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Whole cell biosensors have been extensively used for monitoring toxicity and contamination of compounds in environmental biology and microbial ecology. However, their application in the pharmaceutical and cosmetics industries for preservative efficacy testing (PET) has been limited. According to several pharmacopoeias, preservatives should be tested for microbial activity using traditional viable count techniques; the use of whole cell microbial biosensors potentially provides an alternative, fast, and efficient method. The aim of the study was to construct and develop whole cell microbial biosensors with Pseudomonas aeruginosa ATCC 9027. Constitutive promoters: PlysS, Pspc, Ptat, Plpp and PldcC and the lux-cassette were inserted into plasmid pME4510 and transformed into P. aeruginosa ATCC 9027 cells to produce bioluminescent strains. Plasmids were found to be maintained stably (~50 copies per cell) throughout the growth and death cycle. The novel bioluminescent strains were validated in accordance with the pharmacopoeia using bioluminescence detection and quantification followed by comparison with the traditional plate counting method. The bioluminescent method was found to be accurate, precise and equivalent at a range of 103 – 107 CFU/mL, as compared with plate counting. Recovery of bacterial cells was quantified using bioluminescence; this method proved to be accurate with percentage recoveries between 70-130% for all bioluminescent strains. The method was also more precise (relative standard deviation less than 15%) than the traditional plate counting method or the ATP bioluminescent method. Therefore, the bioluminescent constructs passed/exceeded pharmacopoeial specified criteria for range, limit of detection, accuracy, precision and equivalence. Physiology of the validated bioluminescent strains was studied by assessing the growth and death patterns using constitutive gene expression linked with bacterial replication. Promoter strengths were evaluated at various stages of the growth and death pattern and related to promoter sequences. PlysS, Ptat and Plpp were relatively strong promoters whilst PldcC and Pspc were relatively weak promoters. Relative promoter strength decreased in the order of Plpp>Ptat>PlysS>PldcC>Pspc during the exponential phase whilst Ptat was stronger than Plpp during the stationary phase of growth. Plpp had its highest level of expression during the exponential phase, while Ptat had relatively stable lux expression during the stationary phase. Correlations between relative bioluminescence and CFU at 24 hours were greater than 0.9 indicating a strong relationship for all bioluminescent strains. Reduction in correlation coefficients to approximately 0.6 between relative bioluminescence and CFU and between relative fluorescence and CFU beyond 24 hours indicated that a certain proportion of cells were viable but non-culturable. Tat-pME-lux showed steady bioluminescence compared to CFU count (R>0.9) throughout 28 days of growth. Equivalence analysis showed no significant difference between the bioluminescence and plate count method throughout 28 days of growth for all five bioluminescent strains. Applicability of these novel bioluminescent strains was evaluated for preservative efficacy tests (PET) using bacterial replication and bioluminescence as a measure of constitutive gene expression. PET using benzalkonium chloride and benzyl alcohol showed no significant difference between the bioluminescent method and the plate count method. Good correlations between bioluminescence, CFU count and fluorescence were obtained for benzalkonium chloride (BKC) concentrations (R>0.9) between 0.0003% and 0.0025% against strains lysR25, lppR4 and tatH5. Similarly, good correlations (R>0.9) between the three parameters were obtained for benzyl alcohol (BA) concentrations between 0.125% and 2% against strains lysR25, lppR4 and tatH5. The bioluminescent method and traditional plate counting method were equivalent for concentrations of BKC (0.0003 - 0.02%) and BA (0.25 - 2%) during preservative efficacy tests. These bioluminescent constructs therefore are good candidates for selection for preservative efficacy testing. The bioluminescent method and traditional plate counting method were also found to be equivalent for construct tatH5 at a concentration of 0.125% BA. PET testing with BKC and BA showed that tatH5-pMElux (R>0.9) had consistently high correlation coefficients between CFU and relative bioluminescence. Together with the results from growth and death kinetics, where tatH5 showed the greatest constitutive expression, it can be concluded that P. aeruginosa ATCC 9027 tatH5-pMElux is the best construct for testing various antimicrobial agents. This study has shown that according to the pharmacopoeial requirements, the bioluminescent method is more accurate, precise and equivalent to the traditional plate counting method and therefore can be utilised instead of the traditional plate counting method for the purpose of preservative efficacy testing.

