The innate immune system is a critical first line of defense against invading pathogens. Innate immunity directly detects pathogens, sets up an appropriate adaptive response, and can directly kill pathogens. Drosophila may lack an adaptive immune response, but have a robust innate immune system with a variety of defense effector mechanisms. While the responses to bacteria, fungi, and RNA viruses have been well characterized, not much is known about the response to DNA viruses. My studies have set out to characterize the Drosophila immune response to a DNA virus, utilizing the large dsDNA virus, Invertebrate Iridescent Virus 6 (IIV-6). IIV-6 infection causes shortened lifespan, and in later stages of infection, flies present with abdominal swelling and iridescent blue color. Our objectives were to identify pathways flies use to protect themselves from IIV-6 infection, determine how this protection is mediated, and to identify any immune inhibitors that IIV-6 uses to suppress innate immune signaling. I have found that IIV-6 strongly up-regulates a class of stress proteins with unknown function, termed Turandots, after infection in vivo or in vitro. This induction is dependent upon viral replication, requires JAK-STAT activation, and activation of p38b MAPK. In addition, the unpaireds, which function as JAK-STAT ligands, are upregulated after IIV-6 infection in a p38b-dependent manner. Together, this data suggests that p38b activation leads to production of unpaired cytokines and activation of JAK-STAT signaling to induce Turandots. I have also found that IIV-6 infected cells secrete protective factors. This response is induced within 12 hours of IIV-6 infection, exosome-mediated, and provides robust protection to naive cells challenged with an mCherry-expressing strain of IIV-6. Additionally, IIV-6 inhibits two major immune responses in Drosophila, the IMD and Toll pathways. Stimulation of IIV-6 infected Drosophila S2* cells with either IMD or Toll stimulators results in very poor antimicrobial peptide responses. Yet, IMD and Relish are still cleaved upon stimulation in IIV-6 infected cells, indicating that the block is downstream. In support of this finding, IIV-6 infected flies respond very poorly to infection with the enterobacteria Erwinia carotovora carotovora compared to mock-injected flies.

Yes / AMP-activated protein kinase (AMPK) is a pivotal regulator of metabolism at the cellular and organismal levels. AMPK also suppresses inflammation. We found that pharmacological activation of AMPK rapidly inhibited the Janus kinase (JAK)–signal transducer and activator of transcription (STAT) pathway in various cells. In vitro kinase assays revealed that AMPK directly phosphorylated two residues (Ser515 and Ser518) within the SH2 domain of JAK1. Activation of AMPK enhanced the interaction between JAK1 and 14-3-3 proteins in cultured vascular endothelial cells and fibroblasts, an effect which required the presence of Ser515 and Ser518 and was abolished in cells lacking AMPK catalytic subunits. Mutation of Ser515 and Ser518 abolished AMPKmediated inhibition of JAK-STAT signaling stimulated either by the sIL-6Rα/IL-6 complex or by expression of a constitutively active V658F-mutant JAK1 in human fibrosarcoma cells. Clinically used AMPK activators metformin and salicylate enhanced the inhibitory phosphorylation of endogenous JAK1 and inhibited STAT3 phosphorylation in primary vascular endothelial cells. Therefore our findings reveal a mechanism by which JAK1 function and inflammatory signaling may be suppressed in response to metabolic stress and provide a mechanistic rationale for the investigation of AMPK activators in a range of diseases associated with enhanced activation of the JAK-STAT pathway.

No description available.

