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CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Leishmania braziliensis à o agente etiolÃgico mais comum da leishmaniose cutÃnea no Brasil. Camundongos BALB/c sÃo os mais susceptÃveis à infecÃÃo por L. braziliensis, embora desenvolvam lesÃes pequenas e nÃo ulceradas. As lesÃes em BALB/c infectados com L. braziliensis sÃo acompanhadas pela expressÃo de um amplo espectro de quimiocinas, entre elas CXCL10, que à capaz de atrair a ativar cÃlulas NK e Th1 com produÃÃo de IFN-γ. O objetivo deste trabalho foi avaliar se a quimiocina CXCL10, administrada nos estÃgios iniciais da infecÃÃo, poderia alterar o curso da doenÃa, dimunindo o tempo de cura. Para isso, camundongos BALB/c (n=24) foram infectados com L. braziliensis e divididos em dois grupos: um grupo (n=12) recebeu CXCL10 (100 ng/5ÂL) e o outro (n=12) PBS (5ÂL), nos dias 1, 3, 5 e 7 dias de infecÃÃo. Alguns parÃmetros foram avaliados: a espessura das lesÃes a cada trÃs dias; a carga parasitÃria na pata e no linfonodo de drenagem, a concentraÃÃo de NO, IFN-γ, IL-12p40, TNF-α, IL-4, IL-10 e TGF-β, 15 e 30 dias apÃs a infecÃÃo; e anÃlise histopatolÃgica das lesÃes. Os animais que receberam CXCL10 apresentaram uma reduÃÃo no tempo de duraÃÃo da doenÃa, uma vez que as lesÃes comeÃaram a regredir mais precocemente (12 dias), enquanto que nos animais controle (PBS) a doenÃa evoluiu por mais tempo (15 dias). CXCL10 induziu diferenÃas estatisticamente significativas na carga parasitÃria do linfonodo de drenagem nos dois pontos avaliados, quando comparado com o controle (15 dias: p=0,0353; 30 dias: p=0,0292). Nos animais que receberam CXCL10, nÃo foi observada mudanÃa no nÃmero de parasitos no sÃtio de inoculaÃÃo, mesmo com reduÃÃo da espessura da lesÃo, mostrando que nÃo houve correlaÃÃo entre o tamanho da lesÃo e a carga parasitÃria. NÃo houve diferenÃa na concentraÃÃo de NO entre os grupos, no 15 dia de infecÃÃo, e esta concentraÃÃo manteve-se igual no grupo que recebeu CXCL10 no 30 dia, embora tenha sido menor do que a observada no grupo controle. CXCL10 tambÃm induziu concentraÃÃes mais elevadas de IFN-γ e IL-12 e mais reduzidas de IL-4, IL-10, TGF-β 30 dias pÃs-infecÃÃo, quando comparados aos animais controle. A anÃlise histopatolÃgica das patas mostrou uma inflamaÃÃo muito leve em ambos os grupos, com diferenÃa apenas quanto à ausÃncia de neutrÃfilos nas lesÃes dos animais que receberam CXCL10. Em conjunto, os dados mostraram que CXCL10 apresentou um efeito protetor na infecÃÃo por L. braziliensis em camundongos BALB/c, com reduÃÃo no tempo de evoluÃÃo da doenÃa, associada a maior concentraÃÃo de IFN-γ e menor de IL-4 e IL-10. / Leishmania braziliensis is the more common etiologic agent of the cutaneous leishmaniasis in Brazil. BALB/c mice are more susceptible to L. braziliensis infection, although develop small and not ulcerated lesions. The lesions in BALB/c mice infected by L. braziliensis are accompanied by expression of a broad chemokines spectrum, including CXCL10, which is able to attract and activate NK and Th1 cells with IFN-γ production. The aim of this study was to evaluate whether the chemokine CXCL10, administered in early stages of infection could influence the course of disease, leading to a faster and more effective time of healing. Groups of BALB/c mice (n=24) were infected by L. braziliensis and divided into two groups: one group (n=12) received CXCL10 (100 ng/5μL) and the other (n=12) received PBS (5μL), at 1, 3, 5 and 7 days postinfection. Some parameters were evaluated: thickness of the lesions every three days; parasite burden in both the footpad and the draining lymph node, NO concentration, cytokines production (IFN-γ, IL-12p40, TNF-α, IL-4, IL-10 and TGF-β), at 15 and 30 days after infection; and histopathological analysis of lesions. Animals receiving CXCL10 showed reduced disease duration, since the lesions started to regress earlier (12 days), whereas the control animals developed the disease for longer (15 days). CXCL10 induced statistically significant differences in parasite load in the draining lymph nodes compared with control in both times evaluated (15 days: p=0.0353; 30 days: p=0.0292). In the animals receiving CXCL10 was not observed change in the number of parasites in the site of inoculation, even with reduced thickness of the lesion, showing no correlation