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The pituitary gland takes, together with the hypothalamus, the main lead in coordinating the endocrine system to regulate vital physiological body processes like growth, puberty, reproduction and stress response. Because of this central role, malfunctioning of the pituitary can cause severe disorders like diabetes, cardiovascular disease, osteoporosis, infertility and psychological problems. Pituitary hormonal cell populations must therefore be maintained in a controlled and balanced manner. Previous studies revealed the existence of stem cells in the pituitary gland, mainly based on a side population (SP) and SOX2-expressing phenotype. Their role, however, remains uncertain. Lineage tracing showed that SOX2+ cells can give rise to all pituitary endocrine cell types during postnatal homeostasis, although only at a low degree. After damage in the gland, the pituitary stem cells are activated and appear involved in the subsequent regenerative process. Recently, it was found that the pituitary SP and SOX2+ stem cells are activated and expanded during tumor development and growth in the gland as occurring in the ‘dopamine receptor D2 knockout (Drd2-/-) mouse. Tumorigenesis is the most common pathology of the pituitary. However, underlying mechanisms remain elusive and current therapies are often unsatisfactory because of inoperability or therapy resistance.In the first part of our study, we investigated the role and significance of the pituitary stem cells in the postnatal gland by depleting the SOX2+ cells through diphtheria toxin (DT)-mediated ablation. In the second part, we further characterized the stem cells in the tumorous Drd2-/- pituitary in search of their role in development, progression and therapy resistance of pituitary tumors.Administration of DT to adult Sox2CreERT2/+;R26iDTR/+ mice (after tamoxifen-induced expression of DT receptor or DTR in SOX2+ cells) resulted in 80% obliteration of SOX2+ cells in the endocrine pituitary, coinciding with reduced pituisphere-forming activity. Counterintuitively for a stem cell population, the SOX2+ cell compartment did not repopulate. Considering the more active phenotype of the stem cells during early-postnatal pituitary maturation, SOX2+ cell ablation was also performed in 4- and 1-week-old animals. Ablation grade diminished with decreasing age and was accompanied by a proliferative reaction of the SOX2+ cells, suggesting a rescue attempt. Despite this activation, SOX2+ cells did also not recover. Finally, the major SOX2+ cell depletion in adult mice did not affect the homeostatic maintenance of pituitary hormonal cell populations, neither the cell (corticotrope) remodeling response to adrenalectomy challenge.To characterize the pituitary stem cell population during tumorigenesis, whole-genome expression analysis using RNA-sequencing (RNA-seq) was performed. Analysis revealed upregulation of different stemness factors and pathways including WNT, epithelial-mesenchymal transition (EMT), chemokine signaling and NOTCH in the SP of Drd2-/- pituitary. Transgenic lineage tracing of SOX2+ cells before and during pituitary tumor growth in the Drd2-/- mouse showed that the pituitary stem cells do not, or at least not to a substantial level, give rise to the tumor cells, suggesting rather an indirect, potentially paracrine role. Finally, higher resistance to the chemotherapeutic drug temozolomide was observed in the stem cell fraction of both mouse and human pituitary tumors as analyzed in vitro.In summary, our study shows that pituitary SOX2+ cells fail to regenerate after major depletion which does not affect adult endocrine cell homeostasis and remodeling. Thus, pituitary SOX2+ cells may constitute a copious stem cell reserve or may have other critical role(s) still to be clearly defined. In addition, pituitary stem cells appear activated during tumor formation in the (Drd2-/-) pituitary regarding their stemness molecular phenotype but are not directly linked to the tumor cells. This activation may imply a reaction against tumor development in the gland which may then indirectly affect tumor growth by paracrine signaling. Moreover, the stem cell fraction in pituitary tumors appears more resistant to chemotherapy; further characterization may lead to insights in the underlying mechanisms of therapy resistance and eventually to new therapeutic opportunities.In conclusion, our study adds new elements to the role of pituitary stem cells during homeostasis and tumorigenesis, but more research is clearly needed.

