<p>Migration of T-lymphocytes on a surface coated with extracellular matrix (ECM) components (two-dimensional (2-D) migration) and migration (infiltration) into a matrix (Three-dimesional (3-D) migration) are complex events and the underlying mechanisms are not yet fully understood. Here 2-D and 3-D migration were studied by use of seven leukemic T-cell lines representing discrete differentiation stages, a non-leukemic T-cell clone, and normal peripheral blood T cells. peripheral blood lymphocytes and the T-cell clone produced nanogram quantities of various chemokines, as compared to a production of ≤ 0.05 ng/ml by the T leukemia cell lines. In a Boyden chamber system, the leukemic T-cell lines showed haptotactic migration on fibronectin. The migration was augmented bu exposure to chemokines, including RANTES, MIP-1α, MIP-1β, and IL-8. The T-cell lines showed a peak response at a chemokine concentration of 10-50 ng/ml, whereas the T-cell clone responded optimally at 100 ng/ml. In contrast to a general capability of T-cells to migrate on 2-D ECM, only some of the T-cell lines were capable of 3-D migration into Matrigel or a collagen matrix. The infiltrative capacity was unrelated to the capacity to migrate on or adhere to the substrata. T-cell lines with a capacity to infiltrate produced matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of matrix metalloproteinases-1 (TIMP-1), whereas non-infiltrating cell lines did not produce MMP-9. T-cell lines capable of infiltrating Matrigel or collagen responded to chemokines exposure with increased infiltration, but the chemokines did not render non-infiltrative cell lines infiltrative. Stimulation of infiltration of T-cell lines into collagen by the chemokine SDF-1α was inhibited by somatostatin, a neuropeptide with immunosuppressive properties. In conclusion, the ability to migrate on 2-D substrata and to infiltrate into 3.D substrata was found to be distinguishable properties of T cells. failure of some T-cell lines to infiltrate correlated with the lack of expression of MMP-9. Chemokines stimulated infiltration of infiltrative T-cell lines into collagen and Matrigel but did not render non-infiltrative T-cell lines infiltrative. Finally, a possible physiological mechanism for modulation of the chemokine-stimulated 3-D migration was demonstrated.</p>

<p>Consequences of atherosclerosis may result in a number of diseases of the cardiovascular system that represent serious health problems and major causes of morbidity and mortality worldwide. Although it is initially considered as disease of fibro-lipid and thrombus deposition in the arterial wall, it also involves an ongoing inflammatory response.</p><p>Normally, the inflammatory response is considered as a protective defence mechanism of the body. However, if the inflammation gets out of proportion to the threat it is dealing with, it may then result in a sustained chronic disorder and thus may underlie the initial stage of atherogenesis. The work of this thesis focuses on the expression of cytokines/chemokines and the vascular transcriptional response to inflammation, i.e. LPS in atherosclerosis. This has mainly been studied in animal models of atherosclerosis; consequently, we set out to investigate these events using human material in vitro (human carotid lesions).</p><p>Employing quantitative analysis, we were able to detect a significant induction of protein and mRNA of the cytokines IL-1β, IL-6, IL-10 and TNF-α and the chemokines IL-8 and MCP-1 by LPS in both atherosclerotic and non-atherosclerotic vessels. In contrast, LPS induction of TNF-α, IL-1β and IL-10 was solely observed in the lesions, but not in normal arteries. In addition, the impact of IL-1 gene polymorphism on the risk of myocardial infarction (MI) was estimated by DNA genotyping of 387 survivors of a first MI and 387 sex and age-matched control subjects. We found no statistically significant differences in either genotypic distribution or allelic frequencies of IL-1β (-511) or IL-1Ra (VNTR) polymorphisms between first-time