Activated islet specific T cells are central to the destructive autoimmune response observed in type 1 diabetes (T1D). Not surprisingly, intense focus is placed on understanding how autoreactive T cell responses arise and contribute to disease pathology in the hope of using this information to develop novel therapeutic strategies for treatment of T1D. Here we investigate the mechanisms underlying defective c-Maf binding to the IL-4 promoter in T cells from diabetes prone mice and identify the mechanisms responsible for suppression of T cells by the inhibitory receptor Ly49A. It is not clear why development of protective Th2 cells is poor in T1D. c-Maf transactivates the IL-4 gene promoting Th2 cell development; therefore abnormalities in c-Maf may contribute to reduced IL-4 production by CD4 cells from nonobese diabetic (NOD) mice. Here we demonstrate that, despite normal expression, c-Maf binds poorly to the IL-4 promoter (IL-4p) in NOD CD4 cells. Immunoblots demonstrate that c-Maf can be modified at lysine 33 by small ubiquitin-like modifier-1 (SUMO-1). Sumoylation is facilitated by direct interaction with the E2 conjugating enzyme Ubc9 and increases following T cell stimulation. In addition, c-Maf physically interacts with p65/RelA. This interaction is dependent on the DNA binding domain of c-Maf and phosphorylation of p65 at serine 536. In transfected cells, overexpression of SUMO-1 or p65 decreases c-Maf transactivation of IL-4p-driven luciferase reporter activity, reduces c-Maf binding to the IL-4p in chromatin immunoprecipitation (ChIP) assays and enhances c-Maf localization into promyelocytic leukemia nuclear bodies (PML-NBs) or nucleoli, respectively. Sumoylation of c-Maf and phosphorylation of p65 are increased in NOD CD4 cells compared to CD4 cells from diabetes-resistant B10.D2 mice, suggesting that increased c-Maf sumoylation and interaction with p65 contribute to immune deviation in T1D by reducing c-Maf access to and transactivation of the IL-4 gene. Islet specific CD4 cells expressing inhibitory receptors may be a useful therapeutic tool for treating T1D. Engagement of transgenic Ly49A inhibits CD4 cell activation and delays onset of T1D in mice. However, in vitro studies suggest the inhibitory effect of Ly49A is incomplete. Here we report that following simultaneous T cell receptor (TCR) and Ly49A engagement, phosphorylation of Zap70, Erk1/2 and c-Jun were significantly diminished. Kinetic studies indicated that Ly49A did not simply delay activation but had a long-lasting effect. In contrast, when only costimulatory signals were provided through CD28, Ly49A engagement did not block p38 MapK or Akt phosphorylation. Likewise, expression of the downstream targets Bcl-xl and Baff were unaffected. Together these data suggest that engagement of Ly49A selectively inhibits signals downstream of the TCR but spares those unique to CD28. These results suggest that when considering its use as an immunotherapy, the potency of inhibitory receptors such as Ly49A may be further improved by pairing them with costimulatory blockade. Take together, these studies suggest that abnormal post-translational regulation of c-Maf function is a novel marker of altered T cell function in T1D and use of inhibitory receptors such as Ly49A may be optimized combining this approach with other complementary therapies.

ABSTRACT I CHARACTERIZATION OF A NOVEL NUCLEAR VARIANT OF BMP2Bone morphogenetic protein 2 (Bmp2) is a signaling protein that was first detected by its ability to induce cartilage and bone formation. It has since been implicated in broad variety of developmental, patterning, and disease processes. To date, Bmp2 has only been known to function as an extracellular signaling molecule. However, we have obtained clear evidence for a nuclear form of Bmp2. This nuclear variant, nBmp2, contains a bipartite NLS that overlaps the site of proteolytic cleavage. The NLS remains intact and functional when translation of Bmp2 initiates from a downstream alternative start codon. The resulting protein lacks the signal peptide and is therefore translated in the cytoplasm rather than the endoplasmic reticulum, thus avoiding proteolytic processing and secretion. Instead, the uncleaved protein containing the intact NLS is translocated to the nucleus. Preliminary functional analyses in zebrafish indicate that nBmp2 is critical for proper heart development. To determine if this function is conserved in mammals, we have also generated mice harboring a null allele for nBmp2. ABSTRACT II COORDINATE REGULATION OF COL11A2 AND COL27A1 BY THE TRANSCRIPTION FACTOR LC-MAF During skeletal development, long bones of the body develop from a cartilage template that is progressively replaced by bone. This process of endochondral ossification requires precisely coordinated expression of extracellular matrix proteins such as the cartilage-specific collagens. In this study, enhancer/reporter assays demonstrated that the transcription factor Lc-Maf inhibits the transcriptional activity of a cartilage-specific Col11a2 enhancer element while a cartilage-specific COL27A1 enhancer element was strongly activated by Lc-Maf. Site-directed mutagenesis identified the binding region within the COL27A1 enhancer, and it was found to be unlike any known consensus Maf family binding site. The in vivo significance of these results was examined using immunohistochemistry and in situ hybridization in mouse limbs undergoing endochondral ossification. Taken together, these results suggest that Lc-Maf participates in the developmental transition from proliferating to hypertrophic chondrocytes during endochondral ossification by coordinately downregulating Col11a2 and upregulating Col27a1 collagen gene expression.

