The work presented in this thesis has two parts. The first part deals with methodological developments in the area of solid-state NMR, relevant to the study of partially ordered systems. Liquid crystals are best examples of such partially ordered systems and they are easily oriented by the magnetic field used for the NMR study. They provide spectra rich in information useful for the study of structure and dynamic s of the oriented molecule. Dipolar couplings and anisotropic chemical shifts are relatively easy to obtain for these systems. However, the methodologies used for extracting the required information are constantly undergoing change, with newer ideas being used for optimal use of the technique and increasing the sensitivity of the methodology. In this thesis, existing methods used for obtaining dipolar couplings from oriented liquid crystalline samples are examined in detail and conditions for optimal use of the methods are investigated. Different approaches for enhancing the sensitivity of the techniques are also proposed. Estimation of chemical shift anisotropy of carbons for a molecule that is used as a building block for several mesogens has been obtained and its utility for estimating the order parameters of the system have been examined. The second part of the thesis deals with the application of solid state NMR methods to the study of a number of novel liquid crystalline systems and for the estimation of dynamics, order and orientation of the mesogenic molecules in the magnetic field. Chapter-2 deals with a detailed and systematic study to improve the sensitivity of cross-polarization based separated local field (SLF) NMR spectroscopy techniques such as PISEMA(Polarization Inversion Spin Exchange at the Magic Angle) and PDLF(Pro-ton Detected Local Field). The chapter has been further divided into three sections. Section-A describes the optimization procedure for cross-polarization period for reducing zero-frequency peaks in SLF experiments. Polarization Inversion(PI) is one of the important components of PISEMA and plays a crucial role in enhancing the dipolar cross-peaks and suppressing the axial-peaks. Shortening this period has the advantage of less r.f. power input into the system, thus less susceptibility to sample heating. Therefore it is crucial to arrive at the optimum condition for which maximum sensitivity and resolution are obtained. A detailed experimental investigation of the role of the initial po1arization period has been carried out for two different samples of static oriented liquid crystalline material at two different temperatures and a contact time of 2ms has been found to be optimal for such samples. Insection-B of this chapter, the initial preparation period of the experiment is considered as a possible means of increasing the sensitivity of the experiment. Thus the use of cross-polarization via the dipolar bath by the use of a diabatic demagnetization in the rotating frame(ADRF-CP) has been proposed to be incorporated into PISEMA. To understand the CP dynamics, magnetization in double-and zero-quantum reservoirs of an ensemble of spin-1/2 nuclei and their role in determining the sensitivity the experiments have been theoretically examined. Experimentally, a modification incorporating ADRF-CP is shown to result in enhancement of signal-to-noise by as much as 90% in the case of rigid single crystals of a model peptide and up to 50% in non-rigid, partially ordered liquid crystalline systems. In section-C another useful SLF technique known as PDLF spectroscopy has also been examined. In this case a sweep of one of the r.f. amplitudes(RAMP-CP),rather than ADRF is found to work well. The reason for the different behaviours has been discussed. Chapter-3 highlights two experimental approaches used to extract the chemical shift anisotropy(CSA) tensor information from rotating solids. Section-A is devoted to the measurement of the CSA values of thiophene by using MAS side band analysis, by extracting the principal values from the intensities of just a few spinning side bands. Experiments have been performed on thiophene-2 carboxylic acid and thiophene-3 carboxylic acid samples and the carbon CSA values have been obtained. In section-B, CSA values of carbons of the core unit of the liquid crystal4- hexyloxybenzoic acid (HBA) have been obtained by using the recoupling pulse sequence SUPER(Separation of Undistorted Powder patterns by Effortless Recoupling).HBA belongs to an important class of thermotropic liquid crystals which are structurally simple and often used as starting materials for many novel mesogens. As this molecule could serve as an ideal model compound, high resolution13C NMR studies of HBA in solution, solid and liquid crystalline phases have been also undertaken. The CSA values obtained from the 2D SUPER experiment showed good agreement to those computed by DFT calculations. The CSA values were used for obtaining the order parameter of the system at different temperatures. These matched well the order parameter obtained from the 13C-1 H dipolar couplings in the nematic phase determined by SLF spectroscopy at various temperatures. A knowledge of the CSA of the carbons is thus very useful, as they can be used