Title Promoter Affiliations Abstract "Experimental evolution in Candida biofilms: does drug tolerance facilitate drug resistance development in fungi?Experimental evolution in Candida biofilms: does drug tolerance facilitate drug resistance development in fungi?" "Patrick Van Dijck" "Molecular Biotechnology of Plants and Micro-organisms" "Invasive fungal infections pose a profound and growing public health threat with treatment failure and mortality estimates often surpassing 50%. Only three major antifungal drug classes are available to treat lethal fungemia and the incidence of resistance to these drugs has risen significantly in recent years. Moreover, multidrug resistant (MDR) ‘superbugs’ like Candida auris have emerged. Despite these alarming concerns, fungal infections and the antifungal resistance crisis remain neglected in terms of research, drug development and public attention.C. auris is an opportunistic pathogenic yeast first identified as a novel species in 2009. Since then, it has emerged simultaneously on all inhabited continents and in five distinct clades associated with a primary geographic origin and clade-specific phenotypic characteristics. Given its recent emergence, accurate identification and diagnosis are a significant challenge, complicating epidemiological surveillance and proper management of infections. In addition, C. auris displays a unique propensity for nosocomial transmission, leading to hospital outbreaks of lethal candidemia. But the greatest public health concern regarding C. auris, is the unprecedented extent of antifungal drug resistance and MDR it displays. At the onset of the research outlined in this dissertation, few molecular mechanisms by which C. auris can acquire resistance had been characterized. Therefore, the primary objective of our studies was to explore how C. auris can adapt to treatment and whether insights into these evolutionary responses can be leveraged to curb resistance development.In our first study, we pioneered in vitro experimental evolution of C. auris to study MDR development to drugs of the three major antifungal drug classes. After whole genome sequencing of experimentally evolved strains, we screened for consecutive mutants along the course of experimental evolution by allele specific PCR (AS-PCR). We revealed the accumulation of novel aneuploidies and mutations in both well-known and newly identified genes linked to antifungal drug resistance. We showcased the first acquired amphotericin B resistance mechanism in C. auris, novel FKS1 mutations – including a novel putative FKS1 hot spot – linked to echinocandin resistance and aneuploidies driving azole resistance. Moreover, we illustrated the ability of C. auris to evolve resistance swiftly and often with minor or no fitness trade-offs, in an isolate from a clade not commonly associated with MDR.The AS-PCR-based screening method to identify consecutive mutants in this study proved to be a valuable tool and was therefore subsequently employed in a proof-of-concept study to identify C. auris and type C. auris clades. As C. auris is commonly misidentified in the clinic and few easy-to-use species and clade typing methods are available, we characterized genetic markers and optimized AS-PCR assays to detect and type C. auris fast, cost-effective, and sequencing-free, providing a useful diagnostic method for low-budget research settings.Accumulating research on genetic determinants of resistance in C. auris over the past years showed that especially the molecular mechanisms of amphotericin B resistance in C. auris remained unidentified. Therefore, we conducted a large-scale investigation on how amphotericin B resistance can be acquired. We typed 441 in vitro and in vivo evolved C. auris lineages from four amphotericin B-susceptible clinical strains of different clades and show a great diversity of acquired resistance responses with resistance level and clade dependent fitness trade-offs. Genotyping and membrane sterol analyses of selected lineages revealed four major types of membrane sterol alterations involved in acquired amphotericin B resistance in C. auris. Using a novel, plasmid based CRISPR-Cas9 allele editing method and Cas9-RNP gene deletions, we showed that amphotericin B resistance can be acquired through modification of various sterol biosynthesis regulators including ERG6, NCP1, ERG11, ERG3, HMG1, ERG10 and ERG12. Additionally, duplications of chromosomes 4 and 6 were observed, representing the first case of aneuploid driven polyene resistance. Leveraging fitness trade-off phenotyping and mathematical modelling of the in vivo environment during treatment, we evaluated the potential of different amphotericin B resistance mechanisms to establish resistant infections, stressing the potency of ERG6 modulation in treatment failure. Furthermore, we evidenced the first mechanism of fitness trade-off compensation in antifungal drug resistance. We show that variation in CDC25 compensates for the reduced oxidative stress tolerance and growth rate due to the loss of function of ERG6, hereby increasing the potential to establish a resistant infection. This fitness trade-off compensation mechanism might have facilitated the only reported case of acquired amphotericin B resistance in the clinic.In aforementioned experimental evolution studies, we observed two interesting phenomena that caught our attention: Collateral sensitivity (CS), the process in which drug resistance towards one drug, confers an increased sensitivity to another drug, and cross resistance (XR), the reduced susceptibility to multiple drugs upon exposure to a single drug. Neither CS nor XR had been explored in pathogenic fungi, although they are well-studied in bacteria and cancer, where they have been implicated in treatment redesign to prevent or reduce resistance development. We characterized CS and XR trends by in vitro