M.Sc. / It is generally believed that the development of a completely effective vaccine for the human immunodeficiency virus (HIV) will likely require neutralizing antibodies that react with the diverse strains of cell-free forms of this virus, as well as induce cellular responses in the form of cytotoxic T-lymphocytes (CTL), to eliminate cell-associated virus. Vaccines based on viral envelope proteins attempt to induce the former response, whilst DNA/vector based approaches aim to induce CTL. The membrane proximal external region (MPER) of HIV-1 gp41 is a target of two broadly neutralizing human monoclonal antibodies, 2F5 and 4E10, and is an important lead for vaccine design. It is conserved among several strains of HIV-1, except for subtype C where restricted mutations are found, especially in the epitopes of 2F5 and 4E10. Mono- and polyvalent (homologous and heterologous) synthetic peptide constructs of the epitopes recognised by 2F5 and 4E10, based on HIV-1 subtype C, have been designed and their immunogenicity compared in this study. The peptide constructs, designated MPER 1 / 2, a / b, induced humoral immune responses in mice and rabbits with the use of adjuvants. The homologous constructs (designated a) induced better humoral immune responses than the heterologous versions (designated b) in small animals. However the antibodies generated in rabbits were not potent enough to neutralize isolates of HIV- 1. The induction of neutralizing antibodies may be addressed by further conformational considerations, as conjugation to an octameric lysine core was insufficient. The peptide constructs did induce proliferative and inflammatory immune responses in a murine model. Additionally, the peptide constructs were highly antigenic as neutralizing anti-HIV antibodies present in naturally infected sera were able to recognise and bind to the MPER peptides as antigen in ELISAs. This suggests that the peptide constructs may be of value for characterizing anti-MPER antibody responses in infected individuals. The constructs were further able to mimic the true representation of these regions in vivo, as human monoclonal antibodies 2F5 and 4E10 were able to recognize and bind 3 of the 4 constructs. The human anti-MPER antibodies as well as the recombinant monoclonal antibodies had a higher binding affinity for the heterologous constructs. The MPER constructs exhibited many beneficial characteristics and may therefore hold application as a component in HIV-1 preventative and therapeutic vaccination following further modification.

The development of an effective HIV vaccine is hindered by several obstacles. One of the leading challenges is the antigenic variability of HIV-1 that is exhibited throughout all viral gene products but to greatest extent in the viral envelope proteins. This phenomenon is the result of continuous mutations in the HIV genome and is responsible for the immune escape of viral mutants. Many studies have suggested that a multivalent vaccine that elicits broadly cross-reactive antibodies is required to efficiently target antigenic variability. To this end, we have designed and analyzed a synthetic peptide construct that mimicked the major variability exhibited in the V3 loops of HIV-1 subtype C isolates. The peptide construct, described as a multiple epitope immunogen of the V3 loop with 8 branches and termed MEIV3b8, was shown to be non-toxic but highly immunogenic in experimental animals (mice and rabbits) and produced antibodies that were reactive to V3 loop peptides of various subtypes, variant envelope proteins and whole viral isolates [at antibody titers 1000 in enzyme-linked immunosorbent assays (ELISAs)]. Furthermore, functional antibodies were generated in rabbits that mediated neutralization of a neutralization-sensitive HIV-1 isolate and two distinct primary HIV-1 isolates in several different neutralization assays (at antibody titres 1213). Additionally, the MEIV3b8 induced both proliferative and inflammatory immune responses in a murine model.Finally, antibodies in the plasma of individuals (n = 148) infected with HIV-1 subtype C, subtype B and HIV-2 were found to bind to the MEIV3b8 as antigen in ELISAs. Through these findings, this study demonstrated that the variable MEIV3b8 effectively addressed antigenic variability and provided evidence that this peptide construct may hold application in HIV-1 preventative and therapeutic vaccination as well as HIV immunodiagnosis. / Dr. D. Meyer

No description available.

