Helicobacter pylori are unique in their ability to colonise the human gastric mucosa. They persist lifelong in untreated individuals despite the presence of a continuous and specific immune response being mounted against it. H. pylori O-antigen is thought to be involved in immune-evasion and subversion by the bacteria and expression has been shown to facilitate colonisation and exacerbate pathology in murine models. This study investigates immuno-relevant roles of H. pylori O-antigen as a pathogen-associated molecular pattern (PAMP) and its interaction with two pattern recognition receptors (PRRs); galectin-3 and DC-SIGN. These PRRs possess distinct carbohydrate recognition domain (CRD) structures and binding affinities. Despite this, we have demonstrated that they compete for adhesion to both Lewis antigen glycoconjugates and whole cell H. pylori 26695 in solid phase binding assays. Galectin-3 significantly reduces DC-SIGN adhesion at a 2:1 stoichiometric ratio in both Lex glycoconjugate and whole cell H. pylori 26695 assays, and abrogates carbohydrate-specific binding in Lex glycoconjugate assays at a 22:1 ratio. These results suggest that galectin-3 may play a role in inhibiting or modulating the interaction between H. pylori O-antigen and DC-SIGN in vivo. Supporting this, we have shown that galectin-3 secreted by AGS cells during competitive infection with H. pylori 26695 is sequestered by H. pylori O-antigen. We have demonstrated that competitive infection of the O-antigen deficient mutant H. pylori 26695 galE in DC-SIGN expressing THP-1 cells reveals a significant reduction in intracellular survival at 8 hours compared to H. pylori 26695 Wt. Co-incubation of H. pylori 26695 Wt with 10 µg ml-1 galectin-3 reduced intracellular survival to the levels of H. pylori 26695 galE at 8 hours. Furthermore, H. pylori 26695 galE displayed rapid association of the endocytic markers Rab5 and Rab7 at 15 minutes compared to H. pylori 26695 Wt. Monoclonal antibody-mediated blocking of DC-SIGN in H. pylori 26695 Wt-THP-1 infections resulted in rapid association of the endocytic markers Rab5 and Rab7, corresponding to that of H. pylori 26695 galE, indicating that DC-SIGN-O-antigen interactions alters intracellular processing of the bacteria and reduces the rate at which these markers are recruited. Together these results elucidate novel mechanisms of H. pylori O-antigen and its interaction with galectin-3 and DC-SIGN that warrant further investigation in vivo. The identification of two PRRs competing for the same PAMP is unconventional and inspires a re-evaluation of PRRs in innate immune recognition.

HCMV is a common viral pathogen that infects most of the world's population by early adulthood. It is typically asymptomatic in immunologically healthy individuals but causes severe disease in immunocompromised patients and congenitally infected infants. HCMV glycoproteins are highly polymorphic, and various types of associations have been suggested between glycoprotein types and the pathogenicity of the virus. Several studies on viruses other than HCMV have related the glycosylation of the viral glycoproteins to virulence. This project aimed to determine whether there is a robust relationship between the individual glycoprotein sequence and its glycosylation, how this influences the growth characteristic of the virus and whether this is related to its pathogenicity. Glycosylation patterns of 89 clinical specimens of different infection categories and specimen types were correlated with genetic sequence alterations of the virus glycoproteins (gB, gH, gL, gM, gN, gO), followed by determining whether mutation results in specific changes in glycosylation. The aim was approached using a cell culture model and a quantitative lectin-based assay (ELLA). A significantly increased glycosylation level for the following genotypes: mixed gH, gN4a, gO4, mixed gL was detected. Whereas a decreased pattern was found to be associated with gH1, gH2, gN3a, gO1a and gL2 genotypes (P < 0.05). Glycoproteins of strains isolated from respiratory specimens were significantly highly glycosylated compared to the blood and urine samples, and from blood specimens compared to the urine samples (P < 0.05). Furthermore, strains from congenitally infected infants and urine samples had a significantly higher growth rate than others tested. No direct association between the virus growth and its virulence was found. These findings demonstrate that glycosylation of glycoproteins in HCMV is affected by the glycoprotein polymorphisms and signifies a potentially important mechanism for avoidance of antibody-mediated neutralization, which, in turn, facilitates HCMV pathogenicity. This phenomenon requires further study and may have application for the selection of novel targets for diagnosis, vaccine development and other preventive measures to combat diseases caused by this virus.