experimental evolution of the C. auris type strain in nine antifungal drugs and subsequent susceptibility screening of resistant isolates to 19 antifungal agents. Beyond intra-class XR, we observed XR between azoles and echinocandins, and between azoles and amphotericin B. As these conclude the most important drug classes used in treatment of C. auris infections, the further investigation of the robustness and clinical relevance of XR in treatment failure, is warranted. CS was most evident in amphotericin B resistant isolates, with increased sensitivity to flucytosine, geldanamycin and echinocandins. By comparing the level of resistance development in in vitro experimental evolution during single drug administration and CS-based drug cyclic or combined drug administration, we showed that CS can be leveraged to prevent and reduce resistance development, especially in cyclic treatment of amphotericin B and caspofungin. We optimized a mathematical model to simulate population dynamics in infections during treatment in ‘virtual patients’, which predicted that these CS-based alternative treatment regimens significantly decrease resistance development, especially in short cycling therapies. To investigate whether a drug exerting CS can eliminate established resistant subpopulations, we optimized a competition experimental set-up in which resistant and susceptible subpopulations can be traced by integrating a selective marker in a neutral genomic locus for C. auris we characterized. In vitro competition experiments showed that resistant subpopulations can be outcompeted upon the administration of a CS-exerting drug, in which caspofungin treatment of amphotericin B-resistant isolates showed the most promising results. This suggests that, beyond preventing resistance, CS can be effectively exploited to treat certain resistant infections. These findings hold a significant promise for the application of CS-based antifungal treatments to prevent and/or reduce resistance development in C. auris. The validation of the consistent effectiveness and in vivo relevance of CS-based alternative treatments across different strains and species present a promising avenue for future research and can potentially revolutionize treatment approaches of MDR fungal pathogens like C. auris.In conclusion, this research highlights the potency of antifungal resistance in C. auris by showcasing diverse novel molecular mechanisms of adaptation that can challenge effective treatment. Insight into these evolutionary responses can however offer a promising avenue for developing innovative approaches to combat MDR and encourage future investigations." "A fluorescence-based tool for protein-protein interactions analysis and identification of novel processes of fluconazole susceptibility in Candida species" "Patrick Van Dijck" "Molecular Biotechnology of Plants and Micro-organisms" "Candida species at the same time represent the predominant commensal fungi in the human gut microbiome and are among top 6 most common causes of serious fungal infections worldwide. Our research imperatives are better understanding the biology of these opportunistic pathogens that drives their dual lifestyle as well as finding effective treatments for the eradication of Candida infections.Mapping the complex protein-protein interactions (PPI) contributes to the understanding of virulence signalling and host-pathogen interactions at the molecular level, as well as to identification of novel drug targets at the clinical level. The parasexual mating and alternative codon usage of C. albicans, complicate the fast application of standard genetic methods. Thus, in the first part of this work, we have established an optimized bimolecular fluorescence complementation (BiFC) assay, a robust method for investigation of PPI in C. albicans. We have developed a set of optimized plasmids that enables N- and C-terminal tagging of proteins of interest and the analysis of their interactions in all fusion orientations. After its validation with a proof-of-principle setup, we have applied BiFC on the cAMP-protein kinase A (PKA) pathway, one of the central signalling pathways that governs morphogenesis and virulence in C. albicans. For the first time, we have visualized the in vivo interaction of the upstream components of cAMP-PKA pathway, Gpr1-Gpa2 module, as well as the interaction of Bcy1-Tpk1 and Bcy1-Tpk2, the regulatory and catalytic components of the PKA complex. The application of BiFC for in vivo visualization and investigation of PPI shows excellent promise in elucidating novel molecular mechanisms in C. albicans. In the second part of this work, we sought to identify novel processes involved in the fluconazole susceptibility of C. glabrata. Given the observed epidemiological shifts from more azole-susceptible C. albicans towards more azole-resistant C. glabrata infections, our research focus has been placed on the molecular mechanisms of fluconazole resistance, the most widely used azole drug. By screening a collection of C. glabrata deletion mutants, we have identified two groups of mutants that enhance and suppress the fluconazole susceptibility in C. glabrata. Phenotypic profiling of the identified mutants provides insight into novel processes involved in fluconazole susceptibility and resistance in C. glabrata." "Neuro-TARGET : integrated platform for target identification, validation and drug discovery with applications for neurodegenerative diseases." "Veerle Baekelandt, Yvette Michotte, Sophie Sarre" "University of Antwerp, KU Leuven, Pharmaceutical Chemistry, Drug Analysis and Drug Information" "In light of the ever increasing incidence of neurodegenerative diseases, such as Alzheimer's and Parkinson's disease, in our ageing population in the western world, we propose to establish an integrated platform for target identification, validation and drug discovery to foster