between lesion size and parasite load. There was no difference in NO concentration between groups with 15 days of infection, and this concentration remained the same in the group receiving CXCL10 at day 30, although it was lower than that observed in the control group. CXCL10 also induced higher levels of IFN-γ and IL-12, and reduced IL-4, IL-10, and TGF-β at 30 days postinfection, when compared to control animals. Histopathology analyses of lesions after 15 days postinfection showed a very mild inflammation in both groups, differing only for the presence of neutrophils, which were practically not observed in lesions of animals receiving CXCL10. Taken together, the data from this study showed CXCL10 chemokine presented a protective effect on L. braziliensis infection in BALB/c mice, with a reduction in the progression of the disease, associated with a higher concentration of IFN-γ and lesser of IL-4 and IL-10.

Orientador: Licio Augusto Velloso / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T19:11:15Z (GMT). No. of bitstreams: 1 Morari_Joseane_D.pdf: 2485057 bytes, checksum: 88660e44d267d2bb162402326080d56d (MD5) Previous issue date: 2013 / Resumo: Nas últimas décadas tornou-se claro que indivíduos e animais obesos apresentam um quadro subclínico de inflamação sistêmica. Estudos desenvolvidos em nosso laboratório ao longo dos últimos 10 anos revelaram que a inflamação do hipotálamo está presente em roedores obesos e sua inibição por métodos genéticos e farmacológicos resulta na correção do fenótipo obeso e dos distúrbios metabólicos comumente associados à obesidade. Ácidos graxos saturados de cadeia longa presentes na dieta parecem ser os principais responsáveis pela ativação da resposta inflamatória no hipotálamo. Os primeiros sinais de inflamação podem ser detectados 24 h após a introdução de uma dieta rica neste tipo de gordura. Pelo menos dois mecanismos moleculares foram identificados como potenciais desencadeadores desta resposta inflamatória, sendo eles; a ativação de receptores TLR4, e a indução do estresse de retículo endoplasmático. Durante a exposição precoce a uma dieta rica em gordura saturada, células da micróglia localizadas no hipotálamo, tornam-se ativas e passam a expressar citocinas que, eventualmente, levam a ativação de vias inflamatórias em neurônios da região. A ativação de JNK e IKK em neurônios resulta na indução de resistência hipotalâmica à leptina e insulina, e, subsequentemente na perda do controle coordenado da ingestão alimentar e do gasto calórico. O fato de se observar ativação inflamatória no hipotálamo poucas horas após a exposição aos nutrientes desencadeadores sugere que células da micróglia residentes participem deste processo. Entretanto, com a manutenção da exposição ocorre uma modificação do padrão inflamatório ao longo do tempo, o que sugere que novas células estejam sendo recrutadas. Na primeira etapa deste estudo avaliamos a migração de células monocíticas da medula óssea para o hipotálamo em animais alimentados com dieta rica em gordura saturada. Para tal, transplantes de medula óssea foram realizados para gerar quimeras expressando TLR4 funcional somente em células da medula óssea ou somente em tecidos não originários da medula óssea. A parecença de TLR4 funcional em células de medula óssea foi necessária para expressão completa do fenótipo obeso. Além disso, somente quando TLR4 estava expresso em células da medula óssea, ocorreu infiltração hipotalâmica com células de micróglia oriundas da periferia. Como o recrutamento de células monocíticas da medula óssea para um determinado sítio anatômico depende invariavelmente da expressão de quimiocinas, nós aventamos a hipótese de que nas etapas iniciais da ativação da inflamação hipotalâmica na obesidade alguma(s) quimiocina(s) poderia(m) desempenhar um papel determinante na progressão do processo inflamatório e, consequentemente do fenótipo obeso. Tanto humanos como animais experimentais não consanguíneos apresentam grande variabilidade na expressão fenotípica da obesidade quando expostos a condições ambientais que favoreçam a mesma. Desta forma, buscamos por diferenças no padrão de inflamação do hipotálamo e na expressão de quimiocinas em duas cepas de camundongos com perfis metabólicos e predisposição a obesidade diametralmente opostos, sendo eles: o camundongo Swiss, com grande predisposição a