La tuberculose (TB) est un problème de santé mondiale majeur à l’origine de 10.4 millions de nouveaux cas et 1.8 millions de morts en 2015 selon l’Organisation Mondiale de la Santé (OMS). Cette maladie est causée par la bactérie Mycobacterium tuberculosis (Mtb) qui infecte principalement les poumons et se transmet par l'inhalation d’aérosols contaminés.Le traitement de TB nécessite la prise quotidienne d’antibiotiques pendant 6 mois, dont une mauvaise utilisation peut être à l’origine de l’apparition de souches Mtb multi-résistantes.La nouvelle stratégie de l’OMS, “End TB”, vise à réduire de 90% l’incidence de TB d'ici 2035. Pour y parvenir, il est important de définir de nouvelles approches pour réduire la durée et la toxicité des traitements et améliorer leur efficacité vis-à-vis des bactéries actives et latentes.L’approche abordée lors de ma thèse vise à utiliser des nanoparticules (NP) pour développer de nouvelles thérapies anti-TB. La bibliographie sur le sujet montre que cela pourrait être une stratégie prometteuse. Nous avons par conséquent étudié quatre applications potentielles des NP:1-Vectorisation des médicaments pour les administrer au niveau pulmonaire. L’éthionamide (ETH) est un antibiotique utilisé pour le traitement de TB avec des effets secondaires indésirables. L’ETH est une «pro-drogue» qui nécessite une activation par une monooxygenase bactérienne, dont l’efficacité peut être elle-même augmentée par des molécules chimiques appelées “booster”. Nous avons étudié l’effet de l’ETH et de booster co-encapsulés dans des NP de poly-β-cyclodextrine (pCD) pour le traitement de TB. Nous avons d’abord évalué leur efficacité in vitro sur la croissance extracellulaire et intracellulaire (dans les macrophages) de Mtb grâce à l'utilisation d'un système automatisé de microscopie confocale à haut contenu. Dans les deux essais, nous avons constaté que les médicaments conservaient leur activité après encapsulation et que les NP n'étaient pas cytotoxiques. L’efficacité de ces NP a ensuite été étudiée in vivo chez des souris infectées avec Mtb. La suspension de NP a été délivrée sous forme d’aérosols directement dans les poumons par voie endotrachéale à l’aide d’un Microsprayer Aerosolizer. Une réduction significative de la charge bactérienne dans les poumons de 3 log a été observée après 6 administrations de doses inférieures à celles thérapeutiques.2-Amélioration de la solubilité et biodisponibilité des antibiotiques. La Clofazimine (CLZ) est un antibiotique utilisé dans le traitement de la lèpre et pourrait être, au regard de son efficacité in vitro sur les souches de Mtb multi-résistantes, un candidat potentiel pour celui de TB. La CLZ est extrêmement lipophile, gênant ainsi sa solubilité. Dans notre étude, son encapsulation dans des particules de silice nanoporeuses a stabilisé l'état amorphe de la CLZ et a augmenté radicalement sa solubilité. Après encapsulation ou solubilisation dans le DMSO, la CLZ a d’autre part montré une activité antibactérienne similaire sur Mtb.3-Stabilisation des antibiotiques. La Vancomycine (VAN) est utilisée pour des applications cliniques comme alternative de la pénicilline dans le traitement de Staphylococcus aureus et pourrait être utilisée pour celui de TB. Alors que la VAN présente une faible stabilité dans les milieux biologiques, nous avons montré que l'encapsulation de cet antibiotique à l'intérieur de NP à base de PLGA a amélioré son efficacité tant sur les bactéries Mtb extracellulaires qu’intracellulaires.4-Activité antimycobacterial Intrinsèque de NP. Différentes NP (60 pCD, 1 NanoMOF et 1 NP en argent) ont été évaluées in vitro. Aucune n'a présenté d'activité antituberculeuse intrinsèque prometteuse.En conclusion et au regard des options thérapeutiques limitées pour combattre les souches résistantes et de la rareté de solutions innovantes dans le pipeline de découverte de médicament, ces travaux ont montré que les NP pouvaient constituer une approche anti-TB originale. / Tuberculosis (TB) is a major problem of global health, responsible for 10.4 million new cases and 1.8 million deaths in 2015 according to the World Health Organization (WHO). This disease is caused by inhalation of small aerosol droplets containing Mycobacterium tuberculosis (Mtb), and lungs are usually the major site of infection.TB can usually be treated with a daily six months course of standard, or first-line, anti-TB drugs. If first-line drugs are misused, the onset of multidrug-resistant Mtb can occur.The new WHO global public health strategy “End TB” aims at the reduction of TB incidence 90% by 2035. To reach these ambitious targets, new approaches are urgently needed to get a faster, less harmful and more-efficient treatment for active and latent TB.My thesis focused on the use of nanoparticles (NPs) to develop new anti-TB therapies. Our review of the literature showed that it could be a promising approach. Here, we investigated four potential uses of the NPs.1- Nanocarrier for pulmonary delivery of drugs. Ethionamide (ETH) is a second line antibiotic with high toxicity and several adverse side effects. ETH is a prodrug that requires bioactivation by a