the development of novel therapeutics for neurodegeneration. Using cutting-edge technologies we will identify new disease-related genes from human biosamples and humanized yeast models. Generic and strategic research tools for identification and validation of novel targets will be developed. Functional genomics will be carried out in different model systems, including yeast, mammalian neuronal cell lines and rodent models to unravel the pathogenic pathways and potential interactions of the corresponding proteins with the objective to validate key molecular events and therapeutic targets. Targets that define the onset of pathology will constitute prime markers for early diagnosis. Both validated targets and new assays will foster a drug discovery platform. Although most of the generated models and tools are generic in nature, in this project we will focus primarily on applications for Parkinson's disease (PD). Our proposal is subdivided in 4 work packages. WP1: Identification of novel genes and targets Novel potential disease targets will be provided to the project from two different sources. First, we will exploit the genetic power of humanized yeast model systems to identify novel genes modulating _-synuclein (_-SYN) toxicity and to elucidate the pathogenic pathways of 2 recently identified familial PD genes, PINK1 and LRRK2. Second, we will identify new genes linked to PD from human biosamples using a dual strategy. We will identify novel chromosomal loci and causal genes for PD using a positional cloning strategy in PD families. In addition we will perform genetic association studies in a large group of PD patients to identify novel genetic risk and protective factors, focusing on variants influencing the expression of known PD genes. WP2 Development of novel generic technologies, assays and disease models Validation of new disease targets and testing of novel therapeutic strategies is highly dependent on the availability of quantitative, reliable and sensitive technologies in cell culture and in animal models. * We will develop quantifiable assays to correlate _-SYN phosphorylation and aggregation with cell death in yeast * We will establish a medium-throughput, multiparametric assay using a high-content analyzer to measure _-SYN aggregation, apoptosis, mitochondrial and proteasomal dysfunction and oxidative stress in neuronal cell lines. * We will perform a kinase substrate search using a novel ""chemical genetic"" strategy to identify substrates of two PD-linked kinases, PINK1 and LRRK2. * We will generate two in vivo reporter systems to monitor respectively proteasomal function and apoptosis in the rodent brain. * We will combine (lenti)viral vector technology with micro-RNA based technology to generate novel transgenic mouse and rat models. * Novel disease models will be generated for each novel disease-related gene identified in WP1 WP3: Platform for target validation In the newly developed model systems (WP2) we will validate novel targets from WP1 as well as a number of previously identified, non-validated targets. Initial target validation will be performed in yeast and in mammalian cells. For a limited set of genes final validation will be done in rodent brain. WP4: Platform for drug discovery For each validated target an assay based on a screenable phenotype in yeast or in cell culture will be established for drug testing. We will initiate a drug discovery pipeline in newly developed and available model systems in yeast and neuronal cell lines to identify novel hits from the CD3 library. Medicinal chemistry will support hit to lead development. Toxicology and pharmacokinetics studies will further improve the efficacy of the ligands. We will also incorporate in silico-selected ligands in the drug discovery program. The final stage will consist of testing selected novel compounds in the available animal models and in vivo ADMETox studies. In conclusion, the NEURO-TARGET consortium offers an integrated 'start-to-end' approach to identify novel targets and develop novel models and therapeutics to combat Parkinson's Disease and related neurodegenerative disorders." "Data integration to close the gap on Prediction of MTB Drug Resistance Mutations" "Nancy Janssens, Oren Tzfadia, Bouke de Jong" "Department of Biomedical Sciences, Management, Mycobacteriology" "Improved diagnosis and treatment are designated priorities of the World Health Organization and the Centers for Disease Control to address the antimicrobial resistance challenge. These measures rely on an improved understanding of the mechanisms of resistance acquisition in bacteria.Especially for new anti-tuberculosis drugs, known genetic mutations poorly predict phenotypic resistance testing, yet real-time resistance testing is essentially impossible in the endemic areas most affected. In M. tuberculosis, drug resistance (DR) is caused by protein modifying mutations indrug targets or in pro-drug to drug converting enzymes. However, the possibility that gene regulation plays an important role in antimicrobial resistance has yet to be systematically studied.While most mutations have no impact on drug susceptibility, distinguishing those mutated genes that drive DR in a patient is essential to assign an effective treatment regimen, typically involving 5 or more drugs for 9+ months. Knowledge of genes and pathways on which DR mutations operate can also resolve the discordance between genotypic and phenotypic drug susceptibility testing and promises to finally replace the slow, more expensive and biohazardous assay susceptibility testing." "Omics Data Integration to Predict Drug Resistance in Mycobacterium tuberculosis" "Ronald Buyl" "Internal medicine, Public Health Sciences" "Tuberculosis (TB) remains the deadliest infectious disease worldwide. Improved diagnosis is essential to address increasing TB antimicrobial resistance and to assign an