for gaining knowledge about the system from the chemical shifts obtained from a simple 1D spectrum. In chapter-4, 1-and 2-dimensional13CNMR techniques have been utilized to obtain extensive information about some novel mesogenic molecules. Four molecules of different structure and topology have been taken up for study. These molecules have the following features. Mesogen-1 has a terminal hydroxyl group. Such systems with further modification can result in mesogenic monomers for side chain liquid crystalline polymers. Mesogen-2 has a dimethyl amino group at one end and has three phenyl groups connected by appropriate linking units that form the core. In the third case, mesogen-3, the terminal hydroxyl group of mesogen-1 is replaced with a hydrogen such that13C-1 H dipolar couplings provide directly information on molecular ordering and orientation. In the fourth case, mesogen-4, the core is built with four phenyl rings. Here the fourth ring is linked to other three rings via a flexible chain unit. In each of these cases the 2DSLFNMR experiments have been carried out where13C-1 H dipolar couplings as well as13C chemical shifts were used for obtaining the order parameters of various segments of the molecule. The data provide useful insight into the phase behaviour, ordering and orientation of the molecules. Chapter-5 discusses the applications of the natural abundance 13CNMR techniques to thiophene based mesogens, that have the potential for use in molecular electronics material. Typically, these molecules consist of phenyl rings appropriately connected by linking units with thiophene. Different core units as well as different linking units to thiophene have been considered. The six mesogens thus obtained have been investigated in detail using 1D and 2D NMR methods.13C-1 H dipolar couplings have been used to obtain ordering information, that show interesting correlation to the molecular orientation and dynamics.

The work of this dissertation has been to prove that the coherence of multiphoton excitation can be studied by an appropriately phased and time delayed sequence of pulses. An application of this fundamental study of coherence has been made for the enhancement of third harmonic generation. The coherent recovery of the energy lost to the two photon absorption process enalled a larger propagation distance for the fundamental than in an interaction which is incoherent or coherent, but not using a 90 degree phase shifted pulse pair. Phase matching over this longer propagation distance gave an enhancement of third harmonic generation.

Philosophiae Doctor - PhD / The Subatomic Physics Department of iThemba Laboratory for Accelerated Based Sciences (iThemba LABS) conducts experiments that require a variety of particle beams in order to study nuclear properties (reaction, structure, etc.) of various nuclides. These particle beams are accelerated using the K-200 Separated Sector Cyclotron (SSC) and delivered to different physics experimental vaults. Prior to acceleration, the particle beam is first ionised using an Electron Resonance Ion Source (ECRIS). The main goal of this study is the production of exotic metallic beams of 60Ni8+ and 62Ni8+ using ECRIS4, which are required for the Coulomb excitation experiments approved by the Programme Advisory Committee (PAC) at iThemba LABS. In order to provide the metallic beams of nickel, a development study of organometallic materials containing 60Ni and 62Ni isotopes in a form of metallocene complexes was undertaken. The nickelocene (NiCp2) complex, a member of the organometallic family, was synthesised at the Physics Target Laboratory of iThemba LABS for the first time. Method development involved the use of natural nickel during the multi-step synthesis before the use of enriched nickel-60 (60Ni) and nickel-62 (62Ni). Nine samples of NiCp2 were synthesised; two were isotopically enriched nickelocene (60NiCp2 and 62NiCp2). The percentage yields of the synthesised nickelocene samples ranged between 16 to 50 %, and samples were characterised by investigating their crystal structure and bonding arrangements in the complexes by X-ray diffraction (XRD) , Fourier Transform Infrared (FT-IR) spectroscopy, and Proton Nuclear Magnetic Resonance (1H NMR). The synthesised nickelocene were further used with ECRIS4 for the production of Ni beams on the Q-line of the cyclotron. The Metal Ions from Volatile Compounds (MIVOC) technique was used for the conversion of 60Ni and 62Ni to ion species. The method used the organometallic compounds which are volatile at specific pressures at ambient temperatures. Metallic ion beams of nickel were successfully produced after a carefully pre-sample conditioning in the MIVOC container before connecting the MIVOC set-up to the new injection system of the ECRIS4. Measured beam intensities during the experiment for both 60Ni+ and 62Ni+ were approximately 30 μA, optimum for physics measurements. The development of the MIVOC technique opens up new beam-target combinations with the use of new exotic stable beams for new science cases at iThemba LABS. Reactions in inverse kinematics, multi-step Coulomb-excitation and other types of reactions will immensely benefit from these developments.