L'incidence des maladies hépatiques est en augmentation, ce qui rend urgente la recherche de systèmes de vectorisation d'agents thérapeutiques vers le foie. La maladie sur laquelle porte notre étude est la maladie orpheline de Wilson. Elle induit une accumulation de cuivre (Cu) principalement dans les hépatocytes et les chélateurs de Cu systémiques utilisés à ce jour pour la traiter présentent de nombreux effets secondaires. C'est dans ce contexte que nous proposons une stratégie innovante qui consiste à piéger sélectivement le cuivre en excès dans les cellules hépatiques. Des chélateurs sélectifs du Cu(I), degré d'oxydation du Cu disponible intracellulaire, ont été conçus. Afin de cibler ces derniers vers les cellules hépatiques, nous avons focalisé notre intérêt sur le récepteur aux asialoglycoprotéines (ASGP-R), une lectine abondamment et presque exclusivement exprimée à la membrane des hépatocytes.Deux familles de glycoconjugués contenant une unité de ciblage associée à une unité de vectorisation des hépatocytes ont été développées : l'une possède une plateforme cyclodécapeptidique et l'autre une plateforme pseudopeptidique d'architecture tripodale. Les unités de ciblage sont composées de N-acétylgalactosamines présentés de manière multivalente, permettant une reconnaissance et une endocytose sélective des glycoconjugués dans les hépatocytes par l'intermédiaire de la lectine ASGP R. Les deux familles de composés ont montré leur capacité à entrer dans les cellules hépatiques et à faire diminuer le taux de Cu intracellulaire disponible.L'objectif de ce travail de thèse est l'étude et l'optimisation du système de ciblage. Les efficacités d'internalisation des deux familles de glycoconjugués dans les hépatocytes sont pour cela comparée. En outre, l'influence de la structure moléculaire des glycoconjugués sur l'efficacité de ciblage est étudiée. Plusieurs glycoconjugués appartenant à chacune des deux familles de composés ont donc été synthétisés, et leur propriétés d'internalisation dans les hépatocytes humains analysée par cytométrie en flux. / Regarding the increasing incidence of liver diseases in the past decades, the discovery of drug delivery systems is becoming a major research area. In particular, the Wilson disease is a liver dysfunction, which needs more specific treatments than those available nowadays. This genetic disorder induces a toxic copper overload in the hepatocytes. Because current copper chelating therapies present many side effects due to their lack of specificity, we propose an innovative strategy that would selectively detoxify copper in liver cells. Since excess intracellular Cu is in the +I oxidation state, we figured that a chelator that would enter the hepatocytes and be specific for Cu(I) could represent an efficient strategy. To selectively target the hepatic cells, we focus our interest on a lectin, which is highly and exclusively expressed in the membrane of the hepatocytes: the asialoglycoprotein receptor (ASGP-R).Two different kinds of glycoconjugates that contain a Cu(I) chelating unit associated to liver targeting systems have been obtained : one has a cyclodecapeptide scaffold and the other a tripodal pseudopeptide core. The targeting units are composed of N-acetylgalactosamine (GalNAc) residues multivalently presented, allowing the selective recognition and endocytosis of the glyconconjugates thanks to ASGP-R. These two families of molecules were demonstrated to enter hepatocytes and to chelate intracellular copper.The aim of the present work is to study and optimise the targeting system. We have compared the efficiency of the targeting systems of the two families of molecules. Moreover the influence of several structural parameters on the internalisation efficiency was determined. Several compounds belonging to cyclodecapeptide and tripode families have been synthesised, and their ability to enter in human hepatocytes has been analysed by flow cytometry.

