| 1. |
- Ölander, Magnus
(författare)
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Proteomic and Functional Analysis of In Vitro Systems for Studies of Drug Disposition in the Human Small Intestine and Liver
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- To reach the bloodstream, an orally administered drug must be absorbed through the small intestine and avoid extensive clearance in the liver. Estimating these parameters in vitro is therefore important in drug discovery and development. This can be achieved with cellular models that simulate human organ function, such as Caco-2 cells and primary hepatocytes. No model fits every scenario, however, and this thesis aimed at using proteomic and functional analysis to better understand and increase the applicability of in vitro models based on Caco-2 cells and human hepatocytes.First, the proteome of filter-grown Caco-2 cells was analyzed. This included near-complete coverage of enterocyte-related proteins, and over 300 ADME proteins. Further, by scaling uptake transport kinetics from Caco-2 cells to human jejunum, the importance of considering in vitro-in vivo expression differences to correctly interpret in vitro transport studies was demonstrated.Focus was then turned to hepatocytes, where proteomics was used as a basis for the successful development of an apoptosis inhibition protocol for restoration of attachment properties and functionality in suboptimal batches of cryopreserved human hepatocytes. As a spin-off project, image-based quantification of cell debris was developed into a novel apoptosis detection method.Next, the in vivo heterogeneity of human hepatocytes was explored in an in vitro setting, where it was observed that human hepatocyte batches contain a wide range of cell sizes. By separating the cells into different size fractions, it was found that hepatocyte size corresponds to the microarchitectural zone of origin in the liver. Size separation can thus be used to study zonated liver functions in vitro.Finally, the proteomes of the major types of non-parenchymal liver cells were analyzed, i.e. liver sinusoidal endothelial cells, Kupffer cells, and hepatic stellate cells. The different cell types all had distinctly different proteomes, and the expression of certain important ADME proteins indicated that non-parenchymal cells participate in drug disposition.In conclusion, this thesis has improved the phenotypic understanding and extended the applicability of Caco-2 cells and primary human hepatocytes, two of the most important in vitro models for studies of small intestinal and hepatic drug disposition.
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| 2. |
- Ås, Joel
(författare)
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Genomic Analysis of Adverse Drug Reactions
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Adverse drug reactions (ADRs) pose a significant global challenge, leading to substantial costs, suffering, and even loss of life. Genetic factors can play a role in determining a patient's response to the drug treatments and predicting ADRs. While many genetic associations with ADRs have been identified, there are still numerous ADRs suspected to have genetic components.In Paper I, the collection and curation strategies for ADR cases in the Swedegene biobank are established, presenting a cohort of 2,550 ADR-cases. Paper II presents the association between genetic variations in human leukocyte antigen (HLA) genes and the development of pancreatitis as a response to azathioprine treatment in patients with Crohn's disease. Paper III reports on an international collaboration to investigate the genetic aetiology of atypical femur fractures (AFF) during bisphosphonate treatment. The study found that previously identified genetic variants did not replicate, and --- as the cohort is the largest of its kind --- provides valuable insights into common genetic factors of AFF. Paper IV examines the genetic associations with central nervous system (CNS) toxicity as an ADR to antimicrobial drugs, identifying correlations with three genes linked to suicide and schizophrenia, although the biological connection remains unclear. Finally, Paper V presents a methodology for the experimental design of ADR studies by analysing the known protein interactions of drugs and proteins associated with ADRs. This approach aims to mitigate the impact of competing genetic correlations by identifying common protein interactions to validate the inclusion of drugs and ADRs in the study. These interactions are then ranked based on importance to the selected drugs and ADRs and used to propose genetic targets of interest. Overall, the findings of these studies contribute to the understanding of genetic predispositions to ADRs and provide a novel approach for data-driven experimental design for phenotype and genetic target selection.
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