La SpA est un rhumatisme inflammatoire chronique fréquent, dont la prévalence est de 0,3% en France. Les mécanismes pathologiques qui en sont à l’origine demeurent largement incertains. Néanmoins, l’héritabilité de la maladie est élevée, impliquant de multiples facteurs génétiques, dont la région du complexe majeur d’histocompatibilité (CMH) et plus particulièrement l’allèle HLA-B27 qui y exerce un rôle prédominant. L’objectif de ce travail était d’identifier de nouvelles cibles moléculaires, en vue d’améliorer la compréhension de la physiopathologie de la SpA, par des approches génétiques et de génomiques fonctionnelles.La première partie de mon travail a consisté en l’identification de polymorphismes du CMH associés à la SpA et distinct de HLA-B27. Les études d’association portant sur les données génétiques de 3 cohortes indépendantes nous ont permis d’identifié 5 variants associés à la SpA indépendamment du HLA-B27. Les deux polymorphismes situés à proximité des gènes MICA et MAPK14 semblent particulièrement intéressants pour leur implication potentielle dans la pathogénèse de la SpA. En marge de cette étude, nous avons entrepris de déterminer la prévalence du HLA-B27 dans une cohorte française représentative de la population générale, qui était de 6,9% chez les témoins et de 74,2% chez les sujets atteints de SpA.Les études fonctionnelles conduites sur des cellules dendritiques dérivées de monocytes (MD-DCs) ont permis d’identifier un défaut de réponse proliférative des LT CD4+ stimulés par les MD-DCs de patients atteints de SpA, ainsi qu’une signature transcriptomique de 81 gènes caractéristique des MD-DCs de ces patients. Parmi les gènes validés, la surexpression d’ADAMTS15, de F13A1 et de SELL pourrait jouer un rôle dans l’inflammation liée à la pathologie, alors que la sous-régulation de CITED2 paraitrait corrélée à une dérégulation de la voie Wnt. Enfin, nos investigations sur les MD-DCS nous ont amené à identifier une corrélation entre l’haplotype d’ERAP1 prédisposant à la SpA, et un niveau accru d’expression de ce gène ainsi que de la protéine ERAP1. / Spondyloarthritis (SpA) is a frequent chronic inflammatory rheumatic disorder, with a prevalence of 0.3% in France. Pathological mechanisms leading to SpA remain largely uncertain. Nevertheless, the heritability of this disorder is high, likely involving multiple genetic factors, among which the major histocompatibility complex (MHC) region and particularly the HLA-B27 allele which plays a prominent role. The objective of this work was to achieve a better understanding of SpA physiopathology via genetic and transcriptomic approaches. The first part of my work consisted in identification of MHC polymorphisms associated with SpA, distinct of HLA-B27. Association studies based on the genetic data of 3 independent cohorts have allowed to identify 5 SNPs associated to SpA, independently of HLA-B27. Two polymorphisms localized next to MICA and MAPK14 genes seem particularly interesting for their implication in SpA pathogenesis. In parallel of this study, we characterized HLA-B27 prevalence in a French cohort corresponding to 6.9% in healthy controls and 74.2% in SpA patients. Functional studies on monocyte-derived dendritic cells (MD-DCs) revealed altered capacity to stimulate allogeneic CD4+ T cell responses by MD-DCs from SpA patients and a transcriptomic signature of 81 genes differentially expressed in those cells, as compared to those from healthy controls. Among validated genes, ADAMTS15, F13A1 and SELL could play a role in SpA inflammation, whereas CITED2 seemed to be correlated to Wnt pathway. Finally, a strong correlation between ERAP1 SpA-susceptibility haplotype and an increased expression of this gene and the ERAP1 protein has been identified.

