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

Submitted in partial compliance with the requirements of the Master’s Degree in Technology: Homoeopathy, Durban University of Technology, Durban, South Africa, 2016. / Aim The aim of this study was to compare the nuclear magnetic resonance spectra of Sulphur 12c samples produced by the traditional Hahnemannian method with Sulphur 12c samples produced using sonication as an alternative method of agitation. Sonication, while not widely employed as an agitating technique in the homoeopathic potentisation process, is a highly effective agitation process which produces effects on liquids that closely resemble the effect of traditional Hahnemannian hand succussion (Bhattacharyya et al. 2008). Thus, this study sought to reveal whether or not homoeopathic remedies produced by sonication bore a close enough physicochemical resemblance to traditional hand succussed remedies to be considered as a viable equivalent. Methodology Five sample groups were manufactured for analysis, all by means of serial dilution at the centesimal ratio (1:100) to the 12c potency, and with agitation between dilution levels where applicable. Three of the sample groups were experimental, namely the Sulphur 12c Hahnemannian, Sulphur 12c sonicated and Sulphur 12c both (succussion and sonication). The Sulphur 12c Hahnemannian samples were produced by hand according to the German Homoeopathic Pharmacopoeia (Benyunes 2005), which includes an agitation phase of 10 hand succussions. Sonicated samples were produced according to the Hahnemannian method as far as possible, however the agitation phase consisted of 30 seconds of sonication in a sonication bath at 40Hz in accordance with related studies (Sukul, Sinhabau, and Sukul 1999: 58-59; Sukul et al. 2001a: 187). Sulphur 12c both (succussion and sonication) samples underwent ten hand succussions and 30 seconds of sonication at 40Hz between dilution levels. Two of the sample groups were controls, namely Sulphur 12c unagitated and Lactose 12c unagitated, neither of which underwent agitation between dilution phases but were otherwise produced according to the German Homoeopathic Pharmacopoeia specification (Benyunes 2005). All samples were raised to the 12c potency level in 87% alcohol from a 3CH triturate. The Lactose 12c unagitated control was derived from a 3CH triturate of lactose, while the other samples were all derived from a 3CH triturate of Sulphur. The sample groups were sent for nuclear magnetic resonance (NMR) spectroscopy at the Department of Chemistry at Stellenbosch University. The NMR device used was the Varian UnityInova 600 NMR Spectrometer ®, with a Deuterated DMSO insert added as an instrument frequency lock. Samples were drawn and analysed by Dr D.J. Brand. One sample was drawn from each sample group. The chemical shift and relative integration values for the OH, H2O, CH2, and CH3 peaks of the NMR spectra were captured and tabulated using Microsoft Excel© 2013. The statistical analysis was performed with the aid of SPSS Version 22. The chemical shift and relative integration values for the H2O, OH, CH2 and CH3 peaks were used in the statistical analysis. The Kruskal-Wallis method was performed for the five sample groups to ascertain whether or not a statistically significant difference existed between the five sample groups. Comparisons between individual paired groups were conducted by means of the non-parametric Mann-Whitney test. The significance interval was set at α = Results The chemical shift values of the CH2 peaks of the samples showed a clear similarity between the samples produced by Hahnemannian hand succussion, sonication and both (succussion and sonication) as well as a clear