Chan Sze-ki. / Thesis submitted in: December 2004. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 73-76). / Abstracts in English and Chinese. / ABSTRACT (English Version) --- p.iii / ABSTRACT (Chinese Version) --- p.iv / ACKNOWLEDGEMENTS --- p.v / TABLE OF CONTENTS --- p.vi / LIST OF TABLES --- p.viii / LIST OF FIGURES --- p.xi / Chapter CHAPTER 1 --- INTRODUCTION / Chapter 1.1. --- Structure of Deoxyribonucleic acid (DNA) / Chapter 1.1.1. --- Configuration and Conformation of Deoxyribonucleic acid (DNA) --- p.1 / Chapter 1.1.2. --- Torsion Angle --- p.2 / Chapter 1.1.3. --- Base Pairing --- p.5 / Chapter 1.2. --- DNA Damage --- p.6 / Chapter 1.3. --- The Objective of this Project --- p.11 / Chapter CHAPTER 2 --- theory and Computational Details / Chapter 2.1. --- Computational Theory / Chapter 2.1.1. --- Density Functional Theory (DFT) --- p.12 / Chapter 2.1.2. --- Closed-shell and Open-shell Determinantal Wavefunctions --- p.13 / Chapter 2.1.3. --- Calculation Method --- p.13 / Chapter 2.1.4. --- Basis Set Details --- p.14 / Chapter 2.2. --- Ionization Potential and Electron Affinity --- p.15 / Chapter 2.3. --- Charge Distribution --- p.16 / Chapter 2.4. --- Molecular Orbital --- p.16 / Chapter 2.5. --- Computation Details in this Project / Chapter 2.5.1. --- Calculation Method --- p.17 / Chapter 2.5.2. --- Studied Model --- p.17 / Chapter CHPATER 3 --- Results and Discussion / Chapter 3.1. --- Neutral State / Chapter 3.1.1. --- Bond Length --- p.19 / Chapter 3.1.2. --- Torsion Angle of DNA backbone --- p.19 / Chapter 3.1.3. --- Sugar Ring Puckering Mode --- p.25 / Chapter 3.1.4. --- Natural Population Analysis (NAP) --- p.28 / Chapter 3.1.5. --- Molecular Orbitals --- p.31 / Chapter 3.2. --- Cationic State / Chapter 3.2.1. --- Ionization Potential --- p.33 / Chapter 3.2.2. --- Bond Length --- p.34 / Chapter 3.2.3. --- Backbone Torsion Angles --- p.38 / Chapter 3.2.4. --- Puckering Mode of Sugar Ring --- p.40 / Chapter 3.2.5. --- Charge Distribution --- p.43 / Chapter 3.2.6. --- Molecular Orbitals --- p.43 / Chapter 3.2.7. --- Summary --- p.47 / Chapter 3.3. --- Anionic State / Chapter 3.3.1. --- Ionization Potential --- p.51 / Chapter 3.3.2. --- Bond Lengths --- p.52 / Chapter 3.3.3. --- Torsion Angles of Backbone --- p.54 / Chapter 3.3.4. --- Sugar Ring Puckering Mode --- p.54 / Chapter 3.3.5. --- Charge Distribution --- p.58 / Chapter 3.3.6. --- Molecular Orbital --- p.63 / Chapter 3.3.7. --- Summary --- p.66 / Chapter CHAPTER 4 --- CONCLUSION AND FUTURE WORK / Chapter 4.1. --- Conclusion --- p.68 / Chapter 4.2. --- Future Work --- p.71 / REFERENCE --- p.73

Wu, Feng. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 38-44). / Abstracts in English and Chinese. / Title Page --- p.i / Thesis Committee --- p.ii / Acknowledgment --- p.iv / Table of Contents --- p.v / List of Figures --- p.vii / List of Abbreviations and Symbols --- p.xi / Abstract (English version) --- p.xii / Abstract (Chinese version) --- p.xiii / Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Significance of DNA CCTG repeats --- p.1 / Chapter 1.2 --- Objectives of this work --- p.2 / Chapter 1.3 --- DNA structure --- p.3 / Chapter 2 --- Materials and Methods --- p.5 / Chapter 2.1 --- Sample design --- p.5 / Chapter 2.2 --- Sample preparation --- p.5 / Chapter 2.3 --- NMR spectroscopy --- p.6 / Chapter 2.4 --- Resonance assignment --- p.7 / Chapter 3 --- NMR Structural Studies of (CCTG)3 --- p.9 / Chapter 3.1 --- Overview --- p.9 / Chapter 3.2 --- NMR resonance assignments --- p.9 / Chapter 3.3 --- Formation of two-residue CT-loop in the middle repeat of (CCTG)3 --- p.12 / Chapter 3.4 --- C-bulge and T.T mispair in (CCTG)3 hairpin stem region --- p.13 / Chapter 3.5 --- Summary --- p.15 / Chapter 4 --- NMR Structural Studies of (CCTG)4 --- p.16 / Chapter 4.1 --- Overview --- p.16 / Chapter 4.2 --- Conformational exchange in (CCTG)4 --- p.16 / Chapter 4.3 --- Formation of two-residue CT-loops in different repeats of (CCTG)4 --- p.17 / Chapter 4.4 --- Mutational studies of (CCTG)4 --- p.19 / Chapter 4.4.1 --- Mutational studies on the 1st repeat of (CCTG)4: (CCTG)4-C2T --- p.19 / Chapter 4.4.2 --- Mutational studies on the 2nd repeat of (CCTG)4：(CCTG)4-C6T --- p.21 / Chapter 4.4.3 --- Mutational studies on the 3rd repeat of (CCTG)4：(CCTG)4-C 10T --- p.26 / Chapter 4.4.4 --- Mutational studies on the 4th repeat of (CCTG)4: (CCTG)4-C14T --- p.28 / Chapter 4.5 --- Summary --- p.33 / Chapter 5 --- Conclusions and Future Works --- p.35 / References --- p.38