5 Abstract Products of major histocompatibility complex (MHC) plays key role in immune system of vertebrates. Prior studies on different vertebrate species show, that heterozygosity in MHC genes is linked to more efficient immune system and preferred in mate choice. Results of human studies are ambivalent, which can be due to the effect of some modulating factors like reproductive status. Therefore, our aim was to test heterozygosity related preferences in faces, odor and voice across menstrual cycle. Our sample consisted of 51 men and 52 women, from which 23 used hormonal contraception and 29 had natural cycle. They were genotyped in -A, -B and -DR alleles. All odor stimuli, face photos and voice records were rated on seven-point scale in both follicular and luteal phase. Repeated measures ANOVA was used for the analysis. Changes in ratings across the menstrual cycle and heterozygosity were most discernible on voice ratings. Voices of homozygous males were rated more attractive than voices of heterozygous males especially in follicular phase. Similar shift to higher ratings in follicular phase was manifested in ratings of homozygous male faces, but the difference between homozygous males and heterozygous males was not significant. Women with natural cycle also rated voices higher in their follicular phase...

Indiana University-Purdue University Indianapolis (IUPUI) / The function of major histocompatability complex (MHC) class II molecules is to present antigenic peptides to CD4+ T cells. Typically, MHC class II molecules present peptides derived from exogenous sources. Yet, certain endogenous antigens (Ags) have been found to be presented by class II molecules. Studies suggest that specific heat shock protein family members may play a role in Ag processing and subsequent class II presentation. The studies presented here using B lymphoblasts demonstrate the importance of HSP90α, HSP90β, and possibly HSP70 in selectively regulating MHC class II presentation. Inactivation of HSP90 function using pharmacological inhibitors inhibited class II presentation of exogenous and endogenous GAD, but did not perturb the presentation of several other intra- and extracellular Ags. Individual knockdown of HSP90 isoforms using isoform specific siRNA selectively inhibited GAD Ag presentation. These results demonstrate a requirement for HSP90α and HSP90β in regulating MHC class II presentation of select Ags. Studies to explore mechanistically the roles of HSP90α and HSP90β in regulating GAD Ag presentation were pursued. The pathways of exogenous and endogenous MHC class II presentation of GAD Ag are distinct yet converge with shared terminal processing of GAD within endosomal/lysosomal vesicles. The effect of HSP90 manipulation on various shared components of the MHC class II pathway was examined. The studies presented here suggest that HSP90α and HSP90β regulate MHC class II presentation of GAD Ag at discrete steps most likely involving HSP90 binding to GAD Ag rather than perturbing overall MHC class II function. vi Studying the role of HSP90 in MHC class II presentation in B cells revealed the potential requirement for HSP70 in the presentation of select Ags. The studies presented here demonstrate a possible role for HSP70 in the presentation of Ags such as SMA or Ig kappa by MHC class II molecules. Also included in this work is a study of a rare case of diabetes caused by type B insulin resistance due to development of insulin receptor autoantibodies during the treatment of hepatitis C with interferon alpha and ribavirin. Clinical and laboratory findings in the case are presented.

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Le campagnol roussâtre Myodes glareolus est le réservoir principal de l'hantavirus Puumala (PUUV) responsable de la néphropathie épidémique (NE) en Europe. L'objectif de cette thèse est de décrire la variabilité de la résistance / tolérance de M. glareolus à PUUV et d'explorer son rôle dans la distribution et la transmission de ce virus. Nous émettons l'hypothèse que la tolérance à PUUV favorise sa persistance et sa transmission, ce qui devrait accroître le risque d'émergence de la NE. Nous avons développé une approche gène candidat pour déterminer le rôle de trois gènes de l'immunité dans la résistance / tolérance à PUUV. L'existence d'associations positives entre les allèles du gène Drb et l'infection par PUUV d'une part et les relations négatives détectées entre le niveau d'expression du gène Tnf-α et la prévalence en PUUV d'autre part corroborent l'évolution d'une tolérance en zones d'endémie