205 7 General Discussion In chapter 5, I focused on the development of another in silico model, aimed to provide insight into the effects of amino-acid restriction on the peroxisomal β-oxidation of fatty acids. This model relies on kinetic information of the enzymes involved, including rate equations describing the kinetic behavior of the enzymes, kinetic parameters such as Vmax and Kms, and ordinary differential equations. Kinetic models have been used to understand multiple metabolic pathways as well as to provide insight into the regulation of the pathway, for instance by understanding what enzyme or enzymes have the most control on the flux. One very interesting application of these models, is the combination with proteomics data from in vitro or in vivo studies to approximate it to physiological conditions23,24. The use of proteomics data also leads to the development of personalized models, very important tools in the case of rare diseases25. Moreover, these models can provide information on the effects of different drugs to the pathway and predict potential accumulations of toxic metabolites. While the model of peroxisomal β-oxidation presented in this thesis is a first approximation which needs further optimization, the long-term goal is to characterize the effects of amino-acid restriction on the peroxisomal β-oxidation of fatty acids and combine it with our previously established mitochondrial counterpart. We believe that this combination of both models might shed some light into the metabolism of fatty acids in the liver not only in malnutrition but also in all diseases where FAO is implicated. Moreover, I have performed metabolic control analysis26 under different conditions. The results from these studies pointed towards ACOX1 as the enzyme with the highest control on the pathway, in line with previous studies describing the enzyme as the rate limiting step of the pathway27,28. Moreover, I have used proteomics data from the in vitro hepatic model to understand the effects of amino-acid restriction on the flux control of the different enzymes of the pathway. Limitations of new translational models and future perspectives Although it is undeniable that the discovery of organoids has transformed the field of biomedical research, it is crucial to be aware of their limitations. Most of the early work on organoids focused on the study of developmental biology, recapitulation of the organ of origin, and disease modelling . Some studies have highlighted some limitations of these in vitro models, such as lack of different cell populations or failure to recapitulate some functions of the organ of origin29,30.
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