Les GTPases de la sous famille RHOA participent à la régulation de nombreuses voies de signalisation qui contrôlent la dynamique du cytosquelette cellulaire et une grande diversité de fonctions telles que la prolifération, la division, la migration et la polarité cellulaires. Ce sont de véritables interrupteurs moléculaires qui, en réponse à un stimulus, changent de conformation tridimensionnelle pour activer leurs protéines effectrices cibles. Elles existent donc sous deux formes, une forme inactive liant le GDP et une forme active, liant le GTP. La proportion de forme active est extrêmement régulée au niveau spatial et temporel dans une cellule et représente moins de 10% de sa totalité. Depuis près de 20 ans, le seul outil disponible pour étudier leur activation est constitué par le domaine de liaison d'un effecteur, le RBD. Peu stable, faiblement soluble et peu adaptable, de nouveaux outils sont nécessaires afin de mieux comprendre la fine régulation de ces protéines. Les anticorps à simple domaine, VHH ou nanobodies, sont caractérisés par leur stabilité, solubilité, haut rendement de production et versatilité de fonctionnalisation. A partir d'une nouvelle banque d'anticorps à simple domaine optimisée pour la production d'intracorps, nous avons isolés différents clones capables de reconnaître in vitro et de bloquer in cellulo la forme active de ces protéines. L'un de ces clones permettra le développement d'un nouvel outil de mesure de l'activité de ces protéines in vitro tandis qu'un autre, in cellulo, permettra de mieux comprendre la régulation spatiale et temporelle des protéines endogènes. / RHOA small GTPase belongs to a subfamily acting as a molecular switch activating major signaling pathways that regulate cytoskeletal dynamics and a variety of cellular responses such as cell cycle progression, cytokinesis, migration and polarity. RHOA activity resides in a few percent of GTP loaded protein, which is finely tuned by a crosstalk between regulators of the GTPase cycle. Manipulating a single RHO at the expression level often induces imbalance in the activity of other RHO GTPases, suggesting that more specific tools targeting these active pools are needed to decipher RHOA functions in time and space. We decided to use single domain antibodies, also known as VHH or nanobodies, as a new tool for studying RHOA activation. We produced and screened a novel fully synthetic phage display library of humanized nanobodies (NaLi-H1) to develop conformational sensors of the GTP loaded active conformation of RHO subfamily. We obtained several high affinity nanobodies against RHOA's active form which we characterized as RHO active antibodies in vitro and RHO signaling blocking intrabodies in cellulo. These new tools will facilitate and improve our current knowledge of this peculiar protein subfamily and will be a paradigm for the study of other RHO related small GTPases.

L'axe formé par la chimiokine CXCL12 et son récepteur CXCR4 est conservé chez les vertébrés où il joue un rôle important dans l'embryogenèse et la vie adulte, régule de nombreux processus des réponses immunitaires grâce à ses fonctions dans la migration cellulaire, la survie et la prolifération.En outre, cet axe est impliqué dans les processus pathologiques tels que les cancers (croissance et métastase) et immunodéficiences ainsi que des dysfonctionnements (par exemple l'expression dérégulée, polymorphismes ou mutations) et est également détourné par certains agents pathogènes (par exemple le virus de l'immunodéficience humaine, virus du papillome humain).Un grand groupe de travail est consacré à cette paire comme cible thérapeutique, mais seulement un composé (à savoir Plérixafor) a atteint l'approbation pour une utilisation clinique faisant le potentiel de cet axe comme cible de médicament encore inexploré.Bien que cet axe est l'objet d'un grand intérêt, des questions demeurent quant aux déterminants structurels impliqués dans l'interaction CXCL12/CXCR4.Cependant, la structure récemment résolue par diffraction de CXCR4 a donné quelque indice au sujet de ces questions, et au­ delà, la possible stoichiométrie entre CXCL12 et CXCR4.Plusieurs éléments de preuve appuient le concept que les formes CXCR4 homo- et hétéro- oligomères (qui peut contribuer à la diversité des fonctions de récepteur), telles que la structure de diffraction, le gain de fonction d'un récepteur CXCR4 mutant responsable du syndrome WHIM et la modulation allostérique des fonctions de CXCR4 par CXCR7 (ACKR3), le second récepteur de CXCL12. La possibilité de former des oligomères ouvre de nombreuses questions en matière de CXCL12 et ses interactions avec CXCR4 et CXCR7/ACKR3. La stoichiométrie de cette interaction reste une question ouverte, comme le récepteur est capable de former des oligomères avec le même récepteur ou autre récepteurs, en particulier CXCR7/ACKR3. Ce récepteur, connu comme scavenger, n'a pas de structure résolue et son mécanisme d'interaction avec CXCL12 reste inconnu.Afin d'étudier les interactions CXCL12/CXCR4/CXCR7, nous avons appliqué plusieurs techniques de modélisation moléculaire tels que peptid-peptide docking et simulations de dynamique moléculaire.Objets du projet ont étés : la résolution des possibles formes stoichiométriques de l'interaction CXCR4/CXCL12 (modélisation moléculaire, docking et dynamique); la modélisation de la structure du récepteur CXCR7/ACKR3 et son interaction avec CXCL12 (homology modeling), avec caractérisation des domaines et des résidus clef de l'activation des pathways de signalisation en aval du récepteur (mutants CXCR7/ACKR3); l'étude et la caractérisation de nouveaux outils innovants pour la détection de l'oligomerisation de ces récepteurs en conditions endogènes. (Nanobodies, HTRF)Les résultats du premier objectif ont été publiés en janvier 2016 : PMID 26813575.La modélisation de CXCR7/ACKR3 nous a permit de générer plusieurs mutants du récepteur pour tester nos hypothèses sur l’activation.Les nanobodies caractérisés pour CXCR4 seront utilisé dans une deuxième étude pour l’identification des formes oligomériques du récepteur sur tissus et cellules. / The axis formed by the chemokine CXCL12 and its receptor CXCR4 is conserved in vertebrates where it plays an important role in embryogenesis and adult life, regulates many processes of immune responses through its functions in cell migration, survival and proliferation.In addition, this axis is involved in pathological processes such as cancers (growth and metastasis) and immune deficiencies and malfunctions (eg deregulated expression, mutations or polymorphisms) and is also hijacked by certain pathogens (eg HIV, human papilloma virus).A large working group is dedicated to this pair as a therapeutic target, but only a compound (ie Plerixafor) achieved approval for clinical use by the potential of this area as a drug target unexplored.Although this axis is the subject of great interest, questions remain about the structural determinants involved in CXCL12 / CXCR4 interaction.However, the recently resolved diffraction structure of CXCR4 gave some clue about these questions, and beyond possible stoichiometry between CXCL12 and CXCR4.Several lines of evidence support the concept that forms CXCR4 homo- and hetero-oligomers (which can contribute to the diversity of the receptor functions), as shown in the diffraction structure, the gain function of a mutant CXCR4 receptor responsible for the syndrome WHIM and allosteric modulation of CXCR4 functions by CXCR7 (ACKR3), the second receptor of the chemokine CXCL12. The ability to form oligomers opens many issues of CXCL12 and its interaction with CXCR4 and CXCR7 / ACKR3.The stoichiometry of this interaction still remains an open question, as the receptor is capable to form oligomers with the same receptor or other receptors, particularly CXCR7 / ACKR3. This receptor, known as scavenger, has not solved structure and the mechanism of interaction with CXCL12 is unknown.To study the interactions CXCL12 / CXCR4 / CXCR7, we applied several molecular modeling techniques such as peptide-peptide docking and molecular dynamics simulations.Objectives of this project were: the resolution of the different stoichiometric forms for the interaction of CXCR4 and CXCL12 (molecular modeling, docking and dynamic); modeling the CXCR7 / ACKR3 receptor structure and its interaction with CXCL12 (homology modeling), with the characterization of domains and residues key in the activation of downstream signaling pathways of the receptor (CXCR7 / ACKR3 mutants); the study and characterization of new innovative tools for the detection of oligomerization of these receptors in endogenous conditions. (Nanobodies, HTRF)The results of the first objective were published in January 2016: PMID 26813575.Modeling of CXCR7 / ACKR3 allowed us to generate several mutants of the receptor to test our hypothesis about the activation pathways.Nanobodies were fully characterized for CXCR4 to be used in a second study to identify oligomeric forms of the receptor in tissues and cells.

La maladie du court-noué est principalement causée en Europe par le Grapevine fanleaf virus (GFLV) et l’Arabis mosaic virus (ArMV). Le principal moyen de lutte contre sa dispersion consiste à certifier l’absence de ces virus dans les vignes commercialisées par des méthodes sérologiques tels que le DAS-ELISA. De par leurs propriétés biophysique et structurale exceptionnelles, les Nanobodies (Nb) issus des domaines variables d’immunoglobulines (Ig) composées uniquement de chaînes lourdes, se distinguent avantageusement des Ig conventionnelles. L’objectif majeur de ma thèse était d’établir un test de diagnostic du court-noué à base de Nb. À partir de deux collections de Nb contre le GFLV et l’ArMV, j’ai identifié des Nb reconnaissant un large spectre d’isolats viraux. Leur fusion à une protéine fluorescente ou à la phosphatase alcaline a conduit à l’obtention de réactifs de détection performants du GFLV et de l’ArMV par DAS-ELISA. La structure atomique d’un complexe Nb/GFLV résolue à 2.8 Å par cryomicroscopie électronique a permis de cartographier l’épitope en surface du virus et a révélé une couverture maximale de la particule virale par le Nb. La comparaison des tests Nb à des réactifs sérologiques commerciaux a révélé leur supériorité en terme de sensibilité et de spécificité, ouvrant ainsi la voie à la commercialisation d’un nouveau test de diagnostic des virus du court-noué de la vigne. / The grapevine fanleaf disease is mainly caused in Europe by the Grapevine fanleaf virus (GFLV) and the Arabis mosaic virus (ArMV). The principal mean to limit their spread, is to certify their absence in marketed grapevines by serological methods such as DAS-ELISA. Their unique biophysical and structural properties make the variable domains of heavy chain-only immunoglobulin, called Nanobodies (Nb) a real asset for the development of a diagnostic test against fanleaf disease viruses. I identified Nb able to detect a broad spectrum of viral isolates from two Nb collections against GFLV and ArMV. Their fusion to a fluorescent protein or to a bacterial alkaline phosphatase resulted in the production of efficient DAS-ELISA detection reagents. The atomic structure of a Nb/GFLV complex was solved at 2.8 Å by cryoelectron microscopy, allowing the precise mapping of the viral epitope. This result showed a maximum coverage of the viral particle by the Nb, leading to a maximal signal in DAS-ELISA. The full Nb tests against GFLV and ArMV were compared to commercial reagents and showed the superiority of the former in both sensitivity and specificity, opening the way for the development and commercialization of a new type of serological kits for the detection of grapevine viruses.

Em caso de envenenamento ofídico, o tratamento no Brasil hoje é realizado pela administração de soros geralmente produzidos por equinos, que apresentam eficácia limitada: são úteis para os efeitos sistêmicos, mas não inibem efetivamente a evolução dos danos locais, podem causar reações adversas e apresentam alto custo de produção. De acordo com a Organização Mundial da Saúde (OMS), trata-se de uma doença negligenciada pelas autoridades científicas mundiais. O presente projeto, em parceria com o Instituto de Pesquisas em Patologias Tropicais da Fundação Oswaldo Cruz - Rondônia, propõe a produção por Escherichia coli de fragmentos de anticorpos de cadeia pesada de camelídeos, denominados VHH, contra as toxinas do veneno de Bothrops jararacussu, utilizando biorreator. Neste trabalho há interesse em produzir VHH, através da otimização do crescimento desta E. coli. A cinética do crescimento bacteriano foi realizada em shaker orbital sob diferentes condições, variando tamanho do frasco, rotação do shaker, composição do meio de cultura e concentração de substrato; e em biorreatores, alternando meios de cultura e modo de operação do reator (descontínuo e descontínuo alimentado), alterando a vazão de alimentação (linear e exponencial) O processo cinético é fortemente limitado pela formação de acetato, por condições auxotróficas da célula e pela transferência de oxigênio. Nos ensaios em frascos agitados, uma melhor condição de crescimento foi obtida utilizando frascos de 1 L, sob rotação a 270 rpm e 5,0 g/L de glicose. Nos ensaios em reator, quando operados em batelada obtiveram-se cerca 5,5 g/L de células finais, contra 