effective treatment regimen. The advent of whole-genome sequencing (WGS) promises to overcome slow, more expensive, and biohazardous assay susceptibility testing. However WGS relies on previous understanding of resistance mechanisms in M. tuberculosis (Mtb). To date, WGS has poor performance to predict resistance on new, lessstudied drugs like bedaquiline, failing to distinguish innocuous rare variants from mutations that confer a resistant phenotype in Mtb. We propose an innovative approach to overcome this gap by applying an integrative multi-omics analysis in Mtb to determine the aggregated effect of each mutation on drug resistance, including the contribution of rare mutations and convergent evolution and using computational tools like machine learning and Bayesian networks. Besides improving the accuracy of WGS data interpretation, this integrative analysis will allow us to explore novel cellular mechanisms involved in antimicrobial resistance, close the gap between genotypic and phenotypic drug susceptibility testing, and better understand resistance evolutionary pathways towards Mtb resistance." "Omics Data Integration to Predict Drug Resistance in Mycobacterium tuberculosis" "Ronald Buyl, Bouke de Jong" Mycobacteriology "Tuberculosis (TB) remains the deadliest infectious disease worldwide. Improved diagnosis is essential to address increasing TB antimicrobial resistance and to assign an effective treatment regimen. The advent of whole-genome sequencing (WGS) promises to overcome slow, more expensive, and biohazardous assay susceptibility testing. However WGS relies on previous understanding of resistance mechanisms in M. tuberculosis (Mtb). To date, WGS has poor performance to predict resistance on new, lessstudied drugs like bedaquiline, failing to distinguish innocuous rare variants from mutations that confer a resistant phenotype in Mtb. We propose an innovative approach to overcome this gap by applying an integrative multi-omics analysis in Mtb to determine the aggregated effect of each mutation on drug resistance, including the contribution of rare mutations and convergent evolution and using computational tools like machine learning and Bayesian networks. Besides improving the accuracy of WGS data interpretation, this integrative analysis will allow us to explore novel cellular mechanisms involved in antimicrobial resistance, close the gap between genotypic and phenotypic drug susceptibility testing, and better understand resistance evolutionary pathways towards Mtb resistance." "Validity and feasability of use of a rapid and innovative test for detection of Multi Drug Resistant (MDR) TB cases in Kinshasa, DRC" "Jean-Jacques Muyembe, Marleen Boelaert" "Mycobacteriology, Neglected Tropical Diseases" "Multidrug drug resistant tuberculosis (MDR-TB), defined as resistance to both rifampicin (RMP) and isoniazid (INE), is a major concern for TB control due to the complexity of its diagnosis and treatment. The challenges posed by MDR-TB seriously jeopardize the progress made in recent years to global TB-control. In 2008, less than 5% of existing MDR-TB patients worldwide are being diagnosed as a result of serious laboratory capacity constraints. In the Democratic Republic of Congo (DRC), more than 4900 MDR-TB cases occurred in 2006 according to World Health Organization (WHO) estimates. However, less than 2% have been detected and put on treatment. Conventional methods for diagnosing MDR-TB such as mycobacterial culture and drug susceptibility testing (DST) are slow and cumbersome requiring at least 2 months to isolate MDR-TB strains. This delay increases the risk of the spread of resistant strains throughout the community. This project intends firstly to validate and secondly to assess the feasibility of use of a simple molecular line probe assay: the Genotype MDR-TB plus focused on rapid detection of RMP and INH resistance among patients at increased risk for MDR-TB under field conditions of Kinshasa, DRC. The turnaround time of this probe assay is 2 days, and therefore it may lead to adequate and more rapid detection and treatment ofMDR-TB patients." "Striking Essential Oil: Novel tools and new insights to study the biological activities of essential oils (EOs) and their components (EOCs)" "Patrick Van Dijck" "Molecular Biotechnology of Plants and Micro-organisms, Animal Physiology and Neurobiology" "The increasing level of antimicrobial resistance poses the threat that no suitable antimicrobials will be available for systematic treatment of common diseases. More and more Candida infections are caused by intrinsically and multi-resistant Candida isolates. There is clearly renewed scientific interest in  discovering drugs from natural sources, although natural product-based drug discovery is considered intrinsically complex and requires a highly integrated interdisciplinary approach. Medicinal plants that are used to treat infectious diseases appear to be an abundant source of new bioactive secondary metabolites. One such class of plant-based molecules are essential oils which have been used therapeutically for millennia as they have a broad range of biological activities.Reanalyzing the data of a large antimicrobial screening (2012) of essential oils in microtiter plates systematically showed false-positive hits. We discovered that these false-positives were mediated by the vapor-phase of some essential oils in adjacent wells. The challenge was to circumvent this problem before we could start screening our latest essential oil (component) collection against two potentially disease-causing fungi i.e. C. albicans and C. glabrata. We developed an alternative plate set-up for the new screening in a way that it intrinsically does not modify the standardized protocol, while controlling for false positive results caused by the vapor-phase-mediated antimicrobial activity of the tested essential oils and