survivors of myocardial infarction and their age-matched healthy controls. Incontrast, our results demonstrated a strong association between the IL-1Ra genotype and severity of angiographically determined coronary artery disease in post-MI patients. To further investigate the vascular response to inflammation, we used gene array analysis to evaluate the human vascular transcriptional response to LPS of non-atherosclerotic human renal arteries compared to carotid lesions. In LPS treated renal arteries, 54% of the transcripts gave a detectable signal, where 4% were upregulated and 3.8% down-regulated. In the LPS stimulated carotid lesions, 44% of transcripts were detected. In this latter group, 5.1% of transcripts were increased and 3.3% decreased. Interestingly, a newly identified virus-inducible antiviral protein, CMV inducible gene ₅/viperin (Cig₅), was among the most strongly induced gene in both normal and atherosclerotic biopsies. Single gene analysis revealed viperin in the endothelium of human atherosclerotic lesions. Further, viperin was induced in vascular cells by inflammatory stimuli and CMV infection.</p><p>In conclusion we show that atherosclerotic vessels produce more proinflammatory cytokines/chemokines than normal vessels. Interestingly, our results indicate that LPS enhances the expression of cytokines/chemokines in a similar pattern both in lesions and normal arteries. However, the response is stronger in atherosclerotic lesions. Furthermore, our results suggest that genetic polymorphisms within the IL-1Ra loci may influence the severity of CAD. Finally, the CMV inducible gene ₅/viperin have been identified as a putative culprit molecule in vascular inflammation and atherosclerosis.</p>

Migration of T-lymphocytes on a surface coated with extracellular matrix (ECM) components (two-dimensional (2-D) migration) and migration (infiltration) into a matrix (Three-dimesional (3-D) migration) are complex events and the underlying mechanisms are not yet fully understood. Here 2-D and 3-D migration were studied by use of seven leukemic T-cell lines representing discrete differentiation stages, a non-leukemic T-cell clone, and normal peripheral blood T cells. peripheral blood lymphocytes and the T-cell clone produced nanogram quantities of various chemokines, as compared to a production of ≤ 0.05 ng/ml by the T leukemia cell lines. In a Boyden chamber system, the leukemic T-cell lines showed haptotactic migration on fibronectin. The migration was augmented bu exposure to chemokines, including RANTES, MIP-1α, MIP-1β, and IL-8. The T-cell lines showed a peak response at a chemokine concentration of 10-50 ng/ml, whereas the T-cell clone responded optimally at 100 ng/ml. In contrast to a general capability of T-cells to migrate on 2-D ECM, only some of the T-cell lines were capable of 3-D migration into Matrigel or a collagen matrix. The infiltrative capacity was unrelated to the capacity to migrate on or adhere to the substrata. T-cell lines with a capacity to infiltrate produced matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of matrix metalloproteinases-1 (TIMP-1), whereas non-infiltrating cell lines did not produce MMP-9. T-cell lines capable of infiltrating Matrigel or collagen responded to chemokines exposure with increased infiltration, but the chemokines did not render non-infiltrative cell lines infiltrative. Stimulation of infiltration of T-cell lines into collagen by the chemokine SDF-1α was inhibited by somatostatin, a neuropeptide with immunosuppressive properties. In conclusion, the ability to migrate on 2-D substrata and to infiltrate into 3.D substrata was found to be distinguishable properties of T cells. failure of some T-cell lines to infiltrate correlated with the lack of expression of MMP-9. Chemokines stimulated infiltration of infiltrative T-cell lines into collagen and Matrigel but did not render non-infiltrative T-cell lines infiltrative. Finally, a possible physiological mechanism for modulation of the chemokine-stimulated 3-D migration was demonstrated.