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Myopathies are characterized by muscular weakness and impaired functionality caused by genetic and acquired factors. Muscular Dystrophies (MD) are amongst the most common diseases of the skeletal muscle 2 . MDs are marked by a progressive muscular degeneration, which leads patients to physical disability and shortens life expectancy. No curative treatment is currently available for MDs. Treatment with glucocorticoids and intensive physiotherapy can only alleviate the symptoms and partially delay the progression. A promising approach relies on stem cell therapy. However, issues such as limited availability, control of cell fate and paracrine effects on the host tissue need to be addressed before therapeutic applications may become feasible. Stem cell therapy with donor mesoangioblasts (MABs- vessels associated stem cells) has produced dramatic amelioration in dystrophic mice and dogs 3,4. Recently, induced pluripotent stem cells (iPSCs) have been generated from MABs (MAB-iPSCs). MAB-iPSCs reproduce unique features of pluripotent stem cells, still retaining a biased epigenetic memory towards the myogenic lineage. Interestingly, MAB-iPSC-derived mesodermal progenitors (MiPs) can be used for combined treatment of both skeletal and cardiac muscles in dystrophic mice 5. More recently, human MiPs have been derived and characterized although questions about their in vivo performance remained unanswered.During the current phd training we investigated the in vivo capacity of human MiPs, mainly focusing our attention on their skeletal myogenic commitment. We assessed the translational potential of human MiPs in dystrophic mice. Next, we compared the transcriptional profiles of human fibroblast-derived - and MAB-MiPs, and the miRNAs profile in order to predict a miRNA cocktail functional for increasing the myogenic commitment of the progenitors.In a second part of the project we wished to move forward with a novel therapeutic approach for enhancing muscle regeneration bypassing the use of stem cells. In this light exosomes have been increasingly studied and applied in regenerative medicine fields as delivery methods for tissue regeneration. Therefore we screened the content of exosomes derived from hypertrophic and dystrophic mice and we focused once again on their micro RNA signature with the goal of modulating muscle degenerative diseases in vivo.

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The contribution of an inflammatory component to AD was already described in the 1990's. However, a role for microglia as a driver of neuroinflammation-induced AD has only been recognized more recently, with the identification of SNPs in immune-related genes as risk factors for common sAD. In this respect, SNPs in TREM2 were found to significantly increase the risk of developing multiple neurological disorders. In particular, a 3-fold increased risk for sAD in individuals with a heterozygous TREM2 R47H mutation was observed, which is nearly equal to the APOE4 effect size. In addition, loss of TREM2 surface expression was found to result in early onset dementia with amyloid pathology. Thus, this underscores a causal role of microglia in AD and implies aberrant TREM2 function can result in amyloid pathology. Obviously, the best system to study the role of TREM2 mutations is in human brain-derived microglia, which is difficult due to limited access to human brains. Therefore, we established a human pluripotent stem cell (hPSC) model to enable the study of loss of TREM2 and the heterozygous AD-linked R47H TREM2 mutation in microglia-like cells. Using CRISPR/Cas9, we knocked in the heterozygous R47H mutation (TREM2 +/R47H), created TREM2 +/- and TREM2 -/- cell lines, and differentiated these hPSCs to monocytes and microglia-like cells (tMG). We demonstrated that the hPSC-derived tMG resemble other hPSC-microglia as well as cultured human microglia. In addition, by means of qRT-PCR and RNA sequencing we showed for the first time that in vitro differentiation to so-called monocytes using the Yanagimachi protocol results in cells that are more similar to microglia than in vivo PB-monocytes and showed this is possibly due to in vitro culture. Moreover, RNA sequencing of TREM2 WT vs. TREM2 -/- revealed amongst others an increased inflammatory response and reduced cytoskeletal remodeling. These pathways might underpin the mechanism by which phagocytic activity of TREM2-/- tMG is impaired. This is the first time these findings are replicated in human microglia-like cells and identifies interesting candidate gene networks for future studies, e.g. deranged integrin signaling, which so far have not been addressed.Furthermore, we were to our knowledge the first to address the phagocytic function of TREM2 R47H heterozygous knock-in human microglia-like cells and perform an ex vivo amyloid clearance assay on TREM2 +/R47H, TREM2 +/- and TREM2 -/- human microglia-like cells. We were able to demonstrate that only heterozygous or homozygous loss of TREM2 impaired both phagocytosis of E.Coli and amyloid plaque clearance. As the phagocytic impairment of TREM2 -/- tMG was not more profound than in TREM2 +/- tMG, we hypothesize that this is due to the severe loss of TREM2 mRNA (80 % reduction) observed upon knockout of 1 allele, and thus that phagocytosis in general is directly affected once TREM2 mRNA expression is 80 % decreased (TREM2 +/- tMG) compared to WT tMG.In respect to the lack of a phagocytic phenotype for TREM2 +/R47H tMG, one should take into account that the biggest risk factor of AD, aging, is not recreated in tMG derived from hPSCs. One could consider generating tMG by direct transdifferentiation of fibroblasts from elderly sAD patients with a TREM2+/R47H mutation that may retain such an 'aged signature' (as shown by Mertens et al., 2015 for transdifferentiated neurons), once such protocols become available. Alternatively, TREM2 +/R47H tMG could be allowed to age by grafting in immune-compromised and microglia-depleted hCSF1-expressing AD mice or SAMP8 mice, and then reanalyzed. Transcriptome analysis of such samples might reveal functions affected by this mutation upon aging and help to further unravel why TREM2+/R47H is associated with a 3-fold increased risk for sAD and how it differs from other TREM2 mutants.