The production of Nitric Oxide (NO) under physiological conditions has beneficial roles in acting as a key signaling component of many biological processes as well as having an anti-microbial effect. However its effects following excess production by the inducible NO pathway is potentially detrimental in the pathogenesis of chronic inflammation including sepsis and several other inflammatory diseases. Understanding the mechanisms that regulate the expression of the inducible nitric oxide synthase (iNOS) responsible for producing the excessive amounts of NO in disease states is therefore critical. In this regards, experiments were carried out to identify the signaling pathways that may mediate this process, focusing specifically on the JAK/STAT cascade. The reason for selecting the latter is because our research group, amongst others, has carried out extensive work investigating other signaling pathways, including the mitogen activated kinases (MAPK). Moreover, studies have also been carried out in an attempt to identify the critical role of JAK/STAT signaling for iNOS induction. These studies however failed to conclusively demonstrate whether, as with the MAPKs, the JAK/STATs may also play an essential role. Furthermore there is indeed controversy in the literature with researchers unable to agree whether expression of iNOS does require JAK/STAT activation. Thus, the aim of the project described in this thesis was to establish unequivocally whether activation of the JAK/STATs preceeds induction of iNOS. The studies were extended to L-arginine transport as well because the latter is widely reported to be induced in parallel with iNOS and substrate supply to iNOS may be critical for sustained NO production. Changes in transporter activity as well as their expression profiles were assessed. All experiments were carried out in either rat aortic smooth muscle cells (RASMCs) or in the J774 macrophage cell line. These cell types were selected because RASMCs are one of the prime targets for induced NO production in vascular inflammation and the macrophages are involved in host defence, acting in part through NO production. To establish the role of JAK/STATs, pharmacological and molecular approaches were used. Pharmacologically, two inhibitors were used and these were AG490 and JAK inhibitor I. The former is reported to be a selective JAK2 inhibitor and the other blocks all known JAK proteins. The potential of the GTPases to regulate the induction of iNOS was also examined using selective inhibitor known to regulate these proteins. In addition to these drugs, siRNA targeting JAK2 was also exploited and western blotting was extensively used to detect expression of various proteins including iNOS, native and phosphorylated JAK2 and TYK2. Changes in iNOS activity was monitored by determining nitrite production using the Griess assay and L-arginine transport was monitored using tritiated arginine (L-[3H]arginine). RASMCs were treated with a combination of LPS (100 µg/ml) and IFN- (100 U/ml) and the macrophages with LPS (1 µg/ml) to induce iNOS and transporter activity. Consistent with previous reports, the above treatment of both cell types resulted in the expression of iNOS, production of NO and enhanced transport of L-arginine. These effects were not affected by AG490 but blocked by JAK inhibitor I. Furthermore, although both cell types expressed the key JAKs (JAK2 and TYK2), neither of these proteins were phosphorylated under conditions of induced NO production. Moreover, siRNA experiments showed that JAK2 expression could be abolished without any significant change in NO production, confirming that at least JAK2 may not be required for this process. Whether TYK2 is involved still remains to be resolved as the phosphor-protein could not be detected. However the conclusive siRNA knockdown studies could not be carried out due to time and cost constraints. Apart from iNOS and NO production, changes in induced L-arginine transport were also not significantly affected under the experimental conditions described above suggesting that like with iNOS, induction of L-arginine transport is independent of at least JAK2. Interestingly however, STAT-1 was phosphorylated and this was blocked by JAK inhibitor I but not AG490. Thus, STAT-1 activation may be essential but its activation may be independent of the JAKs. One possible alternate upstream activator of STAT-1 may be the GTPases. Indeed these proteins have been indicated to phosphorylate STAT-1 independent of the JAKs. However, in this project, inhibition of the GTPase pathway enhanced NO production and L-arginine transport suggesting that the GTPases downregulate these processes. In conclusion, the studies carried out in this thesis have shown that induction of iNOS, NO production and L-arginine transport in both RASMCs and J774 macrophages are independent of JAK2 but require STAT-1 activation which may be phosphorylated independently of the JAKs. The role of other JAKs such as TYK2 although unlikely, will need to be resolved using a more specific approach such as siRNA.

