Title Promoter Affiliations Abstract "Structural Biology of the Human Androgen Receptor for next generation drug design" "Frank Claessens" "Laboratory of Molecular Endocrinology" "The aim of this PhD project is to elucidate the mechanism of action of a novel class of human androgen receptor antagonists (MEL6), which were recently discovered in the KU Leuven Molecular Endocrinology Laboratory. Fragments of the human androgen receptor will be expressed in and purified to high purity from E. coli and utilized for crystallization experiments. While the structures of agonist complexes are already available, all efforts have failed to crystallize a complex of the hAR ligand binding domain (AR-LBD) bound to an antagonist. Therefore, we will follow a novel approach. First of all, the novel confirmed class of antagonists is significantly different in shape from the classical antagonists. Molecular modeling of AR-LBD:MEL6 complexes has suggested a binding mode that may stabilize the antagonistic conformation. Therefore, a classical co-crystallization approach will be followed. Secondly, we aim to crystallize the hAR-LBD in the presence of additional facilitator molecules, which can induce different crystal packings that may allow for soaking of antagonists or stabilization of the antagonistic conformation. These molecules are the so-called Poly-Oxometalates (POMs), which are being developed at the KU Leuven, Department of Chemistry, where also the MEL6 derivatives are being produced. Apart from the new MEL6 ligands, I will also attempt to soak the currently used drug molecules with the POM-protein crystal complexes, as structural information of them is still absent as well. In parallel with the two tracks of structural biology work, I will perform biophysical screening experiments to identify more potent ligand derivatives. As more insights are being gathered about the interactions of the N-terminus of the androgen receptor with cellular cofactors, in the final stage of this project, I will also focus on solving the structure of fragments of the androgen receptor N-terminal domain with (fragments of) its cellular cofactors." "Hormones and neuroplasticity: image guided discoveries of molecular mechanisms in neuroplasticity" "Veerle Darras" "Animal Physiology and Neurobiology" "During juvenile development, male songbirds exhibit a high amount of neuroplasticity in the brain regions responsible for song learning and production, called the 'song control nuclei'. Additionally, seasonal learners display a cycle of song control nucleus growth and regression throughout a year: song control nuclei increase in volume when the days get longer in early spring and regress again when days get shorter. Both neuroplastic phenomena are associated with song learning. In zebra finches for example, developmental neuroplasticity is accompanied by the learning of a single song that does not change after they become adults. In contrast, the brain of strong, seasonal learners like European starlings becomes sensitive again to new input every spring, which is associated with song control nucleus growth. Song learning and nucleus plasticity happen more strongly or even exclusively in male birds. Both displays of neuroplasticity (developmental and seasonal) are rare in the animal kingdom and their correlation with speech learning is obvious, making it a highly interesting research topic. Despite the appeal of the phenomena as a basis for research for human applications, the molecular and cellular mechanisms underlying this type of neuroplasticity are still unclear.In this thesis we investigated the possible involvement of thyroid hormones (THs) as regulators in this learning-associated plastic process. THs are essential for many processes and not in the least for neural development, differentiation and maturation. The effects of THs in a given tissue are mainly regulated by the local availability of three types of regulators. TH transporters enable THs to cross the blood-brain barrier and to enter neurons and glial cells through their cell membrane. The four known avian TH transporters are the monocarboxylate transporters 8 and 10, the organic anion-transporting polypeptide 1C1 and the L-type amino acid transporter 1 (LAT1). Deiodinases maintain the necessary amount of active TH by converting the prohormone T4 to the bioactive hormone T3 (deiodinase type 2, DIO2), or by inactivating T4 and T3 (deiodinase type 3, DIO3). Finally, TH receptors (encoded by the THRA and THRB genes) are ligand dependent transcription factors that regulate expression of TH-responsive genes. We used in situ hybridization to study the presence of mRNA of these regulators; firstly in the developing zebra finch brain during the entire period of song learning (10 to 120 days post hatch (dph)) and secondly in the brain of seasonally learning starlings in various photoperiods: photosensitive, photostimulated and photorefractory, corresponding to winter, early spring and summer respectively.In the first part of our research, we showed that in zebra finches, DIO2 expression was high in the endothelial cells lining the brain capillaries in the entire telencephalon at 10 and 20 dph. At 30 dph, when song control nuclei are still growing but the rest of the brain has attained its mature size, DIO2 expression diminished everywhere