L'inflammation est un processus important dans lequel le systeme immunitaire reagit a un irritant. Plus precisement, l'inflammation aide a defendre le corps humain contre les agents pathogenes. Cependant, une inflammation chronique et incontrolee peut entrainer des pathologies telles que l'atherosclerose, l'asthme et le cancer. L'enzyme 5-lipoxygenase (5-LO), codee par le gene ALOX5, est principalement exprimee par les leucocytes et joue un role central dans la reaction immunitaire. C'est la premiere enzyme engagee pour la biosynthese de leucotrienes (LT) a partir de l’acide arachidonique. L'activite biologique des LT est diverse, mais principalement le LTB4 induit une chimiotaxie des phagocytes et la liberation d'effecteurs antimicrobiens, alors que les cysteinyl leucotrienes (LTC4, LTD4 et LTE4) induisent la bronchoconstriction et la permeabilite vasculaire. Cette these porte sur 1) les effets biologiques des metabolites du LTB4 sur les fonctions des neutrophiles et sur 2) les mecanismes par lesquels l’expression de la 5-lipoxygenase est regulee dans les monocytes dans le contexte de la defense de l’hote et de l’atherosclerose. Specifiquement, le LTB4 est rapidement degrade en 20-OH- et 20-COOH-LTB4 par les neutrophiles humains. Bien que ces metabolites se lient au recepteur BLT1 avec une haute affinite, ils sont generalement consideres comme inactifs car compare au LTB4, ils activent considerablement moins les leucocytes. Par consequent, le premier objectif de cette these etait d'evaluer les effets biologiques du 20-OH- et du 20-COOH-LTB4 sur les fonctions des neutrophiles humains telles que la migration, la biosynthese des leucotrienes et la production d'effecteurs antimicrobiens. Ensuite, des etudes anterieures ont montre que la differenciation des cellules myeloides avec la 1,25-dihydroxyvitamine D3 et le TGF-β est associee a une forte augmentation de l'expression de la 5-LO. Etant donne que les lipopolysaccharides (LPS) induisaient egalement la maturation cellulaire entrainant une expression accrue du CD14, nous avons emis l'hypothese que le LPS pourrait moduler l'expression de la 5-LO dans des lignees monocytaires. En consequence, notre deuxieme objectif etait d'etudier l'impact du LPS sur l'expression de la 5-LO et la biosynthese des LT. Fait important, cette etude a ete la premiere a rapporter une induction de l’activite transcriptionnelle du promoteur ALOX5 d’une maniere agoniste-dependante. Enfin, une interaction entre l'apport alimentaire en acides gras polyinsatures (PUFA) et des mutations naturelles du promoteur ALOX5 a ete associee a un risque accru de maladies cardiovasculaires. Par contre, des etudes anterieures utilisant des essais rapporteurs avaient opte pour des modeles cellulaires non-humains ou 5-LO-negatifs afin d'etudier la regulation de la transcription de la 5-LO impliquant les polymorphismes du promoteur. Par consequent, en utilisant notre modele d’essais rapporteurs precedemment valide dans des cellules monocytaires humaines, le troisieme objectif de ma these etait de comparer l'activite transcriptionnelle des variants du promoteur ALOX5 distincts en presence ou en l'absence d'une supplementation en PUFA dans les cellules Mono Mac 6. En conclusion, nous avons cherche a mieux comprendre les mecanismes impliques dans la regulation transcriptionnelle de l'expression de la 5-LO et dans la reponse des neutrophiles aux metabolites du LTB4. Nos donnees contribuent a la comprehension du role crucial de la 5-LO et de ses produits sur la defense de l'hote et les maladies. / Inflammation is an important process where the immune system responds to an irritant. Specifically, inflammation helps defend the human body against pathogens. Chronic and uncontrolled inflammation, however, can lead to pathologies such as atherosclerosis, asthma, and cancer. The 5-lipoxygenase enzyme (5-LO), which is encoded by the ALOX5 gene, is mostly expressed by leukocytes and plays a central role in the immune reaction. It is the first committed enzyme for the biosynthesis of leukotrienes from arachidonic acid. The biological activity of leukotrienes is diverse, but mainly LTB4 induces phagocyte chemotaxis and the release of anti-microbial effectors, whereas the cysteinyl leukotrienes (LTC4, LTD4 and LTE4) increase bronchoconstriction and vascular permeability. This thesis focuses 1) on the biological effects of LTB4 metabolites on neutrophil functions and 2) on the mechanisms by which 5-lipoxygenase expression is regulated in monocytes in the context of host defense and disease. Specifically, LTB4 is rapidly degraded into 20-OH- and 20-COOH-LTB4 by human