1 Abstract Diabetes mellitus is characterized by the dysfunction and reduction of insulin-producing cells, resulting in hyperglycemia, which in long term harms the organism. For future therapy, it is crucial to understand the function of various factors participating in the differentiation and maturation of endocrine pancreatic cells. The aim of this study was to unravel the functional role of ISL1 during the development of the pancreas. ISL1 is expressed in all endocrine cells of the islets of Langerhansbut its function remains unclear, especially during early pancreatogenesis. As the global deletion of this gene is embryonically lethal, we used the tissue specific deletion of Isl1 in Neurod1 possitive cells using the Cre-loxP system. In this work we studied the effect of this deletion on the structure of islets of Langerhans, the formation of endocrine cell types and relative expression of genes during early pancreatic development. A defective achitecture of islets together with postnatal absence of α-cells was found in the Isl1 deletion mutant. Also, the expression of genes important for the specification of α-cell lineage and their subsequent function was decreased. The secondary outcome was the optimalization of a protocol for effective sorting of endocrine cells using fluorescent flow cytometry, which...

Murine polyomavirus is an important member of Polyomaviridae family offering potential applications in gene therapy and immunotherapy. Viral mutant analysis is crucial for study of the virus, however, commonly used methods of its production are laborious and give low yields. This thesis involves development of the new experimental system that can produce intact viral genome from recombinant plasmid in vivo using Cre/loxP-mediated recombination. One loxP site is unavoidably introduced into newly generated viral genome during recombination. Two variants of production plasmids generating wild type viral genome with incorporation of loxP between the poly(A) signal sites of early and late genes or into the intronic region of early genes were prepared. LoxP insertion between the poly(A) signal sites has a dramatic effect on viral gene expression and leads to complete loss of virus infectivity. Conversely, the infectious virus was obtained from the viral genome containing loxP site in the early intronic region. To ensure expression of Cre recombinase I also prepared stably transfected cell lines which can simplify the virus production. This thesis shows that newly designed system gives satisfactory yield of the virus, solves restrictions connected with commonly used methods and can be used for low infectious viral...

Longtemps considérée comme un simple support mécanique, la matrice extracellulaire (MEC) est un élément majeur dans le maintien de l’homéostasie. Ainsi l’élastine, principal constituant de la MEC des gros vaisseaux élastiques, est dégradée au cours du vieillissement, produisant ainsi des peptides d’élastine bioactifs (PE). Plusieurs études ont démontré l'implication des PE en physiopathologies tels que l’invasion tumorale, l’athérosclérose ou l’insulino-résistance (IRes). Ces effets s’expliquent par l’activation du complexe récepteur de l’élastine (CRE), composé par : une sous-unité extracellulaire liant les PE (EBP, elastin binding protein), la cathepsine A (dont le rôle reste inconnu), et la neuraminidase 1 (induisant la signalisation intracellulaire). L'IRes décrite, pourrait être associée à l’activité de désialylation de la neuraminidase-1 sur les chaines de N-glycosylation (Ng-c) du récepteur de l’insuline (RI). Sur la base de cette hypothèse, notre objectif a donc été de confirmer ce mécanisme et ses conséquences in silico (sur le RI), in vitro (pré-adipocytes 3T3-L1) et in vivo (aorte de souris). Nous montrons ainsi in vitro que les PE provoquent un dysfonctionnement de l’autophosphorylation du RI se répercutant sur plusieurs processus cellulaires comme l’entrée du glucose ou encore la différenciation adipocytaire. In silico, nous montrons pour la première fois le rôle des acides sialiques sur le comportement des Ng-c d'une part et sur le RI d'autre part. Enfin, in vivo, cette interaction CRE / IR engendre une hypertension artérielle par une diminution de la vasorelaxation des cellules endothéliales. / Often considered as a simple mechanical support, the extracellular matrix (ECM) is a major element of homeostasis regulation. Thus, elastin, the main constituent of large elastic vessels, is degraded during aging, producing bioactive elastin-derived-peptides (EDP). Several studies have demonstrated the EDP effects in physiopathologies such as tumor invasion, atherosclerosis, or insulin resistance (IRes) development. Those effects are explained by the activation of the elastin receptor complex (CRE), composed of: an extracellular subunit binding EDP (EBP, elastin binding protein), cathepsin A (its role is still unknown) and the sialidase neuraminidase-1 (Neu-1, involved in signaling pathway induction). Interestingly, the lab suggested that IRes may be induced by the desialylation of the N-glycan chains (Ng-c) on the insulin receptor (IR). The aim of this study was to confirm this hypothesis by demonstrating the consequence of desialylation on the IR in silico, on a 3T3-L1 pre-adipocyte cell in vitro, and on vascular complications in vivo. We show that EDP induce in vitro an impairment of IR autophosphorylation, affecting glucose uptake and adipocyte differentiation. In silico approach demonstrates the role of sialic acids on the behavior of Ng-c in the one hand and in other hand of IR. Finally, the IRes induced by ERC-IR interaction increase the vascular complication such as arterial hypertension by endothelial cell impairment. To conclude, Ng-c alteration would likely be responsible for structural changes in the IR at the origin of insulin resistance.

