hypaxial muscles precursors was observed. In conclusion our results establish X. laevis as an ideal model to get new insights into the neuromuscular developmental alterations associated to purine deficiencies.

No description available.

published_or_final_version / Biochemistry / Master / Master of Philosophy

Thesis (Ph. D.)--University of Sydney, 2003. / Title from title screen (viewed Apr. 28, 2008). Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the Dept. of Medicine, Nepean Hospital, Faculty of Medicine. Includes bibliography. Also available in print form.

Thèses (M.Sc.)--Université de Sherbrooke (Canada), 1999. / Titre de l'écran-titre (visionné le 20 juin 2006). Publié aussi en version papier.

Thesis (M.S.)--University of Wisconsin--Madison, 1967. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

The biosynthesis of purine nucleotides in Ehrlich ascites carcinoma cells was investigated under different conditions. In initial studies the effect of actinomycin D was examined. The nucleotides from the acid soluble fraction of Ehrlich ascites carcinoma cells incubated with actinomycin D and ¹⁴C-formate were adsorbed on charcoal and eluted with a mixture of pyridine and ethanol. The eluted nucleotides were separated by two dimensional paper chromatography using isobutyric acid-ammonia-water in the first direction and aqueaus ammonium acetate-ethanol in the second. .The nucleotides were estimated by ultra violet spectrophotometry and the radioactivity incorporated was determined by liquid scintillation counting. As the results of these studies using small amounts of cells were inconclusive due to variations from experiment to experiment, similar studies were carried out using larger amounts of cell suspensions. The acid soluble nucleotides from these experiments were separated by ion-exchange chromatography on DEAE-cellulose and finally by paper chromatography. It was observed that there was an accumulation of acid soluble nucleotides in Ehrlich ascites carcinoma cells incubated with ¹⁴C-formate and actinomycin D. The specific activities of these nucleotides were not significantly different from those of the control experiments. The incorporation of ¹⁴C-formate into nucleic acids was inhibited by actinomycin D in these cells. From these observations it was concluded that actinomycin D did not inhibit the biosynthesis of purine nucleotides in Ehrlich ascites carcinoma cells in vitro. It is suggested that the effect of actinomycin D on nucleic acid metabolism is therefore, at a stage beyond the synthesis of nucleotides. Further studies on the effect of actinomycin D revealed that the antibiotic inhibited the respiration of Ehrlich ascites carcinoma cells in vitro slightly. The glycolysis in Ehrlich ascites carcinoma cells was unaffected by actinomycin D. In experiments where the effect of glucose on the incorporation of radioactive precursors into nucleotides and nucleic acids of Ehrlich ascites carcinoma cells was examined, contrary to the results of many, a decrease in incorporation was observed. This decrease in incorporation was independent of the presence of Ca⁺⁺ ions in the incubation medium, the buffer and of the radioactive precursors used in these incubations. It was observed that there were two factors controlling the incorporation of radioactive precursors in. vitro into the nucleotides and nucleic acids of Ehrlich ascites carcinoma cells in presence of glucose; the concentration of glucose in the medium and the concentration of cells in suspension. In dilute cell suspensions (packed cell volume less than 5%) glucose at a concentration of 5.5 mM decreased whereas in dense cell suspensions (packed cell volume above 8%) the same concentration of glucose increased the incorporation of labelled precursors into both acid soluble nucleotides and nucleic acids. 2-Deoxyglucose, an analogue of glucose at a similar concentration decreased the incorporation of ¹⁴C-formate in dilute as well as in dense cell suspension. Dinitrophenol, an uncoupler of oxidateive phosphorylation, also decreased the incorporation of ¹⁴C-formate in these cells which was more marked in dilute cell suspensions in presence of glucose. It was concluded from these observations that the main factor controlling the incorporation in vitro of radioactive precursors into nucleotides and nucleic acids of Ehrlich ascites carcinoma cells was the transient depletion and regeneration of ATP in these cells in presence of glucose. Glucose increased the incorporation of ¹⁴C-formate into the serine of the acid soluble fraction of Ehrlich ascites carcinoma cells. A marked increase in the incorporation of ¹⁴C-formate into serine was observed in presence of 2-deoxyglucose. These effects were independent of the concentration of cells in suspension. It was suggested that when the availability of the common precursor, N⁵-N¹º methylenetetrahydrofolic acid was increased particularly due to a decrease in incorporation into nucleic acids, more of the labelled precursor may be incorporated into serine. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate