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Tobacco use is a major cause of cardiovascular diseases (CVDs), which begin with endothelial dysfunction. In this thesis, we objectified, by the quantification of specific biomarkers, an elevation of inflammation, oxidative stress, and endothelial activation in smokers, associated with an altered lipid profile. All these processes are involved in endothelial dysfunction and therefore, promote CVDs development. We found that these effects could be reversible on quitting. These improvements were already visible at short term. The role of nicotine, especially of nicotine replacement therapy (NRT), in CVDs progression remains controversial in the literature. Therefore, we assessed the influence of NRT on biomarkers and we demonstrated that they did not affect the biological improvements observed with smoking cessation. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2023

The goal of radiation therapy is to deliver a sufficient dose to cancer cells while keeping the exposure of healthy tissue as low as possible to avoid toxicity. Conventional radiotherapy has benefited from technological advances to deliver more conformal doses to the tumor, yet these techniques are still constrained to the natural interaction of photons with matter. Unlike photons, protons slow down as they pass through the patient, releasing most of their energy at the end of their trajectory in the form of what is known as the Bragg peak. By taking advantage of this feature, it is possible to precisely localize the dose peaks in the tumor, reducing the exposure of surrounding healthy tissue. Radiation therapy treatments are planned based on a pre-treatment image to simulate the patient’s anatomy. However, patients are not static, and variations in the anatomy, such as weight loss, are frequent during a treatment course of several weeks. Proton therapy is especially sensitive to anatomical changes along the proton beam path, which alter the position of the Bragg peaks and can strongly deteriorate the initially planned dose. The adaptation of the initial plan is therefore required to preserve the quality of the treatment. Online adaptation in proton therapy, with the patient in the treatment position, aims to provide a rapid response to anatomical changes to compensate for proton dose distortions ensuring accurate treatment delivery. In this thesis, we investigate different strategies to automatically perform online adaptation to facilitate its clinical implementation in proton therapy. Eventually, online adaptation would allow clinicians to achieve greater control over the delivery of conformal proton dose distributions, reducing toxicity and improving patients’ quality of life. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2023

The aim of my project was to develop and apply focused ultrasound stimulation (FUS) to study the human brain. Recently, TFUS was proposed as an advantageous alternative technique for non-invasive modulation of human brain activity. Compared to transcranial magnetic stimulation and transcranial direct current stimulation, the two most-used techniques for neuromodulation, TFUS is more focal and allow to target deeper brain structures with a reduced effect on surrounding cortical tissue. I first determined the sonication parameters to use according to international safety guidelines. Then, I used the prototype I designed to characterize the effects of TFUS sonication over the primary somatosensory cortex using somatosensory-evoked potentials elicited by transcutaneous electrical stimulation of the median nerve (MN). We report the successful modulation of early SEPs potentials (N20 and P27) for TFUS applied 200ms before the delivery of MN stimulation. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2023

Endometriosis is an inflammatory gynecological pathology that affects approximately 10% of women worldwide and can cause subfertility. In 30% of patients, the endometriotic tissue is resistant to progestin treatment. The objective of my thesis was to characterize and define the potential role of the protein Progesterone Receptor Membrane Component 1 (PGRMC1), described as a "fertility stabilizer", in the human endometrium and endometriosis. A spatiotemporal regulation of PGRMC1 was identified with a predominant expression on the surface of the proliferative endometrium and potentially reduced in endometriosis. My results (i) discredit the AG-205 inhibitor, a major tool in the study of PGRMC1, (ii) demonstrate the repressive activity of PGRMC1 towards MMP-3 and (iii) suggest that it regulates cell proliferation during the menstrual cycle. PGRMC1 could thus control endometrial tissue remodeling, which is crucial for fertility. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2022

Chronic alcohol consumption is one of the leading causes of chronic liver disease and liver-related deaths worldwide. Only up to 20% of patients with an alcohol use disorder (AUD) progress to advanced stages of alcohol-associated liver disease (ALD) from the benign form of the disease, namely simple steatosis. This process is usually silent at early disease stages and the mechanisms underlying disease progression are yet incompletely understood. Recent reports have highlighted the role of the gut-liver axis in ALD. However, data in early human ALD were scarce. Therefore, this PhD work aimed at investigating dysfunctions of the intestinal barrier in AUD patients and their relationship with activation of immune responses contributing to ALD progression. We here revealed a specific diminution of effector immune cells in the duodenal mucosa and the presence of a tolerogenic immune status associated with changes in the gut microbiota and elevated translocation of microbial products in patients with progressive ALD. These microbial products activated circulating monocytes and liver inflammatory responses, thus contributing to systemic inflammation and ALD progression. The results of this PhD thesis support the contribution of gut barrier dysfunctions in human ALD pathogenesis. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2022

Alcohol use disorder (AUD) is a major public health problem affecting 5 to 10% of the population. Chronic alcohol abuse induces alterations in the composition of the gut microbiota, which are correlated with psychological symptoms, suggesting the involvement of the gut-brain axis in the development of addiction. Among dietary component able to modulate the microbiota, dietary fibers are of particular interest. In a first study we investigated the link between dietary fiber intake and psychological symptoms in AUD patients. We found that only a proportion of AUD patients displays alterations in the gut microbiota composition. This dysbiosis is associated with higher craving scores and impaired sociability. Finally, our intervention study aiming to supplement AUD patients with inulin shows that 1) it does not lead to gastro-intestinal intolerance, 2) it induces specific changes in the gut microbiota, 3) it has limited impact on biological and behavioural outcomes, 4) it increases sociability score. If our results show the importance of prebiotic dietary fiber in AUD patients, further studies are needed to define an adapted strategy targeting the gut microbiota to improve metabolic and behavioural alterations in these patients. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2022

Radiotherapy is used in more than 50% of cancer treatments and is thus of the utmost importance in the field of cancer care. The clinical aim of such treatment is to achieve locoregional tumor control with targeted ionizing radiation while minimizing the complication risks for the patient. To this end, the use of ions such as protons is particularly adequate, due to the high dose they deposit at the end of their path. This results in highly conformal dose distributions, with sharp dose gradients around target volumes, enabling excellent sparing of healthy tissues. The generalisation of magnetic steering of protons (pencil beam scanning) and intensity modulated proton therapy further improves the conformity. However, the presence of high dose gradients and localized energy deposition peaks make proton plans particularly sensitive to the uncertainties involved in the preparation and the execution of the treatments. Several strategies exist for managing the uncertainties and mitigate their effect. Among them, accurate methods for computing the dose that will be delivered to the patient are of paramount importance, enabling this way safe treatment planning and reliable quality assurance (QA). For QA, in particular, independent Monte Carlo dose engines are undeniably optimal, providing accurate dose verification in independent conditions. However, their inherent statistical uncertainty can hinder a proper interpretation of QA outputs, and their clinical integration remains challenging, time-consuming and, subsequently, a source of errors. This thesis aims at proposing tools to facilitate a correct and efficient use of independent Monte Carlo dose calculation in proton therapy, so that we can move towards a safer delivery of this treatment modality. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2022

The cell envelope of Gram-negative bacteria is essential for their survival. It serves as their initial line of defense as well as a physical barrier between the cell and its surrounding environment. Furthermore, the cell envelope protects bacteria by allowing them to respond to changes in their environment. This complex structure comprises an outer membrane (OM) and an inner membrane (IM) separated by a viscous compartment, the periplasmic space. The cell wall, a sacculus with a mesh-like structure comprised of peptidoglycan, is found in the periplasm (PG). The Rcs (Regulator of capsule synthesis) system responds to OM and PG damage in Enterobacteriaceae. The OM lipoprotein RcsF senses stress and activates the signaling cascade. The β-Barrel Assembly Machinery (BAM) inserts OM β-barrel proteins (OMPs) in the OM of Gram-negative bacteria. BAM also most likely exports RcsF to the cell surface, probably by forming RcsF-OMP complexes. The main objective of my thesis project was to investigate the relationship between RcsF and its OMP partners. Hereby by dissecting the protein-protein interaction interface (PPI) between OmpA, an abundant E. coli OMP, and RcsF, I identified an unexpected role of OmpA in the Rcs stress response system. In this complex, OmpA, folded as a two-domain protein, interacts via its periplasmic domain with the globular domain of RcsF. This mode of interaction between OmpA and RcsF does not allow OmpA to be the vehicle for the surface exposure of RcsF. Instead, my results suggest that OmpA plays the role of a periplasmic buffer for the Rcs system: The periplasmic domain of OmpA, when interacting with RcsF, prevents the interaction of RcsF with IgaA, the Rcs repressor. To identify the PPI of the OmpA-RcsF complex, I used a site-specific photo-crosslinking method based on non-natural photoactivable amino acids. By doing so, my work identified a flaw in this considered foolproof method, which I investigated to raise the attention of scientists working on protein-protein interaction or protein complexes on the probable existence of artifacts. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2022