Consequences of atherosclerosis may result in a number of diseases of the cardiovascular system that represent serious health problems and major causes of morbidity and mortality worldwide. Although it is initially considered as disease of fibro-lipid and thrombus deposition in the arterial wall, it also involves an ongoing inflammatory response. Normally, the inflammatory response is considered as a protective defence mechanism of the body. However, if the inflammation gets out of proportion to the threat it is dealing with, it may then result in a sustained chronic disorder and thus may underlie the initial stage of atherogenesis. The work of this thesis focuses on the expression of cytokines/chemokines and the vascular transcriptional response to inflammation, i.e. LPS in atherosclerosis. This has mainly been studied in animal models of atherosclerosis; consequently, we set out to investigate these events using human material in vitro (human carotid lesions). Employing quantitative analysis, we were able to detect a significant induction of protein and mRNA of the cytokines IL-1β, IL-6, IL-10 and TNF-α and the chemokines IL-8 and MCP-1 by LPS in both atherosclerotic and non-atherosclerotic vessels. In contrast, LPS induction of TNF-α, IL-1β and IL-10 was solely observed in the lesions, but not in normal arteries. In addition, the impact of IL-1 gene polymorphism on the risk of myocardial infarction (MI) was estimated by DNA genotyping of 387 survivors of a first MI and 387 sex and age-matched control subjects. We found no statistically significant differences in either genotypic distribution or allelic frequencies of IL-1β (-511) or IL-1Ra (VNTR) polymorphisms between first-time survivors of myocardial infarction and their age-matched healthy controls. Incontrast, our results demonstrated a strong association between the IL-1Ra genotype and severity of angiographically determined coronary artery disease in post-MI patients. To further investigate the vascular response to inflammation, we used gene array analysis to evaluate the human vascular transcriptional response to LPS of non-atherosclerotic human renal arteries compared to carotid lesions. In LPS treated renal arteries, 54% of the transcripts gave a detectable signal, where 4% were upregulated and 3.8% down-regulated. In the LPS stimulated carotid lesions, 44% of transcripts were detected. In this latter group, 5.1% of transcripts were increased and 3.3% decreased. Interestingly, a newly identified virus-inducible antiviral protein, CMV inducible gene 5/viperin (Cig5), was among the most strongly induced gene in both normal and atherosclerotic biopsies. Single gene analysis revealed viperin in the endothelium of human atherosclerotic lesions. Further, viperin was induced in vascular cells by inflammatory stimuli and CMV infection. In conclusion we show that atherosclerotic vessels produce more proinflammatory cytokines/chemokines than normal vessels. Interestingly, our results indicate that LPS enhances the expression of cytokines/chemokines in a similar pattern both in lesions and normal arteries. However, the response is stronger in atherosclerotic lesions. Furthermore, our results suggest that genetic polymorphisms within the IL-1Ra loci may influence the severity of CAD. Finally, the CMV inducible gene 5/viperin have been identified as a putative culprit molecule in vascular inflammation and atherosclerosis.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 122-140).

Le récepteur atypique ACKR3 et le récepteur CXCR4 sont des récepteurs couplés aux protéines G appartenant à la famille des récepteurs CXC des chimiokines. Ces deux récepteurs sont activés par la chimiokine CXCL12 et sont surexprimés dans de nombreux cancers comme les gliomes, dont ils favorisent la prolifération et le caractère invasif. Le récepteur CXCR4 active des voies de signalisation qui dépendent de la protéine Gi et des β-arrestines et s’associe à plusieurs protéines impliquées dans la transduction du signal, le trafic et la localisation cellulaire du récepteur. Par contre, les mécanismes de signalisation impliqués dans les effets d’ACKR3 restent mal connus. Le récepteur déclenche une signalisation dépendant des β-arrestines, mais son couplage aux protéines G dépend du type cellulaire ou se fait par un mécanisme indirect via son association au récepteur CXCR4. Le récepteur ACKR3 s’associe également au récepteur de l’EGF pour induire la prolifération cellulaire par un mécanisme indépendant de sa stimulation par un agoniste. Ces données illustrent l’intérêt de caractériser de façon systématique l’interactome de ces récepteurs pour comprendre leurs rôles physiologiques et pathologiques. Cette thèse a poursuivi cet objectif grâce à la mise en œuvre d’une approche