Acute myeloid leukemia (AML) is a genetically heterogeneous disease and represents about 20% of pediatric leukemias. Survival rates vary depending on subtypes but are particularly unfavorable for acute megakaryoblastic leukemia (AMKL), a rare subtype of AML that usually affects children under 3 years old (≤ 30% survival for certain subtypes of AMKL). In pediatrics, genetic rearrangement leading to the expression of a chimeric fusion gene are present in many cases and are considered initiator events in the development of leukemia. In AMKL cases, more than 70% of them exhibit such rearrangement. Several of these chimeric transcripts, such as NUP98::KDM5A and CBFA2T3::GLIS2, occur in a higher proportion of cases. The analysis of the transcriptome from pediatric leukemic cases allowed us to identify new chimeric fusion transcripts in pediatric leukemias. Specifically, we discovered BPTF as a new fusion partner of NUP98 in the case of acute megakaryoblastic leukemia (AMKL), and the ACIN1::NUTM1 fusion in B-cell lymphoid leukemias. These studies have refined the molecular classification of these leukemias and provided tools for diagnosis and disease monitoring. The hypothesis of my thesis is that the NUP98::KDM5A and CBFA2T3::GLIS2 fusions are oncogenic and their expression in normal human hematopoietic and progenitor cells leads to transformation into acute megakaryoblastic leukemia in immunodeficient recipient mice, allowing for the generation of renewable xenograft models. My work has contributed to the generation of AMKL models with NUP98::KDM5A (N5A) and CBFA2T3::GLIS2 (CG2) fusions. To do this, we optimized a pipeline for transducing these chimeric genes in CD34+ cells isolated from cord blood, followed by transplantation into immunodeficient mice. These xenograft models phenocopy the leukemia of patients from a morphological, immunophenotypic, and transcriptomic standpoint. These synthetic AMKL models can be serially transplanted into mice and have a high frequency of leukemic stem cells. I also contributed to the development of a unique patient-derived xenograft (PDX) model derived from primary cells of a patient with an NUP98::BPTF genotype AMKL leukemia. These synthetic and PDX models then served as substrates for my experiments and those of several members of our laboratory. My research has allowed us to identify and characterize new biomarkers specific to NUP98- rearranged and CBFA2T3::GLIS2 positive AMKL. Taking advantage of the biomass generated by these AMKL leukemia models, we conducted transcriptomic and proteomic studies of the membrane surface. These results were compared to normal cells isolated from cord blood to identify several surface proteins specific to each leukemia genotype and shed light on new potential biomarkers. Furthermore, we confirmed the sensitivity of our AMKL models to JAK-STAT pathway inhibitors and performed synergy assays between JAK-STAT and the PI3K-AKT-mTOR pathway inhibitors. These experiments demonstrated the synergistic induction of apoptosis in our models upon the combine exposure to JAK-STAT and PI3K-AKT-mTOR pathway inhibitors. These works allowed us to identify potential therapeutic vulnerabilities of AMKL. Finally, since research on AMKL is affected by the limited number of patient samples, the human models and molecular data presented in this thesis constitute an invaluable resource to accelerate translational research for these high-risk leukemias. / La leucémie myéloïde aiguë (LMA) est une maladie hétérogène sur le plan génétique et représente environ 20% des leucémies pédiatriques. Les taux de survie varient selon les sous- types