Acetyl-CoA carboxylase (ACC) catalyzes the first step of de novo lipogenesis. We previously demonstrated that ACC activation promotes thrombus formation by increasing platelet arachidonic acid (AA)-containing phosphatidylethanolamine plasmalogen (PEP) content and thromboxane A2 (TxA2) generation. Therefore, we hypothesized that ACC deletion could be a protective mechanism against thrombus formation, especially in prothrombotic diseases such as atherosclerosis. We discovered that platelet-specific ACC1 deletion reduced thrombus formation on collagen-coated surface under flow conditions via a decrease in AA-containing PEP content leading to a subsequent decrease in TxA2 and dense granule secretion. Interestingly, two hours treatment with CP640.186, a pharmacological ACC inhibitor, impaired platelet aggregation via an increase in tubulin acetylation level rather than a change in phospholipid composition. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2022

The dose concentration at the end of the proton range has the potential to significantly reduce exposure of healthy tissues to radiation, while maintaining the curative dose to the tumor. However, the beneficial effect of protons comes at the cost of increased sensitivity to treatment uncertainties, which can lead precisely to over-exposure of healthy tissues. The main objective of this thesis is the development of treatment planning methods that ensure robustness, while avoiding the possible loss of plan quality at the moment of delivery. The focus lies on establishing sound confidence levels for treatment planning and the integration of target volume uncertainty, previously unconsidered in the planning process. Overall, the concepts developed in this thesis regarding robust dose optimization and statistical analysis, offer better tools for managing the trade-offs and robustness of treatments. This can bring us one step closer to delivering on promises made in proton therapy. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2022

This thesis aims to study extracellular vesicles (EVs) in thyroid cancer (TC) hoping to understand their role in cancer progression and to identify new biomarkers for the differential diagnosis between TC and benign nodules. Using a mouse model mimicking human TC, we isolated and characterized EVs from control and cancerous thyroid tissues. We identified tumor-derived EV-miRNAs by sequencing. More abundant EV-miRNAs were mostly deregulated in tumor cells and could affect immune-related pathways. We thus propose that tumor cells secrete more EVs containing a deregulated miRNA signature and that this could affect the immune microenvironment. Finally, the diagnostic value of a few miRNA candidates was assessed by quantifying their levels in plasma-EVs from patients with thyroid diseases, suggesting the diagnostic potential of EV-enclosed miR-146b-5p and miR-21a-5p (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2022

Cochlear implant (CI) is the most effective solution for the restoration of hearing function in severe to profound sensorineural hearing loss. The main objective of this PhD thesis is to answer the question: « Does AI-assisted programming of a cochlear implant provide similar or better results than manual programming, with less time investment for the patient and the clinician? ». A first study that was conducted in 4 French-speaking centers in Belgium and in France showed a large variability of CI programming within and between centers. This context supports the use of a tool using artificial intelligence, such as the Fitting to Outcome eXpert® (FOX®). A pilot study on 2 subjects who had limited results with their manually programmed CI showed encouraging benefits. Secondly, FOX® was used on a larger scale in 2 prospective interventional studies. The first study evaluated the benefits of using FOX® in former patients who had been implanted and formerly manually programmed for several years. 89% of the subjects showed better auditory performances (psychoacoustic battery A§E®) with the FOX® map than with the manual map. This significant but not systematic gain is discussed, considering the impact of FOX® as a novelty among other factors. The second study compared the manual and FOX® types of programming in a randomised longitudinal study with a crossover design in newly implanted "naïve" subjects. The crossover design involved each group changing programming type at one year post-activation. The FOX® group achieved better speech test results more rapidly than the manual group and maintained these results at one year post-activation. Due to the crossover, FOX® improved the results of 81% of the patients initially programmed manually. In terms of time gain, the number of programming sessions decreased by 55% over one year in patients initially programmed with FOX® compared to those initially programmed manually. A general discussion and a conclusion nuance the results. AI-assisted programming provides support to clinicians and shows positive results in terms of hearing performance and time gain. Current clinical practice is heading in the direction of developing advanced technological solutions up to remote treatment, which FOX® will enable. L’implant cochléaire (IC) est la solution la plus efficace, en termes de restauration de la fonction auditive, dans le cadre d’une surdité neurosensorielle sévère à profonde. L’objectif principal de cette thèse de doctorat est de répondre à la question : « La programmation d’un implant cochléaire assistée par l’intelligence artificielle donne-t-elle des résultats semblables voire meilleurs que ceux obtenus par la programmation manuelle, avec moins d’investissement en temps pour le patient et le clinicien ? ». Une première étude réalisée auprès de 4 centres francophones en Belgique et en France rend compte de la grande variabilité des programmations d’ICs intra et inter-centres. Ce contexte justifie l’apport d’un outil utilisant l’intelligence artificielle, comme le Fitting to Outcome eXpert® (FOX®). Une étude pilote sur 2 sujets présentant des résultats limités avec leur IC programmé manuellement relève des bénéfices encourageants. Ensuite, FOX® est utilisé à plus grande échelle dans 2 études prospectives interventionnelles. La première évalue son apport chez d’anciens patients implantés et programmés manuellement depuis des années. 89% des sujets présentent de meilleures performances auditives (batterie psychoacoustique A§E®) avec la map FOX® qu’avec la map manuelle. Ce gain significatif mais non systématique se discute, en tenant compte, entre autres, de l’impact du FOX® en tant que nouveauté. La deuxième compare chez de nouveaux sujets implantés « naïfs » les types de programmation, manuelle et FOX®, dans une étude longitudinale randomisée avec un cross-over. Le design cross-over implique que chaque groupe change de type de programmation à un an post-activation. Le groupe FOX® obtient plus rapidement de meilleurs résultats aux tests vocaux que le groupe manuel, résultats qu’il maintient à un an post-activation. Suite au cross-over, FOX® améliore les résultats de 81% patients initialement programmés manuellement. Au niveau du gain de temps, le nombre de sessions de programmation diminue de 55% sur un an chez les patients initialement programmés avec FOX® par rapport à ceux initialement programmés manuellement. Une discussion générale et une conclusion nuancent les résultats. La programmation assistée par l’intelligence artificielle fournit une aide aux cliniciens et montre des résultats positifs en termes de performance auditive et de gain de temps. La pratique clinique actuelle va dans le sens du développement de solutions technologiques de pointe allant jusqu’au traitement à distance, ce que permettra FOX®. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2022

Arginase 1 (Arg1) is a key hydrolase involved in the urea cycle and in the immune system. Arg1 is upregulated in various types of cancers and plays crucial roles in the regulation of tumor growth and metastasis. Thus, Arg1 is an increasingly attractive target for cancer therapy. In this thesis, we aimed to find new scaffolds. A reliable enzymatic evaluation method of chemical libraries such as that of 5-bromo-2-amino-thiazole was set up. A molecular modeling approach (SBDD) led to a better understanding of the possibilities and limitations in the Arg1 inhibitors design for the study of larger scaffolds. A Fragment-Based approach (FBDD) was then developed. A screening of coordinating motif libraries found two hits: 2-furanylboronic acid and cyclohexylboronic acid. Finally, we attempt to optimize these two hits and discussed about the challenges of obtaining such derivatives. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2022