neutrophils. Although these metabolites bind to the BLT1 receptor with high affinity, they are generally considered inactive because they are considerably less capable of activating leukocytes in comparison to LTB4. Therefore, the first objective of this thesis was to evaluate the biological effects of the LTB4 metabolites, 20-OH- and 20- COOH-LTB4, on human neutrophil functions like migration, leukotriene biosynthesis, and production of antimicrobial effectors. Furthermore, previous studies found that the differentiation of myeloid cells with 1,25-dihydroxyvitamin D3 and TGF-β is associated with a strong increase in 5-LO expression. Since the endotoxin lipopolysaccharide (LPS) also induced cell maturation resulting increased CD14 expression, we hypothesized that LPS may modulate 5-LO expression in monocytic cell lines. In consequence, our second objective was to investigate the impact of LPS on 5-LO expression and LT biosynthesis. Importantly, this study was the first to report an agonist-dependent induction of ALOX5 promoter transactivation. Lastly, an association between dietary intake of polyunsaturated fatty acids (PUFA) and naturally occurring mutations in the ALOX5 promoter has been linked to an increased risk of cardiovascular diseases. Unfortunately, prior studies using reporter gene assays opted for both non-humans or 5-LO-negative cell models to study the transcriptional regulation of 5-LO expression in relations to the promoter polymorphisms. Therefore, by using our previously validated gene reporter model in human monocytic cells, the third objective of my thesis was to compare the reporter construct activity of distinct ALOX5 promoter variants in the presence or absence of PUFA supplementation in Mono Mac 6 cells. In conclusion, we sought to better understand the mechanisms involved in the transcriptional regulation of 5-LO expression and in the neutrophil response to LTB4 metabolites. Consequently, our data increases the comprehension of the crucial role of 5-LO and its products on host defense and diseases.

Les médiateurs lipidiques de l'inflammation comprennent plusieurs familles de molécules biologiquement actives, incluant le leucotriène B₄ (LTB₄) et le facteur activant les plaquettes (PAF). Ces médiateurs s'avèrent être particulièrement importants dans la pathogenèse de maladies inflammatoires comme l'arthrite. Effectivement, ils participent directement au recrutement et à l'activation des neutrophiles au site inflammatoire. La biosynthèse du LTB₄ et du PAF implique la phospholipase A₂ de groupe IVA (cPLA₂α); en effet, cette enzyme libère les substrats nécessaires à la biosynthèse de ces médiateurs à partir des phospholipides membranaires. Les mécanismes de régulation de la cPLA₂α sont encore aujourd'hui peu compris. Cette présente étude visait à mieux comprendre et à élucider de nouveaux mécanismes de régulation de la cPLA₂α, première enzyme impliquée dans la biosynthèse du LTB₄ et du PAF dans les neutrophiles humains. Les résultats démontrent que, dans les neutrophiles humains, l'activité de la cPLA₂α est inhibée par son produit, l'acide arachidonique (AA) et que l'AA induit la translocation de l'enzyme aux membranes. Nos résultats suggèrent également une interaction directe du produit (AA) avec l'enzyme et que cette interaction se fait probablement au site catalytique. Les résultats de cette étude ont permis de révéler un nouveau mécanisme de régulation de la cPLA₂α jamais identifié, la régulation négative de la cPLA₂α par l'AA. Le site d'interaction de l'AA sur l'enzyme lors de cette régulation reste à être confirmé.

A television image is formed by a series of parallel luminous lines called a raster. The visual prominence of the raster structure interferes with the extraction of information from the image. The raster may be suppressed experimentally by a deflection process called spot wobble. Experiments were conducted to determine the effects of raster structure suppression on visual sine wave modulation thresholds and dynamic target acquisition performance using normal and noise degraded imagery. Results indicate raster structure suppression and improvements in Sine wave threshold sensitivity are correlated and that a suppressed raster significantly improves target acquisition performance for noise-free conditions. Performance correlations with the modulation transfer function area (MTFA) image quality metric were not as good as were the correlations between observer task performance measures and areas under the threshold functions. A rationale for improving the efficacy of the MTFA image quality metric was postulated. / Ph. D.