difference between these three samples and the two controls. The relative integration values, however, showed no clear trends in support of or detracting from the hypotheses. Conclusion In terms of the CH2 peak chemical shift values it can be concluded that distinct similarities exist between 12c potency level of Sulphur produced by Hahnemannian hand succussion and sonication, and that the two methods of agitation produce similar structural properties in samples of the 12c potency level. Furthermore in terms of the chemical shift values, succussion and sonication develop remedies that are distinct from unagitated remedies of equivalent potency level. Thus, these findings support the use of sonication as a potentially viable alternative to hand succussion as a method of agitation in the potentisation process. Further studies need to be conducted however, with the inclusion of a greater variety of potency levels in order to possibly reveal more trends in terms of the relative integration values as these values were inconclusive in this study. / M

Dissertation (PhD)--University of Stellenbosch, 2006. / ENGLISH ABSTRACT: The partial double bond character of the carbon-nitrogen bond of the (S)C-NRR’ moiety results in unsymmetrical dialkyl-substituted N-alkyl-N-alkyl(aryl)-N’-acylthioureas, R”C(O)NHC(S)NRR’ (HL) displaying E,Z configurational isomerism in solution. The isomerism manifests itself by the duplication of resonances of the N-alkyl groups in the 1H and 13C NMR spectra. In one class of these ligands where R and R’ groups are non-equivalent alkyl groups the isomerism is easily observable at 298 K in chloroform. In the other class where R’ is still an alkyl group and R is a para-substituted phenyl group the isomerism is only observable at much lower temperatures due to a lower barrier to rotation around the (S)C-N(alkyl)(para-X-Ph) bond (X = O-CH3, H and NO2). The electron-withdrawing nature of the nitro group in N-methyl-N-(4-nitro-phenyl)-N’-2,2-dimethylpropanoylthiourea, HL3A and N-pentyl-N- (4-nitro-phenyl)-N’-2,2-dimethylpropanoylthiourea, HL3D result in the E,Z isomerism of these ligands not observable even at 198 K in dichloromethane. The distribution of E and Z isomers of the unbound ligands vary depending on these R and R’ groups. Several E isomers of these ligands have been isolated and structurally characterised and the (S)C-NRR’ bond falls in the range [1.343(3) – 1.329(3) Å] which shorter than the average C-N single bond of 1.472(5) Å. The E,Z configurational isomerism in the unbound ligands is passed on to the Pt(II) chelates derived from these ligands. The presence of cis-[Pt(ZZ-L-S,O)2], cis-[Pt(EZ-L-S,O)2] and cis-[Pt(EE-L-S,O)2] is readily observable by means of 195Pt NMR spectroscopy which shows three well resolved resonances, and this can be confirmed by 1H and 13C NMR spectra of these complexes. The 195Pt nuclei are spatially linked to the 13C nuclei, four bonds away resulting in 4J(195Pt-13C) couplings with N-CH2- or N-CH3 carbons in a W pathway. The 195Pt NMR spectra are also linked to N-CH2- or N-CH3 proton resonances by means of the ZZ, EZ and EE isomer distributions. Assignment of these configurational isomers was then achieved by means of a combination of low magnetic field 13C NMR spectra and high-resolution gHSQC (1H/13C) NMR experiments. 