mais sont particulièrement défavorables pour les leucémies aiguës mégacaryoblastiques (AMKL), un sous-type rare de LMA touchant généralement les enfants de moins de 3 ans (≤ 30% de survie pour certains sous-types d’AMKL). En pédiatrie, les réarrangements génétiques entraînant l’expression d’un gène de fusion chimérique sont présentes dans un grand nombre de cas et sont considérées comme des événements initiateurs à l’origine de la leucémie. Chez les leucémies de type AMKL, c’est plus de 70% des cas qui présentent un tel réarrangement. Quelques-uns de ces transcrits chimériques, tels que NUP98::KDM5A et CBFA2T3::GLIS2, surviennent dans une plus grande proportion des cas. Dans le cadre de mes recherches, l’analyse du transcriptome de leucémies pédiatriques nous ont permis de mettre en évidence de nouveaux transcrits chimériques. Notamment, nous avons découvert BPTF comme étant un nouveau partenaire de fusion de NUP98 dans le cas d’une AMKL, ainsi que la fusion ACIN1::NUTM1 chez des leucémies lymphoïdes à cellules B. Ces travaux ont permis de raffiner la classification moléculaire de ces leucémies et propose de nouvelles approches pour le diagnostic et le suivi de la maladie. L’hypothèse de ma thèse est que les fusions NUP98::KDM5A et CBFA2T3::GLIS2 sont oncogéniques et leur expression chez des cellules souches hématopoïétiques et progénitrices humaines normales entraîne une transformation en leucémie aiguë mégacaryoblastique dans les souris receveuses immunodéficientes, permettant de générer des modèles de xénogreffe. Mes travaux ont contribué à la génération de modèles d’AMKL arborant les fusions NUP98::KDM5A ainsi que CBFA2T3::GLIS2. Pour ce faire, nous avons optimisé un processus de transduction de ces gènes chimériques chez des cellules CD34+ isolées de sang de cordon, suivi de transplantation chez la souris immunodéficiente. Ces modèles de xénogreffe récapitulent la leucémie des patients aux points de vue morphologique, immunophenotypique et transcriptomique. Ces modèles synthétiques d’AMKL peuvent être transplantés de manière sériée en souris et présentent une fréquence élevée de cellules souches leucémiques. De plus, nous avons aussi développé un modèle pdx unique (patient derived xenograft) dérivé des cellules primaires d’un patient atteint d’une leucémie AMKL présentant la fusions NUP98::BPTF. Ces modèles synthétiques et pdx ont ensuite servi de substrats à mes expériences ainsi que celles de plusieurs membres du laboratoire. Mes recherches ont permis d’identifier et de caractériser de nouveaux biomarqueurs spécifiques aux AMKL présentant un transcrit de NUP98 réarrangé et CBFA2T3::GLIS2. Tirant avantage de la biomasse générée par ces modèles de leucémie AMKL, nous avons fait des études transcriptomiques et protéomiques de la surface membranaire de nos modèles. Ces résultats furent comparés aux cellules normales isolées de sang de cordon afin d’identifier plusieurs protéines de surface spécifiques aux leucémies initiées par NUP98 réarrangé et CBFA2T3::GLIS2 afin de mettre en lumière de nouveaux biomarqueurs potentiels. De plus, nous avons aussi confirmé la sensibilité de nos modèles AMKL aux inhibiteurs de la voie JAK-STAT ainsi que démontré l’induction synergique de l’apoptose de nos modèles en présence des inhbitieurs combinés des voies JAK-STAT et PI3K-AKT-mTOR. Finalement, puisque la recherche sur les AMKL est ralentie par la quantité limitante d’échantillons de patient, les modèles humains et les données moléculaires présentés dans cette thèse constituent une ressource inestimable afin d’accélérer la recherche translationnelle pour ces leucémies à haut risque.

