Carbonic anhydrases (CAs) are fundamental and ubiquitous enzymes that catalyse the reversible hydration of CO₂ to form HCO₃^- and H⁺ ions. Evidence derived from heterologous expression systems has led to the proposal of a novel role for CA in intracellular pH regulation, where its physical and functional coupling to membrane H⁺ -equivalent transport proteins appears to enhance their activity. It has yet to be established whether such a functional association occurs naturally in wild-type cells. Additional evidence on CA activity in-vitro, has also suggested that certain CA isoforms are regulated by physiological changes of pH, an effect that may then affect their ability to enhance H⁺ -equivalent transport. No information, however, exists on the pH sensitivity of CA in intact cells. Finally, pharmacological inhibition of CA activity has been reported previously for various compounds, in addition to those designed specifically as CA inhibitors. It is possible that some compounds, currently used to inhibit membrane H⁺ transport, may also target CA. The present work has examined functional aspects of CA activity in ventricular myocytes isolated enzymically from rat heart, focusing on the potential role of C A in controlling sarcolemmal Na⁺/H⁺ exchange (NHE) and sarcolemmal Na⁺-HCO₃^- cotransport (NEC). NHE and NEC activity were estimated from the rate of recovery of intracellular pH (pH_i), following an intracellular acid load in myocytes loaded with carboxy-SNARF-1 (a pH-sensitive fluorescent dye, used to measure pH_i)). In other experiments, in-vitro CA activity was assessed from the time-course of pH change after addition of CO₂-saturated water to a buffered solution containing either CA II or a cardiac homogenate. In further experiments, intracellular CA activity was assessed from the rate of CO₂-induced fall of pH_i. Three major results emerged, (i) In intact myocytes, CA activity doubles acid extrusion on sarcolemmal NBC, but has no effect on NHE activity. Facilitation of NBC activity by CA is likely to be mediated by an intracellular CA isoform. (ii) In-vitro and intracellular CA activity displays strong pH-dependence within the physiological pH range, activity declining with a fall of pH. (iii) The NHE inhibitor, cariporide, the bicarbonate transport inhibitors DIDS (4,4'- diisothiocyanatostilbene-2,2'-disulphonic acid) and S0859 (an experimental compound from Sanofi-Aventis), and the aquaporin blocker, pCMBS (p-chloromercuribenzene sulphonate), all showed strong inhibitory activity towards CA in-vitro, but had no effect on intracellular CA activity. Overall, the work provides the first clear demonstration of a functional role of CA activity in H⁺-equivalent transport in a wild-type cell. CA thus represents an important regulatory mechanism of H⁺ -equivalent transport. The pH sensitivity displayed by in-vitro and intracellular CA activity may also have significant functional consequences for pH_i regulation. CA inhibition by various membrane transport inhibitors highlights the need for careful drug and experimental design, to avoid secondary inhibition of CA activity and its side-effects. The present work thus provides insight into the functional roles of CA, plus important new information on the enzyme's pharmacological properties.

Picornaviruses replicate in association with cytoplasmic membranes of infected cells. Poliovirus 2C and 2BC play an important role in the formation of membranous vesicles, and induce dramatic changes in membrane trafficking. Theiler’s murine encephalomyelitis virus protein 2C was localized in infected cells using an anti-TMEV-2C antibody. Early upon infection, TMEV 2C was localized in the cytoplasm in an ER-like pattern. At later stages, 2C redistributed to a juxtanuclear site, which represents the viral replication site. Co-localization with the Golgi complex could not be observed. TMEV 2C seems to interact in vitro with reticulon 3, a highly conserved ER-associated protein. It was not possible to confirm a previously identified interaction with AKAP10, a protein kinase anchoring protein, presumably reflecting conformational constraints of the interaction. Two mutations in the AKAP10 binding site of TMEV 2C were identified, which inhibit the completion of the infectious cycle of TMEV. The intracellular changes that occur during TMEV infection were observed. Both actin filaments and microtubules may be used at early stages of infection; however both cytoskeleton components accumulate at the periphery of the cell during late stages of infection. A computer- based analysis has demonstrated that TMEV 2C is highly similar to katanin, a microtubule- severing protein, and may play a similar role in the reorganization of microtubules during infection. The Golgi complex turns from a solid, crescent-shaped organelle, into a series of punctuate fluorescent points forming an expanding balloon-like structure surrounding the concomitantly expanding site of virus replication. The remnants of the Golgi complex are finally dispersed throughout the cytoplasm. Live imaging confirmed these findings. It was observed that PKA also undergoes displacement to the cell periphery during infection. However, BIG1 seems to locate to the viral replication site during infection, suggesting it may play a role during viral replication. The localization of PKA and BIG1 in the infected cell may in part explain the observed dispersion of the Golgi complex.

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

A Dissertation Submitted to the Department of Physiology, University of the Witwatersrand, Johannesburg for the Degree of Master of Science. / Extensive disruption of muscle fibres has been shown to occur after short term eccentric exercise where high mechanical forces are generated. This study tested whether downhill running acts as a stimulus for inducing eccentric damage, and results in greater muscle damage and deterioration in muscular performance than an equal workload of uphill running. The study aimed at determining whether an adaptation or training eflect takes place such that the muscle is more resistant to the damaging effects of a repeated bout of the same exercise. In addition, the study aimed at determining whether the lower muscle volumes and forces of muscular contractions in females compared to males, makes females less susceptible to the damaging effects of eccentric contraction. / IT2018

Statistical Mechanics describes the macroscopic behavior of a system through the analysis of its microscopic components. It is therefore a framework to move from a probabilistic, high-dimensional description of a system to its macroscopic description through averaging. This approach, now one of the pillars of physics, has seen successes in other fields, such as statistics or mathematical finance. This broadening of the applications of statistical physics has opened new avenues of research in the field itself. Ideas from information theory, differential geometry, and approximate computation are making their way into modern statistical physics. The research presented in this dissertation straddles this boundary: we start by showing how concepts from statistical physics can be applied to statistical learning, then show how modern statistical physics can provide insights into molecular transport phenomena. The first three chapters focus on building an understanding of statistical learning as a thermodynamic relaxation process in a high-dimensional space: in the same way that a statistical mechanical system is composed of a large number of particles relaxing to their equilibrium distribution, a statistical learning system is a parametric function whose optimal parameters minimize an empirical loss. We present this process as a trajectory in a high-dimensional probability Riemannian manifold, and show how this conceptual framework can lead to practical improvements in learning algorithms for large scale neural networks. The second part of this thesis focuses on two applications of modern statistical mechanics to molecular transport. First, I propose a statistical mechanical interpretation of metabolon formation through cross-diffusion, a generalization of the reaction-diffusion framework to multiple reacting species with non-diagonal terms in the diffusion matrix. These theoretical results are validated by experimental results obtained using a microfluidic system. Second, I demonstrate how fluctuation analysis in motility assays can allow us to infer nanoscale properties from microscale measurements. I accomplish this using computational Langevin dynamics simulations and show how this setup can be used to simplify the testing of theoretical non-equilibrium statistical mechanics hypotheses.

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Typescript (photocopy) / v, 179, viii leaves, [3] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1983

Bibliography: leaves 174-210 / 210 leaves, [5] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Plant Physiology, 1987

Thesis (Ph. D.)--School of Pharmacy. University of Missouri--Kansas City, 2006. / "A dissertation in pharmaceutical sciences and pharmacology." Advisor: Srikumaran Melethil. Typescript. Vita. Title from "catalog record" of the print edition Description based on contents viewed Dec. 20, 2007. Includes bibliographical references (leaves 159-192). Online version of the print edition.