Browsing by Author "Naidoo, Kevin"

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    Combined NMR and simulation study of carbohydrate linkage dynamics
    (2000) Best, Robert; Naidoo, Kevin; Jackson, Graham Ellis
    Biomaterials have recently gained significance as a result of environmental pressures and their increased viability through crop engineering. Starch harvested from maize crops is one example of a cheap and abundant biopolymer, which could substitute conventional polymers such as polyethylene as a material in manufacturing. Its practical application, however, will depend on understanding its physical behaviour, so that intelligent modifications can be made to enhance its properties. The most effective way of changing a polymer's properties is by modification at the chemical level, since ultimately it is this which determines the macroscopic features, such as viscosity, plasticity and tensile strength. Since the origin of these features is difficult to study experimentally, this thesis will tackle the problem by means of computer simulation of small carbohydrate fragments. In particular, the two principal types of carbohydra te linkage (namely 0: (1--+4) and 0: (1--+6)) will be examined in this thesis, since these are the chief elements of conformational flexibility in these molecules. The 0(1--+4) and 0:(1--+6) linkages are studied both separately in the maltose and isomaltose molecules respectively and together in panose and the resultant water structuring and dynamic behaviour are studied. Although the computational approach provides insights not available experimentally, it is nonetheless important to compare the results obtained with an experimental reference as a check on their validity. N!vlR T1 relaxation measurements, which give indirect information on the atomic scale dynamics, have been measured for each of the model fragments and compared with values calculated directly from simulation.
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    Computer simulations of Fréchet dendrimers in solutions
    (2005) Simpson, Stephen Charles; Naidoo, Kevin; Moss, John R
    The structure and dynamics of dendrimers in solution are studied through nanosecond atomistic Molecular Dynamics (MD) simulations of explicitly solvated Fréchet dendrimers, generations G1 to G5. The properties of these dendrimers are investigated in four solvent invironments: vacuum and water (representatives of poor solvents), and tetrahydrofuran (THF) and chloroform (representatives of good solvents). To establish the quality of the solvent on the conformation of the dendrimer, additional nanosecond MD simulations fo the dendrimers are performed, from both inititally folded and unfolded conformations.
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    Development of coupled enzyme assay and in vitro synthetic biology approach for glycosylation pathway characterization
    (2024) Nashed, Abdullateef; Naidoo, Kevin
    Glycans play essential roles in living organisms that encompass a wide range of biological functions ranging from energy metabolism all the way through to intricate cell signalling pathways. They carry out these functions either directly by participating in metabolism and binding or indirectly by altering the structure and nature of their conjugates, for example by being the major posttranslational modifier of proteins. The family of glycans in a biological organism (the glycome) correlates to the system's state. In this thesis the focus is the development of methods and measures to model the correlation of aberrant glycosylation of MUC1 that is associated with certain cancers. Central to this is an accurate MUC1 peptide monomer and associated glycosyltransferases (GTs) able to be deployed in specific construction. However, deconstructing the machinery of the GTsubstrate interactome is challenging because of the complexity of glycan structures and the diversity of GTs acting on them. Two tool sets that can advance our understanding is the development of reliable kinetic assays and constructing biologically relevant substrate arrays. Presently used assays are disadvantaged by either being endpoint assays or continuous ones compromised by lack of specificity and sensitivity, or have high cost. Chemically synthesized glycan arrays often face insurmountable hurdles of complex carbohydrate chemistry. To address these challenges, the work undertaken in this thesis was firstly the development of a continuous enzymatic-coupled assay through the redesign of the pyruvate kinase/lactate dehydrogenase (PK/LDH)-coupled assay that lead to increasing the sensitivity and specificity of detection. Further the assay was adapted to accommodate all classes of GTs. Secondly, an in vitro synthetic biology approach was developed to construct a MUC1 O-glycans model peptide as a proof-of-concept of a seamless “one pot” glycopeptide synthesis strategy. Here a fusion protein, consisting of the MUC1 core peptide and a carrier protein, was designed for expression in E. Coli.. The fusion protein elements were optimized to maximize solubility, facilitate without obstruction the in vitro enzymatic glycosylation of the peptide, and enable the recovery of the products through a simple one-step purification. The peptide in the fusion protein was optimized to undergo in vitro sequential enzymatic glycosylation that replicates the native pathway. The properties of the fusion proteins enabled investigation of the activities of the downstream GTs on the synthesized peptides in their fusion forms, which was used in the investigation of the site specificity of ST6GALNAC1, and its role in the biosynthesis of the breast cancer marker STn.
