Proteomic approach to oxidative stress in Daphnia magna

The keystone aquatic organism Daphnia magna is extensively used to assess the toxicity of chemicals. This has recently lead to an increase in the omics literature focusing on daphnids, an increase fuelled by the sequencing of the Daphnia pulex genome. Yet, no omics study has looked directly at oxidative stress (OS) in daphnids, even though OS is of primary importance in the response of aquatic organisms to their changing environment and is often induced by anthropogenic xenobiotics. This thesis thus focuses on the application of redox-proteomics, the study of the oxidative modification of proteins, to D. magna Specifically, daphnids were exposed to copper or paraquat, two well studied prooxidants, and protein carbonyls were labelled with fluorescein-5-thiosemicarbazide prior to twodimensional electrophoresis (2DE). This showed clearly that both compounds affect a different portion of the proteome. The identified proteins indicated that energy metabolism was affected by paraquat, while copper induced a reduction of the heat shock response (heat shock proteins, proteases and chaperones) a counterintuitive result which may be adaptative to metal toxicity in arthropods. The same approach was then applied to the study of the toxicity mechanism of silver nanoparticles (AgNP), an increasingly utilised form of silver with expected environmental toxicity, and its comparison to silver nitrate. The results demonstrate that, although less toxic than silver ions, AgNP toxicity functions through a different mechanism. AgNP toxicity is thus not a product of silver dissolution and increased protein carbonylation indicates that AgNP cause OS. Interestingly three of the four tested compounds altered vitellogenin levels and oxidation. Vitellogenins could thus represent an interesting subproteome for the detection of stress in daphnids. Finally, an experiment with oxidised BSA demonstrates the applicability of solid phase hydrazide in the enrichment of undigested carbonylated proteins.

The impact of whey protein isolate on energy balance regulation

Using C57BL/6J mice fed whey protein isolate (WPI) enriched high fat (HFD) or low-fat diets (LFD), this study tested the hypothesis that WPI directly impacts on adiposity by influencing lipid metabolism. WPI suppressed HFD-induced body fat and increased lean mass at 8 weeks of dietary challenge despite elevated plasma triacylglycerol (TAG) levels, suggesting reduced TAG storage. WPI reduced HFD-associated hypothalamic leptin and insulin receptor (IR) mRNA expression, and prevented HFD-associated reductions in adipose tissue IR and glucose transporter 4 expression. These effects were largely absent at 21 weeks of HFD feeding, however WPI increased lean mass and cause a trend towards decreased fat mass, with notable increased Lactobacillus and decreased Clostridium gut bacterial species. Increasing the protein to carbohydrate ratio enhanced the above effects, and shifted the gut microbiota composition away from the HFD group. Seven weeks of WPI intake with a LFD decreased insulin signalling gene expression in the adipose tissue in association with an increased fat accumulation. WPI reduced intestinal weight and length, suggesting a potential functional relationship between WPI, gastro-intestinal morphology and insulin related signalling in the adipose. Extending the study to 15 weeks, did not affect adipose fat weight, but decreased energy intake, weight gain and intestinal length. The functionality of protein sensing lysophosphatidic acid receptor 5 (LPA5) in 3T3-L1 pre-adipocytes was assessed. Over-expression of the receptor in 3T3-L1 pre-adipocytes provided a growth advantage to the cells and suppressed cellular differentiation into mature fat cells. In conclusion, the data demonstrates WPI impacts on adiposity by influencing lipid metabolism in a temporal manner, resulting possibly due to changes in lean mass, hypothalamic and adipose gene expression, gut microbiota and gastrointestinal morphology. The data also showed LPA5 is a novel candidate in regulating of preadipocyte growth and differentiation, and may mediate dietary protein effects on adipose tissue.