We present research on the design, development and application of algorithms for DNA sequence analysis, with a focus on environmental DNA (metagenomes). We present an overview and primer on algorithm development for bioinformatics of metagenomes; work on frameshift detection in DNA sequencing data; work on a computational pipeline for the assembly, feature prediction, annotation and analysis of bacterial genomes; work on unsupervised phylogenetic clustering of metagenomic fragments using Markov Chain Monte Carlo methods; and work on estimation of bacterial genome plasticity and diversity, potential improvements to the measures of core and pan-genomes.

Thesis (PhD (Genetics))--Stellenbosch University, 2010. / ENGLISH ABSTRACT: The current understanding of the regulation of carbohydrate accumulation is still under investigation despite the tremendous work done in this subject. Purine and pyrimidine nucleotides have been implicated in many biochemical processes in plants. Amongst others, they are building blocks for nucleic acid synthesis, an energy source, precursors for the synthesis of primary products such as sucrose, polysaccharides, phospholipids, as well as secondary products. With the aim of placing adenine and uridine nucleotides in the context of sucrose and starch metabolism and carbon partitioning in higher plant, we have investigated the transcripts, enzymes and metabolites in carbohydrate metabolism and both de novo and salvage of purine and pyrimidine nucleotides in both sugarcane and tobacco tissues. For that purpose, adenylate kinase (ADK) and UMP synthase were chosen for silencing and over expression as they are rate limiting steps of de novo adenine and uridine nucleotides biosynthesis, respectively. Sugarcane with repressed ADK activity showed significant increase in both the starch and adenylate pools. Increase in starch content was highly correlated with reduced ADK activity. As a result of decreased ADK activity, the salvage pathway was up regulated via the increased activity of both adenosine kinase (AK) and adenine phosphoribosyl transferase (APRTase) which positively correlated with increase in adenine nucleotide contents. In addition hexose phosphates and ADP glucose, the committed substrate for starch biosynthesis positively correlated with changes in starch content. A high ratio of ATP/ADP was observed in all transgenic lines compared with the untransformed wild type and suggested to favour starch synthesis. Over expression of cytosolic ADK in tobacco demonstrated an expression of the enzyme where 2/3 of the total activity was in the direction of ADP production. As a result of over expression of ADK, starch content increased in all transgenic plants and positively correlated with changes in the activity of ADK. Despite changes in adenine nucleotide content, the salvage pathway was not activated and no significant changes in both AK and APRTase acivities were found between the transgenic and the untransformed plants. Sucrose synthase (SuSy) activity in breakdown direction positively correlated with changes in starch content suggesting a contribution in the starch accumulation in tobacco plants. In addition the ratio of ATP/ADP was low in all transgenic lines compared with the untransformed wild type. This was in line with the higher content in ADP compare to ATP in all transgenic lines and was supported by the over expression of ADK, and predominantly in the