Bibliography: pages 163-176. / The immunospecific inactivation of bacteriophage is one of the most sensitive methods available for the detection of very low concentrations of antibody. By chemically modifying the phage coat-protein, this sensitivity can be extended to antibodies against a wide variety of haptens and proteins. Phage particles that have been modified by attaching some molecule onto their surface are sensitive to antibodies directed against the coupled chemical moiety. Furthermore, the inactivation of the modified phage by antibody can be inhibited by free antigen, and this provides a sensitive assay for small quantities of antigen. Antibody and antigens have been detected at the nanogram level by this technique. The modified phage technique can also be used to distinguish antibodies of different specificities and to discriminate between closely related antigens. This technique has not yet been applied to the immunochemical study of viral components and the present work represents such an attempt. Tobacco mosaic virus (TMV) was chosen as a model system since it permits the study of numerous inununological phenomena (Rappaport, 1965; van Regenmortel, 1966). A series of preliminary experiments were performed to obtain experience in the methodology of the technique. These included the inactivation of native T4 phage by phage antiserum and anti phage IgG, the chemical modification of phage with DNP and the attachment of lysozyme to phage under a variety of conditions. The success of the covalent binding of protein to phage particles depends on finding conditions under which a proportion of the phage remains viable and, at the same time, can still be neutralized by anti-protein sera. To this end, different proportions of reactants and three different bifunctional reagents were tested. To prevent aggregation of tobacco mosaic virus protein (TMVP) at the high concentration used, the protein was treated with N-bromosuccinimide. TMVP-phage conjugates which were sensitive to antiserum were prepared using bis-diazobenzidine as the bifunctional reagent.

Bibliography: pages 107-111. / Durham, in a review published in 1974, presented the following hypotheses concerning the factors that control amoeboid movement: (1) actin and myosin are present in all cells that exhibit amoeboid movement and, changes in the internal [Ca⁺⁺] regulate contraction, (2) filaments of actin and myosin form an intimate association with the surface membrane and depending on the local [Ca⁺⁺] , the filaments can cause the membrane to relax or become rigid, (3) Ca⁺⁺ fluxes across the external membrane (viz. efflux and influx) regulate the state of contraction in the proposed actinomyosin-surface membrane network and, (4) such Ca++ fluxes operating across the membrane manifest themselves (especially with slime mould plasmodia) as waves of adhesion running across the undersurface of a cell and aid in movement. A working hypothesis, that encompasses the ideas of Durham, is that Ca⁺⁺ entry and efflux across the external membrane control such cellular processes as extension of pseudopodia, exocytosis, endocytosis and the direction of movement (chemotactic response) of amoeboid cells. In the specific case of slime mould plasmodia, which best exemplify all of Durham's hypotheses, the simplest hypothesis to explain the control of chemotaxis is that attractants (sugars, food; organisms) cause a Ca⁺⁺ efflux across the membrane and a subsequent movement forward. Repellents would act in a reverse manner by causing Ca⁺⁺ entry. This hypothesis also allows for the existence of a Ca⁺⁺-accumulating organelle. This organelle might replace or act in concert with the proposed Ca⁺⁺ fluxes across the external membrane. The investigations reported in this thesis were devised to examine experimentally this hypothesis.

Includes bibliographical references. / Eleven bacterial strains that have been isolated from the ostrich hindgut, have successfully been identified with the aid of PCR amplification of the 16S rDNA genes and sequence analysis using the BLASTN search of the GenBank database. The nucleotide sequences were determined either through direct sequencing of the PCR products or after cloning the products into the plasmid vector pBluescript (SK). The results obtained from the 16S rDNA sequence homologies strongly suggests that the newly identified bacteria should be assigned to three major bacterial genera, namely, Ruminococcus, Buryrivibrio and Bacteroides. Four highly cellulolytic strains were identified as ruminococci while two weakly cellulolytic strains, showed highest homology to members of the genus Butyrivibrio. Another weakly cellulolytic strain showed closest identity to the sulfur reducing bacterial genus Desulfovibrio. Four non-cellulolytic strains showed highest homology to the genus Bacteroides.

Bibliography: pages 122-131. / A method was developed to test the toxicity of several metal compounds to selected bacteria. The technique was based on the agar diffusion method. Reliability and reproducibility were enhanced by careful standardisation of experimental parameters. By quantification of the concentration of metal compounds in the media using sequential strips of agar it was .possible to prepare standard graphs (metal concentration vs. distance from disc). These graphs demonstrated the exponential rate of diffusion of metals. The diffusion rates were metal specific. In the case of lead compounds the diffusion rate was poor. A media was developed which allowed lead chloride to diffuse relatively well.

Bibliography: pages 107-112. / This project was then aimed at finding naturally occurring isolates of CMV, characterising them, producing much needed antisera and to use such antisera in a comparison with other well characterised strains by the use of new contemporary sensitive serological techniques.

Bibliography: pages 102-111. / A method for monitoring the growth of Thiobacillus ferrooxidans independently of iron oxidation was developed. This method involved the analysis of protein using the Cobmassie Blue protein-binding assay. It was found to be free from interference by the inorganic ions present in T. ferrooxidans culture medium. The effect of ferrous iron concentrations and mixtures of ferrous and ferric irons on the rate of iron oxidation by T. ferrooxidans in batch culture was determined. The exponential iron oxidation rate was not affected by the ferric iron concentration per se but rather by the ferrousferric ratio.