their components. However, the alternative plate set-up and its derivatives are not limited to the testing of essential oils and their components but can be used with any volatile compound; nor is the set-up limited to testing antimicrobial activity but can essentially be extended to any biological activity tested in a microtiter plate. Therefore, we recommend these adaptations as good laboratory practices when working with volatiles in microtiter plates.An in vitro assay to test the vapor-phase-mediated antimicrobial activity of volatiles did not exist. From a drug discovery point such an assay is interesting as a model for testing compounds for e.g. the treatment of tract-related infections. We hypothesized that if we can detect false-positives in microtiter plates caused by the vapor-phase of volatiles, it should be possible to develop a microtiter plate-based assay. We developped the qualitative vapor-phase-mediated patch assay, a relatively simple test to detect the vapor-phase-mediated activity of volatiles such as essential oils and their components. We conceptualized a quantitative version of the aforementioned assay, i.e. the vapor-phase-mediated susceptibility assay, which we then developed further. Subsequently we quantified with this assay the vapor-phase-mediated antimicrobial activity of a collection of 175 commercial essential oils and 37 reference essential oil components against C. albicans and C. glabrata. About half of the essential oils and their components had a growth-inhibitory vapor-phase-mediated antimicrobial activity. On average, a stronger activity was observed against the intrinsically more resistant C. glabrata, with essential oil component citronellal showing the largest  significant difference in vapor-phase-mediated antimicrobial activity. In contrast, representatives of each class of antifungals currently used in clinical practice did not exhibit any vapor-phase-mediated antimicrobial activity. The vapor-phase-mediated susceptibility assay can advance the search for novel (applications of existing) antimicrobials. Furthermore, our study is the first comprehensive characterization of essential oils and their components as a unique class of antifungals with properties distinct from existing antifungal classes. We conducted a pairwise comparison between the inhibitory vapor-phase-mediated anti-Candida activity of 33 certified organic essential oils and as many equivalent essential oils without such certification. It is believed among essential oil consumers that essential oils from organic cultivation, which includes plants that are harvested in the wild, are therapeutically more potent than those from conventional cultivation. However, we did not discern a significant difference in inhibitory vapor-phase-mediated anti-Candida activity between certified organic essential oils and those without such certification.Although essential oils have been used therapeutically for millennia they continue to be considered as products mainly used in complementary and alternative medicine. Few have made the transition to drugs despite a shared Nobel Prize in 2015 for essential oil-related research of Dr. Youyou Tu for her discoveries concerning a novel therapy against malaria. In the past decades, natural products such as essential oils and their components have somehow been avoided in drug discovery because of inter alia possible technical problems during processing. However, is this still justified with our current understanding of drug discovery? Therefore, we studied selected physicochemical parameters, used in conventional drug discovery, from the components of a collection of 142 essential oils. We demonstrated that, contrary to generally held belief, most essential oil components satisfy current-day requirements of medicinal chemistry for good drug candidates. Therefore, their therapeutic potential remains vastly under-used and should be more vigorously explored with modern methods. Furthermore, it seems that they offer striking opportunities for lead optimization or even fragment-based drug discovery." "Drug-induced cholestasis: in vitro detection and mechanistic insights." "Pieter Annaert" "Drug Delivery and Disposition" "Drug-induced liver injury (DILI) is the primary cause for the discontinuation of clinical trials and post-marketing withdrawal of drugs, leading to considerable losses for the pharmaceutical industry. Despite the immense financial losses that are related to DILI, its impact on patient healthcare is of much greater importance. Indeed, numerous cases have been reported in literature that illustrate the potentially fatal consequences of DILI. As DILI presents itself in multiple clinical forms (e.g., acute hepatitis, cholestasis and steatosis), the plethora of possible underlying mechanisms is an immense hurdle for the development of next generation prediction tools. Therefore, a better understanding of these mechanisms is an absolute priority for the pharmaceutical industry. During the last decade, both academia and industry have put a great deal of effort into unravelling these underlying mechanisms. Based on this, it has become more and more apparent that the pathological disturbance of bile acid homeostasis plays a key role in the onset of liver injury, making cholestasis one of the major causes of DILI. However, the early detection of drug-induced cholestasis (DIC) remains challenging during the drug development process. Indeed, the majority of currently developed in vitro tools that evaluate DIC are based on the assumption that assessing a drug’s potency to interfere with certain hepatobiliary transporters suffices to accurately predict the drug’s in vivo cholestatic potential. However, as these tools do not adequately mimic the in vivo situation where various types of cellular responses (both adaptive and adverse) regulate bile acid homeostasis, they are unable