bacterial monooxygenase, which can be enhanced by chemical molecules named “boosters”. We investigated the simultaneous delivery of ETH and boosters coencapsulated in biodegradable poly-β-cyclodextrin (pCD) based NPs by the pulmonary route for the treatment of TB. First, we evaluated the in vitro efficacy of the designed formulations on Mtb extracellular growth and intracellular growth inside macrophages using an automated confocal high-content microscopy system. And we found for both assays that the drugs maintained their activity after encapsulation and the pCD were not cytotoxic. Given these promising results, their efficacy was then tested in vivo. The NPs suspension, administered directly into mouse lungs by endotracheal way using a Microsprayer® aerosolizer, was proved to be well-tolerated and led to a 3-log decrease of the pulmonary mycobacterial load after 6 administrations and using lower doses than the therapeutic ones.2- Enhancement of the solubility and the bioavailability of antibiotics. Clofazimine (CLZ) is an antibiotic usually used in a combination therapy for the treatment of leprosy and could be a potential candidate for the treatment of TB because of its in vitro efficacy on resistant Mtb strains. CLZ is extremely lipophilic and has important solubility problem. In our study, its encapsulation in nanoporous silica particles stabilized the amorphous state of CLZ and dramatically increased the drug solubility. On the other hand, CLZ encapsulated in nanoporous silica particles or efficiently dissolved in DMSO showed a similar antibacterial activity on Mtb, validating the assessment of solubility of CLZ by encapsulation.3- Improvement of the antibiotic stabilization. Vancomycin (VAN) is used for clinical applications for nearly 50 years as a penicillin alternative to treat penicillinase-producing strains of Staphylococcus aureus. VAN can be used for TB treatment as a repurpose. While VAN presented low stability in biological media at 37°C, we showed that the encapsulation of this antibiotic inside PLGA-based NPs enhanced its efficacy both on extracellular and intracellular bacteria.4- Intrinsic antimycobacterial activity of NPs. Different NPs (60 pCD, 1 NanoMOF, and 1 silver NP) were tested in vitro but none presented promising intrinsic antitubercular activity. However some pCD were slightly active in vitro on extracellular Mtb but cytotoxic.In conclusion, these works demonstrated that nanoparticles can provide a novel anti-TB approach regarding the limited therapeutic options to fight drug-resistant Mtb and the scarcity of novel antituberculosis drugs in the drug discovery pipeline.

abstract: Euendolithic cyanobacteria have the remarkable ability to actively excavate and grow within certain minerals. Their activity leads to increased erosion of marine and terrestrial carbonates, negatively affecting coral reef and bivalve ecology. Despite their environmental relevance, the boring mechanism has remained elusive and paradoxical, in that cyanobacteria alkalinize their surroundings, typically leading to carbonate precipitation, not dissolution. Thus, euendoliths must rely on unique adaptations to bore. Recent work using the filamentous model euendolith Mastigocoleus testarum strain BC008 indicated that excavation relied on transcellular calcium transport mediated by P-type ATPases, but the phenomenon remained unclear. Here I present evidence that excavation in M. testarum involves an unprecedented set of adaptations. Long-range calcium transport is achieved through the coordinated pumping of multiple cells, orchestrated by the localization of calcium ATPases in a repeating annular pattern, positioned at a single cell pole, adjacent to each cell septum along the filament. Additionally, specialized chlorotic cells that I named calcicytes, differentiate and accumulate calcium at concentrations more than 500 fold those of canonical cells, likely allowing for fast calcium flow at non-toxic concentrations through undifferentiated cells. I also show, using 13C stable isotope tracers and NanoSIMS imaging, that endolithic M. testarum derives most of its carbon from the mineral carbonates it dissolves, the first autotroph ever shown to fix mineral carbon, confirming the existence of a direct link between oxidized solid carbon pools and reduced organic pools in the biosphere. Finally, using genomic and transcriptomic approaches, I analyze gene expression searching for additional adaptations related to the endolithic lifestyle. A large and diverse set of genes (24% of 6917 genes) were significantly differentially regulated while boring, including several master regulators and genes expectedly needed under this condition (such as transport, nutrient scavenging, oxidative stress, and calcium-binding protein genes). However, I also discovered the up-regulation of several puzzling gene sets involved in alternative carbon fixation pathways, anaerobic metabolism, and some related to photosynthesis and respiration. This transcriptomic data provides us with several new, readily testable hypotheses regarding adaptations to the endolithic lifestyle. In all, my data clearly show that boring organisms show extraordinarily interesting adaptations. / Dissertation/Thesis / Doctoral Dissertation Microbiology 2016