Gram-negative ESKAPE pathogens are responsible for increases in multidrug-resistant infections worldwide. Poor permeability and active efflux cause intrinsic resistance to many antibiotics. Besides, these pathogens can also adopt specific lifestyles like intracellular survival and biofilm formation where bacteria can switch to dormant phenotypes poorly responsive to antibiotics. This thesis investigated the capacity of the polyaminoisoprenyl potentiator NV716 to revive disused antibiotics against Gram-negative bacteria using planktonic, intracellular and biofilm models. We showed that NV716 re-sensitizes Gram-negative bacteria to disused antibiotics by binding to the lipopolysaccharides (LPS) of the outer membrane, permeabilizing this membrane as well as downregulating quorum sensing regulated processes. Therefore, our work highlights a strong potential for this compound as an adjuvant therapy against difficult-to-treat Gram-negative organisms, and suggests that NV716 deserves further investigations, notably in in-vivo models of infections. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2022

Tryptophan 2, 3-dioxygenase (TDO2) is a heme-containing enzyme constitutively expressed at high concentrations in the liver and responsible for L-tryptophan (L-Trp) homeostasis. TDO2 catalyses the cleavage of L-Trp’s pyrrole ring to produce N’-formylkynurenine; the first step of the kynurenine pathway. The expression of TDO2 in cancer cells leads to the depletion of L-Trp and an increase of KP catabolites, overall resulting in the suppression of the anti-tumour immune response. The present work describes the development of new (1H-indol-3-yl)-heterocyclic derivatives as TDO2 inhibitors through rational design, inspired from existing inhibitors. Evaluation of these compounds provided new insights regarding the structural modulations that can be accommodated inside the active site of TDO2 and their effect on inhibitory potency. Finally, in this same series of inhibitors, we also investigated the integration of a selenium atom as a bioisostere of lower molecular weight chalcogens. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2021

Medical devices-associated infections are characterised by the formation of a biofilm that encapsulates the microorganisms in an extracellular matrix. The thesis focused on inter-kingdom biofilms, when at least a bacterium and a fungus are present. They are hard to treat infections with high failure rates. Our aim was to determine if the disruption of the biofilm matrix with hydrolytic enzymes could potentiate the activity of the antimicrobials against inter-kingdom biofilms. An in-vitro three-species biofilm model was set up co-culturing the bacteria S. aureus and E. coli, and the fungus C. albicans. The results confirmed that the antimicrobials on their own had a limited activity, but the enzymes permitted the antimicrobials to reduce further the biofilm biomass and C. albicans culturable cells. The inclusion of the strategy proposed in this thesis to current clinical practices could lead to a better outcome of the treatment of inter-kingdom medical device-associated infections. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2021

The infection by the human immunodeficiency virus (HIV) is typically treated using a combination of pharmacological agents targeting different stages of the viral cycle. Darunavir is a second-generation protease inhibitor that occupies a place of choice in this therapeutic arsenal and for which an important inter-individual pharmacokinetic variability has been described in the literature. First, we characterized darunavir pharmacokinetics by developing a population model based on data collected in a large cohort of HIV patients. Alpha-1 acid glycoprotein level, sex, and genetic polymorphisms in the CYP3A5 and SLCO3A1 genes were found to be significant predictors of darunavir pharmacokinetics. The model was thoroughly evaluated using internal and external validation techniques. The model also allowed us to simulate the effect of alternative dose regimens in populations representative of clinical practice. A reduction of the standard 800 mg once-daily dosage to 600 or 400 mg once-daily was found to be safe in a large proportion of patients, based on commonly defined pharmacokinetic targets for this molecule. On the other hand, intermittent therapy (five out of seven days) constituted an unsafe option in most subjects. Whether individual patients could benefit from these alternative regimens could be predicted by our model. Additionally, optimal sampling strategies for darunavir were derived, showing how to best design future studies or how to optimize therapeutic monitoring for this drug. Finally, in vitro experiments helped determine the contribution of polymorphisms in the P-glycoprotein gene to the aforementioned variability. These experiments showed that neither the c.1199G>A polymorphism nor the haplotype defined by c.1236C>T, c.2677G>T and c.3435C>T influence intracellular accumulation of darunavir. L'infection par le virus de l'immunodéficience humaine (VIH) est typiquement traitée par une combinaison d'agents pharmacologiques ciblant différentes étapes du cycle viral. Le darunavir est un inhibiteur de protéase de seconde génération qui occupe une place de choix dans cet arsenal thérapeutique et pour lequel une importante variabilité pharmacocinétique inter-individuelle a été décrite dans la littérature. Premièrement, nous avons caractérisé la pharmacocinétique du darunavir en développant un modèle de population sur base de données collectées dans une large cohorte de patients VIH-positifs. Il a été démontré que le taux d’alpha-1 glycoprotéine acide, le sexe, et des polymorphismes dans les gènes CYP3A5 et SLCO3A1 étaient des prédicteurs significatifs de la pharmacocinétique du darunavir. Le modèle a été évalué en profondeur par le biais de techniques de validation interne et externe. Le modèle a également permis de simuler l’effet de régimes posologiques alternatifs dans des populations représentatives de la pratique clinique. Une réduction de la dose journalière standard de 800 mg à 600 ou 400 mg a été démontrée sure pour une large proportion de patients, sur base de cibles pharmacocinétiques couramment définies pour cette molécule. En revanche, la thérapie intermittente (cinq jours sur sept) ne constituait pas une option sure pour la plupart des sujets. L’existence d’un bénéfice individuel de ces stratégies alternatives pourrait être prédite par notre modèle. Par ailleurs, des stratégies de prélèvement optimales pour le darunavir ont été définies, montrant comment concevoir de futures études de manière optimale ou comment optimiser le monitoring thérapeutique pour ce médicament. Enfin, des expériences in vitro ont permis d’établir la contribution de polymorphismes dans le gène de la P-glycoprotéine à la variabilité précédemment mentionnée. Ces expériences ont montré que ni le polymorphisme c.1199G>A ni l’haplotype défini par c.1236C>T, c.2677G>T et c.3435C>T n’altèrent l’accumulation intracellulaire du darunavir. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2021

The β-amyloid peptide (Aβ) is the major constituent of senile plaques, a characteristic lesion of Alzheimer's disease (AD). Aβ is released by the metabolism of a precursor protein through the action of the γ-secretase complex, whose catalytic activity is ensured by either presenilin 1 (PS1) or 2 (PS2). Following their release as monomers, Aβ peptides are prone to aggregate. Under pathological conditions, this leads to the formation of Aβ fibrils which deposit in senile plaques. However, it is now recognized that the intermediate forms of Aβ, called oligomers, correlate better with AD pathology. In this work, we characterize a specific cell-derived oligomeric form, hexameric Aβ, as a readily formed Aβ assembly that can serve as a nucleus to enhance the aggregation process. More precisely, we identify hexameric Aβ as both a novel biomarker for AD and a potential therapeutic target. Specifically, we propose a direct link between PS2-dependent γ-secretase activity and the cellular release of hexameric Aβ, potentially responsible for its spread. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2021

Amino acids blood levels are remarkably constant, in spite of fasting periods and large variations in diet supply. Systemic homeostasis is particularly important for tryptophan, given its key role in many cellular processes, including immunity and neuronal activity. In humans, tryptophanemia is maintained around 60µM. Here, we showed that tryptophan homeostasis is largely controlled by the post-translational stability of tryptophan 2,3-dioxygenase (TDO), the hepatic enzyme catalyzing the first step of tryptophan catabolism along the kynurenine pathway. TDO, located at the strategic crossroad of the portal and systemic circulation, degrades more than 90% of food-derived tryptophan. High tryptophan levels stabilize the active tetrameric conformation of TDO through binding non-catalytic exosites, promoting rapid blood tryptophan catabolism. In low tryptophan, the lack of tryptophan binding in the exosites destabilizes the tetramer into inactive monomers and dimers, and unmasks a degron. This triggers the polyubiquitination of TDO by SKP1-CUL1-F-box complexes, resulting in proteasome-mediated degradation of TDO and rapid interruption of tryptophan catabolism. Artificial stabilization of tetrameric TDO with the non-metabolizable analog alpha-methyl-tryptophan in vivo stably reduces blood tryptophan levels. Our results uncover a mechanism allowing a rapid adaptation of tryptophan catabolism to ensure quick degradation of excess tryptophan while preventing further catabolism below physiological levels. This ensures a tight control of tryptophanemia, as required for both neurological and immune homeostasis. La concentration plasmatique des acides aminés est remarquablement constante en dépit des variations de nos apports alimentaires. L’homéostasie systémique des acides aminés est particulièrement importante pour le tryptophane, en raison de son rôle clef dans de nombreux processus biologiques, incluant l’immunité et l’activité neuronale. Chez l’homme, la tryptophanémie est en permanence maintenue à 60µM. Nos découvertes révèlent que l’homéostasie du tryptophane est majoritairement contrôlée par la stabilité post-traductionnelle de la tryptophane 2,3-dioxygenase (TDO), l’enzyme hépatique catalysant la première réaction du catabolisme du tryptophane dans la voie métabolique de la kynurénine. Localisée au carrefour de l’artère hépatique et de la veine porte, la protéine TDO occupe une position stratégique pour filtrer le sang et dégrade plus de 90% du tryptophane d’origine alimentaire. Lorsque la tryptophanémie augmente, le tryptophane active la protéine TDO en stabilisant sa conformation tétramérique au moyen de quatre exosites non catalytiques, promouvant ainsi le catabolisme du tryptophane plasmatique. Une fois la tryptophanémie faible, le tryptophane libère les exosites et provoque la déstabilisation du tétramère en dimères et en monomères inactifs, libérant ainsi un motif de dégradation ou dégron reconnu par des ubiquitines ligases SKP1-CUL1-F-box. Elles déclenchent alors la polyubiquitination de TDO, laquelle conduit à la dégradation de l’enzyme par le protéasome et à l’arrêt du catabolisme du tryptophane. In vivo, la stabilisation de la protéine TDO hépatique avec un analogue non métabolisable, l’alpha-méthyl-tryptophane, réduit de manière permanente la concentration plasmatique du tryptophane. Nos résultats décrivent un mécanisme d’adaptation rapide du catabolisme du tryptophane permettant une dégradation de l’acide aminé en excès et prévenant son catabolisme en dessous de sa teneur physiologique. Cette boucle de rétrocontrôle assure une régulation fine de la tryptophanémie dont le maintien est nécessaire à l’homéostasie neurologique et immunitaire. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2021

A growing body of evidence indicates that mitochondria play a key role in many disorders as well as in cancer progression and response to treatment. Next to being the main cellular energy generator through respiration, mitochondria are also the major producer of superoxide and other downstream reactive oxygen species in the cell. In this thesis, we aimed to develop an integrated electron paramagnetic resonance (EPR) toolbox enabling the assessment of mitochondrial (dys)function by measuring the oxygen consumption rate and superoxide production in a same cellular or mitochondrial preparation. This EPR toolbox was used for the benefit of two different projects: discovering potential radiosensitizers in anticancer therapy (like statins) and assessing the impact of agents such as Boscalid and Bixafen, which are succinate dehydrogenase inhibitors (SDHI), on the mitochondrial function of human cells. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2021

Li-ion batteries (LIB) are invading the market and their production and use dramatically increase. LIB are made of relatively insoluble particles, respirable in size and containing toxicologically relevant metal ions such as cobalt or nickel. Workers, and possibly the general population, can be exposed to these particles via inhalation. Information about their respiratory toxicity is, however, very limited. Here, we identified the respiratory toxicity of particles used in LIB in a mouse bioassay, and conclude that they represent a differential respiratory hazard (lung inflammation and fibrosis). Given that a large range of LIB particles with different composition, metal content and synthesis processes are developed by the industry, we investigated the mechanisms of toxicity of these LIB particles. We identified Co and Ni content/bioaccessibility and in vitro/in vivo hypoxia-inducible factor-1 a as determinants and key mediator, respectively, of the inflammatory lung responses induced by LIB particles. These parameters can be monitored in vitro in cell culture to predict the lung inflammatory potential of LIB particles and possibly categorize them. Additionally, we showed that one of the most inflammatory particles, LiCoO2, exerts genotoxic and mutagenic activities in vitro and in vivo with, at least, a primary mechanism and in association with the production of reactive oxygen species. This work provides, for LIB particles, the first toxicological information, and contributes to support stakeholders from academia and industry, in their efforts for a safer and sustainable development of LIB. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2020

Upon traumatic injury (SCI), spinal cord capacity for regeneration is limited and one of the main reasons may be the chronic inflammation. It can be then hypothesized that strategies aiming to resolve inflammation would contribute to spinal cord repair. Among different options to achieve that goal, mesenchymal stem cells (MSC) and extracellular matrix (ECM)-based hydrogels have been both described as being immunomodulatory. The aim of this thesis then was to explore the potential of stem cells of dental apical papilla (SCAP) and ECM hydrogels to mitigate SCI inflammation. SCAP had previously been shown to reduce tumour necrosis factor alpha (TNFα) gene (Tnf) expression in vitro, whilst ECM hydrogels had been shown to induce the M2-like phenotype in naïve monocytes. First, rheological properties of spinal cord and bone ECM hydrogels of 8mg/mL (S8 and B8) were measured. To increase S8 gelation rate and modulus fibrin was added to S8 pre-gel solutions at a 75/25 ratio (S/F). To investigate anti-inflammatory properties of hydrogels and SCAP, a mouse microglia cell line was used (BV2 cells). S8 solubilised, i.e. dispersed in medium before hydrogel formation is completed, reduced LPS-induced increase in inducible nitric oxide synthetase and arginase 1 gene expression ratio (Nos2/Arg1). Solubilised B8 reduced Tnf expression. SCAP reduced Nos2/Arg1 ratio and Tnf expression. S8+SCAP reduced Nos2/Arg1, whilst B8+SCAP reduced Nos2/Arg1 ratio and Tnf expression. To get a better understanding of how SCAP metabolism, free or incorporated in S8 ECM hydrogels, could be influenced by pro-inflammatory cytokines, a metabolomic analysis of cell supernatants was performed. SCAP stimulated with TNFα and interferon gamma (IFNγ) activated kynurenine pathway. Anti-inflammatory lipids were detected in scECM. SCAP+scECM had altered antioxidative amino acid and lipid profile. Still, in SCAP+scECM+stimuli the kynurenine pathway was enhanced and anti-inflammatory molecules were more abundant compared to stimulated SCAP alone. Thus, SCAP combined with ECM hydrogels may be considered as a strategy to mitigate SCI inflammation. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2020

The present thesis is articulated around three main axes. The first one focuses on assessing whether it is possible to obtain reliable motor-evoked potentials in both hands at once using a paired-pulse transcranial magnetic stimulation (TMS) method where the two primary motor cortices are stimulated with an inter-pulse interval as short as 1 ms (double-coil1ms TMS). The second axis assesses the function(s) of preparatory inhibition and more specifically its suggested role for behavioural inhibition. The third one investigates with double-coil1ms TMS whether preparatory inhibition is altered in two distinct populations, that is, in one likely to suffer from alcohol-use disorder (i.e., binge-drinkers) and in one suffering from gambling disorder. Our results are threefold. First, they infer that double-coil1ms is a reliable TMS method that can be used both at rest and in the context of a task. Second, they support and extend previous work on the presence and role of preparatory inhibition. Last, they suggest abnormal preparatory activity in binge-drinkers but not in patients suffering from gambling disorder. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2020