FundaÃÃo de Amparo à Pesquisa do Estado do Cearà / Lectinas sÃo proteÃnas que atravÃs de ligaÃÃes a resÃduos de carboidratos podem interagir com sistemas biolÃgicos elicitando uma diversidade de efeitos. As lectinas vegetais tÃm sido utilizadas como ferramentas no estudo da inflamaÃÃo devido a sua capacidade de reconhecer resÃduos de carboidratos presentes nas membranas das cÃlulas inflamatÃrias atravÃs de seus domÃnios lectÃnicos. Assim, investigou- se neste trabalho o possÃvel efeito prÃ-inflamatÃrio da lectina de sementes de Dioclea rostrata; ligadora de &#945;-metil-D-manosÃdeo sobre a migraÃÃo de neutrÃfilos (MN) [(in vivo e in vitro)]. Os modelos utilizados foram: peritonite, edema de pata e bolsa de ar subcutÃnea (in vivo), quimiotaxia de neutrÃfilos e cultura de macrÃfagos (in vitro). Foi verificado que a lectina apresentava atividade prÃ-inflamatÃria em todos os estudos realizados. O aumento do nÃmero de macrÃfagos atravÃs do prÃ-tratamento dos animais com tioglicolato potencializou a MN induzida por Dros; a depleÃÃo de mastÃcitos atravÃs do tratamento com o composto 48/80 nÃo interferiu na MN da lectina. Sendo assim sugeriu-se o envolvimento de macrÃfagos e desconsiderou-se o de mastÃcitos. Na modulaÃÃo farmacolÃgica no modelo de peritonite feita atravÃs do prÃ-tratamento dos animais com drogas anti-inflamatÃrias, observou-se que indometacina, dexametasona e talidomida inibiram a MN. A lectina induziu quimiotaxia in vitro, estimulou a sÃntese/liberaÃÃo de citocinas como TNF-&#945; e IL-1, IL-10, desta maneira sugere-se que estudos mais detalhados sejam realizados em continuidades a este trabalho para verificar se esta lectina pode ser utilizada em modelos de imunosupressÃo. O &#945;-metil-D-manosÃdeo reverteu a MN induzida por Dros, desta forma parece que a Dros desencadeia resposta inflamatÃria atravÃs da interaÃÃo de seus domÃnios lectÃnicos com aÃucares presentes na membrana de macrÃfagos / Lectins are proteins possessing a carbohidate moiety that are able to interact with biological systems eliciting a variety of effects. Vegetal lectins have been used as tools in the study of inflammation due to its ability to recognize carbohydrate residues in inflammatory cell membranes by means of its lectin domain. The present work studied the pro-inflammatory effects of the lectin from Dioclea rostrata seeds (Dros), a binder of &#945;-methyl-D-manoside, on neutrophil migration ( NM ) [(in vivo and in vitro)]. The models of peritonitis, paw edema, and subcutaneous air pouch (in vivo), neutrophil chemotaxy and macrophage culture (in vitro) were utilized. It was found that Dros showed a pro-inflammatory activity in all of the above models. The increase in the number of macrophages by the pre-treatment of the animals with thioglycolate potentialized the Dros-induced NM. Also, the depletion of mast cells by the use of the substance 48/80 did not interfere in the lectin-induced NM. The above data suggest the involvement of macrophages but not mast cells in the mechanisms studied. The pre-treatment of the peritoneum with anti-inflammatory drugs like indomethacine, dexamethasone and thalidomide inhibited the NM. Dros induced chemotaxy in vitro and stimulated the synthesis / release of cytokines as TNF-&#945; and IL-1 in addition to IL-10 and this way he/she suggests himself that more detailed studies are accomplished in continuities to this work to verify this lectin can be used in imunosupression models. In the paw edema model the lectin promoted an intense edema and a significant increase in the mieloperoxidase activity (when compared to the control group). The &#945;-methyl-D-manoside reversted the Dros-induced NM and so it seems that Dros triggers the inflammatory response by means of the interaction of its lectin domain with sugars in the macrophage membrane. The present data suggest that Dros could be used as tools in biochemical and pharmacological studies.