1H NMR rotational dynamics study showed that the barrier to rotation, ΔG≠, around the (S)C-N(Me)(para-X-Ph) bond in cis-bis(N-methyl-N-(4-methoxy-phenyl)-N’-2,2-dimethylpropanoylthioureato)platinum(II), cis-[Pt(L1AS, O)2]; cis-bis(N-methyl-N-phenyl-N’-2,2-dimethylpropanoylthioureato)platinum(II), cis-[Pt(L2A-S,O)2] and cisbis( N-methyl-N-(4-nitro-phenyl)-N’-2,2-dimethylpropanoylthioureato)platinum(II), cis-[Pt(L3A-S,O)2] complexes was observed to follow the order: (electron-withdrawing group) NO2 < H < (electron-donating group) O-CH3. The ZZ isomer was observed to be favoured over the EZ and EE isomers in this order of the para-substituent on the Nphenyl group. The 1H dynamic NMR trends about the barrier to rotation, ΔG≠, around the (S)C-N(Me)(para-X-Ph) bond were also complemented by DFT linear transit calculations. The isomer distributions were also influenced by solvent polarity and the temperature at which the distributions are determined apart from the electronic influence of the para-substituent of the N-phenyl group. The ZZ, EZ and EE isomers of complexes derived from N-alkyl-N-(para-X-Ph)-N’-acylthioureas with varying Nalkyl substituent (methyl, isopropyl, cyclohexyl and n-pentyl) were determined from the 195Pt NMR spectra which were measured under identical conditions. The ZZ isomer was observed to be favoured over the EZ and EE isomers upon methyl group substitution with a bulkier alkyl group in the order: methyl < isopropyl < cyclohexyl < n-pentyl. Qualitatively it has been shown that a bulkier N-pentyl group increases the barrier to rotation around the (S)CN( alkyl)(para-X-Ph) bond over the N-methyl group and this leads to higher concentrations of the ZZ isomer over the EZ and EE isomers. The combined effects of the electron-donating substituent (X = O-CH3) on the N-(para-X-Ph) group and the bulkier N-alkyl group (n-pentyl) result in highest ZZ concentration (76 %) over EZ and EE isomers in the complex cis-bis(N-pentyl-N-(4-methoxy-phenyl)-N’-2,2-dimethylpropanoylthioureato)platinum(II), cis-[Pt(L1DS, O)2]. The lowest concentration ZZ (27 %) is obtained in the complex cis-bis(N-methyl-N-(4-nitro-phenyl)-N’-2,2- dimethylpropanoylthioureato)platinum(II), cis-[Pt(L3A-S,O)2] when the coordinated ligand has both N-methyl group and N-(4-nitro-Ph) group which both lower the barrier to rotation around the (S)C-N(alkyl)(para-X-Ph) bond. A crystal of the complex cis-bis(N-pentyl-N-(4-methoxy-phenyl)-N’-2,2-dimethylpropanoylthioureato)platinum(II), cis-[Pt(L1D-S,O)2] has been isolated and structurally characterised and was shown to be in the ZZ configuration, which is the major component (76 %) in chloroform. This is the first example of Pt(II) chelates with asymmetrically disubstituted ligands to be reported. / AFRIKAANSE OPSOMMING: Die gedeeltelike dubbelbinding karakter van die koolstof-stikstof-binding van die (S)C-NRR’-moieteit lei tot onsimmetriese dialkiel-gesubstitueerde N,N-dialkiel-N’-asieltioureums, R”C(O)NHC(S)NRR’ (HL) wat E,Zkonfigurasionele isomerie in oplossing besit. Die isomerie is sigbaar in die verdubbelling van die seine van die Nalkielgroepe in die 1H- en 13C-KMR spektra. In een so klas ligande waar R- en R’-groepe nie-ekwivalente alkielgroepe is, is isomerie duidelik sigbaar by 298 K in chloroform. In die ander klas waar R’ steeds ’n alkielgroep is, en R ’n para-gesubstitueerde feniel groep, is die isomerie alleenlik sigbaar by baie laer temperature as gevolg van ’n laer rotasieversperring om die (S)C-N(alkiel)(para-X-Ph)-binding (X = O-CH3, H and NO2). Die elektrononttrekkende aard van die nitrogroep in N-metiel-N-(4-nitrofeniel)-N’-(2,2-dimetielpropanoïel)tioüreum, HL3A en N-(4-nitrofenyl)-N-pentiel-N’-(2,2-dimetielpropanoïel)tioüreum, HL3D lei daartoe dat die E,Z-isomerie van die ligande nie eers by 198 K in dichlorometaan waargeneem word nie. Die verspreiding van die E en Z isomere verskil na gelang van die R en R’ groepe. Verskeie E-isomere van hierdie ligande is geïsoleer en struktureel gekarakteriseer en die (S)C-NRR’-bindingslengte is in ‘n gebied [1.343(3) – 1.329(3) Å] wat korter is as die gemiddelde C-N-enkelbindingslengte van 1.472(5) Å. Die