Development of a mature and functional pancreas is guided by an intrinsic program, but also by interactions with surrounding tissues. In the first part of my thesis, we found that endothelial cells produce abundant laminin-111, an extracellular matrix protein, in a regionalized manner. Laminin-111 repressed acinar differentiation, only allowing expression of the pro-acinar transcription factors Ptf1a and Rbpjl, and thus acinar differentiation, at a distance from blood vessels. In the second part, we studied the tight junction protein MarvelD3 in the pancreas, and investigated its role by gene inactivation. We found that despite a very early expression, MarvelD3 is not required for normal pancreas development and function. However, preliminary data in mouse models of pancreatic diseases suggest that MarvelD3 could impact on acinar-to-ductal-metaplasia upon caerulein-induced pancreatitis, and on precancerous PanIN lesions development upon oncogenic KRas expression. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2020

Small-cell lung cancer (SCLC) is a devastating illness with frequent metastases and poor survival. The molecular steps leading to SCLC development and progression are still poorly understood and this has translated into the absence of effective targeted therapies and a five-year overall survival as low as 5%. Focal Adhesion Kinase (FAK) is a non-receptor tyrosine kinase localized at sites of focal adhesions and playing an important role in signal transduction pathways initiated by integrins and G-protein-coupled-receptors. It is overexpressed in many cancers and contributes to cancer progression through a central role in cell adhesion, migration, invasion, survival, and growth, which are highly relevant in SCLC, known to be very aggressive. Since FAK has been poorly studied in SCLC, we decided to investigate it at the functional level in this cancer. We found that FAK was strongly expressed and activated in primary SCLC tumors compared to non-small-cell lung cancer and normal lung. Moreover, inhibition of FAK activity in SCLC cell lines where FAK is overexpressed and constitutively activated decreased FAK phosphorylation (Tyr397) and resulted in antitumoral effects. Inhibition of FAK activity significantly decreased cell proliferation, induced cell cycle arrest in G2/M phases, decreased DNA synthesis, increased apoptosis, and decreased motility as well as invasion in SCLC cell lines. Altogether, this work demonstrates that FAK activation plays a key role in the invasive behaviour of SCLC and suggests that FAK-targeted therapeutic strategies should be evaluated in clinical trials with the hope of reducing mortality from SCLC. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2020

This thesis studies the activity of antibiotics on S. aureus collected in Vietnam from persistent or recurrent infections, in an attempt to elucidate the reasons for this persistence/recurrence for one specific antibiotic, moxifloxacin. We confirm a high rate of resistance in Vietnam (50% MRSA-83% MDR). Our work on MXF showed that the persister character, as established in-vitro, promotes the evolution of resistance (faster increase in MIC for persistant isolates). Moreover, it is predictive of a reduced efficacy against intracellular forms of infection (more abundant residual inoculum) and of the capacity to produce biofilms with an abundant, enriched in polysaccharides. The persister character of a clinical isolate could be an important determinant in its capacity to adopt persistent modes of life and to escape to antibiotic action, suggesting the interest of detecting this persiter character in routine in the hospital microbiology laboratories. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2020

Nowadays, the prevalence of obesity-related liver diseases is increasing around the globe. However, the efficacy of the available treatments is still limited suggesting that new therapeutic strategies must be discovered. In this thesis we aimed to understand better the etiology of hepatic disorders by examining the impact of two proteins: the first one belonging to the immune system (MyD88) and the second one to the endocannabinoid system (NAPE-PLD). To reach this goal, we investigated the consequence of their deletion in hepatocytes in two distinct mouse models (Myd88∆Hep and Napepld∆Hep). Surprisingly, we discovered that both mutant mice were prone to liver inflammation and hepatic fat accumulation. Additionally, one crucial bioactive lipid family (i.e. bile acids) that regulates host homeostasis and inflammation was strongly altered in both Myd88∆Hep and Napepld∆Hep mice compared to WT. This thesis thereby provides new insights into the regulation of bile acids by two systems that seemed, at first, unrelated to this lipid family and emphasizes the importance of a proper regulation of molecules involved in their regulation to tackle the emergence of metabolic disorders. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2020

IL-22 is a cytokine that plays a deleterious role in psoriasis and a beneficial one in colitis through the activation of JAK/STAT pathway. We demonstrated the presence of an alternative mechanism of STAT3 activation, requiring the C-terminal (C-ter) region of IL-22Ra and the coiled-coil domain (CCD) of STAT3. In vivo, our data show that, in various organs, the C-ter part of IL-22Ra is involved in STAT3 activation mediated by IL-22. In the skin, deletion of the C-ter partially protects mice from psoriatic lesions while it slightly affects the development of colitis. In conclusion, this study suggests that the C-ter part of IL-22Ra is a potential target to partially block the deleterious effects of IL-22 in the skin but leaving intact the beneficial ones. For this purpose, we used a monobody approach targeting the CCD of STAT3. We show that this monobody blocks the alternative activation of STAT3 by IL-22Ra. L’IL-22 est une cytokine qui joue un rôle délétère dans le psoriasis et bénéfique dans la colite via l’activation de la voie JAK/STAT. Nous avons mis en évidence l’existence d’un mécanisme alternatif d’activation de STAT3, dépendant de la queue C-terminale (C-ter) de l’IL-22Ra et du domaine coiled-coil (CCD) de STAT3. In vivo, nos données montrent que, dans certains organes, la partie C-ter est requise pour l’activation de STAT3 induite par l’IL-22. Dans la peau, la délétion de cette partie protège partiellement les souris des lésions de type psoriasis alors qu’elle impacte peu le développement de la colite. En conclusion, cette étude suggère que la queue C-ter est une cible potentielle pour bloquer partiellement les effets délétères de l’IL-22 dans la peau tout en conservant ses effets bénéfiques. Dans cette optique, nous avons utilisé une approche monobody ciblant le CCD de STAT3. Nous avons montré que ce monobody bloque le mécanisme alternatif d’activation de STAT3 par l’IL-22Ra. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2020

Cancer immunotherapies aim at inducing or stimulating anti-tumor immune responses to eliminate tumor cells and trigger tumor regressions in patients with cancer. Approaches under clinical development comprise cancer vaccines, adoptive transfers of immune cells and immunostimulatory antibodies. Several immunostimulatory antibodies were approved by regulatory agencies for the treatment of various types of cancers. Despite impressive clinical successes, response rates to cancer immunotherapies remain very diverse, and only a minority of patients benefit from the currently available approaches. Novel approaches that target other immunosuppressive pathways are needed to increase the clinical efficacy of cancer immunotherapies. Regulatory T cells or Tregs are immunosuppressive cells that are indispensable to prevent auto-immunity, but in cancer patients they are detrimental because they suppress anti-tumor immunity. Our laboratory previously identified a mechanism of immunosuppression by Tregs which implies production and activation of the immunosuppressive TGF-β1 cytokine. Unlike other cells, activation of TGF-β1 by Tregs requires intervention of two transmembrane proteins named GARP and integrin αvβ8. We developed antibodies against GARP:TGF-β1 complexes that allow to selectively block TGF-β1 activation by Tregs. These could provide significant advantages over global TGF-β1 inhibition, which bears the risk of undesired tumor-promoting effects. Indeed, TGF-β1 exerts vastly diverse effects in cancer, supporting either tumor progression by favoring metastases and inhibiting anti-tumor immunity, or tumor suppression by inhibiting malignant cell proliferation. Here, we show that blocking TGF-β1 activation by Tregs with anti-GARP:TGF-β1 antibodies induces regressions of mouse tumors that are otherwise resistant to anti-PD-1 immunotherapy. We found GARP-expressing Tregs and evidence that they produce TGF-β1 in human melanoma metastases. Finally, we provide evidence that upon administration to tumor-bearing mice, anti-GARP:TGF-β1 antibodies directly bind Tregs and reduce autocrine TGF-β1 signals in these cells. Altogether, our work suggests that blocking production of a single TGF-β isoform emanating from a restricted cellular source is sufficient to potentiate anti-tumor immunity. Anti-GARP:TGF-β1 mAb may serve as a new immunostimulatory drug to improve patient responses to cancer immunotherapies. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2020