Janus kinase 3 (JAK3) est un membre de la famille JAK de protéines tyrosine kinase impliquées dans la transduction du signal intracellulaire médiée par les récepteurs de cytokines via la voie de signalisation JAK/STAT. JAK3 s'est avéré exprimé de manière différentielle dans les cellules de la granulosa (GC) des follicules pré-ovulatoires bovins et régulé à la baisse par l'hormone lutéinisante. Ces observations suggèrent que la régulation de JAK3 pourrait moduler la prolifération des GC, l'activité stéroïdienne et l'activation/l'inhibition des cibles en aval. Pour étudier les mécanismes des actions de JAK3 dans GC, nous avons utilisé JANEX-1, un inhibiteur pharmacologique de JAK3, et des traitements FSH et analysé des marqueurs de prolifération, des enzymes stéroïdogènes et la phosphorylation de protéines cibles, y compris STAT3 et les partenaires JAK3 précédemment identifiés CDKN1B/p27Kip1 et MAPK8IP3/JIP3. Les GC en culture ont été traités avec ou sans FSH en présence ou non de JANEX-1. L'ARN total et les protéines ont été extraits et analysés par RT-qPCR, western blot et UHPLC-MS/MS. L'expression de l'enzyme stéroïdogène CYP11A1, mais pas du CYP19A1, était significativement régulée à la hausse dans les GC traités avec la FSH et les deux étaient significativement diminuées lorsque JAK3 était inhibé par rapport au contrôle. Les marqueurs de prolifération CCND2 et PCNA ont été significativement réduits dans les GC traités au JANEX-1 et régulés positivement par la FSH. Les analyses Western blots ont montré que le traitement JANEX-1 réduisait de manière significative les quantités de pSTAT3 tandis que la surexpression de JAK3 augmentait pSTAT3. De même, le traitement à la FSH a augmenté pSTAT3 même dans les GC traités au JANEX-1. Les analyses UHPLC-MS/MS ont montré une phosphorylation et des modifications supplémentaires de résidus d'acides aminés spécifiques dans JAK3 ainsi que ses partenaires de liaison CDKN1B et MAPK8IP3 révélant une activation ou une inhibition possible de JAK3 après des traitements FSH ou JANEX-1, respectivement. L'abondance de la protéine totale JAK3 a augmenté après le traitement par FSH et a diminué de manière significative, avec MAPK8IP3, dans le GC traité par JANEX-1, tandis que l'abondance totale de CDKN1B a été modifiée après FSH et augmentée après JANEX-1. Nous montrons que JAK3 influence l'activité GC par la phosphorylation de protéines cibles en réponse à des stimulations telles que la FSH, ce qui conduit à l'activation de JAK/STAT et module probablement d'autres voies de signalisation impliquant CDKN1B et MAPK8IP3. / Janus kinase 3 (JAK3) is a member of the JAK family of tyrosine kinase proteins involved in cytokine receptor-mediated intracellular signal transduction through the JAK/STAT signaling pathway. JAK3 was shown as differentially expressed in granulosa cells (GC) of bovine preovulatory follicles and downregulated by the luteinizing hormone. These observations suggested JAK3 regulation could modulate GC proliferation, steroidogenic activity and activation/inhibition of downstream targets. To investigate the mechanisms of JAK3 actions in GC, we used JANEX-1, a pharmacological JAK3 inhibitor, and FSH treatments and analyzed proliferation markers, steroidogenic enzymes and phosphorylation of target proteins including STAT3 and previously identified JAK3 partners CDKN1B/p27Kip1 and MAPK8IP3/JIP3. Cultured GCs were treated with or without FSH in the presence or not of JANEX-1. Total RNA and proteins were extracted and analyzed by RT-qPCR, western blotting and UHPLC-MS/MS. Expression of steroidogenic enzyme CYP11A1, but not CYP19A1, was significantly upregulated in GC treated with FSH and both were significantly decreased when JAK3 was inhibited as compared to control. Proliferation markers CCND2 and PCNA were significantly reduced in JANEX-1-treated GC and upregulated by FSH. Western blots analyses showed that JANEX-1 treatment significantly reduced pSTAT3 amounts while JAK3 overexpression increased pSTAT3. Similarly, FSH treatment increased pSTAT3 even in JANEX-1-treated GC. UHPLC-MS/MS analyses showed phosphorylation and additional modifications of specific amino acid residues within JAK3 as well as its binding partners CDKN1B and MAPK8IP3 revealing possible activation or inhibition of JAK3 following FSH or JANEX-1 treatments, respectively. Abundance of JAK3 total protein was increased post-FSH treatment and significantly decreased, along with MAPK8IP3, in JANEX-1-treated GC while CDKN1B total abundance was altered post-FSH and increased post-JANEX-1. We show that JAK3 influences GC activity through phosphorylation of target proteins in response to stimulations such as FSH, which leads to the activation of JAK/STAT and likely modulating other signaling pathways involving CDKN1B and MAPK8IP3.

