Abstract FAP52 (focal adhesion protein, 52 kDa) is a focal adhesion-associated protein composed of a highly α-helical NH2-terminus containing a poorly characterized FCH (Fes/CIP4 homology) domain, unstructured linker region and the COOH-terminal SH3 domain. FAP52 is also known as PACSIN 2 or syndapin II. Together with other PACSINs and syndapins FAP52 shares a common domain architecture. The aim of this study was to characterize FAP52 in structural and functional terms. The function was pursued by identifying binding partners for FAP52, and by overexpressing the recombinant FAP52 in cultured cells. For the structural studies, various physico-chemical methods, such as chemical cross-linking, gel filtration chromatography, circular dichroism and X-ray crystallography were applied. In addition, the histological distribution of FAP52 in chicken tissues was explored. FAP52 binds filamin, a protein that regulates the dynamics of the cytoskeleton by crosslinking actin filaments. The binding site in FAP52 was mapped to the NH2-terminal 184 amino acids, of which the residues 146–184 form the core of the binding. In filamin, the binding site resides in the repeats 15–16 in the rod-like molecule encompassing 24 such repetitive domains. Overexpression of FAP52 or its filamin-binding domain in chicken embryo heart fibroblasts induced the formation of filopodial extensions on the cell surface and reduced the number of focal adhesions, suggesting a role in the organization of the cellular cytoskeleton and in cell adhesion machinery. Experiments utilizing surface plasmon resonance analysis, size exclusion chromatography and chemical cross-linking showed that FAP52 self-associates in vitro and in vivo. The region responsible for the self-association was mapped to the amino acids 146–280, which is predicted to fold into a coiled-coil arrangement. FAP52 was crystallized by using the hanging-drop vapor-diffusion method and ammonium sulfate grid screen. Native dataset was collected from two crystals, which diffracted to 2.8 Å and 2.1 Å resolution. For one form of crystals, phasing was performed using the native dataset and the datasets from two xenon-derivatized crystals. X-ray crystallography studies revealed a dimer in asymmetric unit. Histological and in vitro studies showed that, in liver, FAP52 is preferentially expressed in bile canaliculi. In other tissues, FAP52 showed a specific staining pattern in gut, kidney, brain and gizzard. Together, these data show that FAP52 self-associates in vivo and, probably via its interaction with its binding partner filamin, participates in the organization of the cytoskeletal architecture, especially of the cell surface protrusions, such as filopodia and microvilli of bile canaliculi.

Osteocytes have been proposed as the mechanosensory cells during the process of bone adaption. In this thesis, a microfluidics chamber system (MCS) device was designed, fabricated and tested as a means to maximally simulate the in vivo osteocytic ultrastructure and reproduce the in vivo shear stress experienced by osteocyte, providing an ideal platform for in vitro study on osteocyte mechanotransduction. By employing a micropipette aspiration technique, single osteocyte adhesion and osteocytic process formation were achieved on PDMS with MCS structure. In this study, the involvement of sphingosine-1-phosphate (S1P) signaling pathway in osteocytes responding to oscillatory fluid flow (OFF) was also examined. Firstly, MLO-Y4 osteocytes like cells were demonstrated to express integrated and functional S1P cascade. By modulating S1P cascade components and testing a series of cellular outcomes, it was indicated that exogenous S1P, endogenous S1P and S1P receptor S1P2 were involved in the regulation of loading induced osteocytic responses.

Osteocytes have been proposed as the mechanosensory cells during the process of bone adaption. In this thesis, a microfluidics chamber system (MCS) device was designed, fabricated and tested as a means to maximally simulate the in vivo osteocytic ultrastructure and reproduce the in vivo shear stress experienced by osteocyte, providing an ideal platform for in vitro study on osteocyte mechanotransduction. By employing a micropipette aspiration technique, single osteocyte adhesion and osteocytic process formation were achieved on PDMS with MCS structure. In this study, the involvement of sphingosine-1-phosphate (S1P) signaling pathway in osteocytes responding to oscillatory fluid flow (OFF) was also examined. Firstly, MLO-Y4 osteocytes like cells were demonstrated to express integrated and functional S1P cascade. By modulating S1P cascade components and testing a series of cellular outcomes, it was indicated that exogenous S1P, endogenous S1P and S1P receptor S1P2 were involved in the regulation of loading induced osteocytic responses.