Pt(II) chelate wat afgelei is van die ligande is blootgestel aan die E,Z-konfigurasie isomere van die ongebinde ligande. Die teenwoordigheid van cis-[Pt(ZZ-L-S,O)2], cis-[Pt(EZ-L-S,O)2] en cis-[Pt(EE-L-S,O)2] is maklik waarneembaar deur middel van 195Pt-KMR-spektroskopy wat drie goed geresolueerde seine toon, en dit kan bevestig word met 1H- en 13C-KMR spectra van hierdie komplekse. Die 195Pt kerne is ruimtelik geskakel met die 13C kerne deur vier bindings wat aanleiding gee tot 4J(195Pt-13C)-koppelings met N-CH2- of N-CH3-koolstofatome in ‘n Wkonformasie. Die 195Pt KMR spektra word geskakel met die N-CH2- of N-CH3-protonresonansies in al drie die moontlike ZZ, EZ en EE kompleksisomere. Toekenning van die konfigurasionele isomere is dan bewerkstellig deur middel van ‘n kombinasie van lae magneetveld 13C-KMR spectra en hoë resolusie gHSQC (1H/13C) KMR experimente. 1H-KMR-rotasiedinamiek studie toon dat die rotasiegrens, ΔG≠, om die (S)C-N(Me)(para-X-Ph)-binding in cisbis( N-metiel-N-(4-metoksifeniel)-N’-2,2-dimetielpropanoïeltioureato)platinum(II), cis-[Pt(L1A-S,O)2]; cis-bis(Nfeniel- N-metiel-N’-2,2-dimetielpropanoïeltioureato)platinum(II), cis-[Pt(L2A-S,O)2] en cis-bis(N-metiel-N-(4- nitrofeniel)-N’-2,2-dimetielpropanoïeltioureato)platinum(II), cis-[Pt(L3A-S,O)2] komplekse was met die volgende orde bepaal: (elektron-ontrekkende groep) NO2 < H < (elektron-skenkende groep) O-CH3. Die ZZ-isomeer blyk by voorkeur te vorm bo die EZ- en EE-isomere in dieselfde orde as hierbo wat betref para-substituent aan die Nfenielgroep. Die 1H dinamiese KMR tendencies ten opsigte van die rotasiegrens, ΔG≠, om die (S)C-N(Me)(para-XPh)- binding is gekomplimenteer met DFT-linêre organgs berekeninge. Die isomer verspreidings blyk ook beïnvloed te word deur die oplosmiddel polariteit en die temperatuur waarby die verspreidings bepaal is, buiten die elektroniese invloed van die para-substituent aan die N-fenielgroep. Die ZZ, EZ en EE isomeer verspreiding van komplekse wat afgelei is van N-alkiel-N-(para-X-Ph)-N’-asieltioureums met veranderlike N-alkiel substituente (metiel, isopropyl, sikloheksiel, en n-pentiel) is vasgestel deur middel van die 195Pt KMR wat opgeneem is onder identiese kondisies. Die ZZ-isomeer blyk die verkose isomeer te wees bo die EZen EE-isomere waar die metiel substituent vervang word met ‘n groter alkiel groep in die orde van: metiel < isopropiel < sikloheksiel < n-pentiel. Dit is kwalitatief getoon dat die groter N-pentielgroep die rotasiegrens verhoog vir rotasie om die (S)C-N(alkiel)(para-X-Ph)-binding bo dié van die N-metielgroep wat aanleiding gee tot hoër konsentrasies van die ZZ-isomeer relatief tot die EZ- en EE-isomere. Die gekombineerde uitwerking van die electron-skenkende substituent (O-CH3) op die N-(para-X-Ph)-groep en die groter N-alkiel groep (n-pentiel) gee aanleiding tot die hoogste ZZ-konsentrasie (76%) bo EZ- en EE-isomere in die kompleks cis-bis(N-pentiel-N-(4- metoksifeniel)-N’-2,2-dimetielpropanoïeltioureato)platinum(II), cis-[Pt(L1D-S,O)2]. Die laagste konsentrasie ZZ (27%) is verkry in die kompleks cis-bis(N-metiel-N-(4-nitrofeniel)-N’-2,2-dimetielpropanoïel)tioureato)platinum(II), cis-[Pt(L3A-S,O)2] waar die gekoördineerde ligand beide die N-metiel- sowel as die N-(4-nitro-Ph)-groep, wat albei die rotasiegrens van die (S)C-N(alkiel)(para-X-Ph)-binding verlaag. ‘n Kristalstruktuur van die kompleks cis-bis(N-pentiel-N-(4-metoksifeniel)-N’-2,2-dimetielpropanoïeltioureato) platinum(II), cis-[Pt(L1D-S,O)2] wat geïsoleer is, is struktureel gekarakteriseer en is in die ZZ-konfigurasie, wat die hoofkomponent (76%) is in chloroform. Hierdie is die eerste voorbeeld van Pt(II) chelate met asimmetriese digesubstitueerde ligande om geraporteer te word.

