About the Project
The aim of this DTP is to produce highly motivated and excellently trained postgraduates in the BBSRC priority areas of Agriculture & Food Security (AFS) and World-Class Underpinning Bioscience (WCUB). These are growth areas of the biosciences and for which there will be considerable future demand.
This project is one of a number that are in competition for funding from the South West Biosciences Doctoral Training Partnership (SWBio DTP).
Circulating oxysterols and phospholipids, both inflammatory and anti-inflammatory, correlate with biological age. The aim of this interdisciplinary investigation of (oxy)lipids in monocyte/macrophages is to explore the relationship of their metabolism with inflammation and ageing. Infection with COVID-19 has had most severe outcomes in older adults and those with metabolic disease, possibly due to poorly controlled inflammation. This work will provide new knowledge about the timing and location of (oxy)sterol and phospholipid metabolites relative to inflammatory cell activity. Novel insight into (oxy)lipid effects on mitochondrial activity and nutrient use by monocyte/macrophages from older people will be explored using repurposed drugs that affect (oxy)lipid metabolism to control inflammation.
The first objective is to develop stable knockdown and overexpression of those genes involved in oxysterol and phospholipid metabolism using macrophage cell lines. Transient knockdown of mitochondrial CYP7A1 and CH25H has been successfully achieved in THP1 cells. Changes in gene expression will be confirmed by western blotting and qPCR. The effects of altered gene expression on extracellular and subcellular (oxy)lipid localisation will be explored using MS supervised by 2020 UKRI FLF, Dr Costello. Macrophage metabolism (Seahorse) and function before/after challenge, including phagocytosis, chemotaxis, respiratory burst and cytokine production will be explored in each 33 / 95 model. This will provide new information on (oxy)lipid accumulation, localisation and secretion, in relation to inflammatory cell function. Furthermore, it may help to identify possible lipid metabolism enzyme targets that control inflammation.
The second objective is to establish a metabolic model in macrophages with and without mutations in oxylipid metabolism. We will use stable isotopically-labelled tracer macronutrients and collaborate with the University of Surrey, awarded an ALERT grant for Isotope Ratio Mass Spectrometer for Nutritional and Metabolic Studies. The student will build a metabolic flux map of nutrient metabolism and (oxy)lipid formation at rest and on challenge with inflammatory and inflammation-resolving stimuli. The methods for measuring (oxy)lipids have been previously developed and published by the Griffiths’ lab. This work will identify effects of altered oxysterols on metabolic nodes during inflammatory cell activation and during the resolution of inflammation.
The final objective is to study oxysterol metabolism in healthy older and younger adults using primary monocytes isolated from peripheral blood. We will focus the metabolic nodes and enzymes identified above, at rest and in the presence of inflammatory and inflammation-resolving stimuli. Repurposing of existing activators and inhibitors of critical oxysterol and phospholipid metabolising enzymes will be explored as potential novel antiinflammatory/pro-resolution drugs.
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