Le récepteur de chimiokine CXCR7 a été récemment identifié comme liant la chimiokine SDF-1, anciennement considérée comme ligand exclusif du récepteur CXCR4. Ces deux récepteurs sont exprimés majoritairement dans les mêmes types cellulaires et, ainsi, la découverte de CXCR7 incite à réévaluer les effets respectifs de SDF-1 sur CXCR4. Étant donné son rôle dans le cancer, CXCR4 est une cible de choix pour le développement de molécules thérapeutiques. Également, CXCR7 semble être impliqué dans la croissance tumorale. AMD3100, un antagoniste «sélectif» pour CXCR4, est maintenant commercialisé. Cet antagoniste a été identifié comme liant lui aussi CXCR7. De plus, sur CXCR7, l’AMD3100 agit comme agoniste puisqu’il induit le recrutement de la β-arrestine, à l’opposé de son effet sur. En revanche, AMD3100 n’induit pas le recrutement de la β-arrestine à CXCR4. Basé sur ces résultats, il est nécessaire de revoir la sélectivité d’autres antagonistes synthétiques de CXCR4. À l’aide de la technique de BRET (Résonance d’un transfert d’énergie par bioluminescence), nos résultats montrent que le Tc14012, un autre antagoniste synthétique de CXCR4, et structurellement distinct de l’AMD3100, interagit avec CXCR7. Contrairement à CXCR4, les deux antagonistes de CXCR4 agissent comme agonistes sur CXCR7 en induisant le recrutement de la β-arrestine. Nos résultats suggèrent que l’organisation spatiale du corps du récepteur serait responsable de cet effet opposé. En conclusion, AMD3100 et Tc14012 ne sont pas sélectifs pour CXCR4, puisqu’ils interagissent avec CXCR7. Lors du développement de nouvelles molécules synthétiques ciblant CXCR4, il serait alors nécessaire d’en évaluer leur sélectivité, et leurs effets en les testant aussi sur CXCR7. / ASBTRACT SDF-1 was at first thought to exclusively bind CXCR4, but it was subsequently found to also bind to the chemokine receptor CXCR7. CXCR4 is a promising target for drug development due to its role in cancer. AMD3100 is newly commercialised synthetic antagonist of CXCR4. This drug leads to massive release of hematopoietic stem cell into the peripheral blood. It was found that AMD3100 also binds to CXCR7 and acts as an agonist of β-arrestin recruitment to CXCR7. An antagonist of CXCR4 acts as an agonist on CXCR7. Prompted by this observation, we tested whether this might hold true for other CXCR4 antagonist. Tc14012, a peptidomimetic of T140, has been extensively described as a potent CXCR4 antagonist. We find that TC14012 also interacts on CXCR7. Like AMD3100, TC14012 alone induces β-arrestin recruitment to CXCR7. Thus, two structurally unrelated CXCR4 antagonists, AMD3100 and TC14012, are agonists of the CXCR7-arrestin pathway. This suggests distinct activation mechanisms of the arrestin pathway by CXCR4 and CXCR7. The results we obtained using a BRET (Bioluminescence Resonance Energy Transfer)-based arrestin recruitment assay, suggest that the CXCR7 receptor core is responsible for the recruitment of beta-arrestin in response to AMD3100 and TC14012. The finding that both AMD3100 and TC14012 do not only bind CXCR4, but also CXCR7, with opposite effects on arrestin recruitment, is important for the use of the compounds as tools to dissect SDF-1-mediated effects. This may be a general feature of synthetic ligands of the two receptors, with potential consequences for drug development. Key words: Chemokine receptor, CXCR4 and CXCR7, BRET, β-arrestin recruitement, TC14012, AMD3100 and SDF-1.