The pine engraver beetles (~ spp.:Coleoptera:Scolytidae) may be serious pests depending on certain prerequisite conditions. In their secondary or "normal" role they breed in slash and damaged, dying, and dead trees. The broods emerging from these sources normally attack similar material. When such material is scarce due to cessation of cutting operations in mid-season or when conditions are especially favorable for brood development, an excess of beetles is often produced which, due to the lack of more suitable material, attack healthy trees. Repeated attacks cause these trees to succumb and die. When normally healthy trees are weakened by fire, flood, defoliation, drought, stagnation, etc., then they become more acceptable host material for successful engraver attacks. When the production of a very large number of beetles in "normal" breeding material coincides with physiological stress in "healthy" trees, then population explosions can occur. When large numbers of beetles and low host vigor do not coincide, spot kills cornnon1y occur. According to Thatcher (32), spot kills, although not conspicuous, add up to large volumes of timber loss each year. / Ph. D.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 5 uppsatser.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O influxo celular (IC) à sinÃvia participa na fisiopatologia da artrite reumatÃide (AR). Hà controvÃrsias sobre o papel do Ãxido nÃtrico (NO) na modulaÃÃo do influxo de neutrÃfilos para sÃtios inflamatÃrios, seja reduzindo ou estimulando-o. Esse trabalho investigou o efeito de inibidores de Ãxido nÃtrico sintase (NOS) sobre o IC agudo em animais submetidos à artrite ou peritonite induzida por zymosan (AZy ou PZy) ou lipopolissacarÃdeo (ALPS ou PLPS), bem como a participaÃÃo de leucotrieno B4 (LTB4) e da molÃcula de adesÃo intercelular -1 (ICAM-1). Ratos Wistar receberam 10-1000 micrograma de Zy ou 1-10 micrograma de LPS intra-articular (i.a.). Outros grupos receberam 1 mg de Zy ou 10 Âg de LPS intraperitoneal (i.p.). Camundongos selvagens ou geneticamente manipulados (knock out) para ICAM-1 (ICAM-1-/-) receberam 100 Âg de Zy i.a. ou i.p. Esquema dos prÃ-tratamentos (30 minutos antes da artrite ou peritonite): na AZy, L-NAME (1-30 mg/kg; i.p. ou 0,3-1 micromol; i.a), 1400W (1 mg/kg; i.p.) ou Aminoguanidina (Amino) (50 mg/kg; i.p.) em ratos e camundongos receberam L-NAME (3-10 mg/kg;i.p.) ou NG-nitro-L-arginia (Nitro) (50 mg/kg; i.p.). Na PZy, L-NAME (10-30 mg/kg; s.c. ou i.p.) ou 1400W (1 mg/kg; s.c.) foi administrado em ratos e camundongos receberam L-NAME (30 mg/kg; s.c.) ou Nitro (50 mg/kg; s.c.). Na ALPS, ratos receberam L-NAME (10-30 mg/kg; i.p) e, na PLPS, L-NAME (30 mg/kg; s.c.). Controles receberam veÃculo (grupos NT). ApÃs o sacrifÃcio, foram quantificados o IC e LTB4 nos lavados articulares e peritoneais. Na AZy (10-100 micrograma) em ratos, L-NAME (1-3 mg) reduziu o IC (47,4 - 76,6%), quando comparado aos animais NT (p<0,05). Na AZy 1mg, L-NAME (30 mg), 1400W (1 mg) e Amino (50 mg) diminuÃram o IC (57,4%, 74,8% e 76,6%) (p<0,05). L-NAME (0,3 micromol) i.a. tambÃm reduziu o IC (85,3%) (p<0,05). Semelhante aos ratos, camundongos prÃ-tratados com L-NAME (3-10 mg) ou Nitro (50 mg) apresentaram diminuiÃÃo do IC (67,6%, 53,8% e 39,5%) (p<0,05). Contrariamente, na PZy, L-NAME (10-30 mg) e 1400W (1 mg) aumentaram o IC (566,7%, 495,1% e 470,7%) em ratos e L-NAME (30 mg), em camundongos (155,8%) (p<0,05). Nesses, Nitro aumentou o IC, mas nÃo significativamente (p>0,05). L-NAME (10 mg) i.p. tambÃm aumentou o IC (747,6%) (p<0,05). Na ALPS, L-NAME (30 mg) diminuiu o IC nas duas doses de LPS (50,3% e 52,3%) (p<0,05). Na PLPS, L-NAME (30 mg) aumentou o IC (53,4%) (p<0,05). Os inibidores de NOS nÃo alteraram os nÃveis de LTB4. Animais ICAM-1-/- artrÃticos prÃ-tratados ou nÃo com Nitro apresentaram reduÃÃo do IC (57,8% ou 32,1%)(p<0,05). Nos ICAM-1-/- com peritonite prÃ-tratados ou nÃo com Nitro, houve uma reduÃÃo do IC (9,5% e 22,3%), mas nÃo significativa (p>0,05). O NO, particularmente o produzido pela NOSi, reduz o IC agudo na articulaÃÃo, enquanto que o incrementa no peritÃneo. Esse efeito à independente do estÃmulo, da espÃcie, da via de administraÃÃo e da liberaÃÃo de LTB4, alÃm de envolver cÃlulas residentes e/ou migradas. ICAM-1 parece participar do IC nesses modelos, especialmente na artrite, com o NO modulando sua expressÃo na peritonite. / Cell influx (CI) to synovium participates in physiopathology of rheumatoid arthritis (RA). There are controversies about the nitric oxide (NO) action in modulation of neutrophil influx to inflammatory sites, with NO decreasing or increasing it. This study investigated the effect of nitric oxide synthase (NOS) inhibitors on acute CI in animals submitted to arthritis or peritonitis induced by zymosan (ZyA or ZyP) or lipopolysaccharide (LPSA or LPSP), and the participation of leukotriene B4 (LTB4) and intercellular adhesion molecule -1 (ICAM-1). Rats Wistar received Zy (10-1000 micrograme) or LPS (1-10 micrograme) intraarticular (i.a.). Other groups received Zy (1 mg) or LPS (10 Âg) intraperitoneal (i.p.). Wild or ICAM-1-deficient (ICAM-1-/-) mice received Zy (100 Âg) i.a. or i.p. Animals were pre-treated (30 minutes before arthritis or peritonitis): in ZyA, rats received L-NAME (1-30 mg/kg; i.p. or 0.3-1 micromol; i.a), 1400W (1 mg/kg; i.p.) or Aminoguanidine (Amino) (50 mg/kg; i.p.). Mice received L-NAME (3-10 mg/kg;i.p.) or NG-nitro-L-arginine (Nitro) (50 mg/kg; i.p.). In ZyP, L-NAME (10-30 mg/kg; s.c. or i.p.) or 1400W (1 mg/kg; s.c.) was administrated in rats and mice received L-NAME (30 mg/kg; s.c.) or Nitro (50 mg/kg; s.c.). In LPSA, rats received L-NAME (10-30 mg/kg; i.p) and, in LPSP, they received L-NAME (30 mg/kg; s.c.). Controls received vehicle (NT groups). After sacrifice, CI was counted and LTB4 was measured in articular and peritoneal exudates. In ZyA (10-100 Âg), L-NAME (1-3 mg) reduced CI (47.4 â 76.6%) as compared to NT animals (p<0.05). In ZyA (1mg), L-NAME (30 mg), 1400W (1mg) and Amino (50 mg) reduced CI (57.4%, 74.8% and 76.6%, respectively) (p<0.05). L-NAME (0.3 Âmol) i.a. also reduced CI (85.3%) (p<0.05). Similarly, L-NAME (3-10 mg) or Nitro (50 mg) pre-treated mice showed a CI reduction (67.6%, 53.8% and 39.5%) (p<0.05). In contrast, in ZyP, L-NAME (10-30 mg) and 1400W (1 mg) increased CI (566.7%, 495.1% and 470.7%, respectively) in rats and L-NAME (30 mg) in mice (155.8%) (p<0.05). In mice, Nitro increased CI, but not significantly (p>0.05). L-NAME (10 mg) i.p. also increased CI (747.6%) (p<0.05). In LPSA, L-NAME (30 mg) decreased CI in two LPS doses (50.3% and 52.3%) (p<0.05). In LPSP, L-NAME (30 mg) increased the influx (53.4%) (p<0.05). NOS inhibitors didnât change LTB4 levels. Arthritic pre-treated or not with Nitro ICAM-1-/- animals showed a CI reduction (57.8% or 32.1%)(p<0.05). In peritonitis, pre-treated or not with Nitro ICAM-1-/- showed a CI reduction (9.5% and 22.3%), but not significantly (p>0.05). NO, especially that produced by iNOS, reduces the acute CI in articulation while increases it in the peritoneum. This effect is independent of stimulus, species, route of administration and LTB4 liberation. Beyond, it involves resident and/or migrated cells. ICAM-1 appears to participate in CI in these models, especially in arthritis. Besides, NO modulates the expression of ICAM-1 in peritonitis.