La plus grande famille de récepteurs membranaires est constituée par des récepteurs à sept domaines transmembranaires couplés aux protéines G (RCPG). Ces récepteurs sont impliqués dans un grand nombre de réponses cellulaires physiologiques et pathologiques et représentent la ciblé de une grande majorité des produits thérapeutiques. La fonction d’un récepteur est déterminée par la quantité de récepteur fonctionnel à la surface cellulaire, qui dépend de différents paramètres comme le niveau de biosynthèse, l’export vers la surface cellulaire à partir de stocks intracellulaires, l’endocytose et les modifications post-transcriptionelles (ex. phosphorylation). Le nouveau concept d’export régulé pour les RCPG présent l’importance physiologique de la rétention de récepteurs, leur relargage, leur interaction avec les partenaires chaperonnes et les escortes. Les études présentées ici concernent les mécanismes d’export régulé de deux RCPG, le récepteur métabotropique de l’acide γ-amino butyrique (GABAB) et le récepteur de chimiokines CC 5 (CCR5). GABAB est un récepteur constitué de deux sous-unités GB1 et GB2 et CCR5 est probablement un homo-dimer. GB1 ainsi que CCR sont retenus dans des compartiments intracellulaire (RE et appareil Golgi) d’où ils sont relâchés en réponse à un signal extern (CCR5) ou/et en interagissant avec protéines d’escorte (comme CD4 pour CCR5 et GB2 pour GB1). L’objectif de ces études était de comprendre le mécanisme de rétention de ces récepteurs et leur régulation. Dans ce contexte, nous avons déterminé en utilisant des approches biophysiques et biochimiques que ces récepteurs interagissent de façon spécifique avec les membres de Prenylated Rab Acceptors Family (PRAF). Ces protéines sont résidentes dans le RE (PRAF2 et PRAF3) et dans le appareil Golgi (PRAF1) où elles fonctionnent comme de gatekeepers pour les récepteurs. Nous avons pu démontrer que PRAF2 interagie de manière spécifique avec des motifs de rétention connus pour leur implication dans la rétention de récepteurs. Cette interaction détermine une rétention au niveau de RE donc régule de façon négatif l’export vers la membrane cellulaire. Dans le cas de récepteur GABAB, l’interaction de GB2 avec GB1 permet la libération de GB1 de sa rétention par PRAF2 par simple compétition. La modification de l’équilibre stoichiométrique entre les gatekeepers PRAF et les protéines d’escorte pour les récepteurs induit des modifications de la fonction du récepteur in vitro et in vivo. Les PRAFs sont ubiquitaires et peuvent interagir avec plusieurs RCPG représentant dans ce cas des régulateurs majors de la fonction de RCPG dans des conditions physiologiques et pathologiques. / The largest family of membrane receptors is constituted by conserved seven-membrane domain spanning receptors, the G-protein coupled receptors (GPCRs). They are involved in numerous cell responses and diseases thus being a major drug target. Receptor function is determined by the amount of active receptors at the cell surface, which depends on various parameters, such as the biosynthetic rate, the export to the cell surface from internal stores, the endocytosis and post-transcriptional modifications (i.e. phosphorylation). Only recently, the importance of the regulated export has emerged, shedding new light on the physiological role of receptor retention, release, chaperoning and escorting. This work concerns the regulated export mechanisms of two members of the GPCRs family, the chemokine receptor 5 (CCR5) and the metabotropic receptor of the g amino butyric acid (GABAB). Whereas CCR5 is likely a homo-dimer of 2 identical protomers, GABAB is an obligatory hetero-dimer of 2 distinct subunit known as GB1 and GB2. Both CCR5 and GB1 are retained in intracellular compartments (the ER and the Golgi) from which they are released in response to external signals (CCR5) and/or interaction with “private escort proteins” (CD4 for CCR5 and GB2 for GB1). The main goal of our work was to understand the mechanism of retention of these receptors and its regulation. In this context, we determined using biochemical and biophysical approaches that these GPCRs specifically interact with the members of the Prenylated Rab Acceptor Family (PRAF). These proteins are resident either in the ER (PRAF2 and PRAF3) or in the Golgi apparatus (PRAF1) where they function as receptor gatekeepers. Indeed, we could document for PRAF2 that this protein likely interacts directly with previously identified receptor retention motifs and inhibits receptor egress from the ER and subsequent trafficking to the plasma membrane. In the context of the GABAB receptor, PRAF2-dependent retention of GB1 can be overridden by GB2 via simple competition. Perturbing the stoichiometry of PRAF gatekeepers respective to that of receptors significantly perturbs receptor function both in vitro and in vivo. Because PRAFs are ubiquitous and seem to interact with many other GPCRs, they might represent major regulators of receptor function both in physiological and pathological conditions.