Mise en oeuvre de microréseaux de lectines naturelles et recombinantes dédiés au suivi de production des glycoprotéines d’intérêt thérapeutiqueLes anticorps thérapeutiques, biomédicaments dont le marché est en pleine expansion, sont des glycoprotéines obtenues en bioproduction dans des cellules eucaryotes. Leur N-glycosylation, cruciale pour la modulation des fonctions effectrices des anticorps, confère un effet pro-inflammatoire ou anti-inflammatoire aux anticorps. Les anticorps thérapeutiques recombinants actuellement commercialisés présentent des glycosylations dites tronquées (GlcNAc terminaux) ou non humaines (α-Gal, NeuGc) rendant les anticorps immunogènes et, par conséquent, réduisant notablement leur efficacité. Les Agences de Santé exigent de réduire cet effet secondaire. La société SiaMed’Xpress a développé un savoir-faire pour obtenir une glycosylation complète, incluant la sialylation terminale des N-glycanes par ingénierie des cellules eucaryotes. Il est nécessaire de disposer d’outils d’analyse permettant de suivre la qualité de la N-glycosylation au cours de la production.La modification du milieu de culture par ajout de suppléments nutritionnels permet une modulation supplémentaire de la glycosylation. L’utilisation de suppléments nutritionnels commerciaux augmentent le taux de production mais bloquent la glycosylation au stade G0(F) (GlcNAc terminaux) alors que l’utilisation d’un supplément propre à SiaMed’Xpress permet de poursuivre jusqu’à la galactosylation et la sialylation, mettant ainsi en évidence un carrefour métabolique clé dans la glycosylation. Pour suivre les modifications de glycosylation dans différentes conditions de production, un test utilisant des lectines a été développé. Un jeu de treize lectines naturelles et recombinantes a été sélectionné par analyse bio-informatique et intégré dans un glycotest. L’utilisation de ce glycotest pour l’analyse de la glycosylation de trois anticorps produits en présence de différents suppléments nutritionnels permet d’obtenir de façon rapide, fiable et reproductible les profils de glycosylation des anticorps. Ainsi, le glycotest développé est fonctionnel et permet de caractériser la glycosylation des anticorps thérapeutiques recombinants. / Development of natural and recombinant lectin microarrays for monitoring the production of glycosylated protein drugsTherapeutic antibodies, biologic drugs whose market is growing, are glycoproteins obtained though bioproduction in eukaryotic cells. Their N-glycosylation, which is crucial for the modulation of effector functions, confers a pro-inflammatory or anti-inflammatory effect to antibodies. Currently, marketed therapeutic recombinant antibodies have truncated (terminal GlcNAc) or non human (α-Gal, NeuGc) glycosylation, inducing immunologic reaction and, consequently, reducing their effectiveness. Health agencies demand to reduce such secondary effects. SiaMed’Xpress developped a knowledge to obtained a complete glycosylation, including terminal sialylation of N-glycans by engineering eukaryotic cells for production. It is now necessary to develop analysis tools to monitor the quality of the N-glycosylation during bioproduction.Modification of culture media with feeds allows to further modulate glycosylation. The use of marketed feeds increase the production rate but block the glycosylation process in the G0(F) state (terminal GlcNAc) whereas SiaMed’Xpress feed allows for galactosylation and sialylation, highlighting a metabolic key point during the glycosylation. To follow the glycosylation modifications under different conditions of production, an assay using lectins has been developed. A panel of 13 lectins, recombinants and naturals, has been determined by bio-informatic analysis and integrated into a glycotest. The use of this glycotest to analyse glycosylation of 3 antibodies produced with different feeds allows fast, reliable and reproductible glycosylation profils of these antibodies. So, this developped glycotest is functionnal and this using permits to monitore therapeutic recombinant antibody glycosylation during bioproduction.