obesidade e diabetes; e, o camundongo Balb-c, virtualmente protegido da obesidade e dos distúrbios metabólicos afins. Identificamos a quimiocina CX3CL1 (fractalkina) como aquela, dentre as avaliadas, com maior distinção de sua expressão no hipotálamo entre as duas cepas de camundongos, encontrando-se mais expressa em Swiss que em Balb-c. Na segunda parte do estudo mostramos que a expressão da fractalkina é induzida em neurônios do hipotálamo frente a um estímulo com nutrientes ricos em ácidos graxos saturados. Em cultura de neurônios, a expressão de fractalkina é induzida preferencialmente por TNF, mas também por palmitato. A inibição da fractalkina no hipotálamo com siRNA protege camundongos Swiss da intolerância à glicose e da obesidade induzidas por dieta rica em gordura saturada, além de reduzir o recrutamento de células monocíticas originárias da medula óssea para o hipotálamo. Assim, concluímos que durante as etapas iniciais da indução da obesidade, células da micróglia residentes ativam a produção de fractalkina por neurônios do hipotálamo a qual desempenha papel importante no recrutamento de novas células monocíticas da medula óssea, que perpetuarão o processo inflamatório do hipotálamo caso a exposição à dieta rica em gordura saturada seja mantida. A fractakina constitui-se, portanto, num dos mais precoces sinalizadores durante a instalação da inflamação hipotalâmica associada à obesidade / Abstract: Studies performed during the last 20 years have shown that obese subjects and experimental models of obesity present a systemic subclinical inflammation. Our group has shown that in experimental obesity the hypothalamus is affected by an inflammatory response and the employment of genetic and pharmacological means to inhibit diet-induced hypothalamic inflammation rescues the obese phenotype and the metabolic disarrangements commonly associated with obesity. Long-chain saturated fatty acids present in the diet are amongst the most important inducers of the hypothalamic inflammation in obesity. The first signs of hypothalamic inflammation can be detected as early as 24 h after the initial exposure to dietary fats. At least two molecular mechanisms have been shown to play a role in the induction of the hypothalamic inflammation in obesity; i.e., activation of TLR4 and induction of endoplasmic reticulum stress. During the early exposition to dietary fats, resident microglia cells are activated and express cytokines that eventually can lead to the induction of inflammatory signaling in the neighboring neurons. The activation of JNK and IKK in neurons results in the induction of hypothalamic resistance to leptin and insulin and, thus, to the loss of the coordinated control of food intake and energy expenditure. As hypothalamic inflammation can be induced a few hours after the initial exposure to dietary fats, it has been suggested that resident microglia are involved in this process. However, as the exposure to dietary fats persists, changes in the pattern of inflammation take place, which may indicate the recruitment of new cells to the inflamed site. In the first part of this study, we evaluate the migration of bone marrow-derived monocytic cells to the hypothalamus of obese animals. Chimera mice were generated by the transplantation of TLR4-expressing bone marrow into TLR4 defective mice and vice-versa. The presence of functional TLR4 in bone marrow-derived cells was required for the complete expression of the obese phenotype. In addition, diet-induced hypothalamic infiltration with bone marrow-derived microglia was obtained only in the presence of functional TLR4 in bone marrow-derived cells. Because the recruitment of bone marrow-derived monocytic cells to a given anatomical site depends invariably on the expression of chemokines, we have raised the hypothesis that, during the initial steps of diet-induced hypothalamic inflammation a (some) chemokine (s) could play an important role in the progression of the inflammatory activity and, consequently, of the obese phenotype. Both humans and outbreed experimental animals display a huge phenotypic variability whenever exposed to environmental conditions that favor obesity. With this concept in mind, we searched for differences in the patterns of hypothalamic inflammation in two strains of mice with different predispositions to obesity and metabolic