Inne Xhonneux  

• 12 december 2024, 17u
• CDE, gebouw Q (D.Q.002)
• Promotoren: Prof. Jo Leroy en Dr. Waleed Marei

Abstract

Maternal diet induced obesity (DIO) affects oocyte mitochondrial functions, reducing the quality of the oocyte and subsequent embryo. Further embryonic development in the oviduct and uterus of DIO mothers may reprogram the offspring, a process that continues during lactation. This may increase the offspring’s sensitivity to an obesogenic (OB) diet, exacerbating the direct effects of an OB diet on offspring health and oocyte quality. However, this was never thoroughly investigated. It also remains unknown whether the impact on offspring’s fertility is only apparent at fertile age or may already be inborn. Such information is crucial to determine the need to mitigate or protect against maternal effects in offspring at risk, and to offer fundamental insights for sustainable advice.

We hypothesized that both offspring and maternal OB diets affect adult offspring health and oocyte quality. Moreover, we hypothesized that maternal DIO influences the direct impact of the offspring’s OB diet. We investigated mature oocytes, but also dormant ovarian follicles at adulthood and even earlier at weaning and at birth.

Outbred Swiss offspring from control and OB-fed mothers were weaned on control or OB diets for 7 weeks. At offspring adult age, we confirmed negative effects of the offspring’s OB diet on offspring health and oocyte quality, and showed for the first time that even the dormant primordial follicle pool is hampered by the offspring’s OB diet. Contrarily, maternal DIO did not hamper the offspring’s metabolic profile, or increased the offspring’s sensitivity to an OB diet, but improved their metabolic profile despite morphological alterations in muscle mitochondria. Furthermore, our data suggest that maternal DIO increases the offspring’s oocyte bioenergetic capacity, without signs that are indicative for damage in mature and primordial follicle oocytes. Maternal DIO also did not interact with the direct effect of the offspring’s OB diet. Offspring from DIO mothers had no increased cellular stress levels (Hsp70) in their primordial follicle oocytes at birth or at weaning, but had reduced TFAM expression which may regulate mtDNA replication. More prominent effects of maternal DIO were detected in offspring primordial follicle oocytes after lactation, of which functional implications on subsequent follicular development require further investigation.  

Based on our data, it is unlikely that adult obese women born to obese mothers would require different preconception care strategies to improve their oocyte quality. However, the subtle maternal DIO-induced changes in the offspring’s oocyte bioenergetic capacity may suggest the need for personalized IVF media formulations.

Marina Stroe

• 22 november 2024, 17u
• CDE, gebouw O (D.O.07)
• Promotoren: Prof. Steven Van Cruchten, Prof. Karel Allegaert, Prof. Pieter Annaert en Prof. Anne Smits

Abstract

Pediatric drug therapy presents significant challenges due to the pronounced growth and development during early life stages, affecting drug response. Perinatal asphyxia (PA) is a critical condition in neonates often requiring therapeutic hypothermia (TH) and intensive care to reduce morbidity and mortality. Animal models, such as the neonatal Göttingen Minipig, are valuable for understanding the mechanisms of conditions and therapies in pediatric populations. This study hypothesized that systemic hypoxia, as well as TH, affect drug disposition, including enzyme functionality. Given the effects of TH and PA on physiology, the primary hypothesis was that TH significantly decreases metabolic clearance (CL) in asphyxiated neonates, necessitating drug dosing adjustments. To address this, in vitro and in vivo data from neonatal Göttingen Minipigs will be used to develop a physiologically-based pharmacokinetic (PBPK) model to ensure accurate drug dosing for asphyxiated neonates. An experimental study was carried out to examine the pharmacokinetics of midazolam, fentanyl, phenobarbital, and topiramate under four conditions: therapeutic hypothermia (group TH), hypoxia (group H), hypoxia and therapeutic hypothermia (group H+TH), and controls (group C). Six healthy male Göttingen Minipigs, within 24 hours of birth per condition, were anesthetized for drug administration and blood sampling. Whole-body hypothermia was induced by lowering the Göttingen Minipigs' body temperature to 33.5°C, while systemic hypoxia was induced using a low-oxygen gas mixture. The gene and protein expression, as well as the activity of specific cytochrome P450 (CYP) enzymes were evaluated using in vitro methods. Hepatic CYP expression levels and activity were assessed in both adult and neonatal Göttingen Minipigs, in addition to the 24 experimental Göttingen Minipigs.

The study confirmed the feasibility of conducting complex in vivo procedures in neonatal Göttingen Minipigs, including endotracheal intubation, vascular access, anesthesia, and mechanical ventilation, which are critical for nonclinical animal experimentation. Furthermore, a novel pig model of asphyxia was established, providing valuable insights into PA and related therapies. The drug disposition investigation conducted for 24 hours highlighted several alterations. Fentanyl was mostly affected since TH led to a 66% decrease in CL and approximately a 3-fold longer half-life. The study also explored the pharmacodynamic effects of TH, revealing notable impacts on heart rate in neonatal Göttingen Minipigs, consistent with documented effects in human neonates. These effects are particularly important for high extraction ratio drugs like fentanyl, due to their dependence on liver blood flow for their subsequent metabolism. Additionally, the research provided insights into the influence of age, hypothermia, and hypoxia on CYP enzymes. The vitro studies revealed differences in CYP expression and activity between neonatal and adult Göttingen Minipigs, underscoring the role of maturation in drug metabolism. Additionally, adult liver microsomes exhibited a 36% reduction in CYP activity following in vitro hypothermia exposure. Alterations in the expression of CYP3A29 and CYP2E1 occurred due to hypoxia in neonatal Göttingen Minipigs. Lastly, this PhD thesis underscores the importance of refining and updating the PBPK model to improve its predictive accuracy and clinical relevance, marking a significant advancement in using neonatal Göttingen Minipigs for evaluating pediatric drug therapies.