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

Dissertation submitted in partial compliance with the requirements for the Master's Degree in Technology: Homoeopathy, Technikon Natal, 2001. / The purpose of this study was to analyse and compare the NMR spectra ofa homoeopathic remedy (in this case Natrum muriaticum was used for its easy solubility and purity), that was prepared in the classical single vial Hahnemannian method, and also the widely used multi-vial Korsakovian method. Comparison was made in terms of the chemical shift (8) and relative integration values of the CH3, CH2, H20, and OH signals. A control was employed for both methods. The only difference between control and test remedies was the actual inclusion ofNatrum muriaticum in the latter, and the same weight of solvent as solute in the former. Comparison was made between both test methods, between test and control, and between the two controls. It was hypothesized that the method of dilution plays an important part in the potentisation process, and thus becomes part of the remedy's information content. The evolution of distinct physicochemical identities was hypothesized to occur specific to each method in ascending potency levels. Differences were therefore also hypothesized to exist between both methods at parallel potency levels in terms of chemical shift and relative integration values. The experiment was conducted as per the limitations of the scientific method. Both methods and their controls of Natrum muriaticum were potentised to the 9C, 30C, and 200C potency levels. They were prepared in 16ml volumes and transported to the NMR spectroscopy laboratory in Pietermaritzburg for analysis. / M

Dissertation submitted in partial compliance with the requirements for the Master's Degree in Technology: Homoeopathy, Durban Institute of Technology, 2002. / The purpose of this study was to investigate whether frequency strength is a parameter requiring consideration when conducting NMR spectroscopy studies on hornoeopathic potencies. To this end, samples of Sulphur 30CH and a Lactose control were analysed using NMR spectrometers operating at three different frequency strengths of 80MHz, 200.MHz and 500.MHz. It was hypothesized that differences existed in the spectra of respective Sulphur samples, control samples, and between parallel samples of Sulphur and control. It was further hypothesized that differences between parallel samples of Sulphur and control would be more noticeable at the lower frequencies. This hypothesis was based on the assumption that a higher frequency strength would have more intense resonance effects on the structure of the homoeopathic potency, thereby disturbing the micro-structural changes induced during potentisation. The design of the investigation was that of a scientific experiment. Potencies of Sulphur and a lactose-based control were prepared to a 30CH potency each, in 87% ethanol. The final prepared volumes (10ml) of Sulphur and control were blinded by means of colour codes by a third party prior to analysis. The blinded samples were transported to the University of Natal, Pietermaritzburg, where they were subjected to analysis using the following instruments: 1) A Varian FT80A 80.MHz instrument 2) A Varian Gemini 200MHz instrument 3) A Varian Inova 500.MHz instrument At each instrument NMR spectroscopy was conducted on ten (10) samples from each group (Sulphur and control). The samples were prepared in coaxial tubes using acetone as both an external lock and reference, and NMR spectra were recorded for each sample. All the samples were run at a thermostatically controlled temperature of 24\xB0C (\xB1 O,2\xB0C), and the laboratory was maintained at a constant temperature of 22\xB0C. The spectra and data of all the samples were recorded in terms of the chemical shift and integration values of their respective CH2, H20 and OH signals. / M

Dissertation submitted in partial compliance with the requirements for the Masters Degree in Technology: Homoeopathy, Durban Institute of Technology, 2002. / The purpose of this study was to analyse and compare the NMR spectra of homoeopathic Sulphur (the most well-known and often tested homoeopathic remedy) in two commonly used potencies, namely the centesimal (CH) and decimal (DH) potencies. Both potencies were prepared according to the Hahnemannian method. In order to assess the differences and similarities between these two potency scales, remedies with the same levels of deconcentration, and remedies with the same numbers of succussions were tested. The Control substance used was Water-Ethanol 87% without lactose or Sulphur. The Control substance was prepared in the same way as the Sulphur i.e. potentised as the Sulphur. Chemical shift and relative integration values of the H20, OH and CH2 peaks were recorded, calculated and compared. The investigation was designed as a scientific experiment. Firstly, the Sulphur remedies were compared to the Controls. Secondly, Sulphur remedies were compared to Sulphur remedies, and Controls to Controls. The following criteria were used: o Equal deconcentrations of the centesimal scale were compared to their equivalent decimal scale. o Equal numbers of succussions of the centesimal scale were compared to their equivalent decimal scale. The following potencies were assessed for both Sulphur and Control (Water-Ethanol): / M

In the work undertaken here, NMR has been used to ascertain the structure of some 1,2,4-triazoles. The investigation provides information concerning the structure of potentially tautomeric triazoles such as hydroxy- and aminotriazole. Connected with this aspect of triazole chemistry is the larger problem of mesohydric tautomerism. The present study also yields information for a comparison of substituent effects in triazoles, N-heteroaromatic substances and benzene.