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive cancers with a low survival rate. Pancreatic intraepithelial neoplasia (PanIN) correspond to a preinvasive lesion of PDAC and originate from acinar cells that undergo acinar-to-ductal metaplasia (ADM). Mutated KRAS plays a major role in the formation of these lesions. EGFR is required for KRAS-mediated PanIN development but little is known about the role of its preferred dimerization partner, ERBB2. To explore the role of ERBB2, we used mouse models deleted for ERBB2 with or without mutated KRAS in acinar cells. We found that, unlike EGFR, ERBB2 is not required for ADM or PanIN formation. We also used a mathematical model of EGFR-ERBB2-KRAS interactions that confirmed the essential impact of EGFR and predicted that ERBB2 impacts ERBB signaling only when overexpressed but not when its expression is decreased. In conclusion, EGFR and ERBB2 differentially control ERBB signaling during pancreatic tumorigenesis. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2020

Diffusion MRI is a medical imaging technique allowing to probe the microstructure of biological tissues, non-invasively. It is based on the mobility of water molecules between and within cells. In this context, double diffusion encoding is a novel method that could improve the sensibility to features of cell microenvironments. For instance, this would include mean cell size, cell density or microvascular perfusion. The main objective of the thesis is to implement a double diffusion encoding sequence on a clinical MR system in order to assess its feasibility. Furthermore, we apply the method to experimental tumors undergoing radiotherapy with the aim of validating its outcomes with histology. Finally, we investigate the added value of double diffusion encoding compared to alternative diffusion MRI techniques existing for tumors. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2020

Cystic fibrosis is a genetic disease characterized by the accumulation of viscous mucus in the patient's airways, which favors bacterial colonization by bacterial communities named biofilms. The most common pathogens are Staphylococcus aureus in children and Pseudomonas aeruginosa in adults. Our goal was to develop a relevant artificial sputum medium (ASM) that can be used to grow biofilms of each of these bacteria and study the activity of antibiotics administered to the patients. We showed that the rheological properties of this medium were similar to those of sputa from patients. Biofilms from both species were less responsive to antibiotics in ASM than in a conventional medium. In addition, some antibiotics selected small colony variants in ASM, another mode of bacterial growth involved in the pathology. These results show the importance of using a relevant culture medium to study the activity of antibiotics in the context of this pathology. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2020

We investigated the biological response to new degradable biomaterials for cardiovascular stents. In a first part, we compared a bioresorbable sirolimus-eluting stent (BP-SES) with a bare metal stent (BMS) in the rat aorta without antiplatelet therapy. We highlighted the low early thrombogenicity of BP-SES with an excellent strut coverage at 1 month with localized thrombotic deposits and no impact on platelet activation. These data could help support the safety of a short 1-month dual antiplatelet therapy following BP-SES implantation in the human coronary artery. Furthermore, this study allowed us to validate the procedure of preclinical evaluation of stents in vivo. In a second part, we evaluated the hemocompatibility of a new candidate material for stent: twinning-induced plasticity (TWIP) steel. In vitro, we showed its excellent hemocompatibility with an absence of hemolysis and an anti-thrombogenic effect. Low thrombogenicity of TWIP steel seems to be confirmed in vivo with the model of wire implantation in the rat aorta. Our findings indicate that TWIP steel could be a promising platform for next-generation stent technologies. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2020

Little is known about anti-tumor T cell activity in low antigenic but frequent tumor such as breast carcinoma. We first explored the immunogenicity towards CD8 T cells for 6 primary human breast carcinomas. We screened tumor derived T cell clones for the recognition of tumor specific antigens. We found only 7 T cell clones that were able to recognize mutations. They originate from the same tumor. This demonstrates that anti-tumor T cell activity is possible in primary human breast carcinoma but seems to be rare. Aware of potential selection bias linked to the ability of T cells to grow in vitro and eager to characterize the ex vivo phenotype of the identified anti-tumor T cells, we developed a workflow involving single cell RNAseq analysis of freshly extracted tissue T cells followed by TCR reconstruction in a reporter cell line to evaluate their specificity. This workflow has also been applied to analyze kidney infiltrating T cells from lupus nephritis suffering patients. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2019

Research addressing how Wnt signaling modulates the behaviors of Stem Cells (SCs) has largely focused on β-catenin-dependent canonical Wnt pathway, and very limited information can be found about β-catenin-independent signaling in the SC niche. In this doctoral research, hair follicle (HF) is used as a model system to study the role of Ror2, a non-canonical Wnt receptor, in adult HFSCs. We uncovered that Ror2-dependent signaling is upregulated in activated HFSCs in vivo. Using a loss-of-function approach, we demonstrated that Ror2 is indispensable for HFSC self-renewal and long-term maintenance. We not only confirmed the classical role of Ror2 in mediating Wnt5a-dependent signaling and cell migration, but also uncovered novel functions of Ror2 in regulation of cell proliferation and maintenance of HFSC identity. Finally, we unveiled a compensatory mechanism mediated by Ror2-PKC signaling that keeps stemness of β-catenin-null HFSCs and prevent their loss from differentiation towards wrong fate. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2019

Calreticulin is an ER resident chaperone protein that also plays an important role in buffering the Ca2+ ions in the ER, regulating the calcium homeostasis and calcium-dependent signaling in the cell. In 2013, mutations of the calreticulin encoding gene CALR were shown to be driver mutations of essential thrombocythemia (ET) and myelofibrosis, two diseases that are part of the myeloproliferative neoplasm family. Previous studies in our team have shown that the thrombopoieitin receptor is activated by calreticulin mutants, thereby activating the JAK-STAT pathway of CALR mutated cells. This work focused on establishing a precise comprehension of the thrombopoietin receptor activation mechanisms by calreticulin mutants by studying their interaction in vitro and by establishing a relevant mouse model of disease. In vitro studies have elucidated that calreticulin mutants act as ‘rogue chaperones’ that are capable of escaping the ER and accompanying the thrombopoietin receptor through the secretory pathway. This is shown to be possible through a specific interaction of the lectin domain of calreticulin mutants with the first N-glycan of the thrombopoietin receptor that remains in an immature form even as both proteins progress through the Golgi compartment to the plasma membrane. We also provide evidence of a direct physical interaction between the calreticulin mutant and the thrombopoietin receptor by BRET. We also show that the human and mouse mutant calreticulin proteins are highly similar in their amino-acid sequences, biochemical properties and oncogenic capabilities. This allowed us to create two mutated Calr knock-in mouse models by CRISPR/Cas9 genome editing that reproduce an ET phenotype but with subtle differences that are attributable to the level of STAT5 activation both mutant calreticulin proteins are able to induce through their activation of the thrombopoietin receptor. We also confirm the observation that the calcium buffering role of calreticulin is essential for the myocardium development during embryogenesis and that lack of the C-terminus of the protein is embryonically lethal just like the full knock-out of the protein. This work provides substantial answers to the field of CALR mutated myeloproliferative neoplasms regarding the oncogenic mechanism at the cellular level but also at the level of a whole organism. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2019

Nephropathic cystinosis is a multisystemic lysosomal storage disease due to genetic absence of functional cystinosin gene, which encodes the lysosomal cystine transporter. Suppressed lysosomal exodus leads to cystine accumulation then precipitation as crystals. The earliest clinical manifestation of cystinosis is a renal Fanconi syndrome which reflects global dysfunction of proximal tubular cells (PTCs). I first addressed early pathophysiological changes in PTCs of a cystinosin KO mouse model and identified apical dedifferentiation related to lysosome swelling, prior to crystals and atrophy. I next found that excision of megalin in cystinotic PTCs prevented cystine accumulation and protected kidney structure. This implies that endocytosis is the main source of cystine accumulation, which itself causes kidney damage. In a promising translational approach, I found that megalin inhibition by a simple dietary supplementation in lysine or arginine could slow down disease progression. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2019