Pyrosequencing is a new DNA sequencing method relying on thesequencing-by-synthesis principle and bioluminometric detectionof nucleotide incorporation events. The objective of thisthesis was improvement of the Pyrosequencing method byincreasing the thermal stability of firefly luciferase, and byintroducing an alternative DNA polymerase and a new nucleotideanalog. Furthermore, the development of a new bioluminescentassay is described for the detection of inorganicpyrophosphatase activity. The wild-type North American firefly(Photinus pyralis)luciferase is a heat-sensitiveenzyme, the catalytic activity of which is rapidly lost attemperatures over 30°C. Two strategies for increasing thethermostability of the enzyme are presented and discussed. Inthe first strategy, the solution thermodynamics of the systemis affected by osmolytes in such a way that heat-mediatedinactivation of the enzyme is prevented. In the secondstrategy, the enzyme is thermostabilized by mutagenesis. Bothstabilizing strategies can be utilized to allow bioluminometricassays to be performed at higher temperatures. For instance,both DNA polymerase and ATP sulfurylase activity could beanalyzed at 37°C. The osmolyte strategy was successfully employed forincreasing the reaction temperature for the Pyrosequencingmethod. By increasing the reaction temperature to 37°Cunspecific signals from primer-dimers and 3’-end loopswere reduced. Furthermore, sequencing of a challenging templateat 37°C, which previously yielded poor, non-interpretablesequence signals at lower temperatures was now possible. Introduction of a new adenosine nucleotide analog,7-deaza-2’-deoxyadenosine-5’-triphosphate (c7dATP) reduced the inhibitory effect on apyraseobserved with the currently used analog,2’-deoxyadenosine-5’-O-(1-thiotriphosphate)(dATPαS). Sequencing of homopolymeric T-regions has previously beendifficult with the exonuclease-deficient form of the DNApolymerase I large (Klenow) fragment. By using the DNApolymerase from bacteriophage T7, known as Sequenase, templateswith homopolymeric T-regions were successfully sequenced.Furthermore, it was found that the strand displacement activityfor both polymerases was strongly assisted if the displacedstrand had a 5’-overhang. In contrast, the stranddisplacement activity for both polymerases was inhibitedwithout an overhang, resulting in reduced sequencingperformance in double stranded regions. A firefly bioluminescent assay for the real-time detectionof inorganic pyrophosphatase in the hydrolytic direction wasalso developed. The assay is versatile and has a linearresponse in the range between 8 and 500 mU. Key words:bioluminescence, osmolytes, glycine betaine,thermostability, firefly luciferase, inorganic pyrophosphatase,inorganic pyrophosphate, Pyrosequencing technology, secondaryDNA-structures, Sequenase, Klenow-polymerase, reaction rates,temperature, c7dATP, dATPαS. / <p>QCR 20161027</p>

Interferentes endócrinos (IE) são substâncias capazes de afetar o sistema endócrino causando danos à saúde. Os compostos estrogênicos são um tipo de IE que geram resposta biológica semelhante aos hormônios endógenos, chamada atividade estrogênica, são frequentemente encontrados no ambiente devido à poluição de origem antrópica e ineficiência de processos de tratamento de água e esgoto. O objetivo deste trabalho foi avaliar a atividade estrogênica de águas de mananciais e águas tratadas para abastecimento no Estado de São Paulo por meio de bioensaios. Foram utilizados dois métodos, baseados em diferentes linhagens da levedura Saccharomyces cerevisiae capazes de responder a agentes estrogênicos pela presença do gene que codifica para o receptor de estrogênio humano e sistemas de gene repórter da atividade estrogênica. Uma das linhagens contém o gene luc, método denominado neste trabalho de Lesk, enquanto a outra contém genes lux como repórter, denominado método San. Os métodos foram comparados quanto à especificidade e sensibilidade de resposta aos principais interferentes endócrinos de ocorrência em águas e com dados de análises cromatográficas das respectivas amostras ambientais. Foi observada atividade estrogênica expressiva nas águas brutas coletadas em pontos específicos nas cidades de Campinas, Barueri, Cerquilho e em efluente hospitalar e em todas essas amostras foram detectados compostos estrogênicos-alvo. As amostras de água tratada não apresentaram atividade estrogênica nem compostos estrogênicos, com apenas uma exceção. O método San foi mais sensível que o método Lesk, tanto para compostos químicos puros quanto para amostras ambientais. O teor de substâncias