Rik van den Boom

• 6 november 2024, 17u
• CDE, gebouw O (D.O.07)
• Promotoren: Prof. Dries Knapen en Dr. Lucia Vergauwen

Abstract

Endocrine disrupting chemicals (EDCs) are a group of chemicals that interact with the endocrine system, causing adverse effects. Some EDCs, commonly known as metabolism disrupting chemicals (MDCs), have the potential to disrupt an organism’s energy metabolism. MDCs are known to increase the risk of metabolic disorders, such as diabetes and obesity. Paraoxonase 1 (PON1) is an enzyme with antioxidant functions that has been suggested to play a role in metabolism disruption: it has been associated with the occurrence of human metabolic disorders by genome-wide association studies. In this work, we used the zebrafish, an established model in toxicology, to study the mechanisms of metabolism disruption in different life stages (embryo, larva, adult), including the role and responses of PON1.

By exposing zebrafish embryos to different MDCs we demonstrated how metabolism disruption can lead to adverse effects in the earliest life stages of fish. Our larval and embryonic zebrafish data suggest that PON1 may respond to oxidative stress that is induced by metabolism disruption. In addition, by comparing wild type to PON1 knockout larvae we showed how PON1 may also play a role in metabolism disruption by modulating metabolic processes directly, in addition to its antioxidant role.

Overall, our results provide new insights into the mechanisms underlying the interactions between metabolism disruption and PON1 in zebrafish. This work can support the development of novel approaches and test methods for the identification and characterization of MDCs.

Chloé Bars

• 5 november 2024, 16u
• CDE, gebouw O (D.O.06)
• Promotoren: Prof. Steven Van Cruchten, Prof. Alain Labro en Dr. Kenn Foubert

Abstract

Over the past two decades, zebrafish (Danio rerio) embryos have gained significant interest as an alternative model for developmental toxicity testing, particularly in place of traditional animal studies, which are costly, require long-term monitoring, and involve large numbers of animals. Zebrafish offer numerous advantages and are classified as an in vitro model up to the free-feeding stage (120 hours post-fertilization, hpf) under European Directive. However, further characterization of this model, including its xenobiotic-metabolizing capacity, is essential for a more comprehensive understanding of its applicability to human safety assessment.

This research project focused on a detailed characterization of the transcript profiling of key enzymes involved in xenobiotic metabolism, such as cytochrome P450 (CYP) enzymes, during zebrafish development up to the juvenile stage. This was achieved through quantitative PCR on mRNA extracted from pooled organisms. Additionally, we investigated the ability of zebrafish embryos/larvae (from 5¼ to 120 hpf) to metabolize two known mammalian proteratogens, carbamazepine (CBZ) and phenytoin (PHE), into their respective mammalian teratogenic metabolites: carbamazepine-10,11-epoxide (E-CBZ) and 5-(4-hydroxyphenyl)-5-phenylhydantoin (HPPH). This was done using an ultra-performance liquid chromatography-triple quadrupole mass spectrometry investigation. The gross morphology of the embryos and larvae exposed to these compounds was also observed under a microscope up to 120 hpf. Subsequently, we explored ways to improve the Zebrafish Embryo Developmental Toxicity Assay (ZEDTA) by incorporating a metabolic activation system through a preincubation strategy.

Our investigation of CYP ontogeny revealed that CYP1-3 mRNA expression levels were low until 72 hpf, after which they increased and reached a plateau around 120 hours. Accordingly, the metabolism study showed that zebrafish embryos metabolized CBZ into E-CBZ at the end of organogenesis (96 hpf), but no conversion of PHE to HPPH was detected. In addition, exposure of zebrafish embryos to CBZ and PHE resulted in teratogenic effects, although these were not observed for the mammalian teratogenic metabolites. The enhancements made to the ZEDTA, including centrifugation and dilution steps in the preincubation medium, enabled exposure of embryos to both compounds and their potential metabolites until 120 hpf. Overall, our findings indicate that zebrafish embryos possess a moderate ability to metabolize xenobiotics by the end of organogenesis. However, teratogenicity in zebrafish may follow different pathways compared to mammalian species and further characterization of other metabolic enzymes is necessary before this model can be fully validated. However, the scientific community is shifting toward combining New Alternative Methods with mechanistic investigations, rather than relying solely on the ZEDTA.

Ben Meulders

• 15 oktober 2024, 17u
• CDE, gebouw O (D.O.06)
• Promotoren: Prof. Jo Leroy en Dr. Waleed Marei

Abstract

The global rise in obesity, driven by high-fat/high-sugar diets, is increasingly linked to fertility issues in women and long-term health problems in their offspring. A key factor mediating these effects is the increased level of non-esterified fatty acids (NEFAs) in the ovarian and oviductal microenvironment, which imposes metabolic stress on the oocyte and early embryo. This stress is linked to mitochondrial dysfunction and epigenetic alterations. While the mitochondria’s influence on epigenetic programming in somatic cells (a process termed metaboloepigenetics) is well known, the direct relationship remains poorly understood in oocytes and early embryos. These cells exhibit unique mitochondrial characteristics and undergo dynamic epigenetic reprogramming. In this PhD research, we hypothesized that mitochondrial dysfunction in metabolically compromised oocytes and embryos affects epigenetic programming during early development, with potential long-term consequences for offspring health. Preconception dietary interventions aimed at improving oocyte mitochondrial function may prevent these outcomes.

Using a bovine in vitro model, we showed that increased NEFAs during final oocyte maturation (and early embryo development) induce epigenetic alterations during early embryogenesis and on day 14 of development. Importantly, this was associated with mitochondrial dysfunction. Oocyte ATP production appears to play a crucial role in the mitochondrial-epigenetic link as specific inhibition of bovine oocyte ATP production during final oocyte maturation resulted in altered epigenetic programming in both the oocyte and early embryos. In a complementary in vivo outbred mouse model, we demonstrated that a long-term maternal high-fat/high-sugar diet from the preconception period until weaning (17 weeks) only moderately impacted offspring metabolic health and oocyte quality after being fed chow diet from weaning until adulthood. Switching the mothers to a control diet for four weeks before conception alleviated most of these effects.