The prospect of the work presented in this thesis has been to engineer Cre recombinase to recognize and recombine a sequence from an HIV-1 Long Terminal Repeat (LTR), characterize the recombination proficiency of the evolved recombinase in mammalian cells and explore the potential of the recombinase for a novel antiretroviral strategy.

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Les néoplasies myéloprolifératives (NMP) sont des maladies hématologiques acquises de la cellule souche hématopoïétique. Une mutation activatrice de la protéine de signalisation JAK2, JAK2V617F, a été identifiée chez la moitié des patients atteints de NMP Philadelphie négatives. Il a été rapporté que les patients avec des NMP avaient une augmentation du risque thrombotique et de la densité microvasculaire dans la rate et la moelle osseuse, sans explication physiopathologique claire. Des travaux récents ont mis en évidence la présence de la mutation JAK2V617F non seulement dans les cellules sanguines mais également dans les cellules endothéliales (CE) de ces patients. Nous faisons l’hypothèse que la présence de JAK2V617F dans les CE pourrait modifier leurs propriétés expliquant l’augmentation de l’angiogenèse dans les NMP. Pour répondre à cette hypothèse, nous avons voulu étudier le phénotype angiogénique des cellules endothéliales portant la mutation JAK2V617F. In vitro, nous disposons des particules lentivirales permettant d’obtenir des CE JAK2V617F par transduction lentivirale. In vivo, nous disposons des souris transgéniques exprimant la mutation JAK2V617F de manière conditionnelle (JAK2V617F/WT) grâce à la stratégie Cre-lox. Pour répondre à notre hypothèse, il été nécessaire de travailler avec des modèles murins exprimant la mutation JAK2V617F spécifiquement dans les CE sans atteinte concomitante de la lignée hématopoïétique. Dans un premier temps, nous avons voulu caractériser deux modèles endothéliaux inductibles couramment utilisés, Cdh5(PAC)-CreERT2 et Pdgfb-iCreERT2, en termes d’efficacité et de spécificité de recombinaison dans les cellules endothéliales vis-à-vis du compartiment hématopoïétique. Nous avons démontré que les souris adultes Cdh5(PAC)-CreERT2 pouvaient être utilisées comme modèles endothéliaux spécifiques, avec toutefois la mise en garde que la recombinaison est très variable entre les souris. Nous avons constaté que les souris PDGFB-iCreERT2 sont appropriées pour cibler les cellules endothéliales dans une large gamme d’organes à l'exception du foie, et devraient être utilisées dans les quatre premières semaines qui suivent l'induction, pour cibler un gène d’intérêt au niveau des cellules endothéliales, sans qu’il ait une atteinte concomitante dans la lignée hématopoïétique. Nous avons ensuite étudié les propriétés angiogéniques des cellules endothéliales JAK2V617F, in vitro en utilisant des HUVEC transduites avec un lentivirus permettant l’expression de JAK2V617F, et in vivo avec les souris Pdgfb-iCreERT2;JAK2V617F/WT. Nous avons démontré que les HUVEC JAK2V617F avaient un profil proangiogénique lié à une capacité proliférative élevée, résultant de l’activation de la voie JAK2/STAT3/PI3K. L’avantage hyperprolifératif que confère la mutation JAK2V617F