Les hépatopathies métaboliques regroupent les maladies non alcooliques du foie (NAFLD) et les maladies alcooliques du foie (MAF) causées respectivement par l’obésité ou une consommation excessive d’alcool. Ces pathologies vont de la simple stéatose à des formes aggravées pouvant aller jusqu’au carcinome hépatocellulaire. D’autres facteurs que le surpoids ou l’abus d’alcool jouent un rôle dans la susceptibilité des patients à développer une NAFLD ou une MAF. Cette thèse a pour objectif de clarifier et d'étudier les mécanismes et les facteurs participant à l’installation de l’inflammation dans ces deux pathologies. Mes travaux ont porté d’une part sur le rôle de la cellule de Kupffer dans les étapes précoces de la NAFLD et d’autre part sur l’étude du microbiote intestinal comme cofacteur déclenchant de la MAF. La cellule de Kupffer lors de la stéatose, étape précoce de la NAFLD, présente une dérégulation de son homéostasie lipidique qui participe à son phénotype pro-inflammatoire et favorise l’inflammation hépatique. L’impact du fructose, largement utilisé dans notre alimentation actuelle, a été étudié et aggrave l’inflammation hépatique lors d’un régime hyperlipidique et ceci est associé à une dysbiose spécifique. Dans la MAF, une dysbiose intestinale, une diminution des Bacteroides, a été associée aux lésions hépatiques dans un modèle murin d’alcoolisation. L‘utilisation de traitements permettant de maintenir cette population à des taux élevés a corrigé cette dysbiose et protégé les animaux face aux lésions hépatiques. Ces travaux permettent d‘envisager le MI comme une cible prometteuse permettant de contrôler l’évolution des hépatopathies métaboliques vers des formes sévères. / Metabolic hepatopathies is including Non Alcoholic Fatty Liver Disease (NAFLD) and Alcoholic Liver Disease (ALD) due to an excessive consumption of alimentation or alcohol. The pathologies range from simple steatosis to aggravated forms until hepatocellular carcinoma. Other factors than overweight or alcohol abuse play a role in sensitivity of patients to develop NAFLD or ALD. The aim of this thesis is to clarify and study the mechanisms and factors that lead to the installation of inflammation in those pathologies. My work covered in part on the role of Kupffer cell in the early stages of NAFLD and secondly on the study of intestinal microbiota as a cofactor triggering the MAF.The Kupffer cell role in steatosis, the early stages of NAFLD, showed a deregulation of its lipid homeostasis involved in the pro-inflammatory phenotype and promotes liver inflammation. The impact of fructose, widely used in our current diet, was studied and worsening liver inflammation during high fat diet. This is associated with a specific dysbiosis. In ALD, intestinal dysbiosis, a decrease of Bacteroides, leading to liver damage has been established. The use of treatments to maintain this population with high levels corrected the dysbiosis and has protected animals against liver damages. Both works on the NAFLD and ALD establish MI is a promising target to control the evolution of metabolic liver diseases toward aggravated forms.