Os canais de junções comunicantes (gap junctions, JC) são formados por subunidades chamadas de conexinas (Cx). Estas proteínas têm papel relevante no acoplamento celular, participando da condutância de células nervosas ou gliais e modulando vários processos fisiológicos e fisiopatológicos. O objetivo deste trabalho é avaliar o envolvimento das conexinas na nocicepção aguda, por meio de ensaios comportamentais e estudos de mapeamento da sua expressão em células do gânglio da raiz dorsal de ratos. Para tanto, foi analisado o efeito de carbenoxolone (CBX) e quinina (bloqueadores de JCs), assim como de oligonucleotídeos antisense para conexinas 36 e 43 na indução e manutenção de hiperalgesia induzida por carragenina em ratos. Os resultados mostraram que a carragenina induz uma diminuição do limiar nociceptivo em ratos e que esse efeito hiperalgésico da carragenina foi bloqueado pelo tratamento com carbenoxolone (nas doses 20-50mg) e significantemente inibido por quinina (nas doses 20-50mg), sugerindo uma participação das junções comunicantes (JC) no processo. / Gap junctions channels (GJ) are formed by proteic subunits called connexins (Cx). These proteins have an important role in cellular coupling, participating in the conductance of glial and nerve cells or modulating various physiological and pathophysiological processes. The aim of this study is to evaluate Cx36 and Cx43 involvement in acute nociception through behavioral assays, mapping studies of its expression in rat dorsal root ganglion cells. For this purpose, we analyzed the effect of intrathecal treatment with carbenoxolone (CBX) and quinine (GJs blockers), as well as antisense oligonucleotides for connexins 36 and 43 in the induction and maintenance of carrageenan-induced hyperalgesia in rats. The results show that carrageenan induces a nociceptive threshold decrease in rats. The hyperalgesic effect was blocked by treatment with carbenoxolone (20-50mg doses), Cx43 antisense and inhibited significantly by quinine (at doses 20 -50mg) but no with Cx36 antisense, suggesting an involvement of gap junctions (JC) in the process.

Neste trabalho apresentamos um modelo teórico que busca descrever aspectos do movimento bidirecional apresentado por objetos intracelulares (vesículas, organelas, vírus etc, aos quais iremos nos referir simplesmente como (\"vesículas\"), observado, sobretudo em experimentos in vivo. Este movimento nao-difusivo e caracterizado por inversões rápidas em sua direção e é capaz de gerar gradientes de concentração do objeto transportado. Os fenômenos de transporte intracelular são sabidamente mediados por proteínas motoras (como as kinesinas e dinenas) cujo movimento unidirecional sobre _lamentos protéicos e bem caracterizado (kinesinas se movem em direção a extremidade mais enquanto as dinenas se movem em direção a extremidade-menos dos microtúbulos) e é normalmente entendido através de modelos estocásticos que descrevem o comportamento de uma partícula browniana na presença de um potencial assimétrico que varia no tempo (ver Astumian [26], Adjari e Prost [22], Magnasco [23]). Mais recentemente, surgiram na literatura trabalhos que tentam descrever o movimento de partículas motoras interagentes, uma vez que se percebeu que efeitos coletivos que surgem nestas situações podem ser relevantes para os fenômenos de transporte sobre microtúbulos. Uma abordagem para a descrição do comportamento destes sistemas de partículas motoras interagentes é aquela baseada nos modelos para os sistemas difusivos dirigidos\". Em particular, a versão contínua dos modelos do tipo totally asymmetric exclusion processes\" (TASEP) e asymmetric exclusion processes\" (ASEP) tem sido utilizada para o estudo do comportamento da densidade de motores sobre os microtúbulos, através da analise de soluções estacionarias da equação de Burgers correspondente (Parmeggiani et al. [33]). Até agora, entretanto, não existem na literatura tentativas de