diseases. The Swiss mice which are prone to obesity, and the Balb-c mice, which are protected from obesity and related metabolic conditions. We identified the chemokine CX3CL1 (fractalkine) as the one, among those that we evaluated, with highest distinction on its hypothalamic expression levels between the two mouse strains; being increased in the hypothalamus of Swiss mice. In the second part of the study we demonstrate that fractakine is induced in hypothalamic neurons by fat-rich diet. In culture, neurons express fractalkine in response to a TNF stimulus preferentially, but also in response to palmitate. The inhibition of hypothalamic expression of fractalkine by siRNA protects mice from glucose intolerance and diet-induced obesity. Moreover, under hypothalamic fractalkine inhibition, diet-induced recruitment of monocytic cells from the bone marrow is severely impaired. We conclude that during the initial steps of diet-induced hypothalamic inflammation, resident microglia cells induce the expression of fractalkine by neighboring neurons, which in turn recruit additional monocytic cells from the bone marrow to maintain the inflammatory process under continuous dietary fatty acid exposure. Thus, fractalkine is one of the earliest signals generated in the hypothalamus during the installation of diet-induced obesity / Doutorado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Doutora em Fisiopatologia Médica

Le sepsis est la réaction inflammatoire généralisée secondaire à une infection. C’est une pathologie fréquente et grave qui fait intervenir le système immunitaire. L’action de l’immunité innée se fait par l’activation et le recrutement des monocytes, des cellules mononuclées circulantes qui modulent le phénomène inflammatoire. La mobilisation des monocytes fait intervenir les cytokines chimiotactiques (chimiokines) et leurs récepteurs. Nous nous sommes spécifiquement intéressé dans ce travail au rôle de l’expression monocytaire des récepteurs aux chimiokines CCR2 et CX3CR1 au cours du sepsis. Pour ce faire, nous avons utilisé des modèles murins de sepsis et analysé le rôle d’un polymorphisme génétique de CX3CR1 dans une cohorte de malades atteints de sepsis. Nous avons montré qu’au cours du sepsis, les monocytes présentaient une augmentation de l’adhérence aux parois vasculaire contrôlée par le récepteur CX3CR1. Nous avons également montré que les monocytes inflammatoires jouaient un rôle essentiel dans la régulation du phénomène inflammatoire au cours du sepsis en protégeant le rein des lésions septiques. Cette protection est médiée par l’expression de CX3CR1. L’allèle I249 du gène Cx3cr1, à l’origine d’une augmentation des propriétés adhésives du couple CX3CR1/CX3CL1, est un facteur protecteur dans la survenue d’insuffisance rénale aiguë chez le malade atteint de sepsis. Collectivement, ces travaux confirment un rôle régulateur des monocytes inflammatoires au cours du sepsis et identifient de potentielles nouvelles cibles thérapeutiques. / Sepsis is the generalized inflammatory response secondary to an infection. This is a common and serious condition that involves the immune system. The action of innate immunity in sepsis is mediated by the activation and recruitment of monocytes, which are mononuclear circulating cells, which modulate the inflammatory process. The mobilization of monocytes involves chemotactic cytokines (chemokines) and their receptors. This work was specifically focused on the role of monocyte expression of chemokine receptors CCR2 and CX3CR1 in sepsis. To this end, we used mouse models of sepsis and analyzed the role of a common genetic polymorphism of CX3CR1 in a cohort of patients with sepsis.We have shown that in sepsis, monocytes’ motility was modified with an increase of their adhesion to vascular walls that was controlled in part by CX3CR1. We have also shown that inflammatory monocytes play a key role in the regulation of the inflammatory phenomenon in sepsis and that they protected the kidney from septic lesions via a CX3CR1 mediated adhesion mechanism. The I249 allele of CX3CR1, confering increased adhesive properties to monocytes, is a protective factor regarding the occurrence of acute kidney injury in septic patients. Collectively, these data confirm a a regulatory role for inflammatory monocytes during sepsis and identify potential new therapeutic targets.