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Cystic Fibrosis (CF) is the most common severe hereditary disease in Caucasian populations, affecting approximately 1 in 3500 births. Mutations in the CFTR gene cause abnormal production and/or function of the CFTR protein, an anion channel in the apical membrane with a high expression in most cells lining the airways and the ducts of exocrine glands as the sweat gland, the pancreas or the vas deferens. The abnormal CFTR function results in defective ion and water transport, leading to abnormal viscous respiratory secretions and eventually progressive respiratory insufficiency, high chloride levels in sweat, pancreatic insufficiency, and male infertility. Even with optimal therapy, CF is a life shortening disease with a predicted median survival of around 50 years.^ Although CF is a multisystem disease, more than 90% of patients die from lung disease.(1)Complex genetics necessitate a personalized medicine approach with genotype-specific disease modifying therapiesMore than 2000 different mutations in the CFTR gene have been reported in the CFTR1 database.(2) The most frequent mutation is F508del, for which around half of the patients are homozygous. The other patients carry at least one of the numerous other mutations, which are almost all very rare. In each country, only 5 or 6 mutations apart from F508del are found in more than 1% of the patients.(3)The current treatment for CF is mainly symptomatic: improving mucociliary clearance with mucolytics and physiotherapy, treating lung infection with antibiotics and lung transplantation in end stage lung disease. Eventually, more than 20 years after the discovery of the CFTR gene, treatments that aim at correcting the basic CFTR defect are emerging.^ New ‘disease modifying therapies’ were recently developed to target the basic defect, thereby directly improving or restoring the function of the CFTR protein.(4) Two of these are already on the market, and many more are at varying stages of development. Ivacaftor is a ‘potentiator’ and restores the chloride transport in patients with gating mutations, in which CFTR protein is present at the apical membrane but are not activated. Ivacaftor improves sweat chloride, lung function and weight in patients with gating mutations. Lumacaftor is a ‘corrector’, allowing a proportion of the misfolded F508del protein to traffic to the cell membrane.^ The combination of lumacaftor and ivacaftor resulted in improvement of the sweat chloride value and of the lung function in patients homozygote for the F508del mutation.The diversity of genetic abnormalities in CF results in the need for different compounds targeting each type of mutation, so-called ‘genotype specific personalized medicine’. Clinical trials in patients homozygous for F508del are feasible because sufficient patient numbers are available. Performing clinical trials to prove the effect of a disease modifying therapy becomes much more challenging in patients carrying rare mutations.^ And even in patients with less rare mutations, reliable and precise outcome measures give more robust evidence and allow more efficient use of patients and financial means.With the advent of these disease-modifying treatments the need for outcome parameters that accurately quantify the improvement in the underlying disease defect are more than ever important.Clinical trials and the need for sensitive and accurate outcome measuresClinical trials assess the risk/benefit profile of a compound. Phase 1 trials establish the safety and the tolerability of the new drug in healthy volunteers and allow dose finding. Phase 2 trials assess compound safety in patients, and evaluate ‘proof of concept’ of the effect of the drug on the disease process. At this stage, biomarkers are often used. Given the low number of participants in such trials and the short observation period, a significant effect on clinical or surrogate endpoints is often not achievable.^ In larger-scale phase 3 trials, the beneficial effect of the drug should be demonstrated as a substantial improvement in clinical or surrogate endpoints. Sensitive endpoints, applicable in the population studied, responsive to intervention and adapted to the phase of the trial are thus required. Endpoints or outcome measures can be classified in three categories:- Clinical outcome measures measure how patients feel, function or survive. Increased survival is the result of improvement in the treatment of patients with cystic fibrosis. With a median survival of 35 years, survival is an impossible endpoint to use in clinical trials. Other clinical endpoints are the frequency of respiratory exacerbations and quality of life questionnaires such as the respiratory part of the Cystic Fibrosis Questionnaire-Revised (CFQ-R).- Surrogate outcome measures are lab measurements used as substitutes for clinical end-points.^ To qualify as surrogate outcome, the link between the measurement and eventual clinical disease outcome such as survival must be established. At present, forced expiratory volume in 1 sec (FEV1) is the only FDA approved surrogate endpoint for clinical trials in CF.