9,3 g/L de células em batelada alimentada com vazão constante. Um maior crescimento foi ainda obtido em um reator de 2 L em regime de batelada alimentada exponencialmente. O biorreator varia a agitação do meio e mantém um nível pré-definido de oxigênio dissolvido, evitando a limitação de oxigênio e controlando a oferta de glicose para o crescimento celular. Neste processo, atingimos 25,6 g/L de células e 0,35 g/L de proteína total após purificação, utilizando meio M9 suplementado. / Nowadays in Brazil, the treatment for snakebite poisoning is carried out by the administration of horse sera, which have limited effectiveness: they are useful for systemic effects, but do not effectively inhibit local damage, cause adverse reactions, and present high production costs. According to the WHO, this disease is neglected by the world`s scientific authorities. This project, in partnership with the Institute for Research in Tropical Diseases of Oswaldo Cruz Foundation - Rondonia, proposes the production of heavy chain antibody fragments from camelids, called VHH, using Escherichia coli, to be used against the toxins from the Bothrops jararacussu poison, using a bioreactor. This work is interested in producing VHH through the use of E. coli. The kinetics of bacterial growth were performed in orbital shaker under different conditions, varying vial size, shaker rotation, composition of the culture medium and substrate concentration; and bioreactors, alternating culture media and operation mode of reactor (batch and fed-batch), changing feeding rate (linear and exponential). The kinetic process is statically bound to acetate formation, auxotrophic conditions of the cell and oxygen transfer. In assays in shaken flasks, an output of 270 rpm and 5.0 g/L of glucose. In reactor runs, when operated in a batch 5.5 g/L of final cells were obtained, against 9.3 g/L of final cells in fed-batch with constant flowrate. A larger value was obtained in an exponentially fed-batch reactor of 2 L. The bioreactor varies the agitation of oxygen and controls glucose addition for cell growth. In this process, 25.6 g/L cells and 0.35 g/L total protein after purification were reached, using supplemented M9 medium.

Em caso de envenenamento ofídico, o tratamento no Brasil hoje é realizado pela administração de soros geralmente produzidos por equinos, que apresentam eficácia limitada: são úteis para os efeitos sistêmicos, mas não inibem efetivamente a evolução dos danos locais, podem causar reações adversas e apresentam alto custo de produção. De acordo com a Organização Mundial da Saúde (OMS), trata-se de uma doença negligenciada pelas autoridades científicas mundiais. O presente projeto, em parceria com o Instituto de Pesquisas em Patologias Tropicais da Fundação Oswaldo Cruz - Rondônia, propõe a produção por Escherichia coli de fragmentos de anticorpos de cadeia pesada de camelídeos, denominados VHH, contra as toxinas do veneno de Bothrops jararacussu, utilizando biorreator. Neste trabalho há interesse em produzir VHH, através da otimização do crescimento desta E. coli. A cinética do crescimento bacteriano foi realizada em shaker orbital sob diferentes condições, variando tamanho do frasco, rotação do shaker, composição do meio de cultura e concentração de substrato; e em biorreatores, alternando meios de cultura e modo de operação do reator (descontínuo e descontínuo alimentado), alterando a vazão de alimentação (linear e exponencial) O processo cinético é fortemente limitado pela formação de acetato, por condições auxotróficas da célula e pela transferência de oxigênio. Nos ensaios em frascos agitados, uma melhor condição de crescimento foi obtida utilizando frascos de 1 L, sob rotação a 270 rpm e 5,0 g/L de glicose. Nos ensaios em reator, quando operados em batelada obtiveram-se cerca 5,5 g/L de células finais, contra 9,3 g/L de células em batelada alimentada com vazão constante. Um maior crescimento foi ainda obtido em um reator de 2 L em regime de batelada alimentada exponencialmente. O biorreator varia a agitação do meio e mantém um nível pré-definido de oxigênio dissolvido, evitando a limitação de oxigênio e controlando a oferta de glicose para o crescimento celular. Neste processo, atingimos 25,6 g/L de células e 0,35 g/L de proteína total após purificação, utilizando meio M9 