221 A Summary Chapter 5 describes another in silico model, in this case using detailed kinetics. This model integrates the behavior of the enzymes involved in the peroxisomal β-oxidation of straight-chain fatty acids. Making use of data generated in chapter 4, as well as from the literature, I predicted the behavior of the pathway under amino acid deprivation as well as in the presence of DHA. Further work is required to expand the model to also recapitulate the metabolism of branched-chain fatty acids. In chapter 6, I established an iPSC-derived hepatic organoid model to study MCADD. To do so, iPSCs from MCADD patients were differentiated to organoids and compared with healthy controls. Expandable hepatic organoids (EHOs) have the ability to proliferate and to further develop into mature EHOs (matEHOs) which recapitulate many liver functions. MCADD EHOs accumulated medium chain acyl-carnitines (esterified form of fatty acids), which are known biomarkers of the disease. Interestingly, MCADD and healthy organoids exhibited some differences in peroxisomal coenzyme A metabolism. This in vitro model is a promising tool to further study the role of peroxisomes, as well as the interaction between peroxisomes and mitochondria, in MCADD and to link the different symptomatology of the disease with the different mutations of the donors. Finally, chapter 7 presents a general discussion on the use of in vitro and in silico models to study malnutrition and MCADD. In this chapter I also discussed how these new models can help understand the pathophysiology of different diseases prior to referring to in vivo models, while mentioning some of the limitations and proposed approaches to overcome such limitations. This chapter also focuses on discussing the importance of peroxisomes and mitochondria in health and disease and their potential interplay in both malnutrition and MCADD. Final remarks In this thesis I have demonstrated that in vitro and in silico models are suitable tools for the study of malnutrition and medium chain acyl-CoA dehydrogenase. These models have proven valuable to gain biological insights into both diseases and understand different pathophysiological processes. Moreover, these results can potentially be extrapolated into other diseases. Although the use of animal models remains an essential tool in the field of biology, the emergence of new methodologies should be regarded as a significant step towards a future with reduced reliance on animal experimentation.
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