The novel insights from this research provide a foundation for further exploration of the mitochondrial-epigenetic link in oocytes and embryos under maternal metabolic stress conditions. These findings also offer potential avenues for developing in vitro or in vivo mitochondrial-targeted therapies to safeguard epigenetic programming and improve offspring health.

Kerlijne Moorkens

• 21 juni 2024, 17u
• CDE, gebouw Q (D.Q.002)
• Promotoren: Prof. Jo Leroy en Dr. Waleed Marei

Abstract

Maternal metabolic disorders associated with the consumption of an obesogenic (high fat/high sugar, HF/HS) diet are strongly linked with reduced fertility in women. 

The impact of obesity and an obesogenic diet on the oviductal microenvironment is much less characterized compared to the impact on the follicular environment and oocyte quality. Furthermore, the onset and duration of changes after the start of an obesogenic diet were unclear. In addition, the sensitivity to obesogenic diet-induced obesity and its influence on the metabolic profile and oocyte quality have been shown to be dependent on the genetic background of the mouse model. Therefore, the impact of feeding an obesogenic diet for different time periods on the transcriptome of oviductal epithelial cells (OECs) in Swiss compared to C57BL/6N mice, specifically focusing on genes involved in oxidative and cellular stress levels and inflammatory responses, was tested. Our findings demonstrated, for the first time, that feeding an obesogenic diet for a short period of only three days resulted in acute systemic changes and acute local oxidative stress or mitochondrial stress effects on OECs, evident already before the development of an obese phenotype. The acute effects in the OECs initiated a cascade of transcriptomic changes to control mitochondrial ROS production and endoplasmic reticulum (ER)-stress. However, prolonged, long-term feeding resulted in a persistent upregulation of (mitochondrial) oxidative stress and ER-stress, with ultimate signs of local and systemic inflammation. Furthermore, different responses to the obesogenic diet were observed between Swiss and C57BL/6N mice. 

Since fatty acids play an important role as structural membrane components, in cell signalling and cell-to-cell interactions, alterations to the lipid composition of the oviduct may reduce its ability to support early embryo development, which may have long-lasting consequences. Therefore, the dynamics and the nature of changes in the lipidomic profile of the oviductal epithelium upon and during obesogenic diet feeding over time in an outbred Swiss mouse model was studied. It was demonstrated that feeding the obesogenic diet resulted in acute changes in the lipid profile in the oviductal epithelium already after 3 days on the obesogenic diet. The changes in the lipid profile progressively increased and became more persistent after long-term obesogenic diet feeding. Functional annotation revealed a differential abundance of phospholipids, sphingomyelins and lysophospholipids in particular. 

The different durations of feeding used in this PhD research, allowed a clear distinction of acute responses and later changes after long-term feeding and after the development of the obese phenotype, and the associated unhealthy metabolic phenotype. Our findings in the oviduct illustrate that the mechanism by which the obesogenic diet impacts fertility is not only mediated through reduced oocyte quality but might also directly impact early embryo development in the oviduct, potentially leading to long-term effects on foetal development, pregnancy success and postnatal health through epigenetic alterations. 

In the ovary, it is known that the impact of lipotoxicity, caused by an obesogenic diet, on oocyte quality is mediated by oxidative stress, ER stress and mitochondrial dysfunction. However, the nature and magnitude of these changes may be dependent on the duration of feeding (and thus the development of the obesogenic phenotype). Therefore the impact of feeding an obesogenic diet for different time periods on the ovary and ovarian cells of outbred Swiss mice was tested, with a focus on dynamic changes in granulosa cell transcriptomics and oocyte quality. The results demonstrated that feeding an obesogenic diet resulted in acute changes in the lipid content of oocytes and impacted endoplasmic reticulum and mitochondrial functions in granulosa cells already after short-term feeding of only 24 hours till 1 week. The effects progressively increased over time after prolonged obesogenic diet feeding, with increased mitochondrial abnormalities in oocytes and different affected pathways, linked to DNA repair, cell signalling, UPRs and many other GO annotation biological terms in the granulosa cells. Exposure to an obesogenic diet thus resulted in cell specific changes in oocytes and granulosa cells, already after 24 hours to 1w, which can directly affect oocyte quality and early embryo development, eventually leading to reduced fertility. 

The deep analysis and novel insights of this PhD thesis can be useful for many researchers and form an important fundament for further studies that focus on protecting and enhancing oocyte and embryo development during the very early stages under diet-induced metabolic stressed conditions. Understanding the magnitude and nature of reproductive cell dysfunction after different exposure periods to a HF/HS diet is crucial to develop more efficient preconception care intervention strategies to enhance fertility under diet-induced metabolic stress conditions.

​​Jente Hoyberghs

• 4 juni 2024, 17u
• CDE, gebouw O (D.O.06)
• Promotoren: Prof. Steven Van Cruchten en Dr. Kenn Foubert

Abstract

Within Europe, new approach methodologies (NAMs) for toxicity assessment of xenobiotics become very important. Several pharmaceutical, (agro)chemical and cosmetic companies are currently using zebrafish embryo assays as an alternative for animal testing to screen new compounds for developmental toxicity. The zebrafish embryo assay is considered to be very promising, as it is the only non-animal assay that allows assessment of a vertebrate model during the main organogenesis period with a relatively high accuracy. However, it still suffers from some limitations. Inter- and intra-laboratory discordances in teratogenicity classification of identical compounds, as well as false negative and false positive results are reported for known mammalian teratogens and non-teratogens, respectively. In view of human safety, false negative results are more critical than false positive results, as teratogens may be missed. Causes for these false negative results include: inter-species differences in mode of action, issues with compound uptake, the limited biotransformation capacity and the limited number of morphological endpoints in zebrafish embryo assays. Therefore, the aim of this doctoral thesis was to further standardize and optimize the Zebrafish Embryo Developmental Toxicity Assay (ZEDTA) in order to increase the sensitivity of this screening assay, and as such better predict birth defects caused by drugs during the first trimester of pregnancy. To do so, we: 1) developed a standardized ZEDTA protocol that can be extended with a metabolic activation system, 2) determined the maximal concentration of DMSO that can be safely used as a solvent in the ZEDTA, 3) investigated whether the sensitivity of the ZEDTA could be increased by including a skeletal staining method, and 4) investigated whether anti-epileptic drugs that require bioactivation to exert their teratogenic potential are biotransformed by non-CYP enzymes in zebrafish embryos and young larvae, and whether these metabolites cause developmental toxicity.