abordar, com estes modelos, o transporte bidirecional de vesículas mediado por estes motores interagentes. A idéia que apresentamos aqui é associar este estranho tipo de movimento ao movimento de ondas de choque presentes nas soluções transientes da equa_c~ao de Burgers para algumas condições iniciais. Deste modo, as vesículas acompanhando (\"surfando\") os choques fariam o papel de suas correspondentes microscópicas partículas de segunda classe\", introduzidas h_a um bom tempo na literatura [36], [37], [38] para o estudo da dinâmica microscópica dos choques que estão presentes também na versão discreta dos modelos TASEP e ASEP. Neste sentido, é natural que as condições iniciais consideradas, que seriam perturbações no estado estacionário das partículas, possam ser causadas, no sistema real, pela própria interação com a vesícula. É o caso, portanto, de se propor que a geometria deste objeto tenha um papel importante na determinação da direcional de seu próprio movimento no meio intracelular. Esta parece ser, por exemplo, uma alternativa interessante para explicar aspectos do movimento de vírus no interior das células. / In this work we present a theoretical model to describe aspects of the bidirectional movement performed by intracellular structures (vesicles, organelles, viruses etc, to which we refer here simply as \"vesicles\"), observed essentially at in vivo experiments. This nondifusive movement is characterized by rapid inversions in direction and is capable of creating concentration gradients of the transported cargo. The phenomenon of intracellular transport is known to be mediated by motor proteins (such as kinesins and dyneins) whose own unidirectional motion along protein laments is well characterized (kinesins moves to the plus-end direction while dyneins moves to the minus-end direction of the microtubules) and is usually modeled by a stochastic dynamics describing the behavior of a Brownian particle in the presence of a time dependent asymmetrical potential held (see Astumian [26], Adjari and Prost [22], Magnasco [23]). More recently, it appeared in the literature works attempting to describe the movement of interacting motor proteins, since it was realized that collective e_ects emerging from this situation may be relevant to the transport phenomena along microtubules. An approach to describe the behavior of such interacting motor particles is based on existing models for \\driven di_usive systems\". In particular, the continuum versions of the totally asymmetric exclusion processes\" (TASEP) or the asymmetric exclusion processes\" (ASEP) have been used to study the behavior of motors density along microtubules by analyzing the steady state solutions to the corresponding Burgers equation (Parmeggiani et al. [33]). Up to now, however, there are no attempts in the literature to approach in this context the questions related to the bidirecionality of vesicles transported by these interacting motors. The idea we present here is to associate this odd movement to the movement of shock waves presented by the transient solutions of Burgers equation for certain initial conditions. Accordingly, the vesicles accompanying (sur_ng) the shocks fronts would play the role of their microscopic analogous \\particles of second class\" introduced long ago in the literature [36], [37], [38] to study the kinetics of the shocks that are also present in the discrete versions of the TASEP and ASEP. In this regard, it is natural to think that the considered initial conditions, namely perturbations to the motor density with respect to a steady state, can be created in the real systems simply by the interaction with the vesicle. It might then be the case also to propose that the geometry of the vesicle plays an important role to direct its own movement within intracellular environment. This seems to be, for example, an attractive alternative for explaining aspects of virus movement inside the cell.