by Chan Chun-Chung, Jerry. / The "59" in Co in title is superscript. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 157-170). / DESCRIPTIVE NOTE --- p.iii / Chapter CHAPTER ONE: --- A BRIEF SURVEY OF TRANSITION METAL NMR STUDIES --- p.1 / Chapter 1.0 --- Introduction --- p.1 / Chapter 1.1 --- Solution NMR Of Transition Metals --- p.2 / Chapter 1.1.1 --- Parametrization Model of Chemical Shifts --- p.4 / Chapter 1.2 --- Theoretical Calculation of the Chemical Shielding Constants of Transition Metals --- p.6 / Chapter 1.3 --- Solid State NMR of Transition Metals --- p.7 / Chapter 1.4 --- Scope of the Thesis --- p.9 / Chapter CHAPTER TWO: --- THEORY AND BACKGROUND --- p.10 / Chapter 2.0 --- Introduction --- p.10 / Chapter 2.1 --- The Origin of Chemical Shielding --- p.10 / Chapter 2.1.1 --- The Ramsey Shielding Tensor Equation with Gauge Origin Chosen at the Nucleus --- p.13 / Chapter 2.1.2 --- The Ramsey Shielding Tensor Equation with Arbitrary Gauge Origin --- p.20 / Chapter 2.1.3 --- The Physical Picture Associated with the Ramsey Shielding Equation --- p.21 / Chapter 2.2 --- Ab Initio Shielding Calculation --- p.24 / Chapter 2.2.1 --- Coupled Hartree-Fock Method --- p.25 / Chapter 2.2.2 --- Gauge Dependence Problem --- p.27 / Chapter 2.2.3 --- Post Hartree-Fock Methods --- p.29 / Chapter 2.3 --- Density Functional Theory --- p.30 / Chapter 2.3.1 --- The Hohenberg-Kohn Theorems --- p.30 / Chapter 2.3.2 --- The Kohn-Sham Approach --- p.35 / Chapter 2.3.3 --- Approximation to the Exchange- Correlation Energy --- p.37 / Chapter CHAPTER THREE: --- INTERPRETATION OF 59Co NMR SHIELDING USING THE HARD AND SOFT ACID-BASE CONCEPT -- INSIGHT INTO THE RELATIVE MAGNITUDE OF THE NEPHELAUXETIC AND THE SPETROCHEMICAL EFFECT --- p.39 / Chapter 3.0 --- Introduction --- p.39 / Chapter 3.1 --- Theory --- p.42 / Chapter 3.2 --- Evaluation of the Model --- p.45 / Chapter 3.3 --- Application to the Studies of trans-[Co(en)2X2](3+2n) + in Different Solvents and the Determination of the Spectrochemical Trend --- p.54 / Chapter 3.4 --- "Simultaneous Determination of the Nuclear Quadrupole Coupling Constant, Chemical Shift Anisotropy and Rotational Correlation Time in trans-Na[Co(acac)2(NO2)2]， trans- [Co(acac)2(NH3)2 ]I, trans-[Co(acac)2(CH3NH2)2]I and trans-[Co(acac)2(NH3)(NO2)]" --- p.59 / Chapter 3.5 --- Summary --- p.64 / Chapter CHAPTER FOUR: --- DENSITY FUNCTIONAL STUDY OF THE ELECTRONIC STRUCTURES OF [Co(NH3)5X](3+n)+ USING DIFFERENT POPULATION AND BONDING ANALYSIS METHODS --- p.66 / Chapter 4.0 --- Introduction --- p.66 / Chapter 4.1 --- Computational Details --- p.69 / Chapter 4.2 --- Bond Covalency Analysis of [Co(NH3)5X](3+n)+ --- p.71 / Chapter 4.2.1 --- Mayer Bond Order Analysis --- p.71 / Chapter 4.2.2 --- Natural Population Analysis --- p.73 / Chapter 4.2.3 --- Natural Bond Orbital Analysis --- p.76 / Chapter 4.2.4 --- Mulliken Population Analysis --- p.82 / Chapter 4.3 --- Summary --- p.86 / Chapter CHAPTER FIVE: --- DENSITY FUNCTIONAL STUDY OF 59Co CHEMICAL SHIELDING CONSTANTS --- p.87 / Chapter 5.0 --- Introduction --- p.87 / Chapter 5.1 --- SOS-DFPT-IGLO Calculations of 59Co NMR Shielding Parameters of Hexacoordinated Diamagnetic Co(III) Complexes --- p.90 / Chapter 5.1.1 --- Computational Details --- p.91 / Chapter 5.1.2 --- Basis Sets and XC Functional for 59Co Shielding Calculations --- p.92 / Chapter 5.1.2.1 --- 59Co NMR Shielding Calculation of [Co(CN)6]3- --- p.92 / Chapter 5.1.2.2 --- Nearest Neighbour Effect --- p.94 / Chapter 5.1.3 --- Comparison of the Calculated and Experimental 59Co Chemical Shift Anisotropy and Asymmetry Factor --- p.95 / Chapter 5.1.4 --- Comparison of the Calculated and Experimental 59Co Isotropic Chemical Shifts --- p.97 / Chapter 5.1.4.1 --- Reproducing the Experimental Trend by SOS-DFPT-IGLO? --- p.99 / Chapter 5.1.4.2 --- Local and Non-local Paramagnetic Shielding Contributions --- p.103 / Chapter 5.1.5 --- General Comments of the Calculated Results --- p.104 / Chapter 