aux cellules endothéliales a été confirmé in vivo avec le modèle de la vascularisation post-natale de la rétine, avec toutefois une diminution de la densité du réseau vasculaire due à une augmentation de la régression vasculaire au niveau de la rétine des souris Pdgfb-iCreERT2;JAK2V617F/WT. / Myeloproliferative neoplasms (MPNs) are acquired hematopoietic stem cell disorders. An activating mutation in the JAK2 signaling protein, JAK2V617F, was identified in half of the patients with Philadelphia chromosome-negative MPNs. It has been reported that patients with MPN had an increased risk of thrombosis but also an increased microvessel density in the spleen and bone marrow with no clear pathophysiological explanation. Several recent studies have demonstrated the presence of JAK2V617F mutation not only in blood cells but also in endothelial cells (EC) in MPN patients. We hypothesized that the presence of JAK2V617F in EC could change their properties leading to an increased angiogenesis process in MPNs. To address this question, our aim was to study the angiogenic phenotype of endothelial cells carrying the JAK2V617F mutation. For the in vitro experiments, we used lentiviral transduction of human JAK2V617F in EC. For the in vivo approach, we used transgenic mice (JAK2V617F/WT) that conditionally express JAK2V617F through Cre-lox strategy. To investigate our hypothesis, it was necessary to work with mice that express JAK2V617F specifically in EC without concomitant expression in hematopoietic cells. We first characterized two commonly-used inducible endothelial models, Cdh5(PAC)-CreERT2 and Pdgfb-iCreERT2, in terms of efficiency and specificity of recombination in endothelial cells. We showed that adult Cdh5(PAC)-CreERT2 mice can be used as specific endothelial model with however the wariness that recombination is highly variable among mice. We found that Pdgfb-iCreERT2 mice are appropriate to target endothelial cells in a wide range of organs except liver, and should be used within the four weeks after induction of Cre-mediated recombination to target a gene of interest in endothelial cells, without having a concomitant expression in hematopoietic lineage. We then studied the angiogenic properties of JAK2V617F endothelial cells, in vitro using JAK2V617F transduced HUVECs, and in vivo using Pdgfb-iCreERT2;JAK2V617F/WT mice. We observed that JAK2V617F HUVECs had a proangiogenic profile that was related to a highly proliferative potency, and that this phenotype results from a constitutive activation of JAK2/STAT3/PI3K pathway. The hyperproliferative advantage conferred by JAK2V617F to endothelial cells was confirmed in vivo using the postnatal vascularization model of the retina, with however a decrease in the density of the vascular network due to an increased vascular regression in Pdgfb-iCreERT2;JAK2V617F/WT mice’s retinas.

The prospect of the work presented in this thesis has been to engineer Cre recombinase to recognize and recombine a sequence from an HIV-1 Long Terminal Repeat (LTR), characterize the recombination proficiency of the evolved recombinase in mammalian cells and explore the potential of the recombinase for a novel antiretroviral strategy.