suplementado. / Nowadays in Brazil, the treatment for snakebite poisoning is carried out by the administration of horse sera, which have limited effectiveness: they are useful for systemic effects, but do not effectively inhibit local damage, cause adverse reactions, and present high production costs. According to the WHO, this disease is neglected by the world`s scientific authorities. This project, in partnership with the Institute for Research in Tropical Diseases of Oswaldo Cruz Foundation - Rondonia, proposes the production of heavy chain antibody fragments from camelids, called VHH, using Escherichia coli, to be used against the toxins from the Bothrops jararacussu poison, using a bioreactor. This work is interested in producing VHH through the use of E. coli. The kinetics of bacterial growth were performed in orbital shaker under different conditions, varying vial size, shaker rotation, composition of the culture medium and substrate concentration; and bioreactors, alternating culture media and operation mode of reactor (batch and fed-batch), changing feeding rate (linear and exponential). The kinetic process is statically bound to acetate formation, auxotrophic conditions of the cell and oxygen transfer. In assays in shaken flasks, an output of 270 rpm and 5.0 g/L of glucose. In reactor runs, when operated in a batch 5.5 g/L of final cells were obtained, against 9.3 g/L of final cells in fed-batch with constant flowrate. A larger value was obtained in an exponentially fed-batch reactor of 2 L. The bioreactor varies the agitation of oxygen and controls glucose addition for cell growth. In this process, 25.6 g/L cells and 0.35 g/L total protein after purification were reached, using supplemented M9 medium.

Viral infections are constantly increasing and impose a large threat to the public health. Alphaviruses are responsible for several animal and human diseases and have a large medical importance with few treatments available today. Alphaviruses are small, spherical single stranded RNA viruses, and are most often transmitted by mosquito vectors. Alphaviruses contains a domain of nonstructural proteins that compose the replication machinery. The domain is crucial for viral replication to occur and is therefore an interesting target for antiviral therapy. With the focus on Chikungunya and Semliki Forest Virus this work investigates the events in the cells on molecular level during infections. To do this a panel of Camelid derived single domain antibodies are developed to target the nonstructural proteins of Chikungunya and Semliki Forest Virus. Binding of the produced nanobodies to the viral proteins was investigated by biochemical methods including immunoprecipitations, western blot, and ELISA. Cell lines that express nsP-specific nanobodies in the cytosol were employed for infection- and plaque assays with Semliki Forest Virus in order to determine the antiviral potential of the new nanobodies. Three of the nanobodies proved to bind two different nonstructural proteins of the viruses, providing opportunities for further investigations and a possible use of these nanobodies to identify viral vulnerabilities that could be exploited for antiviral intervention.

Early detection of illness is essential in preventing symptoms from escalating and infectious diseases from spreading. Electrochemical biosensors are a promis- ing tool in healthcare detection. Previously, the collaboration between the Arold and Inal labs has led to the design of organic electrochemical transistors (OECT) capable of rapidly detecting coronavirus in saliva by using nanobody constructs as biorecognition units. In this project, I aimed to prove the versatility of nanobody- functionalized OECT biosensors in detecting other relevant viruses, specifically, Zika and Dengue. Both viruses pose a risk to multiple populations around the world, including the Kingdom of Saudi Arabia. I designed and produced nanobod- ies that are reported to bind to the NS1 glycoprotein, which is released by Zika and Dengue into the blood of the patient. Then, I confirmed the binding of the nanobodies to their associated targets. I also developed a robotic liquid handling script to automate the biosensing operations. Ultimately, this project aims to support the design of a multiplex OECT biosensor for blood-borne pathogens.