Les cellules NKT invariantes (iNKT) sont des lymphocytes T non conventionnels restreints par la molécule CD1d qui présente des glycolipides. Les cellules iNKT expriment un TCR avec une chaîne a invariante, Va14-Ja18 chez la souris et Va28-Ja18 chez l’homme. Elles ont la particularité de produire de grande quantité de cytokines (IFN-? et IL-4) rapidement après leur activation et peuvent à leur tour stimuler d’autres cellules du système immunitaire comme les cellules dendritiques, les cellules NK et les lymphocytes T. Elles représentent ainsi un pont entre les réponses immunitaires innées et adaptatives. Le diabète de type 1 est une maladie autoimmune caractérisée par la destruction des cellules ß pancréatiques productrices d’insuline. Bien que l’apparition de diabète de type 1 soit associée à des polymorphismes génétiques, les facteurs environnementaux ont également été impliqués dans l’étiologie de cette maladie. De nombreuses études suggèrent que les infections virales, en particulier les infections par le virus de coxsackie B4 (CVB4), pourraient être impliquées dans le développement de cette maladie. Notre étude a été réalisée avec des souris NOD qui développent un diabète de type 1 vers 15 semaines d’âge et des souris NOD déficientes pour la proinsulin 2 (Pro-ins2-/-) développant un diabète vers 8 semaines d’âge. Nos résultats montrent qu’après infection par CVB4, la moitié des souris NOD et Pro-ins2-/- développent un diabète accéléré par rapport à des souris non infectées. Toutefois, une injection de l’agoniste des cellules iNKT, la molécule aGalactosylceramide (aGalCer), au moment de l’infection des souris, diminue fortement l’incidence de diabète. L’infection par CVB4 induit un fort recrutement de macrophages dans le pancréas et l’activation des cellules iNKT modifie la fonction de ces macrophages. En effet, les macrophages pancréatiques des souris infectées par CVB4 expriment fortement les cytokines IL-1ß, IL-6 et TNF-a, révélant leur caractère pro-inflammatoire alors que les macrophages des souris infectées et traitées par aGalCer expriment faiblement ces cytokines inflammatoires et fortement des enzymes immunosuppressives iNOS (inducible NO synthase), IDO (Indoleamine 2,3-dioxygenase) et arginase I. L’utilisation d’inhibiteurs de ces enzymes montre que la protection contre le diabète est induite par IDO. Nous avons également observé une forte infiltration de lymphocytes T autoréactifs dans les îlots pancréatiques des souris infectées. De façon intéressante, l’incidence accrue de diabète du groupe CVB4 est associée à une fréquence élevée de cellules T autoréactives produisant de l’IFN-? dans le pancréas, alors que la production d’IFN-? par les cellules T autoréactives est très faible dans les souris du groupe CVB4+aGalCer. Cette inhibition de la production d’IFN-? est dépendante de l’enzyme IDO, car l’utilisation d’un inhibiteur d’IDO augmente fortement la production d’IFN-? par les lymphocytes T anti-îlots et l’incidence de diabète. Dans l’ensemble nos résultats montrent, que l’activation des cellules iNKT lors de l’infection par CVB4 induit des macrophages immunosuppresseurs dans le pancréas, ces cellules inhibant la fonction des lymphocytes T autoréactifs et ainsi le développement du diabète. / INKT cells are non-conventional T lymphocytes that are restricted to glycolipid presenting CD1d molecule. iNKT cells express an invariant TCR a chain (Va14-Ja18 in mice and Va28-Ja18 in humans). Their particularity is to rapidly produce copious amounts of cytokines (IFN-? and IL-4) after activation and to activate other cells of the immune system such as dendritic cells, NK cells and T lymphocytes. iNKT cells, therefore, form a bridge between innate and adaptive immune responses. Type 1 diabetes is an autoimmune disease characterized by the destruction of pancreatic ß cells whose role is to produce insulin. While diabetes development can clearly be associated with genetic polymorphisms, environmental factors were also implicated in the etiology of the disease. Numerous studies suggest that viral infections, particularly infections with Coxsackievirus B4 (CVB4), could be implicated in the development of type 1 diabetes. Our study was performed with NOD mice that develop type 1 diabetes around 15 weeks of age and with proinsulin 2 knockout NOD mice (Pro-ins2-/-) which become diabetic around 8 weeks of age. Our results show that CVB4 infection induces accelerated diabetes in around half of NOD and Pro-ins2-/- mice compared to uninfected mice. However, the activation of iNKT cells with their agonist, aGalactosylceramide (aGalCer), at the time