detectado por análises cromatográficas não foi suficiente para explicar a atividade estrogênica observada nos bioensaios, indicando que concentrações abaixo dos limites de detecção podem gerar o efeito biológico ou que compostos estrogênicos não estudados e suas misturas podem estar presentes gerando efeitos aumentados. / Endocrine disrupting (ED) chemicals are compounds able to interact with the endocrine system causing health adverse effects. Estrogenic compounds are a type of ED that produce a biological response similar to organism\'s natural hormones, called estrogenic activity; they are frequently found in the environment and are usually associated to the pollution from human activities. The goal of this work was to evaluate the estrogenic activity of rivers raw and treated water around the State of São Paulo Brazil - using bioassays. Two different strains of Saccharomyces cerevisiae able to respond to estrogenic compounds, due to the presence of genes that encodes for the human estrogen receptor, were used. One of the strains has the luc gene (method Lesk), a reporter of estrogenic activity and the other contains lux genes (method San) as reporter of estrogenic activity. Both methods were compared regarding their specificity and sensitivity for chemical substances and environmental samples. The results were also compared with the chemical analysis data of target pure compounds in those waters and with the literature. Estrogenic activity was detected in raw water samples collected in Campinas, Barueri and Cerquilho and also in hospital effluent, for all those samples estrogenic compounds were determined. San method showed to be more sensitive to pure chemical compounds and environmental samples. For treated water no estrogenic activity was found, except for one sample from Barueri that presented a low response. The amounts of compounds detected by chromatographic analysis were not sufficient to explain the observed estrogenic activity when bioassays were used, therefore low compounds concentration or other compounds and their mixtures may be responsible for the high biological effet detected by bioassays.

Espécies reativas de oxigênio (EROs) são produzidas normalmente durante o metabolismo de organismos aeróbios. Fungos degradadores de lignina geram estas espécies também no processo de degradação da lignina. As espécies de fungos bioluminescentes Gerronema viridilucens e Mycena lucentipes foram utilizadas para se estabelecer possível dependência entre intensidade de bioluminescência, viabilidade celular e atividades das enzimas de defesa antioxidante e ligninolíticas nos micélios e corpos de frutificação. Verificou-se o efeito de espécies causadoras de estresse químico (metais e fenóis) na emissão de luz, viabilidade celular, defesas antioxidantes e enzimas de respiração celular no fungo bioluminescente G. viridilucens, com o objetivo de conectar a inibição da bioluminescência com danos oxidativos ao organismo. Constatou-se que diferentes espécies de fungos bioluminescentes podem apresentar características diferentes quanto à emissão de luz e proteção antioxidante. Diferenças na intensidade e variação temporal da emissão de luz de diferentes espécies foram observadas nos corpos de frutificação e também no micélio. Isto foi revelado pela irregularidade do perfil de luz reprodutível para a espécie M. lucentipes, ao contrário do observado para G. viridilucens. A viabilidade celular de ambas as espécies varia com o tempo, sendo que no caso de G. viridilucens seu perfil é similar ao da bioluminescência. Os ensaios enzimáticos indicam maior atividade no micélio dos fungos do que nos corpos de frutificação, provavelmente pela função específica reprodutora do corpo de frutificação, enquanto a atividade metabólica do fungo está concentrada no micélio. As enzimas ligninolíticas também apresentam atividade baixa nas culturas estudadas, provavelmente por serem enzimas de degradação extracelulares. O fato de ambas viabilidade celular e bioluminescência do micélio serem reduzidas na presença dos metais (cobre e cádmio) e fenóis (fenol e 2,4,6-triclorofenol) testados atesta a interrelação entre atividade luminogênica e injúria oxidativa aos fungos. Os metais parecem afetar mais negativamente as defesas antioxidantes dos fungos do que os fenóis, os quais possivelmente são eliminados pela atividade da glutationa S-transferase (GST), sem também afetar as demais defesas antioxidantes. No conjunto, estes resultados possibilitam estabelecer uma relação metabólica entre abatimento da bioluminescência e as defesas antioxidantes do organismo. No