Thesis (Ph.D.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / The in vitro relationship of sialic acids to the A, B, M, N, C, D, and E antigens of the human erythrocyte has been studied by means of the Landsteiner hapten inhibition test with the idea that substances that strongly inhibit anti-D might find practical application in the prevention and treatment of erythroblastosis fetalis. Our results suggest that N-acetyl neuraminic acid (NANA) is a major constituent of the D (Rh0), M, and N agglutinogens, a minor constituent of the A antigen, but is probably not a functional portion of the B, C, and E antigenic structures. [TRUNCATED] / 2999-01-01

Antibodies specific for insulin and human immunoglobulin G (HlgG) were attached to controlled pore glass (CPG) particles which had been silanized with a diol-bearing silane. Up to 20 mg of antibody protein could be attached covalently to 1 gram of CPG. Such immobilized antibodies, or immunosorbents, would bind specific antigens, but not unrelated proteins, when used in a high pressure liquid chromatographic configuration. This technique was given the name "high performance immunoaffinity chromatography" (HPIC). The HPIC properties of these immunosorbents were evaluated by an equilibrium theory and were found to be comparable to batch values. An immunosorbent for HIgG antigen showed an HPIC association constant of 10⁷·⁶; the batch equilibrium constant for the same immunosorbent was 10⁷·⁸. Two different anti-insulin immunosorbents retained the intrinsic affinity (10⁶ and 10⁹) of the antibody used to make them. The total active antibody concentrations of these immunosorbents were evaluated by HPIC and batch methods with good agreement between the two. The immobilization reaction was seen to result typically in the loss of 90% of the original antibody activity. HPIC was shown to be applicable to the rapid analysis of antigens at levels as low as ng/mL. This was found to be possible in part because of the rapid forward kinetics which were assessed by HPIC. A forward rate constant of 3 X 10⁷ L·mol⁻¹·sec⁻¹ for the binding of insulin by a specific HPIC column could be determined. The possibility of HPIC fluorescence immunoassays was investigated using a highly sensitive fluorescence detector. An Eimac collimated xenon arc lamp provided sufficient power to detect picomolar levels of fluorescamine labeled insulin and other compounds. The limitations of HPIC in performing picomolar immunoassays were thus shown to be immunochemical rather than instrumental. The ability of immunoaffinity purifications to overcome these limitations was demonstrated.

Multicellular tumour spheroids (TS) are an in vitro model of avascular tumours, and have been widely used to investigate tumour growth, metabolism and hypoxia. The geometry of the TS lends itself to mathematical representation, and theoretical models of TS growth and the development of hypoxia are abundant. With some notable exceptions however, these models have been developed independently of the biological data collection process and are overwhelmingly based upon data from multiple sources. Thus, whilst mathematical modeling has the potential to help explain and guide biological experiments, without reliable data it is unlikely to live up to this expectation. In this thesis, a combination of experimental and theoretical approaches was used to characterize the relationship between proliferation, hypoxia and metabolism during the growth of TS derived from the DLD-1 human colon adenocarcinoma cell line. Experimental data were collected over the entire period of TS growth, generating a high volume of predominantly imaging data. To facilitate the extraction of quantitative information from this, a suite of image analysis software, which is readily applicable to other data sets, was developed. During growth, the DLD-1 TS maintained a macroscopic spherical geometry but at the microscale level the TS boundary was increasingly irregular, with TS disintegrating rapidly after 20 days. Immunofluorescence (IF) studies showed that hypoxia developed soon after TS initiation, followed by the characteristic onset of necrosis. Reduced proliferation was found to be concomitant with the development of hypoxia, although some cells retained proliferative capacity even under severely hypoxic conditions. Towards the end of culture, TS were primarily comprised of severely hypoxic and necrotic cells, a probable cause of disintegration. Mathematical simulation of oxygen gradients in TS using literature-based values for the maximal rate of oxygen consumption was used to estimate the partial oxygen pressure (pO₂) at which the IF marker of hypoxia was bound. Assuming a spatially-invariant rate of oxygen consumption, the model predicted that the onset of hypoxic binding occurs at pO₂ levels similar to those reported in the literature, however the onset of necrosis was overestimated. Mathematical simulations predicted that oxygen consumption decreases as TSs increase in size, supporting previous observations. The Warburg Effect, where glucose metabolism is favoured even under aerobic conditions, is a hallmark of tumours. Although development of the glycolytic phenotype during TS growth was observed in the form of an elevated activity of the lactate dehydrogenase V (LDHV) enzyme, the activity and expression of other glycolytic enzymes, such as hexokinase II (HKII), was unaltered. Whilst the spatial distribution of HKII was unrestricted throughout the TS's viable fraction, LDHV expression was elevated in regions of hypoxia, suggesting constant adaptation of tumour cells to their microenvironment. In addition to the above findings, the data generated have been collected and analysed in the context of the requirements of theoretical modelling at each step; thus, they can be used to parameterise and inform more sophisticated models of tumour metabolism.