5.2 --- A Comparative Study of the Calculation of 59Co NMR Shielding Constants of Hexacoordinated Diamagnetic Co(III) Complexes Using SOS-DFPT-IGLO and Hybrid DFT-GIAO Methods --- p.105 / Chapter 5.2.1 --- Computational Details --- p.106 / Chapter 5.2.2 --- Comparison of DFT-IGLO-Becke/Perdew and DFT-GIAO-Becke/Perdew --- p.106 / Chapter 5.2.3 --- DFT-GIAO-B3LYP --- p.108 / Chapter 5.2.4 --- Summary --- p.111 / Chapter CHAPTER SIX: --- STUDY OF THE SHIELDING CONSTANTS OF DIAMAGNETIC HEXACOORDINATED Co(III) COMPLEXES BY POLYCRYSTALLINE 59Co NMR AND DENSITY FUNCTIONAL THEORY --- p.112 / Chapter 6.0 --- Introduction --- p.112 / Chapter 6.1 --- Solid State NMR Technique for Quadrupolar Nuclei --- p.112 / Chapter 6.2 --- Static Powder Lineshape Analysis --- p.114 / Chapter 6.2.1 --- Excitation of Quadrupolar Nuclei --- p.114 / Chapter 6.2.1.1 --- Selective and Partially Selective Excitation --- p.116 / Chapter 6.2.2 --- Spin Echo Pulse Sequence --- p.117 / Chapter 6.2.3 --- Lineshape Simulation --- p.120 / Chapter 6.3 --- Solid State 59Co NMR Study of Hexacoordinated Co(III) Complexes --- p.124 / Chapter 6.3.1 --- Experimental --- p.124 / Chapter 6.3.2 --- Simulation Details --- p.125 / Chapter 6.3.2.1 --- [Co(NH3)4CO3]NO3 --- p.127 / Chapter 6.3.2.2 --- [Co(en)2CO3]Cl and [Co(en)2NO3](NO3)2 --- p.130 / Chapter 6.3.2.3 --- cis-[Co(en)2(N02)2]NO3 and cis-[Co(en)2(N3)2]NO3 --- p.133 / Chapter 6.3.2.4 --- K3[Co(CN)6] --- p.133 / Chapter 6.3.2.5 --- "Co(acac)3， K3[Co(NO2)6] and [Co(en)3]X3 (X = C1, Br, I)" --- p.137 / Chapter 6.4 --- Dependence of 59Co Shielding Calculation on Basis Sets and Exchange Correlation Functional --- p.143 / Chapter 6.4.1 --- CSA and η calculations of [Co(NH3)4C03] Br --- p.144 / Chapter 6.4.2 --- CSA and η Calculations of [Co(NH3)6]C13 --- p.147 / Chapter 6.4.3 --- Shielding Calculations of Larger Co(III) Complexes at B3PW91/6311+G* Level --- p.149 / Chapter 6.5 --- Summary --- p.153 / Chapter CHAPTER SEVEN: --- CONCLUSION AND FUTURE WORK --- p.154 / BIBLIOGRAPHY --- p.157 / APPENDIX A MATHEMATICAL DETAILS FOR THE DERIVATION OF THE RAMSEY SHIELDING EQUATION --- p.171 / APPENDIX B ANALYSIS OF THE SIGN OF PARAMAGNETIC AND DIAMAGNETIC SHIELDING --- p.178 / APPENDIX C GENERALIZATION OF EQUATION [3.4] TO INCLUDE THE EFFECT OF π-BONDING --- p.181 / APPENDIX D GEOMETRY OPTIMIZATION OF CoH AND CoO --- p.183 / APPENDIX E A NON-LINEAR ITERATIVE LEAST SQUARE FITTING PROCEDURE FOR THE ANALYSIS OF SOLID STATE NMR STATIC SPECTRUM OF QUADRUPOLAR NUCLEUS --- p.187

The structural dynamics of proteins and other macromolecules typically serve crucial roles for their respective biological function. While rigid protein structures are used in classic “lock and key” descriptions of enzymology and receptor-ligand interactions, more and more evidence suggest that the majority of molecular interactions occur on the spectrum between induced-fit binding and conformational selection binding. This model of biomolecular interaction requires, to differing degrees, conformation plasticity and dynamics of the protein itself. To characterize the determinants and implications of protein dynamics, there exists no more suited biophysical technique than nuclear magnetic resonance (NMR) spectroscopy. This method is capable of probing the individual atomic nuclei of proteins in a site-specific manner. Furthermore, NMR spectroscopy is unique in being able to access timescales from picoseconds to seconds, providing information on events from bond vibration and libration to protein folding and ligand binding. The breadth of biophysical information accessible by NMR spectroscopy has led to its widespread use in the study of protein dynamics. The work presented herein involves i) the use of NMR for investigation of structure and dynamics in two separate biological systems that demonstrate a high degree of flexibility for folded proteins and ii) the improvement of pulse sequences and methodology for better characterizing picosecond to