Neointimal hyperplasia (NIH) is a cardiovascular disease characterised by increased smooth muscle cell (SMC) inflammation and proliferation. Suppressor of cytokine signalling 3 (SOCS3) limits Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathways involved in vascular remodelling but is limited by its short biological half-life. Therefore, mutation of all 9 Lys residues that are potential sites of ubiquitylation to Arg should produce a mutated SOCS3 resistant to ubiquitin-mediated proteasomal degradation (“Lys-less” SOCS3). This study hypothesise that enhancing SOCS3 stability and limiting JAK/STAT signalling may provide sustained inhibition of the vascular remodelling in NIH. Lentiviral transduction of WT and Lys-less SOCS3 in human saphenous vein (HSVSMCs) was highly efficient after 48 hours (>97%) and was sustained over 2 weeks. Lys-less SOCS3 was resistant to ubiquitylation contrary to WT-transduced HSVECs, and Lys-less SOCS3 was more stable (t1/2=4h) than WT (t1/2<4h) (n=6, P<0.001) in HSVSMCs. In HSVSMCs, both Lys-less SOCS3 and WT inhibited sIL-6Rα/IL-6 mediated STAT3 activation but not extracellular signal regulated protein kinase 1/2 (ERK1/2) by 80±7% (Lys-lessSOCS3/pSTAT3) and 74±6% (WT/pSTAT3) (n=3, P<0.05) and similarly inhibited PDGF-mediated STAT3 activation but not ERK1/2 by 67±17% (Lys-less SOCS3/pSTAT3) and 72±18% (WT/pSTAT3) (n=3, P<0.05). Functionally, Lys-less SOCS3 and WT were equivalent in inhibiting sIL-6Rα/IL-6 and PDGF-induced proliferation, whilst having no effects on PDGF-induced migration in HSVSMCs. Lys-less SOCS3 can be successfully transduced into primary HSVSMCs. It is more stable than WT yet retains its functional ability to ameliorate pro-inflammatory signalling and SMC proliferation, making it an attractive option for developing treatment of NIH. / University of Botswana

El gen humano 11&#61538;-HSD2 es un modelo para investigar la contribución de los efectos de los receptores de esteroides en células de cáncer de mama. El análisis del promotor mostró que la región distal está implicada en la mayor parte de la activación dependiente de hormona. En respuesta a hormona, STAT5A se recluta a la región distal y PR a las regiones distal y proximal del promotor. El reclutamiento de PR se debe a dos mecanismos diferentes, la unión directa de PR a la región proximal, y la implicación vía JAK/STAT en el reclutamiento a la región distal. La inducción del gen 11&#61538;-HSD2 por hormonas disminuye parcialmente por inhibidores de MAPK y PI3K/Akt y totalmente por inhibidores de JAK/STAT. Así, los efectos citoplasmáticos del PR están implicados en la inducción del gen progesterona. La forma activa de la ARN-polimerasa II es reclutada por la inducción con hormonas a la región distal del promotor 11&#61538;-HSD2 y la región distal tiene respuesta a hormonas por sí misma, indicando que la inducción del gen por hormonas empieza antes del sitio de inicio de transcripción descrito previamente. / The human 11&#61538;-HSD2 gene is a model to investigate the contribution of steroid hormone receptors effects on a progesterone responsive promoter in breast cancer cells. Deletion analysis of the 11&#61538;-HSD2 promoter showed that the distal region is involved in most of the hormone-dependent activation. ChIP showed hormone-dependent STAT5A-recruitment to the distal region and PR-recruitment to the distal and proximal promoter regions. Results suggest two different mechanisms of hormone-induced PR-recruitment, since cells stably expressing PR containing a mutated DNA-binding domain have affected hormone-dependent PR-recruitment to proximal promoter, and JAK/STAT pathway inhibition blocks PR-recruitment to distal promoter. Hormone-stimulated 11&#61538;-HSD2 gene-expression was partially decreased by MAPK and PI3K/AKT pathway inhibitors and totally blocked by JAK/STAT pathways inhibitors, indicating that cytoplasmic PR effects involvement in progestin-induced 11&#61538;-HSD2 expression. Importantly, upon hormone induction active RNA-polymerase II is recruited from the 11&#61538;-HSD2 distal promoter region and the distal minimal promoter has hormone-responsiveness by itself, suggesting that progesterone-dependent 11&#61538;-HSD2 expression starts upstream the previously characterized transcription start site.