Les traitements actuels dirigés contre le VIH ne sont que partiellement efficaces en raison de l'apparition de mutations qui confèrent au virus une grande capacité de résistance aux antirétroviraux existants. Un moyen d'améliorer la lutte contre le virus consiste par conséquent à trouver de nouvelles stratégies d'inhibition. Le complexe de reverse transcription est une des principales cibles pour le développement de traitement anti-SIDA, il catalyse une étape obligatoire du cycle de réplication du virus. Cependant, l'ensemble des inhibiteurs de la transcriptase inverse sont limités par l'apparition rapide de souches résistances. Dans ce contexte, mes travaux de thèse ont permis de développer des inhibiteurs ciblant spécifiquement la reverse transcriptase (RT) du VIH-1, basée sur des fragments d'anticorps dérivés des anticorps chaînes lourdes de dromadaire appelés VHHs ou encore Nanobodies. Associé à une stratégie de vectorisation non invasive basée sur l'utilisation de peptides vecteurs pénétrants, les Nanobodies ont été délivré efficacement dans les cellules et par conséquent ils présentent tous une forte activité antivirale de l'ordre du nanomolaire. L'étude du mécanisme d'action du Nanobody leader NbRT20 montre qu'il agit en tant qu'inhibiteur conformationnel. Il interagit avec la forme intermédiaire inactive de la RT et empêche la mobilité du sous-domaine thumb requis pour le positionnement correct de la matrice/amorce sur la RT et inhibant l'incorporation des nucléotides dans la chaîne d'ADN naissante déstabilisant l'enzyme dans une conformation inactive, non processive. Pris ensemble, ces résultats montrent que la plate-forme Nanobody peut être très efficace pour générer des intracorps extrêmement puissants et sélectifs pour neutraliser la RT et la réplication virale.Mots clés : HIV-1, RT, Nanobodies, peptide vecteur pénétrant / The rapid emergence of drug-resistant viruses against all approved HIV clinical drugs together with inaccessible latent virus reservoirs and side effects of currently used compounds have limited the potency of existing anti-HIV-1 therapeutics. Therefore, there is a critical need for new safer drugs, active against resistant viral strains. Reverse transcriptase (RT) plays an essential role in the replication of human immunodeficiency virus type 1 (HIV-1) and remains a primary target of anti-HIV-1 drugs. To develop specific HIV inhibitors, we have elaborated a new strategy based on short antibody fragments derived from the unique Heavy-chain antibodies present in Camelidae called Nanobodies that targets RT-activation. The immunization of dromedaries with RT has lead to the isolation of a panel of Nanobodies that tightly bind the two subunits of RT and inhibit its DNA-dependent DNA polymerase activity at nanomolar range. From that screen we have elaborated an intrabody (cell penetrating anti-RT Nanobody) NbRT20 that constitutes a potential interesting anti-HIV compound.We demonstrated that NbRT20 inhibits RT polymerase activity and exhibiting a potent antiviral activity with a subnanomolar IC50. NbRT20 binds the thumb subdomain and restricts its flexibility and mobility resulting in an inactive/non processive dimeric conformation of the enzyme. From a mechanistic point of view, we have showed that NbRT20 is a conformational inhibitor. it prevents proper binding of primer/template and of dNTP and destabilizes the enzyme in an inactive/non processive dimeric conformation.Taken together, these results demonstrated that, the Nanobody platform may be highly effective at generating extremely potent and selective intrabody to neutralize RT and HIV proliferation.Key words: HIV-1, RT, Nanobodies, cell penetrating peptide

The myc family of oncogenic transcription factors, which includes c-myc, N-myc and L-myc, control major cellular processes such as proliferation and differentiation by integrating upstream signals and orchestrating global gene transcription. They do this largely through dimerising with Max, which together bind to enhancer (E)-box elements in DNA. Myc proteins function similarly but differ in potency and tissue distribution. For instance, N-myc is expressed predominantly during development in undifferentiated cells of the nervous system, whereas c-myc is ubiquitously expressed in all proliferating cells. Myc proteins, when deregulated, are major drivers of tumourigenesis. Myc deregulation occurs in up to 70% of all human cancers and is often associated with the most aggressive forms. For example, MYCN, the gene encoding N-myc, is