of infection greatly decreases diabetes incidence. CVB4 infection induces a strong recruitment of macrophages into the pancreas. Interestingly, iNKT cell activation modifies the function of these macrophages. Indeed, pancreatic macrophages of CVB4 infected mice strongly express IL-1, IL-6 and TNF-a, indicating their pro-inflammatory character. On the contrary, macrophages of mice infected with CVB4 and treated with aGalCer express low levels of these cytokines, but strong levels of suppressive enzymes iNOS (inducible NO synthase), IDO (Indoleamine 2,3-dioxygenase) and arginase I. The use of inhibitors of these enzymes showed that diabetes prevention is induced by IDO. We have also observed that autoreactive T cells strongly infiltrate the pancreatic islets after CVB4 infection. It is interesting to note that the high diabetes incidence of CVB4 infected mice is associated with an increased frequency of IFN-? producing autoreactive T cells in pancreatic islets. On the contrary, the frequency of these cells is very low in infected mice treated with aGalCer. The inhibition of IFN-? production is dependent on IDO enzyme, since the use of its inhibitor strongly increases IFN-? production by anti-islet T cells and diabetes incidence. To summarize, our results show that iNKT cell activation during the infection with CVB4 induces immunosuppressive macrophages in the pancreas. These cells inhibit the function of autoreactive T cells and prevent diabetes development.

Les récepteurs couplés de protéines G (RCPG) sont des capteurs biologiques polyvalents responsables de la majorité des réponses cellulaires aux hormones et neurotransmetteurs ainsi que des sens de la vue, de l'odorat et du goût. La transduction des signaux est associée à un ensemble de changements dans la structure tertiaire des récepteurs entraînant l'activation de partenaires intracellulaires dont les protéines G. La dimérisation est un élément central du mode de fonctionnement des RCPG ; cependant, son influence sur la façon dont le signal est transmis est encore mal définie.Nous avons utilisé ici le récepteur BLT1 du leucotriène B4 comme modèle afin d'analyser les changements de conformation au cours de l'activation. Pour cela, nous avons produit le récepteur suivant une approche qui consiste à l'exprimer dans les corps d'inclusion bactériens puis à le renaturer à l'aide de détergents et/ou surfactants originaux. L'accès au récepteur purifié nous a permis de montrer que la protéine G induit une asymétrie dans les changements de conformation au sein de l'homodimère de BLT1. De plus, nous avons pu établir que l'activation de la protéine G se fait essentiellement par le protomère ayant fixé l'agoniste (cis-activation). Enfin, nous avons montré que la forme monomérique du récepteur est parfaitement capable d'induire l'activation de la protéine G, même si le dimère apporte une modulation de la réponse. Ceci indique qu'un monomère de récepteur possède tous les déterminants moléculaires nécessaires à la transmission du signal. L'ensemble de ces résultats apporte un éclairage nouveau sur la façon dont les dimères de RCPG fonctionnent et peuvent moduler la réponse biologique. / G-protein coupled receptors are versatile biological sensors that are responsible for the majority of cellular responses to hormones and neurotransmitters as well as for the sense of sight, smell and taste. Signal transduction is associated with a set of changes in the tertiary structure of the receptor that are recognized by the associated intracellular partners, in particular the G proteins. There is compelling evidence that GPCR can assemble as dimers but the way these assemblies function at the molecular level is still under investigation.We used here the leukotriene B4 receptor BLT1 as a model to analyze the conformational changes occurring during activation. To this end, we first produced the receptor in E. coli inclusion bodies and subsequently folded it back to its native state in vitro using original membrane mimetics. Using the purified dimeric receptor, we showed that (i) the G protein induces an asymmetric arrangement of the BLT1 homodimer where each of the protomers is in a distinct conformation, and (ii) the G protein is cis-activated, i.e. the protomer that binds the agonist also activates Gα. Finally, we brought evidence that, although the dimer fully activates its G protein partner, the monomer has per se all the molecular determinant for an efficient functioning. All these data are original evidence that sheds light into the way GPCR dimers are activated and in turn modulate G protein-mediated signaling.

