An interdisciplinary approach to understanding the TTP pathway

Rink, Victoria Veronika ORCID: 0000-0002-4913-6262 (2025). An interdisciplinary approach to understanding the TTP pathway. University of Birmingham. Ph.D.

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Abstract

Dysregulated expression of inflammatory mediators as a product of complex immune and nonimmune cell interactions is a hallmark of chronic inflammatory diseases. Understanding and identifying drivers of dysregulated gene expression in the context of the inflammatory response enables researchers to understand pathogenic mechanisms better and to develop novel therapies. Much of this work previously focused on epigenetic and transcriptional regulators. However, changes in the transcriptome are not solely caused by changes in transcription. Posttranscriptional regulation of the stability and translation initiation of pro-inflammatory and immuno-modulatory mRNAs is a critical factor in determining their expression. The stability of mRNAs is controlled primarily by a set of RNA-binding proteins that recognise cisregulatory elements in the 3' untranslated regions. A prominent member of these proteins, TTP, vividly demonstrated, in several animal models, its role in the production of inflammatory mediators. TTP acts downstream of the p38 MAPK pathway as a regulatory switch between the on- and off-phase of the inflammatory response. Its regulation, however, is highly complex, and the dynamic interplay between the components involved is not yet fully understood.

This thesis aims to improve understanding of the dynamic regulation of the p38 MAPK-TTP signalling axis through mathematical modelling. A model based on ordinary differential equations and mass action kinetics was developed, describing TTP regulation by integrating the known properties of the components and interactions involved. To explore different properties of the model, two previously described p38 MAPK pathway models were adapted for integration with the newly built TTP model.

One of the resulting p38 MAPK-TTP models focuses on describing the pathway dynamics quantitatively, using data from bone marrow-derived macrophages and the murine macrophage cell line RAW274.7. The calibrated model was then challenged by computationally simulating pathway mutants and comparing the results with data obtained from such mutant mice. The results underscore the need for further refinements and extensions to our model. They specifically highlighted the importance of considering the role of TTP in inhibiting mRNA translation, which is often overlooked compared to its mRNA degrading properties. The results further emphasised that the phosphatase DUSP1, while important, is insufficient to describe p38 MAPK regulation within our model accurately. Additionally, the results of inhibitor experiments underscore the need for caution in interpreting previous results and designing future experiments regarding the TTP phosphatase PP2A, as it affects TTP regulation on multiple levels.

The second model qualitatively explores potential oscillatory p38 MAPK activation patterns and their impact on TTP. A set of genetic engineering tools was developed for application in live-cell microscopy to investigate the dynamics of TTP activation experimentally. Captured live-cell microscopy data suggested that the stress induced by the imaging conditions affects TTP subcellular localisation. Consequently, the microscopy conditions need to be optimised before going forward. A fixed microscopy time-course experiment utilising activated cells expressing distinct TTP mutants, which resulted in different TTP activity, showed no differences in subcellular localisation over time in contrast to the literature, which suggests localisation to P-bodies or the nucleus.

With this interdisciplinary work, we have designed mathematical and experimental toolkits to improve our knowledge of the p38 MAPK-TTP signalling axis, which is relevant to various inflammatory diseases.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):