Das Ziel der Studien, die im Rahmen dieser Arbeit vorgestellt werden, war es neue Informationen über die elementare Zusammensetzung des mineralisierten Knochens zu gewinnen. In einer ersten Studie wurden zwei Parameter verglichen, die beide eng mit der Knochenmineralisierung verknüpft sind. So zeigte die Gegenüberstellung des mineral/matrix Raman-Wertes und der Kalziumkonzentration gute Übereinstimmung mit theoretischen Überlegungen. Diese Methoden wurden auch verwendet um Knochengewebe von Mäusen zu charakterisieren bei denen ein genetischer Defekt zu einem Mangel von Sclerostin führte. So war es möglich nachzuweisen, dass eine hierdurch verstärkte Knochenneubildung zu einer veränderten Mineralisationskinetik des Knochens führen kann. Nachdem zukünftig Sclerostinantikörper für die Behandlung von Knochenkrankheiten eingesetzt werden sollen, haben diese Erkenntnisse große medizinische Bedeutung. Es wurde auch die Mineraldichteverteilung eines Mausmodells mit fragilem Knochen (Osteogenesis Imperfecta, OI) untersucht. Die Mäuse wurden mit Sclerostinantikörpern behandelt. Es zeige sich ein signifikanter Knochenzuwachs doch die Mineraldichteverteilung veränderte sich gleichermaßen für gesunde und für OI Mäuse. In einer Studie am humanen Knochen konnten der Zusammenhang zwischen Osteozytennetzwerk und Knochenzusammensetzung untersucht werden. Elemente wie Na, Mg und S wiesen typische Konzentrationsverteilungen auf. Die Routinen wurden auch verwendet um Mineralisationsfronten zu charakterisieren. Es zeigte sich, dass die Konzentrationen von K, Mg, Na und Cl abhängig von dem analysierten anatomischen Ort, stark voneinander abweichen. Abschließend kann gesagt werden, dass durch die Entwicklung neuer Routinen zusätzliche Erkenntnisse über die Knochenmineralisierung und Zusammensetzung gewonnen werden konnten. Die Resultate sind von medizinischer und biologischer Bedeutung und tragen zu aktuellen Debatten über die Knochenentwicklung bei. / The purpose of the presented work was to gain new insight into the elemental composition of mineralized bone matrix at different sites of human bone tissue, and in mouse models linked to human genetic diseases. Using novel tools and routines, human (femur cross sections from healthy adults and children) and murine samples (femur long-and cross sections of two mouse models) were analyzed with focus on the elemental composition. In a methodological study the consistency of matrix mineralization measured by Raman microspectroscopy (e.g. the mineral/matrix ratio) and the Calcium content (wt%Ca) as measured by qBEI was proved. Both methods were applied to a mouse model exhibiting induced bone overgrowth due to a genetic defect causing a lack of Sclerostin, which is a negative regulator for bone formation. We found changes in the mineralization kinetics depending on the anatomical site. This result is of clinical importance since Sclerostin antibodies are suggested for future treatment of diseases characterized by fragile bone. Hence, also a mouse model of a brittle bone disease (Osteogenesis Imperfecta) was analyzed with and without Sclerostin antibody treatment. A significant increase in bone mass was documented while the mineralization pattern revealed no interaction between genotype and treatment. The correlation between OLCN and the composition of the mineralized matrix was examined in the same regions of human compact bone. Characteristic distributions of the minor elements (Mg, Na, S) were found. The developed tools were also used to investigate mineralization fronts, reflecting a critical stage of bone development. Differences in the Ca/P ratio and in the concentrations of K, Mg, Na and Cl depending on the anatomical site were revealed. In conclusion, using newly developed measurement routines, it was possible to gain novel information of bone mineralization and composition. The results contribute to actively debated issues of biological and medical importance.