amplified in 20-30% of neuroblastomas, and amplification strongly correlates with advanced stage and poor prognosis. Myc proteins are therefore considered “most wanted” targets for cancer therapy, but have long been considered undruggable mainly due to challenges in nuclear drug delivery and physically targeting myc directly given that it is a largely disordered protein that lacks discernible clefts and pockets for small molecules to inhabit. Furthermore, c-myc is important in normal tissue maintenance so the effect of its inhibition in humans is difficult to predict. However, recent in vivo studies showed that systemic myc inhibition (using the peptide pan myc inhibitor Omomyc) has mild and reversible side effects and induces tumour regression. This has alleviated concerns about the side effects that myc inhibition might have, and reinforced the promise of myc as a powerful drug target. However, the translation of Omomyc into the clinic has been hindered by poor cellular delivery. In fact, no direct myc inhibitor has yet been approved, indicating that novel approaches are needed. Moreover, inhibitors in development tend to inhibit all myc family proteins. An inhibitor that could specifically target N-myc might improve safety through bypassing c-myc inhibition. This could be used for the treatment of N-myc-driven cancers such as MYCN-amplified neuroblastoma. Nanobodies, camelid-derived single-domain antibodies, are a relatively new drug class. Whilst some are already in clinical trials for a wide range of diseases, these are specific for cell-surface or extracellular targets. However, their properties also make them ideal for use as intracellular antibodies or ‘intrabodies’. For example, they are small (just 12-15 kDa) and highly soluble due to naturally occurring hydrophobic to hydrophilic amino acid substitutions. Their small size and convex shape makes them advantageous in capturing structures in intrinsically disordered proteins and allows them to reach hidden epitopes not accessible to conventional antibodies, which could improve biological activity. Importantly, nanobodies retain the high specificities and affinities of conventional antibodies. Their small, single-domain nature also means they can be engineered with ease to modify aspects of their localisation and/or function. For example, they can be coupled to carrier molecules to facilitate cellular entry, and a nuclear localisation signal (NLS) can be added to drive them into the nucleus. Also, it was recently shown that an F-box domain could also be incorporated into nanobodies to recruit degradation machinery to its antigen, which depletes the antigen from cells via the proteasomal degradation pathway. Due to their highly advantageous properties, nanobodies raised against N-myc might overcome the barriers to targeting N-myc, providing potent and specific means of directly inhibiting N-myc therapeutically, which has not yet been achieved. In this thesis, nine unique nanobodies were raised against N-myc. These included three against the basic helix-loop-helix leucine zipper (bHLH-LZ) domain where Max dimerises, and six against the transactivation domain where numerous regulatory and cofactor proteins bind, such as the E3 ubiquitin ligase Skp2. Nanobodies against the transactivation domain were more specific for N-myc and were shown to inhibit its Skp-2-mediated ubiquitylation. This could provide novel means of eradicating tumours based on a study showing that inhibition of ubiquitylation at this domain triggers a transcriptional ‘switch’ that induces a non-canonical target gene Egr1, leading to p53-independent apoptosis. A nanobody against the bHLH-LZ (Nb C2) was shown to bind both N- and c-myc to similar magnitudes. Its affinity for N-myc bHLH-LZ was superior to that of the small molecule myc inhibitor 10058-F4, which prolongs survival in a MYCN-dependent mouse model of high-risk neuroblastoma. Nb C2 spontaneously transduced cell membranes and its coupling to a novel small molecule carrier (SMoC) enhanced its cellular uptake. Furthermore, the addition of a NLS increased its nuclear localisation. Preliminary experiments showed that Nb C2 might slow proliferation and induce apoptosis in cancer cell lines expressing c-myc, suggesting that Nb C2 might also be effective against cancers characterised by deregulated c-myc. Taken together, data generated in this thesis have revealed intriguing findings that provide a basis for the development of these nanobodies for the treatment of N-myc- and c-myc-driven cancers.