except in HVC, RA and Area X of male birds. Said expression remained high up to 60 dph while expression in the rest of the telencephalon became undetectable. At 90 and 120 dph, when song crystallizes, DIO2 expression in the nuclei also became nearly undetectable. In females, no such local expression was observed; DIO2 expression inside the song control nuclei receded in parallel with that in the rest of the brain at 20-30 dph. Additionally, in males but not females, LAT1 expression was higher in HVC than in the rest of the brain from 30 dph up to our final sampling point at 120 dph. THRA expression was widespread and THRB expression was generally low except in RA in both sexes. Our data clearly indicate that TH regulation, of which mainly TH activation by DIO2, is gender-specific and dynamic over time during developmental neuroplasticity.Secondly, we showed that in seasonal neuroplasticity, DIO3 expression in the starling HVC was strongly elevated after 4 weeks of photostimulation compared to the photosensitive state. This was accompanied by a decrease in expression of LAT1, further substantiating that TH action is actively restricted at that point in the photostimulated state. Assuming HVC has reached its maximal size by that time, we suggest that DIO3 inhibits further TH action and thus neuroplasticity in HVC. This would help in keeping the nucleus stable during breeding, which is necessary for strong, stereotypic song. DIO2 expression was below the detection limit at all stages but may yet be expressed in HVC shortly after photostimulation and thus before our studied time point. Both THRA and THRB were expressed but the observed expression level was constant for the different photoperiods.The present results indicate that regulation of THs, mainly activation by DIO2, plays an important role in developmental neuroplasticity of the song control nuclei. Furthermore, they suggest that active inhibition of TH action by DIO3 after 4 weeks of photostimulation contributes to the stability of HVC necessary for breeding-state song. Whether DIO2 also contributes to song control nucleus growth during seasonal plasticity is yet unclear but is a promising lead for further research. Our data provide essential information on a potentially crucial mechanism in learning-associated neuroplasticity; information that will hopefully help to further elucidate the molecular machinery involved in vertebrate, and especially human, memory processing." "The chicken cerebellum as a model to study the impact of monocarboxylate transporter 8 deficiency on early brain development" "Veerle Darras" "Animal Physiology and Neurobiology" "Thyroid hormones (THs) have been characterised as important regulatory factors of vertebrate brain development. As such, they contribute to a wide variety of neurodevelopmental processes that depend on adequate TH levels as well as a correct timing of TH access to distinct neural cells in order to obtain a fully functional brain. This strict regulation of local TH availability is the result of a complex interplay between TH transporters mediating TH passage across the plasma membrane and deiodinases which intracellularly activate or inactivate THs. While the consequences of TH deficiency on brain development have already been well described, the challenges for a better understanding of the underlying mechanisms of TH action in the developing brain are considerable. In this thesis, we focused on the functional involvement of the monocarboxylate transporter 8 (MCT8) in cerebellar circuit organisation by using a knockdown approach in the chicken embryo.To build a general picture of the dependency of the developing cerebellum on TH signalling, we determined the cell-specific expression pattern of TH transporters and deiodinases. Our results showed that throughout embryonic development, the cerebellum expresses the genes needed for TH traffic between neural cells and TH (in)activation in a tissue- and cell-specific way. The differential expression of TH regulatory genes implies differing spatio-temporal sensitivities to TH signalling. Purkinje cells (PCs), which are the sole output neurons of the cerebellum and known to be TH-sensitive, showed pronounced expression of MCT8 supporting a model where the MCT8 transporter plays a decisive role in the uptake of receptor-active 3,5,3’-triiodothyronine (T3) in central neurons.To study the role of this transporter in TH-dependent cellular events driving early cerebellar development, we developed a strategy to downregulate MCT8 expression and thereby alter local TH levels in the developing cerebellum. We created gene-specific RNA interference (RNAi) vectors based on the pRFPRNAiA and RCASARNAi vectors specifically designed for optimal gene silencing in chicken. The pRFPRNAiA vector is a standard plasmid vector, whereas the RCASARNAi vector is a precursor for a modified RCAS virus. In vitro testing in cell culture showed that both RNAi vectors efficiently silence MCT8 mRNA expression. To use these RNAi vectors in vivo, we developed a method for in vivo transfection of the cerebellum. By performing site-restricted in ovo electroporation at embryonic day 3, we were able to efficiently transfect distinct cerebellar cell types, as checked in embryos between 6 and 18 days old. Whereas the non-viral vector was predominantly