Knochen ist ein lebendes Material, das seine Struktur an die mechanische Umgebung anpasst. Zur strukturellen Anpassung muss der Knochen die mechanische Belastung erfassen. Allerdings sind Knochen mechanisch so steif, dass die lokalen Verformungen zu klein sind um von den Knochenzellen direkt detektiert zu werden. Osteozyten sind Knochenzellen, die ein Zellnetzwerk in der mineralisierten Matrix bilden. Ihre Zellkörper sind in Lakunen untergebracht und ihre Zellprozesse in engen Kanälchen, den Canaliculi. Die Hypothese des Flüssigkeitsflusses besagt, dass der lastinduzierte Flüssigkeitsfluss durch dieses Lakunen-Canaliculi-Netzwerk (LCN) einen Verstärkungsmechanismus bereitstellt, der es den Osteozyten ermöglicht, die dynamische Belastung des Knochens zu erfassen. Wir stellen die Hypothese auf, dass die Architektur des LCN eine wesentliche Rolle in Bezug auf die Mechanosensitivität spielt, da sie den Flüssigkeitsfluss beeinflusst. Das zentrale Ziel dieser Arbeit ist es, diese Hypothese an realen LCN-Architekturen mit einem Modell des lastinduzierten Flüssigkeitsflusses zu testen und den resultierenden Fluss mit der Mechanoreaktion des Knochens zu vergleichen. Wir haben das LCN mithilfe konfokaler Laser-Scanning-Mikroskopie untersucht. Wir haben dann die auf den Kirchhoffschen Gesetzen basierende Schaltungstheorie verwendet, um die Geschwindigkeiten der Flüssigkeit in allen abgebildeten Canaliculi zu modellieren und darzustellen wie sich die verdrängte Flüssigkeit über das LCN verteilen würde. Basierend auf diesen Geschwindigkeiten wurde die Mechanoreaktion des Knochens vorhergesagt. In meiner Studie wurden die Knochen von Mäusen verwendet, wodurch kontrollierte in vivo Belastungsexperimente und die Messung der Mechanoreaktion in Bezug auf gebildeten bzw. resorbierten Knochen unter Verwendung von in vivo µCT möglich waren. Die Flüssigkeitsströmungsmuster durch das LCN korrelierten mit der gemessenen Mechanoreaktion. Das heißt, Knochenbildung wurde in Bereichen nahe höherem Fluss beobachtet, während Knochenabbau in Bereichen nahe geringem Fluss beobachtet wurde. Die Vorhersage der Mechanoreaktion unter Berücksichtigung der Architektur des LCN war quantitativ besser als eine Vorhersage, die nur auf mechanischer Belastung basiert. Qualitativ haben wir festgestellt, dass Gefäßkanäle im Kortex als lokale Senken des Flüssigkeitsflusses fungieren und daher den Fluss an der nahegelegenen Knochenoberfläche reduzieren. Im Gegensatz dazu nahmen die Strömungsgeschwindigkeiten für konvergente Netzwerkstrukturen zu, bei denen die Zahl der Kanäle zur Knochenoberfläche hin abnimmt. In einem zweiten Projekt konzentrierten wir uns auf gesunden, menschlichen osteonalen Knochen. Osteone sind zylindrische Strukturen um Gefäßkanäle, die praktisch vom umgebenden Knochen abgeschottet sind. Wir analysierten acht gewöhnliche Osteone mit einem nahezu homogenen LCN und neun Osteon-in-Osteonen, die durch eine ringartige Zone mit geringer Netzwerkkonnektivität zwischen dem inneren und dem äußeren Teil dieser Osteone gekennzeichnet sind. In Canaliculi, die die beiden Teile des Osteons in Osteonen überbrücken, wurde ein wesentlich höherer lastinduzierter Flüssigkeitsfluss beobachtet als in anderen Canaliculi. Dies führte dazu, dass der durchschnittliche Fluss 2,3-mal höher war als bei normalen Osteonen. Es ist daher wahrscheinlich, dass Osteon-in-Osteon-Konstruktionen besonders zur Mechanosensitivität des kortikalen Knochens beitragen. Die Untersuchungen in dieser Doktorarbeit legen nahe, dass die LCN-Architektur neben der mechanischen Belastung als Schlüsselfaktor für die Knochenanpassung dient. / Bone is a living material, which adapts its structure in response to the mechanical environment. For structural adaptation bone need to sense the mechanical loading. However, bone is so stiff that the local strains are too small to be directly sensed by bone cells. Osteocytes are bone cells that form a cell network located within the mineralized matrix. Their cell bodies are housed in lacunae and their cell processes in narrow canals, the canaliculi. According to the fluid flow hypothesis, load induced fluid flow through this lacunocanalicular network (LCN) provides an amplification mechanism which allows osteocytes to sense dynamic loading of the bone. We hypothesize that the network architecture of the LCN plays an essential role in bone’s mechanosensitivity, as it influences the fluid flow. We aimed to test these hypotheses by using real LCN architectures in a model of load induced fluid flow, and compare the resulting flow with the mechanoresponse of bone. We imaged the LCN using confocal laser scanning microscopy (CLSM). Image processing was then used to describe the LCN as a mathematical network consisting of edges and nodes, representing the canaliculi and their connections respectively. We then employed circuit theory, based on Kirchhoff’s laws, to model the velocities of the fluid in all the imaged canaliculi. Based on these velocities, the mechanoresponse of bone was predicted. Mice were