O diabetes tipo 1 (DT1) é uma doença metabólica associada a uma inflamação sistêmica de baixo grau, responsável por importantes co-morbidades associadas. Nosso grupo demonstrou que esta inflamação depende dos níveis plasmáticos aumentados de leucotrieno-B4 (LTB4), o qual estimula o eixo Myd88/STAT1 amplificando a resposta dos receptores TLR/IL1&#946 em macrófagos. Isso caracteriza o programa pró-inflamatório M1 nestas células, que requer energia proveniente da glicólise e produz substancias tóxicas como espécies reativas de oxigênio (ROS) e óxido nítrico (NO). Isto pode ser minimizado pela respiração mitocondrial desacoplada à síntese de ATP no metabolismo lipídico. Assim, na primeira parte do trabalho os macrófagos de camundongos (C57Bl/6) DT1, expressaram níveis elevados de marcadores de oxidação de ácidos graxos, o que não foi observado em macrófagos de camundongos diabéticos tratados com o antagonista de LTB4 (u75302). Além disso, o u75302 também reduziu a expressão aumentada de CD36, receptor envolvido na captação de lipídios, assim como aumento de lipídios intracelulares nestas células. Os elevados níveis de triglicérides e ácidos graxos presentes no plasma dos diabéticos também foram reduzidos pelo antagonista u75302, e isso foi consistente com o aparecimento de marcadores de lipólise (Prdm16 e Fgf21) no tecido adiposo branco destes animais. Da mesma forma, u75302 reduziu o consumo de oxigênio dos macrófagos de animais diabéticos. Isso foi consistente com os resultados obtidos em macrófagos de camundongos diabéticos deficientes da UCP1, os quais apresentaram maior peso corporal, maior massa de gordura e metabolismo mitocondrial mais baixo do que diabéticos WT. A perda de gordura também foi recuperada pelo tratamento com u75302 indicando o envolvimento do LTB4 na perda de adiposidade e dislipidemia em DT1. Na segunda parte do trabalho, confirmamos a participação dos leucotrienos na inflamação sistêmica em DT1 induzida por estreptozotocina. Camundongos (129SvE) diabéticos apresentaram níveis sistêmicos elevados das citocinas e este aumento não ocorreu em 129Sve deficientes da enzima 5-lipoxigenase (5LO-/-), responsável pela síntese de leucotrienos. A freqüência de monócitos pró-inflamatórios (CD11b&#43Ly6ChighLy6G-) circulantes estava aumentada em camundongos diabéticos WT mas não nos 5LO-/-. Macrófagos peritoneais residentes de camundongos diabéticos também apresentaram um fenótipo semelhante ao M1 classicamente ativado (alta expressão de Nos2 e Stat1, e alta produção de NO), que não foi revertido com o estímulo de IL4 in vitro ou in vivo. Por outro lado, os macrófagos dos 5LO-/- diabéticos apresentaram o fenótipo de macrófagos M2 alternativamente ativados (alta expressão de Ym1 e Arg1, e alta atividade de arginase). Os animais WT diabéticos tiveram cicatrização deficiente que se correlacionou com uma baixa freqüência de macrófagos M2 (CD45&#43F4/80&#43CD206&#43) nas lesões cutâneas comparado com os demais grupos. Juntos, estes dados sugerem que no DT1 os leucotrienos contribuem para a inflamação sistêmica e reprogramação dos monócitos e macrófagos para perfil inflamatório e isto está associado com aumento do metabolismo energético nestas células. Estas alterações induzidas nos macrófagos pelos níveis elevados de leucotrienos, particularmente LTB4, se correlacionam com a cicatrização deficiente, com a perda de gordura e hiperlipidemia nos camundongos DT1, sugerindo que o LTB4 possa ser um alvo terapêutico no diabetes. / Type 1 diabetes (T1D) is a metabolic disease associated to systemic low grade inflammation, which has an important role in co-morbidities. Our group showed that this inflammation depends on the high systemic levels of leukotriene-B4 (LTB4), which stimulateds MyD88/Stat1 axis, amplifying TLR/IL1&#946 response in macrophages. This characterizes pro inflammatory M1 program, which requires energy from glycolysis, and produces harmful molecules, such as reactive oxygen species (ROS) and nitric oxide (NO). This can be mitigated by mitochondrial respiration uncoupled to ATP synthesis in lipid metabolism. Therefore, in the first part of this study, macrophages from T1D mice (C57Bl/6) expressed high levels of fatty acid oxidation markers, which was not observed in macrophages from T1D mice treated with LTB4 receptor antagonis, u75302. Moreover, u75302 also reduced the high expression od