Les cancers, qu’il s’agisse de leucémies ou de tumeurs solides, sont le résultat de proliférations cellulaires anormales et non contrôlées au sein des tissus. Ces proliférations anarchiques sont le reflet d’une surexpression et/ou sur-activation de protéines intracellulaires engendrées par un événement oncogénique. Aujourd’hui encore il est donc nécessaire de trouver de nouvelles molécules à usage thérapeutique ciblant spécifiquement ces protéines. C’est dans ce contexte que les facteurs de transcription STAT5 constituent de véritables cibles de choix puisque ces protéines participent activement à la leucogénèse. L’implication directe des protéines STAT5 dans la génèse des leucémies a été démontrée par l’utilisation de formes mutées constitutivement active de STAT5. Les facteurs de transcription STAT5 jouent un rôle essentiel dans la voie de signalisation JAK/STAT. Cette voie aboutit à la régulation de grandes fonctions biologiques telles que la prolifération cellulaire, la différenciation cellulaire ou encore l’apoptose. L’objectif de ce projet consiste donc à cibler spécifiquement les protéines STAT5 dans le but de rétablir le processus de mort cellulaire et empêcher la prolifération des cellules cancéreuses. Les inhibiteurs spécifiques des protéines STAT5 sont sélectionnés selon la méthode SELEX qui permet d’isoler des ligands structurés de forte affinité pour la protéine. L’affinité et la spécificité de ces inhibiteurs, appelés aptamères, sont caractérisées à partir de modèles cellulaires de leucémies dépendant de l’activité des facteurs de transcription STAT5. Les aptamères sont aujourd’hui de véritables outils thérapeutiques en pleine évolution. / Leukemias are due to abnormal cell proliferation, which is the result of intracellular over-expression or excessive activation of protein due to oncogenic event. Still today, it is necessary to find new therapeutic molecules, which specifically target these proteins. STAT5, via the JAK/STAT signaling pathway, controls fundamental cellular processes, including .cell survival, proliferation and differentiation. To struggle against tumorigenesis, JAK/STAT signaling pathway has to be inhibited. The aim of this project is to target specifically STAT5 factors to restore healthy signal transduction. We generated aptamers by an iterative in vitro selection. Aptamers are short-structured single strand DNAs or RNAs that bind with high affinity and specificity to their target. Once STAT5B recombinant proteins are produced, they are subjected to SELEX process. The number of rounds depends on various parameters. After seven rounds, two sequences are retrieved. The specificity and affinity of these aptamers are assessed by fluorescent immunoassays. Binding affinity and kinetics of interaction are characterized by SPR. Aptamer anti proliferative effects are determined by evaluation of the growth of cells depending on STAT5. Finally, we developed several .assays aiming at understanding the mechanism of an aptamer action on STAT5B such as phosphorylation measurement and EMSA. Aptamers are now emerging therapeutic tools; they exhibit significant advantages relative to protein therapeutics.

No description available.