used in my study, as this allowed a controlled in vivo loading and a measurement of the mechanoresponse in terms of formed/resorbed bone using in vivo µCT. Fluid flow patterns through the LCN of mice correlated with the measured mechanoresponse, i.e., bone formation was observed near surfaces of higher flow, while resorption was observed near surfaces with low flow. The prediction of the mechanoresponse considering the architecture of the LCN was quantitatively better than a prediction based on strains only. Qualitatively, we identified that vascular canals in the cortex act as local sinks of fluid flow and, therefore, reduce the flow at the nearby bone surface. In contrast, flow velocities increased in convergent network structures, where the flow is channeled into fewer canaliculi nearby the surface. In a second project we focused on healthy human osteonal bone. Osteons are cylindrical structures around vascular canals, which are practically sealed off from the surrounding bone. We analyzed 8 ordinary osteons with a rather homogeneous LCN, and 9 osteon-in-osteons, which are characterized by a ring-like zone of low network connectivity between the inner and the outer parts of these osteons. A substantially higher load-induced fluid flow was observed in canaliculi that bridge the two parts of the osteon-in-osteons. This resulted in an average flow, which was 2.3 times higher compared to ordinary osteons. It is therefore likely that osteon-in-osteons particularly contribute to the mechanosensitivity of cortical bone. Based on both studies in this PhD thesis we conclude that LCN architecture should be considered as a key determinant of bone adaptation besides mechanical loading.

La cholestase intrahépatique représente environ 40% des lésions hépatiques induites par les médicaments et se caractérise par une accumulation intracellulaire des acides biliaires (AB). Les mécanismes impliqués sont encore mal connus et sa prédiction reste difficile. Le but de ce travail était de caractériser dans la cholestase d’origine médicamenteuse et de développer des méthodes de screening pour sa prédiction précoce, en utilisant la lignée humaine hépatique HepaRG et les hépatocytes humains. Tout d’abord, nous avons démontré que la motilité des canalicules biliaires (CB) est indispensable à la clairance des AB et requiert une alternance de phosphorylation/déphosphorylation de la chaine légère de la myosine (MLC), contrôlé par la voie Rho-kinase/Myosin Light Chain Kinase (ROCK/MLCK). Nous avons ensuite montré pour la première fois que les médicaments cholestatiques altèrent la voie ROCK/MLCK/MLC et la dynamique des CB. En utilisant la famille des antibiotiques résistant à la pénicillinase, dont fait partie la flucloxacilline, responsable de nombreux cas de cholestase, nous avons observé que la dérégulation de ROCK pouvait se faire par activation de HSP27, associée aux voies de signalisation PKC/P38 et PI3K/AKT. Enfin, nous avons montré une capacité variable des médicaments cholestatiques à moduler les profils des AB. En effet, les médicaments cholestatiques majeurs induisent une accumulation préférentielle des AB hydrophobes toxiques, in vitro, dans les premières 24h, qui résulte d’une inhibition de leur amidation. Au total, l’ensemble du travail a permis de progresser dans la compréhension des mécanismes impliqués dans la cholestase d’origine médicamenteuse et de mettre en évidence de nouveaux biomarqueurs utiles pour sa prédiction. / Intrahepatic cholestasis represents around 40% of drug-induced liver injuries and is characterized by intracellular accumulation of bile acids (BA); mechanisms involved and its accurate prediction remains challenging. The aim of the current work was to characterize the mechanisms involved in drug-induced cholestasis and to develop screening methods for its early prediction, using human differentiated HepaRG and primary human hepatocytes. First, we demonstrated that bile canaliculi (BC) motility is essential for BA clearance and requires alternating phosphorylation/dephosphorylation of myosin light chain (MLC) that is controlled by the Rho-kinase/Myosin Light Chain Kinase (ROCK/MLCK) signaling pathway. Then, we showed, for the first time that cholestatic drugs could alter the ROCK/MLCK/MLC pathway and BC dynamics. Using the penicillinase-resistant antibiotics family, including flucloxacillin that is responsible for many cases of cholestasis, we found that deregulation of ROCK could be modulated by HSP27, associated with PKC/P38 and PI3K/AKT signaling pathways. Finally, we evidenced variable potency of cholestatic drugs to modulate BA profiles. Indeed, the well-known cholestatic drugs induced a preferential accumulation of unconjugated toxic hydrophobic BA in vitro within the first 24h that resulted from inhibition of their amidation. Altogether, these data bring new information on the understanding of the mechanisms involved in drug-induced cholestasis and highlight new morphological and molecular predictive biomarkers of drug-induced cholestasis.