to thoroughly assess human DIC risks. This shortcoming can be overcome by using more in vivo-relevant in vitro models such as sandwich-cultured human hepatocytes (SCHH). Indeed, our group recently developed a SCHH-based in vitro assay which allows for the detection of cholestatic compounds based on their ability to modulate bile acid homeostasis. Although the assay is able to distinguish cholestatic compounds from both non-hepatotoxic and non-cholestatic but hepatotoxic compounds, it was not designed to yield mechanistic insights into drug-induced changes in bile acid homeostasis. Such information is extremely valuable, especially when trying to make an informed decision regarding the cholestatic potential of a given drug. Consequently, the goal of the current doctoral thesis was to investigate human DIC by profiling in vitro bile acid disposition in SCHH and by evaluating these data using both non-compartmental analysis (NCA) techniques as well as a more advanced cellular mechanistic bile acid disposition model. In vitro data were obtained using an improved experimental setup that consisted of incubating 10 μM of the prototypical bile acid chenodeoxycholic acid (CDCA) in absence and presence of therapeutically relevant bosentan concentrations.As cryopreserved human hepatocytes were used throughout this thesis, the aim of the first study was to evaluate whether cryopreserved cells are a viable alternative to freshly-isolated cells. The results from this study clearly showed that cryopreservation does not affect the hepatocyte’s biochemical integrity nor its application potential for drug disposition studies. Moreover, transporter studies, which were conducted using fluorescent probes, indicated that the organic anion transporting polypeptide (OATP) and the multidrug resistance-associated protein 2 (MRP2) activity levels remained unaltered following cryopreservation. In the same study, disposition of telmisartan and telmisartan-glucuronide was evaluated using an in-house developed mechanistic cellular disposition model which allowed us to distinguish between the susceptibilities of the individual disposition pathways to cryopreservation. The model predicted that the relative contribution of uptake, metabolism and efflux of telmisartan and telmisartan-glucuronide remained unchanged following cryopreservation, indicating that cryopreserved hepatocytes are a suitable alternative for freshly-isolated hepatocytes. This result also represents an inspiring example of how optimization of hepatocyte cryopreservation protocols can support implementation of the 3R concept for animal experimentation. In addition to assessing the effects of cryopreservation on hepatocyte longevity and functionality, the first study conducted in this doctoral thesis aimed to establish a generic modelling framework that could be used in future studies. Indeed, in a second study, the framework was utilized to develop a mechanistic model that was able to quantitatively evaluate the effects of bosentan on CDCA and GCDCA disposition in SCHH originating from five different human liver tissue donors. The model consisted of seven compartments to which CDCA and GCDCA could distribute using both linear and non-linear kinetics. Model predictions showed that the amidation of CDCA as well as biliary efflux clearance of GCDCA decreased in presence of bosentan, in line with the non-compartmental analysis results and reports from other research groups. Interestingly, not all tested donors were affected by in vitro bosentan treatment, underlining the importance of using an in vivo-relevant in vitro model when assessing human DIC. Indeed, human hepatocytes, that originate from various donors, allow for the evaluation of interindividual differences in susceptibility to cholestatic compounds, something which cannot be achieved with for instance hepatic cell lines as they do not reflect the interindividual variability. In a last study, we aimed to expand on our study regarding bosentan-mediated cholestasis, by pursuing new insights into the role of Ro 47-8634 (O-demethylation of the phenolic methyl ether of bosentan), Ro 48-5033 (hydroxylation at the t-butyl group of bosentan) and Ro 64-1056 (combination of hydroxylation and O-demethylation of bosentan), the three known phase I metabolites of bosentan. To do so, disposition of CDCA and GCDCA in presence of bosentan was evaluated using SCHH originating from three donors which showed distinct capacities in terms of enzymes that play a role in the metabolic pathways of bosentan. As expected, the donor with greatest metabolic capacity showed increased formation of all metabolites as compared to the other donors. However, this was also the only donor which showed a significant decrease in CDCA uptake and its subsequent conjugation to GCDCA following bosentan treatment, suggesting that formation of Ro 47-8634, Ro 48-5033 and/or Ro 64-1056 could drive the observed effects. Indeed, linear regression analysis indicated that inhibition of CDCA’s uptake could (at least in part) be attributed to formation of bosentan’s metabolites, while inhibition of CDCA conjugation most likely resulted from interaction with the parent.In summary, this doctoral thesis provided new insights into human DIC using an alternative experimental setup that consisted of incubating SCHH with CDCA in presence and absence of therapeutically relevant bosentan concentrations. By quantifying subtle changes in CDCA and GCDCA disposition using NCA techniques and an in-house developed cellular mechanistic disposition model, quantitative insights into the interplay of bosentan’s mechanisms of toxicity were gained. More specifically, based on our results, it has become apparent that bosentan’s metabolites play a role in the inhibition of bile acid uptake, leading to the prehepatic accumulation of bile acids, while, in addition, bosentan decreases the