Triptofano (TRP) é metabolizado por duas vias, a via serotonérgica e a via das quinureninas. Na via serotonérgica, TRP é metabolizado a serotonina (5-HT) e, em algumas células, à melatonina (MLT) que pode ser oxidada à N1-acetil-N2-formil-5- metoxiquinuramina (AFMK) e N1-acetil-5-metoxiquinuramina (AMK) por ação de peroxidases. Na via das quinureninas o TRP é diretamente metabolizado à N formilquinurenina (NFK) e em seguida a quinurenina (QUIN). A enzima indolamina 2, 3 dioxigenase (IDO) é uma das responsáveis por esta reação. Dada a importância da IDO na tolerância imunológica e pelo fato desta enzima ser induzível nos propusemos a avaliar a existência de uma regulação cruzada entre esta enzima e a via serotonérgica. Avaliando a interferência de AMK sobre a ação de IDO e a interferência de QUIN sobre a formação de AFMK por peroxidases, observamos uma possível interação entre as vias. AMK é um inibidor competitivo clássico de IDO e o Ki encontrado foi de 0,98 mM. QUIN é um inibidor acompetitivo linear simples da formação de AFMK e o Ki encontrado foi de 0,1 mM. A inibição da formação de AFMK também ocorre para a peroxidase humana (mieloperoxidase, MPO). Além de representarem uma regulação cruzada utilizada in vivo, as inibições encontradas podem ser relevantes para a proposta de novos inibidores de IDO e MPO na terapia imunomodulatória. Dado o nosso interesse pelas enzimas IDO e MPO, avaliamos ainda a localização intracelular destas enzimas em células de peritônio de camundongo, tanto residente como ativada com concanavalina A (Con A). O estímulo com Con A representa uma ativação de linfócitos T mediado por interferon gama (IFN-γ) e foi usado como modelo experimental para avaliar condições de localização em células ativadas. Por imunocitoquímica verificamos que IDO e MPO localizam-se próxima à membrana plasmática sendo que uma leve dispersão apenas de MPO foi observada em células ativadas com Con A. A localização intracelular das duas enzimas é no citoplasma, vesículas e núcleo. Curiosamente, verificamos MPO em células isoladas e também em agrupamentos celulares de duas ou mais células. Por citometria de fluxo identificamos macrófagos, linfócitos B1 e agrupamentos celulares como células que contém MPO. A mobilização de MPO durante a ativação celular, a presença de MPO em linfócitos e a presença de MPO e IDO em núcleos são informações novas que sugerem novas atividades para estas enzimas. / Tryptophan (TRP) is metabolized by two mains pathways, the serotoninergic pathway and the kynurenine pathway. In the serotoninergic pathway, TRP is metabolized to serotonin (5-HT) and, in some cells, to melatonin (MLT). The later can even be oxidized to acetyl-N1-N2-formyl-5-methoxykynuramine (AFMK) and N1-acetyl-5 -methoxykynuramine (AMK) by peroxidases. In the kynurenine pathway, TRP is metabolized to N-formylkynurenine (NFK) and to kynurenine (KYN). Indoleamine 2, 3 dioxygenase (IDO) is one of those responsible for this reaction. Since IDO is importat in immune tolerance and the fact that this enzyme is inducible by cytokines we proposed whether there is a cross regulation between this enzyme and the serotoninergic pathway. A possible interaction between MLT and TRP oxidation pathways was shown by the AMK influence on IDO activity and QUIN interference on AFMK formation by peroxidases. AMK was shown to be an IDO classical competitive inhibitor with a Ki of 0.98 mM. QUIN was a peroxidase (horseradish peroxidase, HRP) classical uncompetitive inhibitor and Ki was found to be 0,1 mM. AFMK formation inhibition was also found in human peroxidase (myeloperoxidase, MPO). Beyond the in vivo crosstalk, new IDO and MPO inhibitors in immunomodulatory therapy would be proposed by the compounds shown in this study. Given our interest in IDO and MPO, we also evaluated their intracellular localization in both resident and concanavalin A (Con A) activated mice peritoneum cells. Con A stimulation is a IFN-γ mediated T lymphocytes activation and was our experimental model to evaluate activated cells. In light microscopy we observed IDO and MPO localization near the membrane and MPO only had a dispersed localization in Con A activated cells. Cytoplasm, nucleus and vesicles were the intracellular localization of both enzymes. Interestingly, we found MPO in isolated cells and in cell clusters of two or more cells. MPO was founded on macrophages, B1 cells and cell clusters by flow cytometry. The MPO mobilization during cell activation, the presence of MPO in lymphocytes and the presence of MPO and IDO in nuclei are new informations to suggest new activities for these enzymes.