Xu, Xiao-ping. / "March 1999." / Thesis (Ph.D.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (p. 152-166). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

The thesis aims at methodological developments in Nuclear Magnetic Resonance (NMR) and study of oriented samples like liquid crystals and single crystals and powder samples. Though methodological development in solid state NMR (ss-NMR) has gone far ahead, this work attempts to contribute some novel methods in this direction. The work presented here falls into two categories (i) methodological developments for obtaining information on anisotropic interactions and (ii) experiments which utilize the existing methodologies to study systems of interest under static condition and also under sample spinning at the Magic Angle. In the solid state, dipolar couplings play a crucial role. On the one hand these couplings could be used to transfer polarization from an abundant nucleus to a rare nucleus and increase the sensitivity of the rare nucleus. On the other hand, the measurement of dipolar couplings itself is crucial for extracting structural and dynamic information. A third aspect is that dipolar couplings could be used to obtain correlation, say between two different nuclear species or between the same kind of nuclei as in an exchange experiment. A major part of this thesis deals with all three aspects mentioned above. The thesis presents a new heteronuclear polarization transfer scheme which is devoid of some of the short comings of the existing and well-known polarization transfer schemes. This pulse sequence has been found to be useful in different contexts involving both spin ½ and spin 1 nucleus. The use of dipolar couplings for obtaining correlation in both static oriented systems and in powder samples has been illustrated. In the case of the powder sample, the study has been useful in obtaining useful orientation information. Finally, chemical shifts are known to be indicators of finer structural features of molecules in solution and solid state. 13 C MAS NMR studies have been exploited in understanding these structural features of short peptides containing prolines in the solid state and for comparing with their structures in solution. Chapter 1 covers the theoretical aspects required for the experimental work described in the thesis. A brief description of NMR has been followed by the explicit description of various interaction Hamiltonian’s in ss-NMR. Subsequently the experimental and the theoretical tools needed for ss-NMR study like Magic Angle Spinning (MAS), Cross-Polarization, Homo/Heteronuclear decoupling schemes have also been discussed. Chapter 2, describes a new heteronuclear polarization transfer scheme for oriented samples – named DAPT (Dipolar Assisted Polarization Transfer) and its application to different systems. DAPT uses a homonuclear decoupling sequence such as BLEW-12 for effecting heteronuclear polarization transfer. The chapter has been divided into five related parts. Section 2(A) starts with an introduction to the existing heteronuclear polarization transfer schemes. Subsequently the theoretical background of the new sequence is presented. Experimental implementation of the sequence in an oriented system, liquid crystal is presented and is compared with the well-known polarization transfer scheme, Hartmann-Hahn Cross Polarization (HH-CP). In 2(B) the implementation of the sequence as a local field spectroscopy for measuring heteronuclear dipolar couplings is presented. After initial discussion about local field spectroscopy and its relevance in ss-NMR, the improvements made in the earlier mentioned sequence along with its 2D implementation in a liquid crystal sample are described. A comparative study is also presented using DAPT with various other homonuclear decoupling sequences. Chapter 2(C) deals with the extension of DAPT to spin-1 systems. The difficulties in setting up the HH-CP in spin-1 systems are highlighted. Experimental demonstrations on a test sample of oriented CD3 I and also on a deuterated liquid crystal is described. The sequence has been incorporated as part of a 2D correlation experiment, where the F1 dimension provides the quadrupolar couplings of deuterium and the F2 the chemical shifts of the attached carbons. The comparison of the sequence with HH-CP, its merits and demerits are discussed and the potential applications are highlighted. Chapter 2(D) deals with the relatively less studied transition in 14N nucleus, known as the Overtone-Transition (OT). An introduction to OT and its relevance is provided in the beginning followed by the extension of DAPT in exciting and detecting OT. The experiments have been done on a single crystal of a model peptide, N-Acetyl-DL-Valine and are compared with the conventional method. Amide proton chemical shifts are also measured using DAPT in an indirect way. The advantages and the future application in studying OT are also discussed. Chapter 2(E) discusses the extension of DAPT to the single crystal of NAV and in identifying the molecules in the unit cell. The SLF spectrum of NAV is complicated due to the presence of two magnetically in-equivalent molecules in the unit cell and with pairs of splitting for each C - 1H and C - 1H pairs. The dipolar couplings are extracted from the experiment and with the aid of a MATLAB program and by incorporating the crystal coordinates, identification of C-1H and C-1H pairs belonging to a particular molecule have been carried out. Chapter 3 describes a novel and useful modification of the well-known Separated Local Field (SLF) sequence in solid state known as PISEMA (Polarization Spin Exchange at the Magic Angle). PISEMA is a popular technique for measuring heteronuclear dipolar couplings in oriented in oriented biological membranes and in liquid crystals. While it has several advantages such as a large dipolar