Sara Prims

• 27 maart 2024, 17u
• CDE, gebouw O (D.O.02)
• Promotoren: Prof. Chris Van Ginneken, Prof. Xaveer Van Ostade en Prof. Christophe Casteleyn

Abstract

In pig farming, identifying potential stress at the farm has multiple advantages. It is known that stress, especially long-term chronic stress, will lead to reduced welfare, suppress the immune system, reduce zootechnical performances and disturb breeding performances implying economical losses for the farmer. Therefore, a fast, easy, reliable, and objective tool to monitor stress can provide beneficial information for the farmer and benefit welfare. Already numerous methods to assess a pig’s welfare exist, unfortunately, all with their own limitations. As a result, no definitive set of parameters or indicators of animal welfare exists. Therefore, the goal of this research project was to explore saliva, more specific the salivary proteome, as a tool to identify chronic stress in piglets.

Since knowledge of the protein composition of porcine saliva was scarce, the first goal of this thesis was to study and expand the knowledge about the porcine salivary proteome. Because whole saliva, i.e., saliva that is secreted in the oral cavity is mixed with gingival crevicular fluid, buccal cells, microorganisms, and food remnants, gland-specific saliva was collected as ductal secretions. A total of 122 porcine salivary proteins and six mammalian salivary proteins with a predicted porcine homolog were identified in gland-specific saliva.

Once more insight into the porcine salivary proteome was gathered, saliva could be exploited as a tool to monitor chronic stress. According to literature, some factors, like deprivation of cage enrichment, frequent mixing of animals and overcrowding could introduce chronic stress in pigs. However, to verify whether these factors could effectively be considered stressors, cortisol was investigated first. Hair from the stressed group contained significantly higher cortisol concentrations, whereas salivary cortisol concentrations did not significantly differ between groups.

Once the effectiveness of the stressors was confirmed, the salivary proteome of chronically stressed piglets was compared with that of control piglets. Shotgun analysis identified 392 proteins in saliva of 28-day-old piglets. The relative abundance of 20 proteins was affected by three weeks of exposure to multiple stressors. From these 20 proteins, eight were selected for further validation with targeted parallel reaction monitoring. This validation confirmed that alpha-2-HS-glycoprotein was upregulated in the stressed group, while odorant-binding protein, chitinase, long palate lung and nasal epithelium protein 5, lipocalin-1, and vomeromodulin-like protein were present in lower concentrations in the saliva of the stressed pigs.

It can be concluded that besides expanded knowledge on the porcine salivary proteome, differences in the salivary profile of chronically stressed young pigs could by detected. After further validation, the affected proteins could be used as salivary biomarkers to identify stress problems at the farm, elicit transparency regarding animal welfare to the consumer, and facilitate research to optimise rearing conditions.

Kevin Van Tichelen

• 12 februari 2024, 17u
• CDE, gebouw O (D.O.07)
• Promotoren: Prof. Chris Van Ginneken, Prof. Joris Michiels en Prof. Steven Van Cruchten

Abstract

In modern pork production, the selection for larger litters has not only resulted in more piglets per sow, but also in an increased proportion of low birth weight (LBW) piglets which are associated with a higher perinatal mortality. This thesis’ scope was to enhance the resilience (the capacity to recover in terms of performance and survival) of LBW piglets during the pre-weaning period through drenching, the oral administration of substances. During a first study, LBW piglets were sham drenched to evaluate the safety of handling during drenching. No harmful or positive effect of drenching was observed, and thus, it was concluded that drenching is a safe tool that can be implemented in a good pre-weaning management. In a second field-trial, LBW piglets were drenched with bovine colostrum, short-chain fructo-oligosaccharides (scFOS) or quercetin (each dissolved in a plain milk replacer) during the first seven days after birth. None of the supplemented compounds had a positive effect on the LBW piglets’ resilience. Moreover, a negative effect on survival was observed in piglets that were drenched with scFOS. These results showed that the evaluated bioactive compounds, in their given dosages, were unable to improve the LBW piglets’ survival and emphasised the complex, multifactorial origin of pre-weaning mortality. A final study aimed to determine whether the performance of LBW piglets could be improved by drenching a dense, concentrated milk replacer (DMR), and whether the frequency of drenching and the severity of the LBW played a role. Secondly, this study compared the supplementation of the same milk replacer at two farms with different perinatal management. No effect of drenching DMR on the survival or performance of (very) low birth weight piglets was observed, regardless of farm and, apparently, of the applied management. In conclusion, the studies within this dissertation validated drenching as a safe technique for LBW piglets, but could not find a positive effect on the resilience of the administered substances. Thus, drenching did not suffice as a single intervention to improve the resilience of LBW piglets.