direction of ADP production. Repressed UMP synthase in transgenic sugarcane resulted in increases in sucrose, starch and uridinylate. UDP-glucose, hexose phosphates and uridinylate content positively correlated with changes in sucrose content. Transgenic lines had increased sucrose phosphate synthase (SPS) activity and low activity in SuSy, which suggests alteration of carbon flux toward sucrose. As a result of decreased UMP synthase activity, an up regulation of the salvage pathway was observed and predominantly via increased activity of uridine kinase (UK) which positively correlated with changes in the uridinylate pool. In addition to repressed UMP synthase activity, starch content and adenine nucleotides increased in transgenic lines. Tobacco plants transformed with a cytosolic UMP synthase demonstrated an over expression of the enzyme in all transgenic lines. As a result of over expression of UMP synthase, key metabolites were up regulated, amongst them sucrose. Increase in sucrose content positively correlated with both hexoses and hexose phosphates but not the uridinylate pool. SPS activity positively correlated with increase in sucrose content, and accounted for most of the sucrose synthesized in transgenic lines. Despite the increase in the adenylate pool, no significant changes were observed in starch content. The depletion level of UDP-glucose in all transgenic lines was a mere reflection of the higher activity of UDP glucose pyrophosphorylase (UGPase) in the formation of glucose-1-phosphate. In addition, no salvage pathway was up regulated in transgenic lines. / AFRIKAANSE OPSOMMING: Die huidige beskikbare inligting in verband met die reguleering van koolhidraat akkumulasie word steeds ondersoek ten spyte van die groot hoeveelheid navorsing wat reeds in hierdie verband gedoen is. Purien en pirimidien nukleotide speel ‘n rol in baie biochemiese prosesse in plante. Onder andere is hulle boublokke vir nukleïensuur sintese, ‘n energie bron, voorlopers vir die sintese van primêre produkte soos byvoorbeeld sukrose, polisakkariede, fosfolipiede, asook sekondêre produkte. Met die vooruitsig om adenine- en uridiennukleotide in verband te plaas met sukrose en stysel metabolisme en koolstof afskorting in plante, ondersoek ons hier die transkripte, ensieme en metaboliete in koolhidraat metabolisme in beide de novo en berging van purien en pirimidien nukleotide in suikerriet asook tabak weefsel. Vir hierdie doel is adenilaatkinase (ADK) en UMP-sintase gekies vir uitskakeling en ooruitdrukking, juis omdat hulle tempo vermindering stappe van de novo adenine- en uridiennukleotide biosintese is. Suikerriet met onderdrukte ADK aktiwiteit wys betekenisvolle vermeerdering in beide die stysel en adenilaat poele. Verhoging in styselinhoud was hoogs gekorreleerd met verminderde ADK aktiwiteit. As gevolg van ‘n vermindering in ADK aktiwiteit, is die bergingspad opwaards gereguleer via die vermeerdering van beide adenosienkinase (AK) en adenien-fosforibosieltransferase (APRTase) aktiwiteit wat positief korreleer met die vermeerdering in adeniennukleotied-inhoud. Addisioneel word hexosefosfate en ADP-glukose, die toegewysde substraat vir stysel biosintese, positief gekorreleer met veranderinge in styselinhoud. ‘n Hoë verhouding van ATP/ADP was geobserveer in alle transgeniese lyne in vergelyking met die nie-getransformeerde wilde tipe en blyk stysel sintese te begunstig. Ooruitdrukking van sitologiese ADK in tabak demonstreer die uitdrukking van die ensiem waar 2/3 van die totale aktiwiteit in die rigting van ADP produksie was. As ‘n resultaat van ooruitdrukking van ADK, word stysel inhoud vermeerder in alle transgeniese plante en positief gekorreleer met die verandering in die aktiwiteit van ADK. Ten spyte van veranderinge in adeniennukleotide inhoud was die bergingspad nie geaktiveer nie en geen betekenisvolle veranderinge in beide AK en APRTase aktiwiteit was gevind tussen die transgeniese en nie-transgeniese plante