protéomique combinant la purification des partenaires des deux récepteurs par affinité suivie de leur identification par spectrométrie de masse. J’ai ainsi identifié respectivement 19 et 151 partenaires protéiques potentiels des récepteurs CXCR4 et ACKR3 exprimés dans les cellules HEK-293T. Parmi les protéines recrutées par ACKR3, nous nous sommes focalisés sur la connexine 43 (Cx43, une des protéines constituant les jonctions Gap) du fait de la similitude des effets du récepteur et de la Cx43 dans la pénétration des leucocytes dans le parenchyme cérébral, la migration des interneurones et la progression des gliomes. J’ai confirmé par Western blot et par BRET l’association spécifique de la Cx43 à l’ACKR3 et non pas au CXCR4. De la même façon, j’ai montré une co-localisation de la Cx43 et de l’ACKR3 dans des cellules de gliome humain, ainsi que dans les astrocytes de la zone sous-ventriculaire et les pieds astrocytaires entourant les capillaires cérébraux chez la souris, suggérant que les deux protéines forment un complexe protéique dans un contexte biologique authentique. Des études fonctionnelles ont révélé que l’ACKR3 module les fonctions de la Cx43 par différents mécanismes. L’expression de l’ACKR3 dans les cellules HEK-293T (mimant la surexpression du récepteur dans les tumeurs), induit par elle-même une inhibition de l’activité jonctionnelle de la Cx43. De même, la stimulation du récepteur par un agoniste réduit l’activité jonctionnelle de la Cx43 par un mécanisme impliquant l’activation d’une protéine Gi, la β-arrestine2 et l’internalisation de la Cx43. Cette thèse établit donc pour la première fois un lien fonctionnel entre le système constitué par les chimiokines CXCL11, CXCL12 et leur récepteur ACKR3 d’une part et les jonctions Gap d’autre part qui pourrait jouer un rôle critique dans la progression des gliomes. / The Atypical Chemokine Receptor 3 (ACKR3) and CXCR4 are two G protein-coupled receptors (GPCR) belonging to the CXC chemokine receptor family. Both receptors are activated upon CXCL12 binding and are over-expressed in various tumours, including glioma, where they have been found to promote proliferation and invasive behaviours. Upon CXCL12 binding, CXCR4 activates canonical GPCR signalling pathways involving Gαi protein and β-arrestins. In addition, CXCR4 was found to interact with several proteins able to modify its signalling, trafficking and localization. In contrast, the cellular pathways underlying ACKR3-dependent effects remain poorly characterized. Several reports show that ACKR3 engages β-arrestin-dependent signalling pathways, but its coupling to G proteins is restricted to either specific cellular populations, including astrocytes, or occurs indirectly via its interaction with CXCR4. ACKR3 also associates with the epidermal growth factor receptor to promote proliferation of tumour cells in an agonist-independent manner. These examples suggest that the extensive characterization of ACKR3 and CXCR4 interactomes might be a key step in understanding or clarifying their roles in physiological and pathological contexts. This thesis addressed this issue employing an affinity purification coupled to high-resolution mass spectrometry proteomic strategy that identified 19 and 151 potential protein partners of CXCR4 and ACKR3 transiently expressed in HEK-293T cells, respectively. Amongst ACKR3 interacting proteins identified, we paid particular attention on the gap junction protein Connexin-43 (Cx43), in line with its overlapping roles with the receptor in the control of leukocyte entry into the brain, interneuron migration and glioma progression. Western blotting and BRET confirmed the specific association of Cx43 with ACKR3 compared to CXCR4. Likewise, Cx43 is co-localized with ACKR3 but not CXCR4 in glioma initiating cell lines, and ACKR3 and Cx43 are co-expressed in astrocytes of the sub-ventricular zone and surrounding blood vessels in adult mouse brain, suggesting that both proteins form a complex in authentic cell or tissue contexts. Further functional studies showed that ACKR3 influences Cx43 trafficking and functionality at multiple levels. Transient expression of ACKR3 in HEK-293T cells to mimic ACKR3 overexpression detected in several cancer types, induces Gap Junctional Intercellular Communication (GJIC) inhibition in an agonist-independent manner. In addition, agonist stimulation of endogenously expressed ACKR3 in primary cultured astrocytes inhibits Cx43-mediated GJIC through a mechanism that requires activation of Gαi protein, and dynamin- and β-arrestin2-dependent Cx43 internalisation. Therefore, this thesis work provides the first functional link between the CXCL11/CXCL12/ACKR3 axis and gap junctions that might underlie their critical role in glioma progression.