de la NE. Elle pourrait être sélectionnée en réponse au coût de la réponse inflammatoire développée contre PUUV. L'approche de génétique des populations a démontré le rôle de la dynamique des réservoirs dans l'épidémiologie de PUUV. La comparaison du polymorphisme des gènes Drb et Tnf-α et des patrons génétiques neutres semble cependant indiquer une faible influence de la sélection dans le polymorphisme de ces gènes. Ce résultat suggère un effet potentiellement important de la plasticité phénotypique dans les niveaux de résistance/tolérance observés. L'étude de la communauté d'helminthes a confirmé cette hypothèse en révélant l'impact de certaines espèces sur le risque d'infection à PUUV. / The bank vole Myodes glareolus is the main reservoir of Puumala hantavirus (PUUV), the agent of nephropathia epidemica (NE) in Europe. This work aims at describing the variability of M. glareolus resistance / tolerance to PUUV and at exploring its role in the distribution and transmission of the virus. We hypothesized that tolerance to PUUV should favour its persistence and transmission, what could increase the risk of NE emergence.We developed a candidate gene approach to determine the role of three immune genes in the resistance / tolerance to PUUV. Both the detection of positive associations between Drb alleles and PUUV infection and the negative relationship observed between Tnf-α gene expression and PUUV prevalence corroborated the evolution of tolerance in NE endemic areas. The costly inflammatory response activated against PUUV infection could mediate this evolution.Using landscape population genetics, we revealed the role of M. glareolus population dynamics in PUUV epidemiology. The comparison of Drb and Tnf-α genetic differentiation with the neutral pattern detected at microsatellites indicated that selection weakly acted on these immune genes. This result suggested the potential effect of phenotypic plasticity in the balance of resistance/tolerance to PUUV. The study of helminth communities confirmed this hypothesis as it revealed the impact of two nematode species on the risk of PUUV infection

Studies of the evolution of the major histocompatibility complex (MHC) have been central to the understanding sexual selection and pathogen-mediated selection. The European badger Meles meles is well suited for exploring such questions because of its life history characteristics, reproductive biology and mating system. In this thesis, I examined both MHC class I and class II genes. Seven putatively functional sequences were found for class I genes and four for class II DRB genes. Evidence of past balancing selection of both genes was demonstrated by the d_Nd_S ratio, by positive selection at the antigen-binding site (ABS) and by trans-species polymorphism of alleles within other mustelids and carnivores. MHC class I genes also showed evidence of concerted evolution, but domains showed different evolutionary histories. MHC genes may influence microbiota and odour of an individual and influence mating preferences. I examined the bacterial community of the subcaudal gland secretion and demonstrated a high number of bacterial species (56 operational taxonomic units), which cubs exhibited a higher diversity than adults. The microbiota may lead to an individual-specific odour as a cue signaling the MHC genotype of potential mating partners. I report the first evidence for a MHC- based mating preference in carnivores. Female badgers showed a MHC-assortative mate choice towards breeding with males that had functionally similar MHC genes, for MHC class II DRB genes. This applied to neighbouring-group matings. I also found considerable annual fluctuation in the occurrence of MHC-based mate choice. Based on genome-wide background in the same mating randomizations I found no evidence of inbreeding, which indicated that MHC similarity was apparently the actual target of mate choice. In line with MHC-assortative mate choice, MHC heterozygosity had no influence on the co-infection status. Individual MHC alleles did, however, associate with resistance and susceptibility to specific pathogens, suggesting that MHC diversity may be driven and maintained by pathogen-mediated selection through rare-allele advantages and/or fluctuating selection. My study of genetic characteristics, mate choice and pathogen pressures in a wild population revealed past and contemporary evolutionary process of the MHC genes. This increases knowledge of how the MHC may affect mating behaviour and sexual selection, ultimately influencing population processes.