caso dos metais, os sistemas de defesa antioxidante envolvendo a glutationa são bastante importantes, tanto para eliminar peróxidos produzidos na presença de cobre, como na quelação de cádmio pela glutationa. Sob condições normais, a bioluminescência, defesas antioxidantes e respiração celular do organismo estariam funcionando e o NAD(P)H seria mobilizado por todos estes sistemas. Quando o fungo é submetido ao estresse químico, o fluxo de NAD(P)H seria desviado da bioluminescência para sustentar prioritariamente as defesas antioxidantes e a respiração celular, essenciais para a proteção, manutenção e reprodução do organismo / Reactive Oxygen Species (ROS) are normally produced during the metabolism of aerobic organisms. Ligninolitic fungi also produce these oxidizing species also during the lignin degradation process. The bioluminescent species Gerronema viridilucens and Mycena lucentipes were studied aiming to establish a correlation between the temporal profiles of bioluminescence, cellular viability and antioxidant defense enzymes and ligninolitic enzymes in mycelium and fruiting bodies. Chemical toxicants such as metals and phenols were here found to affect light emission, cellular viability, antioxidant defenses and cellular respiration enzymes when administered to G. viridilucens, thereby attesting a metabolic connection between bioluminescence inhibition and fungal oxidative damage. Different species of fungi exhibit different characteristics linked to light emission and antioxidant defenses. Differences in light emission displayed by different species do not resume to fruiting bodies, but the light profile and intensity can also vary in the mycelia. This may explain the irreproducibility of the light profile from M. lucentipes, differently to that observed with G. viridilucens. The cellular viability of both species varies with time, G. viridilucens profile being similar to the time course of bioluminescence. The enzymatic data pointed to higher activities in mycelium than in fruiting bodies, probably due to a main reproductive function of the latter, whereas the metabolic activities are prevalent in the mycelium. Ligninolytic enzymes exhibit low activities in the extracts of fungus samples, probably because they are extracellular degradation enzymes. The inhibition effect of phenols and metals (copper and cadmium) on mycelium viability reinforces the notion that cellular oxidative damage hampers bioluminescence emission. Redox active (copper) and heavy metals (cadmium) were found to display higher impact on antioxidant defenses than phenols (phenol and 2,4,6-trichlorophenol), which are expected to be promptly metabolized and excluded by principally glutathione S-transferase (GST). Notably, a metabolic correlation between bioluminescence inhibition and antioxidant defenses was unveiled by the present work. Regarding the metals, glutathione was found to be a crucial antioxidant, both to eliminate peroxides when in the presence of copper, and to act as a cadmium chelating agent. Under normal conditions, the bioluminescence system, the antioxidant defenses and the cellular respiration sets cooperate through the common demand of reducing power of NAD(P)H. Under chemical stress, the NAD(P)H flux would be deviated from bioluminescence, to principally sustain the antioxidant defenses and cellular respiration, essential for the protection, maintenance and reproduction of the organism.

Interferentes endócrinos (IE) são substâncias capazes de afetar o sistema endócrino causando danos à saúde. Os compostos estrogênicos são um tipo de IE que geram resposta biológica semelhante aos hormônios endógenos, chamada atividade estrogênica, são frequentemente encontrados no ambiente devido à poluição de origem antrópica e ineficiência de processos de tratamento de água e esgoto. O objetivo deste trabalho foi avaliar a atividade estrogênica de águas de mananciais e águas tratadas para abastecimento no Estado de São Paulo por meio de bioensaios. Foram utilizados dois métodos, baseados em diferentes linhagens da levedura Saccharomyces cerevisiae capazes de responder a agentes estrogênicos pela presença do gene que codifica para o receptor de estrogênio humano e sistemas de gene repórter da atividade estrogênica. Uma das linhagens contém o gene luc, método denominado neste trabalho de Lesk, enquanto a outra contém genes lux como repórter, denominado método San. Os métodos foram comparados quanto à especificidade e sensibilidade de resposta aos principais interferentes endócrinos de ocorrência em águas e com dados de análises cromatográficas das