A hiperplasia prostática benigna (HPB) e o câncer de próstata (CaP) são duas condições crônicas muito comuns em homens com idade avançada e têm sido relacionadas a processos inflamatórios. As quimiocinas são reconhecidas como mediadores críticos da resposta inflamatória por regular a migração das células imunológicas através da ativação de receptores de quimiocinas na superfície destas células. As quimiocinas estão relacionadas à patogênese tumoral, embora não seja claro de que modo afetam a progressão tumoral humana. O objetivo desse estudo foi investigar a associação de dois polimorfismos de receptores de quimiocinas, CCR2-64I e CCR5-delta32, com HPB e CaP. Neste trabalho foram genotipadas 385 amostras de DNA genômico de homens do sul do Brasil, predominantemente euro-descendentes, incluindo 130 casos de HPB, 136 casos de CaP e 119 indivíduos controle saudáveis. Para o polimorfismo CCR2-64I a genotipagem foi realizada por PCR-RFLP e para o CCR5-delta32 foi por PCR convencional. As frequências alélicas do CCR2-64I foram 14,0%; 15,8% e 11,1% nos grupos controle, HPB e CaP, respectivamente; enquanto as do CCR5-delta32 foram 5,1%; 7,1% e 6,2%, respectivamente. A mediana referente aos níveis de PSA foi de 0,79; 1,45 e 6,91 ng/mL nos grupos controle, HPB e CaP, respectivamente; diferindo significativamente entre estes (todos p<0,001). A mediana do volume da próstata foi 20,00 cm3 no grupo controle, portanto, menor que dos grupos HPB (35,35 cm3) e CaP (35,80 cm3) (ambos p<0,001); no entanto, não foi observada diferença entre pacientes com HPB e CaP (p=0,172). Algo interessante observado foi CCR2-64I como um fator protetor para CaP quando comparado com HPB (OR=0,550; IC95%=0,311–0,975), mas não quando comparado com o grupo controle (p=0,448). Não foi observada associação do CCR2-64I com os estados clinicopatológicos do CaP (estadiamento tumoral e escore de Gleason) (todos p≥0,308). Não foi observada associação significativa da variante CCR5-delta32 com HPB ou CaP (todos p≥0,072), ou com os estados clinicopatológicos do CaP (todos p≥0,253). Assim, nossos dados sugerem a influência da variante CCR2-64I, observada como fator protetor para CaP quando comparada com HPB, no desenvolvimento do câncer de próstata. / Benign prostatic hyperplasia (BPH) and prostate cancer (PCa) are two chronic conditions very common in aged men and have been related to inflammatory process. Chemokines are recognized as critical mediators of inflammatory responses by regulating the migration of immune cells through the activation of chemokine receptors on the surface of these cells. Chemokines are implicated in tumor pathogenesis, although it is not clear how it affects human tumor progression. The aim of this study was to investigate the association of two chemokine receptor gene polymorphisms, CCR2-64I and CCR5-delta32, with BPH and PCa. In this study were genotyped 385 genomic DNA samples from southernmost Brazilian men, predominantly euro-descendants, including 130 BPH, 136 PCa and 119 healthy control subjects. To CCR2-64I polymorphism the genotyping was performed by PCR-RFLP and to CCR5-delta32 by conventional PCR. The allele frequencies of CCR2-64I were 14.0%, 15.8% and 11.1% in control, BPH and PCa, respectively; while of CCR5-delta32 were 5.1%, 7.1% and 6.2%, respectively. Median of serum PSA levels were 0.79, 1.45 and 6.91 ng/mL in control, BPH and PCa group, respectively (all p<0.001). The prostate volume median was 20.00 cm3 in the control group, thus, lower than BPH (35.35 cm3) and PCa (35.80 cm3) group (both p<0.001), nevertheless no difference was observed between BPH and PCa patients (p=0.172). Interestingly, CCR2-64I was detected as a protective factor to PCa when compared with BPH (OR=0.550; 95%CI=0.311–0.975), but not when compared with control group (p=0.448). No significant associations of the CCR2-64I were observed with PCa clinicopathologic states (tumor stage and Gleason score) (all p≥0.308). No significant associations of the CCR5-delta32 variant were observed with BPH or PCa (all p≥0.072), or with PCa clinicopathologic status (all p≥0.253). Thus, our data suggest a influence of the CCR2-64I variant, that was observed as a protective factor in PCa when compared with BPH, in prostate cancer development.