La différentiation entre le « soi » et le « non-soi » est un processus biologique essentiel à la vie. Les peptides endogènes présentés par les complexes majeurs d’histocompatibilité de classe I (CMH I) représentent le fondement du « soi » pour les lymphocytes T CD8+. On donne le nom d’immunopeptidome à l’ensemble des peptides présentés à la surface cellulaire par les molécules du CMH I. Nos connaissances concernant l’origine, la composition et la plasticité de l’immunopeptidome restent très limitées. Dans le cadre de cette thèse, nous avons développé une nouvelle approche par spectrométrie de masse permettant de définir avec précision : la nature et l’abondance relative de l’ensemble des peptides composant l’immunopeptidome. Nous avons trouvé que l’immunopeptidome, et par conséquent la nature du « soi » immun, est surreprésenté en peptides provenant de transcrits fortement abondants en plus de dissimuler une signature tissu-spécifique. Nous avons par la suite démontré que l’immunopeptidome est plastique et modulé par l’activité métabolique de la cellule. Nous avons en effet constaté que les modifications du métabolisme cellulaire par l’inhibition de mTOR (de l’anglais mammalian Target Of Rapamycin) provoquent des changements dynamiques dans la composition de l’immunopeptidome. Nous fournissons également la première preuve dans l’étude des systèmes que l’immunopeptidome communique à la surface cellulaire l’activité de certains réseaux biochimiques ainsi que de multiples événements métaboliques régulés à plusieurs niveaux à l’intérieur de la cellule. Nos découvertes ouvrent de nouveaux horizons dans les domaines de la biologie des systèmes et de l’immunologie. En effet, notre travail de recherche suggère que la composition de l’immunopeptidome est modulée dans l’espace et le temps. Il est par conséquent très important de poursuivre le développement de méthodes quantitatives au niveau des systèmes qui nous permettront de modéliser la plasticité de l’immunopeptidome. La simulation et la prédiction des variations dans l’immunopeptidome en réponse à différents facteurs cellulaires intrinsèques et extrinsèques seraient hautement pertinentes pour la conception de traitements immunothérapeutiques. / Self/non-self discrimination is a fundamental requirement of life. Endogenous peptides presented by major histocompatibility complex class I (MHC I) molecules represent the essence of self for CD8 T lymphocytes. These MHC I peptides (MIPs) are collectively referred to as the immunopeptidome. Very little is known about the origin, composition and plasticity of the immunopeptidome. Here, we developed a novel high-throughput mass spectrometry approach that yields an accurate definition of the nature and relative abundance of peptides presented by MHC I molecules. Surprisingly, we found that the immunopeptidome, and therefore the nature of the immune self, is biased toward peptides derived from highly abundant transcripts and conceals a tissue-specific signature. Then, we showed that the immunopeptidome is plastic and moulded by cellular metabolic activity. In fact, we found that altering cellular metabolism via the inhibition of the mammalian target of rapamycin (mTOR) results in dynamic changes in the cell surface MIPs landscape. Importantly, we provide the first systems-level evidence that the immunopeptidome projects at the cell surface a faithful representation of biochemical networks and metabolic events regulated at multiple levels inside the cell. Our discoveries open up new perspectives in systems biology and immunology. Indeed, our work suggests that the composition of the immunopeptidome is modulated in space and time. Therefore, it is imperative to further develop and exploit systems-level quantitative methods that will enable modelling of the immunopeptidome’s plasticity. Simulating and predicting variations in the immunopeptidome in response to cell-intrinsic and -extrinsic factors could be relevant to numerous contexts, including the rational design of immunotherapeutic interventions.