Indiana University-Purdue University Indianapolis (IUPUI) / The Cre/loxP system is a tool for targeted recombination of DNA. For applying Cre recombinase-mediated genome modifications, there is a requirement for reliable, high-fidelity, and specific transgenic expression of the Cre recombinase. This study focuses on the reliability of different bone cell specific Cre models in the Cre/loxP system. In this study, DMP1-Cre transgenic mouse which has a transgene driven by DMP1 promotor that allows Cre-expression only in late stage osteoblasts and osteocytes was used. Ctsk-Cre mouse with a driven by Ctsk promoter was used so that only osteoclasts would undergo Cre-mediated recombination. E2A-Cre mouse where the Cre recombinase is driven by a global promoter E2A was also included in this study as a control line to test the Cre reporter line Ai9. Dmp1-Cre, Ctsk-Cre and E2A-Cre mice were crossed to the fluorescent Cre-reporter line—Ai9, which harbors a floxed stop codon, followed by the fluorophoremTomato, inserted into the Rosa26 locus. This construct is expected to give red fluorescence when it recombines with Cre-expressing mouse cells and no fluorescence in non-recombinant mouse cells. Double positive (Ai9+/Cre+) offspring selected by PCR were perfused, and 5mu-m thick section of bone and soft tissues were examined for red fluorescent expression. Cre positive cells were quantitated using ‘ImageJ’ software program. The DMP1-vi Cre mouse results showed significant expression in the targeted osteocytes and osteoblasts. In addition, skeletal muscle tissue also showed significant Cre- expression. Ctsk-Cre mice showed significant expression in targeted osteoclasts. But brain tissue was positive in Cre-expression. Bone-Cre mouse models are expected to express Cre recombinase only in their respective bone cells and they have been used for gene deletion studies in bone cells. However, this study has revealed that the bone cell specific Cre mouse models DMP1-Cre and Ctsk-Cre have unexpected expression in muscle and brain respectively. In order to use these models for targeted gene deletion in bone cells, further testing and studies have to be conducted.

Mammals cannot re-form heavily damaged bones as in large fracture gaps, whereas zebrafish efficiently regenerate bones even after amputation of appendages. However, the source of osteoblasts that mediate appendage regeneration is controversial. Several studies in zebrafish have shown that osteoblasts are generated by dedifferentiation of existing osteoblasts at injured sites, but other observations suggest that de novo production of osteoblasts also occurs. In this study, we found from cell-lineage tracing and ablation experiments that a group of cells reserved in niches serves as osteoblast progenitor cells (OPCs) and has a significant role in fin ray regeneration. Besides regeneration, OPCs also supply osteoblasts for normal bone maintenance. We further showed that OPCs are derived from embryonic somites, as is the case with embryonic osteoblasts, and are replenished from mesenchymal precursors in adult zebrafish. Our findings reveal that reserved progenitors are a significant and complementary source of osteoblasts for zebrafish bone regeneration.

We have investigated the role of the metabotropic glutamate receptor 7 (mGluR7) and the gastrin releasing peptide receptor (Grpr) population that are involved at different levels of itch transmission. We found that mGuR7 deficient mice displayed an anaphylaxis-like behavior when provoked with histamine. Analysis of blood revealed elevated plasma levels of histamine and mouse mast cell protease-1 (mMCP1), two indicators of anaphylaxis, in mGluR7 deficient mice compared with control mice. Inhibition of the neurokinin 1 receptor, by preventing binding of the corresponding ligand substance P (SP), prior to provocation with histamine prevented the development of anaphylaxis in mGluR7 deficient animals. However, blocking GRPR (gastrin releasing peptide receptor) only resulted in decreased itch levels in mGluR7 deficient mice but did not prevent the systemic anaphylaxis-like behavior. Our findings indicate that mGluR7 normally functions as a brake on histaminergic itch that is mediated through GRPR as well as anaphylaxis through Substance P. Grpr has previously been shown to mediate both histaminergic and non-histaminergic itch but little is known about the GRPR neuronal population. We used a BAC cloning strategy to construct a Grpr-Cre line, which we crossed with the reporter lines tdTomato and Viaat-egfp as well as with Vglut2-lox. We could conclude that Grpr-Cre neurons are mainly excitatory interneurons located in lamina II-IV, that convey itch using VGLUT2-mediated glutamatergic transmission to the next, currently unknown, step in the labeled line of chemical itch. To eventually deduce the function of the endogenous opioids dynorphin and enkephalin, which are hypothesized to be involved in gating pain and itch in the spinal cord, we constructed two Cre lines using BAC cloning that targeted the precursor proteins preprodynorphin and preproenkephalin, respectively. Preprodynorphin-Cre neurons were mainly located in lamina II-IV and overlapped to 47% with Vglut2 mRNA, while the co-expression with the inhibitory markers Viaat-egfp and PAX2 was 13% and 28% respectively in the spinal cord. Preproenkephalin neurons were more localized to lamina III in the dorsal horn, furthermore single cell analysis showed that they overlapped to 94% with Vglut2 mRNA while 7% and 13% expressed Viaat-egfp and PAX2 respectively.