La liaison de CXCL12 à ses récepteurs CXCR4 et CXCR7 peut-être bloquée par un neutraligand de CXCL12, la chalcone 4, qui présente une activité anti-inflammatoire dans un modèle d’asthme chez la souris. Notre travail a consisté à proposer des stratégies permettant le développement de molécules biodisponibles et actives localement, et d’étudier le mécanisme d'action in vivo des neutraligands de CXCL12. Nous avons développé un modèle court et reproductible d'asthme allergique chez la souris, adapté à une évaluation rapide de l’activité anti-inflammatoire des nouveaux composés et un développement raisonné des stratégies envisagées. Nous avons synthétisé trois prodrogues solubles, adaptées à une administration locale, inactives et rapidement clivées en chalcone 4 active. In vivo, les prodrogues sont anti-inflammatoires à des doses susceptibles de limiter les effets indésirables. Pour favoriser davantage l'action anti-inflammatoire locale du neutraligand de CXCL12, nous avons synthétisé une ante-drogue, la carbonitrile-chalcone 4, active in vivo par administration locale et rapidement dégradée en deux composés inactifs avant sa distribution dans l'organisme. Enfin, l'étude de la chalcone 4 a mis en évidence une activité antiasthmatique significative, liée à l'élimination rapide de la chimiokine CXCL12 du poumon. La capture de CXCL12 par le neutraligand réduit la différenciation des macrophages M1 et leur libération de cytokines pro-inflammatoires, ainsi que le recrutement des éosinophiles et des lymphocytes CXCR4+. En conclusion, nos travaux ont permis de conforter les rôles de la chimiokine CXCL12 dans l'asthme et présenter deux stratégies aptes à limiter les effets indésirables. / The binding of the chemokine CXCL12 to its receptors CXCR4 and CXCR7 may be prevent by a CXCL12 neutraligand, chalcone 4, having an anti -inflammatory activity in a mouse model of allergic asthma. Our work consisted in proposing strategies for the development of active and bioavailable molecules, able to promote local action, and to study the in vivo mechanism of action of the CXCL12 neutraligands. We initially developed a short and reproducible mouse model of allergic asthma, suitable for a rapid assessment of the anti -inflammatory activity of new compounds, in order to ensure rational development of the proposed strategies. We developped three soluble prodrugs, adapted to local administration, inactive but rapidly cleaved in active chalcone 4. In vivo, the prodrugs have an anti-inflammatory activity at suitable doses to minimize side effects. To promote the benefit of local anti-inflammatory action of CXCL12 neutraligand, we synthesized an antedrug, carbonitrile-chalcone 4, active locally in vivo after local administration and rapidly degraded before its distribution in the body.Finally, the study of chalcone 4 allowed us to highlight a significant asthma activity. An activity related to the rapid elimination of the CXCL12 chemokine from the lung. The trapping of CXCL12 by the neutraligand reduced M1 macrophage activation and their release of pro inflammatory cytokines, as decreases the recruitment of CXCR4+ eosinophils and lymphocytes. In conclusion, our work provided mechanistic elements related to the roles of the chemokine CXCL12 in asthma, and present two interesting strategies adapted to local administration to limit adverse effects.