La cholestase est l'une des manifestations les plus courantes des lésions induitespar des médicaments. Dans 40% des cas elle n’est pas prévisible; une meilleure prédictibilité représente donc un défi majeur. Tout d'abord, nous avons démontré que les cellules hépatiques humaines HepaRG différenciées sont un modèle approprié pour étudier la cholestase induite par les médicaments en comparant la localisation et l’activité des transporteurs d'influx et d'efflux avec les hépatocytes humains primaires. Tous les transporteurs d'efflux et d’influx testés ont été correctement localisés au niveau des membranes canaliculaire (BSEP, MRP2, MDR1 et MDR3) et basolatéral (NTCP, MRP3) et sont fonctionnels. En outre, ces cellules expriment également les enzymes qui métabolisent les acides biliaires (ABs) et ont la capacité de les synthétiser et de les conjuguer avec la taurine, la glycine et le sulfate, à un taux similaire à celui des hépatocytes primaires. Des changements ont été observés sur la répartition des ABs totaux après traitements de cellules HepaRG par un médicament cholestatique, la cyclosporine A (CsA), de manière concentration- dépendante. L'inhibition de l'efflux et de l'influx de taurocholate a été observée après 15 min et 1 h respectivement. Ces premiers effets ont été associés à la dérégulation de la voie des cPKC et l'induction d’un stress du réticulum endoplasmique puis d’un stress oxydant. Nous avons également montré pour la première fois une accumulation intracellulaire d’ABs endogènes avec un médicament cholestatique in vitro. En outre, notre travail apporte des preuves que la motilité des canalicules biliaires (BC) est indispensable à la clairance des ABs. La voie ROCK et le complexe actomyosine sont fortement impliqués. Nous avons fourni la première démonstration que la voie ROCK et les dynamiques des BC sont des cibles majeures des composés cholestatiques. Nos données devraient contribuer à l'élaboration de méthodes de screening pour la prédiction précoce des effets secondaires induits par les médicaments cholestatiques. / Cholestasis is one of the most common manifestations of drug-induced liver injury (DILI). Since up to now it is unpredictable in 40% of all cases its accurate prediction represents a major challenge. First, we validated that differentiated HepaRG human liver cells are a suitable in vitro model to study drug-induced cholestasis, by comparing localization of influx and efflux transporters and their functional activity in these cells and primary human hepatocytes. All tested influx and efflux transporters were correctly localized to canalicular (BSEP, MRP2, MDR1, and MDR3) or basolateral (NTCP, MRP3) membrane domains and were functional. In addition, the HepaRG cell line also exhibits bile acids (BAs) metabolizing enzymes and has the capacity to synthesize BAs and to further amidate these BAs with taurine and glycine as well as sulfate, at a rate similar to that of primary hepatocytes. Concentration- dependent changes were observed in total BAs disposition after treatment of HepaRG cells by the cholestatic drug cyclosporine A (CsA). Inhibition of efflux and uptake of taurocholate was evidenced as early as 15 min and 1 h respectively. These early effects were associated with deregulation of cPKC pathway and induction of endoplasmic reticulum stress that preceded generation of oxidative stress. We also showed for the first time intracellular accumulation of endogenous BAs by a cholestatic drug in vitro. In addition, our work brings evidences that motility of bile canaliculi (BC) is essential for BAs clearance where ROCK pathway and actomyosin complex are highly implicated. We provided the first demonstration that ROCK pathway and BC dynamics are major targets of cholestatic compounds. Our data should help in the development of screening methods for early prediction of drug-induced cholestatic side effects.