In der vorliegenden Arbeit wurde die Rolle der Transkriptionsfaktoren NFATc1/αA bzw. NFATc1/ßC und NFATc2 in NK-Zellen und die Rolle der Faktoren NFATc1-4 und NFAT5 in Keratinozyten analysiert. Die Familie der Nuclear Factor of Activated T-cell (NFAT) Transkriptionsfaktoren besteht aus fünf Mitgliedern, welche entscheidend die Gentranskription bei Immunantworten beeinflussen. Nach Antigenaktivierung wird in Lymphozyten die Expression des Nfatc1-Gens stark induziert. Die Akkumulation der dabei gebildeten kurzen Isoform NFATc1/αA ist für die Zytokinproduktion als Effektorfunktion sowie für die Proliferation und das Überleben aktivierter Zellen verantwortlich. Das kurze NFATc1-Protein unterscheidet sich nicht nur strukturell, sondern auch funktionell von fast allen anderen NFAT-Proteinen. Um neue Erkenntnisse über die Funktion der Isoform NFATc1/αA in Lymphozyten zu gewinnen, wurden KT12 NK-Zellen mit NFATc1/αA bzw. NFATc1/ßC und NFATc2- exprimierenden Vektoren transfiziert, geklont, stimuliert und anschließend auf ihre jeweilige Apoptoserate und die Zytokinsynthese bzw. -expression hin untersucht. Die gewählten KT12-Hybridoma-Zellen erwiesen sich allerdings in ihrer intendierten Funktion als Testzellen für die Über-Expression von NFATc-Proteinen in NK-Zellen als ungeeignet. Fehlregulationen von NFAT-Signalwegen werden mit einer fehlerhaften Entwicklung des Immunsystems, mit der Entstehung von Autoimmunerkrankungen und mit Krebs in Verbindung gebracht. Um die Rolle von NFAT-Faktoren in Keratinozyten besser zu verstehen, wurden HaCaT-Zellen und primäre humane Keratinozyten mit Differenzierungssignalen bzw. UVB-Licht stimuliert. Änderungen der Transkription von NFAT-Faktoren, Keratinozyten-spezifischen Proteinen und Chemokinen wurden mittels qRT-PCR-Assays detektiert und analysiert. Insgesamt konnte die Beteiligung von NFAT-Faktoren am Differenzierungsprozess und an der UV-Antwort von Keratinozyten gezeigt werden. Es zeigte sich tendenziell eine stärkere Induktion der kurzen NFATc1-Isoform im Vergleich zu langen NFATc1-Isoformen, was die Frage nach einer besonderen Funktion der kurzen NFATc1-Isoform in Keratinozyten aufwirft. Generell ließen sich besonders hohe Expressionslevel des Transkriptionsfaktors NFAT5 - verglichen mit anderen NFAT-Faktoren - messen. Für die Entwicklung von Therapien, welche die Ursachen und Folgen einer dysregulierten Hautbarrierenbildung behandeln, könnten sich weitere Studien zu einzelnen NFAT-Faktoren bzw. NFATc1-Isoformen als zielführend erweisen. / In the present work the role of the transcription factors NFATc1/αA or NFATc1/ßC and NFATc2 in NK cells and the role of the factors NFATc1-4 and NFAT5 in keratinocytes were investigated. The nuclear factor of activated T-cell (NFAT) transcription factor family consists of five members that critically influence gene transcription in immune responses. Following antigen activation, expression of the Nfatc1 gene is strongly induced in lymphocytes. Accumulation of the short NFATc1/αA isoform - formed in this process - is responsible for cytokine production as an effector function and for proliferation along with the survival of activated cells. The short NFATc1 protein differs not only structurally but also functionally from almost all other NFAT proteins. To gain new insights into the function of the NFATc1/αA isoform in lymphocytes, KT12 NK cells were transfected with NFATc1/αA, NFATc1/ßC or NFATc2- expressing vectors. The KT12 cells were cloned, stimulated and subsequently analyzed for their respective apoptosis rates together with cytokine synthesis or expression. However, the selected KT12 hybridoma cells proved to be unsuitable in their function as test cells for the overexpression of NFATc proteins in NK cells. Misregulation of NFAT signaling pathways has been associated with defective development of the immune system and autoimmune diseases and cancer. To better understand the role of NFAT factors in keratinocytes, HaCaT cells and primary human keratinocytes