Les récepteurs de chimiokines sont des régulateurs essentiels de la migration cellulaire dans le cadre de la surveillance immunitaire, et le développement. Les récepteurs CCR5 et CXCR4 sont de plus spécifiquement impliqués dans les métastases cancéreuses et l'infection par le VIH. Nous avons développé un système permettant de sur-exprimer ces deux RCPGs. Afin de s'affranchir des problèmes de toxicité inhérents à l'expression des protéines membranaires en bactérie notre approche de production consiste à adresser les protéines vers les corps d'inclusion d'E. coli grâce à une fusion protéique N-terminale permettant de hauts niveaux d'expression. Après purification en conditions dénaturantes, les protéines sont alors repliées en présence de surfactants originaux, les amphipoles. La validation de cette nouvelle approche pour les récepteurs des chimiokines représente un des objectifs principaux de ce travail. Afin de tester la fonctionnalité des protéines repliées, une série d'outils a été développée : des versions modifiées des chimiokines ont été produites (RANTES pour CCR5 et SDF 1a pour CXCR4). La fonctionnalité des chimiokines a été évaluée au niveau moléculaire et cellulaire. L'interaction entre le récepteur replié en amphipole et son ligand a été testé par résonance de plasmons de surface (SPR). Différents types de surfaces fonctionalisées avec le récepteur de chimiokine replié en amphipole ont été explorés au cours de ce travail. A la fin de ce projet, la production des chimiokines et de leur récepteur a été mise au point. L'accès à ces outils ouvre la voie à de futures études moléculaires telles que la compréhension de la dimérisation du récepteur ou la détermination de la stoechiométrie du complexe. / Chemokine receptors are critical regulators of cell migration in the context of immune surveillance, inflammation and development. The GPCRs (G protein-coupled receptors) CCR5 and CXCR4 are specifically implicated in cancer metastasis and HIV-1 infection. An expression system to over-express these two GPCRs was developed. To overcome the toxicity problem of membrane protein expression in bacterial system, the production approach consists in targeting the proteins towards E. coli inclusion bodies thanks to a N-terminal fusion allowing a high yield expression. After purification under denaturing conditions, these GPCRs were then folded using original polymeric surfactants: the amphipols. The validation of this new approach for the chemokine receptor production is one of the goals of this work. In order to assess the functionality of the folded proteins, series of tools have been developed: engineered chemokine ligands (RANTES for CCR5 and SDF1a for CXCR4) were produced. The functionality of chemokines was evaluated at cellular and molecular levels. Interaction between the receptor folded in amphipols and its ligand was evaluated using Surface Plasmon Resonance (SPR) technique. Several types of surfaces, functionalized with the chemokine receptor/amphipol complex have been explored in this work. At the end of this project the productions of chemokines and their receptors has been set up. These established tools open the way to future studies, at the molecular level, in order to, for instance, investigate receptor dimerization and complex stoichiometry.

