A Dissertation Submitted to the Faculty of Science University of the Witwatersrand, Johannesburg In fulfilment of the requirements for the Degree of Doctor of Philosophy / Living cells are highly auto-dynamic entities which means that the underlying biochemistry is equally dynamic, a reality which is ignored by most researchers. Theoretical studies indicate that such a state must be due to the existence of oscillatory variations in the levels and activities of key components in the cell. In this study, the dynamic behaviour of four major, interrelated areas of cell biochemistry (phosphorylation, dephosphorylation, the terminal reaction of glycolysis and the amount of soluble protein) were examined and all systems found to oscillate in murine erythroleukaemic cells (MEL) and, where examined, also in the human HL-6Q leukaemic cell line. certain processes have been shown to be oscillatory for the first time ( phosphorylation potential, the lactate dehydrogenase active isozyme level and aspects of the regulation thereof). While others have been shown to occur at a higher frequency than previously reported (phosphotyrosine phosphatase activity, the activity and apparent isozyme pattern of lactate dehydrogenase, the amount of extractable protein). All rhythms are shown (for the first time) to be complex and to involve several contributing periodicities, some modulating the period and amplitude of the observed oscillation. The frequencies are very high (periods of 1-20 minutes and probably Less) and the amplitudes are equally high (variations in magnitude of as much as a hundred fold). Phosphorylation processes, currently of particular interest with regard to the nature and control of cell proliferation are thus found to be more highly dynamic than previously believed, a fact which throws some doubt on the current ideas on cell proliferation. The actual lactate dehydrogenase (LDH) active isozyme pattern is shown not to be constant (as generally believed) but to vary at high frequency (possibly due to phosphorylation of the the enzyme) while the kinetics and specificity of the lone isozyme in murine erythroleukaemic cells appear to be varying at equally high frequency due to the action of regulators (perhaps arising elsewhere within the glycolytic pathway). Similar results were obtained with HL-60 leukaemic cells with at least two of the isozymes varying in level, to some extent independently. The hormone, insulin, and the inducer of cell differentiation, HMBA (hexamethylenebisacetamide), have been found to affect the dynamics of the four systems although, because of the complexity of the rhythms the actual effects on the dynamics are not easily defined. Insulin has a marked effect on the mean level of the activity of the LDH isozyme. The fact that all oscillations are seen despite no attempt being made to synchronise the cell population suggests the existence of communication between cells but how this can occur when the rhythms are of such high frequency is intriguing. All the results add further support for the long standing view of my supervisor, that the properties and behaviour of cells reflect the internal dynamics and that differentiation, cancer and intracellular signalling occur through changes in the pattern of temporal organisation of cellular oscillations. / AC2018

A Dissertation Submitted to the Faculty of Science University of the Witwatersrand, Johannesburg In fulfilment of the requirements for the Degree of Doctor of Philosophy Johannesburg 1994 / Living cells are highly auto-dynamic entities which means that the underlying biochemistry is equally dynamic, a reality which is ignored by most researchers. Theoretical studies indicate that such a state must be due to the existence of oscillatory variations in the levels and activities of key components in the cell. In this study, the dynamic behaviour of four major, interrelated areas of cell biochemistry (phosphorylation, dephosphorylation, the terminal reaction of glycolysis and the amount of soluble protein) were examined and all systems found to oscillate in murine erythroleukaemic cells (MEL) and, where examined, also in the human HL-6Q leukaemic cell line. [Abbreviated Abstract. Open document to view full version] / MT2017

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Voltage converters or charge pumps find their use in many circuits. They are extensively used in hand held devices as cell phones, pagers, PDA's and laptops. Some of the important issues relating to design of voltage regulators for handheld devices are size, efficiency and noise. Another important factor to be considered is the discharge characteristic of the various batteries used by the handheld devices. This thesis addresses the issues of tones present in the conventional switched capacitor voltage regulator. An alternate architecture with a delta-sigma control loop to eliminate this problem is proposed. Also discussed is a method to compute the efficiency of switched capacitor charge pumps. A test chip implementing the new architecture was fabricated in a 0.72-micron CMOS process. The results of the test chip verify the improved architecture. / Graduation date: 2002