- Biomarkers are an objective measure of a normal or pathogenic biologic process, or of a pharmacologic response to a therapeutic intervention. In CF, biomarkers that reflect CFTR function and ion transport are the sweat chloride concentration, the nasal potential difference measurement or intestinal current measurement. Other biomarkers reflect early pathogenic processes, e.g. the mucociliary clearance. A combination of endpoints can be used during the evaluation of a new therapy in patients with CF.Given the importance of accurately assessing new therapies, our research has focused on 2 types of outcome measures.^ First, outcome measures that reflect the basic CF defect and that are thus especially useful in phase 2 clinical trials with disease modifying drugs: nasal potential difference (NPD) and sweat chloride. Next, outcome measures that reliably quantify CF lung disease, the major cause of death in CF. These parameters are especially useful in phase 3 clinical trials, when proof of clinical benefit must be provided. We explored the value of lung clearance index (LCI) and FEF25-75.Study objectives, methods and results1.Nasal potential differenceA nasal potential difference (NPD) measurement quantifies the voltage across the nasal epithelium resulting from the mucosal ion transport. It directly assesses the CFTR function by measuring chloride transport, taking into account the function of ENaC, a sodium transporter, which is inhibited by CFTR. A catheter is placed in the nose, with a sensing electrode to measure the potential and a channel to perfuse solutions locally on the nasal mucosa.^ A fixed perfusion sequence is used: Ringer solution and Ringer with amiloride assessing sodium transport through ENaC, followed by a zero-chloride solution and subsequent addition of isoproterenol. The change in potential induced by the latter two solutions is called the total chloride response (TCR) and is a measure of chloride transport through CFTR.The NPD differentiates patients with CF from healthy controls. Patients with CF and mild phenotypes show intermediate NPD values reflecting the degree of residual CFTR function.(5) NPD has been used as an exploratory endpoint in trials, measuring the change in CFTR function during treatment with disease modifying therapies.^ With ivacaftor, a dose response was observed: progressive improvement of chloride transport with increasing drug doses.(6)The NPD is thus a unique way to measure CFTR function in the airways, reflecting the first step of the pathophysiological cascade initiating the respiratory illness responsible for most of the morbidity and mortality of CF. The challenges with NPD as endpoint are the lack of standardization and the poor repeatability of the measurements.Therefore, we investigated the impact of several technical aspects of the NPD measurement, with an emphasis on improving repeatability to reduce the sample size in clinical trials.Vermeulen, F., Proesmans, M., Feyaerts, N., & De Boeck, K. (2011). Nasal potential measurements on the nasal floor and under the inferior turbinate: does it matter? Pediatr Pulmonol, 46(2), 145-152.Nasal potential is not uniform over the nasal mucosa. Therefore, the place of the catheter in the nostril could have an impact on the results obtained.^ We examined the influence of the place of the catheter by comparing the values obtained with the nasal catheter placed medially on the nasal floor in one nostril (the ‘European’ floor protocol), and laterally under the inferior turbinate in the opposite nostril (the ‘American’ turbinate protocol). Thirty-four patients with CF, 26 heterozygotes and 61 control subjects underwent simultaneous measurements with both techniques, with a repeat test in 57 to measure repeatability. Both protocols discriminated well between CF and control subjects, and the mean NPD values were not different. Repeatability was similar with the two methods. Sample size projections using the proportion of interpretable measurements, and mean values in CF and in control subjects slightly favored the use of the ‘floor catheter’ (Table 1).Bronsveld, I.*,Vermeulen, F.*, Sands, D., Leal, T., Leonard, A., Melotti, P., European Cystic Fibrosis Society - Diagnostic Network Working, G. (2013).^ Influence of perfusate temperature on nasal potential difference. Eur Respir J, 42(2), 389-393. (*First authors equal contributions)The effect of the temperature of the perfused solutions was evaluated. Different devices were in use to warm the solutions to 34-37°C during local perfusion, while some operators used solutions at room temperature. To assess the effect of the temperature of the solutions, NPD was measured in healthy subjects (CF subjects have virtually absent chloride transport) at different perfusion temperatures: Warmed (34-37°C, W) or room temperature (RT). Two