were stimulated with differentiation signals and UVB light, respectively. Changes in transcription of NFAT factors, keratinocyte-specific proteins and chemokines were detected and analyzed by qRT-PCR assays. Overall, the involvement of NFAT factors in the differentiation process and UV response of keratinocytes was demonstrated. The short NFATc1 isoform tended to be more strongly induced compared with long NFATc1 isoforms, raising the question of a special function of the short NFATc1 isoform in keratinocytes. Particularly high expression levels of the transcription factor NFAT5 - compared to other NFAT factors - could be measured. For the development of therapies that treat the causes and consequences of dysregulated skin barrier formation further studies on individual NFAT factors or NFATc1 isoforms will prove useful.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21632号 / 医博第4438号 / 新制||医||1034(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 生田 宏一, 教授 上本 伸二, 教授 竹内 理 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The CXC chemokine receptor 4 (CXCR4) and the atypical chemokine receptor 3 (ACKR3) are seven transmembrane receptors that are involved in numerous pathologies, including several types of cancers. Both receptors bind the same chemokine, CXCL12, leading to significantly different outcomes. While CXCR4 activation generally leads to canonical GPCR signaling, involving Gi proteins and β‐arrestins, ACKR3, which is predominantly found in intracellular vesicles, has been shown to signal via β‐arrestin‐dependent signaling pathways. Understanding the dynamics and kinetics of their activation in response to their ligands is of importance to understand how signaling proceeds via these two receptors. In this thesis, different Förster resonance energy transfer (FRET)‐based approaches have been combined to individually investigate the early events of their signaling cascades. In order to investigate receptor activation, intramolecular FRET sensors for CXCR4 and ACKR3 were developed by using the pair of fluorophores cyan fluorescence protein and fluorescence arsenical hairpin binder. The sensors, which exhibited similar functional properties to their wild‐type counterparts, allowed to monitor their ligand-induced conformational changes and represent the first RET‐based receptor sensors in the field of chemokine receptors. Additional FRET‐based settings were also established to investigate the coupling of receptors with G proteins, rearrangements within dimers, as well as G protein activation. On one hand, CXCR4 showed a complex activation mechanism in response to CXCL12 that involved rearrangements in the transmembrane domain of the receptor followed by rearrangements between the receptor and the G protein as well as rearrangements between CXCR4 protomers, suggesting a role of homodimers in the activation course of this receptor. This was followed by a prolonged activation of Gi proteins, but not Gq activation, via the axis CXCL12/CXCR4. In contrast, the structural rearrangements at each step of the signaling cascade in response to macrophage migration inhibitory factor (MIF) were dynamically and kinetically different and no Gi protein activation via this axis was detected. These findings suggest distinct mechanisms of action of CXCL12 and MIF on CXCR4 and provide evidence for a new type of sequential signaling events of a GPCR. Importantly, evidence in this work revealed that CXCR4 exhibits some degree of constitutive activity, a potentially important feature for drug development. On the other hand, by cotransfecting the ACKR3 sensor with K44A dynamin, it was possible to increase its presence in the plasma membrane and measure the ligand‐induced activation of this receptor. Different kinetics of ACKR3 activation were observed in response to CXCL12 and three other agonists by means of using the receptor sensor developed in this thesis, showing that it is a valuable tool to study the activation of this atypical receptor and