Ro ribonucleoproteins are antigenic protein-RNA particles that are the major targets of the immune reaction in autoimmune disorders like systemic lupus erythematosus. The Ro protein has been implicated in cellular RNA quality control, due to its preference for binding misfolded non-coding RNAs such as pre5S ribosomal RNAs and U2 small-nuclear RNAs besides binding cytoplasmic RNAs called Y RNAs. Although well characterized in eukaryotes, an understanding of Ro in prokaryotes is lacking. To gain structural insight into Ro-RNA interactions we have determined a high resolution crystal structure of Rsr, a Ro ortholog from the bacterium D. radiodurans. The structure of Rsr reveals two domains- a flexible, RNA binding HEAT repeat domain and a cation binding vonWillebrand factor A domain. Structural differences between Rsr and Xenopus laevis Ro at the misfolded non-coding RNA binding site suggest a possible conformational switch in Ro that might enable RNA binding. Structural and biochemical characterization reveals that Ro binds cytoplasmic small RNAs called Y RNAs with low nanomolar affinity, to form ~700kDa multimers. Formation of these multimers suggests one possible mode by which Ro RNAs may be targeted towards downstream processing events. Metal responsive transcriptional regulators sense specific metals in the cells and regulate the expression of specific operons involved in export, import or sequestration of the metal. CsoR is a copper(I) specific transcriptional regulator of the cso operon which consists of a putative copper export pump, CtpV. In copper limiting conditions, CsoR binds the operator/promoter region of the cso operon. In increased concentrations of copper (I), CsoR binds copper (I) with high affinity and is released from the operator/promoter site, causing derepression of the cso operon. To gain structural insight into CsoR function, we have solved the crystal structure of copper(I) bound CsoR. The structure reveals a homodimer with a subunit bridging copper site. The trigonal planar geometry and the presence of cysteine and histidine ligands at the metal site are favorable for copper(I) binding. The structure reveals a novel DNA binding fold in CsoR, making it the founding member of a new structural class of metalloregulators.

Ro ribonucleoproteins are antigenic protein-RNA particles that are the major targets of the immune reaction in autoimmune disorders like systemic lupus erythematosus. The Ro protein has been implicated in cellular RNA quality control, due to its preference for binding misfolded non-coding RNAs such as pre5S ribosomal RNAs and U2 small-nuclear RNAs besides binding cytoplasmic RNAs called Y RNAs. Although well characterized in eukaryotes, an understanding of Ro in prokaryotes is lacking. To gain structural insight into Ro-RNA interactions we have determined a high resolution crystal structure of Rsr, a Ro ortholog from the bacterium D. radiodurans. The structure of Rsr reveals two domains- a flexible, RNA binding HEAT repeat domain and a cation binding vonWillebrand factor A domain. Structural differences between Rsr and Xenopus laevis Ro at the misfolded non-coding RNA binding site suggest a possible conformational switch in Ro that might enable RNA binding. Structural and biochemical characterization reveals that Ro binds cytoplasmic small RNAs called Y RNAs with low nanomolar affinity, to form ~700kDa multimers. Formation of these multimers suggests one possible mode by which Ro RNAs may be targeted towards downstream processing events. Metal responsive transcriptional regulators sense specific metals in the cells and regulate the expression of specific operons involved in export, import or sequestration of the metal. CsoR is a copper(I) specific transcriptional regulator of the cso operon which consists of a putative copper export pump, CtpV. In copper limiting conditions, CsoR binds the operator/promoter region of the cso operon. In increased concentrations of copper (I), CsoR binds copper (I) with high affinity and is released from the operator/promoter site, causing derepression of the cso operon. To gain structural insight into CsoR function, we have solved the crystal structure of copper(I) bound CsoR. The structure reveals a homodimer with a subunit bridging copper site. The trigonal planar geometry and the presence of cysteine and histidine ligands at the metal site are favorable for copper(I) binding. The structure reveals a novel DNA binding fold in CsoR, making it the founding member of a new structural class of metalloregulators.

Thesis (M.Eng.), Memorial University of Newfoundland, 2000. / Bibliography: leaves 115-118.

published_or_final_version / Electrical and Electronic Engineering / Master / Master of Philosophy

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