respectivas amostras ambientais. Foi observada atividade estrogênica expressiva nas águas brutas coletadas em pontos específicos nas cidades de Campinas, Barueri, Cerquilho e em efluente hospitalar e em todas essas amostras foram detectados compostos estrogênicos-alvo. As amostras de água tratada não apresentaram atividade estrogênica nem compostos estrogênicos, com apenas uma exceção. O método San foi mais sensível que o método Lesk, tanto para compostos químicos puros quanto para amostras ambientais. O teor de substâncias detectado por análises cromatográficas não foi suficiente para explicar a atividade estrogênica observada nos bioensaios, indicando que concentrações abaixo dos limites de detecção podem gerar o efeito biológico ou que compostos estrogênicos não estudados e suas misturas podem estar presentes gerando efeitos aumentados. / Endocrine disrupting (ED) chemicals are compounds able to interact with the endocrine system causing health adverse effects. Estrogenic compounds are a type of ED that produce a biological response similar to organism\'s natural hormones, called estrogenic activity; they are frequently found in the environment and are usually associated to the pollution from human activities. The goal of this work was to evaluate the estrogenic activity of rivers raw and treated water around the State of São Paulo Brazil - using bioassays. Two different strains of Saccharomyces cerevisiae able to respond to estrogenic compounds, due to the presence of genes that encodes for the human estrogen receptor, were used. One of the strains has the luc gene (method Lesk), a reporter of estrogenic activity and the other contains lux genes (method San) as reporter of estrogenic activity. Both methods were compared regarding their specificity and sensitivity for chemical substances and environmental samples. The results were also compared with the chemical analysis data of target pure compounds in those waters and with the literature. Estrogenic activity was detected in raw water samples collected in Campinas, Barueri and Cerquilho and also in hospital effluent, for all those samples estrogenic compounds were determined. San method showed to be more sensitive to pure chemical compounds and environmental samples. For treated water no estrogenic activity was found, except for one sample from Barueri that presented a low response. The amounts of compounds detected by chromatographic analysis were not sufficient to explain the observed estrogenic activity when bioassays were used, therefore low compounds concentration or other compounds and their mixtures may be responsible for the high biological effet detected by bioassays.

<p>Pyrosequencing is a new DNA sequencing method relying on thesequencing-by-synthesis principle and bioluminometric detectionof nucleotide incorporation events. The objective of thisthesis was improvement of the Pyrosequencing method byincreasing the thermal stability of firefly luciferase, and byintroducing an alternative DNA polymerase and a new nucleotideanalog. Furthermore, the development of a new bioluminescentassay is described for the detection of inorganicpyrophosphatase activity.</p><p>The wild-type North American firefly<i>(Photinus pyralis)</i>luciferase is a heat-sensitiveenzyme, the catalytic activity of which is rapidly lost attemperatures over 30°C. Two strategies for increasing thethermostability of the enzyme are presented and discussed. Inthe first strategy, the solution thermodynamics of the systemis affected by osmolytes in such a way that heat-mediatedinactivation of the enzyme is prevented. In the secondstrategy, the enzyme is thermostabilized by mutagenesis. Bothstabilizing strategies can be utilized to allow bioluminometricassays to be performed at higher temperatures. For instance,both DNA polymerase and ATP sulfurylase activity could beanalyzed at 37°C.</p><p>The osmolyte strategy was successfully employed forincreasing the reaction temperature for the Pyrosequencingmethod. By increasing the reaction temperature to 37°Cunspecific signals from primer-dimers and 3’-end loopswere reduced. Furthermore, sequencing of a challenging templateat 37°C, which previously yielded poor, non-interpretablesequence signals at lower temperatures was now possible.</p><p>Introduction of a new adenosine nucleotide analog,7-deaza-2’-deoxyadenosine-5’-triphosphate (c⁷dATP) reduced the inhibitory effect on apyraseobserved with the currently used analog,2’-deoxyadenosine-5’-O-(1-thiotriphosphate)(dATPαS).