β2-microglobulin (β2m) exists free of the major histocompatibility complex class I (MHC I) receptor in many bodily fluids. The amount of protein present in these fluids has been found to be a useful prognostic marker for various diseases but outside of its practical value not much is known about this form of β2m. In fish, soluble β2m has not been studied at all. Another unknown in fish is the effects that plants lectins might have on naturally carnivorous species in aquaculture. These plant proteins which bind to specific sugar groups found on cells have been shown to have a multitude of gastrointestinal and immune effects in mammals and can be found in the plant products being fed to carnivorous, cultured fish making them possible toxicants. The two studies of this thesis set out to pioneer knowledge on these subjects using rainbow trout as a model. The first investigation inspected the various body fluids of these fish for their free β2m content. Soluble β2m was found to be present in the plasma, the seminal fluid, ovarian fluid, and the mucus of the skin and intestines. This distribution shows that β2m could indeed make a good biomarker, not only for disease but also for pheromone release and alludes to some possible functions of soluble β2m while opening the way for future research on this form of the protein. The second study looked at the effects of lectins on the gut of rainbow trout by treating RTgutGC, an intestinal epithelial cell line derived from trout, with plant lectins from wheat (WGA) and soybean (SBA), among others. This study found WGA to be a potent inducer of morphological and cytotoxic effects in these cells while other lectins and plant factors were not. WGA was also observed to effect the expression of β2m and the α-chain of the MHC I receptor. This work suggests WGA ingested by trout through the wheat in their diet might be causing them harm and should be studied further. It is also interesting that both studies related β2m to the intestines of trout. This could allow soluble β2m to serves as a marker of WGA’s effect or for WGA to aid in the study of free β2m.

Thesis (Ph. D.)--Institute of Paper Chemistry, 1982. / Includes bibliographical references (p. 104-108).

Tyrimo metodai: Ląstelių sėjimo metodika; ląstelių tankio nustatymas; ląstelių gyvybingumo nustatymas MTT testu; neuronų žūties įvertinimas fluorescencinės mikroskopijos metodu; viduląstelinių RS kiekio įvertinimas; ekstraląstelinių RS kiekio įvertinimas; statistinė analizė. Tyrimo tikslas: Ištirti įvairių koncentracijų lektinų, išskirtų iš Phaseolus vulgaris L., antioksidacinį/ prooksidacinį poveikį ir įtaką C6 ląstelių gyvybingumui. Tyrimo uždaviniai: 1. Atlikti literatūros duomenų analizę apie lektinų, išskirtų iš Phaseolus vulgaris L., biologinį poveikį; 2. Nustatyti įvairių koncentracijų lektinų, išskirtų iš Phaseolus vulgaris L., antioksidacinį/prooksidacinį aktyvumą; 3. Nustatyti įvairių koncentracijų lektinų poveikį C6 ląstelių kultūros gyvybingumui. Tyrimų rezultatai: 1. Buvo matuojami vandenilio peroksido pokyčiai esant 1 - 10 µg lektino koncentracijoms ir nustatyta, kad skirtingų koncentracijų lektinas neveikia neutralizuojančiai vandenilio peroksido kiekio. 2. Atlikti vandenilio peroksido matavimai DMEM terpėje su 1 - 10 µg lektino koncentracijoms parodė, kad lektinas, priklausomai nuo jo koncentracijos, negeneruoja laisvųjų radikalų. 