Doctoral Degrees (Biochemistry)

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Browsing Doctoral Degrees (Biochemistry) by Author "Barry, Christopher James"

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    Kinetic modelling of the peroxiredoxin system
    (Stellenbosch : Stellenbosch University, 2024-03) Barry, Christopher James; Rohwer, J. M.; Pillay, C. S.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: Hydrogen peroxide is a cellular oxidant that disrupts numerous cellular systems by being readily converted to the hydroxyl radical, which indiscriminately reacts with biomolecules. However, in many contexts, it also serves crucial cellular functions and is intricately connected to maintaining normal functioning. As a result, antioxidant sys- tems find themselves in a delicate balance between two opposing roles: they must defend cells against this cellular toxin without being so effective as to disrupt the normal cellular functions of hydrogen peroxide. The peroxiredoxin protein forms the core of one such antioxidant system and, belying its significance, is found across all domains of life. This versatile and vital protein can switch between several levels of hydrogen peroxide metabolism and possesses its own signalling and chaperone properties. The switch between roles is effectuated through a combination of redox configuration transitions and quaternary structure transformations. The launching-point for this project was a 100-fold change in the ability of peroxire- doxin to neutralise hydrogen peroxide when transitioning from a dimer to a decamer. While this relationship between oligomeric form and peroxidase activity had already been thoroughly established, it had yet to be explored in any dynamic sense. The first major outcome of the research in this dissertation was the formulation of a theoretical framework for the transition between dimers and decamers. This was closely followed by a characterisation of the kinetics for the association and dissociation of a re- duced peroxiredoxin decamer. Armed with these data, the dimer-decamer transition was explored through simulation and found to have an inhibition-like effect on peroxiredoxin activity. Impressively, integrating this process into an established in vivo model resolved an outstanding discrepancy between the experimental and simulated responses of the peroxiredoxin oxidation state to hydrogen peroxide insult. In the course of researching the kinetics of peroxiredoxin, a bias was discovered in the data of multiple independent reports of horse radish peroxidase competition assays. Investigating this bias led to the discovery of a flaw in the data analysis methodology of this assay. This issue was resolved by developing a method of fitting the target rate constant directly to assay trace data instead of through the established fractional inhibition method. We provide tools for researchers to easily apply the improved analysis method in their own work. The final major outcome of this project was the formulation of a model of hydrogen peroxide neutralisation in a Saccharomyces cerevisiae cell culture. The model was parame- terised by deriving several new parameters and the parameter sensitivities of the model were assessed using a Fourier amplitude sensitivity test and metabolic control analysis. This model is capable of simulating the dynamic responses of the peroxidase systems of a S. cerevisiae culture in media exposed to hydrogen peroxide stimulation—a widely used experimental system in redox biology. This dissertation is focused on the exploration of the peroxiredoxin protein using a systems biology approach. This work has culminated in multiple novel findings which contribute significantly to our understanding of the dynamics of hydrogen peroxide metabolism by peroxiredoxin and to redox biology more broadly.