Browsing by Author "Lind, Diane Justine."

Now showing 1 - 2 of 2

Quantification of time-dependent redox signalling in the Tpx1/Pap1 pathway in Schizosaccharomyces pombe.
(2019) Lind, Diane Justine.
Reactive oxygen species (ROS) can damage cellular components leading to cell death, but paradoxically, ROS also play essential roles in metabolism and signalling in eukaryotic cells. Dysregulation of this balance is associated with a range of host diseases and cells have consequently evolved sophisticated signalling networks to sense, detoxify and adapt to changes in ROS levels. Hydrogen peroxide, for example, is reduced by thiol-peroxidases which in turn, can trigger the oxidation of thiol-dependent redox transcription factors. However, the relationship between hydrogen peroxide stimuli and the level of redox transcription factor activation has largely been described in qualitative terms. Because quantitative measures of the redox signal have been lacking, we tested whether three signalling parameters viz. the signalling time, duration and amplitude could be used to quantify the hydrogen peroxide-dependent redox signal in the Tpx1/Pap1 pathway in Schizosaccharomyces pombe. We found significant differences in the signalling time and duration, but not signal amplitude as hydrogen peroxide concentrations were increased from 100 to 1000 μM in our assays. By way of comparison, we also found that the general oxidant, tert-butyl hydroperoxide at 200 μM, decreased signal time and duration in the Pap1 pathway when compared to an equivalent hydrogen peroxide concentration. This method was also used to compare the hydrogen peroxide signalling by OxyR in Escherichia coli and Yap1 in Saccharomyces cerevisiae showing that these measures could be used to characterize and compare redox signalling from different oxidants and in different species. Thus, quantification of time-dependent redox signalling revealed new insights into hydrogen peroxide signalling that could not be readily obtained by qualitative methods and, these measures are expected to facilitate a better understanding of the role of redox signalling in health and disease.
Quantifying redox signals: a tool to investigate adaptive responses to oxidative stress.
(2024) Lind, Diane Justine.; Pillay, Che Sobashkar.
In the context of redox biology's oxidative eustress/distress model, the contrasting roles of ROS, such as hydrogen peroxide, under normoxic and toxic oxidative stress conditions has been more clearly elucidated. However, a fundamental question in the field has been understanding how dynamic redox signalling processes contribute to balancing the cellular response to oxidative eustress/distress.We employed a quantitative approach, evaluating redox signals based on area under the curve (AUC), signal amplitude, time, and duration. These parameters allowed us to investigate how dynamic profiles changed in response to various oxidants across three distinct experimental contexts. First, quantifying the dynamic response of the Tpx1/Pap1 redox-regulated pathway in Schizosaccharoymyces pombe revealed a graded signal and transcriptional response to input peroxide concentrations. Secondly, we applied this method to establish the upper and lower limits of detection for the redox probes HyPer7 and roGFP-TSA2/Tpx1 in Saccharomyces cerevisiae and S. pombe, providing a set of criteria for improved selection and comparison of probes. Thirdly, we quantitatively evaluated the dynamic response of the mammalian NRF2/Keap1 pathway following pre-exposure to DEM and tBHQ. Our findings revealed that, unlike the Tpx1/Pap1 pathway, this pathway showed that the timing of oxidant exposure influenced the NRF2 response. These metrics have therefore provided useful insights to several different pathways in both experimental and analytical contexts. Importantly, these measures allow for further exploratory questions to be asked and provide a fundamental framework for assessing dynamic responses of redoxregulated pathways associated with cell physiology and disease.