3. Buvo matuojamas glioblastomos C6 ląstelių gyvybingumas su MTT po 24 valandų ir nustatyta, kad didėjant lektino koncentracijai ląstelių gyvybingumas sparčiai mažėjo: 5 µg koncentracijos lektinas gyvybingumą sumažino beveik 2 kartus (iki 57,2111.16%), o 10 µg koncentracijos lektinas ląstelių gyvybingumą sumažino beveik 13... [toliau žr. visą tekstą] / Methods: The cell cultivation method; measurement of cell density; determination of cell viability with MTT assay; assessment of neuronal death with fluorescent microscopy; measurement of intracellular RS; measurement of extracellular RS; statistical analysis. The aim of research: To investigate antioxidative/prooxidative effects of different concentrations of the lectin, isolated from Phaseolus vulgaris L., and influence on the viability of the C6 cells. Goals of the study: 1. To perform an analysis of the literature on the biological effects of lectin, isolated from Phaseolus vulgaris L.; 2. To determine antioxidative/prooxidative activity of different concentrations of the lectin, isolated from Phaseolus vulgaris L.; 3. To determine effect on viability of the C6 cell culture using different concentrations. Results: 1. Concentrations of hydrogen peroxide were measured with 1 - 10 µg concentrations of lectin and it was found, that different concentrations of lectin did not neutralize hydrogen peroxide. 2. Measurements of hydrogen peroxide in DMEM medium with 1-10 mg concentrations of lectin showed, that the lectin, depending on it's concentration, did not generate free radicals. 3. Viability of cells was measured in C6 glioblastoma cells with MTT after 24 hours and it was found, that when concentration of lectin increased, viability of cells rapidly decreased: 5 µg concentration of lectin reduced the viability of almost 2 times ( up to 57.21% ± 11.16% ) and at 10 µg... [to full text]

R. Staršelskytės magistro baigiamasis darbas/ mokslinė vadovė prof. N. Savickienė; Konsultantės: dr. Annabella Vitalone, prof. Gabriela Mazzanti, dr. Antonella Di Sotto; Lietuvos sveikatos mokslų universiteto, Farmacijos fakulteto, Farmakognozijos katedra. – Kaunas. Romos universiteto La Sapienza, Fiziologijos ir farmakologijos katedra. – Roma. Darbo tikslas: baltymų frakcijų, praturtintų lektinais, išskirtų iš Urtica doica L. žolės, antimutageninio, citotoksinio ir antioksidacinio aktyvumo įvertinimas. Darbo uždaviniai: 1. Įvertinti Urtica dioica L. baltymų frakcijų įtaką Salmonella typhimurium ir Escherichia coli kamienų mutageniškumui, naudojant AMES testo metodą. 2. Ištirti Urtica dioica L. baltymų frakcijų citotoksiškumą HepG2 ląstelių proliferacijai, naudojant MTT dažo redukcijos reakcijos metodą. 3. Ištirti Urtica dioica L. baltymų frakcijų antioksidacinį aktyvumą, naudojant modelinius ABTS (2,2-azino-bis-(3-etilbenztiazolin-6-sulfono rūgšties)) ir superoksido radikalus. Metodai: 1. Lektinais praturtintų baltymų frakcijų antimutageniškumas tiriamas AMES testo metodu (grįžtamosios mutacijos modeliu in vitro) su S9 metabolinės aktyvacijos sistema (supernatantu iš žiurkių (paveiktų fenobarbitalio/β-naftoflavono mišiniu) kepenų lątelių mitochondrijų) ir be S9 metabolinės aktyvacijos sistemos. Eksperimentui naudojami trys bakterijų kamienai: S. typhimurium TA98, S. typhimurium TA100 ir E. coli WP2uvrA. 2. Citotoksiškumas nustatomas MTT dažo redukcijos reakcijos metodu... [toliau žr. visą tekstą] / Rasa Staršelskytė master thesis/ Supervisor of the research paper: prof. Nijolė Savickienė1 Consultants: PhD Annabella Vitalone, prof. Gabriela Mazzanti, PhD Antonella Di Sotto2 1Department of Pharmacognosy, Faculty of pharmacy, Lithuanian University of Health Sciences, Lithuania 2Department of Physiology and Pharmacology, Sapienza University of Rome, Italy Objective of work: evaluation of antimutagenicity, cytotoxicity and antioxidant activity of lectin-enriched protein fractions from herb of Urtica dioica L. Main tasks: 1. To evaluate antimutagenic activity of lectin-enriched protein fractions by bacterial reverse mutation assay. 2. To determine cytotoxicity of lectin-enriched protein fraction by the tetrazolium dye (MTT) colorimetric assay. 3. To evaluate antioxidant activity of lectin-enriched protein fraction against ABTS-free radical and superoxide-radical. Methods: 1. The antimutagenicity was studied in a bacterial reverse mutation assay (Ames test), both in the absence and presence of an exogenous metabolic activator S9 (the liver postmitochondrial supernatant of rats treated with the mixture phenobarbital/β-naphthoflavone to induce the hepatic microsomal enzymes). A set of three strains, S. typhimurium TA98, S. typhimurium TA100 and E. coli WP2uvrA, was used. 2. Cytotoxicity was determined by the tetrazolium dye (MTT) colorimetric assay in HepG2 human hepatoblastoma cell line. 3. The antioxidant activity was evaluated by ABTS-free radical scavenging activity test... [to full text]