scaling factor, narrow line-widths in the dipolar dimension and ease of setting up etc it suffers from a major problem. The technique is highly sensitive to the proton off-sets which affect the measured dipolar couplings. In the present chapter the origin of this problem has been analyzed in detail and a solution has been proposed. The modification to the experiment has been implemented on a liquid crystal and the off-set independence of the new sequence has been demonstrated. Further studies on a more rigid system such as a solid single crystal has been used to verify the effect of the modification on homonuclear decoupling efficiency and the consequent effects on the line widths in addition to off-set independence. The advantages of the proposed method over the existing one in terms of line-width and robustness in measuring heteronuclear dipolar couplings are demonstrated. Chapter 4 presents a study of deuterium exchange on a di-peptide. Deuterium as well as carbon-13 NMR spectroscopy has been extensively used earlier on static powder samples for studying exchange phenomena. In the present study we have applied the methodology for obtaining relative N-D vector orientation in a di-peptide. The magnetization exchange between deuterium nuclei through the dipolar couplings between them has been monitored. The need to match the quadrupolar split energy levels of two different deuterium’s differently oriented in the magnetic field requires that the sample be spun slowly. Characteristic exchange powder patterns were obtained which were used to infer relative orientation information. Comparison with the crystal structure indicates that the magnetization is likely to be inter-molecular rather than intra-molecular. The chapter follows the following sequence. A brief description about the importance of exchange studies in ss-NMR is presented. A theoretical approach is followed by a discussion of the angular dependence of the frequencies and the visualization of the mutual re-orientation angles. The motivation of the project followed by the experimental techniques, especially the use of slow MAS ~ 100 Hz in exchange studies are also presented. Initial studies have been carried out on di-methyl sulphone to check the reproducibility of the earlier reported results and later the sequence is extended to amide proton deuterated di-glycine. The 2D exchange spectrum recorded under slow MAS is then discussed in the context of the crystal structure and possible amide deuteriums involved in the exchange process are inferred. Chapter 5 discusses the natural abundant 13C and 15N NMR spectroscopy in the solid state in designed tri-peptides containing prolines. Proline is a unique amino acid because of it torsion angle values and is responsible for the turns and the globularity of the proteins. The well studied SH3 domain protein often binds to short peptides containing prolines and hence these study gains importance. Three peptides containing prolines were taken up for study. For peptide (1), the conformation was observed as cis/trans in the solution state and for the other two peptides it was all trans. The X-ray studies showed that peptide (1) has two molecules in the unit cell with both cis conformation. This motivated us to look at the solid state spectra of the peptides. Chemical shifts are signatures of conformers and it was established from the chemical shift differences that there exist two molecules in the unit cell for peptide (1), both in cis conformation. The conformers for the other two peptides predicted by NMR chemical shifts also matched with those obtained from X-ray studies. This opens up the possibility of using simple NMR measurements in the solid state as tools for secondary structure determination in larger peptides and proteins.

Different aspects of natural organic matter composition and cycling have been studied using solid-state 13C and 31P Nuclear Magnetic Resonance (NMR) spectroscopy. Depending on the specific study, complementary analytical techniques such as elemental, isotopic and molecular analyses have also been applied. Samples from a variety of environments were examined including ocean waters, marine sediments and atmospheric aerosols. Studies from all these environments illustrate differences in natural organic matter composition resulting from various factors such as sources, cycling mechanisms and redox conditions. In the marine water column, organic matter of two different size fractions (dissolved and particulate) is found to have distinctly different bulk chemical and isotopic compositions. Overall, this indicates that particulate organic matter does not form from the simple physical aggregation of dissolved organic matter, and dissolved organic matter is not the primary source for particulate organic matter. Comparison of carbon and phosphorus compositional changes with depth in the ocean within the dissolved and particulate fractions reveals differences in cycling mechanisms. In the marine water column, selective mineralization of specific carbon compounds such as carbohydrates and amino acids occurs relative other species such as lipids. Whereas for phosphorus, the relative proportion of the different functional groups are unvarying with depth. In marine sediments, NMR spectroscopy reveals P cycling for specific phases such as polyphosphates is a function of sediment redox conditions. In atmospheric aerosols 13C NMR spectroscopy shows differences in water-soluble organic carbon composition from urban versus biomass burning sources. Urban aerosols have higher aliphatic and lower aromatic compound contents relative to samples derived from biomass burning. The results of these studies provide new insights into carbon and phosphorus cycling in the environment and demonstrate the capabilities of solid-state NMR as a tool for investigating natural organic matter composition.