Allan Valenzuela

• 24 november 2023, 16u
• CDE, gebouw O (D.O.05)
• Promotoren: Prof. Steven Van Cruchten en Dr. Miriam Ayuso Hernando

Abstract

Antisense oligonucleotides (ASOs) belong to a large group of nucleic acid-based therapeutics that utilize synthetic oligonucleotides to modulate RNA translation. As ASOs need to hybridize to their complementary target RNA, nonhuman primates (NHPs) are the preferred non-rodent model due to their close genetic homology and pharmacokinetics with humans. However, the adult Göttingen Minipig has been recognized as a suitable alternative to NHPs in the safety testing of ASOs, as it showed similar pharmacokinetic, pharmacodynamic, and safety profiles in a previous study. Since ASOs are usually indicated to rare genetic conditions that could start early in life, and as the rapid growth and development of the pediatric population can influence the pharmacokinetics/pharmacodynamics of therapeutic agents, leading to potential toxicities, extending the previous work to the juvenile minipigs was deemed necessary. In this thesis, we evaluated the juvenile Göttingen Minipig as a pediatric safety testing model for ASOs. As such, we assessed potential differences in exposure/toxicity and pharmacologic effect of a model ASO (RTR5001) in the juvenile Göttingen Minipig in an 8-week repeat-dose toxicity study (weekly subcutaneous dosing starting at postnatal day 1). Accordingly, the model ASO that had been previously characterized in adult Göttingen Minipigs and NHPs showed comparable clinical chemistry and toxicity profiles in the juvenile minipigs. However, differences in plasma and tissue exposures, and pharmacologic activity were observed compared to the adult data. The ontogeny evaluation of the key nucleases responsible for ASO metabolism and pharmacologic activity revealed a differential nuclease expression and activity, which could affect the metabolic pathway and pharmacologic effect of ASOs in different tissues and age groups. Likewise, to further understand the species translatability of ASO-induced thrombocytopenia, in vitro platelet activity and aggregometry assays were performed using a panel of tool ASOs with different sequences and modifications. Our data on direct platelet activation and aggregation by ASOs in adult minipig samples are remarkably comparable to human data. Moreover, phosphorothioated ASOs bind to platelet collagen receptor glycoprotein VI (GPVI) and directly activate minipig platelets in vitro, mirroring the findings in human samples. The differential abundance of GPVI (and platelet factor 4) in minipigs provides insight into the influence of ontogeny in potential ASO-induced thrombocytopenia in pediatrics. Therefore, the body of data from this thesis is fundamental for selecting the juvenile Göttingen Minipig in assessing safety concerns for ASOs intended for the human pediatric population.

Rohit Arora

• 9 november 2023, 15:15u
• Online
• Promotoren: Prof. Xaveer Van Ostade en Prof. Alain Labro

Abstract

The largest family of membrane receptors, known as G protein-coupled receptors (GPCRs), are essential to cellular signaling and regulate physiological processes. Presently, ~35%–40% of US FDA-approved medications target GPCRs. A subfamily of GPCRs, Mas-related G protein-coupled receptors (MRGPRs), which belong to the δ-group of the rhodopsin-like GPCRs, was discovered two decades ago. MRGPRs are expressed by small non-myelinated sensory neurons of the dorsal root ganglia and trigeminal ganglia, mast cells, neutrophils, and macrophages and are known to play a role in itch, pain, and pseudo-allergic drug reactions. Moreover, MRGPRs have been identified as mediators in the renin-angiotensin system and cardiovascular biology. In addition, literature suggests that MRGPRs are also involved in inflammatory processes. Despite the fact that humans express eight MRGPRs (MRGPRD to G and X1-X4), information about their activation, signaling pathways, and role in inflammation is insufficient, and most of them are still classified as orphans. Since MRGPRs are involved in itch, pain, and inflammation, which are important physiological processes, the goal of this PhD was to: i) examine the role of MRGPRs in inflammation biology; ii) decipher the activation mechanism of MRGPRs; and iii) elucidate the oligomeric interaction of MRGPRs.

Firstly, it was investigated whether β-alanine or alamandine-activated MRGPRD induces interleukin-6 (IL-6) release. It was observed that β-alanine activated MRGPRD-induced IL-6 release via the Gαq/Phospholipase C/NF-kB signaling pathway. Moreover, using IL-6 as a marker for MRGPRD activation, the mechanosensitivity of the MRGPRD and the effect of sterol derivatives, i.e., cholesterol and bile acids, on the activation of MRGPRD were established. Furthermore, it was discovered that the MRGPRD was constitutive (ligand-independent) active. In addition, it was discovered that methyl-β-cyclodextrin, which is known to remove sterols from the plasma membrane, triggered the MRGPRD-mediated IL-6 release.

Secondly, in an effort to deorphanize MRGPRs, it was established that cysteine protease cathepsin S activates MRGPRD and MRGPRF. Lastly, using biophysical and biochemical techniques such as luciferase complementation, bioluminescence resonance energy transfer, and co-immunoprecipitation assays, the heteromeric interactions between MRGPRE and MRGPRF were unambiguously detected.

Overall, in this doctoral thesis, the primary objective was to improve understanding of the involvement of MRGPRs in inflammatory biology, the activation mechanisms of MRGPRs, and the oligomeric interaction of MRGPRs.

Laura Buyssens

• 11 oktober 2023, 17u
• CDE, Aula Fernand Nédée (D.Q.001)
  
• Promotoren: Prof. Steven Van Cruchten en Prof. Chris Van Ginneken
  

Abstract

The pediatric population is one of the most vulnerable age groups within the human population, especially regarding drug development. They comprise approximately 30% of the total human population, but still very little research is conducted in their interest. Although pediatric drug development has been encouraged and efforts are ongoing, knowledge gaps still exist. ADME (i.e., absorption, distribution, metabolism and excretion) properties are particularly understudied possibly resulting in under or overdosing. The neonatal population is especially prone to adverse drug reactions since clinical trials in this group are often perceived as not feasible or unethical. Clinicians are thus required to administer drugs off-label (i.e., without information on safety and efficacy). To overcome this issue, alternatives are explored.

The use of juvenile animal studies, for example, is supported by FDA and EMA to improve our knowledge in the pediatric population. In this regard, the (mini)pig has gained ground as nonclinical species. Newborn piglets share the same size as human neonates and can be assessed in the same way as babies in the neonatal intensive care unit. With regard to hepatic drug metabolism, large similarities have been observed between adult humans and pigs, but data in the human and porcine (preterm) neonatal and juvenile population remain scarce. The goal of this thesis was thus to further characterize hepatic phase I (e.g., cytochrome P450 (CYP)) and phase II (e.g., uridine 5’-diphospho-glucuronosyltransferase (UGT)) enzyme ontogeny in neonatal and juvenile (mini)pigs. 