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    In silico redesign and de novo design of anti-MUC1 monoclonal antibodies
    (2023) Dilsook, Kyllen; Naidoo, Kevin; Barnett Christopher
    Mucin 1 (MUC1) is a cellular membrane-tethered protein which is abnormally glycosylated and overexpressed in several epithelial cancers. This abnormal glycosylation is hypothesized to participate in the hyper-activation of signaling pathways which promote tumor growth and provides a suitable target for detection and treatment of epithelial cancers via antibodies. While anti-MUC1 monoclonal antibodies do exist, none have proven to be effective in clinical trials. Antibody fragments have been identified as possible therapeutic agents since their small molecular weight and improved selectivity allow for better targeting and tissue penetration. Antibodies which do not elicit strong immune responses have also been used in treatment by means of conjugation to cytotoxic drugs. The failure of anti-MUC1 antibodies in targeting MUC1 is due to the inability of majority of existing antibodies to specifically recognize and bind the cancer associated truncated sugar and MUC1 peptide simultaneously. Thus far only one antibody has been shown to bind the cancer associated sugar and peptide (SN-101) however, this is a murine derived structure which is not ideal for treatment in humans. In this thesis, MD, and related analyses (such as structural analysis and free energy calculations) will be used to investigate and optimize the binding of the antigen binding fragment (Fab) region of the SN-101 antibody to Tn-glycosylated MUC1. The Tn O-glycosylated MUC1 glycoprotein was targeted due to previous research indicating that this particular glycosylated variant of MUC1 had been found in increased concentrations in breast cancers. The Fab region was considered as this is the region involved in binding the antigen target. Further structural data is available in the form of an X-ray crystal structure for this region. The utility of MD in rational design of antibodies against glycopeptide antigens is demonstrated by this research. This is of particular interest due to the role glycopeptides play in the pathology of complex diseases, such as cancer and arthritis, as well as viral infections, such as HIV and COVID-19. The automation of this in silico workflow provides a rapid screening approach which could possibly lead to the development of more specific, targeted treatments for glycoprotein related diseases via antibody drug conjugates. This approach can also be used to improve cancer biomarker detection assays.
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    A stereoelectronic and thermodynamic study of b-D-methyl glucose conformational changes related to anomeric centre reactivity
    (2014) Yusuf, Sabena Shaik; Naidoo, Kevin; Barnett, Christopher, Bevan
    The major part of this thesis focuses on investigating the rationale for ring deformation of -D-methyl glucose in glycosidase reactions (for example, cellulose hydrolysis). The investigation is computational and is done in isolation from the enzyme binding pocket and incoming nucleophile. What is the effect of the C1-O1 bond breaking process on key glucose properties is the central question asked and answered in this thesis. A battery of ab initio methods is used to uncover details of the glucose ring pucker free energy volumes. The free energy volumes were computed using the Free Energy from Adaptive Reaction Coordinate Forces (FEARCF) method. The bond stretch of the C1-O1 bond in -D-methyl glucose serves as a sugar model for hydrolysis, following the DN*AN mechanism. The FEARCF method has been employed as it was previously shown to generate molecular sampling traversing all of pucker phase space resulting in a multidimensional free energy surfaces (or volumes). Density functional theory and post SCF analysis have been used to investigate the stereoelectronic changes that occur during ring deformation. In particular, changes involving the anomeric carbon, that is the C1-O1, C1-O5 bond distances, electron densities and charges of the C1, O5 and O1 atoms.