Le cancer de l’ovaire est la cinquième cause de décès par cancer chez la femme en France. La prolifération tumorale dans ce cancer est liée principalement à la dérégulation de l’apoptose. Cette dérégulation joue un rôle clé principalement dans la pathogénèse et la progression tumorale mais aussi dans le développement des résistances aux chimiothérapies existantes. La croissance accrue des cellules tumorales dans le cas du cancer de l’ovaire est liée à une surexpression des protéinesanti-apoptotiques de la famille Bcl-2, en particulier les protéines MCL-1 et Bcl-xL. L’inhibition concomitante de ces deux protéines conduit à la mort des cellules chimiorésistances. A ce jour, seuls les inhibiteurs de Bcl-xL ont démontré une efficacité en clinique, et l’inhibition de MCL-1 reste problématique dans un contexte clinique. Dans ce contexte, l’objectif principal de ces travaux de thèse consiste à caractériser, par RMN et modélisation moléculaire, l’interaction entre la protéine MCL-1 et le Pyridoclax, foldamère oligopyridinique très prometteur, synthétisé au CERMN. La spectroscopie RMN nous a permis de définir le site d’interaction, d’estimer la constante d’affinité et de guider le Docking pour obtenir une structure tridimensionnelle du complexe MCL-1:Pyridoclax. Afin de conforter notre étude sur l’implication du Pyridoclax dans l’interaction, des fragments dérivés de ce dernier ont été utilisés. La caractérisation de l’interaction entre ces fragments et la protéine MCL-1 a permis de relever l’importance du groupement styryle du Pyridoclax dans l’interaction avec la protéine MCL-1. Par ailleurs, nous avons élargi notre étude à d’autres inhibiteurs potentiels de MCL-1 tels que certains médicaments existant sur le marché. La spectroscopie RMN a permis de mettre en évidence et de caractériser l’interaction entre deux de ces médicaments (le Torsémide et le Déférasirox) et la protéine MCL-1. Pour finir, afin d’améliorer l’affinité de ces médicaments pour la protéine MCL-1, des modifications structurales ont été proposés en se basant sur la littérature. Les dérivés proposés semblent avoir une affinité très importante pour la protéine MCL-1. / Ovarian cancer is the fifth leading cause of cancer death of women in France. Tumor proliferation in this cancer is mainly related to the deregulation of apoptosis. This one plays a main role in pathogenesis and tumor progression but also on the term of development of resistance to existing chemotherapies. The increased growth of tumor cells in ovarian cancer is linked to an overexpression of the anti-apoptotic proteins of the Bcl-2 family, in particular the MCL-1 and Bcl-xL proteins. The concomitant inhibition of these two proteins leads to the death of chemoresistance cells. To date, only Bcl-xL inhibitors have demonstrated clinical efficacy andinhibition of MCL-1 remains problematic in a clinical context. In this context, the aim of this thesis is to characterize, by NMR and molecular modeling, the interaction between MCL-1 protein and potential inhibitors including Pyridoclax, an oligopyridine foldamer. NMR spectroscopy allowed us to define the interaction site, estimate the affinity constant and guide Docking to obtain a three-dimensional structure of the MCL-1:Pyridoclax complex. In order to obtain more information on the involvement of Pyridoclax in this interaction, fragmentsderived from it has been used. The characterization of the interaction between these fragments and MCL-1 protein revealed the importance of the styryl group of Pyridoclax in the interaction with MCL-1 protein. Furthermore, we expanded the scope of our study to include some existing drugs able to interact with MCL-1. NMR spectroscopy allowed us to identify and characterize the interaction between two of these drugs (Torsemide and Deferasirox) and the MCL-1 protein. Finally, in order to improve the affinity of these drugs for MCL-1 protein, structural modifications had been proposed based on the literature. The proposed derivatives appear to have a very high affinity for MCL-1 protein.