Le premier but de mon projet de doctorat a ete la caracterisation moleculaire de l'interface entre les recepteurs activateurs des cellules Natural Killer (NK) et de leurs ligands exprimes dans les cellules infectees ainsi que l'implication de cette interaction sur la reponse a l'infection par le cytomegalovirus (MCMV). / J'ai tire un avantage de la variation naturelle au sein des membres lies aux recepteurs Ly49C ainsi que de la disponibilite des structures cristallines des Ly49 afin de comprendre les determinants moleculaires des interactions Ly49H-m157 et egalement identifier les residus des acides amines qui permettent de discriminer entre les recepteurs qui se lient et ceux qui ne se lient pas a m157. Mes decouvertes suggerent que le "site 2" du contact entre le CMH de classe I et Ly49 n'est visiblement pas implique dans la liaison avec m157. Au contraire, les residus localises au niveau de l'interface homodimere-recepteur seraient probablement critiques a la reconnaissance fonctionnelle de la glycoproteine m157. Notre approche fonctionnelle et de modelisation tridimensionnelle suggerent que l'architecture du dimere Ly49H est cruciale pour l'accessibilite de m157 mais non pour les molecules de CMH de classe I et relient la variabilite dans l'homodimerisation des Ly49 a la reconnaissance directe des produits pathogeniques. / Un autre mecanisme de la reponse de l'hote contre MCMV provient de l'etude de la souche de souris MA/My, laquelle, malgre l'absence du gene Ly49h ainsi que la proteine pour laquelle il code, y est hautement resistant. Des etudes anterieures ont demontre qu'une interaction epistatique entre un gene issu du groupe des genes Ly49 sur le chromosome 6 et le CMH (H2) sur le chromosome 17 est associee avec la resistance au virus. Utilisant une methode de co-culture de cellules reportrices NFAT-GFP exprimant les recepteurs activateurs Ly49 et de fibroblastes primaires infectes, j'ai montre que le recepteur activateur Ly49P des cellules NK reconnait specifiquement les cellules infectees par MCMV et que cette reconnaissance depend de la presence de l'haplotype H2k. Ce signal etait bloque par l'utilisation des anticorps anti-H2-D k mais non par anti-H2-Kk. Ces resultats indiquent l'existence d'un nouveau mecanisme des cellules NK implique dans la resistance au MCMV, lequel depend de l'interaction fonctionnelle entre le recepteur Ly49P et la molecule du MHC de classe I, H2-Dk, dans les cellules infectees par MCMV. Comme contribution directe de ce travail, nous avons demontre que la resistance chez MA/My est au moins partiellement dependante des interactions entre le recepteur Ly49P et la molecule H2-Dk modifiee par le virus dans les cellules infectees. Comme MCMV regule negativement l'expression des molecules du CMH de classe I, j'ai confirme la presence de H2-Dk dans les cellules infectees par l'utilisation d'un virus MCMV recombinant portant un gene rapporteur GFP. En permutant la plateforme peptidique de liaison, les domaines transmembranaires et intracellulaires entre les molecules H2-Db et H2-D k, j'ai demontre que la plateforme peptidique de liaison est critique pour la reconnaissance des cellules infectees. Par le criblage d'un panel de mutants MCMV portant des genes impliques dans l'evasion immune, j'ai demontre que l'infection de fibroblastes par le MCMV depourvu du gene m04 (Deltam04) abolit totalement l'activation de Ly49P. (Abstract shortened by UMI.)