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Glycosylation is one of the most abundant protein modifications found in nature. It results from a meticulously orchestrated process involving numerous proteins for the assembly and modification of oligosaccharide chains, and their attachment onto proteins and lipids. The importance of glycosylation is illustrated by a group of diseases called Congenital Disorders of Glycosylation (CDG). To date, almost 100 distinct disorders have been identified encompassing defects in N- and O-linked protein glycosylation, but also in the synthesis of GPI-anchors and glycolipids. Considering the possibility to screen for most deficiencies in protein N-glycosylation by means of isoelectric focusing of serum transferrin, the project focused on this group of disorders.The genetic heterogeneity of CDG, but also the phenotypic overlap between the different disorders is remarkable. A clinical ‘hit and run’ diagnosis forms thus rather the exception than the rule. Therefore, patients with a biochemically proven glycosylation deficiency remain often without a molecular diagnosis. In this study, we aimed to circumvent the bottleneck of a gene by gene approach through the implementation of massive parallel sequencing techniques in as well CDG research as diagnostics (Chapter 3).For the elucidation of novel CDG, whole exome sequencing was performed in 24 individuals with a presumed deficiency in the N-linked glycosylation pathway (Chapter 4 and 5). Once the genetic defect was identified, its pathogenic nature was confirmed using cell biological assays. In this way, a genetic diagnosis could be obtained in nine patients (i.e. 38%), while the most likely candidate gene is still under investigation in seven additional cases (i.e. 30%).In parallel, a targeted assay for a panel of 79 genes was developed to improve CDG diagnostics (Chapter 6). Over a period of two years, the panel was used for molecular testing in a total number of 86 patients with a presumed deficiency in the N-linked glycosylation pathway. A final molecular diagnosis could be obtained in 38 of them (i.e. 44%). Based on these results, we proposed a tentative novel flowchart wherein a patient considered to have CDG first enters a diagnostic setting for gene panel testing. A close collaboration between the diagnostic and research department would then allow those patients, in whom the culprit gene could not be identified or in whom the pathogenicity of a variant needs to be verified, to subsequently enter a research setting for further biochemical testing.During this study, mutations in MAN1B1 were identified to cause a novel CDG-II. The biochemical characteristics of the index case allowed for the rapid identification of 18 additional patients (Chapter 7). All cases displayed a similar phenotype characterized by intellectual disability, delayed motor and speech development, hypotonia, macrocephaly and truncal obesity.During the time span of this PhD project, the intracellular localization of MAN1B1 became the subject of a still ongoing debate. Indeed, besides its role in N-glycan processing, the α(1,2)-mannosidase has been proposed to act as a key factor in ER quality control by targeting terminally misfolded proteins for proteasomal degradation. Since all mediators of ERAD are assumed to reside within the ER, it only seemed natural that MAN1B1 would execute its function within the same organelle. However, today opinions are changing. While some researchers still believe that MAN1B1 resides within the ER, others are convinced that the enzyme localizes to a presumed ERQC compartments or resides within the Golgi apparatus.In Chapter 9, we could clearly demonstrate that the endogenous MAN1B1 in primary skin fibroblasts is localized within the Golgi apparatus, thereby confirming the initial –but still controversial– results of Sifers and coworkers. Our findings were further supported by the observation that MAN1B1 deficient fibroblasts display an aberrant Golgi morphology in the absence of an ER stress response (i.e. UPR or unfolded protein response) (Chapter 8 and 9).While former studies mainly focused on the effect of MAN1B1 deficiency on the fate of misfolded cargo, we investigated –with respect to the phenotype– the effect on secretory proteins that attained their native folding state (Chapter 9). In this way, we could demonstrate that MAN1B1 deficiency does not only enable the intracellular accumulation and partial secretion of nonnative proteins, but in addition impairs the anterograde trafficking of the properly folded cargo.In Chapter 10 of this manuscript, we assumed that the aforementioned accumulation of (mis-)folded proteins within the Golgi apparatus could overwhelm the capacity of the secretory pathway, thereby generating a primary Golgi stress response. Through several pilot experiments we could show that MAN1B1 deficiency generates a mild to moderate transcriptional response that was not only uniformly present among the different patients, but that also differed from the responses observed in other CDG-II cell lines. However, additional investigations are necessary to further address the extent of a possible Golgi stress response in MAN1B1-CDG and to understand how this transcriptional response might impact patient management.