Doctor of Philosophy / Biochemistry and Molecular Biophysics / Om Prakash / John M. Tomich / As part of the ongoing development of a channel forming peptide with the potential to be used clinically to treat cystic fibrosis, a number of structural studies using solution NMR spectroscopy have been carried out on a number of the test sequences. Given their structural similarities of the monomers it is important to evaluate whether or not there is a compelling need to determine the solution NMR structure of next-generation peptides. The determination of the NMR monomeric solution structure of peptides NK₄-M2GlyR-p22 and NK₄-M2GlyR-p20 T17R S20W in TFE solution and SDS micelles sample shows predominantly alpha-helical conformations for both sequences with an extended conformation for the N-terminal lysine residues. The I[subscript max], K[subscript 1/2] and Hill coefficient, derived from data on ion conductance across monolayers of MDCK cells, were used to compare the ion conductance properties of the peptide sequences. Peptide NK₄ M2GlyR p20 T17R S20W has both a higher I[subscript MAX] (43.8 ± 2.8 μA/cm²) and a lower K[subscript 1/2] (58 ± 8 μM) compared to other M2GlyR derived peptides with calculated NMR structures. All available molecular structures calculated by NMR for M2GlyR derived peptides were compared and the correlation of the structural changes observed in the NMR structures with the ion conductance changes was evaluated. The NMR structures were found to have limited predicting potential over the ion conduction data. NMR determined structures have provided an experimentally based starting point for studies of the channels formed by the family of M2GlyR peptides. Computer simulations account for inter peptide interactions and packing effects that are not experienced by the monomeric form of the peptides in the NMR samples that have been used until now. The determination of the structure of the oligomeric peptide channels is deemed needed to improve the relevance of future use of NMR in this project. The use of larger membrane mimicking agents, isotopically labeled (¹⁵N, ¹³C) samples, 3D NMR experiments and potentially solid state NMR would be required to accomplish that task.

Submitted in partial compliance with the requirements of the Master's Degree in Technology: Homoeopathy, Durban University of Technology, 2010. / The aim of this study was to compare the Nuclear Magnetic Resonance (NMR) spectra of homoeopathic potencies prepared according to the orthodox Hahnemannian method with those produced with Radionic instrumentation. Methodology The chemical shift values and relative integration values for the H2O, CH2, CH3 and OH peaks of the 6C, 12C and 30C potencies of Hahnemannian and Radionic Natrum muriaticum were compared. The orthodox Hahnemannian method of preparing potencies involves dilution of the crude substance followed by the dilution and succussion at each subsequent deconcentration (potency) level. The Hahnemannian potencies were prepared according to the German Homoeopathic Pharmacopoeia (GHP) and the potencies diluted using a 1:100 ratio and succussed ten times at each potency level. The Radionic group of potencies were prepared using the ‘Magnetogeometric Potency Simulator’ (a Radionic apparatus). NMR testing took place at the Chemistry Department at the University of KwaZulu Natal, Pietermaritzburg using a Bruker Avance lll NMR spectrometer 500MHz.The samples were dispensed into boro-silicate glass NMR tubes with a co-axial tube containing Dimethyl sulfoxide-d6 (DMSO-d6) which was used as a frequency lock around the tube. Three samples were drawn from each group, including the controls, and analysed using the NMR spectrometer. The NMR spectrometer information was received and the chemical shift and relative integration values of H2O, OH, CH2 and CH3 peaks on the NMR spectra recorded. All the data was entered into a Microsoft Excel© 2000 spreadsheet and then from there transferred into SPSS© software package for statistical analysis. The Kruskal-Wallis test was used to make a comparison between the eight unpaired groups. If a significant difference occurred between the groups iii individual comparisons between groups were made using the non-parametric Mann-Whitney test. The significant value was set at α= 0.05. Results The results of this study revealed significant differences between the Hahnemannian and Radionic samples. The chemical shift values of the parallel potencies showed significant differences for the H2O, CH2 and CH3 peaks. A significant difference for the OH peaks was observed between the 30C potencies. The relative integration values showed a significant difference for the OH and CH3 peaks between the parallel 12C and 30C potencies but not between the parallel 6C potencies. Conclusion From studying the results it can be concluded that the respective manufacture methods resulted in the NMR spectra of the parallel potencies being significantly different (exhibiting distinctive physico-chemical properties) thus confirming the hypotheses of the study. The standardisation of the process of preparing homoeopathic remedies is important as different methods produce potencies with distinct physico-chemical identities. Further studies into different methods should be researched in order to control and standardise the production of potencies.