pharmacologically characterize ligands. No CXCL12‐induced G protein activation via ACKR3 was observed even when the receptor was re-localized to the plasma membrane by means of using the mutant dynamin. Altogether, this thesis work provides the temporal resolution of signaling patterns of two chemokine receptors for the first time as well as valuable tools that can be applied to characterize their activation in response to pharmacologically relevant ligands. / Der CXC Chemokin‐Rezeptor 4 (CXCR4) und der atypische Chemokin‐Rezeptor 3 (ACKR3) sind heptatransmembranäre Rezeptoren, die in zahlreichen Krankheitsbildern eine Rolle spielen, wie in einigen Krebsarten. Beide Rezeptoren werden zwar von dem gleichen Chemokin CXCL12 aktiviert, allerdings mit unterschiedlichen Signalweiterleitungsmustern. Die Aktivierung von CXCR4 führt zu kanonischer GPCR Signaltransduktion über Gi‐Proteine und β‐Arrestine. Die Signalweiterleitung des Rezeptors ACKR3 hingegen, welcher hauptsächlich in intrazellulären Vesikeln vorliegt, erfolgt über ß‐Arrestinabhängige Signalwege. Es ist von großer Wichtigkeit die Dynamik und Kinetik dieser beiden Rezeptoren hinsichtlich der Aktivierung durch ihre Liganden und der Signalweiterleitung zu verstehen. In dieser Arbeit wurden verschiedene Förster‐Resonanzenergietransfer (FRET) Anwendungen kombiniert, um die frühen Phasen der Signal‐Kaskade von CXCR4 und ACKR3 zu untersuchen. Zur genaueren Aufklärung der Rezeptoraktivierung wurden intramolekulare FRET‐Sensoren entwickelt, hierzu wurden die Fluorophore Cyan‐fluoreszierendes Protein und engl. fluorescence arsenical hairpin binder verwendet. Die generierten Sensoren zeigten ähnliche funktionelle Eigenschaften wie die unveränderten Rezeptoren. Liganden‐induzierte Änderungen der Rezeptorkonformation können mittels dieser Sensoren beobachtet werden und stellen die ersten RET‐basierten Sensoren auf dem Forschungsgebiet der Chemokin‐Rezeptoren dar. Weitere FRET‐basierte Methoden wurden zur Untersuchung von Interaktionen zwischen Rezeptor und G‐Protein, Neuanordnung von Dimeren, sowie der G‐Protein Aktivierung eingesetzt und für beide Chemokin‐Rezeptoren etabliert. CXCR4 zeigte einen komplexen Aktivierungsmechanismus nach Stimulation durch CXCL12, bei welchem zunächst eine Neuordnung der Rezeptor‐Transmembrandomäne gefolgt von Neuordnungen zwischen Rezeptor und G‐Protein und zuletzt eine Neuordnung zwischen CXCR4 Protomeren erfolgte. Dies impliziert, dass im Aktivierungsprozess des Rezeptors Homodimere eine Rolle spielen. Zudem wurde eine verlängerte Gi ‐Protein Aktivierung gegenüber der Gq‐Protein Aktivierung bei CXCL12 stimuliertem CXCR4 beobachtet. Hingegen zeigte eine Stimulierung mit dem Macrophage Migration Inhibitory Factor (MIF) bei jedem Schritt der frühen Singal‐Kaskade veränderte Dynamiken und Kinetiken im Vergleich zu CXCL12. Darüber hinaus konnte keine Gi ‐Protein Aktivierung festgestellt werden. Dieser Befund zeigt individuelle Mechanismen für MIF und CXCL12 am CXCR4‐Rezeptor und liefert Belege für eine neuer Art von sequenziellen Signalweiterleitungen an GPCRs. Eine wichtige Beobachtung dieser Arbeit für eine potentielle Medikamentenentwicklung ist das CXCR4 ligandenunabhängige Aktivität zeigt. Um die Aktivierung des ACKR3 Sensors messen zu können wurde durch eine Co‐Transfektion mit K44A Dynamin eine höhere Membranständigkeit erreicht. CXCL12 und drei weiteren Agonisten zeigten am hier entwickelten ACKR3‐Sensor unterscheidbare Kinetiken. Mit diesem wertvollen Werkzeug können Liganden an diesem atypischen Rezeptor pharmakologisch charakterisiert werden. Es konnte keine CXCL12‐induzierte G‐Protein Aktivierung gemessen werden, trotz der stärkeren Präsenz an der Plasmamembran mit Hilfe der Dynamin‐Mutante. In Summe liefert diese Arbeit zum ersten Mal eine zeitliche Auflösung von Signalweiterleitungsmustern von zwei Chemokin‐Rezeptoren sowie wertvolle Werkzeuge zur Charakterisierung der frühen Phase der Signal‐Kaskade durch andere pharmakologisch relevanten Liganden.