A hiperplasia prostática benigna (HPB) e o câncer de próstata (CaP) são duas condições crônicas muito comuns em homens com idade avançada e têm sido relacionadas a processos inflamatórios. As quimiocinas são reconhecidas como mediadores críticos da resposta inflamatória por regular a migração das células imunológicas através da ativação de receptores de quimiocinas na superfície destas células. As quimiocinas estão relacionadas à patogênese tumoral, embora não seja claro de que modo afetam a progressão tumoral humana. O objetivo desse estudo foi investigar a associação de dois polimorfismos de receptores de quimiocinas, CCR2-64I e CCR5-delta32, com HPB e CaP. Neste trabalho foram genotipadas 385 amostras de DNA genômico de homens do sul do Brasil, predominantemente euro-descendentes, incluindo 130 casos de HPB, 136 casos de CaP e 119 indivíduos controle saudáveis. Para o polimorfismo CCR2-64I a genotipagem foi realizada por PCR-RFLP e para o CCR5-delta32 foi por PCR convencional. As frequências alélicas do CCR2-64I foram 14,0%; 15,8% e 11,1% nos grupos controle, HPB e CaP, respectivamente; enquanto as do CCR5-delta32 foram 5,1%; 7,1% e 6,2%, respectivamente. A mediana referente aos níveis de PSA foi de 0,79; 1,45 e 6,91 ng/mL nos grupos controle, HPB e CaP, respectivamente; diferindo significativamente entre estes (todos p<0,001). A mediana do volume da próstata foi 20,00 cm3 no grupo controle, portanto, menor que dos grupos HPB (35,35 cm3) e CaP (35,80 cm3) (ambos p<0,001); no entanto, não foi observada diferença entre pacientes com HPB e CaP (p=0,172). Algo interessante observado foi CCR2-64I como um fator protetor para CaP quando comparado com HPB (OR=0,550; IC95%=0,311–0,975), mas não quando comparado com o grupo controle (p=0,448). Não foi observada associação do CCR2-64I com os estados clinicopatológicos do CaP (estadiamento tumoral e escore de Gleason) (todos p≥0,308). Não foi observada associação significativa da variante CCR5-delta32 com HPB ou CaP (todos p≥0,072), ou com os estados clinicopatológicos do CaP (todos p≥0,253). Assim, nossos dados sugerem a influência da variante CCR2-64I, observada como fator protetor para CaP quando comparada com HPB, no desenvolvimento do câncer de próstata. / Benign prostatic hyperplasia (BPH) and prostate cancer (PCa) are two chronic conditions very common in aged men and have been related to inflammatory process. Chemokines are recognized as critical mediators of inflammatory responses by regulating the migration of immune cells through the activation of chemokine receptors on the surface of these cells. Chemokines are implicated in tumor pathogenesis, although it is not clear how it affects human tumor progression. The aim of this study was to investigate the association of two chemokine receptor gene polymorphisms, CCR2-64I and CCR5-delta32, with BPH and PCa. In this study were genotyped 385 genomic DNA samples from southernmost Brazilian men, predominantly euro-descendants, including 130 BPH, 136 PCa and 119 healthy control subjects. To CCR2-64I polymorphism the genotyping was performed by PCR-RFLP and to CCR5-delta32 by conventional PCR. The allele frequencies of CCR2-64I were 14.0%, 15.8% and 11.1% in control, BPH and PCa, respectively; while of CCR5-delta32 were 5.1%, 7.1% and 6.2%, respectively. Median of serum PSA levels were 0.79, 1.45 and 6.91 ng/mL in control, BPH and PCa group, respectively (all p<0.001). The prostate volume median was 20.00 cm3 in the control group, thus, lower than BPH (35.35 cm3) and PCa (35.80 cm3) group (both p<0.001), nevertheless no difference was observed between BPH and PCa patients (p=0.172). Interestingly, CCR2-64I was detected as a protective factor to PCa when compared with BPH (OR=0.550; 95%CI=0.311–0.975), but not when compared with control group (p=0.448). No significant associations of the CCR2-64I were observed with PCa clinicopathologic states (tumor stage and Gleason score) (all p≥0.308). No significant associations of the CCR5-delta32 variant were observed with BPH or PCa (all p≥0.072), or with PCa clinicopathologic status (all p≥0.253). Thus, our data suggest a influence of the CCR2-64I variant, that was observed as a protective factor in PCa when compared with BPH, in prostate cancer development.