INTRODUÇÃO: O diabetes tipo 1 autoimune (DM1A) está associado a alterações na imunidade inata e adaptativa. A agressão autoimune, órgão específica, determina a destruição das células beta do pâncreas e a deficiência da produção de insulina. O infiltrado inflamatório do tipo linfomononuclear, configurando a insulite, e a escassez ou a ausência das células ?, definem o quadro histológico do DM1A. Os autoanticorpos contra antígenos das células beta, que geralmente se desenvolvem na fase pré-clínica, conferem predisposição para DM1A. No entanto, é difícil definir quando e quais indivíduos progredirão para o diabetes manifesto, justificando a busca de outros biomarcadores que auxiliem nas indicações de tratamentos preventivos. Nesse contexto, sabe-se que os microRNAs (miRNAs), pequenos RNAs que atuam pós transcrição, desempenham papel crucial na regulação de genes, integrando fatores genéticos e ambientais e influenciando o funcionamento de órgãos e tecidos de maneira pontual ou sistêmica. OBJETIVOS: avaliar o envolvimento biológico e a relevância da expressão de miRNAs na resposta imunológica e na função das células ? na patogênese do DM1A. MÉTODOS: analisamos o perfil dos miRNAs séricos em 4 grupos, a saber: pacientes portadores de DM1A, até 6 meses do diagnóstico (DM1A recente), (n=30); pacientes portadores de DM1A com duração de 2-5 anos (DM1A 2-5)(n=26) e indivíduos com autoanticorpos pancreáticos positivos sem diabetes (AcP) (n=25), os quais foram comparados aos indivíduos controles saudáveis(n= 29). A expressão dos microRNAs foi obtida com ensaios individuais TaqMan® MicroRNA Assays 5x primers e TaqMan MicroRNA Human Array Card A, (Applied Biosystems- Forster City CA, USA) constituído por 377 alvos e 4 endógenos. Os dados de expressão foram analisados no Software Cloud, (Thermo Fisher Scientific) e no programa Limma (Linear Models for Microarray and RNA-Seq Data). RESULTADOS: Não houve diferença nas características demográficas, como idade, cor auto referida e sexo entre os grupos (p > 0,05). Pacientes portadores de DM1A (recente e com duração de 2-5 anos), diferiram do grupo controle pelos valores elevados de glicose, hemoglobina glicada, títulos de autoanticorpos pancreáticos, e menores de peptídeo C (p < 0,05) e foram semelhantes entre si. Os portadores de autoanticorpos (AcP) tinham características intermediárias entre os grupos: menores valores de HbA1c e de anticorpo anti-tirosina-fosfatase (anti-IA2) e maiores de peptídeo C em relação aos dois grupos com diabetes. Diferiram dos controles apenas pelos maiores títulos de anticorpo anti-insulina (IAA) e anti-descarboxilase do ácido glutâmico 65 (anti-GAD65). A frequência dos alelos HLA de risco para diabetes (-DR3 ou -DR4 e -DQ2 ou DQ-8) decresceu dos grupos DM1A recente e DM 2-5 para AcP e controles. Foram avaliados 135 miRNAs que estavam expressos em 20% ou mais das amostras dos quatro grupos analisados. Maior expressão foi observada em 13, 4 e 33 miRNAs dos grupos AcP, DM1A recente e DM1A 2-5 respectivamente e menor em 11, 7 e 31 miRNAs destes grupos. Destes, 4 miRNAs foram diferencialmente expressos nos grupos AcP, DM1A recente e DM1A 2-5 em relação ao grupo controle. Os miRNAs: miR -16, miR-195 e miR-454, relacionados com regeneração endócrina do pâncreas, efeito anti-inflamatório e resposta à injúria da célula ? estavam diminuídos nestes 3 grupos. O miR-200a, implicado em apoptose das células beta, estava aumentado nos grupos AcP e DM1A recente e diminuído nos pacientes com maior duração do diabetes (DM1A 2-5), possivelmente devido à escassez destas células. Outros 8 miRNAs apresentaram expressão diferente da do grupo controle em dois dos grupos avaliados, e tendência semelhante no terceiro grupo, sendo 4 deles elevados (miR-193a-5p, miR- 323-3p, miR-423-5p, e miR-92a) e 4, diminuídos (miR-191, miR-19a, miR- 376a, miR-590-5p) ou neutralidade no 3º grupo (miR-15b, miR-100, miR-181a e miR-483-5p) Resposta antagônica foi observada para o miR-25 e miR-485- 3p, diminuídos no grupo AcP e aumentados no DM1A 2- 5. Tais miRNAs estão relacionados com resposta imunológica, secreção de insulina, lesão de células ? e glicotoxicidade, à semelhança do observado para o miR-101-3p, validado por ensaios individuais numa casuística maior. CONCLUSÃO: nossos dados sugerem que miRNAs circulantes podem estar envolvidos na patogênese do DM1A / INTRODUCTION: Autoimmune type 1 diabetes (T1D) is associated with changes in innate and adaptive immunity. The organ-specific autoimmune aggression determines the destruction of beta-cells in the pancreas and the deficient insulin production. The inflammatory infiltration of the lymphomononuclear type, configuring the insulite, and the scarcity or the absence of the beta cells, define the histological picture of T1D. Autoantibodies against beta-cell antigens, which usually develop in the preclinical phase, confer predisposition to T1D. However, it is difficult to define when and which individuals will progress to overt diabetes, justifying the search for other biomarkers that could be indicative of preventive treatments. In this context, it is known that the microRNAs (miRNAs) - small RNAs that act post transcription - play a crucial role in regulating genes and in integrating genetic and environmental factors, influencing the function of organs and tissues in a punctual or systemic way. OBJECTIVES: to evaluate the biological involvement and relevance of miRNA expression in the immune response and ?