expressed in PCs and cerebellar nuclei in one cerebellar half, the RCAS vector showed widespread expression in granule cells (GCs) throughout the cerebellum.To study the effects of MCT8 deficiency on cerebellar circuit assembly, we used the non-viral MCT8 RNAi vector to specifically silence MCT8 expression in developing PCs. Our results showed that the MCT8 transporter seems to be an important factor throughout development in controlling neuronal T3 supply. This in turn regulates key cellular events of cerebellar development such as PC differentiation and GC proliferation via the cross-talk between TH signalling, Retinoic acid receptor-related orphan receptor alpha (RORα) and Sonic hedgehog (SHH). Our strategy thus not only provides novel insights into the molecular pathways possibly disturbed in MCT8-deficient cerebellar development in humans, it also opens up new opportunities to reveal the functional involvement of other TH regulatory genes, leading to a better understanding of the mechanisms underlying TH action in the entire developing brain." "Neuro-immune interaction in postoperative ileus: new targets for treatment" "Guy Boeckxstaens" "Translational Research in GastroIntestinal Disorders" "Postoperative ileus (POI) is the transient inhibition of co-ordinated bowel motility after surgery resulting in delayed transit of intestinal contents, intolerance of oral intake and inability to defecate. The main underlying pathophysiological mechanism is intestinal inflammation. Anti-inflammatory intervention is therefore a potential treatment for POI. We have recently demonstrated that stimulation of the n. vagus has an anti-inflammatory action resulting in attenuation of inflammation and POI. This effect is mediated by acetylcholine (ACh) which inhibits intestinalmacrophages through interaction with α7 nicotinic ACh receptors. It is however unknown how the vagus anatomically interacts with the intestinal immune system. This PhD research focuses on the study of morphologic and functional interaction of the n. vagus and the intestinal immune system the characterization of the cells involved in this pathway and totest ghrelin as pharmacological activator of the CAIP in the murine model of POI." "Post-infectious immune activation and altered visceral pain perception: evidence for neuro-immune interaction in the gut." "Guy Boeckxstaens" "Translational Research in GastroIntestinal Disorders" "Approximately 10 % of patients who suffered from a bacterial gastroenteritis develop persistent mucosal microscopic inflammation associated with prolonged symptoms of abdominal pain and altered defecation. Accumulating evidence shows that low grade inflammation has a major effect on the phenotype and function of afferent nerve fibers, especially nociceptive nerve fibers, leading to abnormal pain perception. The innate immune system is crucial in maintaining tolerance against luminal antigens and protects the epithelial barrier against pathogenic invasions. A bacterial infection activates the mucosal immune system: activated dendritic cells produce a variety of cytokines which polarize naïve T cells to helper T cells (Th)1, Th2 or Th17. This acute immune response to pathogens can result in priming of the immune system against food antigens or commensal flora (= bystander effect). In parallel with clinical studies, we have developed animal models of abnormal visceral pain perception (visceral hypersensitivity) to study the interaction between nerves and the immune system, including a mouse model of post-infectious inflammation. The present project focuses on the immune cells involved and on the potential contribution of commensal bacteria as trigger to maintain the state of immune activation." "Pathophysiology of cholestatic liver dysfunction during critical illness" "Greet Van den Berghe" "Laboratory of Intensive Care Medicine" "The liver is an important and versatile organ, which plays a crucial role in a number of metabolic functions. Liver dysfunction, including cholestasis and biliary sludge, frequently occurs during the prolonged phaseof critical illness. Although it has been shown in literature that thisis associated with an increased mortality and a prolonged stay at the hospital, up until now, liver dysfunction during critical illness is bothclinically and pathophysiologically only poorly characterized. Animal studies have shown that the expression pattern of bile salt transporters is changed during acute sepsis. Hepatic bile salt transporters are responsible for exchanging bile salts between blood and bile. During the pastyears, a considerable progression has been made in characterizing thesetransporters in human.The central hypothesis studied in this research project states that the neuroendocrine changes during acute and prolongedcritical illness induce cholestasis via modification of bile salt transporters, and that an increased glucose burden in the liver causes a (forthe liver at least) protective response in the hepatocytes. This response comprises that bilirubin and bile salts are pumped back to the blood via bile salt transporters, in this way limiting hepatocellular accumulation. In addition, since bilirubin has a strong anti-oxidizing function it is likely to function as an endogenous scavenger and as such counteract cellular damage caused by free radicals."