Alzheimer’s disease (AD) is characterized by cognitive impairments such as apraxia, aphasia and memory decline. The two majors hallmarks of AD are neurofibrillary tangles and senile plaques. The latter are composed of amyloid peptide (Aβ) produced from its precursor, the Amyloid Precursor Protein (APP). The final cleavage of APP occurs on its C-terminal fragment (C99) by γ secretase and produces the APP intracellular domain (AICD) and Aβ. The abnormal production of Aβ leads to its aggregation and finally to its toxic effects on neurons. The work presented in this thesis focuses on the molecular determinants of APP/C99 that drive the production of both Aβ and AICD. We have shown that one particular dimeric orientation promotes strong AICD while another one favors the formation of stable oligomers composed of Aβ peptides. The characterization of these orientations could potentially aid a strategy to target AD by changing the dimerization profile leading to lower pathogenic Aβ production. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2019

Tacrolimus (Tac) is the cornerstone of immunosuppression in pediatric liver transplantation (PLT). However, its use is complicated by side effects and lack of efficacy partly due to high pharmacokinetic (PK) variability. This thesis aims to better individualize Tac therapy in PLT according to factors accounting for this variability. We conducted three retrospective PK studies to identify and model the effect of these factors on Tac PK by using non-linear mixed effects modelling. We developed 3 population PK models. The two first studies focusing either on the one year or fifteen days post PLT showed that apparent clearance (CL/F) increases with graft weight/recipient weight ratio and time post PLT. In the last model, developed with bicentric data, Tac CL/F showed a 30% increase in case of donor CYP3A5 expression and a 29% decrease in case of donor CYP3A4*22 mutation. We also confrimed the inhibiting effect of fluconazole on CL/F according to the CYP3A5 status and proposed an algorithm for tacrolimus dose initiation. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2019

The selective targeting of metabolic differences that allow cancer cells to proliferate appears as a promising strategy to develop novel anticancer agents. In this context, phosphoglycerate dehydrogenase (PHGDH), the first enzyme of the serine synthetic pathway, is a potential target. Our works therefore focused on the development and study of new PHGDH inhibitors. First, α-ketothioamide PHGDH inhibitors were identified and further optimized by chemical modifications. The best inhibitor of the series was also shown to inhibit PHGDH in cell-based models and to decrease cancer progression in vivo. The use of an original photoactivatable diazirine probe together with mutagenesis experiments led to the identification of a new regulatory allosteric site on PHGDH as the putative binding site. In the second part of this thesis, the works focused on the study of disulfiram (DSF), a drug approved for the treatment of chronic alcoholism, as potential anticancer agent. Hence, we demonstrated that the anticancer effect of DSF was, at least in part, due to PHGDH inhibition through disruption of its particular oligomerization state. In conclusion, this work provides a better understanding of PHGDH inhibition and offers new tools to investigate tumor metabolism. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2019

Additive manufacturing techniques are promising technologies to produce patient-specific and effective scaffolds for tissue engineering. Chemical and physical surface modifications are often performed on scaffolds to control cell behavior and foster tissue regeneration. On the other hand, surface mechanical properties are known to influence stem cell proliferation and to direct their differentiation. The aim of this work was to produce degradable poly(lactic acid) (PLA) and poly(epsilon-caprolactone) scaffolds using a so-called gyroid geometry, which features an isotropic and spring-shaped design. This was achieved by adapting the parameters to get closer to the resolution limit of fused deposition modeling, an easy-to-use and low-cost 3D printing technology. A semi-interpenetrating polymer network made with poly(dimethylsiloxane) crosslinked at different densities was then designed to modify the surface mechanical properties of PLA gyroid scaffolds. Finally, the effect of the coating mechanical properties on the proliferation and osteogenic differentiation of stem cells from the apical papilla was evaluated. The production of complex scaffolds was successfully achieved using 3D printing. The coating technology that was further developed paves the way towards the modification of the surface mechanical properties of macroporous scaffolds with a complex shape, with a view to control stem cell behavior. (AGRO - Sciences agronomiques et ingénierie biologique) -- UCL, 2019

Locomotion is a complex behavior regulated by circuits, named central pattern generators, located in the ventral part of the spinal cord. These circuits are composed of motor neurons (MNs) and of different populations of interneurons: dorsal dI6 and ventral V0 to V3 cardinal populations. During development, they are produced from specific progenitor domains distributed along the dorso-ventral axis of the spinal cord. The p2 progenitor domain generates V2 interneurons that diversify at least into five populations: V2a, V2b, V2c, V2d and Pax6-V2 interneurons. This differentiation process involves fine genetic regulations and cross-repressive mechanisms that consolidate cell fate. Indeed, progenitor domains of V2 interneurons and of MNs are closely adjacent during development and share some molecular determinants. Cross and mutual repressions between Ceh-10 homeobox (Chx10) gene, expressed in differentiating V2a interneurons, and Homeobox 9 (Hb9), expressed in early MNs, participate in the consolidation of V2 interneuron versus MN identity by preventing the activation of inappropriate differentiation program. However, Chx10 starts to be expressed in differentiating V2a interneurons. Therefore, we could address the following question: which factor secures the identity of V2 interneurons at early stages of development. Studies demonstrate that the unique paralog gene of Chx10, the Visual System homeoboX 1 (Vsx1), is also present in V2 interneuron compartment in a more medial part of the spinal cord. The aim of my thesis project is to characterize the expression profile of Vsx1 in the spinal and determine whether it may, in collaboration with its paralog Chx10, contribute to the securisation of V2 interneuron identity. In this work, we show that Vsx1 transiently labels an intermediate V2 precursor compartment. We provide evidence that this transcription factor is not necessary for V2 interneuron production but contributes to their development. We show that the unique paralog factor of Chx10 identically prevents motor neuron differentiation in early V2 precursors. Furthermore, combined inactivation unveiled cooperativity between Vsx1 and Chx10 although they are not produced in the same cells. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2019

The envelope of Gram-negative bacteria is a complex compartment that is essential for viability. To ensure survival of the bacterial cells in fluctuating environments, several signal transduction systems, called envelope stress response systems (ESRSs), exist to monitor envelope biogenesis and homeostasis. We show that the Cpx two-component system, an extensively studied ESRS of E. coli, can monitor two essential envelope processes: cell wall synthesis and lipoprotein trafficking. We first show that Cpx is activated when peptidoglycan (PG) integrity is challenged and that this activation provides protection to cells exposed to antibiotics inhibiting PG synthesis. We also found evidence that an outer membrane lipoprotein, NlpE is used by Cpx to detect perturbations in lipoprotein trafficking. The activation of Cpx by NlpE improves fitness in these nefarious conditions. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2019

Hypoxia is the common feature of most solid tumors. The hypoxic environment is associated with the reduced efficacy of cancer treatments, especially radiation therapy. Although modification of tumor hypoxia improves the outcome of radiation therapy, screening for the presence of hypoxia has not been the entry criterion prior to hypoxia modification trials. To bridge the gap between the occurrence of tumor hypoxia and the routine clinical practice, there is an important need to have a method to robustly map tumor oxygenation for patient stratification and treatment adaptation. Among the methods that have been developed to measure hypoxia, MRI endogenous contrast markers, R1 and R2*, appear to be a relevant approach for this purpose. Since there is a lack of study comparing R1 and R2* in response to oxygen modifiers, the aim of this thesis is to address the value of R1 and R2* to assess tumor oxygenation and response to radiation therapy using different oxygen modifiers. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2019