-cell function in the pathogenesis of T1D. METHODS: we analyzed the profile of serum miRNAs of 4 groups, namely: patients with T1D up to 6 months after diagnosis (recent T1D), (n = 30); patients with T1D lasting 2-5 years (T1D 2- 5) (n = 26) and individuals expressing pancreatic autoantibodies without diabetes (AbP) (n = 25), which were compared to healthy controls (n = 29). Expression of the microRNAs was obtained with individual assays TaqMan® MicroRNA Assays 5x primers and TaqMan MicroRNA Human Array Card A (Applied Biosystems-Forster City CA, USA), consisting of 377 targets and 4 endogenous. The expression data was analyzed in the Cloud Software (Thermo Fisher Scientific) and Limma (Linear Models for Microarray and RNASeq Data) program. RESULTS: There was no difference in demographic characteristics, such as age, self-reported color, and sex among groups (p > 0.05). Patients with T1D (both recent and 2-5 years), similar to each other, differed from the control group by high glucose, glycated hemoglobin levels, pancreatic autoantibody titers, and lower C peptide values (p < 0.05) . Pancreatic autoantibodies (AbP) carriers had intermediate characteristics among the groups: lower HbA1c and anti-tyrosine phosphatase antibody (anti- IA2) values and higher C-peptide levels than the two groups with diabetes. They differed from controls only by the higher titers of anti-insulin (IAA) and anti-decarboxylase of glutamic acid 65 (anti-GAD65) autoantibodies. The frequency of high risk HLA alleles for diabetes (-DR3 or -DR4 and -DQ2 or DQ- 8) decreased from the recent T1D and T1D 2-5 groups to the AbP and controls. We evaluated 135 miRNAs that were expressed in 20% or more of the samples from the four groups analyzed. Higher expression was observed in 13, 4 and 33 miRNAs of the Abp, recent T1D and T1D 2-5 groups respectively and lower in 11, 7 and 31 miRNAs of these groups. Of these, 4 miRNAs were differentially expressed in the AbP, recent T1D and T1D 2-5 groups in relation to the control group.The miRNAs: miR -16, miR-195 and miR-454, related to endocrine regeneration of the pancreas, anti-inflammatory effect and response to beta-cell injury were decreased in these 3 groups. miR-200a, implicated in beta-cell apoptosis, was increased in the recent and decreased AbP and T1D groups in patients with longer duration of diabetes (T1D 2-5y), possibly due to the shortage of these cells. Another eight miRNAs showed different expression of the control group in two of the evaluated groups, and a similar trend in the third group, four of them high (miR-193a-5p, miR-323-3p, miR-423-5p, and miR- 92a ) and four, decreased (miR-191, miR-19a, miR-376a, miR-590-5p) or neutrality in the 3rd group (miR-15b, miR-100, miR-181a and miR-483-5p) was observed for miR-25 and miR-485-3p, decreased in the AbP group and increased in T1D 2-5y. Such miRNAs are related to immune response, insulin secretion, ?-cell damage and glycotoxicity, similar to that observed for the miR- 101-3p, validated by individual trials in a larger cohort. CONCLUSION Our data suggests that circulating miRNAs may be involved in the pathogenesis of T1D

The Major Histocompatibility Complex (MHC) encodes proteins critical to the vertebrate immune response; therefore MHC diversity is an indicator of population health. I have (1) Isolated exon 2 of the class II gene DQA in Tursiops truncatus in the North Indian River Lagoon (IRL) (n=17), South IRL (n=29) and adjacent Atlantic waters (n=20), (2) assessed genetic variability between groups, (3) developed a method to genotype individuals, (4) typed 11 unique alleles in 66 individuals, (5) detected geographic patterns of diversity between estuarine and coastal individuals (FST=0.1255, p<0.05), (6) found evidence of positive selection centered in the binding pockets P1, P6 and P9 of the peptide binding region (w=2.08), (7) found that patterns of polymorphism did not closely match patterns of diversity in neutral markers, (8) performed a pilot study with Orcinus orca. The initial findings highlight the need for further comparative work and suggest that silent mutations are not neutral. / by Tatiana Ferrer. / Thesis (M.S.)--Florida Atlantic University, 2013. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.