Bibliography: p. 125-141. / The phytohormone ethylene is essential to many plant developmental processes, of which the control of climacteric fruit ripening is among the best characterised. However this hormone eventually causes fruit rotting which results in a non-marketable product. One approach to reduce ethylene synthesis in plants is metabolism of its immediate precursor, 1-aminocyclopropane-1carboxylic acid (ACC). This can be achieved through degradation of ACC by the enzyme ACC deaminase to form α-ketobutyric acid and ammonia. ACC degrading soil microorganisms were identified by their ability to grow on ACC as a sole nitrogen source. Enzyme assays indicated that Pseudomonas had high ACC deaminase gene-specific primers and probes respectively revealed that only one bacterium, Pseudomonas fluerescens strain 17, had a gene with homology to previously sequenced ACC deaminase genes.

Bibliography : pages 164-179. / Three mutants of Streptomyces cattleya NRRL 8057 with lesions in the DNA repair pathway were obtained by NTG mutagenesis. The mutants were selected on the basis of their resistance or sensitivity to Mitomycin c. Of the three mutants, two, R6 and Rl2, were selected as MMC resistant and one, S26, as MMC sensitive. They were also UVr and UVˢ, respectively. The mutants were subjected to UV irradiation in order to obtain UV kill curves. The curves generated for the two MMCʳ mutants showed induction or derepression of a second repair system. In instability studies performed on them, the two MMCʳ mutants showed increased instability compared to the wild type at low UV dosage. However, the instability dropped to below that of the wild type at high UV dosages. Both these mutants appeared to roughly mimic the plateau shown by the wild type as higher levels of UV irradiation were achieved. S26 in contrast showed a much higher initial incidence of instability, and no plateau was observed at high UV dosages. Single strand breaks were induced in the wild type and mutant strains by incorporation of P³² into their DNA. R6 showed a five-fold increase in instability after this treatment. Rl2 showed no significant change, and S26 showed a slight decrease, which may be due to the increased lethality of DNA damage sustained by this strain due to its relatively labile nature. The strains were further analysed using polyacrylamide gel electrophoresis of total cellular proteins. S26 showed a startlingly different protein profile. R6 was almost identical to the wild type, while Rl2 was extremely similar, showing only the loss of one major band and a significant lowering of the amount of detectable protein in the region below this band.

Immune correlates of protection against Human Immunodeficiency Virus Type 1 (HIV-1) acquisition in humans remain unknown. The role of antibodies in providing protection, either through neutralization to prevent virions from infecting cells or Fragment crystallizable (Fc) effector functions to help clear already infected cells, are poorly understood. Previous work fails to support neutralizing antibodies (nAbs) as an in vivo protective correlate, therefore we sought to investigate whether the Fc effector function, antibody-dependent cellular cytotoxicity (ADCC), was protective. Mother-to-child-transmission (MTCT) cohorts provide a model of passive immunization and viral challenge, because infants acquire antibodies from their mother throughout gestation and breastfeeding and are consistently exposed to HIV. We hypothesized that infants of non-transmitting mothers would be protected due to higher ADCC responses compared to transmitting mother-infant pairs. To address our hypothesis, we developed a cell line susceptible to viruses incorporating HIV envelopes (Envs) isolated from maternal plasma for use in a luciferase-based ADCC assay. Serum samples came from the control arm of the Breastfeeding, Antiretroviral, and Nutrition (BAN) cohort in Malawi where HIV-infected mothers that transmitted (TM) infection to their infants were matched to two non-transmitting (NTM) mothers on parameters known to be important in MTCT, including maternal age, viral load, absolute CD4 count, and time to sample collection. All transmission events occurred during breastfeeding and the samples assessed in this study came from a timepoint prior to documented transmission. HIV-exposed but uninfected (HEU) infants had significantly higher levels of ADCC responses against their corresponding mother’s variants compared to HIV-exposed infected (HEI) infants. Additionally, higher ADCC was associated with reduced morbidity and mortality in infected infants up to 1 year after birth. Although nAbs were previously determined to not play a role in the prevention of MTCT in this cohort, our data suggests that neutralization and ADCC are independent antibody responses that may act in conjunction to prevent transmission. In order to utilize these protective antibodies in a preventative HIV-1 vaccine, the antibodies must recognize a range of Envs to be effective globally. ADCC breadth and potency (ADCCBP) was determined against a panel of heterologous globally diverse variants, unrelated to the infected mother’s Envs. No significant difference in ADCCBP was observed between TM and NTM mother-infant pairs. However, we identified samples with elite ADCCBP, specified as the top 5% of samples tested, and future HIV-1 vaccines may want to elicit the types of antibodies present in these samples. In summary, maternally acquired ADCC correlates with protection against HIV-1 transmission to exposed infants and is associated with lower infant morbidity up to 1 year after birth. Further characterization of antibodies with elite ADCCBP may be helpful in developing an effective vaccine.

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