Submitted in fulfillment of the requirements of the degree of Master of Technology (Biotechnology), Durban University of Technology, Durban, South Africa, 2014. / Microbial enzymes are gaining worldwide attention due to their potential industrial applications. Microorganisms producing thermostable -xylanase and their associated hemicellulases have significant application in the paper and pulp, food, animal feed, and textile industries. The potential of partially purified xylanase from Thermomyces lanuginosus MC 134, Luminase PB 100, Luminase PB 200 (a commercial xylanase) and T. lanuginosus DSM 5826 (Sigma Aldrich) was evaluated in bleaching of bagasse pulp. The temperature and pH optima for all the enzymes were 60°C and pH 6, respectively. The temperature (50- 80°C) and pH (5-8) stability of the enzymes were also assessed. All the enzymes were relatively stable at 60°C and pH 6 for 180 min. T. lanuginosus MC 134 retained 80% of its activity at 60°C and pH 6 for 180 min and PB 200 retained 75% of its activity at 80°C for 180 min. T. lanuginosus MC 134 also exhibited good alkaline stability at pH 8. The commercial xylanases Luminase PB 100, Luminase PB 200, T. lanuginosus DSM 5826 (Sigma Aldrich) were purified to homogeneity using a gel filtration column packed with sephadex G-100 and characterized for Km and Vmax. However extracellular crude xylanases from T. lanuginosus MC 134 was purified to homogeneity using (N )2S04 precipitation and gel filtration column, packed with sephadex G-100. The purified xylanases exhibited a molecular mass of- 26 to 24 kDa, given range as determined by SDS page. The Km and Vmax values of Luminase PB 100, Luminase PB 200, T. lanuginosus MC 134, and T. lanuginosus DSM 5826, xylanases were determined by the Michaelis-Menten equation using birchwood xylan as the substrate. The Km value for Luminase PB 100, Luminase PB 200, T. lanuginosus DSM 5826 and T. lanuginosus MC 134 were, 8.1 mg/mL, 11.7 mg/mL and 14.3 mg/mL respectively. The Vmax for Luminase PB 100, Luminase PB 200, T lanuginosus DSM 5826 and T lanuginosus MC 134 were 232.6, 454.6 and 74.6 !Jl11ol/min/mg. Biobleaching conditions of the xylanases were also optimised and the release of reducing sugars and lignin derived compounds showed that an enzyme dosage of 50U/g of pulp was ideal for biobleaching at pH 6 and 60°C for 180 min. This brightness for T lanuginosus MC 134, Luminase PB 200, Luminase PB 100 was 45.5 ± 0.11%, 44.1 ± 0.007% and 42.7 ± 0.03% respectively at pH 6, compared to untreated samples. Reducing sugars and UV-absorbing lignin-derived compound values were considerably higher in xylanase-treated samples. All the enzymes analysed exhibited similar trends in the release of lignin derived compounds and reducing sugars which indicated their potential in the pulp and paper industry. / PDF Full-text unavailable. Please refer to hard copy for Full-text / M

Thesis (Ph. D.)--University of Missouri-Columbia, 2001. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 2001. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

Thesis (Ph. D.)--Institute of Paper Chemistry, 1969. / Includes bibliographical references (p. 63-65).