CD36, a receptor involved in lipids uptake, and also reduced intracellular lipids in these cells. The high levels of triglycerides in diabetic plasma were reduced by u75302, and this is consistent with lipolysis markers (Prdm16 and Fgf21) in white adipose tissue of these mice. This was also consistent with results obtained in macrophages from diabetic UCP1 deficient mice, which had higher body weight and lower mitochondrial metabolism then WT diabetics. Fat loss was also recovered by u75302 treatment, indicating an involvement of LTB4 in adiposity loss and dyslipidemia in T1D. In the second part of this study, we confirmed leukotrienes participation in systemic inflammation in T1D streptozotocin-induced. T1D mice (129SvE) increased systemic levels of cytokines, which was not observed in T1D 5-lipoxygenase deficient mice (5LO-/-).The frequency of pro inflammatory monocytes (CD11b&#43Ly6ChighLy6G-) was increased in WT diabetic mice, but not in 5LO-/-. Resident peritoneal macrophages in diabetics had a phenotype similar to M1 classically activated (Nos2 and Stat1 highly expressed, and high production of NO), which was not reversed by IL-4 stimulation in vitro and in vivo. On the other hand, macrophages from diabetic 5LO-/- had a phenotype M2 alternatively activated (Ym1 and Arg1 highly expressed, and high arginase activity). WT diabetic mice had a defective wound healing, which was related to low frequency of M2 (CD45&#43F4/80&#43CD206&#43) macrophages in cutaneous wounds, compared to the other groups. All together, our data suggest that in T1D leukotrienes induce systemic inflammation, and reprogram pro inflammatory phenotype in monocytes and macrophages, and this is related to increased energetic metabolism in these cells. These alteration in macrophages due to leukotrienes effects, mainly LTB4, are correlated to defective healing, with fat loss, and dyslipidemia in T1D mice, suggesting that LTB4 can be a therapeutic target in diabetes.

The CHRNA5/A3/B4 genomic cluster encodes for the alpha5, alpha3 and beta4 subunits of the nicotinic acetylcholine receptors (nAChRs). Human genetic studies have revealed a significant association of variants in this genomic region with nicotine dependence. However, the mechanisms through which overexpression of these three subunits may influence smoking-related behaviours is not understood. To gain insight in the possible mechanisms, we used a BAC transgenic mouse model overexpressing this cluster containing the three genes together with their transcriptional regulatory elements. We found that overexpression of the cluster: i) increases sensitivity to the pharmacological effects of nicotine; ii) modifies particular cognitive domains associated to drug addiction and hippocampal neuronal complexity and synaptic plasticity; and iii) shifts the rewarding and aversive properties of nicotine and the manifestation of nicotine-withdrawal syndrome. Our study suggests that the genomic cluster CHRNA5/A3/B4 contributes to genetic vulnerability to nicotine addiction and promotes smoking-related behaviours possibly through hippocampal plasticity changes. / El cluster genòmic CHRNA5/A3/B4 codifica per les subunitats alfa5, alfa3 i beta4 dels receptors d’acetilcolina (nAChRs). Estudis de genètica humana han revelat que variants en aquesta regió genòmica estan significativament associats a la dependencia a nicotina. Malauradament, els mecanismes pels quals la sobreexpressió d’aquestes tres subunitats influencia comportaments relacionats amb el consum de tabac no són del tot coneguts. Per tal d’entendre els possibles mecanismes, hem utilitzat un model de ratolí transgènic que sobreexpressa aquest cluster amb els tres gens i les seus elements de regulació transcripcional. Hem trobat que la sobreexpressió del cluster: i) incrementa la sensibilitat als efectes farmacològics de la nicotina; ii) modifica determinats dominis cognitius associats a l’addicció a droges i la complexitat neuronal i plasticitat sinàpica de l’hipocamp; a més a més iii) canvia les propietats de recompensa i aversió de la nicotina i la manifestació del síndrome d’abstinència. El nostre estudi suggereix que el cluster genòmic CHRNA5/A3/B4 contribueix a la vulnerabilitat genètica a l’adicció a la nicotina i promou comportaments relacionats amb el consum de tabac possiblement a través de canvis de plasticitiat a l’hipocamp.