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In this project, we aim to define the role of CNOT3 in the development of T-ALL. We will investigate if CNOT3 loss of function enhances the malignant transformation of Tcells in mouse leukemia models. In relation to this, we will also study the role of CNOT3 in T-cell development and we aim to define the molecular changes aused by loss of CNOT3 in leukemia cells. These results can provide important insights on the role of RNA metabolism in leukemia development.

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This PhD project will contribute to the elucidation of the functional role of prolyl endopeptidase-like. Inactivation of PREPL results in neonatal hypotonia, growth hormone deficiency and feeding problems. The regulated secretion pathway is disturbed in PREPL deficient patients, somatotrophs of PREPL KO mice and in PREPL knockdown neuroendocrine cells. Also, since PREPL interacts with AP-1, which is essential for biogenesis and maturation of secretion vesicles, we can suggest that PREPL is involved in regulated secretion. Furthermore, this interaction results in an increased membrane dissociation of AP-1. Therefore we hypothesize that PREPL has a phospholipase activity.Unravelling the molecular mechanisms of PREPL can contribute to a treatment that can improve the quality of life of the patients. Therefore, we will perform competition experiments and look into the interactions of PREPL and PI-4-P. Additionally, secretion studies and morphometric and biochemical analysis of the secretory vesicles will be performed to reveal the role of PREPL in regulated secretion.These findings are complemented by the results of Luc Régal (PhD: The role of PREPL in the hypotonia-cystinuria syndrome). The focus is on the clinical aspect, with the identification of PREPL deficient patients and the clinical study for the treatment of hypotonia.

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The Amyloid Precursor Protein (APP) has been extensively studied because of its link to Alzheimer’s disease. Its precise molecular function has long been a widely studied and highly controversial topic, however so far the physiological function of APP in the nervous system remains ambiguous. APP has all the major features expected from a cell surface receptor, but whether it is indeed a receptor and what its ligand may be are unclear.Using both in vivo fly and primary neuronal culture models we first confirm previous observations that APP is a neuronal modulator of the Wnt/Planar Cell Polarity (Wnt/PCP) pathway (Soldano et al., 2013) and we show that this modulation is conserved in mammals. Additionally we find that APP homologues contain a highly conserved Cysteine Rich Domain (CRD) in their extracellular domain that resembles the conserved CRD present in receptors of the Wnt/PCP pathway. More importantly we show that the CRD is necessary for binding to Wnt5, the most important Wnt/PCP pathway ligand. These data explain mechanistically our previous observations that both Drosophila and human APP interact genetically with the classic Wnt/PCP pathway receptor Van Gogh (Vang) (Soldano et al., 2013). Surprisingly, however, we discover that Wnt5 binding to APP reduces its binding to Vang and thus APP acts as a homeostatic regulator of Wnt signaling in neurons. Moreover, deletion of the APP CRD abrogates the Wnt5 effect and creates an activated form of APP, again consistent with its role as a receptor. Additionally we show that the negative effect exerted by Wnt5 on neuronal PCP through its binding to APPL, is compensated for by the positive effect generated through Wnt5 acting the Frizzled receptor.In summary, we have discovered a novel and surprising activity for a major human disease gene as a Wnt receptor. Given the highly conserved and pervasive role of APP in brain development, function and disease, these findings have broad implications, especially that an AD disease variant in the canonical Wnt receptor LRP6 was discovered and functional analysis showed that this variant reduces canonical Wnt signaling (De Ferrari et al., 2007) and it is well established that canonical and non-canonical Wnt activities are tightly balanced and that increases in Wnt5 levels can inhibit canonical Wnt signaling.