La cholestase intrahépatique est une manifestation fréquente des lésions hépatiques induites par les médicaments; Cependant, les mécanismes impliqués sont peu connus. Nous avons cherché à étudier les mécanismes de la cholestase induite par les médicaments afin d’améliorer sa détection précoce en utilisant les cellules HepaRG humaines. Tout d'abord, nous avons prouvé que les canalicules biliaires (BC) subissaient des contractions spontanées, essentielles pour l'efflux d’acides biliaires et nécessitaient des séries d’alternance dans la phosphorylation/déphosphorylation de la chaîne légère de myosine (MLC2). La courte exposition à des composés cholestatiques a révélé que la modulation des BC était associée à des perturbations de la voie de signalisation ROCK/MLCK. Afin de confirmer notre étude, 12 médicaments cholestatiques et six non cholestatiques ont été analysés et nous avons démontré que tous les médicaments cholestatiques classés sur la base de résultats cliniques provoquaient des perturbations dans la dynamique des BC (dilatation ou constriction), et altéraient la voie de signalisation ROCK/MLCK, tandis que les composés non cholestatiques n'avaient pas d'effet. Nous avons également prouvé que ces changements étaient plus spécifiques que la mesure de l'inhibition de l’efflux comme marqueurs prédictifs non cliniques de la cholestase induite par les médicaments. Afin de confirmer et d’étendre ces conclusions, nous avons analysé les mécanismes impliqués dans les effets cytotoxiques et cholestatiques induits par 4 médicaments de la famille des antagonistes des récepteurs de l'endothéline: deux ayant un lien avec des cas cliniques d'hépatotoxicité (sitaxentan) et/ou cholestase (bosentan), et deux n’ayant pas été impliqués dans l’élévation de transaminases hépatiques ou de bilirubine (ambrisentan et macitentan). Les résultats montrent que le macitentan récemment commercialisé et ayant une structure chimique similaire à celle du bosentan, était capable de causer, comme ce dernier, des altérations in vitro des BC. En revanche, l'ambrisentan n’était pas hépatotoxique et le sitaxentan qui a été retiré du marché pour des cas d’hépatotoxicité, n’affectait pas la dynamique canaliculaire. / Intrahepatic cholestasis represents a frequent manifestation of drug-induced liver injuries; however, the mechanisms involved in such injuries are poorly understood. We aimed to investigate mechanisms underlying drug-induced cholestasis and improve its early detection using human HepaRG cells. First, we proved that bile canaliculi (BC) underwent spontaneous contractions, which are essential for bile acid efflux and required alternations in myosin light chain (MLC2) phosphorylation/dephosphorylation. A short exposure to cholestatic compounds revealed that BC dynamics was altered and associated with impairment of the ROCK/MLCK pathway. Then, in order to confirm our study, 12 cholestatic drugs and six noncholestatic drugs were analyzed and we demonstrated that all cholestatic drugs classified on the basis of reported clinical findings caused disturbances of both BC dynamics (dilatation or constriction), and alteration of the ROCK/MLCK signaling pathway, whereas noncholestatic compounds had no effect. We also proved that these changes were more specific than efflux inhibition measurements alone as predictive nonclinical markers of drug-induced cholestasis. To confirm and extend these conclusions, we analyzed the mechanisms involved in cytotoxic and cholestatic effects induced by the 4 main drugs from the endothelin receptor antagonists family: two related to clinical cases of hepatotoxicity (sitaxentan) and/or cholestasis (bosentan), and two that have not been reported to cause elevation of liver transaminases or bilirubin (ambrisentan and macitentan). The results showed that like bosentan, the structurally similar recently marketed drug, macitentan, could cause in vitro major BC alterations. By contrast, ambrisentan appeared as a safe drug and sitaxentan that has been withdrawn from the market for hepatotoxic cases, did not impair BC dynamics.