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Although tremendously better insights in the static 2D characteristics of spinal alignment have been achieved the last decade, these are not the main driver of self-perceived HRQOL in Adult Spinal Deformity (ASD) patients. The current state-of-the-art diagnostic algorithm of patient profiling, which is based on 2D spinopelvic alignment analysis and demographic data, fails therefore to analyze the true impact of a spinal deformity on the adult patient. The WHO’s International Classification of Function, disability and health model (ICF-model) offers a comprehensive and structural way to approach a diagnostic algorithm and to unravel the true impact of a chronic disorder on the individual patient. This doctoral thesis demonstrates, with respect to the ICF model, that better insights in the unique functional fingerprint and 3D body structure of the ASD patient offers potential to identify the true drivers of self-perceived HRQOL in the individual patient.We demonstrated in chapter 1 that individuals with a primary spinal deformity encounter impaired balance control during regular activities of daily life. Furthermore, we investigated the relation between functional impairment and decreased self-perceived HRQOL in ASD patients. To quantify function, we used easy approachable quantitative functional assessments of balance control, like the Balance Evaluation Systems Test (BESTest) and Trunk Control Measurement Scale (TCMS), and investigated whether these clinical tests could complement the current golden standard 2D static spinopelvic alignment analysis and demographics in terms of understanding ASD’s impact on health-related quality of life. We demonstrated that the BESTest has a higher potential to predict HRQOL in the primary ASD population than demographic variables and 2D radiographic spinopelvic measurements. As such, the future introduction of this test during the diagnostic algorithm of patient profiling provides a clear opportunity to unmask functional impairments which are related to HRQOL and unvisible on static X-ray images. Future research should examen whether the BESTest offers potential starting points for novel treatment algoritms including targeted physiotherapy and rehabilitation programs for the ASD patient in a non-surgical, pre-and postoperative setting.To understand the impact of a deformed body structure on an individual with respect to the functional dimension of the ICF model, we examined the relation between function and spinal alignment in chapter 2. Function is quantified with performance on Balance Evaluation Systems Test (BESTest). Spinal alignment is quantified by standard 2D and novel Gravity-Line instrumented 3D (i3D) spinal alignment parameters. Therefore, we described the Transverse Gravitational Deviation Index (TGDI) as a novel spinal alignment parameter which aims to describe the threedimensional spinal deformity in the transverse plane with respect to the gravity line. We demonstrated that an increase in the value of sagittal spinopelvic parameters like T1 Pelvic Angle (TPA) and Global Sagittal Axis (GSA), which quantify the global sagittal spinal malalignment despite the use of compensation mechanisms in pelvis and lower limbs, is related to a decreased performance on BESTest. Furthermore, we demonstrated a significant relation between the TGDI and balance control. Indeed, we reported that adult patients with a spinal deformity resulting in a combined coronal and sagittal malalignment at the level of the L3 vertebra, associated with and quantified by an increasing distance between L3 and the gravity line in the transverse plane, are more likely to suffer from impaired balance performance. Although the clinical relevance should be elucidated in future research which combine transverse plane measurements like Axial Intervertebral Rotation and TGDI, this work suggests that patients with occurrence of rotatory subluxation on the level L3 on L4 and progressive lumbar scoliosis, are more likely to encounter declined balance control and thus lower health-related quality of life. In addition, we demonstrated that age is an important driver of balance control in patients with ASD. While these patients get older, they are more at risk for progressive degenerative structural spinal alignment changes and impaired balance performance which puts them in a downward spiral of disability.In chapter 3, we demonstrated the complementary value of sagittal spinopelvic parameters, TGDI, BESTest and instrumented 3D motion analysis in the diagnostic algorithm. We reported the relation between activity level and self-perceived HRQOL, throughout different types of spinal deformity stratified according to the presence of SRS-Schwab sagittal modifiers. Integrated three-dimensional (3D) motion analysis (iMAS) offers potential to increase insights in the functional fingerprint of our ASD patient by means of kinematic, kinetic and electromyographic data of trunk and lower limbs during walking and other activities in daily life. We demonstrated that besides impaired balance performance on BESTest also a rigid trunk strategy during walking is related to self-perceived HRQOL in adult patients with spinal deformity after correction for covariates. Furthermore, we demonstrated different motion strategies in terms of trunk inclination and pelvic anteversion during walking in comparison to stance throughout different types of spinal alignment with use of sagittal kinematic data of trunk and pelvis. In addition, we attempted to explain these differences in motion strategy during walking with use of Dubousset’s cone of economy in a figurative sense.In conclusion, this doctoral thesis demonstrates the added value of including new biomechanical measurements in the current diagnostic algorithm of adult patients with a spinal deformity to identify the true drivers of self-perceived HRQOL in the individual patient, with respect to the WHO’s International Classification of Function, disability and health (ICF-model).