nie. Sukrose sintese (SuSy) aktiwiteit tydens afbreking korreleer positief met die veranderinge in stysel inhoud en dui moontlik op ‘n bydrae in die stysel akkumulasie in tabak plante. Verder was die verhouding van ATP/ADP laag in alle transgeniese lyne in vergelyking met die nie-getransformeerde wilde tipe. Hierdie bevinding word ondersteun deur die hoër inhoud in ADP in vergelyking met ATP in alle transgeniese lyne en word verder ondersteun deur die ooruitdrukking van ADK, hoofsaaklik in die rigting van ADP produksie. Onderdrukte UMP-sintase in transgeniese suikerriet lei tot verhogings in sukrose, stysel en uridienilaat. UDP-glukose, hexose-fosfate en uridienilaat inhoud korreleer positief met die verandering in sukrose inhoud. Transgeniese lyne het verhoogde sukrose-fosfaatsintase (SPS) aktiwiteit en lae SuSy aktiwiteit wat dui op ‘n verandering in koolstof vloei in die rigting van sukrose. As gevolg van die afname in UMP-sintese aktiwiteit, word ‘n verhoogde reguleering van die bergingspad gesien, en dít hoofsaaklik via verhoogde aktiwiteit in uridienkinase (UK) wat positief korreleer met veranderinge in die uridienilaat poel. Addisioneel tot die onderdrukking van UMP-sintase was stysel inhoud en adenine- nucleotides in transgeniese lyne verhoog. Tabak plante wat getransformeer is met sitologiese UMP-sintase demonstreer verhoogde uitdrukking van die ensiem in al die transgeniese lyne. As ‘n resultaat van ooruitdrukking van UMP-sintase is sleutel metaboliete, onderandere sucrose, oorgereguleer. ‘n Verhoging in sukrose inhoud korreleer positief met beide hexose en hexose-fosfate maar nie met die uridienilaat poel nie. SPS aktiwiteit korreleer positief met die verhoging in sukrose inhoud en verklaar die meeste van die sukrose vervaardig in transgeniese lyne. Ten spyte van die verhoging in die adenilaat poel word geen noemenswaardige veranderinge gesien in die stysel inhoud nie. Die uitputtingsvlak van die UDP-glukose in alle transgeniese lyne was slegs ‘n aanduiding van die hoër aktiwiteit van UDP-glukose pirofosforilase (UGPase) in die formasie van glukose-1-fosfaat. Verder was geen bergingspad opgereguleer in die transgeniese lyne nie. / The South African Sugarcane Research Institute and the Gabonese Government who provided the financial support for this work

In particle induced desorption-ionization mass spectrometry the strength of an analyte's signal under a given set of bombardment conditions is usually considered to be representative of the analytes relative surface activity. This rationale is generally used to explain differences in the technique's sensitivity between and within various classes of compound. In liquid matrix assisted secondary ion mass spectrometry (SIMS) sensitivity enhancement of ionic analytes by pairing with surface active counterions has been demonstrated by several groups. This technique has been utilized in this work to achieve a 10,000 fold enhancement in the signal for ATP on a double focusing magnetic sector instrument and to detect femtomole quantities of nucleoside monophosphates on a time-of-flight instrument. The analyte's signal, however, is dependent on both the analyte bulk concentration and that of the surfactant. Additionally, the surfactant concentration that produces the maximum analyte signal changes with the analyte concentration. In this study, this phenomenon has been modeled in terms of conventional solution equilibria and surface chemical principles. It is assumed that the initial surface composition and the bulk concentration are the boundary conditions of a steady state established by the competing processes of surface sputtering and surface replenishment from the bulk during analysis. Calculated surface excesses correlate well with observed relative ion intensities, suggesting that equilibrium conditions are approached in the sample matrices despite the outwardly dynamic nature of the sputtering processes. / Graduation date: 1994

No description available.