La spondylarthrite ankylosante (SA), la forme la plus commune des spondylarthrites (SpA), est fortement associée à la molécule du CMH de classe I HLA-B27 mais le rôle de cet antigène d'histocompatibilité dans le développement de ces pathologies reste encore inexpliqué. L’étude des rats transgéniques HLA-B27, développant une pathologie inflammatoire spontanée ressemblant aux SpA, a permis de confirmer l’implication directe du HLA-B27 et de corréler l’apparition des symptômes avec une forte expression de cette molécule. De plus, il a été montré que l’HLA-B27 présentait une propension particulière au mauvais repliement et à la formation d’oligomères de chaînes lourdes. L’objectif de mon travail de thèse était de déterminer si le trafic et/ou la formation d’oligomères du HLA-B27 étaient corrélés à sa surexpression. Pour cela, notre équipe a développé des protéines de fusion (HLA-BYFP et HLA-BRLuc) ainsi que la technique BRET afin d’étudier les interactions HLA-B/HLA-B. Au moyen de ce système expérimental, nous avons montré la formation de vésicules intracellulaires riches en protéines HLA-B mal repliées lorsqu’elles étaient fortement exprimées, qu'il s'agisse d'allèles associés à la SA (HLA-B*2702, -05, et -07) ou non (HLA-B*2706, et -09, HLA-B*0702). Cependant, ce phénomène était significativement plus prononcé pour les sous-types associés à la SA. Dans les conditions de forte expression, nous avons également observé que les sous-types associés à la SA formaient des oligomères qui se comportent différemment de ceux formés par la protéine HLA-B7. Ce phénomène ne semble pas être dû au déclenchement de la réponse cellulaire « Unfolded Protein Response » (UPR) et n’est pas abrogé par l’inhibition du protéasome. / Ankylosing spondylitis (AS), the most common form of spondyloarthritis (SpA), is strongly associated with the MHC class I HLA-B27 molecule. Although this association has been largely studied, mechanisms of pathology remain unclear. Development of a spontaneous inflammatory disease resembling human SpA in HLA-B27 transgenic rats confirmed the direct involvement of HLA-B27 and allowed to associate disease development with high expression levels of this molecule. Moreover, the HLA-B27 protein has an enhanced propensity to misfold and form aberrant disulfide linked heavy chain oligomers in the endoplasmic reticulum and at the cell surface. The goal of my thesis work was to determine if the HLA-B27 traffic and/or its ability to form oligomers are involved in this requirement of overexpression. For that, our team has developed fusion proteins ((HLA-BYFP and HLA-BRLuc) and the BRET technique to study, in vitro, the HLA-B/HLA-B interactions. Using this experimental system, we have shown the formation of intracellular vesicles, in which misfolded/unfolded HLA-B proteins accumulated when they were highly expressed, for both AS-associated alleles (HLA-B*2702, -05, et -07) or not (HLA-B*2706, et -09, HLA-B*0702). This phenomenon is strongly pronounced for AS-associated subtypes. For high-level expression, we also observed that the AS-associated subtypes form oligomers that behave differently from those formed by the HLA-B7 control protein. This phenomenon doesn’t appear to be due to unfolded protein response (UPR) triggering and is not abrogated by proteasome inhibition.

Les cellules endothéliales cornéennes (CEC) jouent un rôle essentiel pour le maintien de la transparence de la cornée. Cependant, chez l’homme, elles sont incapables de proliférer en raison d’un arrêt de leur cycle cellulaire en phase G1, ce qui rend la couche endothéliale cornéenne particulièrement vulnérable. La délivrance de molécules thérapeutiques (gènes ou médicaments) représente une solution prometteuse pour maintenir la viabilité des CEC. Néanmoins, la difficulté majeure de cette technique repose sur le fait de traverser la membrane cellulaire, normalement imperméable aux molécules de grande taille. Plusieurs techniques de délivrance de molécules ont déjà été testées sur le tissu cornéen mais aucune d’entre elles ne donnent de résultats suffisamment probants pour être utilisée en applications cliniques. L’objectif de cette thèse est d’adapter et de développer une nouvelle technique de délivrance intracellulaire de molécules, basé sur une perforation cellulaire via un phénomène photoacoustique induite par l’activation de nanoparticules de carbone par laser femtoseconde, sur un modèle d’endothélium cornéen in vitro et ex vivo / Corneal endothelial cells (CEC) are essential for corneal transparency. However, on humans, they are unable of proliferation owing to its arrest of G1 phase of the cell cycle, making corneal endothelial monolayer particularly vulnerable. The gene and drug delivery represents a promising solution to maintain CEC viability. Unfortunately, the major difficulty of this technique is the transport across the cell membrane, normally impermeable to high-size molecules. Several techniques of molecules delivery have already been tested on corneal tissue but none of them gives results sufficiently convincing to be used in clinical applications. The aim of this thesis is to adapt and develop a new technique of intracellular molecules delivery, based on cell perforation via photoacoustic effect induced by the activation of carbon nanoparticles by femtosecond laser, on in vitro and ex vivo models of corneal endothelium