This research project aimed to analyze the anti-trypanosomal and anti-leishmanial potential of essential oils extracted from Vietnamese plants and their constituents. These two important neglected diseases are affecting millions of people either in poorly developed or developing countries despite of many efforts made this past decade to decrease their incidence. Literature data show that some essential oils possessed promising anti-parasitic activity not only in vitro but also in vivo using animal models. 37 Vietnamese essential oils were therefore screened on Trypanosoma brucei brucei bloodstream form and Leishmania mexicana mexicana promastigotes. Their cytotoxicity was also analyzed on two different mammalian cell lines in order to choose the samples showing the best selective effect toward parasites. Among them, 4 essential oils extracted from Litsea cubeba, Curcuma longa, Curcuma zedoaria, and Zingiber officinale revealed selective anti-trypanosomal effect, while the most promising anti-leishmanial one was extracted from Ocimum gratissimum. Their chemical composition was then characterized by gas chromatography coupled with FID and MS detectors. Curlone isolated from Curcuma longa essential oil and curzerene from Curcuma zedoaria one were identified as promising anti-trypanosomal candidates while eugenol was responsible for the selective anti-leishmanial effect of Ocimum gratissimum essential oil. Eugenol was already known for its activity on different Leishmania species. However, its mechanism of action against these parasites remains unknown. As it has been shown that eugenol can interact with ergosterol in Candida albicans membrane, and because ergosterol is also a major sterol in Leishmania plasma membrane, its effect was evaluated on liposomes mimicking Leishmania membrane as well as on Leishmania cells. Our results indicate that eugenol did not induce membrane permeabilization nor membrane fluidization at its IC50, but only at much higher concentrations. We also observed, at this IC50, an inhibition of Leishmania metabolism. Ce projet de recherche a pour but l’étude des potentiels antitrypanosomaux et antileishmaniens d’huiles essentielles extraites de plantes vietnamiennes et leurs constituants. Ces deux maladies négligées touchent des millions de personnes dans des pays peu développés ou en cours de développement et ce, malgré les nombreux efforts consentis durant ces dernières décennies pour réduire leur incidence. Selon les données de la littérature, certaines huiles essentielles présentent une activité antiparasitaire prometteuse, tant in vitro que in vivo sur des modèles animaux. De ce fait, 37 huiles essentielles ont été testées sur la forme sanguine du Trypanosoma brucei brucei et sur les promastigotes Leishmania mexicana mexicana. Par ailleurs, leur cytotoxicité a également été déterminée sur deux lignées cellulaires de mammifères différentes afin de sélectionner les échantillons démontrant un effet sélectif envers les parasites. Parmi ceux-ci, 4 huiles essentielles extraites de Litsea cubeba, Curcuma longa, Curcuma zedoaria, et Zingiber officinale se sont révélées avoir un effet antitrypanosomal sélectif, tandis qu’une huile essentielle extraite de Ocimum gratissimum a présenté l’effet antileishmanien sélectif le plus prometteur. Leur composition chimique a ensuite été caractérisée par chromatographie en phase gazeuse couplée à des détecteurs à spectrométrie de masse et FID. La curlone, isolée à partir de l’huile essentielle de Curcuma longa et le curzerène issu de Curcuma zedoaria ont été identifiés comme des candidats antitrypanosomaux prometteurs alors que l’eugénol s’est révélé être le composant responsable de l’effet antileishmanien sélectif de l’huile essentielle d’ Ocimum gratissimum. Bien que l’eugénol fût déjà connu pour son activité sur plusieurs souches de Leishmania, son mécanisme d’action sur ces parasites reste encore inconnu. Comme il avait été démontré que l’eugénol pourrait interagir avec l’ergostérol dans la membrane Candida albicans et que l’ergostérol est un stérol majeur de la membrane plasmique de Leishmania, son effet a été évalué à la fois sur des liposomes imitant la membrane leishmaniale et sur les cellules de Leishmania elles-mêmes. Nos résultats montrent que l’eugénol n’induit ni une perméabilisation, ni une fluidification de la membrane à sa CI50, mais seulement à de plus hautes concentrations. Par contre, nous avons pu observer une inhibition du métabolisme de Leishmania à cette concentration. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2019

As there is an unmet medical need for the treatment of glioblastoma, the aim of this project is to prevent local tumor recurrences with an anticancer drug-loaded hydrogel directly injected in the tumor resection cavity. In this project, a photopolymerizable hydrogel loaded with paclitaxel nanoparticles was developed and the antitumor efficiency in glioma orthotopic mouse model was investigated. Then the hydrogel was loaded with paclitaxel nanoparticles and free temozolomide. The drug combination achieved synergistic effect in vitro and superior antitumor efficacy in vivo. In addition, the beneficial antitumor efficacy of combination treatments with paclitaxel nanoparticle-loaded hydrogel and oral administration of temozolomide was also demonstrated. In conclusion, hydrogel-based local drug delivery has potential to be included in the standard of care of glioblastoma. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2019

Until now, DNA vaccination against cancer failed to demonstrate a relevant activity in clinic or in a therapeutic setting in preclinical models. This thesis shows two strategies to improve DNA vaccine activity. The first is the optimization of the plasmid itself, by codon optimization and a rational antigen choice. The second is the combination of the vaccines with other complementary therapies: 1) Immune checkpoint blockade in P815 mastocytoma. 2) Oncolytic virus in B16F1 melanoma. 3) Tumor resection in orthotopic GL261 glioblastoma. The common outcome was the dramatic increase in survival in the combination group, explained by the presence of active and antigen-specific T cells in the tumor. Hence, vaccine optimization and rational combined therapies overcome the current limitations of DNA vaccines. This work opens the way to new therapies for the treatment of cancer patients. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2019

Sensory experience is encoded by specific neurons located both in the peripheral and in the central nervous systems to integrate and elicit adequate responses. Sensory neurons, the cell bodies of which reside in dorsal root ganglia (DRG), relay input such as pain, temperature, itch, touch and limb and muscle length. This information is largely integrated and relayed in the spinal cord. During development, distinct neuronal types, including sensory neurons and dorsal interneurons (dIN), differentiate from progenitors into neurons characterized by distinct molecular identities, localizations and connectivity. However, the molecular mechanisms that control those processes remain incompletely characterized. Previous data obtained in our laboratory have shown that the Onecut (OC) transcription factors are present in sensory neurons and in several spinal dIN populations. This suggests that these factors could contribute to the regulation of the sensory neuron and spinal dIN development. My thesis goal is to study OC roles during these processes and to elucidate the underlying molecular mechanisms in the spinal cord. In this work, I provide evidence that the OC factors are necessary for proper diversification of dI5 interneurons and migration of early-born dIN populations. Moreover, I highlight that the OC factors control the expression of spinal Pou2f2. Using gain- or loss-of-function experiments, we show that Pou2f2 contributes to the regulation of dIN distribution. Also, I show that OC factors regulate early differentiation and segregation of distinct sensory subsets. Furthermore, OC are required for proper positioning and for normal projections of sensory neurons into the spinal cord or towards their peripheral targets. Thus, we uncover mechanisms of action of OC proteins in the regulation of spinal dIN development and OC involvement in sensory neuron differentiation, positioning and axonal projections during embryonic development. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2019

Mitochondrial superoxide is widely investigated as its implication was suggested in the development of neurodegenerative diseases, hypertension and cancer. So far, only a few techniques exist to detect this species sensitively and specifically. Fluorescent-based probes (e.g., mitoSOX) are the most widely used tools. However, several concerns about their specificity were highlighted further questioning the validity of the information provided. Electron Paramagnetic Resonance (EPR) is a technique of choice to detect and identify the radicals involved in a given biological process. The aim of the thesis was to develop a new EPR probe targeted to mitochondria. Different EPR techniques exist although they have never been systematically compared to each other. The first study allowed us to select CMH as the most sensitive tool to detect superoxide. Then, we targeted the latter to mitochondria by linking a triphenylphosphonium moiety to form mitoCPH. We next characterized this tool in PBS, lysates and paraquat-stimulated cells. Finally, we attempted to detect mitochondrial superoxide levels in superinvasive cancerous cell lines. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2019

Erythrocytes have a remarkable ability to deform. This deformability depends on a dynamic cytoskeleton but also on surface lipid domains involved in membrane deformation and calcium exchanges. In this work, we investigated whether and how lipid domains are altered and contribute to the pathophysiology of hereditary elliptocytosis and spherocytosis, two erythrocytes diseases caused by cytoskeletal defects. Our results show impairment of the abundance, properties and functionality of the domains involved in calcium exchange. Depending on the disease and its severity, those alterations are accompanied by an overactivation of the plasmatic acid sphingomyelinase, an increase in the intracellular calcium content and an activation of downstream signaling pathways, resulting into impaired membrane asymmetry and increased oxidative stress. This study paves the way towards a better understanding of the molecular mechanisms behind both diseases and opens new perspectives for treatment. (BIFA - Sciences biomédicales et pharmaceutiques) -- UCL, 2019