Nutraceutical modulation of lipotoxicity and the development of a tool to monitor palmitate induced sub-cellular dysfunction. - by Dean Frederick Ashley. Over 64.3% of the UK are currently overweight or obese according to the NHS survey, a number that has continued to increase throughout the beginning of the 21st century. Inherently this increases the use of NHS resources through an increased prevalence of obesity associated diseases, including type 2 diabetes. To address this, changes to individual’s diets are required and a greater understanding of the sub-cellular mechanisms, that lead to insulin resistance and type 2 diabetes, need to be further understood. In this thesis, C2C12 myotubes & S. cerevisiae are treated with tunicamycin or the saturated fatty acid palmitate (Chapter 3), to create models of ER stress and lipotoxicity, respectively. These models are then used in subsequent chapters to study the nutraceutical modulation (Chapters 4 & 5) and sub-cellular aberrations of lipotoxicity (Chapter 6). Palmitate, the most prevalent saturated fatty acid elevated during obesity, was exposed to C2C12 myotubes in combination with the artificial sweetener Acesulfame-K (Ace-K)(Chapter 4) and the polyphenol compound curcumin (Chapter 5). Previous studies have highlighted a need for a more comprehensive understanding surrounding the possible effects of Ace-K. Using rtPCR and mass spectrometry, this thesis shows Ace-K can exacerbate palmitate induced inflammation, lipid intermediate accumulation and insulin resistance, through an AMPK dependant mechanism. Further consolidating the need for more in depth in vivo studies to understand the compound’s risks. The same techniques are then used to study the effects of curcumin, in combination with palmitate, on C2C12 mouse skeletal muscle cells. Previous studies have shown curcumin alleviates palmitate induced inflammation, general lipid accumulation and insulin resistance. However, the specific lipid intermediate responsible for these changes remained unknown. This study demonstrates curcumin decreases diacylglycerol accumulation, through a βoxidative dependant mechanism, whilst simultaneously increasing long chain ceramide concentrations, through PERK signalling. This provides a possible combining mechanism for curcumins anti-inflammatory and cytotoxicity phenotypes. Lastly (Chapter 6), a protocol for monitoring the sub-cellular relocalisation of proteins and lipids is developed using the Localisation of organelle proteins by isotopic tagging (LOPIT) technique. Organelles are separated based on their densities and proteomic and lipidomic LCMS open-profiling is used to quantify proteins and lipids abundances across the differential centrifugation fractions. Known organelle marker proteins are then used by the Bayesian ANalysis of Differential Localisation Experiments (BANDLE) algorithm to assign organelle localisations to proteins and lipids with previously unknown organelle localisations, whilst simultaneously identifying species that have changed location following palmitate treatment. This offers a combined workflow to study the sub-cellular relocalisation of proteins and lipids that can be applied to a range of perturbations within C2C12 mouse skeletal muscle cells.