</p><p>Sequencing of homopolymeric T-regions has previously beendifficult with the exonuclease-deficient form of the DNApolymerase I large (Klenow) fragment. By using the DNApolymerase from bacteriophage T7, known as Sequenase, templateswith homopolymeric T-regions were successfully sequenced.Furthermore, it was found that the strand displacement activityfor both polymerases was strongly assisted if the displacedstrand had a 5’-overhang. In contrast, the stranddisplacement activity for both polymerases was inhibitedwithout an overhang, resulting in reduced sequencingperformance in double stranded regions.</p><p>A firefly bioluminescent assay for the real-time detectionof inorganic pyrophosphatase in the hydrolytic direction wasalso developed. The assay is versatile and has a linearresponse in the range between 8 and 500 mU.</p><p><b>Key words:</b>bioluminescence, osmolytes, glycine betaine,thermostability, firefly luciferase, inorganic pyrophosphatase,inorganic pyrophosphate, Pyrosequencing technology, secondaryDNA-structures, Sequenase, Klenow-polymerase, reaction rates,temperature, c⁷dATP, dATPαS.</p>

RÉSUMÉ Le récepteur de chimiokine CXCR7 a été récemment identifié comme liant la chimiokine SDF-1, anciennement considérée comme ligand exclusif du récepteur CXCR4. Ces deux récepteurs sont exprimés majoritairement dans les mêmes types cellulaires et, ainsi, la découverte de CXCR7 incite à réévaluer les effets respectifs de SDF-1 sur CXCR4. Étant donné son rôle dans le cancer, CXCR4 est une cible de choix pour le développement de molécules thérapeutiques. Également, CXCR7 semble être impliqué dans la croissance tumorale. AMD3100, un antagoniste «sélectif» pour CXCR4, est maintenant commercialisé. Cet antagoniste a été identifié comme liant lui aussi CXCR7. De plus, sur CXCR7, l’AMD3100 agit comme agoniste puisqu’il induit le recrutement de la β-arrestine, à l’opposé de son effet sur. En revanche, AMD3100 n’induit pas le recrutement de la β-arrestine à CXCR4. Basé sur ces résultats, il est nécessaire de revoir la sélectivité d’autres antagonistes synthétiques de CXCR4. À l’aide de la technique de BRET (Résonance d’un transfert d’énergie par bioluminescence), nos résultats montrent que le Tc14012, un autre antagoniste synthétique de CXCR4, et structurellement distinct de l’AMD3100, interagit avec CXCR7. Contrairement à CXCR4, les deux antagonistes de CXCR4 agissent comme agonistes sur CXCR7 en induisant le recrutement de la β-arrestine. Nos résultats suggèrent que l’organisation spatiale du corps du récepteur serait responsable de cet effet opposé. En conclusion, AMD3100 et Tc14012 ne sont pas sélectifs pour CXCR4, puisqu’ils interagissent avec CXCR7. Lors du développement de nouvelles molécules synthétiques ciblant CXCR4, il serait alors nécessaire d’en évaluer leur sélectivité, et leurs effets en les testant aussi sur CXCR7. Mot-clés : Récepteur de chimiokine CXCR4 et CXCR7, BRET, recrutement de la β-arrestine, AMD3100, Tc14012 et SDF-1. / ASBTRACT SDF-1 was at first thought to exclusively bind CXCR4, but it was subsequently found to also bind to the chemokine receptor CXCR7. CXCR4 is a promising target for drug development due to its role in cancer. AMD3100 is newly commercialised synthetic antagonist of CXCR4. This drug leads to massive release of hematopoietic stem cell into the peripheral blood. It was found that AMD3100 also binds to CXCR7 and acts as an agonist of β-arrestin recruitment to CXCR7. An antagonist of CXCR4 acts as an agonist on CXCR7. Prompted by this observation, we tested whether this might hold true for other CXCR4 antagonist. Tc14012, a peptidomimetic of T140, has been extensively described as a potent CXCR4 antagonist. We find that TC14012 also interacts on CXCR7. Like AMD3100, TC14012 alone induces β-arrestin recruitment to CXCR7. Thus, two structurally unrelated CXCR4 antagonists, AMD3100 and TC14012, are agonists of the CXCR7-arrestin pathway. This suggests distinct activation mechanisms of the arrestin pathway by CXCR4 and CXCR7. The results we obtained using a BRET (Bioluminescence Resonance Energy Transfer)-based arrestin recruitment assay, suggest that the CXCR7 receptor core is responsible for the recruitment of beta-arrestin in response to AMD3100 and TC14012. The finding that both AMD3100 and TC14012 do not only bind CXCR4, but also CXCR7, with opposite effects on arrestin recruitment, is important for the use of the compounds as tools to dissect SDF-1-mediated effects. This may be a general feature of synthetic ligands of the two receptors, with potential consequences for drug development. Key words: Chemokine receptor, CXCR4 and CXCR7, BRET, β-arrestin recruitement, TC14012, AMD3100 and SDF-1.