CXCR3 appartient à la famille des récepteurs couplés aux protéines G. Avec ses ligands, les chimiokines CXC, CXCR3 régule diverses fonctions biologiques et participe à de nombreux processus comme l’angiogenèse, l’inflammation et le cancer. La complexité de CXCR3 provient de son épissage alternatif qui conduit à des isoformes distinctes. CXCR3-A est reconnu pour promouvoir la prolifération, la survie et la migration cellulaire tandis que CXCR3-B induit des signaux inhibiteurs de la croissance cellulaire.Le modèle cellulaire U87, dérivé d’un glioblastome humain, a été utilisé afin d’étudier les mécanismes moléculaires régulant l'activité et le trafic des isoformes de CXCR3 dans les cellules tumorales. CXCR3 est le récepteur fonctionnel de l’activité angiostatique de CXCL4 et son variant CXCL4L1. En fonction de leur état d'oligomérisation, ces deux chimiokines ont des interactions préférentielles avec les isoformes de CXCR3. L’activation de CXCR3-A conduit à un important changement conformationnel et induit des voies de signalisation pro-migratoires. L’étude du trafic souligne l’importance de la clathrine et du réseau Trans-Golgi dans l’internalisation et le recyclage de CXCR3-A. Pour la première fois, LRP-1 a été identifié comme nouveau partenaire de CXCR3-A. LRP1 n’est pas seulement reconnu comme un récepteur de l’endocytose mais également comme une protéine de la signalisation. LRP1 interagit avec CXCR3-A au niveau extracellulaire et régule sa conformation, son trafic et son activité pro-tumorale.L'utilisation de modèles cellulaires d'adénocarcinome pancréatique a permis de caractériser CXCL4L1 comme facteur pro-tumoral, via l’activation de CXCR3-A dans les cellules tumorales. CXCL4L1 apparait pour la première fois comme un biomarqueur important dans la progression du cancer pancréatique.Dans les différents modèles, les signalisations chimiokines CXC/CXCR3-A induisent une augmentation des propriétés invasives tumorales. Au niveau moléculaire, l’association de CXCR3 à diverses protéines (ligands et partenaires) est essentielle pour réguler les fonctions biologiques de la cellule tumorale.Les nanoparticules sont désormais connues comme une nouvelle génération d'anticorps thérapeutiques présentant de nombreux avantages par rapport aux anticorps conventionnels. Ainsi, le développement de nanoparticules associées à des inhibiteurs de CXCR3 apparaît comme une nouvelle stratégie thérapeutique anti-tumorale prometteuse. / CXCR3 belongs to the G-protein-coupled receptors family. With its ligands, the CXC chemokines, CXCR3 regulates several biological functions and plays important roles in angiogenesis, inflammation and cancer. The interaction with CXCR3 is rather complex due to the presence of distinct spliced isoforms. CXCR3-A is known to promote cell proliferation, survival, and migration while CXCR3-B leads to cell growth inhibition.The human glioblastoma cell model, U87, was used to study the molecular mechanisms regulating the activity and trafficking of CXCR3 isoforms in tumor cells. CXCR3 has been reported as the functional receptor for the angiostatic activity of CXCL4 and its variant CXCL4L1. Depending on their oligomerization status, these two chemokines present preferential interaction with CXCR3 isoforms. Activation of CXCR3-A leads to an important conformational change and induces pro-migratory signaling pathways. Studies on the vesicular trafficking highlight the importance of the clathrin and the Trans-Golgi network for both internalization and recycling of CXCR3-A. For the first time, LRP-1 is identified as a new partner of CXCR3-A. LRP1 is not only recognized as an endocytic receptor but also as a signaling protein. LRP1 interacts with CXCR3-A via its extracellular α subunit and regulates CXCR3-A conformation, trafficking and pro-tumoral activity.Pancreatic ductal adenocarcinoma cell models were used to characterize CXCL4L1 as a pro-tumoral factor that activates CXCR3-A in tumor cells. For the first time, CXCL4L1 appears as an important biomarker for pancreatic cancer progression.In the different cell models, signaling pathways of CXC chemokine/CXCR3-A lead to an increase in tumor invasive properties. At the molecular level, the association of CXCR3 with various proteins (ligands and partners) is essential to regulate tumor cell biological functions.The nanoparticles are now known as a new generation of therapeutic antibodies with many advantages over conventional antibodies. Thus, the development of nanoparticles associated to CXCR3 inhibitors appears as a new promising pharmacological targeted strategy to treat cancer.