Thesis (MSc)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: The interests in understanding the metabolic processes of microbial systems are numerous. The interest in the species Lactococcus lactis (L. lactis) lies in applications to the food industry and in studies comparing the metabolism of related organisms. The aim of this study was to perform in vivo supply-demand analysis on anaerobically fermenting L. lactis. This was done by perturbing both the supply and demand pathways, then measuring glycolytic flux (by means of 13C NMR spectroscopy) and intracellular ATP/ADP (by means of 31P NMR spectroscopy) at steady state – where the central metabolite, ATP, is produced at the same rate as it is consumed and its concentration thereby remains constant. The L. lactis reference strain MG1363 was supplemented with glucose and analysed “online” by 13C-NMR under anaerobically-fermentative conditions. The rates of glucose consumption and lactate production were determined from this 13C flux. Due to experimental difficulties with the online detection of 31P (possibly due to the low biomass yield and the choice of growth medium), ATP/ADP levels had to be determined offline: from the same batch cultures as the 13C samples, fermentations were performed and halted at time points when the cells had attained a steady state. These fermentation cultures were then subjected to cell lysis and centrifugation in order to extract intracellular metabolites. These cell extracts were analysed offline by 31P NMR in order to determined levels of phosphate metabolites, specifically ATP and ADP. Perturbation of the supply pathway was achieved by utilising a genetically modified strain (the CS8 strain with over-expressed las operon) and comparing it to the reference strain MG1363. This resulted in a slight increase in ATP/ADP, but also yielded a slightly reduced flux, which is contrary to expectations from a mutant with over expressed glycolytic enzymes. The demand pathway was perturbed by two methods: 1) utilisating a genetically modified strain (the BK1506 strain with over-expressed F1-ATPase) and comparing it to the reference strain MG1363, and 2) by treating wild-type MG1363 with sodium acetate and comparing flux and ATP/ADP values to the untreated wild-type. Sodium acetate dissociates in the cytoplasm and causes dissipation of the transmembrane proton motive force, which is re-established by upregulation of membrane-bound H+-translocating ATPases. While the use of genetically modified strains provided only one flux-vs-ATP/ADP data point to compare to the wild-type (not sufficient for complete supply-demand analysis), the treatment of the wild type with uncoupler yielded several data points where flux and ATP/ADP values differed according to the concentration of uncoupler added. The CS8 strain demonstrated a 19 % reduced glucose flux (24 % reduced lactate flux) with respect to the wild type MG1363. The BK1506 strain demonstrated a 72 % increase in glucose flux (33 % increase in lactate flux) with respect to the wild type. The treatment with 2 mM acetate resulted in a 72 % increase in glucose flux (123 % increase in lactate flux), whereas treatment with 4 mM acetate resulted in a 107 % and a 126 % increase in glucose and lactate fluxes, respectively. The treatment with different concentrations of acetate provided several data points with corresponding flux and ATP/ADP, enabling the calculation of the elasticity coefficient of the supply pathway to changes in ATP/ADP (εsupply ) which was found to be -5.6 and -6.3 for glucose and lactate, respectively. ATP/ADP The elasticity coefficient was high compared with values obtained in similar studies on other organisms. Considering that at steady state the supply and demand fluxes are equal, the high supply elasticity (which is easier to measure), when incorporated into control coefficient summation theorems, gives the indication that: 1) a greater amount of control may reside in the ATP demand pathway (the elasticity of which is more difficult to determine experimentally, but which may well be lower than the supply elasticity), and 2) ATP/ADP homeostasis is good, as indicated by a high elasticity of the supply pathway to ATP/ADP. This study represents a basis for further supply-demand analysis with non-growing batch cultures of L. lactis. / AFRIKAANSE OPSOMMING: Daar is groot belangstelling daarin om die metaboliese prosesse van mikrobiese sisteme beter te