First, we characterized hepatic phase I drug metabolism on protein level in fetal, neonatal, juvenile and adult Göttingen Minipigs. Hepatic CYP protein abundance was investigated in liver microsomes using an LC-MS/MS approach. Various ontogeny profiles were discovered which were in line with observations in man. Sex-related differences were detected with highest expression in female compared to male minipigs. CYP protein abundance was correlated with earlier observed CYP enzyme activity.

Next, the translational potential of the preterm pig model for human neonatal drug metabolism was investigated, as preterm-born neonates are still considered to be “therapeutic orphans” within pediatric drug development. The aim of this study was to examine the ontogeny of CYP3A and UGT enzyme activity in the liver of preterm (90% gestation, gestational day 105 – 107) and term-born (100% gestation, gestational day 115 – 117) domestic piglets. In addition, the effect of chronological (i.e., gestational age, and postconceptional age) and postnatal age (i.e., birth effect) on the onset of enzyme activity was examined. UGT activity showed a significant postnatal increase in both preterm and term-born piglets from birth onwards. CYP3A enzyme activity was only detected in preterm-born piglets at postnatal day 26 while a gradual increase was observed in term-born piglets from postnatal day 11 onwards. In both groups, enzyme activity was lower in the preterm compared to the term group, suggesting that postconceptional age rather than postnatal age is affecting CYP3A and UGT enzyme ontogeny in the pig.         

Finally, the data obtained in this thesis contribute to a better understanding of the biotransformation capacity in the (mini)pig. In general, large similarities with human pediatric hepatic drug metabolism were found which encourages the use of the pig as nonclinical species for pediatric drug development. However, it needs to be emphasized that nonclinical species selection for juvenile animal studies is not just driven by hepatic drug metabolism, as other factors are also important. Therefore, species selection has to be considered very carefully on a case-by-case basis.

Anouk Smits

• ​31 maart 2022, 17u
• CDE, auditorium O.07
• Promotoren: Prof. Jo Leroy en Prof. Diane De Neubourg

Abstract

There is a significant negative impact of metabolic health disorders (like obesity) on female fertility, with an important role of reduced oocyte and embryo quality. As a result, more and more research focuses on the possibilities of specific interventions to improve fertility. Such intervention should prevent further damage or should improve and/or recover oocyte quality leading to an optimized oocyte developmental competence and ultimately restored fertility. Can we substantiate these concepts in vitro and which mouse model should be used to investigate the importance of preconception care in vivo?

The consequences of maternal metabolic disorders on oocyte quality are clear. However, it is not known if this metabolically-compromised oocyte can be rescued. An in vitro bovine exposure model was used to obtain metabolically-compromised oocytes. To investigate the impact of post-conception care interventions, the in vitro culture medium was supplemented with anti-apoptotic, antioxidative and mitogenic factors. Results indicated that supportive embryo culture conditions might improve oocyte developmental competence but resulting embryo quality was still aberrant.

Research investigating the impact of obesity on metabolic health (and fertility) often uses the inbred C57BL/6 strain. However, inbred strains are characterized by reduced fertility and might limit translation to human outbred physiology. We showed that control inbred C57BL/6 mice had oocytes with already high cellular stress and significant ultrastructural abnormalities. This deviating profile was only present in outbred Swiss mice after exposure to an obesogenic diet for 13 weeks. These results indicate that Swiss mice are the better choice for research regarding obesity and oocyte quality.

Nowadays, the development of preconception care interventions (PCCI) for obese women to improve their fertility is gaining more and more attention. However, so far there are no clear preconception guidelines. Knowledge on the impact of dietary PCCI on oocyte quality and the necessary duration for this PCCI is lacking. Therefore, the impact of diet normalization or caloric restriction for different time periods on metabolic health and oocyte quality of high fat or high-fat/high-sugar diet-induced obese mice was tested. The impact of the tested PCCI seemed to depend partially on the obesogenic diet used. Overall, the results indicated that switching to an ad libitum control diet for at least four weeks was the most promising approach to improve metabolic health and oocyte quality. However, complete recovery of oocyte quality was not present. These increased fundamental insights can bridge the gap towards clear preconception guidelines for obese women planning for pregnancy.

Anniek Bus

• 20 december 2021, 17u
• CDE, Promotiezaal (Q.002)
• Promotoren: Prof. Peter Bols en Prof. Jo Leroy

Abstract

Nowadays, an emerging need for (human) fertility preservation (FP) strategies is present, in particular when young girls and woman are confronted with cancer treatment. In addition, the preservation of genetic material from endangered animal species or animals with important genetic traits will also benefit from the development of alternative FP strategies. The strategies in this thesis focus on the use of pre-antral follicles (PAF) as, for prepubertal girls and women whose cancer treatment cannot be postponed, there are no other options. As PAFs account for the vast majority of follicles in the ovarian cortex, they represent an untapped potential, which could be cultivated for reproduction, preservation or research purposes. Vitrification is the most successful preservation method for gametes and embryos. However, a negative consequence of this strategy is the increased probability of injuries caused by exposure of cells to other non-physiological conditions that may impair their further development. Research performed on effects of vitrification on the viability of isolated PAFs remains scarce and protocols still need to be optimized for each specific cell type and species. Due to their small size (30 µm), the isolation and processing of isolated PAFs remains a huge challenge in terms of follicular retrieval and manipulation. Research presented in this thesis shows that the vitrification of isolated PAFs is a promising FP strategy. This is particularly relevant for cancer patients at moderate-to-high risk of ovarian metastasis. Different vitrification techniques including straws, follicle embedding, cell strainers and Cryotop were evaluated for follicle survival and applicability in clinical practice. We however, found that all evaluated techniques, next to their advantages also had their downsides. Several methods were used to assess follicle quality, ranging from gross morphology, simple viability stains as Neutral red and Calcein, immunochemical methods to more complex xenografting procedures. Our main finding is that isolated PAFs can survive the cryopreservation process and maintain their intercellular connections which are crucial for further development. In vitro development of PAFs to the pre-ovulatory stage has not yet been achieved in humans and larger animals. However, in vitro culture systems for PAFs are under development and are expected to become available in the near future. Animal in vitro models such as the bovine that rely on unlimited sources of research material and which are largely free of ethical or moral restrictions, have an important role in contributing to make faster progress in the development of these FP strategies.