nanosecond backbone and side-chain dynamics. The organizing principle of this work, which is best exemplified in the structural studies of the piRNA-pathway protein Gametocyte-specific factor 1, is the unmatched capability of NMR spectroscopy to decipher molecular details within dynamic protein systems. First, the molecular structure and RNA-binding properties of gametocyte-specific factor 1 (GTSF1) of the piRNA effector pathway were investigated. A partially disordered protein with two Zn finger domains, the work presented here describes the isolation of a GTSF1 protein construct amendable to study by NMR spectroscopy. Chemical shift assignment of GTSF1 allowed site-specific observation of amide correlations, which established the basis for NMR structure calculation of GTSF1 and the evaluation of binding to candidate RNA sequences, with goal of the identification of an in vivo RNA binding partner for GTSF1. The work presents compelling data that indicate GTSF1 Zn finger 1 specifically binds a motif GGUUC(G/A) RNA, which in this study was found in the T-arm loop of transfer RNA. Zn finger 2 is affected by the interaction with RNA, but the available structural and binding data indicate that the second Zn finger is a more dynamic, breathable entity, supported by cysteine chemical shift and structural differences between the two GTSF1 Zn fingers. Although it’s currently speculative, the function of GTSF1 might first require binding of RNA to the more stable Zn finger 1, which then leaves Zn finger 2 poised for binding to another molecular species. tRNA-derived fragments that include the T-arm TC loop have been recently implicated in silencing of transposable elements in mammalian cells. GTSF1, which was identified in a genetic screen for piRNA-pathway proteins as vitally required for gene silencing, might plausibly act as a sensor of transcription of transposable elements and help initiate Piwi-piRISCs-mediated chromatin modification and heterochromatin formation. Next, NMR spectroscopy is used to investigate protein thermostability in psychrophilic (cold-loving) cytochrome c552. Isolated from the bacterium Colwellia psychrerythraea (Cp), previous work has implicated two conserved Cpcyt c552 methionine residues, which are both conserved across psychrophilic and psychrotolerant cytochromes, as acting in dynamical ligand substitution with a third methionine that is the axial heme ligand. It is proposed that elevated backbone dynamics in these methionine residues and the ability for them swap into the axial ligand position accounts for an uncharacteristically high melting temperature (Tm) compared to meso- and thermophile c-type cytochromes. Progress was made in NMR sample preparation and backbone chemical shift assignment of both redox states of Cpcyt c552, and insight from 1D 1H NMR experiments focused on the heme group bound to Cp cytochrome c552 is discussed. Additionally, chemical shifts are used to predict protein dynamics as a first test of a multiple methionine axial ligand hypothesis. Initial data analysis predicts relatively large measures of Random Coil Index for residues surrounding the native axial heme ligand, and shows the hyperfine shifts localized to the residues surrounding the heme. Future experiments will selectively record methyl group dynamics of methionine residues for elucidation of rate constants of methionine substitution and to determine the structural properties of this minor conformation. Finally, two NMR methodology studies are presented in this thesis: a novel simultaneous-acquisition TROSY pulse sequence for measurement of backbone spin relaxation rates (R1 and {1H}-15N heteronuclear NOE) and a side-chain 2H spin relaxation method for using multifield experimental datasets for better sampling of the spectral density function. Together, these pulse sequences represent significant advancements in NMR measurement of microscopic rate constants and more nuanced detail of protein dynamics.