verstaan. Die belang van die spesie Lactococcus lactis (L. lactis) lê beide in die toepassing in die voedselbedryf en in studies wat die metabolisme van verskeie organismes vergelyk. Die doel van hierdie studie was om in vivo vraag-aanbod analise uit te voer op anaerobies-fermenterende L. lactis. Dit was gedoen deur beide die aanbod en vraag reaksie-blokke te moduleer en dan die glikolitiese fluksie (d.m.v. 13C KMR spektroskopie) en die intrasellulêre ATP/ADP (d.m.v. 31P KMR spektroskopie) in ’n bestendige toestand te meet (wanneer die sentrale metaboliet, ATP, teen dieselfde tempo geproduseer en verbruik word en sy konsentrasie daardeur konstant bly). Die L. lactis verwysing-stam MG1363 is met glukose aangevul en 13C fluksie is aanlyn onder anaerobies-fermenterende kondisies gemeet. Die tempo van glukose verbruik en laktaat produksie is vanaf die 13C fluksie bereken. Eksperimentele probleme met die aanlyn bepaling van 31P (dalk as gevolg van lae biomassa en/of die keuse van groeimedium) moes ATP/ADP vlakke af-lyn indirek bepaal word: fermentasies van dieselfde lot-kulture as die 13C monsters is opgestel en by sekere tydpunte gestop wanneer ‘n bestendige toestand bereik was (waar ATP/ADP konstant bly). Hierdie fermenterende kulture is blootgestel aan sel-lise en sentrifugasie om intrasellulêre metaboliete te onttrek. Dié sel-ekstrakte is deur 31P KMR geanaliseer om die vlakke van fosfaat metaboliete, spesifiek ATP en ADP, te bepaal. Die aanbod blok is gemoduleer deur gebruik te maak van ‘n geneties-gemodifiseerde stam (die CS8 stam met ‘n ooruitgedrukte las operon) en met die verwysing stam MG1363 te vergelyk. Dié gemuteerde stam het ’n effense toename in ATP/ADP getoon, maar het gelyktydig ook ’n afname in glikolitiese fluksie getoon, wat onverwags is vir ’n stam met ooruitgedrukte glikolitiese ensieme. Die vraag blok is met twee metodes gemoduleer: 1) deur gebruik te maak van ‘n geneties-gemodifiseerde stam (die BK1506 stam met ‘n ooruitgedrukte F1-ATPase), en 2) deur die wildetipe MG1363 met natrium asetaat te behandel en daardeur ATP verbruik van biomassa produksie te ontkoppel en die vraag na ATP te vermeerder. Daarna word die fluksie en ATP/ADP waardes met die onbehandelde wildetipe vergelyk. Natrium asetaat dissosieer in die sitoplasma en verswak die transmembraan elektriese potensiaal, wat dan weer versterk word deur membraan-gekoppelde H+-ATPase ensieme wat protone uit die sitoplasma uit pomp. Terwyl die gebruik van geneties-gemodifiseerde stamme net een fluksie-tot-ATP/ADP datapunt voorsien om met die wildetipe te vergelyk (wat nie voldoende is vir totale vraag-aanbod analise nie), het die behandeling van die wildetipe met ontkoppelaar meerdere datapunte voorsien waar fluksie en ATP/ADP waardes verskil volgens die konsentrasie van ontkoppelaar wat bygevoeg is. Die CS8 stam het ’n 19 % verminderde glukose fluskie getoon, asook ’n 23 % verminderde laktaat fluksie, in vergelyking met die wilde tipe MG1363. Die BK1506 stam het ’n 73 % toename in glukose fluskie getoon, asook ’n 34 % toename in laktaat fluksie, in vergelyking met die wilde tipe. Behandeling met 2 mM natrium asetaat het ’n 64 % toename in glukose fluksie veroorsaak, sowel as ’n 124 % toename in laktaat fluksie, en behandeling met 4 mM natrium asetaat het 108 % toename in glukose fluksie en 127 % toename in laktaat fluskie veroorsaak. Behandeling met verskillende konsentrasies natrium asetaat het genoeg data punte (fluksies met toepaslike ATP/ADP waardes) verskaf om die berekening van elastisiteits-koëffisiënt van die aanbod reaksie-blok tot veranderinge in ATP/ADP (εsupply ) te bereken. Die waardes was -5.6 vir glukose fluksie en -6.3 vir laktaat fluksie. ATP/ADP Die elastisiteits koëffisiënt was relatief hoog in vergelyking met waardes wat in soorteglyke studies op ander organismes bepaal is. Aangesien die fluksies van die aanbod en vraag reaksie blokke by ’n bestendige toestand dieselfde tempo het, kan die hoë waarde van die aanbod elastisiteits-koëffisiënt (wat die makliker een is om te meet) na die volgende afleidings lei: 1) meer kontrole mag in die ATP verbruikende reaksie-blok geleë wees (die elastisiteits-koëffisiënt is moeiliker om eksperimenteel te bepaal maar mag wel laer as die aanbod-elastisiteit wees), en 2) ATP/ADP homeostase word goed gehandhaaf, soos aangetoon deur die hoë aanbod-elastisiteit. Hierdie studie dien as ’n basis vir verdere vraag-aanbod analise in nie-groeiende L. lactis lot-kulture.