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Magnesium alloys have poor formability at room temperature, due to a limited number of slip systems owing to the hexagonal closed packed structure of magnesium. One possibility to increase the formability of magnesium alloys is to refine the grain size. A fine grain magnesium alloy shows high strength and high ductility at room temperature, hence an improved formability. In addition to grain refinement, the formability of Mg alloys can be improved by controlling crystallographic texture. Severe plastic deformation (SPD) processes namely, equal channel angular pressing (ECAP) and multi-axial forging (MAF) have led to improvement in room temperature mechanical property of magnesium alloys. Further, it has been reported that by adding rare earth elements, room temperature ductility is enhanced to nearly 30%. The increase in property is attributed to crystallographic texture. Many rare earth elements have been added to magnesium alloys and new alloy systems have been developed. Amongst these elements, Ce addition has been shown to enhance the tensile ductility in rolled sheets at room temperature by causing homogeneous deformation. It has been observed that processing of rare-earth containing alloys below 300°C is difficult. Processing at higher temperatures leads to grain growth which ultimately leads to low strength at room temperature. The present thesis is an attempt to combine the effect SPD and rare earth addition, and to examine the overall effect on microstructure and texture, hence on room temperature mechanical properties. In this thesis, Mg-0.2%Ce alloy has been studied with regard to the two SPD processes, namely, ECAP and MAF. The thesis has been divided into six chapters. Chapter 1 is dedicated to introduction and literature review pertaining to different severe plastic deformation processes as applied to different Mg alloys. Chapter 2 includes the details of experimental techniques and characterization procedures, which are commonly employed for the entire work. Chapter 3 addresses the effect of ECAP on the evolution of texture and microstructure in Mg-0.2%Ce alloy. ECAP has been carried out on two different initial microstructure and texture in the starting condition, namely forged and extruded. ECAP has been successfully carried out for the forged billets at 250°C while cracks get developed in the extruded billet when ECAP was done at 250°C. The difference in the deformation behaviour of the two alloys has been explained on the basis of the crystallographic texture of the initial materials. The microstructure of the ECAP materials indicates the occurrence of recrystallization. The recrystallization mechanism is identified as “continuous dynamic recovery and recrystallization” (CDRR) and is characterized by a rotation of the deformed grains by ~30⁰ along c-axis. The yield strengths and ductility of the two ECAP materials have been found quite close. However, there is a difference in the yield strength as well as ductility values when the materials were tested under compression. The extruded billet has the tension compression asymmetry ~1.7 while the forged material has the asymmetry as ~2.2. After ECAP, the yield asymmetry reduces to ~1 for initially extruded billet, while for the initially forged billet the yield asymmetry value reduces to ~1.9. In chapter 4, the evolution of microstructure and texture was examined using another severe plastic deformation technique, namely multi axial forging (MAF). In this process, the material was plastically deformed by plane strain compression subsequently along all three axes. In this case also two different initial microstructures and texture were studied, namely the material in as cast condition and the extruded material. The choice of initial materials in this case was done in order to examine the effect of different initial grain size in addition to different textures. By this method, the alloy Mg-0.2%Ce could be deformed without fracture at a minimum temperature of 350⁰C leading to fine grain size (~3.5 µm) and a weak texture. Grain refinement was more in the initial cast billets compared to the initial extruded billet after processing. The mechanism of grain refinement has been identified as twin assisted dynamic recrystallization (TDRX) and CDRR type. The mechanical properties under tension as well as under compression were also evaluated in the present case. The initially extruded billet has shown low tension compression asymmetry (~1.2) than cast billet (~1.9), after MAF. Chapter 5 addresses the exclusive effect of texture on room temperature tensile properties of the alloy. Different textures were the outcomes of ECAP and MAF processes. In this case, in order to obtain an exact role of texture, a third of deformation mode, rolling, was also introduced. All the processed materials were annealed to obtain similar grain size but different texture. A similar strength and ductility for ECAP and MAF, where the textures were qualitatively very different, was attributed to the fact that texture of both the ECAP and MAF processed materials, was away from the ideal end orientation for tensile tests. In chapter 7, the final outcomes of the thesis have been summarized and scope for the future work has been presented.

This master thesis deals with issues of development of applications for augmentative and alternative communication (AAC) in environment of special education for mentally challenged pupils with communication disorders. Thesis presents issues of development contemporary applications and follows on design principles of Computer therapy project, which compliance during the design and implementation should prevent the current issues. The aim of the thesis is creation of application for AAC of YES/NO type and i-CT framework - framework simplifying creation of similar apps for described environment. Both apps works on iOS and Android systems and were tested in an environment of people with mental disabilities.