The long-term goal of this project is to develop novel methods for the detection of nucleotides, oligonucleotides, and modified nucleotides such as DNA adducts by mass spectrometry. DNA adducts are important because they are formed during chemical carcinogenesis as well as during anti-cancer chemotherapy. However, DNA adducts are not routinely monitored due to difficulties associated with their detection. Mass spectrometry is a promising method for the detection of DNA adducts because it can detect almost any type of adduct, and in addition mass spectrometers can provide structural information. The work presented here shows successful detection of nucleotides and oligonucleotides of various sizes. Specific sizes detected include mononucleotides, 6-mer, 8-mer, 1 O-rner, and 16-mer oligonucleotides, and enzyme digests of genomic DNA and oligonucleotides. Through researchinvolving several separation methods (HPLC, TLC, and PAGE) and alternative detection methods (32P postlabeling and mass spectrometry), a novel method for the separation and detection of DNA adducts has been developed. The present research has shown promising results for tracking nucleotides in TLC using biomimetic dyes in order to eliminate the need for radioactive isotopes. In addition, progress has been made involving elution of nucleotides from a TLC plate and subsequent detection of these nucleotides by mass spectrometry. Together, these results will facilitate future studies that involve testing samples that contain altered DNA by different mass spectrometers, which are expected to be particularly useful for the detection and identification of mixed or novel DNA ' modifications.

Ion channels are fundamental to the functioning of life, regulating processes as diverse as neural signaling, homeostasis, and environmental sensing, across the complexities of life from bacteria to the most advanced organisms. Among this vast diversity of ion channels, cyclic-nucleotide gated (CNG) channels hold particular significance and play a pivotal role in the sensory transduction across a variety of species. They transduce chemical signals into electrical signals, linking the external environment and our sensory perceptions. CNG channels were discovered almost 40 years ago and much knowledge has been gained on their physiological roles, biophysical properties, molecular characteristics, and channelopathies. However, the structural details of these channels remained elusive for a long time, mainly due to the lack of a full-length channel structure. It was only recently that atomic-resolution structures of full-length CNG channels became available, and structures of native mammalian CNG channels were only determined within the last two years. In my thesis, I use single particle cryogenic electron microscopy (cryo-EM) to determine the structures of native human cone CNGA3/CNGB3 channel in different biochemical environments and in different states, spanning the full spectrum of channel activation by its natural ligand cGMP. In addition, I use cryo-EM, electrophysiology, calcium imaging, and other biochemical techniques to characterize both wild-type and disease-associated mutant (DAM) CNG channels. Collectively, my thesis work contributes to a deeper understanding of the structural determinants of CNG channel properties, provides a detailed dissection of the CNG channel gating mechanism, demonstrates a potential CNG channel pathogenic mechanism, and calls for an interdisciplinary reevaluation of CNG channel DAMs.