Doctor of Philosophy / Department of Biology / Stephen K. Chapes / Silvia Mora Fayos / My work focused on understanding adipocyte function and regulation because of the importance to diabetes. In addition to being a fat storage depot, adipose tissue is an endocrine tissue. Adiponectin and leptin are two adipokines that control insulin sensitivity and energy balance. In spite of their importance, there are still questions about their secretion. I hypothesized that leptin and adiponectin follow different secretory routes. I found adiponectin localized in Golgi and the trans Golgi Network, while leptin mostly localized in ER during basal metabolisms. Common requirements for their secretion were the presence of class III Arf proteins and an intact Golgi apparatus, since BFA treatment inhibited secretion of both adiponectin and leptin. I found that trafficking of adiponectin is dependent on GGA1 coated vesicles. Endosomal inactivation significantly reduced adiponectin, but not leptin, secretion in both 3T3L1 and isolated rat adipocytes. Also, adiponectin, but not leptin, secretion was reduced in cells expressing non- functional form of Rab11 and Rab5 proteins. However, secretion of leptin, but not adiponectin was inhibited in cells expressing mutants of Protein Kinase D1. These results suggest that leptin and adiponectin secretion involve distinct intracellular compartments and pathways. Insulin resistance is associated with macrophage infiltration into adipose tissue and elevated levels of IL-6, TNF-alpha and IL-1beta Therefore, the second part of my dissertation tested the hypothesis that the interaction of macrophages and adipocytes causes insulin resistance. To test this hypothesis, I co-cultured macrophages and adipocytes. I found that mouse elicited peritoneal macrophages significantly decreased insulin-stimulated GLUT4 translocation to the plasma membrane in a contact-independent manner. IL-6 was the most inhibitory cytokine in reducing GLUT4 translocation, GLUT4 expression, Akt phsphorylation and reducing adipocyte differentiation compared to TNF-alpha and IL-1beta. These data suggest that IL-6 is the most effective cytokine secreted by macrophages involved in insulin resistance. Lastly, I tested the impact of adipocytes on macrophage differentiation in vitro and in vivo. I found that C2D macrophages isolated from the peritoneal cavity had increased IL-6 transcript levels after co-culture with 3T3L1 adipocytes in vitro. After i.p. injection, C2D macrophages isolated from WAT increased expression of mature macrophage surface markers and transcript levels of proinflammatory cytokines compared to C2D cells in vitro. However, macrophages isolated from BAT expressed low levels of cytokines and macrophage surface markers.

Aging is the main risk factor for cognitive decline. The hippocampus, a brain region critical for learning and memory formation, is especially vulnerable to normal and pathological age-related cognitive decline. Dysregulation of both insulin and intracellular Ca2+ signaling appear to coexist and their compromised actions may synergistically contribute to neuronal dysfunction with aging. This dissertation focused on the interaction between insulin, Ca2+ dysregulation, and cognition in hippocampal neurons by examining the contributions of insulin to Ca2+ signaling events that influence memory formation. I tested the hypothesis that insulin would increase cognition in aged animals by altering Ca2+-dependent physiological mechanisms involved in learning. The possible effects of insulin on learning and memory in young and aged rats were studied. In addition, the effects of insulin on the Ca2+-dependent afterhyperpolarization in CA1 pyramidal hippocampal neurons from young and aged animals were compared. Further, primary hippocampal cultures were used to examine the possible effects of insulin on voltage-gated Ca2+ channel activity and Ca2+-induced Ca2+-release; mechanisms known to influence the AHP. We found that intranasal insulin improved memory in aged F344 rats. Young and aged F344 rats were treated with Humalog®, a short-acting insulin analog, or Levemir®, a long-acting insulin analog. The aged rats performed similar to young rats in the Morris Water Maze, a hippocampal dependent spatial learning and memory task. Electrophysiological recordings from CA1 hippocampal neurons revealed that insulin reduced the age-related increase in the Ca2+-dependent afterhyperpolarization, a prominent biomarker of brain aging that is associated with cognitive decline. Patch clamping recording from hippocampal cultured neurons showed that insulin reduced Ca2+ channel currents. Intracellular Ca2+ levels were also monitored using Fura-2 in response to cellular depolarization. Results indicated that a reduction in Ca2+-induced Ca2+-release from intracellular stores occurred in the presence of insulin. These results suggest that increasing brain insulin levels in aged rats may have improved memory by reducing the AHP and intracellular Ca2+concentrations. This study indicates a possible mechanism responsible for the beneficial effects of intranasal insulin on cognitive function absorbed in selective Alzheimer’s patients. Thus, insulin therapy may reduce or prevent age-related compromises to Ca2+ regulatory pathways typically associated with cognitive decline.