​Evelyn Stinckens​

• 22 september 2020
  
• Promotoren: Prof. Dries Knapen, Dr. Hilda Witters en Dr. Lucia Vergauwen

Abstract

Thyroid hormones (THs) play a crucial role in the regulation of vertebrate development and homeostatic processes. A growing number of environmental pollutants are known to adversely affect the TH system. Disruption of this system is increasingly being recognized as an important endocrine disrupting mode of action that can cause a wide variety of adverse effects.

Major gaps have been identified in the tools available for the hazard and risk assessment of TH disrupting substances. The scientific community is currently challenged with developing new or improved testing approaches to evaluate TH disruption in fish. Therefore, the overall aim of this thesis was the development of a tiered testing strategy for the assessment of TH disruption in fish. We used the Adverse Outcome Pathway (AOP) framework for guiding our work.

First, we constructed AOPs leading from thyroperoxidase (TPO, essential for TH synthesis) and deiodinase (DIO, critical for TH activation) inhibition to impaired swim bladder inflation of zebrafish and fathead minnow through decreased thyroid hormone concentrations, ultimately affecting survival probability and population growth.

Next, we provided additional biological and toxicological data supporting the mechanisms and processes captured in the AOPs. We performed in chemico assays targeting the specific molecular initiating events, DIO and TPO inhibition, and we evaluated the use of the resulting in chemico data for predicting higher level in vivo endpoints. We demonstrated that the combined information of the enzyme inhibition assays can be used as a tool to reliably predict the biological effects on swim bladder inflation with only few outliers. This work thus provides an example of how the AOP framework and associated data generation can address current TH disruption testing challenges in the context of fish early-life stage assays, and fish tests in general.

Finally, we demonstrate how different assays covering essential biological processes along the continuum of the AOP network can be implemented in a tiered screening and testing approach for TH disruption in fish. The addition of the associated assays to existing test guidelines allows to increase their diagnostic value for the assessment of TH disruption. Further expanding the applicability domain of our AOP network would be of great value for screening and testing of TH disrupting compounds.

​Evy Verbueken​

• 16 december 2019
  
• Promotoren: Prof. Steven Van Cruchten en Prof. Chris Van Ginneken

Abstract

The zebrafish embryo is increasingly used as an alternative model to screen new drug candidates and environmental pollutants for developmental toxicity (i.e. teratogenicity). Since the zebrafish is not considered to be a test animal until it reaches the stage of independent feeding, the zebrafish embryo developmental toxicity assay (ZEDTA) fits within the 3Rs (i.e. Replacement, Reduction and Refinement) concept as described within laboratory animal sciences.

The externally developing zebrafish embryo cannot rely on maternal metabolism unlike the mammalian embryo. Hence, the zebrafish embryo gets directly exposed to the parent compound and depends on its own drug–metabolizing capacity for the detoxification or bioactivation of xenobiotics. In this respect, knowledge of the intrinsic biotransformation capacity during zebrafish organogenesis, which coincides with the exposure window of the ZEDTA, is key in order to correctly interpret the outcome of the ZEDTA. However, the overall results of studies described in literature regarding the xenobiotic–metabolizing capacity of zebrafish embryos are contradictory.

Hence, the main goal of this doctoral project was to characterize drug disposition in zebrafish during organogenesis with a main focus on cytochrome P450 (CYP)–mediated metabolism since the latter enzymes are responsible for the oxidation of the majority of marketed drugs. To this end, the thesis investigates the ontogeny of CYP enzymes on mRNA as well as on activity level, and to a lesser extent also of the expression levels of two major phase II enzymes and a drug transporter, i.e. abcb4, at different time–points during zebrafish organogenesis and beyond.

This project mainly showed that CYP–mediated biotransformation of xenobiotics appears to be immature during a major part of the ZEDTA exposure window (i.e. 4–120 h post–fertilization (hpf)). Moreover, the mRNA expression levels of the phase II enzymes and abcb4 reached maximum expression levels by the end of zebrafish organogenesis. These findings can have a profound impact on the predictivity of the ZEDTA for human safety assessment in the drug development process, especially in case of proteratogenic compounds that require bioactivation to exert their teratogenic potential.

A solution to overcome the immature biotransformation capacity of zebrafish embryos is to co–incubate the ZEDTA with a human–derived external metabolic activating system (MAS), such as human liver microsomes, during the entire exposure window of the ZEDTA. However, the co–incubation method with the external MAS needs to be further optimized and validated before it can be used in regulatory developmental toxicity testing.

​Ellen Michiels​

• 6 december 2019
  
• Promotoren: Prof. Dries Knapen en Prof. Steven Van Cruchten

Abstract

The presence of endocrine disrupting compounds (EDCs) in the environment is a major concern. EDCs directly interact with the endocrine system, leading to important adverse effects in humans and wildlife (e.g, related to reproduction, energy metabolism and growth, behaviour, etc.). To identify these compounds, several regulatory strategies and frameworks have been developed in many parts of the world. Various assays are available to assess the endocrine disrupting potential of compounds, and several methods are used to administer the compounds. Within the context of non-mammalian assays, fish are often used as model organisms.

