Browsing by Author "Riekert, Brendon"

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    Quantitation of the effects of cellular glucocorticoid receptor concentration on dimerization
    (Stellenbosch : Stellenbosch University, 2021-04) Riekert, Brendon; Louw, A.; Rohwer, J. M.; Stellenbosch University. Faculty of Science. Dept. of Biochemistry.
    ENGLISH ABSTRACT: The glucocorticoids (GCs) are a group of steroids affecting virtually every cell in the body. The ability of the GCs to modulate the effects of the immune system is of particular interest therapeutically. Due to this characteristic, it is not surprising that the GCs have become the front line treatment in addressing a diverse array of inflammatory diseases ranging from sepsis to asthma. The GCs are known to function by means of their cognate receptor, the glucocorticoid receptor (GR), which resides in the cytoplasm as a monomer, complexed to various chaperone proteins. Once activated by ligand binding, the GR is able to act via two classical pathways. In the first, the activated GR remains in its monomeric form and acts in a trans-repressive manner to attenuate pro-inflammatory signalling and is associated with the positive therapeutic effects of the GCs. Alternatively, the activated GR is able to form a homodimer and activate metabolic pathways through transactivation. This form of activation is associated with the negative side effects of the GCs. The concentration of the GR in the body varies substantially, not only between different tissue types, but also within the same tissue in different disease states. Studies on GR concentration by Robertson et al. [1] has given rise to the ligand-independent dimerization model. This model suggests that higher concentrations of the GR lead to ligand independent dimerization that improves the sensitivity of tissues to GC stimulation through conformational changes that increase affinity of the GR for the ligand. Subsequent research by Barry [2] produced an in silico model of the GR dimerization cycle highlighting the importance of fluctuations in koff as opposed to the kon in affecting the affinity of the receptor for ligands at increasing GR concentrations, stressing the importance of ligand residence time in ligand receptor interactions. The experiments of Robertson et al. [1] were completed using the human GR. However, another pharmacologically sound model is that of the mouse GR, which at present offers the advantage of the recently developed monomeric GR construct, the GRmon and offers superior dimerization resistance when compared to the classical GRdim construct. Furthermore, the new fluorescence correlation spectroscopy technique for the observation of oligomers, the number and brightness assay (N&B), offers a unique opportunity to observe concentration dependent GR oligomerization by a confocal microscope [3]. The current project illustrates a comprehensive means of translating the previously defined physiologically relevant GR concentrations to the confocal microscope using median fluorescence intensity measurement. Furthermore, a method is proposed to use the N&B technique to observe ligand independent dimerization in the mouse model. Finally, a pilot investigation was conducted using the N&B method to capture 30 minutes of the oligomerization behaviour of the mouse GR following stimulation with the GR agonist dexamethasone, and some challenges which could be overcome in future research are highlighted. To our knowledge, this is the first time the N&B technique has been attempted on the African continent.