further conjugation of unconjugated bile acids to their conjugated forms." "Understanding Drug-Induced Cholestasis: Mechanistic exploration of hepatic bile acid disposition" "Pieter Annaert" "Drug Delivery and Disposition" "Drug-induced liver injury (DILI) is the most frequent reason for drug withdrawal from the market (Xu et al., 2004; Atienzar et al., 2016). Hence, DILI poses a major risk for patient health and pharmaceutical industry. Nowadays, pharmaceutical industry is still confronted with the poor prediction of DILI in non-clinical safety environment during drug development. The current non-clinical in vivo animal tests are not able to reliably detect DILI, as several key pathways involved in DILI differ between animals and human. In addition, taking the 3R principle (replacement, refinement, reduction) into account, non-clinical safety assessment is eagerly searching to substitute the conventional in vivo animal tests and implement new and reliable in vitro tools. These in vitro methods are pivotal for the accurate prediction and screening of compounds that can cause DILI. Unfortunately, the quest for the development of new in vitro tools is hampered with the limited mechanistic knowledge concerning DILI. Therefore, it is crucial to enhance our understanding regarding the toxicity pathways and thus to address the challenge to improve our apprehension about DILI.Drug-induced cholestasis (DIC) accounts for about half of the DILI cases. DIC implies a disturbance of bile acid homeostasis, which eventually results in toxicity. Therefore, it is important that the pathways, responsible for bile acid homeostasis in vivo, are mimicked appropriately in vitro. In fact, over the past decades, the inhibition of the Bile Salt Export Pump (BSEP) by drugs served as the prime mechanism of DIC. As bile acid homeostasis is a complex interplay of several (other) hepatic transporters and metabolizing enzymes, there is growing interest in expanding our mechanistic knowledge concerning other causes of DIC, apart from BSEP inhibition. Furthermore, an improved insight in the different aspects of bile acid homeostasis will expand our horizon on the development of more advanced in vitro and in silico models. The overall goal of the current thesis research aimed to shed more light on the mechanistic understanding of interferences with bile acid homeostasis, caused by drugs. Therefore, sandwich-cultured rat or human hepatocytes (SCRH or SCHH) were applied to mimic the bile acid disposition pathways in vitro. Sandwich-cultured hepatocytes preserve all hepatocyte-specific functions, together with the maintenance of hepatic transporters and metabolic enzymes. In that respect, an hepatocyte-based in vitro DIC assay, that was previously developed by our research team, was optimized and validated using an extended set of test compounds (Aim 1). This in vitro DIC assay is based on the co-incubation of a cholestatic compound with a physiological bile acid mixture, followed by the measurement of urea production to determine the cell functionality. In a next phase, this in vitro DIC assay was used as a starting point to improve our knowledge about the role of (other) hepatic bile acid transporters in DIC (Aim 2). As such, this thesis research provided an answer to following specific research questions:Can cryopreserved hepatocytes inter-changeably be applied in the in vitro DIC assay, next to freshly-isolated hepatocytes? (Chapter 3; part of Aim 1).Is the in vitro DIC assay able to reliably predict cholestatic compounds in vitro and translate it to risk for DIC in vivo? (Chapter 4; part of Aim 1).Is there a link between domperidone-indiuced modulation of OATP1B1-mediated uptake transport in hepatocytes and domperidone-induced alteration of bile acid homeostasis? (Chapter 5 & 6; part of Aim 2).What are the predominant bile acid disposition pathways affected by bosentan, when evaluated in SCHH with chenodeoxycholic acid (CDCA) as a prototypic bile acid?  (Chapter 7; part of Aim 2).The first study demonstrated that hepatocytes before or after cryopreservation can inter-changeably be applied in the in vitro DIC assay. Using SCRH from freshly-isolated and cryopreserved sources, we demonstrated the robustness of the model. Cyclosporin A and troglitazone were used as model compounds for cholestasis. Comparable Drug-Induced Cholestasis Index (DICI) values were obtained, irrespective of the cryopreservation history. The DICI reflects the ratio of the urea produced in the conditions where the model compounds were co-incubated with bile acids and the urea produced in the conditions where the model compounds were incubated alone (without bile acids challenge). A DICI value lower than 0.80 indicates that a compound at a particular concentration is able to disturb bile acid homeostasis in the SCRH. As an important outcome of this study, we proposed to normalize the urea production for the confluence rate of the cultures as SCRH from cryopreserved hepatocytes yielded a lower attachment efficiency, compared to SCRH from freshly-isolated hepatocytes.To answer the second research question, 14 test compounds (set of hepatotoxic, cholestatic and non-hepatotoxic compounds) of the EU-EFPIA IMI project MIP-DILI consortium were applied to investigate the performance of the in vitro DIC model and thus to expand on validation of the model. Several batches of human hepatocytes in sandwich-culture were qualified for DIC assessment by verifying the bile acid-dependent increase in susceptibility to the toxic effects of cyclosporin A. To account for the high-interindividual variability between donors, “Cyclosporin A DICI-positive” human batches were introduced to exclude human hepatocyte donors which did not reveal a disturbance in bile acid homeostasis in presence of cyclosporin A. The 14 test compounds were classified based on the safety margin (SM)-concept. Using