Almost all current fish assays use adult animals, but there is increasing public pressure to reduce or even avoid the use of animal testing. Therefore, significant efforts are being made to develop new, alternative testing methods as well as to optimize existing methods for the assessment of endocrine disrupting compounds. The development of a fish embryo test for the detection of EDCs may offer a particular advantage in this context, since fish embryos are not considered test animals until the stage of free feeding while they do offer the biological relevance gained from using a model organism with an intact endocrine system.

Assays using fish and fish embryos are mostly performed via aquatic exposure. However, many EDCs are hydrophobic compounds. Aquatic exposure may therefore not always be the most biologically relevant exposure method. In addition, the use of hydrophobic compounds in an aquatic exposure set-up brings several practical challenges as well. In the present work, we used an existing fish embryo test guideline as a basis for the development of an alternative test system for the assessment of  EDCs.

Specifically, the Fish Embryo Acute Toxicity (FET) Test (OECD Test Guideline 236) was supplemented with a number of specific endpoints relevant to the screening and testing of estrogen disrupting compounds. In addition, we suggest a combination of aquatic exposure (i.e, as currently required in the FET test) and administration of the compound under consideration via micro-injection. Micro-injection into the yolk of the embryo can possibly mimic maternal transfer, and it helps to solve a number of practical challenges that are associated with the use of hydrophobic compounds in aquatic exposure scenarios. Overall, we developed a new approach allowing us to advance the zebrafish embryo test for use in the assessment of estrogen disrupting compounds.

​Karolien Desmet​

• 19 november 2019
  
• Promotoren: Prof. Jo Leroy en Prof. Peter Bols

Abstract

Lipolysis-associated maternal metabolic disorders, like obesity and diabetes type II in women and negative energy balance in dairy cows, are a major risk factor for subfertility. These altered metabolic states are reflected in the follicular and oviductal micro-environment with emphasis on elevated concentrations of non-esterified fatty acids (NEFAs). Although information is available about the toxic effects of elevated NEFA concentrations on oocyte and embryo developmental competence, there remains a lack of information regarding the underlying mechanisms. In this dissertation, the bovine model was used to study the relation between lipolysis-linked maternal metabolic disorders and fertility.

We showed that elevated NEFA concentrations during either in vitro oocyte maturation or early embryo development impact the resultant blastocysts’ transcriptomic and epigenetic profiles. This disturbance in epigenetic reprogramming may render the embryo susceptible to changes during further development. We observed that good quality blastocysts produced from metabolically compromised oocytes have defective post-hatching development after embryo transfer to a recipient cow. These embryos were retarded in growth, exhibited disturbed metabolism and transcriptomic profile, and displayed a reduced capacity to signal its presence to the mother.

Also the oviductal environment plays a vital role in the functionality of and interaction between the oocyte and sperm cells. We showed that although sperm functionality was affected under lipolytic conditions, adequate numbers of viable sperm cells retained their fertilizing ability. In contrast, the fertilization process was affected as well as subsequent embryo development.

Metabolic disorders and sub-/infertility are both increasing in men and women. The combination of having both obese parents on fecundity has received minimal attention although most couples share lifestyle and thus obesity in both persons is more likely to occur. We investigated if metabolic conditions of couples seeking assisted reproductive services in an IVF-clinic could be linked to embryo quality and metabolism. Aberrations in follicular fluid, female and male waist circumference had an effect on embryo quality and metabolism. Combining BMI of both parents led to an association between parental BMI and embryo metabolism. Only male BMI was associated with pregnancy outcome.

We concluded that lipolytic conditions cause subfertility through impairment of oocyte maturation, fertilization and embryo development. We also substantiated an effect of the parents’ metabolic health on quality and metabolism of their embryos in a human setting. In characterizing these effects, we may increase the awareness of the importance of parental metabolic health in relation to fertility and carryover effects persisting in the offspring.

​Isabelle Gabriëls​

• 6 juni 2019
  
• Promotoren: Prof. Dries Knapen, Prof. Marc De Loose en Dr. Lucia Vergauwen

Abstract

In the EU, a mandatory part of the safety assessment of genetically modified crops (GMOs) consists of a 90-day rodent feeding trial. As a relevant addition to the current assessment strategy, our aim was to develop a complementary zebrafish feeding trial specifically designed to address a number of shortcomings of existing test methods, using maize as a case study.

First, to ensure the use of nutritionally balanced experimental diets, we investigated whether substitution of a non-GM maize into the zebrafish diet causes component-related effects. High dietary maize percentages resulted in an overall disturbed uptake and processing of carbohydrates, influencing processes such as growth rate, and affected mRNA transcriptional liver patterns of genes involved in key metabolic processes. Balancing the physiological tolerance of the fish and achieving a toxicologically relevant dose of the GMO, 15% was selected as the optimal maize substitution level to be used for all experiments.

Next, a subchronic zebrafish feeding trial was developed for the toxicological safety assessment of GMOs. Endpoints included general health parameters, effects on energy metabolism, and reproductive effects including transgenerational effects. As a key aspect of our experimental design, we investigated the importance of different non-GM comparators. Our results highlight the importance of distinguishing between potential effects caused by the transformation process (i.e., the process of genetically modifying the plant) and those caused by the presence of the transgenic event itself. Indeed, most differences (in general health endpoints as well as transcriptional liver profiles) were identified between the conventional wild type control (i.e., the original genetic starting material) and null segregant (i.e., the plant progeny created during the transformation process but lacking the transgenic event) on the one hand, and between wild type and GM maize on the other hand, while differences between GM maize and null segregant were limited. To assess the toxicological relevance of biological responses, we further optimized a method allowing the interpretation of results relative to the natural response variation, i.e., the normal biological range in endpoint responses of the fish. We established natural response variation datasets for various zebrafish endpoints and discussed equivalence testing as a conceptual approach using examples originating from the subchronic/transgenerational feeding trial.

Overall, this work forms a strong foundation for advancing zebrafish feeding trials and, to facilitate the development of further strategies, main conclusions and recommendations have been summarized in a Guidance Document for the safety evaluation of GM crops using the zebrafish model.