the SM, which is the ratio of the lowest compound concentration with a DICI ≤ 0.80 to the Cmax,total, we were able to determine the cholestatic risk of each compound in vivo. We unambiguously classified all 14 compounds for their risk of DIC in vivo, based on in vitro predicted SM values and in vivo incidence reports as provided in literature.The next two studies focused on the mechanistic understanding of the drug-induced disturbances of bile acid homeostasis. In a first study, the role of hepatic uptake as mediated by the Organic Anion Transporting Polypeptide 1B1 (OATP1B1) was explored with respect to bile acid homeostasis. We hypothesized whether domperidone, for which modulation of OATP1B1-mediated transport has been shown in vitro, would be able to modulate the uptake of bile acids and as such, disturb bile acid homeostasis. Initially, we further explored the stimulatory effect of domperidone on OATP1B1-mediated uptake of two fluorescent probe substrates (sodium fluorescein and cGamF) using different hepatic in vitro models namely, OATP1B1-transfected cell lines, rat and human hepatocytes in suspension. In addition, the role of OATP was investigated in the hepatic uptake of domperidone in rat and human hepatocytes. Overall, it was shown that the stimulatory capacity of domperidone was substrate- and isoform- dependent. In particular, domperidone was able to stimulate sodium fluorescein uptake in OATP1B1-transfected cell lines and human hepatocytes, while it resulted in inhibition of sodium fluorescein uptake in rat. Also, our findings indicated a possible involvement of the Organic Cation Transporter (OCT) in the domperidone uptake in rat and human hepatocytes. Secondly, the in vitro DIC assay was applied to investigate the effect of domperidone on bile acid homeostasis. Again a species-dependent effect could be determined, showing that domperidone clearly disturbed bile acid homeostasis in human, but not in rat hepatocytes. Next to decreases in DICI values by domperidone in SCHH, domperidone was able to decrease the levels of glycine conjugated bile acids, while increases were observed in the amount of unconjugated bile acids in a concentration-dependent manner. The Bile Acid Disturbance Index (BADI) was introduced to comprehensively express the capability of a compound to disturb bile acid homeostasis in vitro. The BADI represents a ratio of the relative difference in the accumulation of unconjugated bile acids in the culture medium and the relative difference in the accumulation of intracellular conjugated bile acids. Both domperidone and the cholestatic reference drug cyclosporin A were able to increase the BADI in a similar fashion while also decreases in DICI values were seen for both compounds. Furthermore, it was observed that exposure to domperidone induced a decrease in the levels of endogenous bile acids. The study concluded that domperidone and cyclosporin A were able to alter bile acid disposition by mechanisms that are not fully elucidated yet. However, the modulation of bile acid uptake transport in human hepatocytes could potentially serve as a potential mechanism playing part in DIC. Moreover, the reliable measurement of endogenous or exogenously added bile acids in the culture medium and cells serves as an interesting and sensitive approach to investigate alterations in bile acid homeostasis in vitro. In that respect, our study confirms that a disturbance in bile acid homeostasis is a key event of DIC, but this does not necessarily imply an increased intracellular accumulation of bile acids in particular.In the final study of this thesis project, we hypothesized that mechanisms, other than BSEP inhibition, could potentially be involved in bosentan-induced cholestasis. Therefore, we studied the effects of clinically relevant concentrations of bosentan on the disposition of endogenous bile acids, but also on the disposition of externally added CDCA in SCHH. We showed that bosentan was able to inhibit the uptake of CDCA, inhibit the conversion of CDCA to GCDCA and alter both sinusoidal and canalicular efflux clearances, as concluded from the disturbances caused by bosentan on the CDCA disposition. In addition, inhibition of the bile acid synthesis by bosentan was observed by a decreased amount of endogenous bile acids in presence of bosentan in SCHH. In conclusion, we gained insights in the overall influence of bosentan on bile acid disposition in SCHH. Therefore, these results may provide a mechanistic perspective on the interference of bosentan with bile acid disposition in human hepatocytes.In summary, this doctoral research aided in the development and improvement of new and currently existing in vitro tools for a better prediction of DIC in vivo. The in vitro DIC assay enables early identification of drug candidates (or their metabolites) with a potential to cause DIC in vivo and supports adequate decision-making on the DIC assessment. Furthermore, the developed in vitro tool served as a starting point for gaining more insights in mechanisms involved in drug-induced alterations of bile acid homeostasis, other than BSEP inhibition, by proposing the impact of uptake transporters in DIC. A disturbance of bile acid homeostasis, but not necessarily an intracellular accumulation of bile acids is playing part in bile acid-mediated toxicity, as shown by measurement of bile acids in medium, cell and bile compartments of SCH. Therefore, bile acid measurements serve as valuable endpoints to mechanistically determine pathways, altered by a cholestatic compound, in bile acid disposition. Although many fundamental questions in this field remain to be answered, the obtained results will be useful in the further development of other, more advanced in vitro models to predict DIC and emphasizes the importance to sharpen our knowledge concerning the key pathways playing roles in the onset of DIC."